Synthesis and Anti-cancer Studies of 2 , 6-disubstituted Benzothiazole Derivatives

On the basis of exhaustive literature review, it has been found that benzothiazole derivatives have good potential to exhibit anticancer activity15-17. So the present study involves the synthesis of 2,6-disubstituted benzothiazole followed by preliminary cytotoxicity screening against three human cancer cell lines( MCF-7, HeLa and MG63) using MTT assay at 48 h of exposure.


INTRODUCTION
The development of new anticancer therapeutic agents is one of the fundamental goals in medicinal chemistry.A dose of anticancer drug sufficient to kill tumour cells is often toxic to the normal tissue and leads to many side effects, which in turn, limits its treatment efficacy.In recent years, there has been a concerned search for the discovery and development of novel selective antitumor agents.
Benzothiazoles are bicyclic ring system with multiple applications.2-substituted benzothiazole scaffold is one of the privileged structure in medicinal chemistry 1,2 and reported cytotoxic on cancer cells [3][4][5] .In a recent years number of such derivatives have exhibited interesting anticancer activities [6][7][8][9][10][11] .Among the anti-tumor drugs doublets), t (triplet), q (quartet) and m(multiplet).Infrared measurements were recorded in the range 400-4000 cm _1 by Perkin Elmer.Elemental analysis was carried out using Perkin Elmer CHNS.Mass spectra were recorded on a Thermo LCQ Deca XP MAX at70eV.Thin layer chromatography (TLC) analysis was carried out on 5x20 cm plate coated with silica gel GF 254 .

Synthesis of N-(6-amino-1,3-benzothiazol-2-yl) acetamide (2)
A solution of compound 1(10g,0.0421mol) in 80 ml 12N HCl ,SnCl 2 .2H 2 O(47.4g,0.210mol) was added at RT.The reaction mixture was stirred at RT for 2 hr.The reaction was monitored by TLC.The reaction mixture was diluted with 300 ml of cold water.The solution was basified to pH 9 with 40% NaOH and the aqueous layer was extracted with ethyl acetate (3x150 mL),washed with water (2×200 mL), Brine(1x250mL) and dried over anhydrous Na 2 SO 4 .The solvent was concentrated under reduced pressure to give the compound

Procedure for synthesis N-(6-{[(4c y c l o h e x y l p h e n y l ) s u l f o n y l ] a m i n o } -1 , 3benzothiazol-2-yl)acetamide (4b)
Prepared as reported above for 4a starting from compound 2 and 4-cyclohexylbenzene-

Procedure for synthesis N-(6-{[(4-fluorophenyl) c a r b a m o y l ] a m i n o } -1 , 3 -b e n z o t h i a z o l -2yl)acetamide (5a)
To the solution compound 2 (0.3g,0015 mol) in 6 mL THF, 4-fluorophenylisocyanate (0.24g,0.0017mol) in 3 mL dichloromethane was added at 0 o C. The reaction mixture was allowed to RT and stirred for 1 hr.The reaction mixture was concentrated under reduced pressure and partitioning between ethyl acetate (50mL) and water (40mL

Cell lines
The human breast adenocarcinoma (MCF 7), human cervical adenocarcinoma (HeLa) and human osteosarcoma (MG63) cell lines were obtained from National Centre for Cell Science (NCCS), Pune and grown in Minimum Essential Medium containing 10% fetal bovine serum (FBS).The cells were maintained at 37 0 C, 5% CO 2 , 95% air and 100% relative humidity.Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

In vitro cytotoxic activities (MTT assay)
The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspension and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x10 5 cells/ml.One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 0 C, 5% CO 2 , 95% air and 100% relative humidity.After 24 hr, the cells were treated with serial concentrations of the test samples.They were initially dissolved DMSO, and an aliquot of the sample solution was 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 100 µl of these different sample dilutions were added to the appropriate wells already containing 100 µl of medium, resulting in the required final sample concentrations.Following sample addition, the plates were incubated for an additional 48 h at 37 0 C, 5% CO 2 , 95% air, and 100% relative humidity.The medium containing without samples were served as control and triplicate were maintained for all the concentrations.After 48 h of incubation, 15 µl of MTT (5mg/ml) solution was added to each well and incubated at 37 0 C for 4 h.The medium with MTT was then flicked off, and the formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using a micro plate reader.The % cell inhibition was determined using the following formula.% Cell Inhibition = 100-Abs (sample)/Abs (control) x100.

Chemistry
The route for the synthesis of the intermediates and target compounds are shown in the scheme (1-3).The Intermediate 1 was prepared by using the reagent acetic anhydride and pyridine.The FT-IR value at 1701 cm -1 (for C=O), 1 H NMR value  2.23 (3H, singlet) and 13 C NMR  170.1(C=O) which confirms that there is the formation of the acetylated product.The intermediate 2 was synthesized by using the reagent stannous chloride and Con.HCl.The FT-IR value at 3417,3298(for NH2) and 1 H NMR  5.13 (2H, broad peak) which confirm that there is the formation of reduction product.The resulting primary amine also confirmed by ninhydrin activity in TLC.The compound 2 was taken as a common scaffold for the synthesis of the new series of 2, 6-disubstituted benzothiazole derivatives.
The sulfonamide derivatives 4a, 4b, 4c was prepared by using corresponding sulfonyl chloride and compound 2. The reaction was carried out in the presence of pyridine base at 90 o C.The FT-IR stretching frequency 1320-1350 cm -1 and 1150-170 cm -1 (S=O) confirm that there is the formation of sulfonamide derivatives.The sulfonamide NH chemical shift value in 1 H NMR observed at 10-10.5 as a broad singlet.The corresponding isocyanate and isothiocyanate respectively was used to prepare urea and thiourea derivative 5a and 5b.

Biology Cytotoxicity
In vitro cytotoxicity of the synthesized compounds were assessed by standard 3-(4, 5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in MCF-7, HeLa, and MG63 cell lines.Among the synthesized compounds 3a, 3f, 3g, and 4b showed cytotoxic effects on all the three cell lines after 48 h exposure.Maximum cytotoxicity was obtained for the sulfonamide derivative 4b in all the three cell lines when compared to others, and the IC 50 was 36, 44 and 34 µM against MCF7, HeLa and MG63 respectively.The amide derivative 3a also showed cytotoxicity in all the three cell lines and the IC 50 was in the range of 48 to 53 µM.The remaining urea and thiourea derivatives did not show any cytotoxicity activity at the tested concentrations.For standard cisplatin was used.All the results are showed in Table 1.
The known numbers of cells (1x10 5 cells/ ml) were incubated for 24 h in a 5% CO2 Incubator at 37 o C in the presence of different concentrations of test compounds.The medium with MTT was then flicked off, and the formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using a micro plate reader.

CONCLUSIONS
A series of new 2,6-disubstitued benzothiazol derivatives were synthesized, and their anti-cancer activities were evaluated in vitro.The results showed that the cyclohexyl benzene sulphonamide derivative of benzothiazole(4b) showed good activity against MCF-7, HeLa, and MG63 cells.Further amide derivatives 3a,3g,3h also show good activity against MCF7, HeLa, and MG63.These data suggested that the compound 4b may be powerful anticancer agent and be worth being further investigated as a potential of an anticancer agent.