Design, synthesis and biological activity studies of 1-aryl-3-(4-methoxybenzyl)ureas as proposed irreversible GSK-3 inhibitors in Alzheimer’s disease therapeutic development

Venter, Jana (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Alzheimer’s disease (AD) is a progressive neurodegenerative disease, characterised by memory loss and cognitive decline. No cure has been found for the disease yet, therefore the development of disease-modifying therapeutics (DMTs), which can target the underlying mechanisms of AD, is necessary. Glycogen synthase kinase 3 (GSK-3) has become a promising CNS target, since GSK-3 dysregulation has been shown to play a central role in the multifactorial neuropathogenesis of AD. In this project, a proposed library of structurally-related, irreversible GSK-3β inhibitors was modelled, synthesised, characterised and biologically tested as potential AD drug candidates. The library contained two sets of 1-aryl-3-(4-methoxybenzyl)ureas wherein the incorporated aryl group was a benzothiazole or benzimidazole scaffold, respectively. Different electrophilic warheads were incorporated onto the scaffolds, with the potential to form a covalent, irreversible bond with nucleophilic Cys199 in the GSK-3 ATP pocket. Targeting of Cys199 was suggested to provide increased GSK-3 selectivity, since Cys199 is exchanged with other amino acids in structurally-related enzymes. A library of 10 covalent inhibitors containing the nitrile, halomethylketone (HMK), vinyl ketone, ethynyl ketone and acrylamide electrophilic warheads was successfully synthesised, as well as the reference GSK-3 inhibitor, AR-A014418 (AstraZeneca). The synthetic route commenced with the preparation of the 6-substituted 2-aminobenzothiazoles and 6-substituted 2-aminobenzimidazoles in good yields. Thereafter, the scaffolds were coupled to 4-methoxybenzylamine through carbonyldiimidazole-mediated urea formation, which afforded excellent yields for the benzothiazoles and moderate yields for the benzimidazoles, proposedly due to tautomeric effects in the latter. These ureas were further modified in position 6 of the benzazole scaffolds, to incorporate the respective electrophilic warheads. The GSK-3β inhibitory activity results were promising, with high activities measured for the nitrilesubstituted ureas and the HMK-substituted benzimidazole urea. In comparison to the reference GSK-3 inhibitor, which displayed an IC50 value of 0.072 ± 0.043 μM in the assay, the best IC50 value obtained in the library was 0.086 ± 0.023 μM, observed for 1-(6-cyano-1H-benzo[d]imidazol-2-yl)-3-(4- methoxybenzyl)urea. In general, the benzimidazole series displayed better IC50 values than the equivalent inhibitors in the benzothiazole series. Although an initial assay was carried out to ascertain whether the newly synthesised inhibitors were in fact irreversible inhibitors, the results remain ambiguous and further study is required to confirm this hypothesis. In conclusion, highly active GSK-3β inhibitors were successfully developed and may potentially contribute to future AD drug development.

AFRIKAANSE OPSOMMING: Alzheimersiekte (AD) is ‘n progressiewe neurodegenerasiesiekte, gekenmerk deur geheueverlies en kognitiewe kwyning. Daar is tans geen geneesmiddel beskikbaar vir die siekte nie, daarom is die ontwikkeling van siekte-wysigingsmiddels, wat instaat is om die onderliggende siekteverwekkende meganismes van AD te teiken, noodsaaklik. Die glikogeen sintase kinase 3 (GSK-3) ensiem is ‘n belowende teiken in die sentrale senuweestelsel, aangesien daar getoon is dat abnormale GSK-3 vlakke bydra tot veelvoudige faktore in die neuropatogenese van AD. In hierdie projek is ‘n versameling van struktuurverwante, onomkeerbare GSK-3β inhibeerders voorgestel, waarna dit gemoduleer, gesintetiseer en biologies getoets is as potentiële AD geneesmiddelkandidate. Die versameling het bestaan uit twee stelle 1-ariel-3-(4- metoksibensiel)ureumverbindings, waarin die arielgroep onderskeidelik ‘n bensotiasool- of bensimidasool-struktuur verteenwoordig het. Hierdie sentrale strukture is elk aangepas met ‘n verskillende elektrofiliese reaktiewe groep, aangesien hierdie substituent potensieel ‘n kovalente, onomkeerbare binding met sisteïen 199 (Cys199) in die ATP bindingsetel van GSK-3 kan vorm. Cys199 word vervang met ander aminosure in struktuurverwante ensieme, daarom kan ‘n kovalente binding met Cys199 moontlik die selektiwiteit van die GSK-3 inhibeerders verbeter. ‘n Versameling van 10 kovalente inhibeerders, met onderskeidelik die nitriel, halometielketoon (HMK), vinielketoon, etenielketoon en akrielamied elektrofiliese reaktiewe groepe, is suksesvol gesintetiseer, asook die kontrole GSK-3 inhibeerder, AR-A014418 (AstraZeneca). Die sintesepad het ‘n aanvang geneem met bereiding van die 6-gesubstitueerde 2-aminobensotiasool- en 6-gesubstitueerde 2-aminobensimidasool-verbindings, met goeie opbrengste. Hierdie verbindings is daarna gekoppel aan 4-metoksibensielamien met behulp van karbonieldiimidasool, om die bensotiasool-ureumverbindings te vorm met puik opbrengste. Die opbrengste van die bensimidasoolureumverbindings was gematig, moontlik a.g.v. toutomeriese effekte. Die ureumverbindings is verder aangepas by posisie 6 van die bensasool-basisstruktuur, om sodoende die relevante elektrofiliese reaktiewe groepe te koppel. GSK-3β inhiberingsaktiwiteit resultate was belowend en die beste aktiwiteit is gemeet vir die nitrielgesubstitueerde ureumverbindings en die HMK-gesubstitueerde bensimidasool-ureumverbinding. Die aktiefste inhibeerder was 1-(6-siano-1H-benso[d]imidasol-2-iel)-3-(4-metoksibensiel)ureum, met ‘n IC50 waarde van 0.086 ± 0.023 μM. Hierdie waarde het ook goed vergelyk met die aktiwiteit van die kontrole GSK-3 inhibeerder (IC50 waarde = 0.072 ± 0.043 μM). Die bensimidasool reeks het oor die algemeen better IC50 waardes vertoon as ooreenstemmende inhibeerders in die bensotiasool reeks Alhoewel ‘n aanvanklike ensiemtoets uitgevoer is om die onomkeerbare bindingsmeganisme van die nuut-gesintetiseerde inhibeerders te bevestig, was die resultate van die kontrole GSK-3 inhibeerder onverklaarbaar en word verdere navorsing benodig om hierdie hipotese te bevestig. Ten slotte, hoogs aktiewe GSK-3β inhibeerders is suksevol ontwerp en kan potensieel bydra tot toekomstige AD geneesmiddel ontwikkeling.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105654
This item appears in the following collections: