Citation
Drug Discovery from Marine Cyanobacteria Symploca spp. and Phormidium spp.: Novel Structures and Bioactivities of Secondary Metabolites

Material Information

Title:
Drug Discovery from Marine Cyanobacteria Symploca spp. and Phormidium spp.: Novel Structures and Bioactivities of Secondary Metabolites
Creator:
Salvador, Lilibeth A
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences
Medicinal Chemistry
Committee Chair:
Luesch, Hendrik
Committee Members:
James, Margaret O
Qi, Xin
Chen, Sixue
Graduation Date:
5/4/2013

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Cells ( jstor )
Cyanobacteria ( jstor )
Hydroxy acids ( jstor )
Ions ( jstor )
Metabolites ( jstor )
Molecules ( jstor )
Polyketides ( jstor )
RNA ( jstor )
Viability ( jstor )
antiproliferative-activity
cyanobacteria
drug-discovery
elastase
natural-products
phormidium
symploca

Notes

General Note:
Four marine cyanobacteria collections were prioritized for the discovery of novel secondary metabolites, based on their antiproliferative activity against HT29 human colorectal adenocarcinoma cells and unique HPLC-MS dereplication profiles. Bioactivity- and 1H NMR-directed purification yielded the elastase inhibitors symplostatins 5–10 (1–6), and the antiproliferative agents veraguamides A–G (7–13), caylobolide B (18), and amantelides A and B (19, 20). Total structure elucidation was done using 1D and 2D NMR spectroscopy, mass spectrometry and enantioselective analysis.   Symplostatins 5–10 (1–6) are cyclic depsipeptides bearing the modified amino acids 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid. Comprehensive protease profiling of 1 indicated potent and selective elastase inhibition. Structure-activity relationship (SAR) studies on 1–6, together with the related compounds lyngbyastatins 4 and 7, identified critical and tunable structural elements. This was corroborated by the X-ray cocrystal structure of lyngbyastatin 7–porcine pancreatic elastase. The effects of symplostatin 5 (1) on the downstream cellular effects of elastase was probed using an epithelial lung airway model system. Compound 1 attenuated elastase-mediated receptor activation, proteolytic processing of adhesion molecule ICAM-1, NF-kB activation and global transcriptome changes, leading to cytoprotection against elastase-induced cell death, detachment and inflammation.   Veraguamides A–G (7–13) are cyclic hexadepsipeptides bearing a C8-polyketide-derived ß-hydroxy acid, an invariant proline residue, multiple N-methylated amino acids and an a-hydroxy acid. Compounds 7–13 together with the semisynthetic derivative tetrahydroveraguamide A (14) displayed weak to moderate antiproliferative activity against HeLa cervical carcinoma and HT29 cells, modulated by several sensitive positions in the veraguamide scaffold. Flow cytometry indicated that veraguamide D (10) caused a dose-dependent increase in cell populations at sub-G1 and G2.  Caylobolide B (18) and amantelides A and B (19, 20) are structurally-related polyketides characterized by a polyhydroxylated macrolactone ring bearing an alkyl pendant side chain. Amantelide A (19) displayed sub-micromolar IC50s against HT29 and HeLa cells, while 18 and 20 showed weaker activity. These cyanobacterial polyketides potentially exert their cytotoxic effect through interaction with the cell membrane.

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Source Institution:
UFRGP
Rights Management:
All applicable rights reserved by the source institution and holding location.
Embargo Date:
5/31/2015

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1 DRUG DISCOVERY FROM MARINE CYANOBACTERIA S YMPLOCA SPP A ND P HORMIDIUM SPP .: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY METABOLITES By LILIBETH APO SALVADOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVER SITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Lilibeth Apo Salvador

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3 To my mom, my brothers, and my husband

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4 ACKNOW LEDGMENTS I am greatly indebted to my mentor Professor Hendrik Luesch, for giving me the opportunity to be a part of his group and work on numerous research projects. I appreciate h is immense support, guidance and patience throughout my graduate studies. I am also thankful to Dr. Luesch for helping me recognize my capabilities and b ringing out more than my best. I appreciate his insights and critiques, which all contributed to the success of this study. I also thank Professor Si xue Chen, Professor Margaret O. James and Professor Xin Qi, for graciously agreeing to be members of my dissertation committee. I appreciate their insightful comments in the prepa ration of this manuscript and their timely response to my inquiries I am grateful to our collaborators, Dr. Valerie J. Paul and Dr. Jason S. Biggs for providing the cyanobacteria collections and lending their expertise. I thank them for their support and fruitful discussions during the preparation of our manuscripts for publication. I thank all the former a nd current m together with Ms. Gudrun Schelegel who contributed to the collection and extraction of cyanobacteria samples for screening and dereplica tion. I am also grateful to Diane Littler for identifying several of the s ample collections, the Fort Zachary Taylor State Park and J. Qui ata of Cetti Bay Agat Station for permission to obtain the samples. I acknowledge Dr. Jean Jakoncic Dr. David A. Ostrov and Ms. Kanchan Taori, who conducted the experiments and data analysi s for the cocrystallization of lyngbyastatin 7 porcine pancreatic elastase I thank them for their help i n the discussion and preparat ion of figures for the X ray analysis. I also thank th e Bioinformatics Core of the Interdi s ciplinary Center for Biotechnol ogy Research for assistance in the

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5 transcriptome data analysis and heat map generation. I am also grateful to M r. James Rocca for lendin g his expertise and passion for NMR I thank Jim for his patience and invaluable help in NMR training and troubleshootin g More so, I am grateful for his friendship and constant reminder to smell the roses I wou ld also like to acknowledge current and former members of the Luesch Lab Ms. Fatma Al A wadhi, Ms. Michelle Bousquet, Ms. Weijing Cai, Dr. Qiyin Chen, Dr. Jason C. Kwan, Dr. Yanxia Liu, Dr. Susan Matthew, Ms. Kamolrat Metavarayuth, Ms. Rana Montaser, Dr. Ranjala Ratnayake, Dr. Rui Wang and Dr. Wei Zhang for their help at variou s stage s of my graduate studie s; from sett ling down and starting with my projects, movin g forwar d with my research and finishing up. I appreciate the technical expertise that everyone has provided as well as insightful di scussions and unforgettable experie nces during our collection trips. I also thank the staff of the Medicinal Chemistry Depa rtment Mr. David Jenkins, Ms. Jan Kallman and Mr. Brian Karcinski for making sure that all necessary requirements for my studies we re taken care of. I am also grateful to my friends and prayer warriors, Dr. Ma. Pythias Espino, Mr. Krisnakanth Kondabo l u, Dr. Francesca Diane Liu, Dr. Mario Edgar Moral and Dr. Ra njala Ratnayake. They shared with me the joys and hardships of graduate school, made Gainesville more memorable and lent their advice on embracing this endeavor working it through and taking the plunge to a new career. I also acknowledge my former supervisors, Professor Gisela P. Concepcion and Professor Amelia P. Guevara, for introducing me to natural products chemistry, for constantly believ ing in my capabilities, for their support and encourage ment.

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6 Finally, I thank my whole family for tons of love, patience, under standing and faith throughout this journey I thank my mother, Emilia, who serves as my inspiration and role model for hardwork and perseverance. I am gra t e ful to my husband, Joeriggo, for sharing this dream with me and for being by my side from day one of graduate school Joeriggo has been one of my toughest cr itics, but he has also been the mo st patient And to my Shepherd I am very much thankful ; and all that is mine to give is for your glory.

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7 TABLE OF CONTENTS page ACKNOWLEDGMEN TS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 19 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .................. 21 Natural Product s in Drug Discovery ................................ ................................ ........ 21 Drugs from the Sea ................................ ................................ ................................ 22 Marine Cyanobacteria: Source Organisms of Novel Molecules .............................. 24 Mechanism of Action of Bioactive Cyanobacterial Metabolites ............................... 25 Interference with Microtubule Dynamics ................................ ........................... 25 Inhibition of Histone Deacetylase ................................ ................................ ..... 26 Inhibition of Proteases ................................ ................................ ...................... 27 Objectives and Specific Aims of the Study ................................ .............................. 29 2 PROBING THE CHEMICAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF CYANOBACTERIAL COLLECTIONS ................................ ............................... 35 Introduction ................................ ................................ ................................ ............. 35 Screening of Cyanobacteria Collections ................................ ................................ 37 Antiproliferative Assay as Preliminary Screening for Bioactivity ....................... 38 Dereplication using an HPLC MS Approach ................................ ..................... 38 Prioritization of Sample Collections ................................ ................................ .. 39 Validation of th e Dereplication Method ................................ ................................ ... 40 Conclusion ................................ ................................ ................................ .............. 40 Experimental Methods ................................ ................................ ............................ 41 General Experimental Procedures ................................ ................................ ... 41 Biological Material ................................ ................................ ............................ 41 HPLC MS Profiling ................................ ................................ ........................... 42 Cell Viability Assay ................................ ................................ ........................... 4 2 Validation of Dereplication Method ................................ ................................ ... 43 3 POTENT ELASTASE INHIBITORS FROM CYANOBACTERIA: ST RUCTURAL BASIS AND MECHANISMS MEDIATING CYTOPROTECTIVE AND ANTI INFLAMMATORY EFFECTS IN BRONCHIAL EPITHELIAL CELLS ...................... 48

