| dc.description.abstract | Alzheimer’s Disease (AD) is the most prominent of all the types of dementias. It’s a neurodegenerative disease that affects the central nervous system (CNS) and general symptoms includes a decline in cognitive abilities (memory, problem-solving, and paying attention) and non-cognitive side effects such as anxiety, depression, apathy and psychosis. AD can be divided into early-onset familial AD (EOFAD) and late-onset AD (LOAD), caused by gene mutations and with CNS changes during aging respectively. The latter route cause is found to be the most prevalent. In 2013, an estimated 5.2 million Americans of all ages were diagnosed with AD. The number of AD cases is rapidly escalating and it’s approximated that by the mid-21st century, an individual in the United States will develop AD every 33 seconds.
On a cellular level, AD is characterised by the presence of extracellular plaques containing the beta-amyloid protein (Aβ), intracellular neurofibrillary tangles (NFTs) of hyper-phosphorylated tau protein and microgliosis (neuro-inflammation). The most dominant neuronal loss is in the cholinergic system but dysfunctions of the dopaminergic systems have been found to be contributing factors. The current clinically available medicinal agents for the treatment of AD include the three acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, galanthamine) and one non-competitive N-methyl D-aspartate (NMDA) antagonist (memantine). However, because of the existence of compensating parallel pathways in such a complex disease, these drugs don’t address the underlying mechanisms and thus only provide symptomatic relief of AD.
This study focused on a strategy based on the rationale that a single compound may have the ability to interact with multiple disease contributing targets, also known as multi-target-directed ligands (MTDLs). This may deliver desired synergistic/potentiating effects, of which AChE and monoamine oxidase-B (MAO-B) inhibition are fundamental in this study.
Coumarin analogues (4- and 7-substituted) were conjugated to selected structures (morpholine, piperidine, erucic acid) via etherification and esterification using conventional and microwave-assisted methods. The microwave-assisted method proved more feasible in both yield and reaction time. The final products were obtained as amorphous waxes or solids through chromatographic and/or crystallisation procedures using appropriate organic solvents. NMR and MS spectroscopic methods were implemented to confirm the correctness of the final newly synthesized structures.
The synthesised derivatives were evaluated for their in vitro activities as MAO-B and AChE inhibitors. A fluorometric assay using kynuramine as substrate was used to determine the MAO-B activities of the compounds. Recombinant hMAO-B was used as the enzyme source and the results of the enzyme inhibition were expressed as IC50-values. The acetylcholinesterase from Electrophorus electricus / electric eel hydrolysis (EE AChE) of DTNB [5,5’di thiobis(2-nitrobenzoic acid] was utilised to test compounds for AChE activity. The EE AChE enzyme inhibition results were expressed as percentage at both 1 μM and 100 μM concentrations.
Computer aided molecular modelling studies were conducted using Accelrys® Discovery Studios® V3.1.1 software utilising the published hMAO-B (2V61) and hAChE (4EY7) crystal structures. The prepared proteins were typed with the CHARMm forcefield, ionised, protonated (pH 0 – 14) and energy minimised. The structures of the test compounds were docked in the active sites of the enzymes using the CDOCKER® module.
The coumarin-morpholine ether conjugate, BPR 10 (4-[2-(morpholin-4-yl)ethoxy]-2H-chromen-2-one) proved to be the most promising hMAO-B inhibitor with an IC50 of 0.372 μM. The coumarin-piperidine conjugates, BPR13 (4-methyl-7-[2-(piperidin-1-yl)ethoxy]-2H-chromen-2-one) and BPR12 [(7-[2-(piperidin-1-yl)ethoxy]-2H-chromen-2-one)] were the most potent inhibitors of EE AChE with an inhibitory activity of 57.43 % at 100 μM and 30.90 % at 1 μM respectively.
The docking studies, showed that the morpholino-coumarin compound, BPR 10 was able to occupy both the entrance and substrate cavities of the active site of MAO-B. The results demonstrated that the coumarin moiety occupies the substrate cavity while the morpholine moiety is present in the entrance cavity. BPR10 shows Pi-interactions with residues CYS172, LEU171 and ILE198, and a relatively strong H-bond is present between the pyrone ring and CYS172. The coumarin entity of the compound is positioned in the “aromatic cage” of the substrate cavity. BPR13 occupied both the peripheral anionic site (PAS) and the catalytic anionic site (CAS) of hAChE, with the coumarin positioned in the PAS region, the linker in the gorge (between the PAS and CAS regions) and the piperdine entity in the CAS region. BPR13 formed Pi-interactions with TRP286 and TYR341, and a H-bond with TYR72 in the PAS. It is concluded that the coumarin structure serves as an effective pharmacophoric multi-target-directed ligand scaffold and that conjugated compounds of coumarin has the potential to exhibit both MAO-B and AChE inhibition. This multi-target-directed approach may have the potential to delay the incidence and/or the progression of AD and serves as a basis for further studies, amongst others, in vivo investigations. | en_US |
