A series of benzo [1] thiophene derivatives (3a-f), (4a-f) and (5a-f) were synthesized and characterized by spectroscopic and elemental analysis. All compounds were subjected to one dose anticancer screening in NCI- America, but the only high results were further subjected to five dose screening. An outstanding result of compound 4f (GI50 = 0.15, TGI= 1.14 μM) and 4c (GI50 = 1.09, TGI = 10.19, LC50 = 100 μM). To explore mechanism of cytotoxicity, compound 4f and 4c were allowed to affect cell cycle using HT-29 cell line for (24 and 48 hr). They caused induction of apoptosis causing preG1 apoptosis and cell growth arrest at G2/M in a time dependant manner inhibiting CDK-2. IC50 of compound 5d was 0.32 μM, IC50 of 6 was 0.15 μM while IC50 of erlotinib reference was 0.3 μM. Finally we synthesized a series of benzo [1] thiophene derivatives having a good cytotoxic activity suggesting promising anticancer derivatives.