A multidisciplinary functional proteomics-aided strategy as a tool for the profiling of a novel cytotoxic thiadiazolopyrimidone

•A small set of [1,3,4] thiadiazolopyrimidones has been efficiently synthesized and then decorated by Suzuki-Miyaura cross-coupling reaction, which provided rapid access to different 2-arylated thiadiazolopyrimidones.•A multi-disciplinary strategy has been used to disclose the potential biological targets of the most active thiadiazolopyrimidone, namely compound 1, using a label-free mass spectrometry based platform.•Functional proteomics, coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring, disclosed Annexin A6 as the most reliable target.•A consistent inhibition of calcium transition, cell migration and invasion of cancer cells has been demonstrated due to Annexin A6 modulation by the thiadiazolopyrimidone-based compound 1.•The first disclosed small-molecule binder targeting Annexin A6 may pave the way to the development of novel anticancer agents.