Triquinylamines as regulators of calcium homeostasis of neuronal cells / Lois-Mary Bezuidenhout
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Neurodegenerative diseases include common and debilitating disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and post-stroke neurodegeneration. Among these disorders, PD and AD have especially drawn attention because of their devastating impact on the elderly, their families, the health care system and society. These disorders are characterised by progressive and irreversible loss of neurons from specific regions of the brain. Among the mechanisms implicated to be responsible for neuronal cell death we focused our interest on excitotoxicity, which initiates a cascade of events resulting in neuronal injury as a result of excessive influx of Ca2+ through the N-methyl-D-aspartate receptor (NMDAR) and voltage gated calcium channels (VGCC). The focus of the current study was to develop a novel group of multifunctional therapeutic agents that can be used in the treatment and/or prevention of neurodegenerative diseases. Several triquinylamine derivatives containing an endocyclic nitrogen atom (ma-bridged) and select side chains were synthesised by thermal [2 + 2] cycloreversion of the symmetric cage compound pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8.11-dione. To be able to perform the thermal fragmentation reactions we designed and built a pyrolysis apparatus based on descriptions found in the literature. With this technology, the reaction was optimised to a yield approaching 80%. The symmetric cis-syn-cis triquinane scaffold obtained from the thermal fragmentation reaction underwent subsequent catalytic reduction, amination and hydride reduction to generate the series of compounds under study. The synthesised compounds were characterised using NMR, MS and IR techniques. Reductive amination produced very low yields of the desired products and we were also unable to isolate the N-methyl derivative, N-methyl-3,11-matricyclo[ 6.3.0.02,6]undecane. A computational study was initiated to explain the apparent selectivity in the formation of the benzylamine derivative over the methylamine derivative. The computational study revealed that a significantly lower energy of formation and more favourable HOMO-LUMO overlap (indicating a stronger covalent bond) might be an explanation for the selectivity in formation of the 3-benzyliminotricyclo[6.3.0.02,6]undecanIe-11-one intermediate over that of the 3- methyliminotricyclo [6.3.0.02,6]undecane-11-one intermediate. In order to afford protection against excitotoxicity, new drug candidates have to attenuate the induced Ca2+ flux through the L-type calcium channels and demonstrate binding affinity for the NMDAR channels. Fluorescent microscopy was utilized to monitor Ca2+ flux through the L-type calcium channel after KCI-induced depolarisation (KCI at 140 mM) in the Mag-fura-2/AM preloaded N2a mouse neuroblastoma cell line. The N-(3-methoxybenzyl)-3,11-azatricyclo[ 6.3.0.02,6]undecane compound proved to be the most potent experimental compound with a reduction in fluorescence of approximately 55.9% and was the only compound that showed a statistically significant (p < 0.05) attenuation of Ca2+ flux. This data indicate that the introduction of electronic effects, such as the inductive effect with significant electron-withdrawing properties of the N-(3-methoxybenzyl)- 3,11-azatricyclo[6.3.0.02,6]undecane compound, significantly influences the L-type calcium channel binding characteristics. The N-(phenylpropyl)-3,11-azatricyclo[ 6.3.0.02,6]undecane showed a reduction in fluorescence of 42.9 %, which indicated that, an increase in chain length leads to a commensurate increase in activity when compared to the benzylamine derivatives. Radioligand binding studies were used to measure the displacement of [3H]MK-801 from NMDA/glycine-activated murine synaptoneurosomes by the triquinylamine derivatives. The study indicated N-benzyl-3,11-azatricyclo[6.3.0.02,6]undecane to be the compound with the highest affinity, with an IC50 value of 1.93 uM (p < 0.05), which is comparable to the clinically used drug memantine(IC50 = 0.54 uM). None of the other triquinylamine compounds tested showed significant displacement, which indicated that these compounds do not strongly interact with the PCP binding site and possibly have a different site of interaction within the NMDA receptor/ion channel complex. Our results demonstrate that the triquinylamines have the ability to simultaneously block both major neuronal calcium channels. Different binding characteristics were however found to be important for the two channels. Of the structure-activity relationship parameters studied, geometric or steric constraints for interaction both at the VGCC and NMDAR channel appear to be dominant. However, binding characteristics for the VGCC were greatly improved with the introduction of inductive electronic effects. We conclude that the triquinylamines tested represent a novel group of dual-mechanistic agents that have potential as therapeutic agents in the treatment of neurodegenerative diseases.
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