Natural Products from Australian Celastraceae Plants and Their Leucine Transport Inhibition in Prostate Cancer Cells
Embargoed until: 2019-11-16
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Natural products are the main source of approved medicines with more than half of the drugs on the market today being either natural products or natural product derivatives. Moreover, a significant number of these drugs are of plant origins. However, it has been estimated that of the >422,000 plant species currently described, only 15% of this plant diversity have been phytochemically explored. Therefore, there still exists a huge potential in the plant kingdom for the discovery of new chemistry, some of which may result in the identification of lead compounds or drugs for the pharmaceutical industry. Several notable examples of plant-derived natural product drugs are the anticancer compounds vinblastine, vincristine, and paclitaxel, the antimalarial agents artemisinin and quinine, and the Alzheimer drugs galantamine and huperzine A. In regards to plant-derived lead compounds, two pertinent examples include camptothecin, which lead to the development of the anticancer agents, topotecan and irinotecan, and papaverine, which was the lead model for the antihypertension verapamil. The Celastraceae plant family is found worldwide, especially in tropical and subtropical regions; currently there are 88 genera and 1300 species belonging to this flowering plants family. Various molecules possessing numerous biological activities have been reported from Celastraceae. One noteworthy example of an interesting natural product and cancer lead compound from Celastraceae is the alkaloid maytansine, which was further developed into the antibody drug conjugate treatment, transtuzumab emtansine, and is currently used for breast cancer treatment. However, the characteristic secondary metabolites of Celastraceae are the dihydro-β-agarofuran sesquiterpenoids, which have been reported in the literature as both chemotaxonomic markers and privileged structures. The L-type amino acid transporters (LATs) are responsible for the uptake of various amino acids (including leucine) into cells. Leucine is a regulator amino acid of the mTORC1 signalling pathway and one of the essential amino acids that is transported by LATs. Though LATs are expressed in both normal and cancer cells, the overexpression of LATs has been reported in various cancer cells, including those associated with prostate cancer. Thus, the inhibition of leucine uptake may be a novel drug target for cancer treatment. To date, very few natural product LATs inhibitors have been identified. Venulosides, which are all monoterpenoid glycosides, were the first and only natural products (reported by Quinn et al.) that had been reported to inhibit LATs prior to these PhD studies. Their recent discovery highlights the potential of natural products in the discovery of new LAT inhibitors. This knowledge coupled with an interest in the chemistry of the hitherto under-investigated Australian plant family, Celastraceae, motivated us to identify new small molecules from this particular biota and evaluate all compounds isolated or semi-synthesised for their ability to inhibit leucine uptake on the human prostate cancer cell line, LNCaP. The plants that were investigated during these PhD studies included Maytenus bilocularis, Denhamia pittosporoides, and Celastrus subspicata and Denhamia celastroides. In Chapter 2, phytochemical studies of the leaves of the Australian rainforest plant Maytenus bilocularis led to the identification of three new dihydro-β-agarofurans, bilocularins A–C, and six known compounds, namely celastrine A, 1α,6β,8α- triacetoxy-9α-benzoyloxydihydro-β-agarofuran, 1α,6β-diacetoxy-9α-benzoyloxy-8α- hydroxydihydro-β-agarofuran, Ejap-10, 1α,6β-diacetoxy-9β-benzoyloxydihydro-β- agarofuran, and Ejap-2. Bilocularin A was used to generate four semisynthetic ester analogues. The absolute configuration of bilocularins A and B was established by X-ray crystallography study; for bilocularin C, the absolute was determined by comparison of ECD spectra. All compounds were found to be inactive in a cytotoxicity assay against the human prostate cancer cell line LNCaP. However, several compounds were found to exhibit similar potency to verapamil in reversing multidrug resistance in the human leukemia cells (CEM/VCR R). Moreover, 1α,6β,8α-triacetoxy-9α-benzoyloxydihydro- β-agarofuran was shown to inhibit leucine uptake in LNCaP cells with IC50 value of 15.5 μM, which was more potent than the leucine analogue 2-aminobicyclo[2.2.1]- heptane-2-carboxylic acid (BCH). Motivated by these findings with dihydro-β-agarofurans from the leaves of M. bilocularis, we extended our studies (see