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8 Introduction ................................ ................................ ................................ ............. 48 Isolation and Structure Elucidation ................................ ................................ ......... 50 Enzyme Inhibition ................................ ................................ ................................ ... 54 Molecular Basis for Elastase Inhibition by Lyngbyastatins and Symplostatins ........ 55 Biological Activity Evaluation ................................ ................................ .................. 57 Cytoprotective Effects of Symplostatin 5 ( 1 ) Against Elastase Induced Antipro liferation and Apoptosis ................................ ................................ ...... 57 Cytoprotective Effects of Symplostatin 5 ( 1 ) Against Elastase Induced Cell Detachment and Morphological Change ................................ ....................... 59 Attenuation of Global Transcript Changes Induced by Elastase ....................... 62 Conclusion ................................ ................................ ................................ .............. 65 Experimental Methods ................................ ................................ ............................ 65 General Experimental Procedures ................................ ................................ ... 65 Biological Material ................................ ................................ ............................ 66 Extraction and Iso lation ................................ ................................ .................... 66 Enantioselective Analysis ................................ ................................ ................. 67 In Vitro Protease Assay ................................ ................................ .................... 69 Cocrystallization of Lyngbyastatin 7 with Porcine Pancreatic Elastase ............ 70 In Vitro Cellular Assays ................................ ................................ .................... 71 General cell culture proc edure ................................ ................................ ... 71 Cell viability assay ................................ ................................ ...................... 71 Cell detachment and morphology change ................................ .................. 71 Caspase activation measurement ................................ .............................. 72 Measurement of sICAM 1 levels ................................ ................................ 72 Immunoblot analysis of mICAM 1 levels ................................ .................... 73 Isolation of nuclear and cytoplasmic proteins ................................ ............. 73 B p65 translocation ............... 74 RNA isolation and reverse transcription ................................ ..................... 75 Real time quantitative polymerase chain reaction (qPCR) ......................... 75 Transcriptome profiling ................................ ................................ .............. 76 4 VERAGUAMIDES A G: CYTOTOXIC CYCLIC HEXADEPSIPEPTIDES WITH A C 8 POLYKETIDE HYDROXY ACID MOIETY FROM CETTI BAY, GUAM ................................ ................................ ................................ ................... 100 Introduction ................................ ................................ ................................ ........... 100 Isolation and Structure Elucidation ................................ ................................ ....... 101 Biological Activity Studies ................................ ................................ ..................... 106 Conclusion ................................ ................................ ................................ ............ 108 Experimental Methods ................................ ................................ .......................... 108 Biological Material ................................ ................................ .......................... 108 Extraction and Isolation ................................ ................................ .................. 109 Hydroge nation of 7 ................................ ................................ ......................... 110 Acid Hydrolysis of Veraguamides and Enantioselective Analysis ................... 111 Methanolysis of 7 ................................ ................................ ........................... 113 Preparation of MTPA Esters of 15 ................................ ................................ .. 114 Biological Activity Assays ................................ ................................ ............... 115

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9 Cell viability assay ................................ ................................ .................... 115 Cell cycle analysis by flow cytometry ................................ ....................... 115 5 CAYLOBOLIDE B AND AMANTELIDES A AND B: ANTIPROLIFERATIVE POLYKETIDES FROM MARIN E CYANOBACTERIA ................................ ........... 132 Introduction ................................ ................................ ................................ ........... 132 Isolation and Structure Elucidation ................................ ................................ ....... 133 Caylobolide B ( 18 ) ................................ ................................ .......................... 133 Amantelides A and B ( 19 20 ) ................................ ................................ ......... 136 Configurational Analysis ................................ ................................ ....................... 138 Biological Activity Studies ................................ ................................ ..................... 139 Antiproliferative Activity ................................ ................................ .................. 139 Elucidation of the Mechanism of Action of Cyanobacterial Polyketides .......... 140 Conclusion ................................ ................................ ................................ ............ 142 Experimental Methods ................................ ................................ .......................... 142 General Experimental Procedures ................................ ................................ 142 Biological Material ................................ ................................ .......................... 143 Extraction and Isolation ................................ ................................ .................. 143 Caylobolide B ( 18 ) ................................ ................................ ................... 143 Amantelides A ( 19 ) and B ( 20 ) ................................ ................................ 144 Acetylation of amantelide A ( 19 ) ................................ .............................. 145 ESIMS/MS Fragmentation of Caylobolide B ( 18 ) and Amantelide A ( 19 ) ....... 145 Cell Viability Assay ................................ ................................ ......................... 146 6 GENERAL CONCLUSION ................................ ................................ .................... 160 APPENDIX A CELL MORPHOLOGY AT 3 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR) ................................ ................................ ................................ .......... 164 B CELL MORPHOLOGY AT 6 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR) ................................ ................................ ................................ .......... 165 C CELL MORPHOLOGY AT 1 2 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR) ................................ ................................ ................................ .......... 166 D CELL MORPHOLOGY AT 2 4 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR) ................................ ................................ ................................ .......... 167 E ICAM1 TRANSCRIPT LEV ELS AT 3 h AND 6 h ................................ .................. 168 F NMR SPECTRA OF ISOLATED COMPOUNDS ................................ .................. 169 LIST OF REFERENCES ................................ ................................ ............................. 257 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 266

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10 LIST OF TABLES Table page 2 1 Antiproliferative activity (IC 50 nM) of known Symploca sp. metabolites ............. 47 3 1 NMR data of symplostatin 5 ( 1 ) and symplostatin 8 ( 4 ) in DMSO d 6 .................. 84 3 2 NMR data of symplostatin 6 ( 2 ) and symplostatin 9 ( 5 ) in DMSO d 6 .................. 87 3 3 NMR data of symplostatin 7 ( 3 ) and symplostatin 10 ( 6 ) in DMSO d 6 ................ 89 3 4 Antiproteolytic activity of Abu containing cyclic depsipeptides from marine cyanobacte ria ................................ ................................ ................................ ..... 92 3 5 Non inflammatory elastase inducible genes ................................ ....................... 93 3 6 Relevant genes involved in NOD and MAPK signaling pathways signifi cantly modulated by elastase ................................ ................................ .... 94 3 7 Symplostatin 5 ( 1 ) inducible genes potentially independent of elastase ............. 95 3 8 Reaction condi tions for protease assays ................................ ............................ 96 3 9 Crystallography data and refinement statistics ................................ ................... 99 4 1 NMR data for veraguamide A ( 7 ) in CDCl 3 ................................ ....................... 121 4 2 NMR data for veraguamide B ( 8 ) and veraguamide C ( 9 ) in CDCl 3 .................. 123 4 3 NMR data for veraguamide D ( 10 ) and veraguamide E ( 11 ) in CDCl 3 .............. 125 4 4 NMR data for veraguamide F ( 12 ) in CDCl 3 ................................ ..................... 127 4 5 NMR data for veraguamide G ( 13 ) and tetrahydroveraguamide A ( 14 ) in CDCl 3 ................................ ................................ ................................ ................ 129 4 6 Antiproliferative activity (IC 50 M) of natural and semisynthetic veraguamides 131 5 1 NMR data of cayl obolide B ( 18 ) in DMSO d 6 ................................ .................... 155 5 2 NMR data of amantelide A ( 19 ) and amantelide B ( 20 ) in DMSO d 6 ................ 157 5 3 Cytotoxic activity (IC 5 0 M) of the isolated cyanobacterial polyketides ( 18 21 ) ................................ ................................ ................................ .................... 159

