This study successfully developed synthetic pathways for novel pyrimidine-based compounds for their potential anti-diabetic α-amylase inhibitory activity, aiming at potential antidiabetic applications. Molecular docking techniques showed that compounds 1, 6, and 8 showed the highest molecular docking in silico binding affinity, followed by in vitro estimation. Compound 1 showed the strongest inhibitory in vitro amylase effect, in comparison to the reference drug acarbose. According to in silico and in vitro combination, a novel radiolabeled compound, 131Iodine-Compound 1, was chosen for further labeling study. The radiolabeling factors, including ligand concentration, pH, and reaction time, were adjusted to achieve maximum radiochemical yield and stability. For the in vivo behavior, biodistribution studies were estimated in mice up to a 36 hours period. The labelled compound showed significant and prolonged …