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dc.contributor.advisorLourens, A. C. U.
dc.contributor.authorLourens, Margit
dc.date.accessioned2018-02-02T07:42:58Z
dc.date.available2018-02-02T07:42:58Z
dc.date.issued2017
dc.identifier.urihttp://hdl.handle.net/10394/26235
dc.descriptionMSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2017en_US
dc.description.abstractAlzheimer’s disease (AD), the most common neurodegenerative disorder, affects about 10% of the population over the age of 65 years. The disease is typified by symptoms such as memory loss and impairment in abilities including attention, concentration, orientation and judgment. Two hypotheses exist regarding the pathogenesis of AD: the first proposes that a decrease in the production of acetylcholine (Ach) in the synaptic junction (cholinergic hypothesis) is the main causative factor, while the second suggests that the disease is largely due to the aggregation of toxic amyloid-β (Aβ) peptide in the brain (amyloid hypothesis). Ach is a neurotransmitter which plays an important role in attention, cognitive processing and other cognitive functions. Consequently, cholinesterases are very important enzymes as they modulate the levels of Ach in the brain. Acetylcholinesterase (AChE) inhibitors including donepezil, rivastigmine and galantamine, inhibits the metabolism of Ach, thus increasing the Ach available for binding. These agents are therefore most commonly used in the symptomatic treatment of AD. Currently, no AD agent is registered as neuroprotective, and this neurodegenerative disorder progresses unhindered with an associated decrease in quality of life. Therefore, the discovery of novel drugs that can slow or stop its progression is of great importance. Parkinson’s disease (PD), on the other hand, is the second most common neurodegenerative disorder and is characterised by the loss of dopamine (DA) in the nigrostriatum. The symptoms of PD can by classified as either motor or non-motor symptoms. Motor symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Non-motor symptoms include sleep disturbances, autonomic dysfunction and sensory abnormalities. Parkinson’s disease dementia (PDD), which is typified by cognitive decline, is often experienced during the later stages of the disease. With the increase in life expectancy throughout the Western world, it is expected that PDD will become more prevalent in the future. The symptomatic treatment of PD usually involves levodopa use. Its continuous use is unfortunately associated with motor complications that impair the quality of life. Additional therapies, which include catechol-O-methyltransferase inhibitors, DA agonists and monoamine oxidase (MAO) B inhibitors are further used in PD treatment. The MAO enzymes (MAO-A and MAO-B), play an important role during the oxidative degradation of amine neurotransmitters including DA, serotonin (5-HT) and epinephrine. The inhibition of these enzymes decreases DA metabolism in the brain, resulting in an increase in the DA concentration. It is also likely to be neuroprotective as the production of harmful metabolic by-products such as hydrogen peroxide is decreased. Since the use of non-selective, irreversible MAO inhibitors are associated with severe side-effects, current efforts are aimed at designing selective, reversible MAO inhibitors. The MAO-B enzyme is of particular significance in PD because it is more active than MAO-A in the basal ganglia which is mainly responsible for the catabolism of DA in the brain. The treatment of the cognitive deficits experienced during the later stages of PD, and in PDD, in particular is not adequate and treatments that address the motor and non-motor aspects simultaneously are urgently required. It is postulated that a dual MAO-B and AChE inhibitor would improve motor symptoms of PD while improving cognitive deficits at the same time. Such an agent would not only be useful in the treatment of PD, but also in PDD and AD as the MAO inhibition component has the possibility of offering neuroprotection. The dihydroquinolinone scaffold has been shown to be privileged with regards to the inhibition of MAO, while the carbamate moiety often features in the structures of AChE inhibitors. AIM The aim of this study was therefore to design, synthesise and evaluate novel carbamates as dual inhibitors of MAO and AChE. METHODS Compounds were synthesised using a one step literature procedure. 