Raymond Andersen

The discovery of novel natural products continues to be critical for the development of new pharmaceuticals. Innovative methods to discover novel natural products can reveal previously overlooked chemical diversity. One such method is genome mining, where sequenced bacterial genomes are assessed for the presence of biosynthetic gene clusters. Another such method is bioassay-guided fractionation. Following either of these approaches, the bacteria must be grown and harvested, and novel natural products must be isolated and characterized. In the first part of this thesis, a nitrogen-NMR guided approach was developed to retrieve genetically predicted natural products from bacterial cultures. Piperazic acid (Piz)-containing natural products were targeted because this unique amino acid is often found in peptidic natural products with biological activity and impressive chemical structures. Piz contains a unique nitrogen-proton NMR correlation targeted through ¹H-¹⁵N HSQC. The unique N-H correlation also gave access to Piz’s diagnostic spin system through ¹H-¹⁵N HSQC-TOCSY NMR experiments. These two ¹⁵N NMR experiments were used to monitor for the presence of peptides containing Piz in culture extracts of genome-mined bacteria. Through the application of these ¹⁵N NMR experiments to guide isolation of Piz natural products, four novel compounds were discovered from Streptomyces incarnatus NRRL 8089. Three of these were isolated and structure’s elucidated as part of this thesis work: dentigerumycin F (4.2), dentigerumycin G (4.1) and incarnatapeptin A (4.3). 4.3 demonstrated a unique bicyclic moiety not previously seen in chemical structures, and a fourth compound, incarnatapeptin B (4.4), has in vitro cytotoxicity. In the second part of the thesis, bioassay-guided fractionation is used to screen a small library of marine bacteria in various assays. Using this method, known natural product molecules were uncovered, along with the discovery of two novel natural products from the marine bacterium Salinispora arenicola RJA3005. These two compounds, 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4), were isolated from extracts of wild-type bacteria for the first time. Feeding studies and analysis of ¹³C splitting patterns suggest that these compounds were biosynthesized from bacterium through phosphoenolpyruvate and erythrose precursors. Altogether, this thesis's work develops and harnesses various natural product discovery methods to uncover diverse natural products.

View record

Natural products are a rich resource for the discovery and development of chemical tools which can be used to study biological processes. The marine environment contains unique niches where organisms have the ability to adapt in producing chemical compounds that are shown to have various biological utilities. In the following chapters, the synthesis of various analogues of Clionamine B, a biologically useful aminosteroid isolated from the marine sponge Cliona celata and their use as chemical biology tools are outlined. Clionamine B was previously synthesized in the Andersen Lab and was found to potently stimulate autophagy and clear latent Mycobacterium tuberculosis (Mtb) from human macrophages. In chapter 2, a more efficient semi-synthesis of analogues of clionamine B is presented, using environmentally benign reagents, in order to perform structure-activity relationship (SAR) studies. Using this methodology, ten analogues of clionamine B were synthesized, all of which were found to stimulate autophagy and clear Mtb to varying degrees. One of the analogues synthesized, an N-benzyl derivative of clionamine B was found to stimulate autophagy very potently compared to the natural product. In addition, the molecular target of the clionamines was identified as PIK1, a phosphatidylinositol 4-kinase which is known to regulate trafficking to and from the Golgi; and play an important role in the mechanism of autophagy and affect Atg9 trafficking. Chapters 3 and 4 highlight efforts towards the synthesis of analogues with reduced lipophilicity and their effect on autophagy stimulation and Mtb inhibition. This was done systematically by introducing heteroatoms in the lactone side chain. However, the effects of reducing the lipophilicity on the autophagy stimulating and Mtb clearing activities of analogues of clionamine B were not conclusively determined. Chapter 5 highlights efforts towards the synthesis of various molecular probes of the analogue of clionamine B displaying the most desirable combination of autophagy stimulating and Mtb clearing activities observed so far. The synthesis of a biotinylated molecular probe was achieved, and was shown to be effective in corroborating PIK1 as the molecular target of the clionamines using yeast cell lysate.

