Abstract Breast cancer is one of the most common cancers that occur in women in the United States. Depending on the expression of estrogen receptor α (ERα), breast cancer can be classified as ER-positive or negative. ER-negative (ER-) breast cancers are often more aggressive and have poorer prognosis compared with the ER-positive (ER+) subtype. Consequently, there is a great need to develop more targeted therapeutic options for ER- breast cancer. One such compound is SHetA2, a flexible heteroarotinoid (flex-hets) that has been shown to inhibit growth of multiple cancer types including breast cancer. However, the clinical utility of SHetA2 is limited by its high lipophilicity (LogP). The goal of this study is to screen eleven 2nd generation analogs of SHetA2 with lower LogP values— SL1-22, 24, 27, 29, 30, 32, 36, 37, 38, 39, and 40— on different breast cancer cell lines to identify potential lead compounds that show the highest anti-growth activities against ER- breast cancer cells and further delineate the mechanism by which these compounds affect breast cancer cell growth. Results from this study demonstrate that SL1-38 [1-(3-chloro-4-methylphenyl)-3-(4-nitrophenyl)thiourea] and SL1-39 [1-(4-chloro-3-methylphenyl)-3-(4-nitrophenyl)thiourea], inhibit ER-negative breast cancer cells (MDA-MB-231, MDA-MB-453 and MDA-MB-468) effectively at micromolar concentrations. In order to understand the mechanisms of action, we evaluated the effects of SL1-38 and 39 on the expression of key cell cycle regulators. Our results show that these two analogs down-regulate the expression of cyclin A, cyclin B, cyclin D1, cyclin E and cdk2 and block S-phase progression. Taken together, these preliminary results suggest that SL1-38 and 39 may be further developed as anti-cancer agents for treating ER-negative breast cancer. Citation Format: Hongye Zou, Emily Ginn, Sabah M. Francis, Shengquan Liu, Maggie Louie. Targeting estrogen receptor negative breast cancer cells using diarylthiourea analogs of SHetA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2090. doi:10.1158/1538-7445.AM2017-2090