Janthitrems are believed to be involved in the observed sporadic cases of AR37- infected perennial ryegrass staggers. Investigations into the role of janthitrems in perennial ryegrass staggers are difficult as isolation of the compounds from the ryegrass is hindered by the inherent instability of these compounds. Therefore attempts were made to isolate janthitrems from an alternative source, allowing these janthitrem analogues to be used as surrogates for endophyte produced janthitrems. Analysis of a series of Penicillium janthinellum cultures revealed the presence of janthitrems in a number of strains, including janthitrem B, janthitrem C and two novel janthitrem compounds. Detailed one- and two-dimensional NMR and mass spectral techniques identified the two novel compounds as 11,12- epoxyjanthitrems B and C, which were subsequently given the trivial names janthitrems A and D, respectively. Janthitrems B and C were isolated and identified by NMR and revisions of some previously reported chemical shift assignments were proposed. In addition to the janthitrems, penitrems were also identified in two strains of P. janthinellum. The isolated janthitrem B was utilised for the development of efficient extraction procedures, and for the determination of ideal storage conditions for janthitrem compounds. A method for the extraction and isolation of janthitrem B from a P. janthinellum culture was developed and optimised to yield 6 mg of janthitrem B from 900 mL of fungal culture in two days. Stability studies of janthitrem B indicated the ideal storage condition which minimised degradation was dry at −80 C where only 7% sample loss was observed over 300 days. Bioactivity studies of janthitrems A and B found these compounds to be tremorgenic to mice, with janthitrem A (an epoxyjanthitrem) inducing more severe tremors than janthitrem B. Insect testing also showed that both janthitrems A and B displayed anti-insect activity to porina larvae. Since the epoxyjanthitrems, which are associated with AR37 endophyte-infected ryegrass, were also shown to be tremorgenic and to display anti-insect activity, the insect resistance and the sporadic cases of ryegrass staggers displayed by AR37 may be related to the presence of epoxyjanthitrem compounds. LC-UV-MS analysis of janthitrems A-D, penitrems A-F, lolitrem B, paspalinine, paxilline and terpendole C found these indole-diterpenoids to be more sensitive by analysis using an APCI source as opposed to an ESI source. APCI negative ion LC-UV-MS required source induced dissociation in combination with increased collision energy to suppress an acetate adduct peak, sourced from the acetic acid buffer. Negative ion MS2 and MS3 data produced more informative fragments compared to the conventional positive ion MS2 and MS3 data. The availability of both positive and negative ion LC-UV-MS methodologies will allow future endophyte products to be more thoroughly screened for different classes of secondary metabolites. Extracts of mouldy walnuts were analysed for the presence of tremorgenic mycotoxins after a dog was found to exhibit symptoms characteristic of tremorgenic mycotoxicosis. LC-UV-MS analysis of the mouldy walnuts identified the tremorgenic mycotoxins penitrems A-F, thus confirming the veterinarian's tentative diagnosis of canine tremorgenic mycotoxicosis the first reported case in New Zealand.

In order to search for bioactive compounds, we have studied the chemical constituents from the organic extracts of soft coral Klyxum molle. In this study, fifteen eunicellin-type diterpenoids, including eleven new compounds, klymollins K¡VU (1¡V11), along with four known compounds 12¡V15 were isolated. Compounds 16 and 17 were prepared by chemical synthesis. The structures of all compounds were established by spectroscopic methods and comparing the spectral data with known compounds. Compound 5 represents the first eunicellin-type compound with phenylacetyl group. The cytotoxicity of compounds 1¡V17 against K562 (human erythro myeloblastoid leukemia), Molt-4 (human acute lymphoblastic leukemia), and T47D (human breast earcinoma) were determined. Compounds 1, 2, and 3 exhibited weak cytotoxicity against Molt-4 (with ED50 11.52 ¡Ó 2.75, 20.41 ¡Ó 2.92, and 13.11 ¡Ó 3.87 £gg/mL). Compound 5 was found to exhibt significant cytotoxicity toward K562, Molt-4, and T47D (with ED50 4.32 ¡Ó 1.38, 2.36 ¡Ó 0.34, and 4.65 ¡Ó 0.93 £gg/mL). Compound 5 also displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils at 10 £gg/mL (with Inh % 81.56 ¡Ó 3.23, and 89.16 ¡Ó 5.77).

