Abstract A novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin, Adva-27a, had been designed and synthesized. The growth inhibitory properties of this drug compared to the currently used Topoisomerase II inhibitor etoposide was evaluated in MCF-7 breast cancer cells and in a multi-drug resistant (MDR) variant of this cell line. These cells were cultured in the presence of the drugs at different concentrations (0.1-100μM) for various times (1-9 days) and analyzed for viability using a mitochondrial metabolic activity dye, Alamar blue versus solvent (DMSO) treated cells. The MCF-7 cells were sensitive to Adva-27a and etoposide, resulting in an IC50 of less than 10μM for etoposide and 20μM for Adva-27a, while the IC50 for the MDR MCF-7 cells was 25μM with Adva-27a, but greater than 100μM for etoposide. The timing for killing by Adva-27a of the MDR MCF-7 cells appeared significantly different than for the MCF-7 parent cells. In this case, 100μM drug resulted in less than 50% of viability at day 3 for the MDR MCF-7 cells, while the same concentration of drug required 5-6 days to cause the same level of killing of the parent cells. To determine the mechanism of action of the drug, we analyzed the Adva-27a treated cells by flow cytometry using propidium iodide staining. We found that after 24 hours with 30μM of the drug, both MCF-7 and the MDR MCF-7 cells were accumulating in the G2/M phase of the cell cycle. Both cells are similarly killed after 7 days by this concentration of the drug. Etoposide is known to cause accumulation of cells at a similar phase, implying the Adva-27a has a similar drug target. Thus, the mechanism for the difference in sensitivity of the MDR MCF-7 cells for Adva-27a relative to etoposide is not yet understood, but will be explored in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1758. doi:1538-7445.AM2012-1758