Design, synthesis, and biological evaluation of N-(4-substituted)-3-phenylisoxazolo[5,4-d]pyrimidin-4-amine derivatives as apoptosis-inducing cytotoxic agents

Abstract

A library of new 3-phenylisoxazolo[5,4-d]pyrimidines (8-10) was designed based on a scaffold hybridization technique incorporating the important pharmacophoric features of 4-aminopyrimidine and phenyl isoxazole scaffold which is renowned for its BET inhibition activity. The designed molecules were synthesized and evaluated with the NCI-60 cell line panel. Examination by NCI-60 cell lines at single-dose and the five-dose study showed that compound 10h exhibited promising growth inhibitory effects with GI₅₀ values on various cancer cell lines such as HCT-15 (Colon Cancer)-0.0221 μM, MDA-MB-435 (Melanoma) - 0.0318 μM, SNB-75(CNS Cancer)-0.0263 μM, and MCF7 (Breast Cancer)-0.0372 μM. Further studies to know the mechanism of action of 10h based on the phase-contrast microscopic evaluation, DAPI, acridine orange/ethidium bromide (AO/EB) staining, and annexin V-FITC assays revealed that elevation in the intracellular ROS leads to alteration in mitochondrial membrane potential which in turn induced the apoptosis in BT-474 cancer cells, which could be the plausible mechanism of action for compound 10h.

Keywords: 4-aminopyrimidine; Apoptosis; Isoxazole; Isoxazolo[5 4–d]pyrimidine; NCI-60 cell line panel.