Natural products play a historical role in the discovery of medicine but present unique challenges for chemical isolation, identification and production. In this work we describe the identification of twenty novel diterpenoids. These were isolated by use of chromatography, and the structures determined by spectroscopic methods, primarily 1D and 2D NMR. Six of these possess unprecedented diterpenoid skeletons and two of them show significant growth inhibitory effects on cancer cell lines in vitro (GI50 < 10 μM). The biomimetic semisynthesis of diterpendoids and analogues is also presented. Access to the bielschowskyane carbon skeleton by dearomatization of a furanocembranoid precursor is described. Highlights include a stereoselective alkene epoxidation, a novel kinetic furan dearomatization method, and an efficient [2+2] photochemical cycloaddition. The role of conformational steering was studied spectroscopically using VT 1H-NMR and NOESY as well as quantum chemical calculations at the DFT level of theory. We also disclose a biomimetic synthesis of providencin using a photochemical Norrish-Yang cyclization. This provided the absolute configuration by chemical correlation with the precursor bipinnatin E, the latter determined by x-ray diffraction. An unexpected, regioisomeric byproduct was observed and a possible mechanism is proposed. A biomimetic synthesis of the diterpene alkaloid aceropterine is also described, using an epoxidation-rearrangement cascade. This work led to a revised structure of aceropterine, formulated by spectroscopic methods. Finally, the isolation and structure elucidation of a novel, cyclic lipopeptide from Pseudomonas sp. is described. The compound was obtained using a unique antibiotic crowd sourcing approach and the structure determined by spectroscopic methods and advanced Marfey’s analysis. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

ABSTRACT The Genus Casearia is a rich source of cleordane - type diterpenes. To search for practical sources of potential cleordane - type diterpenes, and to study the structure and activity relationship of cleordane - type diterpenes, the twigs and leaves of Casearia membranacea Hance ( Flacourtiaceae ) was collected for phytochemical and anti-tumor investigation. Bioassay-directed fractionation of an ethyl acetate layer of Casearia membranacea has resulted in the isolation of six new clerodane-type diterpenes. The structures of these cleordane-type diterpenes were established and designated as Caseamembrins G¡ãL¡]1¡ã6¡^and their derivatives compounds 7 and 8. The structures of compounds 1¡ã8 were determined by application of NMR techniques included 1H NMR, 13C NMR, DEPT, COSY, HMQC, HMBC, NOESY and another physical methods which include MS, UV, IR and optical rotation, and the published reports about the data of related compounds. The spectral data of 1¡ã8 are in conformity with the basic skeleton of cleordane-type diterpenes previously isolated from Casearia membranacea Hance. The basic structures of 1¡ã8 contain two 6- menbered ring. The structures were identified as rel-(2S,5R,6R,8S,9S,10R, 18S,19R)-2-hydroxy-6-butanoyloxy-18,19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene (Caseamembrin G, 1¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S, 19R)-2-hydroxy-6,18-dibutanoyloxy-19-acetyloxy-18,19-epoxy-cleroda -3,13(16),14-triene (Caseamembrin H, 2¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S ,19R)-2-(2-methylbutanoyloxy)-6-hydroxy-18-methoxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]Caseamembrin I, 3¡^¡Brel-(2R,5R ,6R,8S,9S,10R)-2-(2-methylbutanoyloxy)-6-hydroxy-cleroda-3,13(16),14 -triene-18,19-dicarboxaldehyde ( Caseamembrin J, 4¡^¡Brel-(2S,5R,6R,7R, 8S,9S,10R)-2,7-diacetyloxy-6-hydroxy-cleroda-3,13(16),14-triene-18,19-dicarboxaldehyde¡]Caseamembrin K, 5¡^¡Brel-(2S,5R,6S,7R,8S,9S,10R, 18S,19R)-2-butanoyloxy-6,7-dihydroxy-18-butanoyloxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]Caseamembrin L, 6¡^¡A¤Îrel-(2S,5R ,6R,8S,9S,10R,18S,19R)-2-O-acetyl-6-butanoyloxy-18,19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene ( 7¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S,19 R)-2-O-acetyl-6,18-dibutanoyloxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]8¡^. It is worthy to mention that this is the first report of the isolation of 1¡Ð6 from a natural source and their derivatives compounds 7 and 8. Compound 1 exhibited moderate cytotoxicity against¡]Hepa59T/VGH¡^ ,¡]KB¡^and ¡]Hela¡^cancer cells , but compound 3 and 4¡B5 show no activity against those cancer cell lines.

