Synthesis and Evaluation of Bioactivable Nitric Oxide (NO) Donors

Abstract

Nitric oxide (NO) is an endogenously produced important biomolecule that is involved in various physiological processes that range from vasodialation to immunoregulation. NO alongwith other reactive nitrogen species react with majority of biomolecules (DNA, proteins and lipids) that leads to nitrosative and oxidative stress in cells. The induction of nitrosative and oxidative stress is an important component of immunogenic role of NO. Though, the immunogenic role of NO in containing pathogens and in controlling growth of cancer cells have been studied for long time but yet not fully characterized. In order to precisely study the role of NO inside cells, reliable sources of NO that can selectively enhance intracellular NO levels are required. Although numerous methodologies for NO delivery have been reported, they are associated with certain limitations such as lack of specificity and tunability, poor cell permeability and lack of a reporter for intracellular NO release. The work in this thesis describes different approaches for reliably enhancing NO inside cells in a site specific manner and also monitoring intracellular NO with reporter based NO donor. For selective delivery of NO inside cells, firstly the bioreductive environment existing in cancer cells was exploited and enzyme activated NO donors which are substrates for bioreductive enzymes were synthesized. DT-Diaphorase (DT-D) is a bioreductive enzyme known to be over-expressed in certain solid tumors. Using indolequinone scaffold, INDQ/NO was designed and synthesized as a possible substrate for DT-D. INDQ/NO was found to be activated by DT-D to generate NO. Cellular assays revealed the capability of this compound to permeate cells to generate NO and damage DNA. Secondly, nitroreductase (NTR), a bacterial enzyme was chosen as a possible enzyme to activate NO donors. NTR has been extensively used in designing prodrugs for the directed enzyme prodrug therapies and is thus of therapeutic interest. 4-Nitrobenzyl based NO donors, possible substrates for NTR were synthesized and studied. These compounds were found to produce NO in the presence of NTR. The suitability of this compound to enhance NO within bacteria including mycobacteria was demonstrated. Current techniques to monitor NO inside cells depend solely on secondary assays. To eliminate the need for secondary assays for detecting NO, we have developed a NTR-activated NO donor with a fluorescence reporter for NO. Lastly, for studying the possibilities of tunability of release, a new indole-based scaffold was designed, synthesized and release rates were studied. Using palladium as a chemical trigger that rapidly deprotects allyloxy carbonyl (alloc) group on indole scaffold, we found that the release rate could be modulated by simple structural modifications.