Pironetin is a natural product with potent antiproliferative activity against various cancer cell lines including ovarian cancer. Pironetin is proposed to inhibit cell division via formation of a covalent adduct with α-tubulin. The disruption of microtubule polymerization dynamics via binding to α-tubulin is a novel mechanism of action since current chemotherapeutics that target tubulin bind β-tubulin. α-Tubulin is an attractive target for ovarian cancer since the overexpression of the α-tubulin isoform TUBAC3 has been reported to be a predictor for short-term survival in ovarian cancer patients who did not respond to platinum/paclitaxel chemotherapy. While pironetin displays potent in vitro activity, it had poor efficacy in a previous in vivo study in mice bearing P388 murine leukemia cells. Animals treated with pironetin also suffered from severe weight loss. To evaluate if pironetin would be a good candidate to develop as a chemotherapeutic agent, we evaluated the natural product’s pharmacological properties. The natural product was found to have poor metabolic stability and form covalent adducts with other proteins and/or biomolecules containing a reactive thiol. We hypothesized these properties were the cause of the natural product’s poor in vivo efficacy in the previous study. We therefore decided to synthesize pironetin analogs to improve upon the natural product’s pharmacological properties and explore the SAR at different parts of the molecule. We evaluated the SAR at the α,β-unsaturated lactone of pironetin, which is involved in the binding between pironetin and α-tubulin. We found that modifying the different stereocenters of the α,β-unsaturated lactone resulted in a loss of biological activity. We also explored the addition of a functional group at the α-position of the α,β-unsaturated lactone to decrease off-target covalent adduct formation. The functionalization at this position resulted in a 100-1000 fold decrease in biological activity. We also evaluated the SAR of the substituent at the γ-position of the α,β-unsaturated lactone in pironetin. We found that groups with similar steric properties as the ethyl group in the natural product are tolerated at this position. Along with evaluating the SAR at positions of the α,β-unsaturated lactone in pironetin, we also synthesized an analog to potentially improve upon the natural product’s metabolic stability. We exchanged the non-conjugated olefin in the natural product, which is the primary site of metabolism, with a phenyl group. The phenyl-containing analog has similar antiproliferative activity as the natural product, but did not have improved metabolic stability. We have identified additional sites of metabolism of pironetin and the phenyl-containing analog that will need to be modified to improve upon the natural product’s metabolic stability.
University of Minnesota Ph.D. dissertation. 2016. Major: Medicinal Chemistry. Advisor: Gunda Georg. 1 computer file (PDF); 323 pages.
The Synthesis and Evaluation of Pironetin and Pironetin Analogs as Ovarian Cancer Chemotherapeutic Agents.
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