Investigation on the chemical constituents of the EtOH extract of the soft coral Lobophytum sarcophytoides, collected off the coast of Dongsha Atoll, Taiwan, has led to the isolation of ten natural compounds 1¡V10, including four new cembrane-type diterpenoids, Sarcophytolins A¡VD (1¡V4) and one new polyhydroxylated steroid 23, 24-Dimethyl-cholesta-5, 16-diene-3£], 20£\-diol (5) along with five know compounds 6¡V10¡CThe structure of compounds 1¡V10 were established by detailed spectral data analysis (IR, MS, 1D, 2D NMR) and by comparison of the spectral data with those of the related known compounds. The relative stereochemistries of compound 5 was further confirmed by X-ray single-crystal diffraction analysis. The cytotoxicity of compounds 1¡V10 against the Daoy (human medulloblastoma), Hep-2 (human laryngeal carcinoma), MCF-7 (human breast adenocarcinoma), CCRF-CEM (human T-cell acute lymphoblastic leukemia), and DLD-1/WiDr (human colon adenocarcinoma) tumor cell lines were determined. Compounds 6, 8, 9, and 10 exhibited moderate cytotoxicities against the tested tumor cells. Furthermore, compounds 1, 2, 4, 6, and 10 were found to show significant activity against the accumulation of the pro-inflammatory iNOS protein at 10 £gM.

Six new compounds were isolated from the aerial and root parts of S. viridis L. cv. Blue Jeans. Two new triterpenoids, lup-20(29)-ene-2α-acetate-3β-ol, and lup-20(29)-ene-2α-ol-3β-acetate were found in the aerial part together with lup-20(29)-ene-2α-3β-diol, ursolic acid, oleanolic acid, β-sitosterol and β-sitosterol glucoside. Three new diterpenoids, 1-oxomicrostegiol, viroxocane, viridoquinone, together with five known diterpenoids, ferruginol, salvinolonyl 12-methyl ether, microstegiol, 7α-acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione and 7α,14-dihydroxy-8,13-abietadiene-11,12-dione were found in roots. 1-Docosyl ferulate, 2'',3''-di-O-acetyl-martynoside and a mixture of 2-(4'-alkoxy-phenyl) ethyl alkanoates were also isolated from roots. Seven caffeic acid derivatives, five flavonoid glycosides, and salidroside were found in the crude aerial fraction. Four caffeic acid derivatives were known phenylpropanoids, i.e. trans-, cis-verbascoside, leucosceptoside A and martynoside, which are now reported in the genus Salvia for the first time. The others were caffeic acid, rosmarinic acid and 6-O-caffeoyl-glucose. A new flavonoid glycoside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside was also identified in the aerial part with four known flavone glycosides: luteolin-7-O-β-glucopyranoside, luteolin-7-O-β-galactopyranoside, luteolin-7-O-rutinoside and apigenin-7-O-β-glucopyranoside. Verbascoside (acteoside), which is a major component in this plant, showed a significant protective effect against UVA induced damage in a human skin fibroblast model in vitro. It exhibited 1.4 fold protective effect against UVA induced necrosis with 1.4 fold higher in cell survival. 50 μM Verbascoside showed the same protective effect as 100 μM DFO at a high intensity UVA dose (500 kJ/m2). Further determination of organelle specific protection suggested a mechanism of action in mitochondria. Two terpenoids, lup-20(29)-ene-2α-acetate-3β-ol and 7α,14-dihydroxy-8,13-abieta-diene-11,12-dione, exhibited antibacterial activity against Enterococcus faecalis with MIC 50 μM. Microstegiol was also active against Staphylococcus aureus with MIC 50 μM. Ursolic acid, oleanolic acid and ferruginol showed appreciable antibacterial activity against three Gram-positive bacteria, Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus with MIC 12.5-50 μM. The other diterpenoids were active against all three Gram-positive bacteria with MIC 100-200 μM. None of crude fractions was active against three Gram-negative bacteria, Klebsiella pneumoniae, Proteus vulgaris, and Escherichia coli.

