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Biologically active secondary metabolites from tropical marine invertebrates Dewi, Ariyanti Suhita 2009

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  BIOLOGICALLY ACTIVE SECONDARY METABOLITES FROM TROPICAL MARINE INVERTEBRATES  by ARIYANTI SUHITA DEWI B.Sc. Brawijaya University, Indonesia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES  (Oceanography)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2009 © Ariyanti Suhita Dewi, 2009    ii ABSTRACT   In our effort to discover promising anticancer agents, we have screened a series of compounds for their activities as indoleamine-2,3-dioxygenase (IDO) inhibitor and SH- containing inositol 5-phosphatase (SHIP1) activator. In comparison to aaptamine (2.1) and demethylaaptamine (2.2), isoaaptamine (2.4) from Aaptos cf. suberitoides appears to be the most promising IDO inhibitor with an IC50 of 0.00215 mg/mL, owing to the presence of hydroxyl group at C9 position and the methylation at N1 position. A study on the sponge extract of RJA 55275 for its SHIP activator yielded theonellapeptolide Id (3.4), the first peptide that enhanced the SHIP with 25% activity at concentration 124 μM, thus makes it the most potent SHIP activator known to date. The third project studied the crystals of a novel eunicellin-based diterpenoid (4.39) with a modest SHIP activity from an unidentified Micronesian soft coral RJA 47686. The X-ray analysis illustrated that the crystals are monoclinic, space group P21/b, with a = 9.3711(14) A; α = 90 0 ; b = 13.5349(17) A; β = 99.142(7)0; c = 10.9891(17) A; γ = 90 0 ; V = 1376.1 (3) Å3; Z value = 2; Dcalc 1.189. 10-3 g/cm3; F000 536.00; Cu (MoKα) 0.84 cm-1. Based on the NMR and x–ray data 4.39 was shown to possess (1R*, 2R*, 3R*, 6R*, 7S*, 10R*, 14R*, 18R*)-configuration with an ether linkage connecting C2 and C6.         N HN H3CO H3CO 2.1 N H3CN H3CO HO 2.4 NH O O N O HN O NH N N H O O N O O N NH O H N O O HN O ON O HN O O 3.4 H H OO OH H O OOO H H H 4.39  iii TABLE OF CONTENTS  Abstract........................................................................................................................................... ii Table of Contents........................................................................................................................... iii List of Tables ................................................................................................................................. vi List of Figures............................................................................................................................... vii List of Schemes.............................................................................................................................. ix List of Abbreviations ...................................................................................................................... x Dedication.................................................................................................................................... xiii Acknowledgements...................................................................................................................... xiv  CHAPTER I Marine natural products....................................................................................... 1 1.1 A brief history of natural products and traditional medicines .............................................. 1 1.2 Marine natural products........................................................................................................ 3 1.3 Marine sponges: potential resource of bioactive metabolites............................................... 6 1.4 Cancer chemotherapeutics: focus on IDO and SHIP.......................................................... 11 1.5 Summary............................................................................................................................. 21  CHAPTER II Identification of IDO inhibitors from the Indonesian marine sponge Aaptos cf. suberitoides ............................................................................................................................. 22 2.1 Introduction ........................................................................................................................ 22 2.1.1 Introduction to IDO ..................................................................................................... 22 2.1.2 Review of IDO inhibitor agents................................................................................... 24 2.1.3 Review of Aaptos sp. and known metabolites from Aaptos sp.................................... 26 2.2 Results and discussions ...................................................................................................... 29 2.2.1 Isolation of IDO inhibitor compounds......................................................................... 29 2.2.2 Structure elucidation.................................................................................................... 31    2.2.2.1  Aaptamine (2.1) .................................................................................................... 31    2.2.2.2  Isoaaptamine (2.4) ................................................................................................ 39 2.2.3 Comments on aaptamine (2.1) and isoaaptamine (2.4) ............................................... 46 2.2.4 Biological activity (SAR) of aaptamine and isoaaptamine.......................................... 46 2.3 Conclusions ........................................................................................................................ 49  iv 2.4 Experimental....................................................................................................................... 49 2.4.1 General experimental procedures ................................................................................ 49 2.4.2 Animal material ........................................................................................................... 50 2.4.3 Extraction and isolation ............................................................................................... 50 2.4.4 Aaptamine (2.1) physical data ..................................................................................... 51 2.4.5 Isoaaptamine (2.4) physical data ................................................................................. 51 2.4.6 IDO inhibitory assay.................................................................................................... 51  Chapter III: A SHIP activator from the Indonesian marine sponge RJA 55275 ................. 53 3.1 Introduction ........................................................................................................................ 53 3.1.1 Introduction to SHIP.................................................................................................... 53 3.1.2 Review of SHIP activator agents................................................................................. 56 3.2 Results and discussions ...................................................................................................... 57 3.2.1 Isolation of SHIP activator compound ........................................................................ 57 3.2.2 Structure elucidation of theonellapeptolide Id (3.4) .................................................... 60 3.2.3 Comments on theonellapeptolide Id (3.4) ................................................................... 78 3.2.4 Biological activity of theonellapeptolide Id (3.4)........................................................ 78 3.3 Conclusions ........................................................................................................................ 80 3.4 Experimental....................................................................................................................... 80 3.4.1 General experimental procedure.................................................................................. 80 3.4.2 Animal material ........................................................................................................... 81 3.4.3 Extraction and isolation ............................................................................................... 81 3.4.4 Theonellapeptolide Id (3.4) physical data ................................................................... 82 3.4.5 SHIP activity assay...................................................................................................... 82   Chapter IV: X-ray crystallographic study of a novel euniellin-based diterpenoid from the Micronesian soft coral RJA 47686 ............................................................................................ 84 4.1 Introduction ........................................................................................................................ 84 4.1.1 Introduction to X-ray crystallography ......................................................................... 84 4.1.2 Review of eunicellin-based diterpenoids..................................................................... 85 4.2 Results and discussions ...................................................................................................... 89  v 4.2.1 Crystal structure and absolute configuration of 4.39................................................... 89 4.2.2 Comments on diterpenoid 4.39 crystals....................................................................... 92 4.3 Conclusions ........................................................................................................................ 92 4.4 Experimental....................................................................................................................... 93 4.4.1 Crystallization method................................................................................................. 93 4.4.2 Data collection............................................................................................................. 93 4.4.3 Data reduction.............................................................................................................. 93 4.4.4 Structure solution refinement ...................................................................................... 94  Chapter V: General Conclusions .............................................................................................. 95  References.................................................................................................................................... 98                  vi LIST OF TABLES  Table 1.1 Sponge-derived compounds in production and clinical or preclinical trials................ 10 Table 1.2 Selected molecularly target agents in clinical development ........................................ 15 Table 2.1 NMR data for aaptamine recorded in DMSO-d6......................................................... 33 Table 2.2 NMR data for isoaaptamine recorded in DMSO-d6 .................................................... 40 Table 3.1 1D and 2D data for theonellapeptolide Id recorded at 600 MHz (1H) and 150 MHz (13C) in CDCl3 .............................................................................................................................. 67 Table 4.1 Crystal data and structure refinement of diterpenoid................................................... 90                    vii LIST OF FIGURES  Figure 1.1 Early medicines. Morphine, salicylic acid and penicillin ............................................ 2 Figure 1.2 Anticancer drugs from terrestrial plants. Paclitaxel and camptotechin........................ 3 Figure 1.3 The East Indies triangle................................................................................................ 8 Figure 1.4 Examples of bioactive compounds from marine sponges. Avarol; halichondrin B; discodermolide; laulimalide............................................................................................................ 9 Figure 1.5 An anticancer agent from Caribbean sponge Cryptotethya crypta, spongouridine and its synthetic analogs Ara-C........................................................................................................... 12 Figure 1.6 Marine natural product-based drugs. Aplidine and Yondelis..................................... 13 Figure 1.7 The process of immunoediting................................................................................... 17 Figure 1.8 Most potent IDO inhibitors known to date. Exiguamine A and annulin C................ 19 Figure 1.9 An example of SHIP activator. Pelorol. ..................................................................... 20 Figure 2.1 The biochemistry of IDO ........................................................................................... 22 Figure 2.2 Naturally occurred aaptamines. Aaptamine, isoaaptamine, 4-methyloxyaaptamine, demethylaaptamine, demethyl(oxy)aaptamine, bisdemethylaaptamine  and bisdemethylaaptamine-9O-sulfate ................................................................................................ 26 Figure 2.3 Bisdemethylaaptamine, a proposed biosynthetic precursor of the aaptamines .......... 27 Figure 2.4 Additional metabolites from Aaptos sp.  Aaptosine, ciliatamides A-C. .................... 29 Figure 2.5 Selected HMBC and COSY correlation for compound 2.3 ....................................... 32 Figure 2.6 600 MHz 1H NMR spectrum of aaptamine recorded in DMSO................................ 34 Figure 2.7 150 MHz 13C NMR spectrum of aaptamine recorded in DMSO.............................. 35 Figure 2.8 600 MHz COSY NMR spectrum of aaptamine recorded in DMSO.......................... 36 Figure 2.9 600 MHz HSQC NMR spectrum of aaptamine recorded in DMSO.......................... 37 Figure 2.10 600 MHz HMBC NMR spectrum of aaptamine recorded in DMSO....................... 38 Figure 2.11 Selected HMBC and COSY correlation for compound ........................................... 40 Figure 2.12 600 MHz 1H NMR spectrum of isoaaptamine recorded in DMSO......................... 41 Figure 2.13 150 MHz 13C NMR spectrum of isoaaptamine recorded in DMSO ....................... 42 Figure 2.14 600 MHz COSY NMR spectrum of isoaaptamine recorded in DMSO ................... 43 Figure 2.15 600 MHz HSQC NMR spectrum of isoaaptamine recorded in DMSO ................... 44 Figure 2.16 600 MHz HMBC NMR spectrum of isoaaptamine recorded in DMSO .................. 45 Figure 2.17 IDO inhibition assay................................................................................................. 46  viii Figure 2.18 The IDO inhibition activity of aaptamines............................................................... 47 Figure 3.1 Enzymatic synthesis and degradation of PI-3,4,5-P3................................................. 53 Figure 3.2 The active analogues of pelorol as SHIP activators ................................................... 56 Figure 3.3 Structure of MN100.................................................................................................... 57 Figure 3.4 Structure of theonellapeptolide Id .............................................................................. 60 Figure 3.5 600 MHz of 1H NMR spectrum for theonellapeptolide Id recorded in CDCl3 .......... 61 Figure 3.6 150 MHz of 13C NMR spectrum for theonellapeptolide Id recorded in CDCl3 ......... 62 Figure 3.7 600 MHz of COSY NMR spectrum for theonellapeptolide Id recorded in CDCl3.... 63 Figure 3.8 600 MHz of HSQC NMR spectrum for theonellapeptolide Id recorded in CDCl3.... 64 Figure 3.9 600 MHz of HMBC NMR spectrum for theonellapeptolide Id recorded in CDCl3... 65 Figure 3.10 600 MHz of TROESY NMR spectrum for theonellapeptolide Id recorded in CDCl3............................................................................................................................................ 66 Figure 3.11 Selected COSY and HMBC key correlations of fragment C1-C16 of theonellapeptolide Id (1H and 13C assignments shown) ............................................................... 71 Figure 3.12 Selected COSY and HMBC key correlations of fragment C17-C27 of theonellapeptolide Id (1H and 13C assignments shown) ............................................................... 72 Figure 3.13 Selected COSY and HMBC key correlations of fragment C28-C42 of theonellapeptolide Id (1H and 13C assignments shown) ............................................................... 74 Figure 3.14 Selected COSY and HMBC key correlations of fragment C43-C51 of theonellapeptolide Id (1H and 13C assignments shown) ............................................................... 75 Figure 3.15 Selected COSY and HMBC key correlations of fragment C52-C64 of theonellapeptolide Id (1H and 13C assignments shown) ............................................................... 76 Figure 3.16 SHIP activity assay................................................................................................... 79 Figure 3.17 The SHIP activity of theonellapeptolide Id compared to MN100............................ 79 Figure 4.1 Selected eunicellin-based diterpenoids from soft corals and gorgonians................... 86 Figure 4.2 The proposed biosynthetic relationship between cembrane and asbestinin skeletons ....................................................................................................................................... 87 Figure 4.3 Bioactive eunicellin-based diterpenoids .................................................................... 88 Figure 4.4 ORTEP diagram of diterpenoid.................................................................................. 91 Figure 4.5 Diterpenoid from soft coral RJA 47686 with (1R, 2R, 3R, 6R, 7S, 10R, 14R, 18R)- configuration................................................................................................................................. 91   ix LIST OF SCHEMES  Scheme 2.1 Isolation procedures for IDO inhibitor from Aaptos cf. suberitoides....................... 30 Scheme 3.1 Isolation procedures for SHIP activator from RJA 55275 ....................................... 