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11 LIST OF FIGURES Figure page 1 1 Representative examples of natural products that inf luenced modern medicine ................................ ................................ ................................ ............. 30 1 2 Marine natural products and analogs that have reached the clinic ..................... 31 1 3 The linear peptides sympl ostatin 1 and dolastatin 10 are potent antiproliferative agents that disrupt tubulin polymerization ................................ 32 1 4 Largazole is a cyclodepsipeptide prodrug that targets canonical histone deacetylases ................................ ................................ ................................ ....... 33 1 5 Representative examples of non cytotoxic metabolites from marine cyanobacteria that target proteases ................................ ................................ ... 34 2 1 Summary of ch emical space and bioactivity profiles of Symploca spp. and Phormidium spp. collections ................................ ................................ ............... 44 2 2 Representative HPLC MS profile of the simultaneous monitoring of largazole, dolastatin 10 and symp lostatin 1 ................................ ................................ ........ 45 2 3 Prioritization scheme of cyanobacteria collections and the corresponding secondary metabolites isolated ................................ ................................ .......... 46 3 1 Elastase inhibitors from marine cyanobacteria and the clinically approved human neutrophil elastase inhibitor sivelestat ................................ .................... 78 3 2 Selectivity profile of Abu containing cyclic depsipeptides from marine cyanobacteria ................................ ................................ ................................ ..... 7 9 3 3 Cocrystal structures of natural cyclic depsipeptide elastase inhibitors ............... 80 3 4 Changes in cell viab ility and caspase activation mediated by elastase and effects of inhibitors ................................ ................................ .............................. 81 3 5 Elastase acts as a sheddase and promotes cell morphology change and desquamation ................................ ................................ ................................ ..... 82 3 6 Elastase caused a global change in transcript levels via, in part, an NF B dependent pathway ................................ ................................ ............................ 83 4 1 Structures of veraguamides A G ( 7 13 ) and the se misynthetic tetrahydroveraguamide A ( 14 ) ................................ ................................ .......... 117

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12 4 2 MS/MS fragmentation of veraguamide A ( 7 ), veraguamide D ( 10 ), and veraguamide E ( 11 ) ................................ ................................ .......................... 118 4 3 Assignment of absolute configuration of veraguamide A ( 7 ) using ................................ ............. 119 4 4 Cell cycle analysis of HT29 and HeLa cells treated with vary ing concentrations of veraguamide D ( 10 ) ................................ .............................. 120 5 1 Caylobolide B ( 18 ) and closely related compound caylobolide A ..................... 147 5 2 Key HSQC TOCSY correlations for caylobolide B ( 18 ) ................................ .... 148 5 3 ESI MS/MS of caylobolide B ( 18 ) ................................ ................................ ..... 149 5 4 Amantelides A and B ( 19 20 ) and the s emisynthetic derivative peracetylated amantelide A ( 21 ) ................................ ................................ ............................. 150 5 5 Partial structure of amantelide A ( 19 ) derived from NMR experiments in DMSO d 6 ................................ ................................ ................................ .......... 151 5 6 ESI MS/MS fragmentation of amantelide A ( 19 ) ................................ ............... 152 5 7 Assignment of relative configuration of caylobolide B ( 18 Universal NMR Database (Database 2) ................................ ........................... 153 5 8 Time course antiproliferative activities of amantelide A ( 19 ) and amphotericin B against cancer cells ................................ ................................ ....................... 154

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13 LIST OF ABBREVIATIONS Angstrom 20 D Speci fic optical rotation Abu 2 amino 2 butenoic acid Ac A cetyl Ahp 3 amino 6 hydroxy 2 piperidone Ala Alanine ANOVA A nalysis of variance Arg Arginine APCI/ESI Atmospheric pressure chemical ionization/electrospray ionization A RID1B AT rich interactive domain 1B Asp Aspartic acid BCA B icinchoninic acid BEAS 2B Bronchial epithelial cell line BEBM B ro nchial epithelial basal medium Br Hmoya 8 bromo 3 hydroxy 2 methyl 7 octynoic acid br q Broad quartet c Concentration in g/100 mL 13 C NMR Carbon 13 nuclear magneti c resonance spectroscopy calcd Calculated CDCl 3 Deuterated chloroform cDNA Complementary deoxyribonucleic acid CE Collision energy CEP Collision cell entrance potential CH 2 Cl 2 Methylene chloride

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14 CH 3 Methyl C=O Carbonyl COPD C hronic obstructive pulmonary di sease COSY Correlation spectroscopy CrO 3 Chromium trioxide CSNK1A Casein kinase 1, alpha CUR Curtain gas CuSO 4 Copper (II) sulfate CXP Collision cell exit potential 1D One dimensional 2D Two dimensional Chemical shift (in ppm) d Doublet D Configurational descriptor (Fisher system) dd Doublet of doublets dt Doublet of triplets DAP3 Death associated protein 3 DDIT4 DNA damage inducible transcript 4 Dhoya 2,2 dimethyl 3 hydroxy 7 octynoic acid DP Decluste ring potential DMEM DMSO Dimethyl sulfoxide DMSO d 6 Deuterated dimethyl sulfoxide ELISA Enzyme linked immunosorbent assay EP Entrance potential

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15 ESIMS Electrospray ionization mass spectrometry EtOAc Ethyl acetate EtOH Ethano l FACS Fluorescence activated cell sorting FBS Fetal bovine serum g Gravity g Gram GAPDH G lyceraldehyde 3 phosphate dehydrogenase GAS1 Growth arrest specific 1 Gln Glutamine Glu Glutamic acid Gly Glycine GS1 Gas 1 GS2 Gas 2 h Hour H 2 Hydrogen gas HCl Hydro chloric acid HCOOH Formic acid HDAC Histone deacetylase 1 H NMR Proton nuclear magnetic resonance spectroscopy Hiva 2 hydroxyisovaleric acid HMBC Heteronuclear multiple bond correlation spectroscopy Hmoaa 3 hydroxy 2 methyl 7 octa noic acid Hmoea 3 hydroxy 2 methyl 7 octe noic acid Hmoya 3 hydroxy 2 methyl 7 octynoic acid

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16 Hmpa 2 hydroxy 3 methylpentanoic acid HNE Human neutrophil elastase HPLC MS Tandem h igh pressure liquid chromatography mass spectrometry HPLC UV Tandem h igh pressure liquid chromatography ult raviolet spectroscopy HRESIMS High resolution electrospray ionization mass spectrometry HSQC Heteronuclear single quantum correlation spectroscopy IC 50 Half maximal inhibitory concentration ICAM 1 I nt ercellular adhesion molecule 1 IFN I NF IL1A Interleukin 1A IL1B Interleukin 1B IL1R1 I nterleukin receptor, typ e 1 IL8 Interleukin 8 Ile Isoleucine i PrOH Isopropanol IS Ionspray voltage IVT In vitro transcription n J Coupling constants via n bonds max Wavelength maximum LRESIM S Low resolution electrospray ionization mass spectrometry m Meter m multiplet (NMR) M Molar

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17 MALDI TOF Matrix assisted laser desorption/ionization time of flight mass spectrometry MAP2K5 M itogen act ivated protein kinase kinase 5 MAPK M i togen activated prot ein kinase MeCN Acetonitrile MeOH Methanol MHz Megahertz mICAM 1 M embrane bound int ercellular adhesion molecule 1 min Minute MRM M ultiple reaction monitoring MTPA Methoxy(trifluorophenyl)phenylacetic acid MTT 3 (4,5 Dimethylthiazol 2 yl) 2,5 diphenyltetraz olium bromide MyD88 M yeloid differentiation pr imary response gene (88) Na Sodium n BuOH n butanol NH 4 OAc Ammonium acetate nM Nanomolar N Me Ile N methyl isoleucine N Me Phe N methyl p henylalanine N Me Val N methyl valine NF B Nuclear factor kappa B NFIB Nuclear factor I/B NOD N ucleotide binding oligomerization domain OMe Methoxy PAR Proteinase activated receptor PDB ID Protein Databank Identification

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18 Pd Palladium PECAM P latelet endo thelial cell adhesion molecule Phe Phenylalanine Pla Phenyllactic acid PPE Porcine pancreatic elastase PTK2 P rotein tyro sine kinase 2 PVDF Polyvinylidene difluoride RBM14 RNA binding motif protein 14 RNA Ribonucleic acid RT qPCR Reverse transcription followed by quantitative polymerase chain reaction s singlet SAR Structure acti vity relationship SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Ser Serine sICAM 1 S oluble int ercellular adhesion molecule 1 SIK2 Salt inducible kinase 2 SV 40 Simian vacuolating virus 40 t R Retention time TEM Temperature Thr Threonine TNF Tumor necrosis factor TOCSY Total correlation spectroscopy M Micromolar Val Valine VCAM V ascular cell adhesion protein

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19 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DRUG DISCOVERY FROM MARINE CYANOBACTERIA S YMPLOCA SPP A ND P HORMIDIUM SPP .: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY METABOLITES By Lilibeth Apo Salvador May 2013 Chair: Hendrik Luesch Ma jor: Pharmaceutical Science s Medicinal Chemistry Four marine cyanobacteria col lections were prioritized for the discovery of novel secondary metabolites, based on their antiproliferative activity against HT29 human colorectal adenocarcinoma cells and un ique HPLC MS dereplication profile s Bioactivity and 1 H NMR directed purification yielded the elastase inhibitors symplostatins 5 10 ( 1 6 ), a nd the antiproliferative agents veraguamides A G ( 7 1 3 ), caylobolide B ( 18 ), and amantelides A and B ( 19 20 ). Tot al structure elucidation was done using 1D and 2D NMR spectroscopy, mass spectrometry and enantioselective analysis. Symplostatins 5 10 ( 1 6 ) are cyclic depsipeptides bearing the modified amino acids 3 amino 6 hydroxy 2 piperidone and 2 amino 2 butenoic ac id. Comprehensive protease profiling of 1 indicated potent and selective elastase inhibition. Structure activity relationship (SAR) studies on 1 6 together with the related compounds lyngbyastatins 4 and 7, identified critical and tunable structural eleme nts. This was corroborated by the X ray cocrystal structure of lyngbyastatin 7 porcine pancreatic elastase. The effects of symplostatin 5 ( 1 ) on the downstream cellular effects of

