| Summary: | A novel series of polycyclic amines, containing nitrogen monoxide donating moieties, were synthesised and tested for possible neuroprotective activity. The premise for this study was the- recent development of a series of nitro containing memantine derivatives, called the nitromemantiries. These nitrogen monoxide donating moieties transfer a nitrogen monoxide to the thiol group of certain crucial cysteine residues in the receptor channel, a process known as S-nitrosylation, which then leads to disulfide bond formation, allosteric modification and desensitisation of the receptor. It was argued that these nitromemantines show better Ca2+ channel activity, and neuroprotective promise, than memantine alone. Because of the structural similarity of the pentacycloundecylamines and memantine, it was thus decided to investigate the influence of nitrogen monoxide donating moieties on the channel activity of the pentacycloundecylamines.
After a thorough structure-activity relationship overview and planning, a series of pentacycloundecylamines were synthesised, each containing a nitrogen monoxide donating group. Sufficient steric freedom was provided by the incorporation of a linker between the cage structure and the aromatic moiety. Esterification was done using the activating agent, 1-ethyl-3-(3'-dimethylamino)carbodiimide (EDC). The synthesised compounds could be classified into two groups, based on their nitrogen monoxide donating moieties: the unsaturated nitro compounds (3.1, 3.2 and 3.3) and the nitro esters, or nitrates (3.4, 3.5 and 3.6). The nitrates were obtained via the reaction of hydroxyl groups with thionylchloride nitrate. Structure elucidation was done using one and two-dimensional NMR spectroscopy, as well as IR absorption spectrophotometry and mass spectrometry.
The S-nitrosylation assay was performed by allowing the synthesised compounds to react with cysteine in a controlled environment. S-nitrosylation took place based on the compounds' ability to donate nitrogen monoxide and the unreacted cysteine residues were methylated using MMTS. Biotin-HPDP was added and the nitrosylated cysteines formed disulfide bonds with it, splitting off pyridine-2-thione, and absorbing UV-radiation at a wavelength of 343 nm. The intensity of this absorption was used as an indication of the extent to which S-nitrosylation took place. All of the compounds synthesised exhibited very significant (p < 0.01) S-nitrosylation capacity, with the nitrate compounds showing better S-nitrosylation than the unsaturated nitro compounds.
The channel activity of the polycyclic amines was evaluated using a Ca2+ flux assay. Fresh synaptoneurosomes were prepared from rat brain homogenate and incubated with the fluorescent ratiometric indicator, Fura-2/AM. The synaptoneurosomes were incubated with the synthesised compounds after which 100 mM KCl solution was added to depolarise the cell membranes and allow Ca2+ to enter. The decrease in fluorescent intensity relative to a (100 %) control was related to the ability of the test compounds to block the Ca2+ channels. Two positive controls were introduced; NGP1-01, the lead pentacycloundecylamine, and the dihydropyridine L-type Ca2+ channel antagonist, nimodipine. At concentrations of 10 uM, all the compounds exhibited better Ca2+ channel antagonism than NGP1-01, with compounds 3.1 (96.3 %) and 3.3 (88 %) showing a very significant inhibition (p < 0.01) and 3.4 (92.2 %) showing significant inhibition (p < 0.05) when compared to the control. Compounds 3.1 and 3.3 also exhibited significant inhibition (p < 0.01) at concentrations of 1 and 0.1 uM. At concentrations of 10 and 1 uM, compound 3.1 exhibited better Ca2+ channel blockade then the commercially available nimodipine. Compounds 3.3 and 3.4 also showed comparable results to that of nimodipine at higher concentrations.
Although no clear correlation was observed between the S-nitrosylation capabilities of the compounds and their Ca2+ channel activity, it is possible that other factors might play a more decisive role in the mechanism of pentacycloundecylamine channel antagonism. This could include the geometric and steric bulk considerations that have been proven to contribute to the Ca2+ channel activity of the pentacycloundecylamines (Malan et al., 1996:125 & 2000:10). The steric freedom brought on by the linker also seemed to have an influence on the channel activity. All the compounds synthesised exhibited promising Ca2+ activity and therefore show promise as potential agents against neurodegeneration. The compounds were also proven to be S-nitrosylators and can, therefore, have possible implications in other studies and/or fields of science === Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2009.
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