59                           x LIST OF ABBREVIATIONS  0 -degree 1D -one-dimensional 1MT -1-methyl tryptophan 2D -two-dimensional aa -amino acid Ac -acetate Ala -alanine APC -antigen presenting cell β -beta b -broad BMmΦs -bone-marrow-derived macrophages BMMCs -bone-marrow-mast cells Bs -broad singlet BuOH -butanol 13C -carbon-13 0C -degree celsius C-18 -octadesylsilane calcd/calc -calculated CDCl3 -deuterated chloroform CH -methine CH2 -methylene CH3 -methyl COSY -two-dimensional correlation spectroscopy d -doublet D -dextrorotatory D -density DCM -dichloromethane dd -doublet of doublet δ -chemical shift in parts per million DMSO-d6 -deuterated dimethylsulfoxide ED50 -effective dose 50% Et -ethyl ET-743 -ecteinascidin 743 EtOAc -ethyl acetate F -structure factor g -gram 1H -proton H2O -water HCl -hydrochloric acid  xi HeLa -Henrietta lacks HIV -human immunodeficiency virus HT-116 -human colon cancer cell line HMBC -two-dimensional heteronuclear multiple bonds correlation HSQC -two-dimensional heteronuclear single quantum coherrence HPLC -high performance liquid chromatography HRAPCIMS -high resolution atmospheric pressure chemical ionization mass spectrometry HRESIMS -high resolution electrospray ionization mass spectrometry Hz -hertz I-1,3,4,5-P4/IP4 -inositol-1,3,4,5-tetraphosphate IC50 -inhibitory concentration 50 IDO -indoleamine-2,3-dioxygenase Ile -isoleucine iNOs -inducible nitrous oxide synthase J -coupling constant L -levorotatory L1210 -mouse lymphocytic leukimia cell line LC -liquid chromatography LC50 -lethal concentration 50% Leu -leucine LPS -lipopolysacharide LRAPCIMS -low resolution atmospheric pressure chemical ionization mass spectrometry LRESIMS -low resolution electrospray ionization mass spectrometry m -multiplet M -molar concentration M -macrophages Me -methyl MeCN -acetonitrile MeOAc -methoxyacetyl MeOH -methanol mg -milligram MHz -megahertz Ml -millilitre MS -mass spectrometry MW -molecular weight m/z -mass to charge ratio NaCl -natrium chloride NMR -nuclear magnetic resonance NO -nitrous oxide NOESY -nuclear overhauser effect spectrometry np -normal phase  xii O2 -oxygen ODS -octadesylsilane P -primitive P388 -lymphocytic leukimia cell line from mice PI3K -phosphatidylinositol-3’-kinase PI-3,4-P2 -phosphatidylinositol-3,4-biphosphate PI-3,4,5-P3 -phosphatidylinositol-3,4,5-triphosphate PI-4,5-P3 -phosphatidylinositol-4,5-triphosphate PTEN -phophatase and tensin homologue PLA2 -phospholipase A2 ppm -parts per million ORTEP -oak ridge thermal ellipsoid program q -quartet R -residual/reliability factor R -rectus ROESY -rotating frame Overhauser effect enhancement spectrometry S -singlet S -sinister SAR -structure activity relationship SCUBA -self-contained underwater breathing apparatus SH2 -src homology 2 SHIP -src homology 2 domain containing inositol 5' phosphatase sp. -species t -triplet TDO -tryptophan-2,3-dioxygenase T min/max -absorption correction minimum/maximum TFA -trifluoro acetic acid Thr -threonine TLC  -thin layer chromatography TROESY -transverse rotating frame Overhauser effect spectrometry μg -microgram μl -microlitre UV -ultraviolet V -volume Val -valine       xiii DEDICATION          To my beloved parents, Subarianto and Putu Sutiani            “...Allah will raise up to (suitable) ranks (and degrees), those of you who believe and have been granted Knowledge.  And Allah is well-acquainted with all you do.”  (Al Mujadaalah: 11)   xiv ACKNOWLEDGMENTS    I am highly obliged to my government for the opportunity to obtain this M.Sc. degree. I would like to acknowledge first and foremost my research supervisor, Prof. Raymond Andersen for his invaluable insights into marine natural product chemistry from which I have gain more knowledge and expertise in the field. I am grateful to the following people: Prof. Dr. Hari Irianto, Dr. Ekowati Chasanah and Ir. Yusro Fawzya, M.Si. for initially coordinating my studies; Dr. David Williams for the diterpenoid crystal and the demethylaaptamine; Mike Le Blanc for collecting the sponges; Dr. Brian Patrick for his assistance with X-ray crystallography; also to Wendy Tay, Sarah Andersen and Ashraf Amlani for the countless biological assays. I would also like to acknowledge Dr. Wendy Strangman and Kate Woods for helping me with the thesis editing. My sincere thanks to all my fellow grad students of Andersen lab (past and present) for the chemistry discussions and the friendships. And of course, my utmost appreciation to all my family and friends for their continuous supports and prayers.                    1 Chapter I: Marine Natural Products  1.1 A Brief History of Natural Products and Traditional Medicines  Throughout the ages, humans have relied on nature to fulfill their basic needs for the production of food, shelter, cloth and medicine. The use of natural substances, particularly plants, to control disease dates back thousands of years and has led to the discovery of more than half of all modern pharmaceuticals. Recorded documentation of natural medicine was initially started as early as 2600 B.C. by the ancient Egyptians.1 Amongst the approximately 1000 plant derived substances that they used were oils from species of Cedrus (cedar) and Cupressus sempevirens (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh) and Papaver somniferum (poppy juice); all of which are still in use today for the treatments of ailments ranging from cough and cold to parasitic infections and inflammation.1 A thousand year later, around 1500 B.C., Ebers Papyrus was constructed by the ancient Egyptians and it documented over 700 drugs which were derived mostly from plants. The study of natural medicine and pharmacy was subsequently developed by the ancient Chinese, Indian, and Greek civilizations.1 Natural products are often defined as secondary metabolites of plants and microbes. Many of them are highly potent and selective as a result of evolutionary selection. They are used as signaling molecules, defense mechanism, as well as to maintain survival and the reproductive fitness of the producing organisms. Their derived compounds may also serve as biochemical tools that demonstrate the role of specific pathways in diseases.2 Since they have been evolved within the living systems, secondary metabolites are often perceived as showing more “drug- likeness and biological friendliness than totally synthetic molecules”, making them prospective candidates for further drug development.3  2 In the modern world, natural products continue to play an important role in disease treatment. The introduction of natural products into the modern pharmaceutical industry was marked by the isolation of two natural products, morphine (1.1) and salicylic acid (1.2). Morphine was discovered by E. Merck in 1826 and became the first commercially available pure natural product, followed by the launching of a semi-synthetic drug based on salicylic acid called aspirin by Bayer in 1899.3 Thirty years later, the serendipitous discovery of penicillin (1.3) by Fleming revolutionized drug discovery by revealing the potential of soil microorganisms as a new source of pharmaceutical drugs.3 When compared with natural products from plants and animals, microbial natural products have the benefit of increased renewability since many cultures can be grown in the laboratory. There are now over 130 commercial drugs of microbial origin and 67 microbial or microbial-derived compounds are currently in various stages of clinical development in the areas of antibacterial, antifungal, antiparasitic, antiviral, anti- inflammatory, anticancer, neurological, cardiovascular, metabolic and immunological disease treatment.4         Despite competition from other drug discovery methods, natural products are continued to be one of the main sources of state-of-the-art drugs in the pharmaceutical industry. This is demonstrated by the fact that natural product-derived drugs are still well represented in the top Figure 1.1 Early medicines. Morphine (1.1), salicylic acid (1.2) and penicillin (1.3) HO O HO N S H N O O OH O R 1.1 1.2 1.3 N H HO HO O  3 35 worldwide bestselling drugs of 2000, 2001, and 2002.5 Additionally, the percentage of natural product-derived drugs was 40% of the total drugs launched in the market during 2000-2001 and 26% in 2002.5 The sales of two categories of plant-derived cancer chemotherapeutic agents, namely paclitaxel (1.4) and its semi-synthetic analogue, docetaxel, as well as the derivatives of camptothecin (1.5), irinotecan and topotecan were responsible for approximately one third of the total anticancer drug sales worldwide, or just under $3 billion in 2002.3 Furthermore, at least 70 natural product-related compounds were in clinical trials in 2004 and the exploration of the bioactivity of natural products continues to provide novel chemical scaffolds for further drug discovery.6          1.2 Marine Natural Products  In addition to terrestrial plants and soil microorganisms, another source of natural product chemical diversity is the marine environment. Unlike that of terrestrial resources, the recorded history of traditional medicines from marine organisms is quite rare. Nevertheless, it was acknowledged that the ancient Phoenicians used the chemical secretion from marine mollusks to produce purple dyes for woolen cloth. Although the world’s ocean covers 70% of Figure 1.2 Anticancer drugs from terrestrial plants. Paclitaxel (1.4) and camptothecin (1.5) NH O OH O H O O OH O O O O O OH O H 1.4 N N O O O 1.5  4 the earth’s surface, the marine ecosystem remained a largely unknown and untapped resource in terms of drug discovery until the 1970’s. Thanks to the development of reliable SCUBA techniques and submarine expeditions roughly 50 years ago, serious attempts to exploit the vast potential of marine organisms as sources of bioactive metabolites for drug development are now possible.7 The isolation of novel marine metabolites bearing unusual chemical architecture and appealing pharmacological activities has prompted an emerging interest of both chemists and biologists in marine natural product research. To date, the majority of marine natural products have been obtained from mostly sessile soft-bodied invertebrates, such as sponges and tunicates. Novel metabolites isolated from these organisms are most often assumed to have a defensive function. Other theories also suggest additional roles such as prevention of fouling, inhibition of overgrowth, and protection from ultraviolet radiation.2,8 Since the 1970’s, more than 15,000 structurally diverse natural products with different bioactivities have been discovered from marine microbes, algae, and invertebrates.2 Although marine compounds have demonstrated promising activities to be developed as drug candidates, a serious challenge often arises when trying to obtain enough material for advanced biological investigations. Marine sponges, tunicates, and other chemically interesting invertebrates naturally often exist in low numbers, resulting in a very limited amount of biomass available from wild stocks.9 Concentrations of many highly bioactive compounds in marine invertebrates are often minute, sometimes less than 0.0001% of the wet weight. It is obvious that this task could never be completed without risking the environment and extinction of the respective species.10 As a result, a number of multidisciplinary researches are currently targeting the development of new technologies for mariculture of plants and marine invertebrates for the environmentally compatible production of pharmaceutical relevant species.11 From a  5 biotechnological point of view, in situ culture is the most reliable and the least expensive method to produce sponge biomass. However, the success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Therefore, other cultivation methods such as in vitro sponge culture and cell culture are being developed to optimize these techniques.12 Other avenues to address the supply issue include organic synthesis as a key option for producing significant amounts of important drug candidates for preclinical and clinical studies. However, because natural products often have exquisitely complicated architectures, the synthetic routes are usually lengthy with long development times, low overall yields and impracticality of commercial scale up. These issues can be somewhat alleviated through semi- synthetic approaches that have been developed to provide simplified analogs more rapidly.13 Over the last decade, research has demonstrated the likelihood that many metabolites, particularly polyketides and non-ribosomal peptides, are not actually produced by the macroorganisms themselves but instead by their associated microbial symbionts. Some sponges are filter feeders and, therefore, it is not surprising to learn that they contain spectacular numbers of symbiotic bacteria that may comprise up to 40% of the animal biomass.14 It is also somewhat interesting that many species of bacteria seem to be fairly specific to sponges. A comparison of specimens of two symbiont-rich sponge species collected from the Mediterranian Sea, the coast of Japan, the Red Sea and the island of Palau suggested that the bacterial composition is largely independent of sponge taxonomy or locality of collection. The symbiont hypothesis has raised considerable interest as it implies that an animal-independent system based on bacterial fermentation processes could be created.14 However, many bacterial inhabitants in sponges appear to be highly selective with regard to culture media and conditions which probably reflect their evolutionary adaptation to the environment provided by the host. Therefore, extensive efforts in designing growth media and culturing conditions for sponge-associated bacteria will  6 have to be invested in order to make symbiotic bacteria culturable under laboratory conditions. Should these efforts be successful, they will present a significant contribution towards solving the natural products supply problem. 9  1.3 Marine Sponges: Potential Resource of Bioactive Metabolites  Although recent advances in microbial cultures and biosynthesis are opening new avenues of marine natural product research, sponges continue to dominate as a source of new compounds.11 Among marine invertebrates, sponges are essential and highly diverse components of marine benthic communities, ranging from the euryhaline estuarine and the intertidal to the deep sea. Roughly 7,000 species of sponges worldwide have been described, although potentially many more remain uncharacterized as evidenced by the huge largely unidentified collections in the world’s museums. These collections, together with molecular studies that have detected a number of cryptic sibling species, suggest that this biodiversity might be twice that presently recognized.15  Sponges are primitive metazoans that were probably the starting point of metazoan explosion during the Precambrian.11 These rather primitive animals are very efficient filter feeders. It has been estimated that some are able to filter their own body volume every 5 seconds.  This water current supplies food and oxygen as well as removes metabolic waste products. The majority of the sponge biomass consists of a gelatinous matrix containing free- floating, non-differentiated cells, called the mesohyle, which also contains spicules and spongin, the skeletal elements of the body. Spicules are needle-like structures made of either silicon or calcium carbonate and spongin is collagenous fibres.16 Sponges are grouped into three major classes, Hexactinellida, Calcarea and Demospongia based on the materials they use to reinforce the mesohyle and in some case to produce skeletons.  7 The latter class contains a vast majority of extant sponges living today.  Sponges inhabit every type of marine environment, from polar seas to temperate and tropical waters, and also thrive and prosper at all depths. They show an amazing variety of sizes, shapes and colors. Although they are eukaryotes, sponges share many functional features with unicellular protozoa, such as nutrition, cellular organization, gas exchange, reproduction and response to external stimuli. They also share genes and proteins with higher eukaryotes that are highly homologues to vertebrate analogues.11 Intensive evolutionary pressures from competitors that threaten overgrowth, poisoning, infection and predation have led sponges to develop an arsenal of potent chemical defensive agents. Studies have shown that many sponges are rich in steroids and terpenoids, which are thought to function in antipredation, spacial competition and control of epibiont overgrowth.2,8,11 Ecological research has also revealed that predation in tropical areas are in fact higher than that in temperate habitats. Consequently, tropical species have evolved more effective defenses to deter predators.17 Therefore, marine natural products research is currently more concentrated on marine organisms from tropical sources, for instance, Indonesian waters. As part of the East Indies triangle (Figure 1.3), the Indonesian region is believed to be the center of origin for most of Indo-Pacific marine fauna and thus it is unique in terms of diversity and ecology.18 The Indonesian archipelago comprises more than 17,000 islands and consequently has an extended shallow water region, which makes it the largest and the most important coral reefs in the world’s biodiversity. Approximately 850 species of sponges, the main component of marine diversity, have been identified from Indonesian waters, although the sponge taxonomy remains incomplete in this region.19 It is proposed that the biodiversity in this area is due to natural selection being involved in reproductive isolation.18    8               Investigations in sponge chemical ecology have discovered that the secondary metabolites not only play roles in the metabolism of the producer but also in their strategies in the given environment.16 In fact, more than 10% of extracts from the investigated marine sponge species exhibited cytotoxic activity.20 These molecules include derivatives of amino acids and nucleosides as well as macrolides, porphyrins, aliphatic cyclic peroxides and sterols. Numerous of these bioactive compounds have been evaluated as potential pharmaceutical agents. Some examples of bioactive metabolites isolated from marine sponges are presented below (Figure 1.4). Many of these bioactive metabolites work by inhibiting certain enzymes, which often mediate or produce mediators of intracellular or intercellular messengers involved in the pathogenesis of a disease. As this process usually occurs as a part of a cascade of reactions inside Figure 1.3 The East Indies triangle18  9 the cell or tissue, many enzymes in the cascade are targets for potential therapy. The different enzymes in the cascade can be structurally unrelated proteins. Therefore, a wide range of metabolites can be used for the treatment of a disease. This approach has been described most completely in cancer, which has been shown to have many different enzymatic factors in its pathogenesis.21 Further discussion on the current cancer therapies is described in subchapter 1.4.                 