Corals are marine animals from the class Anthozoa and are widely distributed in tropical and subtropical seawaters. They are considered as an important source of lead compounds for drug discovery. For evaluating the medicinal activities of briarane-type diterpenoids (BrDs) from marine coral Briareum excavatum, the regulation of a group of briarane-type diterpenoids (BrDs) on dendritic cell (DC) function, TPA-induced dermatitis and type 1 diabetes was investigated. The results show that the BrD excavatolide K (BrD2) remarkably suppressed the activation of human DCs, especially the expression of IL-12 p40. This inhibitory effect was mediated apparently by interference with the rictor-mTOR/Akt-mediated signaling network, resulting in persistent-phase activation of NF-kB and Erk1/2 signalings. In addition, the 8,17-epoxide of BrDs was observed to play a crucial role in inhibition of IL-12 p40 expression. Replacement of the C-12 hydroxyl group with longer esters in BrDs gradually decreased this inhibitory activity in human DCs. BrD excavatolide B (BrD1) effectively suppressed the capacity of mouse bone marrow-derived DCs to induce an antigen-specific Th1, response via the inhibition of IL-12 expression. Moreover, excavatolide B prevented the onset of autoreactive T cell-mediated diabetes in NOD/SCID mice. Furthermore, excavatolide B remarkably suppressed TPA-induced vascular permeability and edema in test skin tissues. At the biochemical level, excavatolide B inhibited TPA-induced expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase-9, the key indicators of cutaneous inflammation. This inhibition is apparently mediated by interference with the Akt/NF-kB-mediated signaling network. Together, these studies demonstrate that BrDs from specific marine corals can effectively regulate defined molecular and cellular functions of dendritic cells, suppress TPA-induced dermatitis, and prevent type 1 diabetes in murine models suggesting that BrDs may warrant further investigation as natural immunomodulatory agents or therapeutics.

In order to discover marine bioactive compounds, we investigated the chemical constituents from the organic extracts of soft coral Klyxum molle. Repeated column chromatography on the ethyl acetate extract of soft coral Klyxum molle, resulted in the isolation of nine new eunicellin-type diterpenoids, klymollins V¡ÐZ (1¡Ð5) and klyxumollins A¡ÐD (6¡Ð9), and four known compounds (10¡Ð13). The structures of new compounds were determined on the basis of extensive spectroscopic data (IR, MS, 1D and 2D NMR) and by comparison of the spectral data with those of the related known compounds. The cytotoxicity of compounds 1¡Ð13 was evaluated against five tumor cell lines including CCRF-CEM (human T cell lymphoblast-like cell line), K562 (human erythromyeloblastoid leukemia cell line), Molt 4 (human acute lymphoblastic leukemia cell line), T47D (human ductal breast epithelial tumor cell line) and DLD-1 (human colorectal adenocarcinoma cell line). Compound 2 exhibited weak cytotoxicity toward three cancer cells (CCRF-CEM, Molt 4 and T47D) and compounds 4 and 9 showed weak cytotoxicity against CCRF-CEM cancer cells. Compound 11 was found to exhibit stronger cytotoxicity toward CCRF-CEM cancer cells (ED50 values of 4.2 £gg/mL). On the other hand, compound 3 showed weak activity against the accumulation of the pre-inflammatory cytokine IL-6 in LPS-induced macrophages, at a concentration of 25 £gM.