View record

Natural products are one of the most important resources for the discovery anddevelopment of new pharmaceuticals. Organisms from different terrestrial and marine environments produce secondary metabolites with unique biological activities that could be utilized by humans medicinally and in other ways. In an effort to search for new potential lead compounds, we use bioassay-guided fractionation and structure elucidation tools to identify new biologically active natural products. The work presented in Chapters 2 and 3 is in an effort to search for new small molecules that activate HIV-1 provirus expression. Novel sesterterpenoids alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24), along with the known sesterterpenoids ansellones A−C (2.86−2.88), were isolated from a Phorbas sp. sponge collected in Howe Sound, British Columbia. The isolation and structure elucidations of these novel sesterterpenoids are presented. These compounds are a family of highly functionalized sesterterpenoids that have the alotane, ansellane, anvilane and phorane carbon skeletons. The unique ansellane and anvilane carbon skeletons have so far only been found in this particular sponge Phorbas sp. Several classes of natural products produced by this genus of sponge are reviewed. These sesterterpenoids are able to activate HIV-1 provirus expression. The most active analog, alotaketal C (3.9) showed 13.8 % activation of HIV-1 provirus expression at a concentration of 30 μM. Chapter 4 describes the identification of two anti-onchocerciasis natural products isolated from the extracts of traditional medicinal plants. Lantadene A (4.4) and morindicinone (4.5) were isolated from the extracts of Lantata camara and Morinda lucida, respectively. The filaricidal properties of lantadene A (4.4) and morindicinone (4.5) were investigated using an in vitro anti-Onchocerca assay.

View record

3,6,7-trihydroxycoumarin C11 (2.14) was first isolated from the green alga Dasycladus vermicularis in 1983. C11 and 3,7,8-trihydroxycoumarin C21 (2.15), alongside their precursors C12 (2.18) and C22 (2.20), were synthesized for a target-based screen for anti-HCV drugs, where ideal hits eliminate fluorescence signals by inhibiting the proteolytic activity of HCV NS3pro/Pep4A against a synthetic peptide “BS-IQFS”. With C12 and C22 serving as negative controls, C11 and C21 inhibited the NS3pro/Pep4A activity in vitro. The IC₅₀’s of C11 and C21 were 3.07 μM and 2.10 μM, respectively.A bioassay-guided fractionation identified sintokamides A – E (3.11 – 3.15) from extracts of the sponge Dysidea sp. in 2008. In a phenotypic screen, the chlorinated dipeptides showed strong to modest inhibition of luciferase activity caused by AR NTD transactivation in LNCaP cells. Larger quantities of sintokamides A and B were isolated from the sponge for further biological study. After developing a practical synthetic route, a comprehensive SAR of the sintokamides was available from the in vitro activities of 29 synthetic analogues/precursors based on a 1,17-dinorsintokamide skeleton. LPY26 [(4R,10R)-3.233] showed the best biological activity in the synthetic analogues prepared to date and it was selected as a drug lead. Mechanism of action study using synthetic probes LPY30 (4.7) and LPY31 (4.8) revealed that the hexachlorinated 1,17-dinorsintokamides covalently bound to the AR, but not to the same AF1 region in the AR NTD as EPI-001 (3.8).The structure of latonduine A (5.1) isolated from the sponge Stylissa carteri and its total synthesis were published in 2003. Later, latonduine A was shown to be active in a phenotypic screen to find drug leads for the treatment of cystic fibrosis caused by the F508del mutation. Latonduine A could efficiently correct immunofluorescent F508del-CFTR trafficking from the endoplasmic reticulum to the plasma membrane in the engineered cells. Synthetic latonduine A and N-biotinylated latonduine A (5.17) were prepared to support biological studies aimed at identifying its cellular protein target(s). These studies culminated in the finding that latonduine A is an inhibitor of PARP-3 with an EC₅₀ of 400 pM in CFBE41o­- cells.