Figure 1.4 Examples of bioactive compounds from marine sponges. Avarol (1.6); Halichondrin B (1.7); Discodermolide (1.8); Laulimalide (1.9) O O O O O O O O O O O O O O CH3 O O HO HO HO H H CH3 H HH3C H2C H H HH H H 1.7 OH H OH 1.6 O OH OH O NH2 O O OH HO 1.8 O OH O OH O O O 1.9 HH CH3  10    Compound Sponge Disease area Status Production Company Ara-A (derivative of spongothymidine) Cryptotethia crypta Antiviral In use Microbial fermentation of analog GlaxoSmithKline Ara-C (derivative of spongouridine) Cryptotethia crypta Antileukimia In use Chemical synthesis of analog Pfizer KRN-7000 (derivative of agelasphin) Agelas mauritianus Anticancer Phase I  Kirin Brewery Avarol Dysidea avara Anti-HIV Withdrawn Wild harvest Bengamide Ijaspis sp. Anticancer Withdrawn from Phase I Chemical synthesis of analog Novartis Discodermolide Discodermia dissolute Anticancer Phase I Chemical synthesis  Novartis Girolline Pseudaxynissa cantharella Anticancer Withdrawn from Phase I  Rhone-Poulene Rorer Halichondrin B Lissodendoryx sp. Anticancer Phase I Chemical synthesis of analog E7389 Eisai Isohomohalichondrin B Lissodendoryx sp. Anticancer Preclinical Aquaculture Pharmamar Laulimalide Cacospongia mycofijiensis Anticancer Preclinical Chemical synthesis Peloruside A Mycale hentscheli Anticancer Preclinical Salicylhalimide A and B Haliclona sp. Anticancer Preclinical Neoamphimedine estospongia sp. Anticancer Preclinical Manoalides Lufffariela variabilis Anti-inflammatory (psoriasis) Withdrawn from Phase II Wild harvest Allergan IPL-576092 (derivative of contignasterol) Petrosia contignata Anti-inflammatory (asthma) Phase II  Inflazyme&Adventis Hemiasterlin A and B Siphonochalina sp. Anticancer Phase II   Wyeth-Ayerst Table 1.1 Sponge-derived compounds in production and clinical or preclinical trials in 2004 21  11  1.4 Cancer Chemotherapy and Immunotherapy: focus on IDO (Indoleamine-2,3- dioxygenase) and SHIP (SH2 containing inositol-5-phosphatase)  Cancer is a major public health problem in the United States and other developed countries. Currently, one in four deaths in the United States is due to cancer. A total of 1,444,920 new cancer cases and 559,650 deaths from cancer are projected to occur in the US in 2007.22 A continuing increase in the incidence rate is shown by 0.3% increase per year in women and a 13.6% total decrease in age standardized cancer death rates among men and women combined between 1991 and 2004.22 Parkin et al. added that the most common cause of cancer death in the world is lung cancer (1.18 million), followed by stomach cancer (700,000) and liver cancer (598,000).23 Chemotherapy is the term broadly employed for the use of pharmaceutical agents that possess cytotoxic or cytocidal activity.  Towards cancer cells, this form of cancer treatment has evolved over more than half century.24 Traditional medicinal chemistry approaches to the development of new chemotherapeutic agents are based largely on the identification of structure activity relationships from which basic pharmacophores can be optimized for specificity and potency against disease specific therapeutic targets.25 Despite surgical treatment and irradiation, chemotherapy remains one of the most important means to treat cancer.26 Most chemotherapeutic agents arose from empirical screening procedures where natural products or natural products-derived compounds were tested for cytotoxic potency against murine and/or human cancer cells grown in cell culture or in rodent tumor models.25 Therefore, many metabolites from plants, microbes and marine invertebrates with potential anticancer activity have been isolated and identified because of the prevalence of these cytotoxicity-based assay screening methods among natural products research programs.27 Marine natural product research has long been focusing on the discovery and  12  development of chemotherapeutic drugs and pharmaceutical agents. The first success story began in 1950’s when Bergmann and co-workers isolated spongouridine (1.10), a nucleoside containing a rare arabinose sugar rather than ribose, from the Caribbean sponge Cryptotethya crypta (Tethylidae). Spongouridine was found to exhibit potential antitumor properties.28 This discovery subsequently led researchers to synthesize an analog known as Ara-C (Cytarabine, Alexan®, Udicil®, 1.11) with improved activities.29          Recently, more marine compounds have progressed into clinical trials and some of them even have launched to the market. Synthetically derived aplidine, a depsipeptide collected from Mediterranian tunicates Aplidium albicans is currently in Phase II clinical trials for the treatment of multiple myeloma under the PharmaMar trade name Aplidin® (1.12). In partnership with Johnson & Johnson, PharmaMar also develops ET-743 or Yondelis® (1.13), an antitumor agent from the tunicate Escteinascidia turbinata which is now produced semi-synthetically.30    Figure 1.5 An anticancer agent from the Caribbean sponge Cryptotethya crypta, spongouridine (1.10); and its synthetic analogs Ara-C (1.11) N NH2 N O OH OH HO O 1.11 HN O N O OH OH HO 1.10 O H  13           Unfortunately, in most cases, chemotherapy remains a treatment modality with severe side effects due to the toxicity of the drugs. Bone marrow depletion and toxic effects in rapidly proliferating cells such as intestinal cells can cause life-threatening infections and side effects that seriously limit the quality of life of the patient.26 Although modern cancer drug discovery aims to enhance cancer specific toxicity with minimum damage against normal cells, the majority of existing compounds do not possess these levels of desirable selectivity. In addition, cancer cells are very adaptive and the probability of developing drug resistance is significantly higher if a single clinical agent is used over a period of time.24 It is undeniable that cytotoxicity-based methods have had a certain degree of success in cancer therapy. However, these approaches only indirectly reflect the considerable potential of designing new techniques that specifically target the unique physiological, biochemical and molecular differences between normal and malignant cells.27 This is because cancer is not merely an entity but a dynamic process involving changes in the patient, aberrant expression of normal growth related proteins, and insensitivity to growth control signals. A critical ratio between cell growth and cell death ensures normal homeostasis and an increase in this ratio due Figure 1.6 Marine natural product-based anticancer drugs. Aplidin® (1.12) and Yondelis® (1.13) N N O N H H N O B OH OH 1.12 N O O OMe HO Me OH H OAc Me O O S H NH MeO HO Me 1.13  14  to either enhanced proliferation or defective death signaling could result in abnormal accumulation cells or tumor formation. There are several functional characteristics that are developed by most cancer cells, i.e.: self sufficiency in growth signals, insensitivity to anti- growth signals, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis and evading apoptosis. Designing therapies to directly target pathways that lead to an alteration in this homeostatic ratio would be the most logical and specific means to inhibit cancer formation and growth.24 In order to reach this particular goal of altered homoeostasis, a transformation has occurred in the selection of antitumor molecular targets and a shift in research focus toward disease-specific mechanism-based screening methods. Many biochemical pathways have been investigated in order to find significant differences between normal cells and tumor cells in attempts to identify suitable targets. The goal is to develop an effective chemotherapy treatment by targeting enzymes and/or pathways which are over-expressed in tumor cells and have only limited importance in non tumorous tissues. Thus, inhibiting these enzymes or pathways might cause malignant cells to undergo apoptosis or necrosis with relatively limited side effects.26, 27 Today, antitumor marine natural products discovery efforts are more heavily focused on recently discovered therapeutic targets that have the capacity to arrest tumor cell growth, stimulate tumor cell differentiation, cut off tumor blood supply, prevent metastasis spread and inhibit the resistance of tumor cells to treatment.27         15   Compound Source organism Chemical class Molecular target Current status Escteinascidin 743 (Yondelis) Escteinascidia turbinate (tunicate; possible bacterial source) Tetrahydroisoquinolone alkaloid Tubulin Phase II Dolastatin 10 Dolabella auricullaria/ Sympioca sp. (mollusk/cyanobacterium) Linear peptide Tubulin Phase II Bryostatin 1 Bugula neritina (bryozoan) Macrocyclic lactone PKC Phase II Synthadotin (Dolastatin 15 derivative) Dolabella auricullaria/ Sympioca sp. (synthetic analogue) Linear peptide Tubulin Phase II Kahalalide F Elysiarufescens/ Bryopsis sp. (mollusk/green algae) Cyclic depsipeptide Lysosomes/ erbB pathway Phase II Squalamine Squalus acanthias (shark) Aminosteroid Phospholipid bilayer Phase II Dehydrodidemnin B (Aplidine) Trididennum solidum (tunicate, synthetic; possible bacterial/cyanobacterial source) Cyclic depsipeptide Ornithin decarboxilase Phase II Didemnin B Trididennum solidum (tunicate) Cyclic depsipeptide FK-506 bp Phase II (discontinued) Cemadotin (Dolastatin 15 derivative) Dolabella auricullaria/ Sympioca sp. (synthetic analogue) Linear peptide Tubulin Phase II (discontinued) Soblidotin (Dolastatin 10 derivative) Dolabella auricullaria/ Sympioca sp. (synthetic analogue) Linear peptide Tubulin Phase I E7389 (halichondrin B derivative) Halichondria okadai (sponge, synthetic) Macrocyclic polyether Tubulin Phase I NVP-LAQ824 (Psammaplin derivative) Psammaplysilla sp. (sponge, synthetic) Indolic cynnamyl hydroxamate HDAC/DNMT Phase I Discodermolide Discodermia dissolute (sponge) Lactone Tubulin Phase I HTI-286 (Hemiasterlin derivative) Cymbastella sp. (synthetic analogue of sponge meatabolite) Linear peptide Tubulin Phase I LAF-389 (Bengamide B derivative) Jaspis digonaxea (sponge, synthetic) ε-Lactam peptide derivative Methionine aminopeptidase Phase I KRN-7000 (Agelasphin derivative) Agelas mauritianus (sponge, synthetic) α-Galacosyl ceramide Vα24+NKT cell activation Phase I Curacin A Lyngbya majuscula (cyanobacterium) Thiazole lipid Tubulin Preclinical DMMC Lyngbya majuscula (cyanobacterium) Cyclic depsipeptide Tubulin Preclinical Salinosporamide A Sakinospora sp. (bacterium) Bicyclic γ-lactam-β- lactone 20S proteasome Preclinical Laulimalide Cacospongia mycofijiensis (sponge) Macrolide Tubulin Preclinical  Table 1.2 Selected molecularly target agents in clinical development in 2005 31  16  Compound Source organism Chemical class Molecular target Current status Vitilevuamide Didemnin cucliferum/ Polysyncration lithostrotum (tunicate) Cyclic peptide Tubulin Preclinical Diazonamide Diazona anulata (tunicate) Cyclic peptide Tubulin Preclinical Eleutherobin Eleutherobia sp./ Erythropodium caribaeorum (soft corals) Diterpene glycoside Tubulin Preclinical Sarcodyctin Sarcodyction roseum (sponge) Diterpene Tubulin Preclinical Peloruside A Mycale hentseli (sponge) Macrocyclic lactone Tubulin Preclinical Salicylihalimides A and B Haliclona sp. (sponge) Polyketide Vo-ATPase Preclinical Thiocoraiine Micromonospora marina (bacterium) Depsipeptide DNA-polymerase Preclinical Ascididemin Didemnum sp. (sponge) Aromatic alkaloid Caspase- 2/mitochondria Preclinical Variolins Kirkpatrickia varidosa (sponge) Heterocyclic alkaloid Cdk Preclinical Lamellarin D Lamellaria sp. (mollusk and various soft corals) Pyrrole alkaloid Topoisomerase I/ mitochondria Preclinical Dictyodendrins Dictyodendrilla cerongiformis (sponge) Pyrrolocarbazole derivative Telomerase Preclinical ES-285 (Spisulosine) Mactromeris polynyma (mollusk) Alkylamino alcohol Rho (GTP-bp) Preclinical Dolastatin 15 Dolabella auricularia (mollusk) Linear peptide Tubulin Preclinical (discontinued) Halichondrin B Halichondria okadai (sponge) Macrocyclic polyether Tubulin Preclinical (discontinued)  Although the established cancer therapies have reached a certain degree of success, cancer still causes 25% of mortalities in the US, indicating a serious need for additional research. As an alternative treatment method, cancer immunotherapy has emerged over the last decade as a method for preventing the metastatic spread of cancer and improving the quality of life of affected individuals. Approaches in immunotherapy are based on the complementation or stimulation of the immune system via a plethora of systems, such as lymphokines, vaccines, in vitro-stimulated-effector cells of the immune system or antibodies.32 An integrated model of oncogenesis and immunoediting provides a deeper understanding of the “fight to a draw” between the tumor and the immune system resulting in tumor dormancy in which tumor can persist in a dynamic and viable yet occult pattern in many years (Figure 1.7). The generation of occult or benign tumors represents the achievement of a state of immune  17  equilibrium by the tumor in which it can stave off but not defeat immune surveillance. In such a state, further iteration of mechanisms to strengthen localized and peripheral immunosuppression could be crucial, since the achievement of immune escape may license invasion and metastasis. Thus, cancer as a clinical disease may be better understood as a progressive disruption of the immune system rather than a disorder of the cell growth, survival and movement.33               Research in cancer immunotherapy initially began in the 1890’s by William Coley, a leading New York surgeon, who noted that feverish infections in cancer patients were occasionally associated with cancer remission. Coley developed a substance known as Coley’s toxin, a Streptococcus pyogenes culture provided by Robert Koch to treat cancer patients, and observed tumors regression in some cases. His findings were published in 1893, with his paper Figure 1.7 The process of immunoediting 33  18  being the first that describes a serious attempt in cancer immunotherapy. During the next 110 years, there have been at least five dramatic fluctuations in attitude toward cancer immunotherapy. As science advances, however, it now appears that both the innate and adaptive immune responses can recognize and eliminate tumors.34 Other studies confirmed that tumor immunity can be demonstrated in experimental animal models. Further research showed that the immune system often appears to recognize tumors, as reflected by the accumulation of immune cells at tumor sites, which correlates with improved prognosis. Today, antitumor immune responses can now be directly detected from many cancer patients. 35 A relatively recent therapeutic target was identified when a relationship between cancer and elevated tryptophan catabolism was initially recognized in the urine of patients with bladder cancer in the 1950’s. Additional studies demonstrated similar findings of elevated urinary tryptophan metabolites in breast cancer, prostate cancer, Hodgkin’s lymphoma and leukemia.36 These findings led to the discovery of indoleamine-2,3-dioxygenase (IDO), a cytosolic monomeric hemoprotein that is responsible for catalyzing the first step of tryptophan catabolism by the kynurenine pathway. IDO also plays an important role in the regulation of T-cell mediated immune responses.37, 38 A recent study showed how small molecule inhibitors of IDO can be used to leverage the efficacy of traditional chemotherapy drugs that are used to treat cancer in the clinic. By promoting antitumor immune responses in combination with cytotoxic chemotherapy drugs, IDO inhibitors may offer a drug based strategy to more effectively attack systemic cancer.39 Our research group in collaboration with Prof. Grant Mauk’s at the Department of Biochemistry and Molecular Biology UBC has begun screening marine natural product extracts for their IDO inhibition activity. Recent success includes the isolation of two IDO inhibitory agents, exiguamine A (Ki 40 nM, 1.14) from the marine sponge Neopetrosia exigua 40, 41 and  19  annulin B (Ki 120 nM, 1.15) from the marine hydroid Garveia annulata, 42 which are claimed to be the most potent IDO inhibitors known to date (Figure 1.8). Further studies of IDO inhibitors isolated from the Indonesian marine sponge Aaptos cf. suberitoides are described in Chapter 2.          Another approach for improving cancer therapy involves the restraining of the phosphatynidylinositol-3-kinase (PI3K), a critical component of growth factor signaling. Phophoinositides, particularly phosphatynidylinositol-3,4,5-triphosphate (PIP3) generated by PI3K are important secondary messengers involved in the regulation of diverse cell functions such as cellular survival, proliferation, adhesion/migration, glucose transport metabolism and energy homeostasis. Thus, the inhibition of PI3K is currently a molecular target in anticancer drugs discovery.43 Approximately 50% of human cancers contain biallelic inactivating mutations of the ubiquitously expressed tumor suppressor PTEN (phosphatase and tensin homolog) illustrating the importance of this phosphatase in preventing uncontrolled cell growth. 