Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity
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This project presented a strategy to select a subset of prefractionated fractions for screening. Marine and plant biota samples were chosen based on their rare taxonomies and mass spectroscopic data. A method to reduce 1155 fractions generated from 105 selected samples to 330 UV active fractions was developed. Cancer cell-based screening of 330 prefractionated fractions against four cancer cell lines (A549, HeLa, LNCaP and PC3) and non-cancer cells (HEK) resulted in nineteen active fractions belonging to fourteen biota samples (two plants and twelve marine organisms). One plant and six marine animals were chosen for further investigation. Subsequent mass-guided isolation led to the identification of forty-four secondary metabolites, nine of which were not previously reported. Structures of these compounds were elucidated by spectroscopic methods (1D and 2D-NMR, MS, CD and specific optical rotation) and chemical methods. In the plant sample Neolitsea dealbata, a total of nine alkaloids (55-63) were isolated. A new aporphine, normecambroline (55), showed selective activity against HeLa cells with an IC50 of 4.0 μM while its analogue, roemerine (56), displayed non-selective activity against all four cancer cell lines (A549, HeLa, LNCaP and PC3) and two non cancer cell lines (HEK, NFF). One of the marine samples, a specimen of the Potter Reef marine sponge Diacarnus sp., showed the presence of terpene peroxides. Three known peroxide compounds, sigmosceptrellin (27), deacarperoxide (28) and methyldiacarnoate (29), were identified. Compound 27 inhibited cytotoxicity against all six cell lines with IC50 values ranging from 0.4 to 3.1 μM while the other two compounds were not active. Chemical investigation of a marine specimen from Houghton Reef, Neopetrosia exigua, resulted in the isolation of two cytotoxic compounds, mortuporamine C (42) and a new 3-alkylpyridinium alkaloid, dehydrocyclostellettamine A (43). These two compounds displayed activity against four cancer cell lines with IC50 values in micromolar concentrations (3.0-13.7 μM). Three new cyclodepsipeptides, neamphamide B (86), neamphamide C (87) and neamphamide D (88), were found as constituents of a rare marine sponge Neamphius huxleyi collected at Milln Reef, off Cape Grafton. Their structures were elucidated by NMR spectroscopy and multiple stages of accurate mass measurements (ESI-FTICR-MSn). Stereochemistry of residues of the peptides was determined by the Marfey amino acid method and J-based configurational analysis. These compounds inhibited the cell growth with IC50 values ranging from 91.3 to 366.1 nM. Two previously unreported milnamide E (116) and hemiasterlin D (117) together with nine known small peptides were isolated from a new sponge genus, Pipestela candelabra. Compound 117 was identified as the first peptide skeleton discovered in nature with a side chain containing 2-hydroxyacetic acid, tert-leucine and N-methylvinylogous valine residues attached to the indole nitrogen. This compound exhibited activity against HeLa cells with an IC50 of 1.8 nM. Cytotoxic results indicated all hemiasterlin derivatives were approximately 100 fold more active against cancer cell lines than the milnamide family.
Master of Philosophy (MPhil)
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