View record

Marine organisms are a potential source of lead compounds for drug discovery. Complex and unique chemical structures isolated from marine invertebrates and their associated microorganisms can interact with specific cellular targets and selectively modulate biological pathways that play an important role in the pathogenesis of various diseases such as diabetes and tuberculosis (TB). The isolation, structure elucidation, and total synthesis of bioactive marine natural products are described herein. Alotaketals A (2.1), B (2.3), D (2.4), and E (2.5) are a new class of sesterterpenoids isolated from the marine sponge Hamigera sp. that activate the cAMP cell signaling pathway. Here, the chemical structures of alotaketals and their unprecedented "alotane" skeleton are described. The ability of alotaketal A (2.1) to activate cAMP signaling (EC50 = 18 nM) can be attributed to direct activation of adenylyl cylcase. Alotaketal A (2.1) is a chemical tool that can be useful for the study of cAMP signaling function in diabetes and other diseases.Clionamines A (5.1), B (5.2), C (5.3), and D (5.4) are a new class of aminosteroids isolated from the marine sponge Cliona celata that strongly stimulate autophagy in MCF-7 human breast cancer cells. Clionamine A (5.1) was tested for its ability to clear Mycobacterium tuberculosis (Mtb) from infected THP-1 cells and it gave a clear dose response with complete clearance at 5 μM and an IC50 of ≈ 3 μM. The anti-TB activity of clionamines confirms the role of autophagy in the response of human macrophages against Mtb infection. Clionamine B (5.2) was synthesized starting from the steroidal sapogenin tigogenin (5.30) in 12 steps with ≈ 2% overall yield. Synthetic clionamine B (5.2) strongly stimulates autophagy at 30 μg/mL and inhibits Mtb proliferation in humane macrophages via autophagy activation. The clionamine pharmacophore was identified by structure-activity analysis of unnatural clionamines analogues that were synthesized starting from the steroidal sapogenin sarsasapogenin (5.5). Among these synthetic analogues, N-benzyl-3,5-epi-clionamine B (5.24) was found to be a potent inhibitor (MIC = 5 μg/mL) of Mtb proliferation in THP-1 human monocytic cells, indentifying N-benzyl-aminosteroids as a new class of potent antimicrobial compounds that are able to kill Mtb.

View record

Natural products offer an unparalleled resource for the discovery and development of chemical tools to be used by humans. The terrestrial and marine environments contain unique niches where organisms chemically adapt to produce compounds that have proven useful medicinally and beyond. In the following chapters, several classes of novel natural products from terrestrial fungi and marine sponges will be presented. In some cases, synthetic methodology, biological activity and enzymatic target identification will also be presented. Chapters 2 and 3 highlight ramariolides A–D (2.18–2.21) and dhilirolides A–N (3.9–3.22), two new fungal derived compound classes isolated from terrestrial sources. The structure elucidation of these compounds will be presented alongside their biological activities as antimycobacterial and insecticidal agents, respectively. Chapter 4 contains the structure elucidation of three new members of the xestoquinone family of compounds, xestolactone A (4.19), xestosaprol O (4.20), and xestosaprol P (4.21) along with their potent inhibitory effect on human indolamine 2, 3-dioxygenase (hIDO). A new method for synthetic access to derivatives of these compounds is demonstrated in Chapter 5 along with a brief structure activity relationship (SAR) study. Lastly, Chapter 6 discusses latonduine A (6.9), a sponge-derived alkaloid, which has shown promise as a lead structure for the correction of cystic fibrosis (CF). Probes derived from latonduine A (6.9) have led to identification of poly (ADP-ribose) polymerase (PARP) as the enzymatic target. Methodology for the probes’ construction and SAR studies resulting in simplified synthetic analogues of latonduine will be presented.

View record

Natural products play a central role in drug discovery. The Andersen lab focuses its efforts on the isolation and structure elucidation of compounds from the marine environment. Many of these compounds possess biological activity, and often their total synthesis is undertaken, to provide structure-activity relationship (SAR) studies for new pharmacophores, and to provide material to probe in vivo biological effects. Several projects probing the biological activities of natural products and their analogues by synthesis were completed.It has been proposed that small molecule activators of SHIP1 may be used as a novel therapy for hematopoietic malignancies as well as inflammatory disorders. Activation of the SHIP1 enzymatic pathway may also provide an alternative to PI3K inhibition. An SAR study based on the SHIP1 activating marine natural product pelorol was completed. The goal of the SAR study was to construct water-soluble analogues for the purpose of enhancing drug-like properties. The study yielded analogues that were active in vitro and in vivo.Small molecule antagonists of the N-terminal domain (NTD) of the androgen receptor (AR) are an appealing avenue of exploration for treating CRPC, an advanced form of prostate cancer resistant to current therapies. The marine natural products niphatenone A and B represent a novel NTD-AR antagonist pharmacophore. Their total syntheses were completed to aid in structure determination and provide additional material for biological testing. Furthermore, a click chemistry probe was constructed and it was shown that the natural product covalently binds to the NTD of the AR. Small molecule AR antagonists are currently used as a therapeutic treatment for prostate cancer. Studies towards the total synthesis of a terpene marine natural product discovered to be an AR antagonist are described.The biological role of cathepsin K in bone resorption has led to the development of inhibitors of cathepsin K as potential therapeutics to combat osteoporosis. Lichostatinal, a novel peptide-aldehyde natural product isolated from cultures of a terrestrial actinomycete was found to be a potent inhibitor of cathepsin K. Synthetic efforts towards lichostatinal, in order to verify its structure and to provide additional material for biological testing is described.

