title: Chemical and Biological Investigations of Australian Crinoids
Creators: name: Lum, Kah Yean
Publisher: name: Griffith University
Year: 2020
Subjects: subject: polyketide constituents
subject: crinoids
subject: Australian waters

description: Natural products (NPs) are commonly defined as the secondary metabolites derived from plants, microorganisms, fungi, insects, and marine invertebrates, as the result of adaptation to the environment or as defense mechanisms against predators. Throughout history, the use of NPs has been described in the form of traditional medicines in different cultures for the treatment of ailments. Apart from their role in medicinal applications, the structures of NPs can also act as lead molecules to inspire the design of new drugs. Of the 1562 new approved drugs by U.S. Food and Drug Administration (FDA) from 1981–2014, 791 (51%) were NPs, NP derivatives or NP-inspired drugs. Since the development of SCUBA in the mid-twentieth century, the marine environment, which contains an incredible diversity of organisms, has been described as the most desired source of NPs for drug discovery research. To date, approximately 35,147 marine natural products (MNPs) had been identified from various organisms, such as marine invertebrates (e.g. sponges, crinoids and ascidians), microorganisms and algae. Many of these MNPs have been found to exhibit a wide variety of pharmaceutically relevant bioactivities, such as anticancer, antimicrobial, antiviral, and anti-inflammatory activities. There are currently 12 marine-derived compounds that have been approved as therapeutic drugs for the treatment of cancer, viral infections, hypertriglyceridemia, and analgesia; while 27 drug candidates are currently in phase I, II, or III clinical trials. While numerous marine invertebrates have been well explored for bioactive MNPs in the last seven decades, crinoids belong to the phylum Echinodermata remained under investigated for their chemistry. Crinoids are the most primitive group of presentday echinoderms. They are known to produce diverse polyketide-derived pigments, which are not only responsible for their colourful appearance, but also have demonstrated significant activity in a range of biomedical assays. There are approximately 700 crinoid species that have been identified worldwide, however, only 36 species have been chemically investigated and only 91 new compounds have been reported to date. Owing to our continuing research interest on crinoid chemistry, the main aim of this PhD project was to identify new chemistry from crinoids sourced from Australian waters and subsequently screen the isolated compounds in a variety of biological assays. The first crinoid project of this thesis focused on the chemistry of the feather star Capillaster multiradiatus since no studies had been undertaken on this Australian species. Capillasterin A, a novel pyrano[2,3-f]chromene, together with seven known naphthopyrones including comaparvin, TMC-256C1, 6-methoxycomaparvin 5-methyl ether, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, 5,8- dihydroxy-6,10-dimethoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256A1 and 6-methoxycomaparvin were isolated from an EtOH/H2O extract of C. multiradiatus collected by collaborators from the Queensland Museum. The structures of all the compounds were determined by detailed spectroscopic (1D/2D NMR and MS) data analysis. As previous studies demonstrated that HIV gene expression is dependent on the host transcription factor complex NF-B and naphthopyrones were reported to inhibit NF-B signalling pathway, the six known naphthopyrones isolated from this crinoid, together with capillasterin A were screened in an anti-HIV assay. Five known naphthopyrones were observed to display moderate inhibition of in vitro HIV-1 replication in a T cell line with EC50 values ranging from 7.5 to 25.5 μM without concomitant cytotoxicity. The three most abundant compounds, capillasterin A, 6- methoxycomaparvin 5-methyl ether, and TMC-256A1 were also tested for their ability to stimulate the proliferation of GFP-expressing immortalised mouse olfactory ensheathing cells (mOEC) using a cell proliferation assay; none of the compounds showed a significant increase in mOEC viability at 10 μM after 24 hours of treatment. The AIMS Bioresources Library, which consisted of over 3000 marine samples, has recently been transferred to the NatureBank biota repository, which presented us with the opportunity to explore several new crinoid samples from a chemical perspective. Hence, the second PhD project, two AIMS-derived Australian crinoid Comatula rotalaria specimens collected from different locations on the Great Barrier Reef were selected for potential new chemistry, since preliminary UHPLC analysis of these crinoid extracts suggested the presence of new anthraquinone chemistry; only four acyl derivatives of anthraquinones had been identified from this species prior to our studies. Five new taurine-conjugated