Date of Completion

12-11-2014

Embargo Period

12-10-2015

Keywords

DHFR, MRSA, Propargyl linked antifolates, Candida albicans, Candida glabrata, Escherichia coli, Klebsiella pneumoniae, cyclopamine, tandem metathesis

Major Advisor

Dr. Dennis L. Wright

Associate Advisor

Dr. Amy C. Anderson

Associate Advisor

Dr. M. Kyle Hadden

Associate Advisor

Dr. Amy R. Howell

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Resistance to antibiotics is on the rise at an alarming pace. The problem is exacerbated by the lack of new class of therapeutic agents and targets. Modes of resistance include alteration of drug target, modification of ligand, and reduction of the intracellular concentration of drug. Dihydrofolate reductase (DHFR) is a validated drug target for the treatment of infectious diseases and cancer. The binding of ligands to DHFR inhibits the downstream folate pathway that is vital for purine and thymidylate synthesis. Trimethoprim (TMP), a clinically used antifolate exhibits resistance in both gram-positive and gram-negative bacteria. In this thesis, methods have been refined to expedite the synthesis of propargyl linked antifolates for lead optimization. As a result, novel antifolate inhibitors with better pharmacokinetic profile have been designed and synthesized for methicillin resistant Staphylococcus aureus (MRSA). Ligands with minimal inhibitory concentration (MIC) of 0.3 - 0.6 µg/mL exhibiting an in-vitro half life of 60 minutes were achieved. Similarly, to overcome resistance to azole antifungal agents, propargylic linked antifolates exhibiting dual inhibition against Candida albicans and Candida glabrata were developed. Antifolate activity of less than 0.1 µg/mL has been achieved against the clinically indistinguishable pathogens. In the development of antifolates for gram negative bacteria – Escherichia coli and Klebsiella pneumoniae, studies were carried out to understand the factors that influence the permeability and efflux of the ligands. With a 1,3 benzodioxole moiety of the antifolate exhibiting low efflux activity, new leads are in progress to synthesize ligands with potent antibacterial activity.

In another project, simplified analogs of the natural product, cyclopamine, have been synthesized. Cyclopamine inhibits the smoothened (Smo) transmembrane of the hedgehog signaling pathway. The synthesis utilizes a furan based Diels Alder reaction to generate a functionalized oxabicyclic adduct. The adduct eventually undergoes a cascade reaction involving a tandem ring-opening, ring closing metathesis reaction to generate the spirocyclic core of cyclopamine.

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