Chapter 3) to the roots of this plant, which had never been phytochemically explored. Six new dihydro-β-agarofuran sesquiterpenoids (bilocularins D–I), together with three known compounds, namely 1α,2α,6β,15- tetraacetoxy-9β-benzoyloxydihydro-β-agarofuran, pristimerin, and celastrol were successfully isolated. Moreover, the absolute configuration of bilocularin D was established by single-crystal X-ray diffraction analysis. Bilocularins D and G, 1α,2α,6β,15-tetraacetoxy-9β-benzoyloxydihydro-β-agarofuran, and celastrol inhibited leucine uptake in the LNCaP cells with IC50 values ranging from 2.5–27.9 μM. This study identified bilocularins D–F as the first dihydro-β-agarofurans possessing a hydroxyacetate group. Chapter 4 details the identification of two previously undescribed dihydro-β- agarofurans, denhaminols I and J together with four related and known metabolites, 1α,2α,6β,15-tetraacetoxy-9α-benzoyloxy-8-oxodihydro-β-agarofuran, wilforsinine F, 1α,2α,6β,8α,15-pentaacetoxy-9α-benzoyloxydihydro-β-agarofuran, and 1α,2α,6β,15- tetraacetoxy-9β-benzoyloxydihydro-β-agarofuran. These metabolites were purified from the CH2Cl2 extract of the leaves of D. pittosporoides. The structure of denhaminol I was further confirmed by X-ray crystallography analysis, which also established its absolute configuration. Denhaminol I and wilforsinine F were shown to exhibit leucine transport inhibitory activity in LNCaP cells with IC50 of 51.5 μM and 95.5 μM, respectively. Chapter 5, reports on the chemical investigation of CH2Cl2 extract of the leaves of the Australian endemic vine, Celastrus subspicata, which afforded seven previously unknown dihydro-β-agarofurans, celastrofurans A–G and two known compounds, (1S,4R,5S,7R,9S,10S)-9-benzoyloxy-1-furoyloxydihydro-β-agarofuran and (1R,2R,4R,5S,7R,9S,10R)-2-acetoxy-9-benzoyloxy-1-furoyloxydihydro-β-agarofuran. X-ray diffraction and ECD studies were undertaken to define the absolute configurations of celastrofurans A–D. All the isolated compounds from this vine were found to inhibit leucine transport in the human prostate cancer cell line LNCaP with IC50 values ranging from 7.0 to 98.9 μM, which were more potent than the L-type amino acid transporter (LAT) family inhibitor, BCH. Finally, Chapter 6 describes the development of an analytical method using UHPLC-MS that was applied to 16 crude CH2Cl2 extracts from Australian Celastraceae plants. A subset of the available Celastraceae plants from Grifitth University’s NatureBank resource was accessed during these studies that included three barks, one fruit, one leaf, seven roots, two twigs and two mixed samples all of which were collected from the State of Queensland. The data generated were analysed and dereplication performed using scientific databases such as the Dictionary of Natural Products and SciFinder in order to identify new natural products from Celastraceae plants. These investigations led to the large-scale extraction and isolation work on the prioritised Denhamia celastroides fruits sample, which resulted in the purification of the new natural products, denhaminol O–R. and a known analogue denhaminol G. In summary, this thesis describes the isolation and characterisation of 18 new natural products and 12 known metabolites from three Australian Celastraceae plants. The chemical structures of all compounds were determined by detailed interpretation of 1D/2D NMR and MS data and X-ray crystallography studies. Full spectroscopic and spectrometric characterisation of all new compounds was performed using NMR, UV, IR, ECD and specific rotation. The compounds were screened for their leucine uptake inhibition in LNCaP cells and several compounds were identified as leucine uptake inhibitors, which were more potent than the LAT family inhibitor and currently used positive control, BCH. This thesis describes for the first time the inhibition of leucine uptake in prostate cancer cells by dihydro-β-agarofurans; the most potent inhibitor of LAT (bilocularin G, IC50 = 2.5 μM) to date was also reported during these studies. Finally, the new UHPLC-MS methodology developed during these studies was used to rapidly analyse 16 Celastraceae plants, and subsequently prioritise three samples for future metabolomics investigations. The fruits of D. celastroides were chosen for chemical investigation due to most encouraging data. An additional four new compounds (denhaminols O–R) were successfully isolated from the CH2Cl2 of the fruits of D. celastroides. This study exemplified the advantage of UHPLC-MS in combination with scientific databases data analysis in natural products dereplication.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
The author owns the copyright in this thesis, unless stated otherwise.
Leucine transport inhibition