44 Like PTEN, SHIP (SH2 containing inositol-5-phosphatase) also hydrolyzes PIP3 and prevents mast cell deregulation.44 Figure 1.8 Most potent IDO inhibitors known to date. Exiguamine A (1.14) and annulin B (1.15) OH O O O O O O 1.15 O N N H O O H2N N N O O 1.14  20  SHIP was originally cloned by three independent groups in 1997. 44 An additional more widely expressed SHIP-like protein, known as SHIP2 was cloned soon after. Due to their PIP3 inactivation ability, the SHIP family of proteins has the potential to regulate many, if not all, PI3K induced events. Since SHIP is restricted to hemopoietic cells and negatively regulates mast cell degranulation, enhancing its activity could also prove beneficial for patients with atopic disorders, such as asthma.44         In 2005, with the partnership of collaborators from Jack Bell Research Center, our laboratory identified pelorol (1.16), a meroterpenoid from the tropical marine sponge Dactylospongia elegans 45 as a SHIP activator (Figure 1.9). Investigation of the structure relationship of pelorol and its analogues with their SHIP activity showed that the presence of the phenol group is required for activity and the replacement of methyl ester at C-20 with a methyl enhanced the activity of pelorol.46 An extensive discussion of SHIP activating peptide and diterpenoid is described in Chapter 3 and 4, respectively.    Figure 1.9 An example of SHIP activator, Pelorol (1.16) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 OCH3 19 O 20 21 22 H 23 H OH OH 1.16  21  1.5 Summary  In an attempt to discover bioactive compounds from marine invertebrates that have potential as anticancer agents, we have screened a library of marine natural product extracts. The bioactive extracts were subsequently purified and the structures of the active compounds were elucidated by NMR (Nuclear Magnetic Resonance), MS (Mass Spectrometer) and x-ray crystallography. The studies described herein are focused on three specific areas. The following chapter describes the isolation and identification of promising indoleamine-2,3-dioxygenase (IDO) inhibitors from the Indonesian marine sponge Aaptos cf. suberitoides. In the third chapter, the isolation and identification of a SHIP (SH2 containing inositol-5-phosphatase) activating peptide from a marine sponge RJA 55275, is discussed. And finally, the last chapter will describe the x-ray crystallographic study of a eunicellin-based diterpenoid from the Micronesian soft coral that modestly activates the SHIP in order to solve the relative configuration of the molecule as well as to determine the arrangement of atoms within the crystals.                     22  Chapter II: Identification of IDO Inhibitors from the Indonesian Marine Sponge Aaptos cf. suberitoides  2.1 Introduction 2.1.1 Introduction to IDO (Indoleamine-2,3-dioxygenase)  IDO (indoleamine-2,3-dioxygenase) is a cytosolic monomeric hemoprotein of 403 aa long (MW = 45,324 kDa) that catalyzes the first step in tryptophan catabolism by the kynurenine pathway.37 It was initially called D-tryptophan pyrrolase in 1963 and is expressed ubiquitously at high levels in lung, gut, epididymis, and thymus.38 Crystallographic studies on human IDO revealed a two domain structure of α-helical domains with a centrally located heme group. IDO is dependent on heme and an electron donor for its activity. The heme moiety of IDO provides a superoxide anion to donate oxygen to tryptophan.36 IDO is induced by tumor growth and by pathogens that promote inflammatory responses in cancerous and infected host tissues.38            Figure 2.1 The biochemistry of IDO 38  23  A relationship between cancer and elevated tryptophan levels was initially observed in the urine of bladder cancer patients in 1950’s. Subsequent studies also found similar phenomenon in breast cancer, prostate cancer, Hodgkin’s lymphoma, and leukemia.36 In single- celled organisms, nutrient depletion is a common biological strategy to control proliferation of competing cells. In mammals, lymphocytes and T-cells are very sensitive in the presence and concentration of particular amino acids in their local environment and their usage or degradation can cause altered immune function.37 In the late 1990’s, Munn and Mellor reported that IDO activity was crucial to prevent allogeneic fetal rejection due to maternal T-cells immunity in mice. Recent studies on IDO have broadly extended an immunosuppressive role of IDO in supporting a variety of chronic infections, including viral, parasitic and bacterial infections such as human immunodeficiency virus (HIV), malaria, hepatitis C, Toxoplasma gondii, and Chlamydia.36 Expression of IDO by cancer cells has been detected in the tumor infiltrating zone, in the peritumoral stroma, and in tumor draining lymph nodes and is responsible in promoting tumor immune escape. As a result, IDO expression has also been utilized as clinopathological marker with prognostic significance.37 There are two mechanisms that explain how the expression of IDO in tumor cells or by host’s APCs (antigen presenting cell) and tumor infiltrating cells shields the tumor from an immune response against it. The first refers to a direct expression of T-cell function and/or expansion and the second to effects imposed upon T-cells by tumor infiltrations APC.37 The activation of IDO has been proposed to lead to the establishment of immune tolerance through either localized depletion of tryptophan or accumulation of toxic catabolites. This process is immunosuppressive because T-cells undergoing antigen-dependent activation are exquisitely sensitive to local tryptophan catabolism, which can cause tumor cells to arrest in G1, become  24  anergic and die.39 However, the biological significance of IDO-mediated tryptophan depletion and production of kynurenines in tissues are not known with certainty as biochemical changes due to IDO expression are difficult to measure in tissues.38 According to Munn (2006), IDO has two potential roles in cancer treatment, as a primary molecular target for cancer immunotherapy and an endogenous counter-regulatory mechanism that can secondarily antagonize the effectiveness of other immunotherapy strategies. However, emerging evidence suggest that this might be more than a theoretical concern.47 As a possible drug development target, IDO has several appealing features. First, IDO is highly tractable for developing small molecule inhibitors compared with most other therapeutic cancer targets. Secondly, the only enzyme that catalyzes the same reaction, TDO2, has more restricted substrate specificity, mitigating “off target” issues posed by novel agents. Third, bioactive and bioorally available “lead” inhibitors exist that serve as tools for preclinical studies. Fourth, IDO inhibitors are most likely to produce manageable mechanism-based toxicities. Fifth, pharmacodynamic evaluation of IDO inhibitors can be done easily by examining the blood serum levels of tryptophan and kynurenine, the substrate and the product of IDO reaction, respectively. Lastly, small molecule inhibitors of IDO likely offer substantial cost advantages relative to biological or cell based therapies that aim at modulating immunity. IDO inhibitors may be useful not only in cancer but also in other pathologic settings, where it is desirable to relieve immune suppression and/or break immune tolerance (e.g. chronic viral infections).39  2.1.2 Review of IDO inhibitor agents  Two initial studies have demonstrated that 1-methyl tryptophan (1MT) can limit the growth of tumors where IDO is overexpressed. 1MT by itself was unable to trigger tumor  25  regression, suggesting limited efficacy as a monotherapy.36 This is presumably because the original tolerogenic mechanisms rapidly restore tolerance following each cycle of chemotherapy.47 On the other hand, combining 1MT with paclitaxel or several other cytotoxic chemotherapy agents used in breast cancer treatment in the clinic caused rapid regression of established tumors in mice. Similarly, inhibitors of IDO based on the natural product brassinin, a plant phytoalexin with known cancer prevention activity in rodents, display antitumor efficacy in the same patterns as 1MT.33 This is might occur because the combination of IDO and chemotherapy was able to break tolerance to established tumors, allowing therapeutic immune responses that formerly might not have been possible to occur.47 Most studies in the literature have used the natural racemic mixture of 1MT, but in some biologic systems, D-1MT seems to be more active form of 1MT.36 This phenomenon may be caused by the fact that the L-isomer becomes inhibitory or toxic to T-cells at high concentrations, whereas the D-isomer does not. 47 Taking this into consideration, D-1MT entered phase I clinical trials in the fall 2007.36  In 2006, our laboratory isolated 2 classes of compounds, exiguamine A and annulins A-C that were claimed to be the most potent IDO inhibitors known to date, compared to tryptophan analogues (Ki ~10 μM or greater).  Exiguamine A, a complex hexacyclic alkaloid was isolated from the Papua New Guinean marine sponge, Neopetrosia exigua and it inhibited IDO in vitro with a Ki of 40 nM.40, 41 Additionally, the polyketides annulins A-C were isolated from a marine hydroid, Garveia annulata, collected from the Northeastern Pacific. They showed an activity against IDO in vitro with a Ki of 120-690 nM.42    26  2.1.3 Review of Aaptos sp. and its known metabolites   The marine sponge Aaptos sp. is classified as part of the order Hadromerida and family Suberitidae. They are known to have a unique class of alkaloids as major components namely the aaptamines, which bear a unique 1H-benzo [de] [1,6] naphthyridine structure. Aaptamines have been extensively investigated by many research groups over the past two decades. Aaptamine (2.1), along with demethylaaptamine (2.2) and demethyloxyaaptamine (2.3), was initially isolated by Nakamura et al. in 1987 from the Okinawan marine sponge Aaptos aaptos and reported to possess α-adenoreceptor blocking activity on vascular smooth muscle and potent cytotoxicity against HeLa tumor cells.48, 49 Shortly after that, Fedoreev et al. reported the discovery of isoaaptamine (2.4) from a sponge of the genus Suberites50 and later by other groups from Aaptos aaptos51 and Hymeniacydon sp.52 The aaptamine family was subsequently enriched by the finding of 4-methyloxyaaaptamine (2.5) from Aaptos aaptos (Figure 2.2).51            Figure 2.2 Naturally occurred aaptamines. Aaptamine (2.1), demethylaaptamine (2.2), demethyl(oxy)aaptamine (2.3), isoaaptamine (2.4), 4-methyloxyaaptamine (2.5), bisdemethylaaptamine (2.6) and bisdemethylaaptamine-9O-sulfate (2.7) N HN H3CO H3CO 2.1 2.4 N HN H3CO O 2.2 N HN H3CO HO 2.5 1 2 3 4 5 6 3a 7 8 9 9a 6a N HN HO HO N HN HOSO2O HO 2.6 2.7 N N H3CO HO CH3 2.3 N N H3CO HO9b H3C  27  Recently, Herlt et al. added bisdemethylaaptamine (2.6) and its sulfated form, bisdemethylaaptamine-9O-sulfate (2.7) from the Indonesian sponge Aaptos sp.53 Prior to its isolation, bisdemethylaaptamine was proposed as the biosynthetic precursor of the aaptamines (Figure 2.3).53          Soon after, the cytotoxicity of aaptamines was extensively studied by many research groups. Demethylaaptamine (2.2) was proven to be significantly toxic against P-388 cell line with an IC50 value of 0.3 μg/ml 54, and active towards KB cells along with aaptamine (2.1) and isoaaptamine (2.4).55, 56 The anticancer activity of aaptamine against murin colon tumor cells57; sea-urchin eggs 58 and p21, a target protein in human tumors 59 were also reported.  The potential development of aaptamines as anticancer agents motivated Shen and co- workers to investigate the correlation between the methylation pattern of aaptamine and isoaaptamine and their cytotoxicity against P-388 cell line and. It was revealed that the presence of hydroxyl group at the C-9 position and aromaticity in ring B were found to be critical for activity. The oxidation of the hydroxyl group was found to give rise to activity, on the other hand, the acylation of the C-9 OH reduced the activity.55 Additionally, Gul et al. reported that NH2 HO HO HN COOH H2N CHO NH HO HO COOH bisdemethylaaptamine L-dopa beta-alanine aldehyde HN N HO HO aaptamine Figure 2.3 Bisdemethylaaptamine (2.6), a proposed biosynthetic precursor of the aaptamines 53  28  the elongation of side chain at the C9 para position of isoaaptamine increased its activity against leukemia cell line K-562.60 In addition to their anticancer properties, several other papers also described the other biological properties of aaptamines. It was found that aaptamines exhibited significant antiviral activity 61, antimicrobial activity against Gram negative S. aureus, Gram positive V. anguillarum, a fungus C. tropicalis, a protozoan Plasmodium falciparum, and Mycobacterium tuberculosis 56, 57, 62, antifouling against zebra mussels 63 and anti-HIV.57 Due to the common occurrence of aaptamine from marine sponges of the genus Aaptos, this compound was once considered as the chemotaxonomic marker of Suberitidae family.64 However, this point of view was proven to be false when the isolation of novel class of aaptamines from the Indonesian marine sponge of the genus Xestospongia 56 and isoaaptamine from the Singaporean marine sponge Hymeniacydon sp. 52 were reported, suggesting the likelihood of the true producer of aaptamines is a microbial source.57 Other than aaptamines, several papers also reported the isolation of novel compounds from Aaptos sp. Aaptosine (2.8) was discovered by Rudi et al. from the Red Sea sponge Aaptos aaptos in 1993.65 Lately, Nakao and co-workers isolated ciliatamides A-C from the deep sea sponge Aaptos ciliata. Ciliatamides A (2.9) and B (2.10) were found to be antileishmanial, whereas ciliatamide C (2.11) exhibited marginal cytotoxicity against HeLa tumor cells (Figure 2.4).66       29             In this chapter, the elucidation of IDO inhibitory agents from the Indonesian marine sponge Aaptos cf. suberitoides is discussed. Their IDO inhibitor activity in comparison to exiguamine A is also presented.  2.2 Results and discussions 2.2.1 Isolation of IDO inhibitor compounds   As part of a general collection of marine invertebrates, specimens of a marine sponge were collected by SCUBA from the Manado coasts, North Sulawesi, Indonesia in 1994. Frozen samples of A. cf. suberitoides (350 g weight) were thawed and exhaustively extracted by methanol over a period of several days. The combined methanolic extract then concentrated in vacuo to give a dark brown gum. A sample of this crude extract showed an inhibition activity against human IDO in vitro. The crude extract was subsequently suspended in water and Figure 2.4 Additional secondary metabolites from Aaptos sp. Aaptosine (2.8), Ciliatamides A-C (2.9-2.11) H N O HN N O Me O 2.9 H N O HN N O Me O 2.10 H N O N O Me 2.11 HN O N H N 2.8  30  partitioned against ethyl acetate and n-butanol, respectively. Bioassay guided-fractionation on these extracts showed that only the aqueous extract was active as an IDO inhibitor (see Scheme 2.1).                    Fractionation of the active extract (200 mg) was performed using Sephadex-LH20® and pure methanol to give three fractions. The IDO activity was only observed in the second fraction. From here, approximately 5 mg of the fraction was subsequently subjected to high performance Scheme 2.1 Isolation procedures for IDO inhibitor from Aaptos cf. suberitoides Frozen Aaptos cf. suberitoides Crude MeOH extract H2O EtOAc n-BuOH Fraction B Fraction A Fraction C 2.1 (1.8 mg) 2.4 (2.4 mg) LH20 (100% MeOH) Reversed-Phase HPLC (20%MeCN-80% H2O (1% TFA)  31  liquid chromatography using isocratic elution 20% MeCN-80% H2O (1% of TFA). This purification process yielded pure samples of 2.1 (1.8 mg) and 2.4 (2.4 mg) corresponding to two major HPLC peaks at 17 and 19 minutes. At this stage, the bioassay was focused on determining the potency of the IDO activity of both compounds.  2.2.2 Structure elucidation of IDO inhibitor compounds  The structures of aaptamine (2.1) and isoaaptamine (2.4) were elucidated by analysis of 1D and 2D NMR spectroscopic data that was recorded in dimethyl sulfoxide (DMSO-d6) at 600 MHz. Low and high resolution APCI-MS gave supporting information about the molecular weight and formula of the compounds. Proton spin systems were identified from COSY data and proton-carbon attachments were determined by HSQC data. The HMBC data provided information about long range proton-carbon connectivity and the assignments of any quaternary carbon resonances.  2.2.2.1 Aaptamine (2.1)        Aaptamine (2.3) was isolated as a yellow-greenish oil that gave an [M + H]+ ion in the HRAPCIMS at m/z 229.0972 (calcd for 229.0977) appropriate for a molecular formula of N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  32  C13H12N2O2 and 9 degrees of unsaturation. A quick analysis of the 1D and 2D NMR data indicates the presence of seven signals for protons and two exchangeable protons at δ 12.44 ppm (H1) and δ 12.24 ppm (H4), demonstrating the presence of aaptamine in a protonated form. A singlet at δ 3.82 ppm (H8) and δ 3.99 ppm (H9) integrating for three protons, suggested the presence of two methoxys.  Beginning with the exchangeable proton at δ 12.44 ppm (H1), COSY correlation was observed to a proton at δ 7.85 ppm (H2), appropriate for an olefin proton α to pyridine nitrogen. This proton was found to be coupled to another proton at δ 6.36 ppm (H3) and exhibited an HMBC correlation to the quaternary carbon at δ 149.7 ppm (C3a). The other exchangeable proton at δ 12.24 ppm (H4) established a vicinal coupling to its neighboring proton at δ 7.43 ppm (H5), which subsequently showed a COSY correlation to the adjacent proton at δ 6.89 ppm (H6) (Figure 2.5).             