Synthesis of novel staurosporine analogues as potential kinase inhibitors

Van der Westhuyzen, Aletta Elizabeth (2015-03)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Protein kinases are enzymes that promote phosphorylation – transferring a phosphate group from ATP to a substrate protein. Due to the central involvement of kinases in growth factor signaling, cell cycle control, apoptosis and angiogenesis, they have been linked to cancer development and are attractive drug targets for cancer therapeutics. Staurosporine is a natural, potent kinase inhibitor, initially isolated from the bacterium Streptomyces staurosporeus. It does not serve as a viable therapeutic drug since it exhibits poor selectivity. However, this natural product has been widely used in research and serves as a “structural muse” for the design of protein kinase inhibitors with improved specificity and selectivity. This project involved the synthesis of potential reversible and irreversible kinase inhibitors inspired by the natural product staurosporine. A primary objective was to develop compounds with improved selectivity, while maintaining the potency of staurosporine. The design strategy incorporated a driving portion, resembling the structural features of staurosporine, carrying a specific warhead to form a reversible or irreversible interaction within the kinase domain. This project comprises of two main parts – the design of pyrrolocarbazole structures and the generation of structures based on the “open” form of the indolocarbazoles. The development of the pyrrolocarbazole compounds was motivated by the encouraging results exhibited by 6-(3-hydroxypropyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (29), previously synthesized in our group. The major steps involved in the synthesis were the reduction, oxidation, Wittig, Diels-Alder and aromatization reactions. Introducing different warheads, allowed for the generation of unaromatized and aromatized pyrrolocarbazole target compounds having an N-tether hydroxyl, propargyl and nitrile functionality. The synthesis of the “open” form structures utilized click chemistry. The synthetic methodology towards the bisaryl maleimide structures involved a “double click” approach, which presented various challenges. In addition, the synthesis of indolyl maleimide click products also proved to be difficult. Finally, a promising route towards bisamino maleimide compounds led to the development of two target compounds containing the acryloyl and propargyl warhead functionality respectively. Biochemical screening by German collaborators against the wild type, and two mutant forms of the EGFR kinase, was undertaken with the target compounds, utilizing the HTRF KinEASE-TK assay. Overall, only the aromatized pyrrolocarbazole N-two carbon linker hydroxyl target compound exhibited inhibitory activity in the μM range. Finally, a number of optimization measures are described to potentially improve the inhibitory activity of both the pyrrolocarbazole compounds and bisamino maleimides.

AFRIKAANSE OPSOMMING: Proteïn kinase is ensieme wat fosforilasie bevorder – deur ń fosfaat groep van ATP na ń substraat proteïn oor te plaas. As gevolg van die sentrale betrokkenheid van kinase in groei-faktor seine, beheer oor sel siklus, apoptosis asook angiogenesis, is hulle gekoppel aan kanker sel toename en is ń aantreklike middel vir kanker-terapie. Staurosporine is ń natuurlike, sterk kinase onderdrukker, aanvanglik geïsoleer van die bakterium Streptomyces staurosporeus. Ongelukkig dien dit nie as ń bruikbare geneesmiddel nie as gevolg van die tekort aan selektiwiteit – vele kinases word geteiken. Nietemin, word die natuurlike produk algemeen gebruik in navorsing en dien as ń “strukturele muse” vir die ontwerp van proteïn kinase onderdrukkers met verbeterde selektiwiteit en meer spesifiek. Die projek behels die sintese van potensiёle omkeerbare en onomkeerbare kinase onderdrukkers wat geïnspireer is deur die natuurlike produk staurosporine. ń Primêre doel was om verbindings te ontwikkel met beter selektiwiteit, wat die effektiwiteit van staurosprine behou. Die ontwerp strategie inkorporeer ń dryfdeel wat ooreenstem met die struktuur van staurosporine en ń spesifieke aanvalpunt wat hopelik ń omkeerbare of onomkeerbare interaksie binne die kinase-omgewing sal vorm. Die projek bestaan hoofsaaklik uit twee dele nl. die ontwerp van pyrrolokarbasoolstrukture, asook strukture wat die “oop” vorm van die indolokarbasole namaak. Die ontwikkeling van die pyrrolokarbasool samestellings was gemotiveer deur die bemoedigende resultate vertoon deur 6-(3-hydroxypropyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (29) wat voorheen in ons navorsings-groep gesintiseer was. Die hoofsaaklike stappe betrokke gedurende die sintese was reduksie, oksidasie, Wittig, Diels-Alder en aromatisering reaksies. Die bekendstelling van verskeie aanvalpunte, maak die ongearomatiseerde en aromatiseerde pyrrolokarbasool teikenprodukte met ń N-koolstof binding met hidroksiel, propargiel en nitriel funksionele groepe moontlik. Die sintese van die “oop” vorm strukture gebruik “click” chemie. Die metode om die sintese van die bisariel-malimied strukture na te streef behels ń “dubbele click” wyse, wat baie uitdagings gehad het. Verder het die sintese van die indolyl-malimied “click” produkte ook verskeie problem opgelewer. Uiteindelik het ń baie belowende roete na bisamino-malimied verbindings gelei, en die ontwikkeling van twee teiken produkte, met ń akryloyl en propargiel aanvalpunt funktionele groepe, was bereik. Biochemiese keuring deur Duitse medewerkers teen die wilde tipe, asook twee mutant vorms van die EGFR kinase, was onderneem teen die teiken verbindings, met gebruik van die HTRF KinEASE-TK toets. Oor die algeheel, was die aromatiese pyrrolokarbasool met die N-koolstof verbinding met die hidroksiel groep, die enigste verbinding wat onderdrukkende aktiviteit in die μM grens getoon het. Verskeie optimisering strategieё was bespreek om die potentiёle onderdrukkings aktiwiteit van die pyrrolokarbasool en bisamino-malimied verbindings te verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96632
This item appears in the following collections: