The overall goals of this research are to design and synthesize novel neotanshinlactone analogues, to evaluate their antitumor activity and elucidate structure-activity relationships (SAR), to discover novel chemical entities, and to explore the mechanism(s) of action. Neo-tanshinlactone (90) and its previously reported analogues are potent and selective in vitro anti-breast cancer agents. In a continuing study, a highly efficient synthesis of 4-ethyl neo-tanshinlactone 91 was accomplished with fewer steps and higher overall yield than those previously reported. This synthetic route was applied to develop new lead compounds and establish SAR of 90. SAR studies on these compounds revealed the key molecular determinants of this family of anti-breast agents. Several analogues (108-110 and 113) displayed potent and selective anti-breast cancer activity. We further explored how the individual rings in the molecule of 90 influence the anti-breast cancer activity. We designed and synthesized five new classes of compounds derived from neo-tanshinlactone. The results led to the discovery of two novel classes of anti-breast cancer agents, 2-(furan-2-yl)naphthalen-1-ol and 6-phenyl-4H-furo[3,2-c]pyran-4- one derivatives. Preliminary SAR of 2-(furan-2-yl)naphthalen-1-ol derivatives was established. Compounds 172, 173, and 181 were developed as new anti-breast cancer agents with better selectivity than 91. Interestingly, 178 showed broad in vitro cytotoxicity against human cancer cell lines. Further development led to tetrahydronaphthalene-1-ol derivatives, a novel class of antitumor agents. 186 displayed potent activity against most tumor cell lines tested. Novel 6-phenyl-4H-furo[3,2-c]pyran-4-one derivatives were synthesized and evaluated as novel anti-breast cancer agents. We explored the SAR and optimized the substituents. Promising lead compounds 198-201, 211, 213, and 215 showed potent inhibition against the SK-BR-3 breast cancer cell line. Importantly, 213 and 215 showed the highest cancer cell line selectivity, being approximately 100- to 250-fold more potent against SK-BR-3 (0.08, and 0.14 μg/mL, respectively) compared with other cancer cell lines tested. Enzyme assays suggested that 91 significantly suppressed CK2α1, ABL, and AKT1, and 178 showed higher inhibition activity against CDC42BPB, PKG1β, and SGK1. The results will guide us to further explore the mechanisms of action. In addition, we demonstrated that 91 is a potent and selective anti-breast cancer agent both in vitro and in vivo.