Figure 2.5 Selected HMBC and COSY correlations for compound 2.1. (1H and 13C assignments shown) N N H3CO H3CO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H H H H HH H COSY HMBC 12.24 7.18 3.99 6.89 7.43 6.36 7.85 12.44 3.82 149.7 131.4 133.7 116.4 N N H3CO H3CO N HN H3CO H3COH H H H HH H 142.1 98.2 129.9 112.7101.0 156.9 56.5 132.6 60.4  33  The quaternary carbon at δ 131.4 ppm (C6a) was determined by observing its HMBC correlation from the aromatic proton at δ 7.18 ppm (H7), which also established a three-bond HMBC correlation to the quaternary carbon at δ 156.9 ppm (C8). A similar HMBC correlation was also observed to C8 resonance from its adjoining methoxy proton at δ 3.99 ppm (8-OMe). The other methoxy proton at δ 3.82 ppm (9-OMe) also established a three-bond HMBC correlation to the quaternary carbon at δ 132.6 ppm (C9). The quaternary carbon at δ 133.7 ppm (C9a) was determined by observing its HMBC correlation from H2. The similar correlation was also observed from H3, H6 and H7 to determine the quaternary carbon at δ 116.4 ppm (C9b) (Figure 2.5). The conjugated system of aaptamine is consistent with the molecule displaying a strong UV absorbance at 255 nm.   The 1D and 2D NMR data of 2.1 were found to be in complete agreement with those previously reported for aaptamine.48 A summary of all NMR assignments and correlations for aaptamine can be found in Table 2.1.   Position δ 1Ha (J value in Hz) δ 13Cb COSY correlations HMBC correlations 1-NH 12.44 (brs) - H2 - 2 7.85 (d, J = 6.9Hz) 142.1 1-NH, H3 H3 3 6.36 (d, J = 6.9 Hz) 98.2 H2 H2 3a - 149.7 - H2, H5, H7 4-NH 12.24 (brs) - H5 - 5 7.43 (t, J = 6.9 Hz) 129.9 4-NH, H6 H6 6 6.89 (d, J = 6.9 Hz) 112.7 H5 H5, H7 6a - 131.4 - H7 7 7.18 (s) 101.0 - H6 8 - 156.9 - H7, 8-OMe 8-OMe 3.99 (s) 56.5 - - 9 - 132.6 - H3, H5, 9-OMe 9-OMe 3.82 (s) 60.4 - - 9a - 133.7 - H2 9b - 116.4 - H3, H6, H7 a Recorded at 600 MHz    b Recorded at 150 MHz  Table 2.1 NMR data for aaptamine (2.1) recorded in DMSO-d6  34                  Figure 2.6 600 MHz 1H NMR spectrum of aaptamine (2.1) recorded in DMSO 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  35                   Figure 2.7 150 MHz 13C NMR spectrum of aaptamine (2.1) recorded in DMSO 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 Chemical Shift (ppm) N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  36                  Figure 2.8 600 MHz COSY NMR spectrum of aaptamine (2.1) recorded in DMSO 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 F 1  C h e m i c a l  S h i f t  ( p p m ) N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  37                  Figure 2.9 600 MHz HSQC NMR spectrum of aaptamine (2.1) recorded in DMSO 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm) 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 F 1  C h e m i c a l  S h i f t  ( p p m ) N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  38             Figure 2.10 600 MHz HMBC NMR spectrum of aaptamine (2.1) recorded in DMSO 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm) 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 F 1  C h e m i c a l  S h i f t  ( p p m ) N HN H3CO H3CO 2.1 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b  39  2.2.2.2 Isoaaptamine (2.4)       Isoaaptamine (2.4) is the isomer of aaptamine ([M + H]+ m/z 229.0974 (calcd for 229.0977, C13H12N2O2) that also occurs in protonated forms but differs from aaptamine by the presence of N-methyl and hydroxyl group at C1 and C9 positions, respectively. A singlet resonance was observed at δ 4.05 ppm (N-Me) integrating for three protons, suggesting the presence of N-methyl (N-Me). From here, the neighboring protons at δ 7.75 ppm (H2) and δ 6.17 ppm (H3) established a three-bond and a two-bond correlation to the quaternary carbon at δ 149.5 ppm (C3a), respectively. The only exchangeable proton at δ 12.24 ppm (4-NH) showed a vicinal coupling to the next aromatic proton at δ 7.28 ppm (H5), which subsequently established the same coupling to its neighboring proton at δ 6.82 ppm (H6). Additionally, H5 also demonstrated an HMBC correlation to the quaternary carbon at δ 129.5 ppm (C6a) (Figure 2.11). A singlet at δ 7.18 ppm (H7) integrating for one proton was observed in the proton spectrum, appropriate for an aromatic proton in between two quaternary carbons. This is confirmed by the HMBC data that showed the correlation of H7 to the carbon at δ 153.8 ppm (C8). The similar correlation to C8 was also observed from its methoxy proton at δ 3.97 ppm (8-OMe). The neighboring quaternary carbon at δ 132.5 ppm (C9) was determined by observing the HMBC correlation from the adjoining hydroxyl proton at δ 9.50 ppm (9-OH). From this proton, another HMBC correlation to the next quaternary carbon at δ 129.5 ppm (C9a) was also 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  40  observed. Finally, the establishment of the last quaternary carbon at δ 118.3 ppm (C9b) was resolved from H3, H6 and H7 in the HMBC data (Figure 2.11).         The 1D and 2D NMR data of 2.4 is found to be in complete agreement with those previously reported.50 A summary of all NMR assignments and correlations for isoaaptamine can be found in Table 2.2.   Position δ1Ha (J value in Hz) δ13Cb COSY correlations HMBC correlations N-Me 4.05 (s) 46.2 - H2 2 7.75 (d, J = 6.9Hz) 149.3 H3 N-Me, H3, H5 3 6.17 (d, J = 6.9 Hz) 97.6 H2 H2 3a - 149.5 - H2, H3, H5 4-NH 12.24 (brs) - H5 - 5 7.28 (t, J = 6.9 Hz) 128.0 4-NH, H6 H3, H6 6 6.82 (d, J = 6.9 Hz) 113.4 H5 H5, H7 6a - 129.5 - H3, H5 7 7.18 (s) 101.7 - H6 8 - 153.8 - H7, 8-OMe, 9-OH 8-OMe 3.97 (s) 56.8 - - 9 - 132.5 - H7, 9-OH 9-OH 9.50 (s) - - - 9a - 129.5 - H2, H3, N-Me, 9-OH 9b - 118.3 - H3, H6, H7 a Recorded at 600 MHz    b Recorded at 150 MHz  Table 2.2 NMR data for isoaaptamine (2.4) recorded in DMSO-d6 Figure 2.11 Selected HMBC and COSY correlations for compound 2.4 (1H and 13C assignments shown) 12.24 7.18 3.97 6.82 7.28 6.17 7.75 4.05 9.50N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H H H H HH3C H N N H3CO HO N N H3CO HO HH H H H HH H3C COSY HMBC H3C 46.2 149.3 97.6 149.5 128.0 113.4 129.5 101.7 153.8 56.8 132.5 129.5 118.3  41                  Figure 2.12 600 MHz 1H NMR spectrum of isoaaptamine (2.4) recorded in DMSO-d6 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm) 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  42                  Figure 2.13 150 MHz 13C NMR spectrum of isoaaptamine (2.4) recorded in DMSO-d6 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 Chemical Shift (ppm) 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  43                  Figure 2.14 600 MHz COSY NMR spectrum of isoaaptamine (2.4) recorded in DMSO-d6 14 13 12 11 10 9 8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 F 1  C h e m i c a l  S h i f t  ( p p m ) 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  44                  Figure 2.15 600 MHz HSQC NMR spectrum of isoaaptamine (2.4) recorded in DMSO-d6 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm) 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 F 1  C h e m i c a l  S h i f t  ( p p m ) 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  45                Figure 2.16 600 MHz HMBC NMR spectrum of isoaaptamine (2.4) recorded in DMSO-d6 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 F2 Chemical Shift (ppm) 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 F 1  C h e m i c a l  S h i f t  ( p p m ) 2.4 N N H3CO HO 1 2 3 4 5 6 3a 7 8 9 9a 6a 9b H3C  46 2.2.3 Comments on aaptamine (2.1) and isoaaptamine (2.4)  Disagreement of the presence of an exchangeable proton at δ 12.24 ppm in 1H spectrum with their molecular weight suggests that the protonated form of aaptamine and isoaaptamine is unstable. The instability of both compounds also showed by the alteration of their color from yellow-green to dark brown oil due to oxidation process.  2.2.4 Biological activity aaptamine (2.1) and isoaaptamine (2.4)  A series of aaptamine class compounds have been evaluated for their IDO inhibition activity in comparison to exiguamine A (1.14), the most potent IDO inhibitor known to date. Demethylaaptamine (2.2) was obtained from the previous isolation work. The IDO activity assay in this experiment is based on Takikawa’s method.67            Figure 2.17 IDO inhibition assay NH3 N H O O HN O NH3 O O O IDO 30% w/v TCA 650 C for 15 min NH3ONH2 O O O NNH3ON O O N detected at 480 nm p-dimethyl aminobenzaldehyde L-tryptophan N-formyl kynurenine kynurenine  47 The assay is described as follow. IDO catabolizes tryptophan into N-formyl kynurenine which is subsequently turned into kynurenine via hydrolysis reaction. The assay detects the formation of N-formyl kynurenine and quantifies the activity by adding p-dimethylaminobenzaldehyde to convert the kynurenine present in the reaction mixture to a yellow adduct that can be detected at 480 nm (Figure 2.17). The percent inhibition of IDO was determined by dividing the difference of the average positive control and the specific well absorbance with the difference of the average control and the negative control of the particular sample.                  AAPTAMINE Concentration (mg/mL) 0 0.15 0.3 0.45 % in hi bi tio n of ID O 0 10 20 30 40 50 ISOAAPTAMINE Concentration (mg/mL) 0 0.15 0.3 0.45 % in hi bi tio n of ID O 35 45 55 65 75 85 95 105 115 ( )68.276.6618exp29.5164.51 xy ×−−= ( )75.058.29exp05.9236.115 xy ×−−=  48           The above figure shows the relationship between the percent inhibition of IDO and the concentrations tested, started from 0.5 mg/mL and double diluted up to 0.001 mg/mL. Aaptamine (2.1) inhibited IDO with the least activity by showing 51% inhibition at 10-times dilution and a rapid decline after 160-times dilution (IC50 0.06 mg/mL). Meanwhile, demethylaaptamine (2.2) exhibited a stronger activity by inhibiting IDO with IC50 of 0.03 mg/mL. The strongest inhibition against IDO was shown by isoaaptamine (2.4) which demonstrated 50% inhibition against IDO at concentration 0.00215 mg/mL. The preliminary SAR study suggests that substitution of the methoxy at C9 position with a hydroxyl group appears to be the crucial aspect for the IDO activity of aaptamines. Additionally, methylation of N1 position is also important to enhance the inhibition activity against IDO. Taking these results into consideration, hydroxylation of C9 position and methylation of N1 position provide the best structure for IDO inhibition activity in this series of compounds.   DEMETHYLAAPTAMINE Concentration (mg/mL) 0 0.15 0.3 0.45 % in hi bi tio n of ID O 10 20 30 40 50 60 70 80 90 100 Figure 2.18 The IDO inihibition activity of aaptamines ( )14.229.807exp04.7346.92 xy ×−−=  49 2.3 Conclusions  The toxicity and side effects of current chemotherapeutic agents have led to an increasing need for alternative therapies and treatments to alleviate, if not eliminate, these harmful impacts. Therefore, indoleamine-2,3-dioxygenase (IDO) as drug development target has been developed to improve the immune surveillance of patients with cancer and trigger tumor regression by restraining the conversion rate of tryptophan into N-formyl kynurenine. The exhibition of IDO inhibition activity by aaptamine (2.1), isoaaptamine (2.4), and demethylaaptamine (2.2) adds another perspective of the previously reported anticancer activities from aaptamine-class compounds. A preliminary structure-activity relationship (SAR) study through comparison of three aaptamine compounds revealed that isoaaptamine (2.4) is the most potent IDO inhibitor with an IC50 of 0.00215 mg/mL, owing to the substitution of the hydroxylation at C9 position and the methylation of N1.  2.4  Experimental 2.4.1. General Experimental Procedure   HPLC grade solvents were used exclusively in this work. Solvent concentration was carried out using a rotary evaporator equipped with a cold finger condenser cooled by acetone- dry ice and water bath temperature. 1D and 2D NMR spectra were recorded on a Bruker AV-600 spectrometer with a cryoprobe. Chemical shift (δ) values expressed in parts per million (ppm) are referenced to the residual solvent signals with resonances at δH/δC 2.50/39.51 (DMSO-d6). All NMR solvents were obtained from Cambridge Isotope Laboratories. All NMR data was processed using Bruker XWINNMR® software.  50 All chromatography was performed using HPLC grade solvents from Fisher Scientific without further purification. TLC (Kieselgel F254) aluminium baked sheets were obtained from Merck. Water was purified using a Millipore MQ filter system. HPLC was performed using a Waters 2487 HPLC system with dual absorbance detector attached to Waters 515 HPLC pump and a reversed-phase column CSC-Inertsil® ODS2. Low and high resolution APCI-MS analyses were measured on Bruker Esquire LC mass spectrometer.  2.4.2 Animal material   Specimens of Aaptos cf. suberitoides (Bronsted, 1934) were collected off the coast of Manado, Indonesia in 1994. Samples were harvested by hand, frozen on site, and delivered to Vancouver over ice. The samples were subsequently freeze dried and kept frozen until extraction. The organism was identified as Aaptos cf. suberitoides by Dr. R. W. M. (Rob) van Soest. Voucher samples are stored both at the University of British Columbia and the Zoological Museum of Amsterdam (Ref. No. ZMAPOR 19868).  2.4.3 Extraction and isolation   Samples of frozen sponge (350 g) were exhaustively extracted with methanol at room temperature over a period of several days and concentrated in vacuo to give dark brown gum of methanolic crude extract. The extract was subsequently suspended in water and partitioned against ethyl acetate and n-butanol to yield an ethyl acetate extract and an n-butanol extract, respectively. Bioassay-guided fractionation of these three extracts retained a maximum of activity in the aqueous extract.  51  Approximately 200 mg of aqueous extract was subjected to Sephadex-LH20® column chromatography and eluted with pure methanol to give three fractions. About 5 mg of the second fraction was furthermore separated using reversed-phase HPLC (CSC-Inertsil® ODS2 column, 2 mL/min flow rate, dual wavelength 210 nm and 254 nm UV detection) eluted with an isocratic mixture of 20% MeCN-80% H2O (1% TFA) as a solvent system. Two major peaks were observed at 17 min and 19 min, corresponding to isoaaptamine (2.4) and aaaptamine (2.1), respectively. The HPLC fractions were evaporated to dryness and dried under high vacuum prior to NMR and MS analysis.  2.4.4 Aaptamine (2.1) Physical Data  Yellow-greenish oil (1.8 mg).  HRAPCIMS: [M + H]+ m/z 229.0972 (calcd for C13H12N2O2, 229.0977). For 1D and 2D NMR data see Table 2.1.  2.4.5 Isoaaptamine (2.4) Physical Data  Yellow-greenish oil (2.4 mg). HRAPCIMS: [M + H]+ m/z 229.0974 (calcd for C13H12N2O2, 229.0977). For 1D and 2D NMR data see Table 2.2.  2.5 IDO Inhibitory Assay 67  IDO enzymatic activity was detected with a reaction mixture (100 μL total volume) that contained potassium phosphate buffer (50 mM, pH 6.5), ascorbic acid (20 mM), catalase (200 μg/mL), methylene blue (10 mM), L-tryptophan (400 mM) and purified recombinant IDO. The reaction was allowed to proceed for 40 minutes (37 0C) and stopped by the addition of 20 μL of  52 30% (w/v) trichloroacetic acid. The N-formyl kynurenine formed from tryptophan in the reaction mixture during this time was then converted to kynurenine by incubating the reaction mixture at 650 C for 15 minutes. After cooling the reaction to room temperature, an equal volume of 2% (w/v) p-dimethyl amino benzaldehyde in acetic acid was added to convert the kynurenine present in the reaction mixture to a yellow adduct that can be detected at 480 nm. A standard curve was constructed with the use of standard solutions prepared from authentic L-kynurenine. Protein concentration was determined by the Coomassie blue dye-binding method of Bradford with bovine serum albumin as a standard.                   53 Chapter III: A SHIP activator from the Indonesian Marine Sponge RJA 55275  3.1 Introduction 3.1.2 Introduction to SHIP   In 1997, three groups independently purified and cloned a 145 kDa-protein that selectively hydrolyzed the 5-phosphate from inositol-1,3,4,5-tetraphosphate (I-1,3,4,5-P4) and phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3) into phosphatidylinositol-3,4-biphosphate (PI-3,4-P2) both in vitro and in vivo. This protein was found to be unique among 5-phosphatases as it was the only enzyme that had a src homology 2 (SH2) domain, and thus it was later called SHIP for SH2-containing Inositol-5-Phosphatase.44, 68 SHIP demonstrated similar function to that of PTEN (phophatase and tensin homologue), a ubiquitously expressed tumor suppressor gene in most human cancers, that hydrolyzes 3-phosphate from PI-3,4,5-P3 and suppress the phosphatidylinositol-3’-kinase (PI3K) pathway (see Figure 3.1).69        Phosphatidylinositol-3’-kinase (PI3K) is a critical component of cell signaling by growth factors. Phosphoinositides, particularly phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3), generated by PI3K are important second messengers involved in the regulation of diverse cell functions, such as proliferation, differentiation, apoptosis, end cell activation, cell movement and Figure 3.1 Enzymatic synthesis and degradation of PI-3,4,5-P3 70  54 adhesion. Deregulation of the PI3K pathway thus underlies the development of cancer and hence several components of this pathway have been targeted for anticancer drug discovery.43 In addition to SHIP, the Kerr group discovered another protein namely sSHIP that lacks the SH2 domain and is only expressed in embryo stem cells and hematopoietic (blood) stem cells. Pesesse et al. also cloned SHIP2, a more widely expressed 150-kDa protein that is present in varying degrees in some hematopoietic cells. Both sSHIP and SHIP2 have a similar function to that of SHIP.70  The ability of SHIP to break down PI-3,4,5-P3 enables this protein to limit normal and prevent inappropriate mast cell degranulation. Therefore, SHIP has the potential to regulate many, if not all, PI3K induced events. Since SHIP is restricted to hemopoietic cells and negatively regulates the mast cell degranulation, enhancing its activity could prove useful for patients with atopic disorders, such as asthma, and could be employed in the treatment and prevention of human tumors.44  Further investigations on SHIP revealed that it is not only important mediator of cancer but also of inflammation. SHIP takes part in LPS (lipopolysacharide) mediated macrophage- activation and programming. LPS or endotoxin is a major glycolipid in the outer membrane of Gram-negative bacteria that potently triggers inflammation by stimulating immune system cells to produce various pro-inflammatory molecules, such as pro-inflammatory cytokines and NO. Although these molecules are crucial