A novel series of 3,5,6-trisubstituted pyrazolo[4,3-d]pyrimidin-7-one derivatives, especially 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-ones were synthesized and evaluated for their in vitro anticancer activities against various human cancer cell lines. The inhibitory activities for several kinases have also been tested. The prepared compounds library exhibited significant anticancer activity towards HT-29 colon and DU-145 prostate cancer cell lines. The structure-activity relationships of the 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one scaffold at R(1), R(2) and R(3) have been elucidated. Among the synthesized compounds, 12b was the most active compound with GI(50) value of 0.44microM and 1.07microM against HT-29 and DU-145 cell lines, respectively, and 13a was the most selective compound towards colon cancer cell line.