A variety of novel thieno[3,2-d]pyrimidines with different decorating functional groups were synthesized as a part of a study aiming to enrich the arsenal of chemotherapeutic agents for the treatment of cancer. The design of synthetic molecules based on DNA-interchelating properties by hydrogen bond formation. The reported compounds herein are: 4-aminothienopyrimidine derivatives 4a, b and their 4-substituted phenylamino analogues 8a, b; 4-thienopyrimidin-4-ones 5a, b; N-alkyl thienopyrimidin-4-ones 6a–g; 4-chlorothienopyrimidines 7a, b and thienopyrimidoquinazolinones 9a, b which are the structural mimics of 8a, b. The synthesized molecules were evaluated for their in vitro cytotoxic activity against human breast cancer cell line (MCF-7). Biological screening revealed varying cytotoxic potencies of the tested molecules compared with Doxorubicin as a reference drug. The cytotoxicity results from the study suggested that the synthesized molecules are potential antitumor agents and compound 4a was the most potent with an IC ₅₀ 2.04 nm.