View record

Clionamines A-D (2.6-2.9) are new aminosteroids isolated from South African specimens of the sponge Cliona celata. All four compounds (2.6-2.9) are activators of autophagy in MCF-7 cells. Autophagy is a catabolic process that plays an important role in maintaining cellular homeostasis. Autophagy is also directly involved in the removal of bacterial and viral antigens and in the development of cancerous tumors.The novel sesterterpenoid ansellone A (3.4) was isolated from the nudibranch Cadlina luteomarginata and was later found to have been sequestered by the nudibranch from the sponge Phorbas sp. Ansellone A (3.4) is an activator of the cAMP signalling pathway. Following the isolation of 3.4, the novel sesterterpenoids ansellones B-D (4.3-4.5) as well as alotaketal E (4.6) were isolated from the sponge Phorbas sp. and were found to also be activators of the cAMP signalling pathway.Several bacterial isolates were obtained from the sponge Phorbas sp. in order to investigate the possibility that the ansellones and the alotaketals isolated from this sponge were biosynthesized by a bacterial symbiont. Since these sesterterpenoids were activators of the cAMP signalling pathway, the investigation was conducted using bioassay guided fractionation of the bacterial isolates. The new meroterpenoid phorbasolic acid (5.1) was isolated, but no sesterterpenoids were found in the bacterial isolates.In an effort to identify molecules with antibiotic properties, a biological assay was designed to screen for inhibitors of the citrate synthase type II enzyme. One aspect of this enzyme that is of therapeutic interest is that Gram-negative bacteria possess a very different isoform of the enzyme than Gram-positive bacteria and eukaryotes. Therefore, an antibiotic specific to type II citrate synthase would target Gram-negative bacteria selectively. An extract from a culture of Bacillus pumillus inhibited the enzyme in the assay. Although the molecule responsible for this effect has yet to be identified, the new aliphatic amide 12-methyl tridecanamide (6.1) was isolated.

View record

Bioassay-guided fractionation of the marine sponge Spongia irregularis led to the isolation of a new sulfated sesterterpenoid, irregularasulfate (2.16), along with two known sulfated sesterterpenoids, halisulfate 7 (2.14) and hipposulfate C (2.15). All three compounds (2.14-2.16) inhibit the related phosphatases calcineurin, PP1 and PP2A. The analogue 2.23 was synthesized and showed similar phosphatase inhibitory activity to the natural products. One new bafilomycin analogue, bafilomycin F (3.2), along with three known bafilomycin analogues, bafilomycin A1 (3.1), bafilomycin B1 (3.3) and bafilomycin D (3.4), were isolated from a marine-derived bacterium identified as Streptomyces sp. All four compounds (3.1-3.4) are extremely potent inhibitors of autophagy. Indoleamine 2,3-dioxygenase (IDO) is a relatively new and promising cancer drug target. Synthetic analogues of exiguamine A, the most potent IDO inhibitor reported to date, were prepared and evaluated for their ability to inhibit IDO in vitro and in vivo. The most potent of these analogues (4.32, 4.38, 4.39, 4.43 and 4.52) inhibit IDO in vitro with potency comparable to exiguamine A. A new exiguamine analogue, exiguamine C (5.2), was isolated from the crude extract of Neopetrosia exigua. Exiguamine B (5.1) was also isolated from this crude extract in order to confirm the structure, and the relative configuration was determined with the aid of synthetic exiguamine B. Bioassay-guided fractionation of the marine fungus Plectosphaerella cucumerina led to the isolation of three new alkaloids, plectosphaeroic acids A-C (6.1-6.3). All three compounds inhibit IDO with approximately the same potency, while the related compound T988 A was completely inactive. Cinnabarinic acid was synthesized in order to aid with the structure elucidation of plectosphaeroic acids A-C. Cinnabarinic acid and analogues were also active against IDO and represent a new pharmacophore for IDO inhibition. The depsipeptides turnagainolide A (7.3) and turnagainolide B (7.4) were isolated from Bacillus sp. Both of these compounds activate the enzyme SHIP1 in vitro. Total syntheses of turnagainolides A and B were accomplished using solid-phase peptide synthesis, and comparison of the synthetic material with the natural products confirmed their structures. Two novel compounds, the peptide 8.1 and the carotenoid 8.7, were isolated from two unidentified marine sponges. The structure of 8.1 was confirmed by a total synthesis using solid-phase peptide synthesis. Analogues of 8.1 were also prepared and showed moderate cytotoxicty against T98G cancer cells.