for counteracting the growth of bacteria, overproduction could lead to endotoxin shock. However, if animals or isolated macrophages are first exposed to a low, non-lethal dose of LPS, this induces a 2-3 week state of cell refractoriness to a second high LPS exposure such that far less pro-inflammatory cytokines and NO are produced after the second exposure and the animals survive. This phenomenon, referred as endotoxin tolerance, is supposed to protect the host from cellular damage and represents the adaptation to bacterial infection.71  55 In order to observe the function of SHIP in vivo, Krystal and co-workers generated mice containing a homozygous deletion of SHIP (SHIP -/- mice). The down-regulation effect of SHIP on PI3K products causes the SHIP -/- mice to have an increased numbers of neutrophils and monocyte/macrophages because their progenitors display enhanced survival and proliferation. These mice also suffer from osteoporosis because of an increased number of hyperactive osteoclast and a signifant infiltration of neutrophils into the lungs.44 Interestingly, SHIP -/- bone-marrow-derived macrophages (BMmΦs) and bone-marrow- mast cells (BMMCs) do not display endotoxin tolerance. Moreover, an initial LPS treatment of wild type BMmΦs and BMMCs increases the level of SHIP approximately 10 fold which is critical for the refractoriness to subsequent LPS stimulation. This result showed that SHIP -/- BMmΦs and BMMCs are consistent with PI3K pathway being a positive regulator of LPS- stimulated events and its down regulation is essential for endotoxin tolerance.44  Macrophages can be subdivided into ‘killer’ (classically activated M1) and ‘healer’ (alternately activated M2) subgroups. M1 macrophages are characterized by having a high inducible nitrous oxide synthase (iNOs) levels and producing a large amount of NO to combat bacterial infection and to destroy tumor cells. M2 macrophages, which are characterized by a high arginase level are supposed to play an important role in cleaning up after an infectious agent is destroyed by phagocytosing cellular debris and stimulating host cell proliferation and collagen synthesis. However, in vivo derived SHIP -/- peritoneal and alveolar macrophages produce 10- fold less NO than wild-type macrophages, suggesting that these macrophages display a skewed development away from M1 macrophages toward M2 macrophages and mount a constitutive anti-inflammatory phenotype to avoid septic shock by reducing NO production and increasing arginase activity. These results suggest that therapeutic manipulation of PI3K pathway could be beneficial in treating patients with infection and inflammatory disorders.44   56 3.1.2 Review of SHIP activator agents   In an effort to discover new anti-inflammatory agents, our lab has screened a library of marine natural product extracts. From this screens and subsequent bioactivity-guided isolation, we reported pelorol (1.16) as an in vitro activator of the inositol-5-phosphatase SHIP.46 Pelorol is a meroterpenoid from the tropical marine sponge Dactylospongia elegans that was originally isolated in 2000 by Goclik and co-workers.45  To study the structure activity relationship of pelorol, Yang synthesized pelorol and analogues, where the methyl ester at C20 was replaced by alkyl residues, and tested them in vitro for SHIP activator activity. It was later shown that pelorol (1.16) and its ethyl (3.1) and methyl analogues (3.2) were active in the assay, while the rest of the analogues were not. It was also observed that the methyl analogue had a higher activity than that of pelorol, suggesting that the replacement of the methyl ester with a methyl enhanced the activity. In addition, the presence of at least one phenol in pelorol was found to be crucial for its activity.46         Later, it was suggested that the catechol moiety of pelorol may cause a problem as it would exhibit activities independent of their specific protein pocket binding interactions. The catechol could bind metal or be oxidized into an orthoquinone, which can lead to covalent Figure 3.2 Pelorol (1.16) and its analogues (3.1 and 3.2) as potential SHIP activators 46 3.1 3.2 HO OH HO OH O HO OH O 1.16  57 modifications of protein (Ong, 2007). Based on these considerations, our lab developed MN100 (3.3), a second generation synthetic analogue of pelorol containing only one phenol.73 MN100 (3.3) enhanced the SHIP activity in vitro and it showed 50% maximum inhibition against TNFα from SHIP +/+ but not SHIP -/- 72, making it the most potent SHIP activator known to date.          In this chapter, the isolation and structure elucidation of a SHIP activator from the Indonesian marine sponge RJA 55275 is discussed. Its activity to enhance SHIP compared with MN100 is also presented.  3.2 Results and discussions 3.2.1 Isolation of SHIP activators from sponge RJA 55275   Sponge samples RJA 55275 were collected from Manado, Indonesia and kept frozen until extraction. Frozen sample (54 g) was then thawed and exhaustively extracted with methanol for several days. The combined methanolic extracts were concentrated in vacuo.  A sample of this crude methanolic extract was shown to be active in the SHIP bioassay. The first fractionation was conducted by suspending the crude extract in water and partitioning against ethyl acetate and subsequently against n-butanol, which were each concentrated in vacuo. OH 3.3 Figure 3.3 Structure of MN100 73  58 Bioassay-guided fractionation showed that the ethyl acetate extract retained the maximum activity in the SHIP assay.  The ethyl acetate extract was fractionated via size exclusion chromatography on Sephadex-LH20® and eluting with pure methanol to afford seven fractions. The second fraction was subsequently purified by a reversed-phase Sep-Pak® using a step gradient mixture of 45 mL volume of from 20% methanol-water to 100% methanol with 10% increments. An additional step of 100% dichloromethane was used afterward to rinse the column, resulting in 10 fractions. From here, 1H NMR characteristics were used to pool the fractions of interest.  The eighth fraction from 90% methanol-water elution was shown to be active and thus a further purification was performed using a reversed-phase HPLC and isocratic elution with a mixture of DCM: MeCN: H2O (1:20:4) to yield one major peak identified as theonellapeptolide Id (5.2 mg, 3.3) at 33 min. The isolation procedure is described in Scheme 3.1.   59                  Scheme 3.1 Isolation procedures for SHIP activator from RJA 55275  60 3.2.2 Structure elucidation of theonellapeptolide Id (3.4)   1D and 2D NMR spectroscopic data recorded on a Bruker AV600 MHz in CDCl3 were used to elucidate the structure of theonellapeptolide Id. The molecular weight of the substance as well as its molecular formula were determined by low and high resolution ESI MS. COSY data was employed to determine the proton spin systems, whereas HSQC was used to determine the proton and carbon attachments. Long range proton-carbon couplings were measured by HMBC, while proton-proton couplings through space were solved by TROESY.   Figure 3.4 Structure of theonellapeptolide Id (3.4) NH O O N O HN O NH N N H O O N O O MeOAc L-Val D-Me-Leu L-Thr D-allo-Me-Ile D-Leu Beta-Ala L-Me-Ala L-Me-Val D-allo-Ile Beta-Ala L-Me-Ile D-Leu Beta-Ala 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 3839 40 41 42 N NH O H N O O HN O 43 44 45 46 48 49 50 51 52 53 56 57 5859 60 47 1 54 55 61 62 63 64 ON O HN O O 3.4 H H H  61  Figure 3.4 600 MHz of 1H NMR spectrum for theonellapeptolide Id recorded in CDCl3 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm) NH O O N O HN O NH N N H O O N O O MeOAc L-Val D-Me-Leu L-Thr D-allo-Me-Ile D-Leu Beta-Ala L-Me-Ala L-Me-Val D-allo-Ile Beta-Ala L-Me-Ile D-Leu Beta-Ala 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 3839 40 41 42 N NH O H N O O HN O 43 44 45 4648 49 50 51 52 53 56 57 5859 60 47 1 54 55 61 62 63 64 ON O HN O O 3.4 H H H  62             Figure 3.5 150 MHz of 13C NMR spectrum for theonellapeptolide Id recorded in CDCl3 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm) NH O O N O HN O NH N N H O O N O O MeOAc L-Val D-Me-Leu L-Thr D-allo-Me-Ile D-Leu Beta-Ala L-Me-Ala L-Me-Val D-allo-Ile Beta-Ala L-Me-Ile D-Leu Beta-Ala 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 3839 40 41 42 N NH O H N O O HN O 43 44 45 46 48 49 50 51 52 53 56 57 5859 60 47 1 54 55 61 62 63 64 ON O HN O O 3.4 H H H  63                   Figure 3.6 600 MHz of COSY NMR spectrum for theonellapeptolide Id recorded in CDCl3 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 F2 Chemical Shift (ppm) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 F 1  C h e m i c a l  S h i f t  ( p p m )  64                   Figure 3.7 600 MHz of HSQC NMR spectrum for theonellapeptolide Id recorded in CDCl3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 F2 Chemical Shift (ppm) 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 F 1  C h e m i c a l  S h i f t  ( p p m )  65                   Figure 3.8 600 MHz of HMBC NMR spectrum for theonellapeptolide Id recorded in CDCl3 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 F2 Chemical Shift (ppm) 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 F 1  C h e m i c a l  S h i f t  ( p p m )  66                   Figure 3.9 600 MHz of TROESY NMR spectrum for theonellapeptolide Id recorded in CDCl3 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 F2 Chemical Shift (ppm) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 F 1  C h e m i c a l  S h i f t  ( p p m )  67  Amino Acid No. 13C  1H  (mult., J/Hz) COSY HMBC (1H to 13C) TROESY L-Thr 1 168.6     H2, 51-NH  2 57.8 4.38 (t, 9.5) 2-NH, H3 H4  3 69.3 5.16 (d, 6.4) H2, H4 H2, H4, 6-NMe, H10  4 17.6 1.12 (d, 5.8) H3  2-NH  8.34 (brs) H2 D-allo-Me-Ile 5 170.6   H3, H6  6 69.3 5.16 (d, 6.4)  6-NMe, H10  7 34.2 2.43 (m)   H10  8 28.4 1.80 (m)  H9, H10  8'  1.80 (m)  H9, H11  9 11.9 0.97 (t, 6.8)  10 14.8 0.77 (d, 6.8)   H6, H8  6-NMe 39.3 3.24 (s)   H12 D-Leu 11 174.5   6-NMe, H12  12 48.1 5.04 (m) 12-NH, H13  6-NMe  13 40.4 1.61(t, 12) H12  13'  1.26 (brt) H14  14 25.2 1.73 (m) H13', H15  15 23.6 0.96 (brd) H14  16 20.1 0.93 (d, 6.8)  12-NH  8.12 (d) H12 Beta-Ala 17 170.6   12-NH, H18  18 37.0 2.38 (m) H18'  18'  2.12 (brdd) H18, H19  19 36.9 3.72 (m) H18'  19'  3.30 (m) H19  19-NH  6.66 (d, 9.0) L-Me-Ala 20 169.1   19-NH, H22  21 56.0 4.91 (t, 6.3) H22 21-NMe, H22 H22  22 14.9 1.36 (d, 6.5) H21  H21  21-NMe 28.9 2.76 (s)  H21   Table 3.1 1D and 2D data for theonellapeptolide Id (3.3) recorded at 600 MHz (1H) and 150 MHz (13C) in CDCl3  68  Amino Acid No. 13C 1H  (mult., J/Hz) COSY HMBC (1H to 13C) TROESY L-Me-Val 23 170.5   21-NMe  24 58.2 4.93 (dd, 12.8) H25 H26, H27, 24-NMe H26  25 28.4 2.38 (m) H24, H26 H24, H26, H27 H26, H27, 24-NMe  26 19.9 0.92 (d, 6.8) H25  H24, H25  27 19.7 0.89 (brd)   H25  24-NMe 32.0 3.33 (s)   H25 D-allo-Ile 28 176.6   H24, 24-NMe, H29  29 52.8 5.44 (d, 6.4) 29-NH H31, H33 24-NMe  30 37.2 1.76 (m) H31  31 26.8 1.47 (m) H30, H31' H30  31'  1.23 (s) H31, H32 H30  32 12.3 0.98 (t, 6.8) H31' H30  33 14.2 0.72 (d, 6.8)  H29, H30  29-NH  8.26 (brs) H29 Beta-Ala 34 171.6   H35'  35 35.5 2.43 (m) H35'  35'  2.24 (dd, 15) H35  36*  4.20 (brs) 36-NH, H36'  36'  3.09 (brt) H36  36-NH  6.86 (d, 9) H36 L-Me-Ile 37 169.9   36-NH, H38  38 60.8 5.00 (brt) H39 38-NMe, H42 38-NMe, H42  39 32.5 2.11 (m) H38  38-NMe  40 25.2 1.29 (brd) H40'  40'  0.91 (m) H40  41 10.4 0.85 (t, 6.8)  H42  42 15.9 0.97 (d, 6.8)   H38, 38-NMe  38-NMe 31.7 3.19 (s)   H38, H39, H42 D-Leu 43 175.2   38-NMe  44 48.4 5.11 (t, 12.8) H45'  45 40.6 1.61 (t, 15) H44, H45 H44, H47  45'  1.26 (brt) H45' H44, H47   Table 3.1 (continued)  69  Amino Acid No. 13C 1H  (mult., J/Hz) COSY HMBC (1H to 13C) TROESY  46 25.2 1.73 (m) H47 H47  47 23.6 0.95 (brd) H46  48 21.4 0.99 (d, 6.8)  44-NH  8.09 (brs) Beta-Ala 49*  50 37.2 2.28 (m) H50' H50'  50'  2.12 (m) H50, H51'  51 36.9 3.72 (brt) H51'  51'  3.30 (m) H50', H51  51-NH  6.50 (brt) D-Me-Leu 52 173.4   H2, 53-NMe  53 55.8 5.17 (d, 9.6) H54 53-NMe, H54, H55  54 38.2 1.93 (t, 12) H55  54'  1.93 (t, 12) H55  55*  1.36 (m) H54   H59  56 23.7 0.95 (brd)   53-NMe  57 20.8 0.80 (d, 6.8)  H54  53-NMe 31.7 3.19 (s)   H56 L-Val 58 174.0   H59, 59-NH  59 53.8 4.98 (brd) 59-NH H61, H62  60 32.0 2.03 (m) H61, H62  H61, H62  61 21.1 0.99 (d, 6.8) H60  H60  62 17.1 0.86 (brd) H60  H60  59-NH  7.22 (d, 9.0) H59 MeOAc 63 169.4   H59, 59-NH, H64, H64'  64 72.0 3.96 (d, 12.8)  64-OMe  64'  3.88 (d, 12.8)  64-OMe  64-OMe 59.5 3.39 (s)  H64    Table 3.1 (continued) * The 1H/13C chemical shift was unable to be determined from the HMBC data because of the broadness of the exchangeable proton peaks  70 Analysis of NMR data suggested that the molecule was a peptide based on the presence of α-methines producing resonances at δ 4-5 ppm as well as N-H protons at δ 7-9 ppm and N- methyls at δ 3-4 ppm in the 1H spectrum. A more detailed analysis (HSQC, COSY, HMBC and TROESY) revealed that theonellapeptolide Id consists of several amino acid fragments. Each amino acid is numbered separately for simplicity. Discussion of the spectra will first examine the elucidation of each amino acid residue followed by the connection to the adjacent residue.  Theonellapeptolide Id (3.4) was isolated as white solid with quasimolar [M + H]+ ion peaks at m/z of 1404.9479, calculated for 1404.9446 and suitable for molecular formula C70H125N13O16.  The proton NMR spectra (1H-NMR) showed signals corresponding to the presence of eight NH-amide groups, five N-methyls and a methoxyacetyl moiety. The occurrence of thirteen amide carbons with 2 carbons overlapping, a lactone carbonyl and a methoxyacetatyl group were observed in carbon spectra (13C-NMR). The structure elucidation of theonellapeptolide Id can be divided into five segments and is started from the threonine residue. Analysis of the first segment from C1-C16 is started from the threonine carbonyl at δ 168.6 ppm (C1) that was detected through HMBC correlation from the α-methine at δ 4.38 ppm (H2) (Figure 3.11). COSY correlation was also observed from the α-methine at δ 4.38 ppm (H2) to both the amide proton at δ 8.34 ppm (2-NH) and the adjacent methine proton at δ 5.16 ppm (H3). From here, H3 exhibited a vicinal coupling to the methyl proton at δ 1.12 ppm (H4). The presence of lactone linkage was revealed through the HMBC data from H3 to the neighboring lactone carbonyl at δ 170.6 ppm (C5) which belongs to the methyl isoleucine residue (Figure 3.11).  Another HMBC correlation to the lactone carbonyl (C5) was also observed from the isoleucine α-methine proton at δ 5.16 ppm (H6) (Figure 3.11). The methine proton at δ 2.43 ppm (H7) was detected from the methyl proton at δ 0.77 ppm (H10) through the TROESY correlation. The methylene carbon at δ 28.4 ppm (C8) was observed from the methyl proton at  71 δ 0.97 ppm (H9) through the two-bond HMBC correlation. The other methyl proton at δ 0.77 ppm (H10) was detected from the TROESY correlation with H6, H7 and H8. Additionally, the N-methyl proton at δ 3.24 ppm (6-NMe) showed TROESY correlation to another α-methine at δ 5.04 ppm (H12), suggesting the presence of leucine residue  (Figure 3.11).             The attachment of the leucine to the methyl isoleucine was observed from the HMBC data which showed the correlation of the leucine carbonyl at δ 174.5 ppm (C11) from the methyl isoleucine’s N-methyl proton at δ 3.24 ppm (6-NMe) (Figure 3.11). A two-bond HMBC correlation to the carbonyl at δ 174.5 ppm (C11) was detected from the α-methine δ 5.04 ppm (H12). A vicinal coupling from the amide proton doublet at δ 8.12 ppm (12-NH) to H12 was also observed. Furthermore, COSY data showed connectivity between the α-methine at δ 5.04 ppm (H12) and the adjacent methylene proton at δ 1.61 ppm (H13). H13 also displayed a geminal coupling to the other methylene proton at δ 1.26 ppm (H13’), which subsequently showed a vicinal coupling to the methine proton at δ 1.73 ppm (H14). The COSY correlation from H14 O O N O HN 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 H N O 1 NH COSY HMBC TROESY L-Thr D-allo-Me-Ile D-Leu Figure 3.11 Selected COSY and HMBC key correlations of fragment C1-C16 of theonellapeptolide Id (1H and 13C assignments shown) 6.50 168.6 4.38 5.16O O N O HN H N O NH O O N O HN H N O 1.12 8.34 5.16 2.43 1.80 1.80 0.97 0.77 3.24 5.04 1.61 1.26 1.73 0.960.93 8.12 H H H H H H 57.8 69.3 17.6 170.6 69.3 34.2 28.4 11.9 14.8 39.3 174.5 48.1 40.4 25.2 23.620.1  72 was observed only to the methyl proton at δ 0.96 ppm (H15). 2D NMR data was unable to determine the correlation of the other methyl proton at δ 0.93 ppm (H16) to the leucine residue. It was resolved from the previous data reported (Figure 3.11).  HMBC connectivity from the amide proton at δ 8.12 ppm (12-NH) to the adjoining carbonyl at δ 170.6 ppm (C17) links the first fragment to the second (C17-C27) (Figure 3.12). A two-bond HMBC correlation to the carbonyl (C17) was also observed from a methylene proton at δ 2.38 ppm (H18). As one would expect, the methylene proton at δ 2.38 ppm (H18) showed a COSY connectivity to its geminal proton at δ 2.12 ppm (H18’), which later demonstrated a vicinal coupling to the nearby methylene proton at δ 3.72 ppm (H19). Another COSY coupling established connectivity from H19 to its geminal proton at δ 3.30 ppm (H19’), thus finishes the elucidation of β-alanine residue (Figure 3.12).              Figure 3.12 Selected COSY and HMBC key correlations of fragment C17-C27 of theonellapeptolide Id (1H and 13C assignments shown) O NH N N O O HN O NH N N O O HN 170.6 37.0 36.9 169.1 56.0 14.9 28.9 170.558.2 28.4 19.9 19.7 32.0 2.38 2.12 3.72 3.30 6.66 4.91 1.36 2.76 4.93 2.38 0.92 0.89 3.33 H H H H COSY HMBC TROESY O NH N N O O 17 18 19 20 21 22 23 24 25 26 27 HN 12 Beta-Ala L-Me-Ala L-Me-Val  73 β-alanine and the nearby methyl alanine residue were connected through a two-bond HMBC correlation from the amide proton at δ 6.66 ppm (19-NH) to the alanine carbonyl at δ 169.1 ppm (C20) (Figure 3.12). The N-methyl proton at δ 2.76 ppm (21-NMe) exhibited an HMBC correlation to the α-methine proton at δ 56.0 ppm (C21). A vicinal coupling was also observed between the α-methine at δ 4.91 ppm (H21) to the methyl resonance at δ 1.36 ppm (H22) (Figure 3.12).  