anthraquinones, named comatulins A−E, together with 11 known metabolites, rhodocomatulin 7-methyl ether, 12-desethylrhodocomatulin 7-methyl ether, rhodocomatulin 5,7-dimethyl ether, 12-desethylrhodocomatulin 5,7-dimethyl ether, rhodocomatulin, rhodolamprometrin, 6-methoxyrhodocomatulin 7-methyl ether, rheoemodin, 6-methoxycomaparvin, 6-methoxycomaparvin 5-methyl ether, and 5,8- dihydroxy-6,10-dimethoxy-2-methyl-4H-benzo[h]chromen-4-one were identified. The structures of all the compounds were elucidated by detailed spectroscopic and spectrometric data analysis. The first X-ray crystal structure of a crinoid-derived acyl anthraquinone, rhodocomatulin 5,7-dimethyl ether, was also obtained. Ten compounds together with two additional naphthopyrone derivatives (comaparvin and 6- methoxycomaparvin 5,8-dimethyl ether) were evaluated for their ability to inhibit HIV-1 replication in vitro; none of the compounds were active at 100 μM. Furthermore, a subset of compounds was tested for their nematocidal activity against Haemonchus contortus, which is a highly pathogenic parasite of small ruminants. The semi-synthetic compound, 6-methoxycomaparvin 5,8-dimethyl ether, showed an inhibitory effect on larval motility (IC50 = 30 μM) and development (IC50 = 31 μM) and induced the eviscerated (Evi) phenotype. In Chapter 4, since none of the crinoid-derived polyketides identified during this PhD had been evaluated for their ability to increase phagocytic activity of human OECs (hOEC), six naphthopyrones and eight anthraquinones were screened using an hOEC phagocytosis assay that has recently been developed by the Clem Jones Centre for Neurobiology and Stem Cell Research group. In addition, microthecaline A and its acetylated, methylated and pivaloylated derivatives, together with antimalarial drug amodiaquine obtained from the in-house Davis compound library, were incorporated into the screening. Results from the primary screening demonstrated that four compounds including 6-methoxycomaparvin 5-methyl ether, 5,8-dihydroxy-6,10-dimethoxy-2- methyl-4H-benzo[h]chromen-4-one, comatulin A, and amodiaquine were found to significantly increase the phagocytic activity and the phagocytic efficiency of hOECs. These findings warrant further investigations in the near future to further expand the preliminary biology results and gain insights in compound specificity and potency. Encouraged by our findings in Chapter 3, we developed a dereplication method using UHPLC-MS for the identification of new sulphur-containing metabolites from Australian crinoids, the details of which are described in Chapter 5. The n-BuOH soluble material of 16 crinoids, including the two C. rotalaria samples described in Chapter 3, were subjected to UHPLC-MS profiling using an optimised method. These crinoids were all sourced from Griffith University’s NatureBank biota repository. The generated UHPLC-MS data were analysed based on the characteristic fragment ions of sulphated compounds in conjunction with scientific database mining; SciFinder Scholar and MarinLit databases were used in this particular study. These investigations led to the large-scale extraction and isolation work on the prioritised crinoid Dichrometra flagellata, which resulted in the isolation of a previously undescribed sulphated compound, which we have tentatively assigned as 5,10-dihydroxy-6–methoxy-8- sulphate-2-propyl-4H-naphtho[2,3-b]pyran-4-one. In summary, this thesis describes the isolation of seven new polyketide constituents and 17 known compounds from four crinoids collected from Australian waters. The chemical structures of all compounds were determined by detailed spectroscopic and spectrometric data analysis. Among all the tested crinoid metabolites, comaparvin was the most active compound in an anti-HIV replication assay, with an IC50 of 7.5 ± 1.7 μM; 6-methoxycomaparvin 5,8-dimethyl ether displayed an inhibitory effect on larval motility (IC50 = 30 μM) and development (IC50 = 31 μM), and induced the Evi phenotype in an anthelmintic assay; 6-methoxycomaparvin 5-methyl ether, 5,8- dihydroxy-6,10-dimethoxy-2-methyl-4H-benzo[h]chromen-4-one, and comatulin A significantly increased the phagocytic activity and the phagocytic efficiency of hOECs. All compounds isolated during this PhD project will be deposited into the Davis Open- Access Compound Library, which is located at Compounds Australia, Griffith University. Compounds Australia makes this academic library available for biological evaluations by both local and international researchers. In addition, the UHPLC-MS methodology developed during these studies enabled the rapid identification of new sulphur-containing compounds from n-BuOH soluble material derived from 16 crinoids, which resulted in the isolation of a new sulphated compound from the prioritised crinoid Dichrometra flagellata; this is the first report of NP chemistry from this crinoid genus. These findings further highlight the importance of UHPLC-MS as a dereplication tool in NP research.