Synthesis and biological investigation of pyrimido[1,2-a][1,3,5]triazine and its analogues

Abstract

The monocyclic 1,3,5-triazine heterocycle is an extensively used scaffold for anticancer drug design. Examples of clinically proven antineoplastic agents possessing 1,3,5-triazine include the alkylating agent, hexamethylmelamine (HMM) and DNA methyltransferase inhibitor, decitabine. Besides the monocyclic triazines, numerous fused 1,3,5-triazines have been reported to possess promising antiproliferative activity. In continuation of the efforts of our research group to find antiproliferative lead compounds in the area of fused 1,3,5-triazine systems, the interest has been extended to include the pyrimido[1,2-a][1,3,5]triazine system and its benzofused analogues with the hypothesis that appropriate substitutions around these heterocycles would exhibit potential antiproliferative leads. 109 derivatives of pyrimido[1,2-a][1,3,5]triazine as well as benzofused pyrimido[1,2-a][1,3,5]triazine were prepared using an approach that annulated 1,3,5-triazine ring onto a pyrimidine scaffold. This 1,3,5-triazine ring annulation was achieved by facile 5+1 heterocyclization of pyrimidyl/quinazolinyl guanidines using one carbon synthons like benzaldehydes and orthoesters. The selected method provided easy means of inserting different substituents at positions 2, 4, 6 and 8 of the pyrimido[1,2-a][1,3,5]triazine ring. Moreover, a rapid microwave-assisted protocol was also developed for the synthesis of the N?-substituted N-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)guanidine which served as an intermediate in the formation of desired pyrimido[1,2-a][1,3,5]triazines.

The antiproliferative activity of the synthesized derivatives was evaluated against breast (MDA-MB-231), lung (A549) cancer cell lines and MRC-5 (human normal lung fibroblasts) using MTT assay. Six compounds, two from the 8-methyl pyrimido[1,2-a][1,3,5]triazin-6-one series and four from the benzo fused pyrimido[1,2-a][1,3,5]triazin-6-one series were identified as bioactive leads as they exhibited IC50 values in the range of 4.9-7.4?M against A549 cells in the MTT assay. The results from biological study concluded that the insertion of a NH2 group at position 2 and benzo group fusion at position 4 and 8 of the pyrimido[1,2-a][1,3,5]triazin-6-one ring system could generate antiproliferative compounds against the A549 cell line. Western blot analysis was used to identify the potential mechanism of the cell death observed in MTT assays for these six lead compounds. The appearance of only faint bands of cleaved PARP in comparison to the positive control (Staurosporine) seemed to suggest that the mechanism of cell death might be majorly caspase independent. ?Reverse/ Inverse? Virtual screening approaches viz. INVDOCK, pharmMapper and Reverse Screen 3D were then applied to identify putative anticancer targets for these lead compounds. Four targets were identified based on Tanimoto similarity scores between lead compounds and their known ligands/inhibitors. It was found that phosphatidyl inositol-3-kinase, MAP kinase, dihydrofolate reductase and Bcr-Abl tyrosine kinase could be the potential target(s) for benzofused pyrimido[1,2-a][1,3,5]triazin-6-ones, while Bcr-Abl tyrosine kinase could be the potential target for bicyclic pyrimido[1,2-a][1,3,5]triazinone (149b). These targets could serve as starting points for experimental validation.