Based on the hit 5-hydroxy-2-methyl-10-propyl-2,3-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione(1), a series of pyrano[2,3-f]chromene-4,8-dione derivatives was designed and synthesized using phloroglucinol as starting material. Meanwhile, a regioselective synthetic route was developed for 5-methoxy-2,3-dihydro-4H,8H-pyrano-[2,3-f]chromene-4,8-dione products(11a-11f), and their structures were further confirmed by nuclear Overhauser effect(NOE). The evaluation of anticancer activities of these compounds against four human cancer cell lines, including human glioma cell line (SHG-44), human lung cancer cell line(H1299), breast cancer cell line(MCF7) and human colon carcinoma cell line(HCT-116) in vitro shows that 5-methoxy-2,2-dimethyl-9-chloro-10-trifluormethyl-2,3-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione(11e) possesses the best anticancer activities with IC50 values of 6.68, 7.90, 5.16 and 4.82 μmol/L, respectively. Finally, the preliminary structure-activity relationships(SARs) were summarized, which could pave the way for generating more potent anticancer agents with drug-like properties.