Triquinylamines & aza-cage compounds as neuronal calcium flux modulators
Domingo, Olwen Charlotte
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The group of disorders that are generally characterised by changes in the normal function of neurons, also referred to as neurodegenerative conditions, are known to follow various underlying molecular pathways. With the pathogenesis of these molecular pathways gradually being elucidated, targets are being identified for the prevention of several diseases that involve the death of neurons. One of these molecular pathways involves the overload of neuronal cells with intracellular calcium ions. Since this may eventually lead to excitotoxicity, the block of calcium flux through the ion channels with the highest calcium ion permeability may thus serve as a major step towards the prevention of neurodegeneration. In this study, the /V-methyl-D-aspartate receptor (NMDAR) ion channel and voltage-gated calcium channels served as primary targets for an inhibition of the flux of calcium ions into neuronal cells. The triquinylamines share structural similarities with existing NMDAR antagonists. These similarities include the three linearly fused cyclopentane rings that serve as lipophilic moiety, as well as the nitrogen atom that forms a hydrogen bond with amino acids in the binding site. In addition to these characteristics that might define them as NMDAR antagonists, the triquinylamines also share structural similarities to the pentacycloundecane cage compounds. In view of previous findings on the NMDAR and calcium channel blocking activity of several pentacycloundecyl amines, it was thus envisaged that the triquinylamines would present similar results. The main aim of this investigation was thus to perform structure-activity analyses between the pentacycloundecylamines and triquinane derivatives with regard to their respective NMDAR and calcium channel blocking activities. Flash vacuum pyrolysis of the cage dione and subsequent hydrogenation yielded the saturated form of the triquinane dione. Reductive amination of this dione, using sodium borohydride, yielded the aza-triquinylamines. Aza-cage compounds were also obtained by reductive amination, but by using sodium cyanoborohydride as reducing agent instead. An additional compound, the lactol derivative of the triquinanes, was synthesised by reducing the triquinane dione with sodiumborohydride. Purification of reaction mixtures was done by means of either open column chromatography or by using a Versaflash system. Identification of the compounds was achieved by means of Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS) and Infrared (IR) techniques. Confocal laser scanning microscopy, together with Fluo-5N and the SHSY-5Y cell line to measure calcium flux, unfortunately did not deliver reproducible results. The ratiometric dye, Fura-2/AM, in combination with a microplate reader with undifferentiated PC-12 rat phaeochromocytoma cells proved to be more effective and adequate in measuring the extent of calcium flux into the cells after KCI-stimulated depolarisation. An interesting observation made with all the aza-bridgehead compounds as well as with NGP1-01, was their tendency to increase intracellular calcium levels at low concentrations. This was accompanied by calcium flux suppression at higher concentrations. This dualistic feature of these compounds can be attributed to activity on both the sigma receptors and calcium channels, as the PC-12 cells distinctly express both these pharmacological entities. Another surprising observation was the increased calcium influx in the presence of increasing lactol triquinane derivative concentrations. These results suggest that the lactol / hemiacetal might have the ability to antagonise ovreceptors and / or stimulate o2-receptors, as both these effects could lead to calcium level increases. In order to determine a more specific site of action for these compounds, a competition radioligand binding study was performed. The potent phencyclidine binding site blocker, [3H]MK-801, served as reference compound in this study. The binding of [3H]MK-801 remained fairly constant in the presence of increasing concentrations of the test compounds, indicating that they exert their calcium modulating effect by binding to a site other than the phencyclidine binding site of the NMDA receptor ion channels. This study confirms the potential of the polycyclic structures as lead compounds in the search for molecules that prevent neuronal death due to apoptosis. Their dualistic effects, as well as the possibility of sigma receptor activity provide new avenues for investigation in the search for biological lead compounds amongst the polycyclic compounds. This places an emphasis on the necessity of further studies on these potential therapeutic agents as neuroprotectors. With the new observations in biological activity for the triquinanes and pentacycloundecylamines in the current study, the possibility of a diversion in structure-activity relationship studies can also be expected in future investigations on this group of molecules.
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