Silyl ethers analogs of SN-38 with unique blue-fluorescent properties exhibit potent anti-cancer activity through topoisomerase I inhibition

Topoisomerase I inhibitors, such as deruxtecan and irinotecan, represent an important class of cell cycle inhibitors in the treatment of cancers. Among these, silylated topoisomerase inhibitors, including DB-67 and karenitecin, have exhibited increased antiproliferative potency. In this study, we prepared and evaluated the biological activity of four synthetic SN-38 analogs bearing the tert-butyldimethyl, tert-butyldiphenyl, triisopropyl, and dimethylthexyl siloxy groups, blocking a key metabolic site and imbuing bright blue-fluorescent properties in such compounds. When evaluated against colorectal, TN breast cancer, and ovarian, non-small cell lung, and liver cancer models, we observed comparable or greater antiproliferative activity. Our dimethylthexyl siloxytecan demonstrated the greatest potency among the four analogs, with a 96 hour IC50 of 6 nM in HCT-116 cells. Correspondingly, analysis by flow cytometry indicates that the siloxytecans, compared to their parent compounds, display comparable apoptotic activity and increased cell cycle G2 arrest. Uniquely, we demonstrate that the enhanced fluorescence of these compounds enables real-time, quantitative visualization of the dynamics and selectivity of intracellular uptake through fluorescence microscopy without the need for extensive sample preparation or installation of auxiliary fluorophores, with the dimethylthexyl siloxytecan displaying the greatest rate of cellular uptake. Additionally, quantification of relaxed and nicked DNA levels via cell-free biochemical studies suggests that these siloxytecans largely exhibit increased topoisomerase inhibition from SN-38. Further computer modeling studies validated that differences in potency and permeability were not attributed to the siloxytecans' positioning within the Topoisomerase I-DNA binding pocket. This initial SAR is further supplemented by an evaluation of these compounds' DMPK properties and metabolic stability, with an assessment of different in-vivo delivery strategies. Moreover, direct tissue injection of the siloxytecans has demonstrated exceptional tissue retention and durability, establishing the dimethylthexyl siloxytecan as a preclinical lead for in vivo evaluation. Collectively, these studies demonstrate improved intracellular delivery of topoisomerase inhibitors with the introduction of silicon-containing motifs and highlight the utility of these compounds for enhanced anticancer activity with unique lipophilicity and fluorescent properties that enable quantitative tracking of compound distribution in cancer cells, which is currently being investigated for in vivo activity in triple negative breast cancer studies.