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2 0 elastase was probed using an epithelial lung airway model system. Compound 1 attenuated elastase mediated receptor activation, proteolytic processing o f adhesion molecule ICAM 1, NF B activation and global tran sc riptome changes, leading to cytoprotection against elastase induced cell death, detachment and inflammation. Veraguamid es A G ( 7 13 ) are cyclic hexadepsipeptides bearing a C 8 polyke tide hydroxy acid, an invariant proline residue, multiple N methylated amino acids hydroxy acid. Compounds 7 13 together with the semisynthetic derivative tetrahydroveraguamide A ( 14 ) displayed weak to moderate antiproliferative act ivity against HeLa cervical carcinoma and HT29 cell s, modulated by several sensitive positions in the veraguamide scaffold. Flow cytometry indicated that veraguamide D ( 10 ) caused a dose dependent increase in cell populations at sub G1 and G2. Caylobolide B ( 1 8 ) and amantelides A and B ( 19 20 ) are structurally related polyketides characterized by a polyhydroxylated macrolactone ring bearing an alkyl pendant side chain. Amantelide A ( 19 ) displayed sub micromolar IC 50 s against HT29 and HeLa cells, while 18 and 2 0 showed weaker a ctivity These cyanobacterial polyketides potentially exert their cytotoxic effect through interaction with the cell membrane

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21 CHAPTER 1 GENERAL INTRODUCTION Natural Products in Drug Discovery Natural products are small molecules, typical ly less than 2,000 Da in size, produced by terrestrial and marine macro and microorganisms via enzymatically assisted biosynthesis. Also referred to as secondary metabolites, these compounds have indirect and specialized function in the survival of produc ing organisms, but are deemed nonessential in primary metabolic pathways. Natural products have evolved out of functional necessity and are regarded to act as chemical defenses against predators, parasites or diseases and may also fulfill intrinsic physiol ogical functions for the producing organisms 1 Similar to primary metabolites, natural products are derived from ubiquitous precursor molecules such as acetyl CoA and proteinogenic amino acids, but differ from th e latter by being species specific, rather than prevalent across organisms. 1, 2 And while primary and secondary metabolites utilize the same precursor molecules, higher structural d iversity is observed in the latter due to the involvement of evolutionary processes in the elaboration of biosynthetic enzymes of secondary metabolites. 1 Natural products are distinguished by the presence of a la rge number of ring systems, functionalized mainly by oxygen and hydrogen bonding donor moieties. 3 An unprecedented feature of secondary metabolites is sterical complexity possessing a high number of stereoc enters as these compounds are produc ts of and target three dimensional protein systems. 3 Comparison of natural products and synthetics indicated that these compounds occupy complementary chemical spaces. 3 Secondary metabolites are also able to bind to different unrelated molecular targets and are thus,

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22 regarded as privileged structures. 4 Hence, natural products repres ent structurally diverse compounds which have been evolutionary optimized to their molecular targets. It is for these reasons that man has relied on Nature to discover new drugs The earliest documented use of purified secondary metabolites as therapeutic s dates back to the early 19 th century, with the discovery of morphine from opium poppy for the alleviation of pain. 5 Several centuries later, natural products continue to be recognized as a validated source of ne w drugs and regarded as one of the most successful strategy in the development of small molecule therapeutics. In a survey of agents introduced for clinical use from 1980 2010, ~50% are derived from natural products 6 The secondary metabolite itself may not be the final drug entity, but rather serve as template for the design of best in class small molecule therapeutics. The majority of these are anti infectives and anticancer agents. 6 Examples of these are the antibiotic penicillin, antimalarials quinine and artemisinin, and antimitotics vinblastine and paclitaxel (Figure 1 1). Drugs from the Sea Terrestrial plants and microorganisms have been the traditional source of natural p roducts. Technological advancements in underwater exploration have paved the way for the utilization of marine organisms as source organisms in drug discovery. 7 Oceans cover the e and harbor rich biodiversity. Each milliliter of seawater is estimated to contain millions of viruses and bacteria, together with thousands of fungi and microalgae. 8, 9 Complex ecological relations hip also exists in these environments, such as endosymbiosis, 10 and there is intense competition for space. These ecological factors can then be expected to imp act the secondary metabolite production in marine organisms. 9

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23 Six marine natural products or their derivatives have successfully reached the clinic and several more at different stages of clinical trials. 7 11 C linically appro ved marine natural products include ziconotide for chronic pain management, antiviral agent vidarabine conceived based on spongouridine from the sponge Tethya crypta and the anticancer agents cytarabine, an anal og of the spongothymidine also from the sponge Tethya crypta ecteinas c idin 743 (ET 743), eribulin mesylate inspired by the sponge compound halichondrin B and brentu x imab vedotin designed based on the sea hare/cyanobacterial metabolite dolastatin 10 (Figur e 1 2 ) Ziconotide is a linear polycationic peptide conotoxin from the cone snail Conus magus characterized by 25 amino acid residues including six Cys, that forms three disulfide linkages (Figure 1 2) 12 This compo und is u tilized by the source organism to immobilize its prey, and in mammalian system targets N type voltage sensitive calcium channels. 12 ET 743 from the sea squirt Ecteinascidia turbinata was approved for use in the European Union for refractory soft tissue sarcoma. The core structure of ET 743 (Figure 1 2) consists of fused tetrahydroisoquinoline rings that are deemed essential in binding to and covalently modifying DNA. 13 Th e clinically approved agent eribulin mesylate for breast cancer treatment is a truncated version of halichondrin B (Figure 1 2) 14 Halichondrin B was initially isolated from the sponge Halichondria okadai and subsequ ently from several more sponge species such as Axinella and Phakellia carteri 15 Halichondrin B binds to the Vinca domain of tubulin. 16 The low yield and high structural complexit y of halinchondrin B limited its clinical development. Simplified analogs of halichondrin B, as in the case of eribulin mesylate, showed similar bioactivities as the natural product and tapped for drug development. 14 Brentu x imab vedotin is an antibody drug conjugate

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24 Brentu x imab vedotin consists of a CD33 targeting antibody, a cathepsin cleavable linker and the drug monomethyl auristatin E ( Figure 1 2) 17 Monomethyl auristatin E is an analog of the sea hare/cyanobacterial metabolite dolastatin 10 (Figure 1 2) which also binds to and disr upt microtubule proteins 18 Mar i ne Cyanobacteria: Source Organism s of Novel Molecules Cyanobacteria or blue green algae are primitive organisms that have existed for billions of years, despite lacking any morphological defense structures such as spines, spicules or shell. Thus, these pr imitive prokaryotic organism s are thought to have evolved an arsenal of bioweapons for chemical defense. Since the pioneering studies of Professor Richard Moore, close to 1 000 secondary metabolites have been isolated from these organisms. 19 22 Marine cyanobacteria utilize polyketide synthases, nonribosomal peptide synthetases and hybrids of these two biosynthetic pathways to produce diver se secondary metabolites. 23 The m ajority of these were isolated from the genera Lyngbya Oscillatoria Phormidium and Symploca The complex ecological relationship among marine organisms and the production of secon dary metabolites can be observed in cyanobacteria as the true producers of bioactive natural products isolated from mollusks and ascidians. Sea hare derived dolastatins 10 15 were originally isolated from these herbivores in low quantities. 24 For example, 1 mg o f dolastatin 10 required 2 ton s of sea hare. 24 A c omparable amount of dolastatin 10 was isolated from a Guamanian Symploca sp., and required only 5 g of drie d cyanobacteria. 25 The significantly enriched amounts of dolastatin 10 together with the isolation of closely related compounds and other sea hare derived metabolites from marine cyanobacteria indicated that the t rue producers are marine cyanobacteria and