View record

Montbretins A-E were isolated from the corms of Crocosmia sp., an invasive perennialplant. The montbretins are inhibitors of human pancreatic α-amylase (HPA). Montbretin A (2-30) is a competitive inhibitor of HPA with a K₁ of 1.3 nM. The activity of the other familymembers varied significantly and provided structure-activity information. Saturation TransferDifference (STD) NMR spectroscopy was used to determine that the caffeic acid region of themontbretins is important for binding. HPA is involved in the breakdown of complexcarbohydrates; inhibition of this enzyme could help with regulation of blood sugar levels after ameal.In the lungs of cystic fibrosis patients, the activation of Toll-Like Receptor 5 (TLR5) inthe presence of flagellin leads to inflammation and obstruction. Girolline (3-1), a knownalkaloid, was isolated from a Phonpeian sponge following potent inhibition of the flagellininitiated TLR5 activation. No activity was observed in any synthetic analogues of girolline. The massacreones are a new family of ecdysteroids isolated from an unidentified Dominicancnidarian. The extract of the cnidarian had good TLR5 activity, but the massacreones – namelymassacreone A (3-25) and massacreone B (3-26) have only moderate activity and a smallwindow of activity before they are toxic.The algal pigment caulerpin (4-29) was isolated from Caulerpa sp. as a compoundshowing good activity in a yeast growth restoration assay designed to identify inhibitors ofhuman indoleamine-2,3-dioxygenase (IDO). Caulerpin did not show any activity in a free enzyme IDO assay. IDO is involved in immune escape, which prevents the immunological rejection of tumors.

View record

Natural products have long been a source of inspiration for many drugs in human use. The Andersen lab examines compounds from marine sources that can be used as lead structures for drug discovery. Synthetic studies, structure-activity relationships (SAR) and biological findings of two such compounds are described in this thesis. The first is pelorol, a meroterpene isolated from a tropical sponge Dactylospongia elegans. Pelorol is a small molecule activator of SHIP 1, a phosphatase that is a negative regulator of the P13K pathway in hematopoetic cells. Using a synthetic route from a previous co-worker, Lu Yang, a series of SHIP 1 activating compounds based on pelorol were synthesized. These compounds were evaluated for selectivity, potency, and efficacy in a series of biological studies, leading to the discovery of 2.27 as a preclinical lead compound. Water-soluble prodrugs of the SHIP 1-activating compounds were also synthesized and their properties reported.The second compound examined is ceratamine A, an alkaloid isolated from the sponge Pseudoceratina sp. from Papua New Guinea. Ceratamines A and B are microtubule stabilizing antimitotic agents that may be useful in cancer chemotherapy. The core imidazo[4,5,d]azepine heterocycle of the ceratamines has no precedent among known synthetic or natural compounds. The relatively simple structure of the ceratamines and the novel antimitotic phenotype they generate makes them attractive targets. Desbromo ceratamine A (3.44) was synthesized by an efficient and scaleable route, confirming the structure of ceratamine A and validating the biological activity of the core pharmacophore. Synthetic efforts towards ceratamine A were ultimately thwarted by the inability to install the bromine atoms present in the natural product. A significant finding is that the bromine atoms in ceratamine A contribute significantly to the antimitotic potency of the compound necessitating a bioisosteric approach to more potent antimitotic ceratamine-based agents.

View record