The presence of a valine residue was detected by observing an HMBC correlation from a sharp singlet at δ 2.76 ppm (21-NMe) to the carbonyl at δ 170.5 ppm (C23) (Figure 3.12). The α-methine proton at δ 4.93 ppm (H24) showed a COSY correlation to the methine proton at δ 2.38 ppm (H25). COSY and TROESY correlations were also observed from H25 to the methyl protons at δ 0.92 ppm (H26) and δ 0.89 ppm (H27), respectively (Figure 3.12).  The N-methyl proton of methyl valine residue (24-NMe) exhibited TROESY correlation to the α-methine proton of isoleucine at δ 5.44 ppm (H29), linking the second fragment to the third (C28-C42) (Figure 3.13). Both protons (24-NMe and H29) displayed HMBC connectivity to the carbonyl at δ 176.6 ppm (C28). A COSY correlation was also observed between the α-methine (H29) and a broad singlet at δ 8.26 ppm (29-NH), which corresponds to the amide proton of the isoleucine residue. Another COSY coupling revealed the connectivity of the β-methine at δ 1.76 ppm (H30) to the methylene protons at δ 1.47 ppm (H31) and δ 1.23 ppm (H31’). H31’ subsequently displayed a vicinal coupling to the neighboring methyl proton at δ 0.98 ppm (H32). The other methyl carbon at δ 14.2 ppm (C33) was detected from both α- and β-methine at δ 5.44 ppm (H29) and β-methine at δ 1.76 ppm (H30), respectively, through HMBC correlations (Figure 3.13).  Unlike the previous fragments, the HMBC correlation between the amide proton of isoleucine residue and the neighboring carbonyl was not observed because the amide proton peak was very broad. However, the HMBC data showed a correlation between the β-alanine’s  74 carbonyl δ 171.6 ppm (C34) from the methylene proton at δ 2.24 ppm (H35’), which also displayed a COSY correlation to its geminal proton at δ 2.43 ppm (H35). Geminal and vicinal couplings were also observed from the corresponding methylene proton at δ 4.20 ppm (H36) to its geminal proton at δ 3.09 ppm (H36’) and the amide proton at δ 6.86 ppm (36-NH), respectively (Figure 3.13).            The presence of methyl isoleucine residue was detected from HMBC data that connecting the amide proton of β-alanine at δ 6.86 ppm (36-NH) to the methyl isoleucine’s carbonyl at δ 169.9 ppm (C37) (Figure 3.13). The similar HMBC correlation was also observed from the α-methine triplet at δ 5.00 ppm (H38). From here, H38 showed a vicinal coupling to the adjacent methine at δ 2.11 ppm (H39). Another COSY correlation was also observed between two geminal protons at δ 1.29 ppm (H40) and δ 0.91 ppm (H40’). The presence of a methyl residue at δ 10.4 ppm (C41) was detected from the neighboring methyl proton at δ 0.97 ppm (H42) through the HMBC correlation, completing the elucidation of the third fragment (Figure 3.13).  N N H O O 28 29 30 31 32 33 34 35 3637 3839 40 41 42 O HN N 24 COSY HMBC TROESY D-allo-Ile Beta Ala L-Me-Ile Figure 3.13 Selected COSY and HMBC key correlations of fragment C28-C42 of theonellapeptolide Id (1H and 13C assignments shown) N N H O O O HN N N N H O O O HN N 176.6 52.8 37.2 26.8 12.3 14.2 171.6 35.5 35.3 169.960.832.5 25.2 10.4 15.9 31.7 32.0H H H H H 5.44 1.76 1.47 1.23 0.98 0.72 8.26 2.432.24 4.20 3.09 6.86 5.00 2.11 1.29 0.91 0.85 0.97 3.19 HH H  75   The linkage of the third fragment to the fourth was connected from the N-methyl proton of methyl isoleucine at δ 3.19 ppm (38-NMe) to the carbonyl of leucine residue at δ 175.2 ppm (C43) (Figure 3.14). The α-methine at δ 5.11 ppm (H44) showed a vicinal coupling to the methylene proton at δ 1.26 ppm (H45’), which subsequently showed another COSY correlation to its geminal proton at δ 1.61 ppm (H45). A vicinal coupling was also observed from the methine proton at δ 1.73 ppm (H46) to the corresponding methyl proton at δ 0.95 ppm (H47). The methyl proton at δ 0.99 ppm (H48) was not clear due to the congestion of methyl proton peaks in the HMBC data (Figure 3.14).             Because of the peak width, the expected HMBC correlation from the leucine’s amide proton to the β-alanine’s carbonyl was not observed. Therefore, the establishment of the rest of this residue was derived from the COSY data (Figure 3.14). The methylene proton at δ 2.12 ppm (H50’) showed COSY correlation to its geminal proton at δ 2.28 ppm (H50) and vicinal proton at δ 3.30 ppm (H51’). Another geminal coupling was observed between two methylene protons at COSY HMBC TROESY N NH O H N O O 43 44 45 4648 49 50 51 47 1 38 D-Leu Beta-Ala Figure 3.14 Selected COSY and HMBC key correlations of fragment C43-C51 of theonellapeptolide Id (1H and 13C assignments shown) N NH O H N O O N NH O H N O O 175.2 48.4 40.625.2 23.6 21.4 37.2 36.9 168.6 H H H H 5.11 1.61 1.26 1.73 0.95 0.99 8.09 3.19 2.28 2.12 3.30 3.72 6.50 H H  76 δ 3.30 ppm (H51’) and δ 3.72 ppm (H51). The amide proton at δ 6.50 ppm (51-NH) subsequently revealed the attachment of β-alanine to threonine residue by establishing a two- bond HMBC connectivity to the threonine carbonyl at δ 168.6 ppm (C1) (Figure 3.14).  The last fragment of theonellapeptolide Id (C52-C64) is the side chain that is attached to the ring via the amide of the threonine residue (Figure 3.15). The attachment of the methyl leucine to the threonine was detected through the HMBC correlation from the α-methine of threonine at δ 4.38 ppm (H2) to the methyl leucine carbonyl at δ 173.4 ppm (C52). A COSY correlation connected two vicinal protons, the α-methine proton at δ 5.17 ppm (H53) and the β- methylene proton at δ 1.93 ppm (H54), which subsequently showed COSY correlation to the adjoining methine at δ 1.36 ppm (H55). The presence of methyl residue at δ 0.95 ppm (H56) was detected from the N-methyl proton at δ 3.19 ppm (53-NMe) through the TROESY correlation, whilst the other methyl residue at δ 20.8 ppm (C57) was observed from H54 through the HMBC correlation (Figure 3.15).             Figure 3.15 Selected COSY and HMBC key correlations of fragment C52-C64 of theonellapeptolide Id (1H and 13C assignments shown) NH O N O N H O O 57.8 173.4 55.8 38.2 23.7 20.8 31.7 174.053.8 32.0 21.1 17.1 169.472.0 59.5 NH O N O N H O O 5.17 1.93 1.36 0.95 0.80 3.19 4.98 2.03 0.99 0.86 7.22 3.96 3.88 3.39 H H 4.38 8.34 52 53 56 57 5859 60 54 55 61 62 63 64 2 NH O N O N H O O COSY HMBC TROESY D-Me-Leu L-Val MeOAc  77 The valine’s carbonyl at δ 174.0 ppm (C58) was observed via correlations from both the α-methine at δ 4.98 ppm (H59) and the amide proton at δ 7.22 ppm (59-NH) in the HMBC data (Figure 3.15). A vicinal coupling connected the β-methine proton at δ 2.03 ppm (H60) to the neighboring methyl protons at δ 0.99 ppm (H61) and δ 0.86 ppm (H62), completing the elucidation of the valine residue (Figure 3.15).  The last fragment is terminated by a methoxyacetyl group which contains distinctive features in the NMR spectra (Figure 3.15). The attachment of the valine residue and methoxyacetyl was observed from an HMBC correlation between the amide proton of valine at δ 7.22 ppm (59-NH) to the methoxyacetyl’s carbonyl at 169.4 ppm (C63). COSY correlation between two geminal protons at δ 3.96 ppm (H64) and δ 3.88 ppm (H64’) was also observed. The methoxy proton singlet at δ 3.39 ppm (64-OMe) was correlated to the methylene carbon at δ 72.0 ppm (C64) in the HMBC spectrum, finishing the elucidation of the last fragment of theonellapeptolide Id (3.4) (Figure 3.15).  Theonellapeptolides (Ia-e) were initially isolated by Kitagawa and co-workers in 1986 from the Okinawan marine sponge Theonella sp. (Theonellidae) along with some other minor peptolides (theonellapeptolides Ia, Ib, Ic and Ie).74 They were also reported from the Indonesian marine sponge Theonella swinhoei by Roy et al. in year 2000.75 These compounds are rare examples of a peptolide, characteristically comprising N-methyl amino acids, D-amino acids and β-amino acids in a high ratio.74, 76 Theonellapeptolides from series II were reported afterward from the same source,77 whereas those from series III were isolated from the New Zealand marine sponge, Lamellomorpha strongylata.78      78 3.2.3. Comments on theonellapeptolide Id (3.4)  Unlike most peptides, theonellapeptolide Id was negative to the nynhidryn test. This is a characteristic of peptolides in which the N-terminal amino group is protected with a methoxyacetyl group and the C-terminal is connected through a lactone linkage to the β-hydroxyl group of threonine.74, 77  3.2.4. Biological activity of theonellapeptolide Id (3.4)   It was previously reported that theonellapeptolide Id (3.4) inhibited the development of the fertilized eggs of the sea urchin Hemicentrotus pulcherrimus at concentration 50 μg/mL.74 Further investigation of theonellapeptolide Id (3.4) found that it also exhibited moderate cytotoxic activity toward L1210 in vitro with IC50 2.4 μg/mL,79 ion transport activity for Na+ and K+ ions, Na+ and K+-ATPase inhibitory activity, and immunomodulatory activity.75  In this project, we screened theonellapeptolide Id for its SHIP activity in comparison to MN100 (3.3), the most potent SHIP activator known to date, based on Ong’s method.72 The assay was designed to observe the ability of the sample to activate SHIP’s ability to hydrolyze PIP3 into PIP2. The amount of organic phosphate released was assessed by the addition of malachite green reagent and absorbance was measured at 650 nm (see Figure 3.16). A standard curve subsequently constructed and the activation percentage of theonellapeptolide Id (3.4) compared to MN100 (3.3) is presented in Figure 3.17.      79         Theonellapeptolide Id (3.4) was tested at concentration 0.9-142.6 μM in comparison to MN100 (3.3) that exhibited 25% activation of SHIP at concentration 307 μM. The titration curve below shows that theonellapeptolide Id (3.4) activated the SHIP with the same activity at concentration 124 μM. This outcome demonstrates that theonellapeptolide Id (3.4) is the most potent SHIP activator known to date.             Figure 3.16 SHIP activity assay OO OH O OH P OO O OO POPO O O P OO O O O O CO R1 CO R2 PIP3 OO OH O OH P OO O OO PHO P OO O O O O CO R1 CO R2 PIP2 SHIP activator R2 = C19H30 R1 = C17H34 P O O OO + malachite green detected at 650 nm Figure 3.17 The SHIP activity of theonellapeptolide Id (3.4) compared to MN100 (3.3) -5% 0% 5% 10% 15% 20% 25% 30% 35% 0.1 1 10 100 1000 Concentration (uM) of Test Compound P e rc e n t C h a n g e  C o m p a re d  to  C o n tr o l 55725 Et-B8 P3 MN100  80 3.3. Conclusions  Immunotherapy is an innovative approach to chemotherapy, aiming to alleviate cancers by targeting certain molecular pathways. Phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3), generated by phosphatidylinositol-3’-kinase (PI3K) is an important second messengers involved in the regulation of diverse cell functions, such as proliferation, differentiation, apoptosis, end cell activation, cell movement and adhesion and its deregulation underlies the development of cancer. Therefore, a cloned protein, known as SHIP, was developed to deregulate the PI3K pathway by selectively hydrolyzing the 5-phosphate from inositol-1,3,4,5-tetraphosphate (I-1,3,4,5-P4) and phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3). Theonellapeptolide Id (3.4), a rare peptolide containing N-methyl amino acids, D-amino acids and β-amino acids in high ratio, from the Indonesian marine sponge RJA 55275 was found to enhance the SHIP with 25% activity at concentration 123.92 μM, makes it the most potent SHIP activator known to date. This is also the first discovery of a peptide as a SHIP activator.  3.4. Experimental 3.4.1. General experimental procedure   HPLC grade solvents were used exclusively in this work. Evaporation was carried out using a rotary evaporator equipped with a cold finger condenser cooled by acetone-dry ice and water bath temperature. 1D and 2D NMR spectra were recorded on a Bruker AV-600 spectrometer with cryoprobe system. Chemical shift (δ) values expressed in parts per million (ppm) are referenced to the residual solvent signals with resonances at δH/δC 7.27/77.0 (CDCl3). All NMR solvents  81 were obtained from Cambridge Isotope Laboratories. All NMR data was processes using Bruker XWINNMR® software. All chromatography was performed using HPLC grade solvents from Fisher Scientific without further purification. TLC (Kieselgel F254) aluminium baked sheets were obtained from Merck and reversed-phase Sep-Pak® was obtained from Waters. Water was purified using a Millipore MQ filter system. HPLC was performed using Waters Breeze HPLC system consisting of a Waters 1525 Binary HPLC and Waters 2487 Dual Wavelength Absorbance Detector interfaced to a PC and reversed-phase column CSC-Inertsil® ODS2. Low and high ESI-MS analyses were measured on Bruker Esquire LC mass spectrometer.  3.4.2. Animal material   Specimens of RJA 55275 were collected off the coast of Manado, Indonesia and transported over ice to Vancouver, Canada. A voucher specimen is available both at the University of British Columbia and the Research Center for Marine and Fisheries Product Processing and Biotechnology, Jakarta, Indonesia. Specimens have not yet been identified.  3.4.3. Extraction and isolation   Sponge samples were frosted on site and designated by code as RJA 55275. The samples were kept frozen during its delivery to Vancouver and subsequently freeze dried. Dried sample of RJA 55275 (54 g) was exhaustively extracted with methanol for several days. The methanolic extract was subsequently concentrated in vacuum to yield crude extract with dark brown color. A sample of this crude methanolic extract was shown to be active in SHIP bioassay. The first fractionation was conducted by suspending crude extract in water and partitioning against ethyl  82 acetate and n-butanol, three of which were concentrated in vacuo. Bioassay-guided fractionation showed that the ethyl acetate extract retained the maximum activity in SHIP assay.  Approximately 266 mg of ethyl acetate extract was subjected in size exclusion column (Sephadex-LH20®) with pure methanol to afford seven fractions. The most active fraction was subsequently purified with a reversed-phase C18-chromatography (Waters 2g Sep-Pak®). Sample was dissolved in methanol and eluted with a step gradient mixture of 45 ml volume of methanol-water with 10% increments (i.e. 45 ml of 20% MeOH-H2O, then 45 ml of 20% MeOH-H2O and so on). Additional 45 ml steps of 100 % methanol and 100% dichloromethane were used afterward to rinse the column, resulting in 10 fractions. From here, the 1H NMR characteristics were used to pool the fractions of interest.  The eighth fraction was shown to be active and thus a further purification was performed using a reversed-phase HPLC (CSC-Inertsil® ODS2 column, 2 mL/min flow rate, single wavelength 205 nm UV detection) using isocratic mixture of DCM: MeCN: H2O (1:20:4) yielding one major peak of theonellapeptolide Id (5.2 mg, 3.4) at 33 min. The HPLC fractions were evaporated to dryness and dried under vacuum prior to NMR and MS analysis.  3.4.4. Theonellapeptolide Id (3.4) Physical Data  White solid (5.2 mg, 3.4). HRESIMS: [M+H]+ m/z 1404.9479 (calculated for 1404.9446, C70H125N13O16). For 1D and 2D NMR data see Table 3.1.  3.4.5. SHIP Activity Assay 72  SHIP enzyme assays were performed in 96-micro titer plates with 0.04 μg diluted to 10 µL enzyme per well with 25 mM Tris HCl and 150 mM NaCl. About 5 µL of test compound  83 (provided in DMSO) was subsequently added. The mixture was incubated for 10 minutes at room temperature before the addition of 10 µL 50 μM Inositol-1,3,4,5-tetraphophate (IP4; Echelon Biosciences, Salt Lake City, UT). After 15 minutes at 370 C, the amount of organic phosphate released was assessed by the addition of malachite green reagent and absorbent measurement at 650 nm.                     84 Chapter IV: X-ray crystallographic study of a novel eunicellin-based diterpenoid from the Micronesian soft coral RJA 47686  4.1. Introduction 4.1.1. Introduction to X-ray crystallography   It is well recognized that most biologically active compounds controlling physiological function are chiral. The biological and molecular functions of bioactive compounds are closely related to their three dimensional shape. Therefore, determination of absolute configuration is a first major issue in biomolecular and molecular science. The second issue is chiral synthesis of target compounds and how efficiently the desired enantiomers can be synthesized.80 Ever since the suggestion in 1874 by van’t Hoff and LeBel that individual molecules possess a three- dimensional structure that may give rise to dissymmetry, the exact orientation of atoms in space i.e. the absolute configuration have been an important problem to be solved.81 X-ray or neutron diffraction and nuclear magnetic resonance (NMR) are currently the most important methods used to “see” the three dimensional architecture of matter at the atomic level. Both techniques are complementary. Diffraction methods allow determination of molecular structure at a very high precision, resolution and accuracy but only in the crystalline state. NMR is the method of choice for molecules in solution. Its potential for observing dynamical processes over a large range of timescales more than compensates for its limited partial spatial resolution.82 X-ray diffraction of single crystals has the capacity to distinguish the enantiomorphs of a chiral molecule. The technique may be applied to compounds with a vast range of chemical compositions.  