Synthesis and biological activity studies of new triazolothiadiazine derivatives

Gliomas pose a severe danger with a huge death rate among central nervous system tumors. One of the mechanism causing cancer occurrence and development is considered to be cyclooxygenase pathway, which takes an important role in arachidonic acid metabolism. In many studies, it has been revealed that blocking arachidonic acid metabolism prevents the occurence and development of cancer in cells. In this study initially, 4-amino-5-[(5-methoxy-2-methyl-1-(4-chlorobenzoyl)-1H-indol-3-yl)methyl]-4H-1,2,4-triazol-3-thione was synthesized via the solvent-free reaction of indomethacin with thiocarbohydrazide. Then the ring closure reaction of this compound with 2-bromoacetophenone derivatives afforded new 3-[5-methoxy-2-methyl-1-(4-chlorobenzoyl)-1H-indole-3-yl)methyl]-6-(4-substituedphenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives (1-8). The structures of these compounds were elucidated by IR, 1H-NMR, 13C-NMR, mass spectroscopic methods and elemental analysis. The effects of the obtained compounds on the proliferation of T98 human glioma cell line were investigated with MTT assay. Besides, possible apoptotic activities and the measurement of COX-2, caspase 3, 8 and 9, cytochrome c mRNA relative quantity of the effective compounds were performed with flow cytometry and RT-PCR method, respectively. T98 cells were treated with 1, 5, 10, 25, 50 and 100 µM doses of the compounds for 24 hours. It was determined that 3-[5-methoxy-2-methyl-1-(4-chlorobenzoyl)-1H-indole-3-yl)methyl]-6-(4-methylphenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (8) reduced cells' survival rates at 50 and 100 µM doses with 25% and 40% reasonable values, respectively. The apoptosis stimulating percentages of compound 8 in comparison with the control group (5%) at 50 and 100 µM doses were calculated as 11% and 12%, respectively. Besides, in RT-PCR analysis, it was determined that compound 8 reduced COX-2 mRNA levels significantly when compared with the control group, whereas it did not cause any significant change in other parameters (Caspase 3, 8, 9, cytochrome c). This study revealed that compound 8 showed dose-dependent anticancer activity via the inhibition of COX-2 pathway. Key Words: Triazole, triazolothiadiazine, T98 glioma, anticancer, apoptosis, COX-2.