Phenotypic screening of high quality compound library is one of the most effective strategy to obtain novel bioactive compounds. Recently, our group have constructed a Wogonin-scaffold library with substituents diversity and successfully obtained a series of potent compounds. Herein, we reported the synthesis of these compounds and evaluated the in vitro antitumor activity against a panel of human tumor cell lines. Most of them showed good activity with a broad spectrum and preliminary structure-activity relationship for the substitutions were obtained. Further biological assays showed that the most potent compounds 18n and 20b could significantly enhance the intracellular ROS level and induce the cell apoptosis at low micromole level. Through similarity searching, CDK9 was identified as the potential target for 20b, which could be a start point for next structure-based drug design.