Essential chemical information such as the molecular geometry, bond angles, distances, and the packing of the molecules in the crystal are part and parcel of the results of the  85 analysis.83 The direct method by Bijvoet, which relies on the anomalous scattering effect produced by a heavy atom, is still regarded as the most reliable method to determine the absolute configuration of chiral molecules.81 However, this technique has limitations. It excludes compounds containing only light atoms since there are no significant differences in diffraction intensity between two crystal structure models of opposite chirality. Additionally, since x–ray diffraction requires a single crystal, which most of the time is difficult to build, it may be necessary to perform derivatization of the compound to get a crystalline product. If a chiral reagent is used for this purpose, a second stereogenic center of known absolute configuration is introduced and the x-ray structure obtained is then unambiguous and gives the absolute configuration of the first compound. This indirect method has been developed and successfully applied.81  4.1.2. Review of eunicellin-based diterpenoids   Eunicellin (4.1), a diterpenoid bearing a heterocyclic skeleton and an ether linkage was initially isolated by Kennard and co-workers in 1968 from the petroleum extract of the gorgonian Eunicella stricta collected off Banyuls-sur-Mer, France.84 A decade later, two eunicellin-type diterpenoids i.e. cladiellin (4.2) and acetoxycladiellin (4.3) were discovered from the Australian soft coral genus Cladiella sp.85, thus they are also known as cladiellins. Twenty years after that, many more eunicellin-based diterpenoids were reported from the same genus (4.4-4.7) 86, 87, namely cladiellisin (4.8)88, cladiellanes (4.9-4.15)89, 90, cladiellaperoxide (4.16).91 Lately, the eunicellins family has been extended by the isolation of klyxumines A-B (4.17-4.18)92, epoxycladin A-D (4.19-4.22)92 and australin A-D (4.23-4.26)93, suggesting the predominance of eunicellin-based diterpenoids as secondary metabolites in Cladiella sp. (Figure 4.1).93   86                          O H HH H H OH OOH H 4.16 O OAc H H H OH O O 4.11 O O H H H OH H O 4.12 O H H OAc OH 4.13 O H H OAc O 4.14 O H H OH OH 4.9 H H O H H OH OAc H H HO 4.10 O H H OAc O  4.15 H H H H H H H H H H 4.1 AcO OAc O 4.2 AcO H H OAcAcO H H OAcH O 4.3 AcO O H HH H H OH OH H 4.8 O OH H H H H OH OH 4.4 O OAc H H H H OH OH O OAc H H H H OH OAc O O OH H H H H OAc 4.5 4.6 4.7 H H 1 2 3 4 5 678 9 10 11 12 13 14 OAc 16 H 18 H OAcAcO H H OAcH 20 19 O 17 15 O H HH H H OH OAc OHAcO OH 4.17 O H HH H H OH OAcAcO OH 4.18 O H H H H OAc OH OH AcO 4.19 O O H H H H OH OAc OH AcO O 4.20 AcO H H AcO H O O OH H OH 4.23 O H H H H OAc OH AcO O 4.22 O H HH O H OH O 4.24 O H H H H OAc OH HO O 4.21 HO OAc H  87       Although eunicellins (cladiellins) are abundantly found in Cladiella sp., some researcher also reported their occurrence in other species, such as the gorgonian Muricella sp.94, 95, the soft coral Sinularia flexibilis96, the gorgonian corals Briaerum sp.97, the octocoral Briaerum asbestinum 98, the gorgonian Solenopodium stechei 99, the soft coral Alcyonium patagonicum 100, and the soft coral Litophyton sp.101 Due to their quite widespread occurrence, eunicellins have been proposed by Faulkner et al. as logical biosynthetic intermediates on the pathway from a cembrane carbon skeleton to the asbestinin diterpenes found in Briaerum sp. (Figure 4.2).98             Figure 4.2 The proposed biosynthetic relationship between cembrane and asbestinin skeleton by Faulkner et al. 98 O 8 3 2 11 C2-C11 O O 12 11 3 16 eunicellin briarellin 1,2 Me shift O O 3 16 asbestinin O cembrane briarein Figure 4.1 Selected eunicellin-based diterpenoids from soft corals and gorgonians H H O O OH H O H O H HO 4.26 HO O H H H H OAc O H OH O 4.25  88  Several interesting bioactivities have been reported for the eunicellins (Figure 4.3). Bloor et al. reported the cytotoxicity of solenopodin A (4.27) obtained from the gorgonian Solenopodium stechei against murine leukemia P388 cells.99 Litophynol A (4.28) and B (4.29) from the soft coral Litophyton sp. were found to exhibit hemolytic activity.101 Briarellin A (4.30) and labiatin B (4.31) also showed cytotoxicity against HeLa cell lines 98 and human colon cancer cells (HT-116)102 with IC50 of 20 μg/mL and ED50 of 0.85 μg/mL, respectively. Cladiellaperoxide (4.16) was found to exhibit toxicity in the brine shrimp lethality test at a 30 ppm concentration,91 whereas, moderate cytoxicity against human leukemia cell line (LC50 of 0.9 µg/mL) and 49% inhibitory activity against PLA2 were observed from muricellin (4.32), a cladiellin-class compound from the gorgonian Muricella sp. 104 Additionally, australin B (4.24) was found to possess moderate cytotoxicity against several breast and liver cancer cell lines.93  A preliminary SAR study of six cladiellane-type compunds from Muricella sp. (4.33- 4.38), which exhibited significant brine shrimp lethality and toxicity against several human cancer cell lines, showed that the presence of acetoxyl group at the C13 position is required for the activity. Long-chain acyl residues appeared to decrease the activity of the compounds.104           H H OAcHO O H 4.27 O OH AcO OAc OAc 4.31 H H H O H H O OH OH O 4.28 H O H H O OH OH O OH 4.29 H H HAcO NAc H O OAc H O OAc AcO OAc H H OAc 4.32 O OAc AcO OAc H H 4.33 4.34 O O H H HO H H OCOC7H15 OH H 4.30  89       In this chapter, the determination of relative configuration of a novel eunicellin-based diterpenoid from the Micronesian soft coral RJA 47686 by x-ray crystallography is discussed.  4.2 Results and discussions 4.2.1 Crystal structure and relative configuration of diterpenoid 4.39   The structural study of diterpenoid 4.39 was conducted for the following reasons: 1) a detailed investigation of the stereochemistry of 4.39 will be a forerunner to the study of other SHIP activators which might facilitate the understanding of its biological activity; and 2) the complete relative configuration of 4.39 will also give a better perceptive to design an effective route for future synthesis work. Diterpenoid 4.39 was isolated from an unidentified Micronesian soft coral designated as RJA 47686 and reported to exhibit a modest SHIP activity. The constitution and partial relative configuration was elucidated by means of NMR and reported elsewhere.105 However, the position of the ether linkage and the relative configuration of C7 could not be concluded from the NMR data. Therefore, an x–ray diffraction analysis was undertaken to establish a complete relative configuration of 4.39.  Colorless needle crystals were obtained by slow evaporation using a solvent mixture of dichloromethane and methanol (1:1) at a temperature of 40 C. Crystals of 4.39 (molecular Figure 4.3 Bioactive eunicellin-based diterpenoids 4.38 O OAc AcO H H O OAc H H 4.35 O OAc AcO OAc H H 4.36 O OAc H H 4.37  90 formula C28H44O7) are monoclinic, space group P21 with cell dimensions a = 9.3711(14) A; b = 13.5349(17) A; c = 10.9891(17) A; α = 900; β = 99.142(7)0; γ = 900; V = 1376.1 (3) Å 3 . Table 4.1 provides information about the crystal data and parameters used in the refinement of the structure.   Empirical Formula C28H44O7 Formula Weight 492.63 Crystal Color, Habit colorless, needle Crystal Dimensions 0.12 X 0.25 X 0.50 mm μ(MoKα) 0.84 cm-1 Crystal System monoclinic Space Group P 21/b Lattice Parameters a = 9.3711(14) A       α = 90 o b = 13.5349(17) A     β = 99.142(7) o c = 10.9891(17) A      γ  = 90 o V = 1376.1 (3) Å3 Z value 2 Dcalc 1.189. 10-3 g/cm3 F000 536.00 Crystal Dimensions 0.12 X 0.25 X 0.50 mm 2θmax 51.86o No. of Reflections Measured Total: 19162 Unique: 5292 (Rint = 0.0231) Corrections Absorption (Tmin = 0.87; Tmax= 0.99) Lorentz-polarization Reflections Collected 19162 Independent Reflections  5292 (Rint = 0.0231) Refinement method Full-matrix least-squares on F2 Goodness of Fit Indicator 1.058 Residuals (refined on F): R1; wR2 0.0502; 0.1374 Residuals (refined on F2 all data): R1; wR2 0.0577; 0.1444 Maximum peak in Final Diff. Map 0.609 e-/Å3 Minimum peak in Final Diff. Map -0.381 e-/Å3   The x–ray structure showed that the basic eunicellin skeleton consists of six-membered and ten-membered rings that are cis-fused at C1 and C10. An ether bridge connecting C2 and C6 was also observed with a cis configuration. The six-membered ring was in a distorted chair conformation with a flattening at C1-C10 and a deep tucking at C13-C12-C17. The dihedral angle between the butyrate chain C1’-C4’ and the ten-membered ring is 53.90, whereas that Table 4.1 Crystal data and structure refinement of diterpenoid 4.39  91 between the butyrate chain C1’-C4’ and the methyl residue C15 is 61.50. Additionally, the dihedral angle between the second butyrate chain C1”-C4” and the methyl residue C20 is 67.90. The hydrogen atom at C2 dips into the plane and forms an angle toward the ten- membered ring of 106.30. The methyl and hydroxyl group at C7 forms an angle of 105.60. A dihedral angle of 46.40 toward the viewer was observed from the residual chain at C14 against the six-membered ring. An ORTEP diagram of the molecular structure and atomic numbering of 4.39 is show in Figure 4.3. Based on the X-ray data and NMR data, the diterpenoid 4.39 is found to possess the (1R*, 2R*, 3R*, 6R*, 7S*, 10R*, 14R*, 18R*)-configuration (Figure 4.4).                  Figure 4.4 ORTEP diagram of diterpenoid 4.39 H H O O OH H O OOO 1 2 3 4 5 6 7 8 91011 12 13 14 15 16 17 18 19 20 1' 2' 3' 4' 1" 2" 3" 4" H H H 4.39  92   Diterpenoid 4.39 was assigned to establish a similar configuration to that as suggested of australin D, whose relative configuration was determined by NOESY.93 However, the C7 position has the opposite relative stereochemistry to that of australin D, with the methyl group (C16) is almost planar and the hydroxyl group (7-OH) is away from the viewer.  4.2.2 Comments on diterpenoid 4.39 crystal   During the experiment, elongated ellipsoid forms were observed from atoms at position C1”-C4”, suggesting the presence of dynamic thermal motion of the ester chain.  4.3 Conclusions   The diterpenoid 4.39 was isolated from a soft coral collected off the coast of Pohnpei, Micronesia as colorless needle crystals possessing a eunicellin-based skeleton and a modest SHIP activity. X–ray analysis of 4.39 suggested that the crystals are monoclinic, space group P21/b, with a = 9.3711(14) A; α = 900; b = 13.5349(17) A; β = 99.142(7)0; c = 10.9891(17) A; γ = 900; V = 1376.1 (3) Å3; Z value = 2; Dcalc 1.189. 10 -3 g/cm 3 ; F000 536.00; Cu (MoKα) 0.84 cm -1 .  The structure of 4.39 was refined using least-squares methods to final R value of 0.0502 from 5292 reflections. Based on the X-ray data and NMR data, the diterpenoid 4.39 is found to possess the (1R*, 2R*, 3R*, 6R*, 7S*, 10R*, 14R*, 18R*)-configuration.    Figure 4.5 A eunicellin-type diterpenoid from the Micronesian soft coral RJA 47686 with (1R*, 2R*, 3R*, 6R*, 7S*, 10R*, 14R*, 18R*)-configuration  93   4.4 Experimental 4.4.1 Crystallization method   Isolation of 4.39 was conducted by Williams.105 A colorless needle crystal of 4.39 was obtained by slow evaporation of a solvent mixture dicholoromethane-methanol (1:1) at temperature 4 0C. Crystallographic data for this sample is currently deposited at UBC X-ray Crystallographic Database.  4.4.2 Data collection   A crystal of C28H44O7 having approximate dimensions of 0.12 x 0.25 x 0.50 mm was mounted on a glass fiber. All measurements were made on a Bruker X8 APEX II diffractometer with graphite monochromated Mo-Kα radiation. The data were collected at a temperature of -100.0 + 0.10 C to a maximum 2θ value of 51.860. Data were collected in a series of φ and ω scans in 0.500 oscillations with 12.0 second exposures. The crystal-to-detector distance was 36.00 mm.  4.4.3 Data Reduction   Of the 19162 reflections that were collected, 5292 were unique (Rint = 0.0231); equivalent reflections were merged.  Data were collected and integrated using the Bruker SAINT Software package. The linear absorption coefficient, μ, for Mo-Kα radiation is 0.084 mm -1 . Data were corrected for absorption effects using the multi-scan technique (SADABS), with minimum  94 and maximum transmission coefficients of 0.87 and 0.99, respectively.  The data were corrected for Lorentz and polarization effects.  4.4.4 Structure Solution and Refinement   The structure was solved by direct methods. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in calculated positions but were not refined. The final cycle of full-matrix least-squares refinement on F 2  was based on 5292 reflections and 327 variable parameters and converged (largest parameter shift was 0.00 times its esd) with unweighted and weighted agreement factors of:  R1 =Σ ||Fo| - |Fc|| / Σ |Fo| = 0.052 wR2 = [Σ ( w (Fo2 - Fc2)2 )/ Σ w(Fo2)2]1/2 = 0.098   The standard deviation of an observation of unit weight was 1.05. The weighting scheme was based on counting statistics.  The maximum and minimum peaks on the final difference Fourier map corresponded to 0.609 and -0.381 e-/Å 3 , respectively.  Neutral atom scattering factors were taken from Cromer and Waber. Anomalous dispersion effects were included in Fcalc; the values for Δf' and Δf" were those of Creagh and McAuley. The values for the mass attenuation coefficients are those of Creagh and Hubbell. All refinements were performed using the SHELXTL crystallographic software package of Bruker- AXS.    95 Chapter V: General Conclusions   Cancer is a group of fatal diseases that affects one out of three people in the world. Although current cancer therapies have reached a certain degree of success, they cause severe side effects to the patients and cancer still causes 25% of mortalities in the United States. This demonstrates that these cancer therapies are flawed. Therefore, a transition has occurred in the selection of antitumor molecular targets, and a shift has begun in research focus toward disease- specific and mechanism-based screening methods.  As an alternative treatment method, cancer immunotherapy has emerged over the last decade as a method for preventing the metastatic spread of cancer and improving the quality of life of affected individuals. The goal is to develop an effective treatment by targeting enzymes and/or pathways which are over-expressed in tumor cells but have only limited importance in non tumorous tissues.  A relatively recent therapeutic target was identified when a relationship between cancer and elevated tryptophan catabolism was initially recognized in the urine of patients with bladder cancer, breast cancer, prostate cancer, Hodgkin’s lymphoma and leukemia. These findings led to the discovery of indoleamine-2,3-dioxygenase (IDO), a cytosolic monomeric hemoprotein that is responsible for catalyzing the first step of tryptophan catabolism by the kynurenine pathway and eventually initiates an altered immune function. Hence, an IDO inhibitor agent would restrain the depletion of tryptophan and prevent the cancer’s escape from the immune system.  Despite competition from other drug discovery methods, natural products continue to be one of the main sources of state-of-the-art drugs in the pharmaceutical industry. This is demonstrated by the fact that natural product-derived drugs are still well represented in the top 35 worldwide bestselling drugs of 2000, 2001, and 2002. In addition to terrestrial plants and soil  96 microorganisms, the marine environment especially marine invertebrates offer a great source of natural products as potential pharmaceuticals.  Marine invertebrates produce defensive compounds to survive with intensive evolutionary pressures from competitors. Further ecological research has also revealed that predation in tropical areas is higher than that in temperate habitats such as Indonesian waters. As part of East Indies triangle, Indonesian archipelago is unique in terms of diversity and ecology, making it the most important coral reef in the world’s biodiversity. Therefore, marine natural product research is currently focused on this area. Bioassay guided fractionation of the methanolic extract of the Indonesian marine sponge Aaptos cf. suberitoides in an IDO inhibitor assay led to the isolation of aaptamine (2.1) and isoaaptamine (2.4). Assay results showed that isoaaptamine was the most potent, exhibiting the strongest activity against IDO with an IC50 of 0.00215 mg/mL. Preliminary structure-activity relationship (SAR) studies indicate that the increased activity is likely due to hydroxylation at the C9 position and methylation of the N1 position. However, advanced SAR studies are needed to obtain more active analogs.  Another approach for improving cancer therapy involves the down regulation of the phosphatynidylinositol-3-kinase (PI3K) by inhibiting its second messenger, phosphatynidylinositol-3,4,5-triphosphate (PIP3). SHIP (SH-2 containing inositol 5- phosphatase) hydrolyzes the 5-phosphate from the PIP3 into PI-4,5-P2 and hence restrains the PI3K pathway. SHIP is currently a potential molecular target for the development of anticancer agents.  Theonellapeptolide Id (3.4), isolated from the unidentified Indonesian marine sponge RJA 55275 was tested for its SHIP activity in comparison to MN100 (3.3), the most potent SHIP activator known to date. It was shown that theonellapeptolide Id (3.4) exhibited 25% activation of SHIP with a concentration of 128 μM, less than half concentration of MN100 (3.3) required to  97 achieve the same activation. This outcome demonstrates that theonellapeptolide Id (3.4) is the most potent SHIP activator known to date, as well as the first peptide ever reported as a SHIP activator.  A colorless needle-shaped crystal showing a modest SHIP activity was also isolated from an unidentified Micronesian soft coral by Dave Williams from our lab. The structure elucidation and most of the partial relative configuration of the compound were determined by NMR and were completed by means of X-ray crystallography. Analysis of the crystal showed that the molecule possessed a eunicellin-based skeleton, with an ether linkage connecting C2 and C6 position. Further analysis of 4.39 suggested that the crystals are monoclinic, space group P21/b, with a = 9.3711(14) A; α = 900; b = 13.5349(17) A; β = 99.142(7)0; c = 10.9891(17) A; γ = 900; V = 1376.1 (3) Å3; Z value = 2; Dcalc 1.189. 10 -3 g/cm 3 ; F000 536.00; Cu (MoKα) 0.84 cm -1 . Based on the X-ray data and NMR data, the diterpenoid 4.39 is found to possess the (1R*, 2R*, 3R*, 6R*, 7S*, 10R*, 14R*, 18R*)-configuration.  To conclude, the results of this thesis show that natural products, especially those from tropical waters, continue to be an excellent source of interesting molecules with extremely potent anticancer activities in a wide range of biological assays. These results also represent that marine natural products research plays critical roles in the development of drug discovery, particularly in cancer immunotherapy. 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