6-Hydroxy-3,4-dihydro-2(1H)-quinolinone, 7-hydroxy-3,4-dihydro-2(1H)-quinolinone, 3- and 4-acetamidophenol were reacted with commercially available carbamoyl chlorides under basic conditions to yield the target compounds. Compounds were characterised by using NMR and IR spectroscopy as well as mass spectrometry. Purity was determined by HPLC and melting points were determined. The potential of the synthesised compounds to inhibit MAO enzymes were expressed as IC50 (50% inhibition concentration) values and the SI (selectivity index) was determined. A fluorometric assay, using kynuramine as substrate was employed. AChE inhibitory activity was determined by measuring the rate of thiocholine production, as generated by the hydrolysis of acetylthiocholine, which served as substrate in a spectrophotometric assay. RESULTS AND DISCUSSION Twenty eight novel compounds were successfully synthesised, albeit in low yields. Generally, most of the synthesised compounds exhibited weak to no inhibition of both MAO-A and MAO-B. Compound 8g, 2-oxo-1,2,3,4-tetrahydroquinolin-7-yl methyl(phenyl)carbamate, was the most potent MAO-B inhibitor of the current series with an IC50 value of 3.73 μM, and was MAO-B selective. It is postulated that the rigidity of the carbamate side chain is responsible for the loss of activity observed for the compounds of this study, when compared to the highly potent dihydroquinolinone derivatives of a previous study. Disappointingly, none of the synthesised compounds inhibited AChE, possibly due to the replacement of an ionisable amine group with an amide. Although the biological results of this study were disappointing, useful information was obtained regarding structural requirements for binding to both MAO and AChE. Alzheimer se siekte, die mees algemene neurodegeneratiewe siekte, affekteer ongeveer 10% van die bevolking bo die ouderdom van 65 jaar. Die siekte word gekenmerk deur simptome soos geheueverlies en inkorting van vermoëns soos aandag, konsentrasie, oriëntasie en oordeel. Twee hipoteses bestaan aangaande die patogenese van Alzheimers se siekte: volgens die eerste is 'n afname in die produksie van asetielcholien in die sinaptiese spleet (cholinerge hipotese) die belangrikste veroorsakende faktor, terwyl die tweede hipotese voorstel dat die siekte grootliks te wyte is aan die aggregasie van giftige amiloïed-β peptied (Aβ) in die brein (amiloïede hipotese). Asetielcholien is 'n neurotransmitter wat 'n belangrike rol in aandag, kognitiewe prosessering en ander kognitiewe funksies speel. Gevolglik is cholienesterases baie belangrike ensieme aangesien hulle die vlakke van asetielcholien in die brein moduleer. Asetielcholienesterase (AChE)-inhibeerders wat donepesil, rivastigmien en galantamien insluit, inhibeer die metabolisme van asetielcholien, en verhoog sodoende die asetielcholien wat beskikbaar is vir binding. Hierdie is dus die mees gebruikte middels in die simptomatiese behandeling van Alzheimers se siekte. Tans is daar geen anti-Alzheimer’s middel wat geregistreer is as neurobeskermend nie, en die verloop van hierdie neurodegeneratiewe siekte gaan dus ongehinderd voort met 'n gepaardgaande afname in lewenskwaliteit. Die ontdekking van nuwe middels wat die siekte kan vertraag of stop, is dus van groot belang. In teenstelling daarmee is Parkinson se siekte die tweede mees algemene neurodegeneratiewe siekte en word dit gekenmerk deur ’n verlies aan dopamien in die nigrostriatum. Die simptome van Parkinson se siekte kan geklassifiseer word as óf motories of nie-motoriese simptome. Motoriese simptome sluit bradikinesie, spierstyfheid, rustremore en verswakte posturale balans in. Nie-motoriese simptome sluit weer slaapversteurings, outonome disfunksie asook sensoriese abnormaliteite in. Parkinson-se-siekte-demensie, wat gekenmerk word deur kognitiewe agteruitgang, word dikwels ervaar gedurende die latere stadiums van die siekte. Met die toename in lewens verwagting regdeur die Westerse wêreld, word daar verwag dat Parkinson-se-siekte-demensie meer algemeen in die toekoms sal wees. Die simptomatiese behandeling van Parkinson se siekte behels gewoonlik die gebruik van levodopa. Ongelukkig lei die langdurige gebruik daarvan tot motoriese komplikasies wat lewensgehalte benadeel. Addisionele terapie, wat die gebruik van katesjol-O-metieltransferase-inhibeerders, dopamienagoniste en MAO B inhibeerders insluit, word verder gebruik in die behandeling van die siekte. Die MAO ensieme (MAO-A en MAO-B), speel 'n belangrike rol in die oksidatiewe afbraak van amienneurotransmitters soos dopamien, serotonien en epinefrien. Die inhibisie van hierdie ensieme verminder dopamienmetabolisme