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25 are acquired by these herbivores through their diet. 26 Cyanobacteria have also been demonstrated to affect secondary metabolite production of other marine organisms through e ndosymbiosis. For example, the production of patellamides by the Didemnidae family of tunicate is dependent on the obligate cyanobacterial symbiont Prochloron spp 27 It is then estimated that 35% of marine derive d anticancer agents are products of cyanobacteria, based on structural similarity. 21 The production of natural products from microbes and microbe interaction with the host organism where the compound was isolated ha s emerged as a pivotal concept in natural products discovery. 6,8, 10 Mechanism of Action of Bioactive Cyanobacterial Metabolites Marine cyanobacteria are well documented to be prol ific produ cers of antiproliferative agents 21 The m ajority of these are actin and tubulin poisons, with the marine cyanobacteria Symploca sp. being the source organism s of the potent tubulin poisons dolastatin 10 and symplostatin 1 24,25,28 29 In addition secondary metabolites with atypical and remarkable m echanisms of ac tion have also been isolated from this marine cyanobacteria genus, such as largazole which inactivates histone deacetylases (HDACs) 30 Protease inhibition is perhaps the major theme among marine cyanobacterial metabolites and are commonly encountered in va rious genera 21 Interference with Microtubule Dynamics Dolastatin 10 and symplostatin 1 are closely related linear pentapeptides characterized by modified amino acids dolaphenine, dolaproline, and dolaisoleucine, together with Val and a terminal N N dime thylated amino acid (Figure 1 3 ). 24,25, 28 Dolastatin 10 and symplostatin 1 are d ifferentiated by their N terminal amino acid residue, N N dimethylVal and N N dimethylIle, respectively (Figure 1 3) These compounds were both demonstrated to have broad spectrum cytotoxicity towards an

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26 array of cancer cell lines, with pico to nanomolar IC 50 s. 29 A dose dependent incre ase in cell populations at G2 and concomitant formation of abnormal mitotic spindles were observed in sym plostatin 1 and dolastatin 10 treated cells. A10 and HeLa cells treated with symplostatin 1 had disrupted cellular microtubule network as evidenced by immunostaining using monoclon tubulin antibody. 29 Dolastatin 10 and symplostatin 1 were both shown to directly interact with tubulin, with the former demonstrated to inhibit the binding of radiolabeled Vinca alkaloid. 18, 29 Molecular docking experiments proposed that dolastatin 10 binds to a distinct region, close to the Vinca domain and inhibited tubulin dependent GTP hydrolysis and nucleotide exchange, processes that are crucial for tubulin assembly. 3 1 Sy mplostatin 1 retarded the growth of colon adenocarcinoma 38 and mammary adenocarcinoma 16/C cells in vivo at dosages of 0.25 1.25 mg/kg. 29 Symplostatin 1, however, caused tissue damage at the site of injec tion and test animals showed 3 1 5% body weight loss, depending on the dosing schedule. 29 Dolastatin 10 reached Phase II clinical trials for prostrate cancer treatment but was discontinued due to observed peripheral neuropathy among patients and weak therapeutic activity as a single agent. 3 2 Several analogs of dolastatin 10 were synthesized to impr ove the in vivo potency and safety profile. On August 2011, FDA approved a dolastatin 10 analog, monomethyl a uristatin E conjugated to a CD33 lymphoma and anaplastic large cell lymphoma treatment. 17 Inhibition of Histone Deacetylase Largazole is a cyclic depsipeptide that is characterized by several unique structural features such as a 4 R methylthiazoline that is fused to a thiazole ring, and a 3 S hydroxy 7 mercapto 4 hept enoic acid linked to an n octanoyl group that serves as

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27 the prodrug moiety (Figure 1 4 ). 3 0 33 Largazole requires a protein assisted hydrolysis to liberate the active species largazole thiol. 34, 35 Cytotoxicity testing showed potent activity against cancer cell lines with superior selectivity index. 3 0 Largazole is the first marine cyanobacteria derived agent demonstrated to target HDACs, with superior class I isoform selectivity. 34 The m ajority of known HDAC inhibitors were derived from terrestrial microorganisms. 36 The reported co crystal structure of largazole and HDAC8 2+ catalytic ion in a tetrahedral arrangement. 37 This optimum interaction is facilitated by the rigid depsipeptide macrocyle arising from the fused thiazole thiazoline rings. NCI60 screening on largazole showed particular susceptibility of colon cancer cell lines to treatment and an HCT116 xenograft mouse model was adopted. 35 In this in vivo animal model, largazole did not show significant toxic effects and was well tolerated. Largazole was able to retard tumor growth in test animals compared to control group, and caused an upre gulation of the cyclin dependent kinase inhibitor p15 and pro apoptotic effector caspase 3, while prosurvival proteins HER2, cyclin D1, IRS 1, and pAKT were downregulated in tumor sections. 35 Inhibition of Proteases From marine cyanobacteria, s everal non cytotoxic metabolites have been demonstrated to be potent protease inhibitors, particularly targeting the serine proteases elastase, chymotrypsin and trypsin. 21,38, 39 The macrocycle of these cyanobacterial serine protease inhibitors is distinguished by an N methylated aromatic amino acid residue, a small nonpolar amino acid such as Val or Ile and a characteristic ester linkage formed by the condensation of the secondary hydroxy group of Thr. The Thr residue is also modified on its N terminus by one to three amino acid residues, and

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28 capped by a terminal fatty or polar acid such as butanoic, hexanoic or glyceric acid, giving rise to the pendant side chain of these cyclic depsipeptides. Lyngbyastatins 4 10 (Figure 1 5 ) and the related compounds so mamide B and molassamide, which bear a modified Th r residue 2 amino 2 butenoic acid adjacent to the Ahp residue on the N terminal showe d potent elastase inhibition. 40 44 A related compound kempopeptin A from a Lyngbya sp. collection bears a Leu residue instead of Abu, and potently inhibited elastase and chymotrypsin (Figure 1 5 ) 45 Its analog kempopeptin B (Figure 1 5 ) bearing a Lys residue inhibited trypsin. 45 Thus, it is evident that the residue on the N terminal side of the Ahp moiety modulates the activity of these inhibitors for different serine proteases. 38,41, 46 These serine protease inhibitors have been demonstrated to function as digestion inhibitors and feeding deterrents of herbivores, fishes and urchins and may also possibly modulate the biosynthesis of other cyanobacterial secondary metabolites 47 49 amino hydroxy acid) containing modified linear peptides from marine cyanobacteria on the other hand, are potent inhibitors of aspartic proteases. Grassystatins A C (Figure 1 5 ) isolated from a Floridian Lyngbya cf. confervoides selectively inh ibited the aspartic protease cathepsin E at pico to nanomolar concentrations and concurrently prevented cathepsin E mediated antigen presentation of dendritic cells. 50 These c ompounds bear a leucine derived statin e unit (4 amino 3 hydroxy 6 methylheptanoic acid), critical for cathepsin inhibition whil e residues adjacent to this moiety confer selectivity towards cathepsin E The related linear peptide, tasiamide B (Figure 1 5 ) 51, 52 on the other hand bears a phenylalanine derived statine site APP Cleaving Enzyme Type 1

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29 (BACE1), an enzyme which has been shown to be central to the formation of amyloid plaques a 53 Tasiamide B served as the template in the d esign of new inhibitors of BACE 1 with potent cellular activity and in vivo efficacy. 53 Objective s and Specific Aims of the Study Wi th marine cyanobacteria being validated source organism s of structurally and pharmacologically diverse secondary metabolites, we aimed to utilize novel chemical entities from these organisms for potentia l biomedical applications as antitumor agents and modulators of elastase mediated pathologies This study focused on the under explored marine cyanobacteria genera of Symploca and Phormidium which yielded several of the best in class antitumor agents. Thi s study aimed to: 1. Prioritize collection s of Symploca and Phormidium using a preliminary profiling of bioactivity and ch emical space 2. Perform a bioactivity guided purification on cyanobacterial collections which demonstrated antiproliferative activity to iso late the bioactive constituent(s) 3. Perform a 1 H NMR guided purification to discover novel secondary metabolites from non cytotoxic cyanobacterial collections 4. Determine the structure of isolated compounds from prioritized collections using combinations of sp ectroscopic techniques such as 1D and 2D NMR spectroscopy and mass spectrometry 5. Elucidate the biological activity and mechanism s of action of identified cyanobacterial secondary metabolites in mammalian cellular systems.

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30 Figure 1 1 Representative exam ples of natural products that influenced modern medicine.

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31 Figure 1 2. Marine natural products and analogs that have reached the clinic.

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32 Figure 1 3. The linear peptides symplostatin 1 and dolastatin 10 are potent antiproliferative agents that disrupt tubulin polymerization.

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33 Figure 1 4. Largazole is a cyclodepsipeptide prodrug that targets canonical histone deacetylases.

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34 Figure 1 5. Representative examples of non cytotoxic metabolites from marine cyanobacteria that target proteases.

