Synthesis, Spectral Characterisation and Cytotoxic Evaluation of Substituted Sulfonamide Schiﬀ Bases

Objectives: To synthesize Schiﬀ bases i.e 4-((2-hydroxybenzylidene)-amino–N-(5-methyl-1,2-oxozol-3- yl)benzene sulphonamide (L1), 4-((2-hydroxy benzylidene)-amino-N-(thiazol-yl)benzene sulphonamide (L2), 4-((2-hydroxybenzylidene)amino-N-(pyridin-2-yl)benzene sulphonamide by the action of 2-hydroxybenzaldehyde with sulfathiazole/ sulfamethoxazole/ sul-fapyridine in ethanolic media. Methods: The Schiﬀ bases obtained were characterized by analytical data, IR, UV, 1 H- NMR, 13 C-NMR, Mass spectrum and monitored for cytotoxic activity against human breast cancer cell [MDA MB -231] line. Findings: The Schiﬀ bases behaves as a bidentate ligand with oxygen and nitrogen as chelating positions and coordinates via phenolic oxygen and azomethine nitrogen. The composition of the ligands has been established by elemental analysis. Structural features and bonding mode of the Schiﬀ bases have been proposed by spectral methods. The evaluated synthesized ligand shows excellent cytotoxic activity towards breast cancer cell line. Novelty: The evaluated highly biologically active L1 and L3 shows desirable cytotoxic activity towards [MDA MB -231] breast cancer cell line. The better IC 50 value of the Schiﬀ base ligands upgrades as chemotherapeutic agents which leads to drug formulation and induces DNA binding studies.

Previously a number of biologically important Schiff bases have been reported by our group. (11)(12)(13) To broaden the scale of investigation on the Schiff bases, the present investigation records the synthesis followed by characterization of Schiff bases derived from sulpha drugs.

Materials and Methods
All chemicals and reagents used were of AR grade except ethanol which was purified prior to use. Solvents were purified and dried according to the standard procedures. Elemental analysis of the ligands was obtained using EL CHN rapid analyzer. IR spectra of the complexes were recorded as KBr pellets on a SHIMADZU 8000-FT IR spectrophotometer. The 13 C NMR and 1 H NMR spectra of the ligand was recorded with a Bruker Spectro spin advance (DPX-400) using TMS as internal standard and DMSO&#8209;d6 as solvent. Melting points were determined by open capillary method (silicon bath electric melting point apparatus) and uncorrected. The electronic spectra of the ligands in UV -visible region was measured by using Perkin Elmer Lambda 35 spectrometer provided with quartz cells.

Synthesis of Schiff-Base ligands
To an ethanolic solution of 2-hydroxybenzaldehyde (0.01mole) an ethanolic solution of sulphamethoxazole /sulphathiazole/sulphapyridine (0.01mole) was added. The reaction mixture was refluxed for 4 to 5 hours. The coloured solid mass formed during refluxing was cooled, filtered, washed thoroughly with ethanol and dried a compound in a desiccator. The compound was recrystallisation from ethanol.

In Vitro cytotoxicity assay
The in vitro cytotoxicity of the newly synthesized ligands was carried out in human breast tumor cell lines. Cell line namely human breast cancer cell line [MDA MB -231] (NCL-Pune) was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) assay (14) . In Vitro cytotoxicity assay was carried out in the Laboratory of South India Textile Research Association (SITRA), Coimbatore, Tamil Nadu, INDIA.

Cell treatment procedure
The monolayer cells were detached with trypsin ethylene diamine tetra acetic acid (EDTA) to make single cell suspensions and viable cells were counted by trypan blue exclusion assay using a hemocytometry. The cell suspension was diluted with medium containing 5%FBS TO GIVE FINAL DENSITY OF 1X10 5 cells/ml. one hundred microliters per well of cell suspensions were seeded into 96-wellplates at plating density of 10000 cells/well and incubated to allow for cell attachment at 370 o C, 5%CO 2 , 95% air and 100% relative humidity. After 24 hrs the cells were treated with serial concentrations of the test samples. They were initially dissolved in neat dimethyl sulfoxide (DMSO) and diluted to twice the desired final maximum test concentration with serum free medium. Additional four, serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100µl of these different sample dilutions were added to the appropriate wells already containing 100µl of medium, resulted the required final sample concentrations. Following drug additional the plates were incubated for an additional 48 hrs at 370 o C, 5%CO 2 , 95% air and 100% relative humidity. The medium without samples were serving as control and triplicate was maintained for all concentrations.
After 48hrs of incubation, 15µl of MIT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370 o C for 4 hrs. the medium with MIT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570nm using micro plate reader.
The cytotoxic activity of the synthesized Schiff base were tested into five series of dilutions. The concentration of the compounds at percentage cell inhibition growth was calculated.
The percentage cell inhibition was determined using the formula.
The ligands were characterized by • Analytical data-colour, melting point, elemental analysis.

Analytical data
The analytical data and physical characteristics of the Schiff base ligands are indicated in the Table 1 .

IR Spectrum
The ligands used in the present investigation contains five donor sites 1. phenolic oxygen 2. azomethine nitrogen 3. sulfonamide oxygen 4. sulfonamide nitrogen 5. ring nitrogen The vibrational spectra of the ligands shows a band in the region of 1616 cm -1 -1627cm -1 corresponds to ν (>c=N−) group 18 and another broad band between 3418 cm -1 -3471 cm -1 which is the characteristic frequency of hydrogen bonded phenolic ν (O−H) stretching vibration (18,19) .

1 H-NMR spectrum
The proton magnetic resonance spectrum of the Schiff bases was taken in DMSO -d 6 solvent was shown in Figures 1, 2

Mass Spectrum
The mass spectral data of the ligand is consistent with the formulation corresponds to [M+3] and M peaks respectively shown in (Figures 7 and 8 Based on the above spectral studies the following structure is proposed for the Schiff bases,

Cytotoxic Activity
The cytotoxic activity of the synthesized ligands is shown in the Figures 9, 10 and 11. Tables 2, 3    The predominant activity of the ligands derived from L1 and L3 may be accounted for in terms of the presences of oxazole and thiazole units in the synthesized ligands. The concentration of the complex at 50% cell growth was inhibited and IC 50 was calculated as shown in Table 4.

Conclusion
The bidentate coordination ability of the newly synthesized azo Schiff bases was proved by IR, UV, NMR and mass spectra which confirms two donor sites: azomethine nitrogen and phenolic oxygen. The synthesized Schiff bases were subjected to anticancer activity against human breast cancer cell [MDA MB -231] line. The order of activity is Ligand(L1) =Ligand (L3)> ligand (L2). The predominant activity of the ligands derived from L1 and L3 is due to the presence of oxazole and thiazole units in the synthesized ligands. The better IC 50 cytotoxic activity of the ligands against breast cancer cell [MDA MB-231] line may pose a significant role in metallodrug formulation in the field of bioinorganic chemistry. The biological activity of the Schiff bases was enhanced by complexation with metal ions.