Abstract Breast cancer (BC) is the most common malignancy in women. Approximately 70% BC are Estrogen receptor (ER) positive, However, most patients develop resistance to current therapies and progress to incurable metastases. We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized, and tested over 500 analogs of ERX-11 in multiple models of BC. Methods: In vitro activity was tested using cell titer glo, MTT, and apoptosis assays. The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts, patient derived xenografts (PDX), and patient derived xenograft explants (PDEx) were used for testing the utility of ERX analogs. Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER-positive BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 50-200 nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines but had potent activity (IC50 ranging from 50 -100 nM) against ovarian cancer cell lines. Through iterative changes, we have identified lead compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, CRISPR-Cas9 based KO screening revealed that these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative BC. In several xenograft and models, including pancreatic cancer and ER-negative BC, the activity of the compounds have been confirmed by oral administration of the ERX analogs in vivo. We have also validated the activity of these analogs using PDX and PDEX models. Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Shi-Hong Ma, Gangadhara Reddy Sareddy, Tae-Kyung Lee, Mei Zhou, Yiliao Liu, Xihui Liu, Dede N. Bahun-Wilson, Kara Kassees, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Generation and characterization of potent analogues of ERX-11 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 21.