Browsing by Author "Van der Westhuyzen, Alet E"

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    Bioactive compounds inspired by nature: synthesis, stability & delivery
    (Stellenbosch : Stellenbosch University, 2020-01) Van der Westhuyzen, Alet E; Van Otterlo, Willem A. L.; Klumperman, Bert; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.
    ENGLISH ABSTRACT: Historically, natural products (NPs) have served as a dominant contributor to the discovery of medicinal agents. Their invaluable role in modern drug discovery is emphasized by the major impact NP-inspired scaffolds and NPderivatives/mimics have had on the inception of clinically approved drugs. Evidently, Nature continues to influence human innovation in the design and development of new therapeutic agents. Since NPs have been programmed to interact with biological targets, they are thought of as evolutionary-chosen, privileged structures. In this regard, NPs serve as a structural ‘muse’ for the design of new biologically active small molecules. Moreover, the unique characteristics of NPs can be leveraged and fine-tuned to enhance potency and drug-like properties of therapeutic candidates. Owing to the tremendous impact of NPs in the field of medicinal chemistry, the inspiration gained from their scaffolds has provided a strong impetus for the development of new antiproliferative agents. The research explored in this dissertation comprised a multi-disciplinary project with the golden thread throughout the various scientific ventures being the development of bioactive anticancer agents inspired by Nature. In its entirety, the research was aimed at investigating two common challenges experienced during drug development –poor aqueous solubility and hysicochemical instability of NP/NP-inspired compounds. The two NP families whichmotivated these endeavours were the rigidins and sphaeropsidins (specifically SphA). The rigidins are marine-derived alkaloids which have been developed and optimized into a potent class of 7-deazahypoxanthines. A major breakthrough regarding the synthesis of the rigidins and their NP-inspired analogues, involved a crucial multi-component reaction (MCR) furnishing the tetrasubstituted 2-amino pyrrole precursor. The implementation of the MCR has permitted the rapid assembly of molecular diversity, serving as an attractive strategy towards the 7-deazahypoxanthines. These rigidin-inspired analogues are capable of disrupting microtubule dynamics in cancer cells and cell lines associated with MDR. However, like many other anticancer agents, the potent 7-deazahypoxanthines suffer from poor aqueous solubility. To address this impediment, two strategies were envisaged – polymeric micelle encapsulation and the introduction of solubilizing appendages. The first approach was directed towards the synthesis of a specific 7-deazahypoxanthine analogue and its covalent conjugation onto a hydrophilic polyvinylpyrrolidone (PVP) polymer via a Michael addition reaction. Although the resulting drug-polymer conjugate was successfully furnished, research efforts evolved towards the design of a more amphiphilic PVP polymer capable of loading multiple 7-deazahypoxanthine molecules. This led to the attempted synthesis of an α-substituted Nvinylpyrrolidone monomer. The next strategy aimed at enhancing the aqueous solubility of the 7-deazahypoxanthines involved incorporation of water-solubilizing groups (WSGs) within the pyrrolo[2,3-d]pyrimidine backbone. Building on a previously identified lead compound, the synthetic strategy was directed towards the addition of WSGs on the aryl rings prior to the MCR. Evaluation of the final compounds revealed that the introduction of WSGs derived from the sulfonamido acetophenone components led to a retention or improvement of nanomolar activity. Inspection of the growth inhibitory activity (GI 50 ) against HeLa cells identified a new front-runner amongst the family of rigidin-inspired analogous. This particular 7-deazahypoxanthine, 6-[4-(2-ethoxyethoxy)benzoyl]-2-(pent-4-yn-1-yl)-5-phenyl-1,7- dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, bearing a glycol ether solubilizing appendage, exhibited an exceptional GI 50 of 15 nM against HeLa cancer cell lines. Although the aqueous solubility of the most potent 7-deazahypoxanthines were assessed based on their hydrophobicity (log D), it was suspected that the poor solubility was rather attributed to the solid-state characteristics arising from the network of strong intermolecular forces. Building on this work, a collaborative project on the prodrug behaviour of N9-substituted 7-deazahypoxanthines was undertaken. Stability studies conducted in cell medium via LC-MS analysis revealed the conversion of the N9-substituted 7-deazahypoxanthines into their unsubstituted potent variants. Thereby, substitution at the N9 position of the 7-deazahypoxanthine presented an additional potential tactic for aqueous solubility enhancement. The other NP which motivated this project is the fungal metabolite SphA. This tetracyclic diterpenoid demonstrates potent cytotoxicity towards apoptotic- and multidrug-resistant cancer cell lines. SphA’s unique mechanism of action has been ascribed to the disruption of ion homeostasis in cancer cells through the impairment of regulatory cell volume increase (RVI). Unfortunately, the potential success of SphA as an anticancer agent is hindered due to its poor physicochemical stability in cell medium. As such, the research of this section was directed towards two objectives – analysis of the degradation behaviour of SphA and the impetus towards improved stability. Initially, SphA was incubated in cell medium with sample analysis taken at varying time intervals using LC-MS. Owing to the complexity of the LC-MS data, a complementary simplified buffer solution was implemented. Extensive LCMS analysis revealed possible SphA degradation metabolites based on their molecular ions, with several corresponding to SphA degradation product-amino acid adducts. Most noteworthy was the presence of a major SphA degradation metabolite (SphA-Met), which increased in concentration over extended incubation periods. Subsequently, scale-up degradation experiments and use of preparative HPLC resulted in the isolation of SphA-Met. Finally, successful structural elucidation of this major degradation metabolite was achieved via extensive 2D NMR spectroscopy and innovative NMR spectroscopy protocols. Bioactivity evaluation of SphA-Met revealed a loss in activity compared to the parent SphA NP, thereby further motivating the necessity to address its undesired degradation behaviour. With the hope of enhancing antiproliferative activity, as well as stability, a semi-synthetic SphA derivative was furnished using olefin cross-metathesis. Although significant improvement in potency was established by our collaborators, stability issues of the semi-synthetic derivative in cell medium persisted. Thus, a final research thrust was directed towards the synthesis of an amphiphilic block copolymer capable of physical encapsulation and protection of the SphA derivative. Pleasingly, the desired PVP-b-PDLLA copolymer was successfully generated and polymeric micelle formation was confirmed. Ultimately, this dissertation encompassed several scientific disciplines focussed on the advancement of rigidin- and SphA-inspired research. In addition to contributions made to the small-molecule synthetic arena, integrated knowledge from separation science, structural elucidation and polymer chemistry was applied towards the different research ventures. Considering the findings, the development of these NP-inspired compounds as efficacious anticancer agents holds much promise.