Seven new eunicellin-based diterpenoids, krempfielins K¡ÐQ (1¡Ð7), along with six known compounds, litophynin I (8), krempfielin A (9), krempfielin D (10), sclerophytin F (11), litophynol B (12), and litophynol I monoacetate (13), have been isolated from the soft coral Cladiella krempfi which was collected from Penghu Island of Taiwan. Their structures were determined through extensive spectroscopic data (IR, MS, 1H NMR, 13C NMR, HSQC, HMBC, COSY, and NOESY) and by comparison of the spectral data with those of the related known compounds. The structure of compound 1 is rare due to the presence of a tetrahydropyran ring constituted from C2¡ÐC6 and oxygen atom. Compound 7 is the first discovery of eunicellin-based dimer. The anti-inflammation activity of twelve compounds (1¡V6 and 8¡V13) at 10 £gM was studied. The results showed that compound 4 inhibited more than 70% express of elastase released in FMLP/CB-induced human neutrophils .

This dissertation mainly presented the investigation of natural products from two different Formosan gongonian soft corals, Briareum violacea and Junceella fragilis. Their extracts were examined by intensive chromatographic methods. Eighty-four compounds including fifty new briaranes were isolated, and parts of their biological activities were studied. Natural products investigation of the Taiwanese gorgonian octocoral Junceella fragilis found fifteen new briarane-type diterpenes, namely frajunolides E-S (1-15), ninteen known briaranes, eg. junceellin, junceellolides A-E¡Bjunceellolide K, 11£\,20£\-epoxy-4-deacetoxyjuncellolide D, umbraculolide A, junceellonoid A, juncin P, juncins Y-ZI, frajunolides A-D and praelolide, and a sterol, ergosterol peroxide. Soft coral B. violacea (Quoy and Gaimard) of Taiwan has isolated thirty-five new briarane-type diterpenoids, briaviolides A-Z (16-41) and viobrianolides A-I (42-50), in addition to fourteen known diterpene lactones, stylatulide lactone, excavatolide A, 9-deacetylstylatulide lactone, 4£]-acetoxy-9-deacetylstylatulide lactone¡B Brianthein Z¡B, renillafoulin A, braexcavatolide E, braexcavatolide I, minabein-4, minabein-6, milolide K, solenolide A, and solenolides D-E. Structures of all above compounds were examined by physical and spectroscopic analyses including mass, IR, UV spectra, optical rotation and 1D, 2D NMR, as well as in comparison with the published data. Moreover, the structures of compounds 16, 32 and prarlolide were further confirmed by single X-ray crystallographic analysis. The stereochemistry of these compounds was investigated by NOESY method, and the configuration of compounds 32-38 were determined by Circular Dichroism spectroscopic analysis. Cytotoixcity and in vitro anti-inflammatory activities were studied by Dr. Kuo, Yao-Haur and Dr. Hwang, Tsong-Long, respectively. Junceellin, junceellolide B and juncin ZI exhibited weak cytotoxicity against Hep2 (Human laryngeal carcinoma), Doay (Human medulloblastoma), WiDr (Human colon adenocarcinoma), Hela (Human cervical epitheloid carcinoma). The in vitro anti-inflammatory activities of 1-7 were evaluated for inhibition of elastase release and for the generation of superoxide anion, as tested on human neutrophils. Compounds 1 and 6 exhibited weak inhibition of elastase release and superoxide anion at 10 £gg/mL. The biological activity analysis of compounds 16-50 are in progress.