Abstract A007: Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer
Authors: Farris Natalie , Batoon Lena , Muthyala Rajeev S. , Remmel H. Lawrence , Hammer Sandra S. , Hawse John R. , Quay Steven C. 
Published: December 2024

Abstarct
Introduction: (Z)-endoxifen (ENDX) is a potent orally bioavailable selective estrogen receptor modulator (SERM) and the most active metabolite of tamoxifen. Given its effective anti-estrogen activity and favorable safety profile, ENDX is being developed as a therapeutic solution in breast health. During its synthesis, two chemical biproducts (compound AT416 and AT402) with Z- and E- isomeric forms are produced. This study aimed to assess the anti-estrogenic activity of these four ENDX-related compounds with ENDX in ERα+ breast cancer. Methods: Compounds AT416E/Z are formed by acetylation of the final API with ethyl acetate during Step 3 of the manufacturing process of ENDX. Compounds AT402 E/Z are formed by coupling of impurity AT300 with propiophenone as a side reaction during Step 2 of the ENDX manufacturing process. The anti-cancer activities of the four ENDX-related compounds were tested across multiple ERα+ breast cancer cell lines (MCF7, T47D, UCD12), including those with ESR1 mutations (Y537S and D538G) known to confer endocrine resistance. Studies included cell proliferation, migration and invasion assays, cell cycle progression assessments, induction of apoptosis, activity of an estrogen response element (ERE) luciferase reporter construct and RT-qPCR analyses of ERα target gene expression. Results: In estrogen-containing media, ENDX exhibited strong anti-proliferative activity in MCF7 cells, regardless of ESR1 mutations. In MCF7 cells cultured in estrogen-deficient media, the four compounds demonstrated more pronounced anti-proliferative effects than ENDX. In T47D cells with the ESR1 Y537S mutation, AT416E showed superior anti-proliferative activity compared to ENDX, while in T47D cells with the D538G mutation, these two compounds exhibited comparable effects. In UCD12 cells, ENDX displayed the strongest anti-proliferative effects. Regarding the inhibition of migration and invasion, AT416E consistently outperformed ENDX in both T47D and MCF7 cells. ENDX was the most effective at inducing G1 phase cell cycle arrest followed. Both AT402E and AT402Z elicited pro-apoptotic effects comparable to ENDX in MCF7 cells, while in T47D cells, ENDX was the most effective at inducing apoptosis, followed by AT402E and AT402Z. In T47D and MCF7 cells, luciferase-based transactivation assays showed that ENDX was the most potent anti-estrogen followed by AT402Z. Finally, based on RT-qPCR analyses, ENDX was a more effective inhibitor of estrogen-induced ERα target gene expression, compared to the biproducts. Summary: These studies have revealed that four novel compounds derived from the manufacturing process of ENDX confer notable anti-tumor activity, with some performing comparably to or better than ENDX in several cancer-relevant cellular readouts. Future studies will validate these findings in additional in vitro/in vivo breast cancer models with the goal of identifying the basis for their varied anti-estrogenic and anti-cancer effects. Such discoveries should allow for the rational design of novel molecules with more potent therapeutic efficacy.
Citation Format: Natalie Farris, Lena Batoon, Rajeev S. Muthyala, H. Lawrence Remmel, Sandra S. Hammer, John R. Hawse, Steven C. Quay. Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A007.