The objective of the proposed research is to discover and develop new classes of anti-breast cancer agents from higher plants, which have been and still remain a significant source of new drugs. We will identify structurally unique hits from rare sources of plants, particularly rainforest extracts with confirmed cytotoxic activity as screened b the National Cancer Institute, as well as medicinal plants used for cancer-related therapy. Lead compounds, optimized using medicinal chemistry approaches, will be subjected to critical preclinical in vivo assessment, not only with conventional xenograft mouse models, but also with an innovative and unique genetically engineered spontaneous breast cancer mouse model having conditional mutations on tumor suppressor genes Brca1 and p53. Furthermore, we will explore possible mechanism(s) of action based on chemical profiling and effects on responsible pathways, including sex steroid receptors, cell cycle arrest, kinase inhibition, or signal transduction pathways. The overall goals of our program are to identify and develop clinical trial candidates, especially for treating breast cancers. The following specific studies will be carried out to accomplish our goals. Specific Aim-1: In vitro screening and bioactivity-directed fractionation and isolation to identify breast cancer-selective compounds starting from plant extracts with confirmed activity and counter-screened in-house on the basis of susceptibility to breast cancer cell lines. Specific Aim-2: New lead optimization using synthetic modifications to improve pharmacological profile. Structural characterization of new active leads (Aim 1) and modified compounds (Aim 2) will be accomplished by chemical, physical, and spectroscopic techniques. Specific Aim-3: Two-step in vivo evaluations to confirm activity in mouse models, both conventional xenograft mouse models and innovative breast cancer Brca1/p53-Crec mouse model. Specific Aim-4: Mechanism of action studies to identify druggable targets and compounds for breast cancer treatment. Advantages of our program include 1) an excellent supply of highly active lead compounds and promising cytotoxic plant species, including rainforest species from the NCI Natural Product Repository Program, (2) excellent productivity in isolation and structural modification of new leads with new mechanisms of action as clinical trials candidates, which in turn could lead to innovative methods for cancer chemotherapy, and (3) superior prospects for the successful development of a clinically useful drug, based on promising in vivo results with neo-tanshinlactone analogs against breast cancers. An emphasis of our study will be on finding optimal analogs from these different compound classes to advance to anticancer clinical trials during the next grant period.