Design, synthesis, and biological evaluation of 2,4-dihydropyrano[2,3-c]pyrazole derivatives as autotaxin inhibitors
Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134 nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site.