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35 CHAPTER 2 PROBING THE CHEMI CAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF CYANOBACTERIAL COLLECTIONS Introduction Filamentou s marine cyanobacteria are a validated source of antiproliferative agents, having yielded several of the best in class inhibitors of malignan cies. 20, 21 Cytotoxins from ma rine cyanobacteria also display not just a variety in structure, but mechanism s of action as well. A ctin targeting agents, with sub nanomolar IC 50 s against cancer cells, include lyngbyabellins, 54 56 dolastatin 11 57, 58 and hectochlorin. 59 T he marine cyanobacteria Lyngbya spp. afforded the cyclic depsipeptides apratoxin s A G that are also potent cytotoxins 60 64 with apratoxin A prevent ing cotra nslational translocation leading to downregulation of receptors and growth factor ligands. 65, 66 The marine cyanobacteria Sympl oca spp. and Phormidium spp. yielded several modified linear peptides that target tubulin polymerization. 25, 28,29,67, 68 The most potent among these are the related dolastatin 10 18 and symplostatin 1, 29 with the former serving as the template for the design of the clinically appr oved anti cell lymphoma drug brentu x imab vedotin. Another novel agent from Symploca sp. is the histone deacetylase inhibitor largazole, which displayed potent activity in preclinical evaluations. 33 With the abundance of novel antitumor agents from marine cyanobacteria, it is thus attractive to employ a primary screening of antiproliferative activity against cancer cells for crude extracts. Measurements of cell viability can be done using colorimetric or fluorometric reagents to measure cellular metabolism, protein activity and interactions, membrane permeabilization and cellular respiration. 36 Reproduced with permission from J. Nat. Prod ., submitted for publication. Unpublished work copyright 2013 American Chemical Society.

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36 The isolation of a large number of antiprolife rative agents from marine cyanobacteria, however, also increases the possibility of reisolating known compounds as bioactive components. Thus, it is advantageous to employ a screen of the chemical space as well. Sev eral dereplication methods identificati on of known metabolites from sample collections with the least effort and resources have been developed for both terrestrial and marine cyanobacteria, employing UV spe ctroscopy and mass spectrometry. To distinguish known bioactive compounds in a screen f or phorbol debutyrate receptor binding activity a HPLC UV dereplication was utilized. 69 Members of the aplysiatoxin class of compound are known to be phorbol debutyrate receptor binders, and comparison of the re tention time and UV profile of authentic debromoaplysiatoxin allowed the identification of this compound as the active principle for several Lyngbya majuscula collections. 69 This method also accounted for debromo aplysiatoxin as the bioactive constituent of seagrasses and macroalgae, possibly due to cyanobacterial contamination. 69 More compound specific techniques emerged with the development of new technologies in mass s pectro metry such as MALDI TOF and ESI MS. The initial utilization of MALDI TOF for dereplication was a serendipitous discovery, but nonetheless, demonstrated the presence of microcystins, micropeptin and anabaenopeptolin from collections of Microcystis Ana baena and Oscillatoria 70 The application of MALDI TOF for dereplication has been extended to determine the spatial distribution of secondary metabolites in cyanobacteria themselves and other marine organisms, in a ddition to identification. 71 Structure determination of nonribosomal peptides have also greatly benefited from mass spectrometry, with tandem mass

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37 spectrometry yielding the identity of these compounds via chara cteristic fragmentation pattern. Recently introduced is comparative dereplication using tandem mass spectrometry and spectral alignment algorithms to identify identical compounds and related analogs. 72 The requirement f or minimal material to perform mass spectrometry analysis and its amenability to high throughput format makes this method an attractive choice for dereplication. Here, an HPLC MS dereplication method utilizing multiple rea ction monitoring was developed to improve the resolution of known cytotoxins in collections of marine cyanobacteria Symploca and Phormidium This, together with antiproliferative screening against HT29 col orectal adenocarcinoma cells was utilized to prioritize cyanobacterial collections f or further studies. Screening of Cyanobacteria Collection s A total of 38 marine cyanobacteria samples were collected in Florida, Guam and the U S Virgin Islands from 2007 2009. These collections were mainly Symploca spp., Phormidium spp. and several taxonom ically unidentified organisms characterized by puffy ball gross morphology characteristic for Symploca sp p Collected organisms were lyophilized and extracted with either CH 2 Cl 2 MeOH (1:1) or EtOAc MeOH (1:1) to yield the nonpolar extracts These extracts were further subjected to a C18 solid phase extraction (SPE) c leanup using a MeOH H 2 O elution. Initial elution using 25% MeOH removed the majority of the salts and ensured minimal non specific bio activity and interference in HPLC MS arisi ng from these pola r compounds. The fraction collected from 100% MeOH elution was tested for antiproliferative activity against HT29 colorectal adenocarcinoma cells and concurrentl y profiled by HPLC MS

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38 Antiproliferative Assay as Preliminary Screening for Bioactivity Antipr oliferative activity was assessed based on the fractional survival of HT29 cancer cells detected using the MTT reagent Extracts which caused < 60% survival of HT29 cells were considered bioactive at the specified concentration From the 38 samples screen ed for antiprolifer ative activity, only two sample collections were inactive at all concentrations tested (Figure 2 1 A) Thirteen sample collections exhibited moderate antiproliferative activity against HT29 cells at concentrations of 1 ,000 and 1 0,000 n g/m L (Figure 2 1 A) The remaining 60% of the screened cyanobacteria collections exhibited antiproliferative activity at concentrations of 10 and 100 ng/mL (Figure 2 1 A) With the large number of cyanobacterial collections showing antiproliferative activity, a dditional information for prioritization of sample collections are needed. Also, with potent cytotoxins such as dolastatin 10, symplostatin 1 and largazole being produced by Symploca s p p. and Phormidium spp. collections, determin ation of the contribution o f these known compounds to the bioactivity should be assessed at an early stage of the discovery process Dereplication using an HPLC MS Approach The dereplication method for the known compounds largazole, dolastatin 10 and symplostatin 1, consisted of a g radient HPLC run using CH 3 CN H 2 O (+ 0.1% HCOOH) and multiple reaction monitoring (MRM) as MS detection mode. This allowed for sensitive, specific and high throughput fo rmat for dereplication of previously isolated metabolites from Symploca spp. and Phormid ium sp p. sample collections The MRM mode relies on the de tection of both the parent ion mass (Q1) and a specific daughter ion resulti ng from fragmentation (Q3), giving a significant reduction in background, improvement in signal to noise ratio and limits of detection. This dereplication format

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39 permitted automation, short run times per sample (< 20 min) and simultaneous monitoring of largazole, symplostatin 1 and dolastatin 10 (Figure 2 2). This method does not have specific structural requirements and can be done using commonly available mass spectrometers. However, authentic standards are needed for optimization of the HPLC MS parameters. Since MRM is also a compound specific detection, no information on the presence of related congeners may be derived usi ng this method. Based on the HPLC MS dereplication, the majority of the sample collections with antiproliferative activity at 10 and 100 ng/mL contained combinations of dolastatin 10, largazole or symplostatin 1 (Figure 2 1 A, B). Except for one sample coll ection, all other bioactive cyanobacterial collection at concentration of 10 ng/mL contained these three antiproliferative agents at biologically relev ant concentrations (Figure 2 1 A). Extracts containing symplostatin 1 or dolastatin 10 alone or lower conc entrations of these metabolites in combination showed activity at a higher concentration of 100 ng/mL. Interestingly, largazole was consistently detected in combination with dolastatin 10 and symplostatin 1, whenever present (F igure 2 1 A, B). Samples witho ut detectable levels of largazole, symplostatin 1 or dolastatin 10 showed varied antiproliferative activity and thus presented as prioritized candidates for both bioactivity and 1 H NMR guided purification (Figure 2 3 ) Prioritization of Sample Collections The bioactivity data together with the dereplication results and available material of the cyanobacteria collection were considered in the prioritization of sample collections for further purification (Figure 2 3) Bioactive c ollections at concentration s < 10 ,000 n g/mL with sufficient amounts of lyophilized cyanobacteria and/or nonpolar extract were g iven highest priority Non cytotoxic or weakly cytotoxic samples were

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40 further subjected to a silica SPE and 1 H NMR profiling to check for rele vant functionali ties such as N CH 3 O CH 3 hydrogens. From the 38 profiled cyanobacteria collections, four priority samples were further pursued (Figure 2 3) A bioactivity guided purification was und ertaken to obtain the antiproliferative agent (s), while cyano bacteria collections with weak cytotoxic activity were purified via a 1 H NMR guided purification. Validation of the Dereplication Method To validate our current HPLC MS dereplication method, l argazole and dolastatin 10 wer e isolated from a Symploca sp. co llection from Pickles Reef in Florida usin g a HPLC MS guided purification. Monitoring by HPLC MS requir ed minimal amounts of sample, while still permitting sensitive detection. Using this approach, sub m illigram quantitie s of largazole and dolastatin 10 we re isolated The identities of the purified compounds were verified using 1 H NMR and LRESIMS measurements, and comparison with literature values (Appendix F). The isolation of symplostatin 1 is presented in Chapter 5. The antiproliferative activities of th e purified largazole and dolastatin 10 again s t HeLa human cervica l adenocarcinoma, HCT116 human colorectal carcinoma and HT29 cells were also tested and in accordance with the literature values (Table 2 1) Con clusion The bioactivity and chemical space of crude extracts of 38 cyanobacterial collections belonging mainly to Phormidium and Symploca cyanobacteria genera, were screened using the MTT cell viability assay and HPLC MS based derep lication method, respectively. The m ajority of the screened cyanobac terial collections with potent bioactivity contained combinations of the cytotoxins largazole, do lastatin 10 and

