A series of 2,6-disubstituted-4-morpholinothieno[3,2-d]pyrimidine derivatives were synthesized and their cytotoxic activity against H460,HT-29,MDA-MB-231,U87MG and H1975 cancer cell lines were evaluated in vitro.Most of the target compounds exhibited moderate to excellent activity to the tested cell lines.The most promising compound 23 (0.84 μmol/L,0.23μmol/L,2.52 μmol/L,1.80 μmol/L) was 1.0,2.9,29.3 and 4.3 times more active than GDC-0941 (0.87 μmol/L,0.66 μmol/L,73.8 μmol/L,7.77 μmol/L) against H460,HT-29,MDA-MB-231 and U87MG cell lines,respectively.