in die brein, wat gevolglik lei tot 'n toename in die dopamienkonsentrasie. Dit is waarskynlik ook neurobeskermend aangesien die produksie van skadelike metaboliese byprodukte soos waterstofperoksied verminder word. Aangesien die gebruik van nie-selektiewe, onomkeerbare MAO-inhibeerders geassosieer word met ernstige newe-effekte, is huidige navorsingspogings gemik op die ontwerp van selektiewe, omkeerbare MAO-inhibeerders. Die MAO-B ensiem is van besondere belang in Parkinson se siekte, aangesien dit meer aktief is as MAO-A in die basale ganglia wat hoofsaaklik verantwoordelik is vir die katabolisme van dopamien in die brein. Die behandeling van die kognitiewe defekte wat voorkom gedurende die later stadiums van Parkinson se siekte en in Parkinson-se-siekte-demensie, is veral onvoldoende en behandeling wat die motoriese en nie-motoriese aspekte gelyktydig kan aanspreek is uiters nodig. Daar word gepostuleer dat 'n tweeledige MAO-B- en AChE-inhibeerder die motoriese simptome van Parkinsons se siekte sal behandel, terwyl verbetering van kognitiewe tekorte terselfdetyd sal plaasvind. So 'n middel sal dus nie net gebruik kan word in die behandeling van Parkinson se siekte nie, maar ook in Parkinson-se-siekte-demensie en Alzheimer se siekte, aangesien die MAO-inhibisie komponent die bykomende voordeel van neurobeskerming kan bied. In vorige studies is aangetoon dat die dihidrokinolonoonkern geassosieer word met die inhibisie van MAO, terwyl die karbamaatgroep dikwels voorkom in die strukture van AChE-inhibeerders. DOEL Die doel van hierdie studie was dus om karbamate te ontwerp, te sintetiseer en te evalueer as tweeledige inhibeerders van MAO en AChE. METODES Verbindings is gesintetiseer deur gebruik te maak van 'n eenstap literatuurmetode. Die teikenverbindings is verkry deur 6-hidroksi-3,4-dihidro-2(1H)-kinolinoon, 7-hidroksi-3,4-dihidro-2(1H)-kinolinoon, 3- en 4-asetamidofenol met kommersieel beskikbare karbamoїelchloriede onder basiese toestande te laat reageer. Verbindings is gekarakteriseer deur gebruik te maak van KMR en IR-spektroskopie asook massaspektrometrie. Suiwerheid is bepaal deur HPLC en smeltpunte is bepaal. Die vermoë van die gesintetiseerde verbindings om die MAO ensieme te inhibeer is uitgedruk as IC50 (50% inhibisie konsentrasie) waardes terwyl die SE (selektiwiteitsindeks) ook bepaal is. ’n Fluorometriese toets, met kinuramien as substraat, is gebruik. AChE inhiberende aktiwiteit is bepaal deur die tempo van tiocholien produksie te bepaal, wat ontstaan as gevolg van die hidrolise van die asetieltiocholiensubstraat, in 'n spektrofotometriese toets. RESULTATE EN BESPREKING Agt-en-twintig verbindings is suksesvol gesintetiseer, alhoewel opbrengste oor die algemeen laag was. In die algemeen het die meeste van die gesintetiseerde verbindings swak of geen inhibisie van beide MAO-A en MAO-B getoon. Verbinding 8g, 2-okso-1,2,3,4-tetrahidrokinolien-7-iel metiel(feniel)karbamaat, was die mees potente MAO-B inhibeerder van die huidige reeks met 'n IC50 waarde van 3.73 μM, en die verbinding is MAO-B selektief. Daar word gepostuleer dat die rigiditeit van die karbamaatsyketting verantwoordelik is vir die verlies aan aktiwiteit wat waargeneem is vir die verbindings van hierdie studie, in vergelyking met die hoogs potente dihidrokinolinone wat gesintetiseer is in ‘n vorige studie. Nie een van die gesintetiseerde verbindings het ongelukkig AChE geïnhibeer nie, moontlik as gevolg van die vervanging van 'n ioniseerbare amiengroep met 'n amied. Hoewel die biologiese resultate van hierdie studie teleurstellend was, is nuttige inligting verkry met betrekking tot strukturele vereistes vir binding aan beide MAO en AChE.en_US
dc.description.sponsorshipNational Research Foundation (South Africa)en_US
dc.publisherNorth-West University (South Africa) , Potchefstroom Campusen_US
dc.subjectCarbamateen_US
dc.subjectDihydroquinolinoneen_US
dc.subjectAcetylcholinesteraseen_US
dc.subjectMonoamine Oxidaseen_US
dc.subjectAlzheimer's diseaseen_US
dc.subjectParkinson's diseaseen_US
dc.subjectParkinson's disease dementiaen_US
dc.subjectKarbamaaten_US
dc.subjectDihidrokinolinoonen_US
dc.subjectAsetielcholienesteraseen_US
dc.subjectMonoamienoksidaseen_US
dc.subjectAlzheimer se siekteen_US
dc.subjectParkinson se siekteen_US
dc.subjectParkinson se siekte demensieen_US
dc.titleDesign and synthesis of dual acting carbamate inhibitors of acetylcholinesterase and monoamine oxidaseen_US
dc.typeThesisen_US
dc.description.thesistypeMastersen_US


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