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41 symplostatin 1. These compounds were rapidly identified as the major cytotoxic consti tuent th rough comparison of the HPLC MS profiles with auth entic standards using multiple reaction monitoring The dereplication method wa s further validated using large scale isolation from a dolastatin 10 and largazole containing cyanobacterial collection. By combining dereplication information, antiproliferat ive activity prof ile s against HT29 cancer cells, availability of material and /or initial 1 H NMR profile, four cyanobateria collections were prioritized for the discovery of novel bioactive secondary metabolites. Experimental Methods General Experimental Pr ocedures 1 H NMR spectra were recorded in CDCl 3 or CD 2 Cl 2 on a Bruker Avance II 600 MHz spectrometer equipped with a 5 mm TXI cryogenic probe using residual solvent signals [(CDCl 3 : H 7.26), (CD 2 Cl 2 : H 5.32 )] as internal standards. LRESIMS measurements, MRM analysis and MS/MS fragmentation were done on an ABI 3200Q TRAP. Biological Material Symploca spp. or Phormidium spp. cyanobacteria collections were collected by hand at various sites in Guam, Florida, and the US Virgin Islands. Samples were kept froze n at 20 C after collection. A voucher specimen, which is preserved in 100% EtOH or formaldehyde, is deposited in the University of Guam Herbarium and at the Smithsonian Marine Station, Fort Pierce, FL. Frozen cyanobacteri a samples were lyophilized prior to extraction. The freeze dried cyanobacteri a were extracted with EtOAc MeOH (1:1) or CH 2 Cl 2 MeOH (1:1) to yield the nonpolar extract s

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42 HPLC MS Profiling Nonpolar cyanobacteria extracts (10 20 mg) were purified b y a C18 SPE column using a MeOH H 2 O elution. The fraction from 100% MeOH was dried under N 2 weighed and methanolic stock solution (1 mg/mL) was prepared. A d ilution (10 ,000 n g/mL) of the stock solution was prepared in MeCN and spiked with the internal standard harmine and was used as test solution injected for HPLC MS analysis, using the following conditions: column, Kinetex (100 2.1 mm), Phenomenex; linear gradient of 0.1% HCOOH in MeCN 0.1% HCOOH in H 2 O [ 50% 100% MeCN in 10 min and then 100% MeCN for 5 min, flow rate, 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan) ] The retention times ( t R min; 7.6), lar Cell Viability Assay HT29 colorectal adenocarcin oma medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone) under a humidified environment with 5% CO 2 at 37 C. HT29 (12, 500) cells were seeded in 96 well plates These were treated with varying concentratio ns (10, 100, 1, 000, 10,000 n g /mL) of the nonpolar extract dissolved in EtOH 24 h post seeding C ells were incubated for an additional 48 h before the additi on of the MTT reagent. Cell s (Promega Madison, WI ). Antiproliferative activity of purified largazole and dolastatin 10 was determin ed using the same procedure employing the cancer cell li nes HT29 human colorectal adenocarcinoma (12,500 cells/well) HeLa human cerv ival carcinoma (3,000 cells/well ) and HCT116 human colorectal carcinoma (10,000 cells/well)

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43 Validation of Dereplication Method A Symploca sp. c ollection from Pickles Reef Florid a was lyophilized and the dried material (13.3 g) was extracted with EtOAc MeOH (1:1) to yield the nonpolar extract (1.7 g). The nonpolar extract was adsorbed o n a Diaion HP 20 resin and eluted with 100% H 2 O, 25%, 50%, 75% and 100% MeOH and 50% CH 2 Cl 2 in M eOH. Each fraction was monitore d for the presence of largazole, symplostatin 1 and dolastatin 10 using the HPLC MS method. The fraction eluting from 50% CH 2 Cl 2 (33 mg) showed peaks corresponding to largazole and dolastatin 10 and was applied onto a silica SPE column, eluting with increasing gradients of i PrOH in CH 2 Cl 2 until 100% i PrOH. The fractions eluting from 10% i PrOH and 20% i PrOH contained largazole and dolastatin 10, respectively based on HPLC MS profiling. These fractions were further purifie d by semipreparative HPLC (Phenomenex Synergi using a linear gradient of MeOH H 2 O (70% 100% MeOH in 60 min and then 100% MeOH for 15 min). The 10% i PrOH fraction yielded l argazole ( t R 41.7 min, 0.3 mg). Using the same chromatographic condition, the 20% i PrOH fractio n afforded dolastatin 10 ( t R 40.0 min, 0.2 mg). The 1 H NMR and LRESIMS of the isolated compounds were identical to th ose of the literature values. Largazole: colorless, amorphous solid; 1 H NMR spectrum is identical to that of an authentic sample, 30 see Appendix F ; LRESIMS m/z 623.0 [M + H] + Dolastatin 10: colorless, amorphous solid; 1 H NMR spectrum is identical to that of an authentic sample, 28 see Appendix F ; LRESIMS m/z 785.6 [M + H] +

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44 Figure 2 1. Summary of chemical space and bioactivity profiles of Symploca spp. and Phormidium spp. collections. (A) The majority of the cyanobacteria collections displayed antiproliferative activity against HT29 human color ectal adenocarcinoma cells as assessed using the MTT reagent. The majority of potent bioactive extracts showed combinations of dolastatin 10, largazole and symplostatin 1. (B) Distribution of the three known antiproliferative agents in profiled cyanobacter ial collections.

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45 Figure 2 2. Representative HPLC MS profile of the simultaneous monitoring of largazole, dolastatin 10 and symplostatin 1.

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46 Figure 2 3. Prioritization scheme of cyanobacteria collections and the corresponding secondary metabolites is olated.

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47 Table 2 1 Antiproliferative a ctivity (IC 50 nM) of known Symploca sp. m etabolites a Compound HT29 HCT116 HeLa Dolastatin 10 0.4 0.01 1.8 0.02 0.2 0.01 Largazole 10 0.6 7.0 1.4 12 1.1 a Data are presented as mean SD (n = 2).

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48 C HAPTER 3 POTENT ELASTASE INHIBITORS FROM CYANOBACTERIA: STRUCTURAL BASIS AND MECHANISMS MEDIATING CYTOPROTECTIVE AND ANTI INFLAMMATORY EFFECTS IN BRONCHIAL EPITHELIAL CELLS Introduction Cyanobacteria, whether of marine, terrestrial or freshwater origin, h ave consistently yielded serine protease inhibitors characterized by a conserved 19 membered cyclic hexadepsipeptide core bearing the modified glutamic acid residue 3 amino 6 hydroxy 2 piperidone (Ahp) and a highly variable pendant side chain. 21,38, 39 The isolation of over 100 members of this group of cyanobacterial metabolites, together with antiproteolytic activity data primarily against the serine proteases elas tase, chymotrypsin, and trypsin, has provided insights into the importance of the Ahp moiety and the adjacent residue on its N terminal s ide which confer selectivity. 38, 46 The role of these moieties was elegantly demonstrated through X ray cocrystallization of A90720A trypsin and scyptolin elastase complexes. 73, 74 Not found in terrestrial or freshwater cyanobacteria is the 2 amino 2 butenoic acid (Abu) moiety, which is hypothesized t o contribute to higher potency. 41 The majority of the marine derived cyanobacterial metabolites in this class bear s the Abu moiety adjacent to the Ahp resi due. These compounds, which include lyngbyastatins 4 10, showed potent antiproteolytic activity against elastase with low nanomolar IC 50 s, and are perhaps among the most potent small mol ecule inhibitors of elastase. 40 42 Therefore, these small molecules are attractive therapeutics for elastase mediated pathologies, as well as molecular probes to elucidate critical interactions for effective enzyme inhibition and to int errogate specific Reproduced with permission from Salvador, L.A.; Taori, K.; Biggs, J. S.; Jakoncic, J.; Ostrov, D. A.; Paul, V. J.; Luesch, H. J Med Chem 2013 56 1276 1290 Copyright 2013 American Chemical Society.

