Utility of 6-aza-2-thiothymine in the synthesis of novel [1,2,4]triazolo[4,3-b][1,2,4]triazin-7-one derivatives: synthesis, structure elucidation, molecular docking and in vitro anti-lung cancer activity†

Abstract

Using 6-aza-2-thiothymine (ATT) as a suitable precursor, a novel series of [1,2,4]triazolo[4,3-b][1,2,4]triazin-7-one derivatives (7a–j) was prepared by refluxing 6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (3) with hydrazonoyl halides (1a–j) in chloroform in the presence of triethylamine. The structures of the newly synthesized compounds 7a–j were confirmed using spectral data, elemental analyses, and single-crystal X-ray diffraction results. All the synthesized triazolotriazin-7-one derivatives (7a–j) were evaluated as in vitro anti-cancer agents against PC3 (prostate cell line), A549 (lung carcinoma), PACA2 (pancreatic cancer cell line) and BJ1 (normal skin fibroblast) cell lines using MTT assay. Compounds 7a and 7g showed greater efficacy and low IC₅₀ values (36.6 and 40.1 μM, respectively) compared to the reference drug, which exhibited an IC₅₀ value of 43.8 μM on the lung cell line, and demonstrated safe mortality effect on the normal cell line (BJ1) with cytotoxicity percentages of 3.5% and 2.8%, respectively. These compounds (7a and 7g) were the most active compounds of the synthesized triazolotriazin-7-one derivatives (7a–j). They were further investigated to ascertain their mechanism of action using DNA fragmentation, DNA damage and gene expression (BCL-2, BAX, and p53 genes). Results indicated a significant increase in the expression levels of BCL-2 and a reduction in the expression of p53 and BAX genes in negative lung cancer cell lines. However, the treatment of negative cell lines with 7g improved the expression of the tested genes to a greater extent than that with 7a. Additionally, the DNA damage and DNA fragmentation levels were significantly elevated in the lung cancer cell line samples treated with 7a much more than 7g. Molecular docking was employed to explore the potential interactions between the most active compounds (7a and 7g) and two key enzymes, human 3-phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1), which play vital roles in the progression of lung cancer.