A natural product scopoletin, which also contains an ortho-substituted phenolic structure in its skeleton, was found in some medicinal plants. In this study, to develop scopoletin-based autophagy activators, various aryl substitutes were introduced at the 3-positon or 4-position of scopoletin skeleton with the ortho-substituted phenolic structure retained. A total of twenty-three derivatives were synthesized, evaluated for their antiproliferation activity against four cancer cells (MCF-7, HeLa, PC3, and MGC803), and discussed for their structure-activity relationships (SARs). Among these derivatives, 5c was the most potent compound with an excellent improvement of antiproliferation activity against PC3 and MGC803 cells compared to the parental scopoletin. 5c displayed up to 17.9- and 5.7-fold improvement of antiproliferation activities against PC3 and MGC803 cells compared to 5-FU (IC<sub>50</sub> = 0.14 μM vs IC<sub>50</sub> = 2.50 μM, IC<sub>50</sub> = 1.02 μM vs IC<sub>50</sub> = 5.81 μM), respectively. Moreover, 5c showed excellent selectivity between cancer cells and one normal cell (GES-1). Further mechanism investigations confirmed that 5c inhibited PC3 and MGC803 cell proliferation via inducing autophagy. Interestingly, 5c also induced mitochondria-mediated apoptosis in PC3 cells but not in MGC803 cells. Moreover, 5c possessed the ability to suppress colony formation and migration of PC3 and MGC803 cells. In addition, 5c arrested the cell cycle at the G2/M phase of PC3 cells.