In this study, some of new thiophenyl thienopyrimidinone derivatives 2-15 were prepared and tested as anti-cancer agents by using thiophenyl thieno[2,3-d]pyrimidinone derivative 2 as a starting material, which was prepared from cyclization of ethyl ester derivative 1 with formamide. Treatment of 2 with ethyl- chloroacetate gave thienopyrimidinone N-ethylacetate 3, which was reacted with hydrazine hydrate or anthranilic acid to afford acetohydrazide 4 and benzo[d][1,3]oxazin-4-one 5, respectively. Condensation of 4 with aromatic aldehydes or phenylisothiocyanate yielded Schiff base derivatives 6,7, and thiosemicarbazise 10, which were treated with 2-mercaptoacetic acid or chloroacetic acid to give the corresponding thiazolidinones 8, 9, and phenylimino-thiazolidinone 11, respectively. Treatment of 4 with ethylacetoacetate or acetic acid/acetic anhydride gave pyrazole 12 and acetyl acetohydrazide 13 derivatives, respectively. The latter compound 13 was reacted with ethyl cycno-acetate or malononitrile to give 14 and 15, respectively. In this work, we have studied the anti-cancer activity of the synthesized thienopyrimidinone derivatives against MCF-7 and MCF-10A cancer cells. Furthermore, in vivo experiments showed that the synthesized compounds significantly reduced tumor growth up to the 8th day of treatment in comparison to control animal models. Additionally, the synthesized derivatives showed potential inhibitory effects against pim-1 kinase activities.