Background: Glioblastoma multiforme (GBM) is the most malignant form of brain tumors with a median survival rate of 15 months. A more effective approach seems to be targeting the tumor’s cell cycle through inhibition of mitotic kinases. Kinesin spindle protein (KSP) are essential for mitosis by mediating centrosome separation. Inhibition of KSP function results in prolonged mitotic arrest leading to cell death. Bioinformatic analyzes of the GBM genome revealed an overexpression of KSP. Several KSP inhibitors are currently investigated in clinical trials. However, their clinical use is severely limited by their high toxicity against rapidly proliferating non-cancerous cells. Objective: Given their high potencies and affinities, we contend that KSP inhibitors can be repurposed for clinical efficacy by adopting bioactivation themes common to conventional chemotherapeutic prodrugs such as pH and enzymatic activation. This study focuses on the synthesis and in vitro biological evaluation of derivatives of the KSP inhibitor ispinesib. Methods: Several phosphonium and phosphoramidate derivatives of ispinesib were synthesized and characterized by UPLC-MS and NMR. Cell proliferation was assayed through crystal violet staining. Cells were seeded at 1500 cells/well in 96-well plates and treated with a dilution series of inhibitors in duplicates after 24h. After 5d cells were fixed with 10% formalin and stained with crystal violet, washed with 10% acetic acid, and absorbance was read at 595nm. The results were plotted using GraphPad Prism 8. The stability of the inhibitors was monitored in phosphate buffer at pH 3.3, 7.0 and 8.8 at 10min, 30min, 60min, 120min and 24h via UPLC-MS. Results: Phosphonium derivatives were stable in pH 3.3 and 7.0 phosphate buffer for 24h, whereas at pH 8.8 they hydrolyzed into ispinesib and phosphine oxide within 60min. Phosphoramidates were stable in all pH ranges within 2h, but they degraded after 24h. Ispinesib derivatives inhibited the cell growth with nanomolar IC50 values. Conclusion: Novel Ispinesib prodrugs have been synthesized and showed potencies similar to ispinesib. The rapid degradation of phosphonium derivatives in alkaline pH implies an intracellular release of the parent drug. The stability of phosphoramidates for at least 2h also implies a sufficient in vivo distribution time in order for the prodrug to reach the target. The prodrugs will advance to in vivo studies.

Design and synthesis of novel ispinesib prodrugs

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.