| Summary: | In recent years polycyclic compounds have been shown to exhibit pharmacological profiles of importance in the symptomatic and proposed curative treatment of neurodegenerative diseases (e.g. Parkinson's and Alzheimer's disease). These structures also show modification and improvement of the pharmacokinetic and pharmacodynamic properties of drugs in current use. The use of fluorescent techniques have found widespread applicability in receptor pharmacology and offers an attractive alternative to the use of radioligand studies such as multicolor detection, stability, sensitivity, low hazard and lower cost. Fluorescent ligands can be used to determine receptor properties like receptor internalization and sub-cellular localization, the thermodynamics and kinetics of ligand binding and to assess the nature of the microenvironment of the ligand binding sites.
Nitric oxide (NO) is a molecular messenger involved in a number of physiological processes in mammalians. It is synthesised by nitric oxide synthase (NOS) from L-arginine and its overproduction could lead to a number of neurological disorders. The aim of this study was to synthesise a series of novel indazole, indole and other fluorescent derivatives conjugated to polycyclic structures for evaluation in NOS assays. NOS is a target system where fluorescent techniques and fluorescently labelled NOS inhibitors, such as 7-nitroindazole (7-NI), can be used for detecting the biophysical properties of enzyme-ligand interactions and thus facilitate development of novel inhibitors of neurodegeneration. This could lead to a greater insight into the neuroprotective mechanism and a possible cure/treatment for neurodegenerative diseases.
A series of compounds incorporating polycyclic structures such as 3-hydroxy-4-aza-8-oxoheptacyclo[9.4.1.02,10.03'14.04'9.09'13.012'15]tetradecane and amantadine as well as suitable fluorescent moieties were selected for synthesis. The polycyclic structures were conjugated to the fluorescent moieties by amination and esterfication or amidation using activation chemistry with the carbodiimides, CDI and DCC. The fluorescent groups were selected on the basis of their spectroscopic properties, ease of synthesis and
structure-activity relationship requirements for NOS inhibition. The groups included TV-methylanthranilic acid, indazole-3-carboxylic acid, l-fluoro-2,4-dinitrobenzene, 1-cyanoisoindole, 1-thiocyanatisoindoleand 1-nitroisoindole.
In the biological evaluation the oxyhemoglobin (oxyHb) assay was employed to determine the activity of the novel compounds at an enzymatic level of NOS. This assay is principally based on the reaction of NO with oxyHb and the formation of methemoglobin (metHb). The slope of the absorption difference between 401 nm and 421 nm versus time is an indication of NOS activity. From this inhibition data the IC50 values were calculated and compared.
The compounds showed inhibition of the NOS enzyme at a micro molar range. Of the compounds that were tested, only the isoindole and indazole compounds showed significant inhibition at low concentrations. 100 % enzyme inhibition could not be achieved, because of solubility problems. The polycyclic structures conjugated to the indazole moiety increased the NOS activities, when compared to the unconjugated indazole-3-carboxylic acid. The anthranilic and dinitrobenzene derivitaves only showed low or no inhibition of the NOS enzyme.
3-{4-aza-8-oxo-heptacyclo[0.4.1.02J0.03'14.04'9.09'13.012'15]tetradecyl}-l/f-indazole-3-car-boxylate and 7V-(l-cyano-2i/-isoindol-2yl)adamantan-l-amine gave the best inhibition at a concentration of 250 uM, inhibiting the enzyme 83.7 % and 89.9 %, respectively. The compounds containing indole and indazole systems clearly have higher affinity for the NOS enzyme than other structures evaluated in this study.
We have thus identified a series of fluorescent structures with moderately high affinity for the NOS enzyme, which may be utilized for further in vitro and in vivo studies using modern imaging techniques. In view of the increase in lipophilicity originating from the pentacycloundecyl cage structure, it is expected that the new structures will display an increase in blood brain barrier permeability when compared to 7-NI. The novel compounds also represent a new class of NOS inhibitors and provide the foundation for potential therapeutic agents. These compounds thus have potential as useful pharmacological tools to investigate enzyme-ligand interactions in the quest for effective neuroprotective strategies. === Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.
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