Synthesis and Evaluation of a Metabolically Stabilized Analog and Conjugates of Pironetin

Abstract

Molecules that bind to tubulin and disrupt tubulin dynamics are known as microtubule targeting agents. Treatment with a microtubule targeting agent leads to cell cycle arrest followed by apoptosis. Tubulin inhibitors have been highly effective in the clinical treatment of a variety of tumors and are being investigated for treatment of several other diseases. Currently, all FDA approved microtubule inhibitors bind to β-tubulin. Given the overall success of tubulin-binding agents in anticancer chemotherapy, α-tubulin is an attractive and unexplored target. Herein, we will discuss the natural product pironetin, the only compound known to bind α-tubulin. Despite the potent in vitro activity against ovarian cancer cell lines both sensitive and resistant to current chemotherapeutics, pironetin was only marginally effective at high doses in mice and resulted in severe weight loss, indicating poor pharmacokinetic/pharmacodynamic (PK/PD) properties and significant off target toxicities. The research presented in this dissertation seeks to improve the therapeutic properties of pironetin by addressing the PK/PD and off target binding concerns. In Chapter 2, we determined that pironetin has a short half-life in liver microsomes and identified pironetin’s major site of metabolism in human liver microsomes to be the unconjugated olefin utilizing tandem mass spectrometry. We then confirmed the identity of the major metabolite as epoxypironetin through semi-synthesis and identified a very minor metabolite, demethylpironetin, with similar potency as pironetin. With the knowledge that the unconjugated olefin is the major site of metabolism, we sought to block this site through total synthesis. We thus completed the total synthesis of 4-fluorophenyldemethylpironetin (19 linear steps, 31 total steps) with the goal of improving the metabolic stability while maintaining potency (Chapter 3). This represents the first total synthesis of demethylpironetin analogs. Finally, in Chapter 4 we describe our efforts to establish a proof-of-concept method for the targeted delivery of pironetin to ovarian cancer cells by targeting the folate receptor. Our results suggest that pironetin conjugates do not enter the cell in a folate receptor mediated manner, therefore necessitating exploration of addition means of targeting for pironetin delivery.