|dc.description.abstract||Natural products (NPs) have been a major source of chemical and structural diversity that has been a driving force for drug discovery over the past century. NPs have displayed a significant and vast range of biological activities, providing therapeutic agents in the areas of oncology, inflammation, infectious diseases, hypercholesteremia and tissue rejection in organ transplantation, to name a few. Many drugs on the market are of NP origin, either as unmodified or modified NPs. In 2016, it was reported that approximately 50% of new chemical entities approved between 1981 and 2014 were of NP origin, as unaltered NPs, NP derivatives or synthetic drugs that were inspired by NP pharmacophores. NPs also represent a repository of lead molecules that have played important roles in various drug development programs. Notable example includes pleuromutilin, a fungal metabolite which was a lead compound for the development of the antibiotics tiamulin, valnemulin, retapamulin, and BC-3781. Another remarkable example is the semi-synthetically derived antifungal drugs caspofungin, anidulafungin, and micafungin, which were all developed based on the NP lead compounds echinocandin B and pneumocandin B.
While chemists have used motifs derived from natural sources as starting points for the synthesis of bioactive compounds, the semi-synthetic approach is gradually gaining popularity as targeted scaffolds become easier to obtain. Many research groups are now utilising isolated NPs as scaffolds for the generation of semi-synthetic analogue libraries rather than pursuing the total synthesis of a bioactive NP, since this has the advantages of saving both cost and time. Also, the use of NP scaffolds for drug discovery has the tendency of increasing the probability of finding new and unique lead compounds for different diseases. Thus, including molecules with a NP template or NP-like template into screening libraries will increase hit rates, and hopefully translate into more, and better quality leads or drugs.
Owing to the success of NPs in drug discovery programs, the main aim of this PhD project was to identify unique NP scaffolds with suitable physicochemical parameters and use them for the generation of chemically diverse libraries, evaluate these libraries against a range of biological targets and if possible delineate structure-activity relationships (SARs). The scaffolds chosen for this project were prioritised based on factors such as abundant quantities, low molecular weight, availability of chemical handles for medicinal chemistry studies, and if possible the presence of stereogenic centres, which confers unique 3D shape on the molecule. Using these selection criteria, the plant NP valerenic acid was used in the generation of a novel screening library that consisted of 11 amide analogues. This library was semi-synthetically obtained using parallel solution-phase synthesis followed by C18 HPLC purifications. The chemical structures of all semi-synthetic analogues were elucidated following analysis of the spectroscopic and spectrometric data (NMR and MS). Two of the analogues afforded crystalline products and their structures were confirmed by single X-ray crystallographic analysis. All analogues together with the NP scaffold were evaluated for their ability to inhibit the release of IL-8 and TNF-α. Six analogues showed moderate activity in the IL-8 assay with IC50 values of 2.8–8.3 μM, while none of the tested compounds showed any significant effect on inhibiting TNF-α release.
The second NP scaffold project was based on the naturally occurring hormone found in plants and fungi, gibberellic acid. Treatment of this NP scaffold with a variety of primary amines afforded a total of 12 drug-like secondary amides. The structures of all the analogues were elucidated by NMR and MS analysis. Slow evaporation of a solution of one of the analogues in MeOH resulted in crystals suitable for X-ray diffraction, whose structure was confirmed by single X-ray crystallography. All compounds were evaluated in vitro for cytotoxicity and deregulation of lipid metabolism in LNCaP prostate cancer cells. While no cytotoxic activity was identified at the concentrations tested, five analogues were able to substantially reduce cellular uptake of free cholesterol in prostate cancer cells when compared with the parent compound, which suggests a novel role of gibberellic acid derivatives in deregulating cholesterol metabolism.
For the third PhD project, the plant alkaloid papaverine was subjected to some recently developed late stage functionalisation chemistry. Initially, with the hope of expanding and applying C-H late stage funcationalisation chemistry (through the use of sulfinate reagents) to the NP field, a search of the in-house Davis compound library led to the identification of eight abundant NPs (3-chloro-4-hydroxyphenylacetic acid, boldine, 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone, xanthurenic acid, the 4-methylumbelliferone, norharmane, papaverine hydrochloride, and DL-kavain. These prioritised compounds had been synthesised, isolated or purchased and were available in quantities, that would allow for preliminary sulfinate reactivity and eventual optimisation studies. Preliminary reactions on these eight NPs with zinc trifluoromethanesulfinate identified papaverine hydrochloride as the most promising NP scaffold for this type of late stage functionalisation chemistry. Reaction of papaverine hydrochloride with 12 other sulfinate reagents showed that this chemistry is more favourable with smaller sized sulfinates, compared to the larger molecular weight and bulkier reagents. The chemical structures of the seven analogues generated were confirmed by detailed analyses of both the 1D/2D NMR and MS data analysis. A library of seven analogues was generated and will be randomly screened in a variety of bioassays in the near future.
Finally, the marine natural products, thiaplakortones A and B were used for the generation of several halogenated analogues. This was achieved by reacting the marine NP scaffolds with N-bromosuccinimide and N-iodosuccinimide. The regio-isomer of thiaplakortone B, which was present in abundant quantities due to previous synthetic work that was performed in the Davis labs, was likewise used as a scaffold to generate additional analogues. This resulted in the generation of 16 semi-synthetic compounds. Late stage innate one-pot C-H functionalisation reactions using sulfinate reagents resulted in the synthesis of ten additional analogues. The structure of all analogues was determined following 1D/2D NMR and LRESIMS data analysis. These thiaplakortone-based semi-synthetic compounds are currently undergoing in vitro anti-malarial screening.
Altogether, this thesis entitled “Use of Natural Product Scaffolds in the Generation of Screening Libraries for Drug Discovery” describes the generation of novel compound libraries using four different NP scaffolds. The chemical structures of all the semi-synthesised compounds were elucidated following interpretation of extensive NMR and MS data. The libraries generated were screened in a variety of bioassays (e.g. anti-inflammatory, anti-cancer, anti- lipogenesis) with some of the analogues showing moderate to high activities when compared with the parent molecule. All compounds isolated or semi-synthesised during this PhD project will be deposited into the Davis Open-Access Compound Library, which is located at Compounds Australia (Griffith University). Compounds Australia will make these unique libraries available for biological evaluations by both local and international researchers. This Open Access deposition means that these novel compounds will be a lasting legacy of this PhD project where they may potentially impact pharma and chemical biology research in the future.||en_US