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49 intracellular and extracellular molecular targets of elastase. However, limited SAR and a lack of information beyond enzymatic assay data hinder further development of these compounds as small molecule therapeutics. Elastase is a broad sp ectrum enzyme that preferentially cleaves on the C terminus of small hydrophobic amino acids such as Gly, Ala, and Val and degrades collagen, elastin, fibronectin and component s of the extracellular matrix. 75 Elast ase has been linked to several diseases involving chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and systemic inflammatory response syndrome, where there is a prote ase antiprotease imbalance. 75, 76 The canonical role of elastase in degrading the extracellular matrix has been documented, as have the stimulating effects of elastase on signaling pathways through direct or indirect re ceptor activation. The resulting changes in transcript and protein levels have been linked to possible disease progression. 77 Current therapies for these diseases are aimed at alleviating the symptoms but not disease progression, which may be re lated to the role of elastase. 78 Sivelestat is the only app roved drug targeting elastase; 79 however, clinical approval in the United St ates and Europe has been stalled due to marginal clinical effects. 80 Finding new small molecule therapeutics for COPD is of importance since the disease has been recognized as a major public health problem an d the fourth lea ding cause of death worldwide. 81 Intratracheal instillation of elastase in animal models showed changes such as enlargement of alveolar space, thickening of alveolar septae and mucus hypersecretion, compar able to clinical observations. 82 This enzyme has also been implicated in cell death, transcriptional and translational modulation and processing of pro inflammatory

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50 cytokines, chemokines and adhesion molec ules, which also dictate downstream c ellular effects. 76 Development of elastase inhibitors has been particularly challenging because of overlapping functions of elastase with those of other serine proteases, as wel l as limited information on the role of elastase in the progression of disease. Here we aimed to determine the potential utility of symplostatin 5 ( 1 ) and related compounds in alleviating the cellular effects downstream of elastase release and compared the cellular potency to sivelestat. Isolation and Structure Elucidation The lyophilized red cyanobacterium collected from Cetti Bay, Guam was extracted with EtOAc MeOH (1:1) to afford the nonpolar extract. Liquid liquid partitioning of the nonpolar extract y ielded the hexanes n BuOH and H 2 O soluble fractions. The 1 H NMR spectrum of the n BuOH fraction showed characteristic resonances for peptides and modified peptides. This fraction was further purified by silica column chromatography and reversed phase HP LC to give six new Ahp containing cyclic depsipeptides, termed symplostatins 5 10 ( 1 6 ) (Figure 3 1). The major compound, symplostatin 5 ( 1 ) (Figure 3 1), showed a pseudomolecular ion of 1044.3981 [M + Na] + suggesting a molecular formula of C 47 H 64 N 7 O 15 SNa LRESIMS using negative ionization showed a loss of 46 amu ( m/z 998.5 [M Na] ) relative to the pseudomolecular [M + Na] + ion. This corresponds to loss of 2 Na + ions and supported that 1 was present as a sodium salt. The 1 H NMR spectrum of symplostatin 5 ( 1 ) showed characteristic signals for peptides and modified peptides such as secondary amide protons ( H 8.18, 7.71, 7.40, 7.34), N CH 3 protons ( H protons for amino acids ( H 3.80 5.10). Analysis of the COSY, TOCSY,

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51 HSQC and HMBC data acquired in DMSO d 6 established the presence of Val, Thr, Ile, N Me Phe, Phe and the modified amino acids Ahp and Abu (Table 3 1). Among the three remaining spin systems, one is a distinctive methine quartet ( H 6.50) that showed a COSY correlation to a CH 3 doublet ( H 1.47) (Table 3 1). HMBC correlations of the latter to a carbonyl at C 162.9 and a quaternary sp 2 C ( C 130.0), together with a TOCSY correlation to a broad NH singlet ( H 9.24), established this unit as Abu. The observed low field methine signal at C / H 73.4/5.03 together with a hydroxy proton resonating at H 6.05 in 1 are distinctive for the Ah p unit. The presence of this cyclized amino acid residue was further supported by COSY and HMBC correlations (Table 3 1). The remaining spin systems consisted of a low field methine ( C / H 79.9/3.98), an oxygenated diastereotopic methylene ( C / H 66.1/3.90 3.73) and an OCH 3 group ( C / H 57.1/3.33). From COSY and HMBC analysis, this moiety corresponds to a modified glyceric acid, where the C 2 and C 3 positions are methoxylated and sulfated, respectively (Table 3 1). The linear sequence of 2 O CH 3 glyceric acid sulfate Val Thr Abu Ahp N Me Phe Phe Ile was established using HMBC and NOESY correlations. In order to fulfill the molecular formula requirements and to account for the low field 1 H NMR chemical shift of the vicinal methine of Thr ( H 5.52), additio nal anisotropic effect from a carbonyl group must be present, and this indicated cyclization of symplostatin 5 ( 1 ) via the carbonyl group of Ile and the hydroxy group of Thr. Comparison of the 1 H NMR spectrum of 1 2 and 3 revealed differences in the split ting pattern of signals in the methyl region ( H 0.75 0.90). No methyl triplet arising from Ile was observed in symplostatin 6 ( 2 ). Instead, two pairs of methyl doublets were

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52 present, suggesting the presence of 2 Val units, which was corroborated by the HSQC spectrum of 2 Hence, the Ile unit presen t in the core ring structure of 2 is replaced by Val, where the vicinal methine ( C / H 30.7/2.00) showed COSY and HMBC correlations to two methyl groups ( C / H 19.0/0.89, 17.1/0.76) ( Table 3 2 ), in agreement with molecular formula of C 46 H 62 N 7 O 15 SNa deduced from HRESIMS. Symplostatin 6 ( 2 ) is reminiscent of dolastatin 13 83 with the primary difference being the modification of the 2 O CH 3 glyceric acid unit, and is the sulfated analog of dolastatin 13 (Figure 3 1). Symplostatin 7 ( 3 ) showed 14 amu mass difference with symplostatin 5 ( 1 ) and has a molecular formula of C 48 H 66 N 7 O 15 SNa. The 1 H NMR spectrum of 3 showed 2 CH 3 triplets ( H 0.92, 0.80) which correlated to two high field carbons ( C 11.3, 10.7) based on the HSQC spectrum ( Table 3 3 ). Hence, 3 has Ile moieties in both the pendant and macrocycle (Figure 3 1). Comparison of the 1 H NMR spectrum of 1 and 3 corroborated this resu lt. Except for 1 H NMR resonances belonging to the additional Ile unit, no significant differences were observed between the two spectra. The 1 H NMR spectra of 1 and 4 2 and 5 and 3 and 6 were highly similar, except for the splitting pattern and chemical shifts of aromatic protons ( H 6.77 7.40). These pairs also showed a difference of 16 amu in their HRESIMS spectra, corresponding to an additional oxygen atom in 4 6 Comparison of the HSQC spectra of these compounds showed an upfield shifted sp 2 C at C 115.2, which correlates to a proton at H 6.77. COSY correlation between H 6.77 and H 6.99, together with their doublet splitting pattern and 3 J H,H of 7.8 Hz, indicated a 1,4 disubstituted phenyl ring ( Tables 3 1 3 3 ). The upfield shifted 1 H and 13 C NMR resonances and the presence o f a broad singlet at H 9.34 for a hydroxy group in the 1 H NMR spectrum of 4 6 supported the

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53 presence of an N Me Tyr instead of an N Me Phe in the macrocycle. Hence, symplostatins 8 10 ( 4 6 ) are the N Me Tyr congeners of 1 3 (Figure 3 1), consistent with t he molecular formula requirements. Enantioselective HPLC MS analysis of the acid hydrolysates of 1 6 established the configuration of the amino acids (Val, Phe, N Me Phe, N Me Tyr, Thr) as L by comparison to authentic standards. L allo Ile was detected fo r compounds having this unit in the macrocycle alone ( 1 4 ), while 3 and 6 which have Ile in both the macrocycle and pendant chain showed peaks corresponding to L allo Ile and L Ile at ~1:1 ratio. Comparison of the 1 H and 13 C NMR chemical shifts of the ma crocyclic Ile of 1 3 4 and 6 showed no significant differences and suggested the same configuration. Hence, the pendant side chain Ile moiety would account for the peak corresponding to L Ile. The presence of L allo Ile in the macrocycle is also support ed by comparison of the 13 C NMR chemical shifts of C 5 and C 6 of the macrocyclic Ile with similar compounds bearing the same amino acid residue. Zafrir and Carmeli reported that the 13 C NMR chemical shifts of C 5 and C 6 of L allo Ile are distinctive, 1 1. 4 and 14.3 ppm, respectively. 84 1 3 4 and 6 which are proposed to bear an L allo Ile in the macrocycle, also displayed these characteristic 13 C NMR resonances (Table s 3 1, 3 3 ). Oxidation of 1 using CrO 3 prior to acid hydrolysis converted the Ahp unit to Glu. Enantioselective analysis of the acid hydrolysate of the oxidation product showed a peak corresponding to L Glu and hence, the Ahp unit would have the same configuration at C 3. The configuration at C 6 of Ahp is deduced to be R in comparison with the NMR chemical shifts with the related compounds symplostatin 2 and lyngbyastatins 4 10. 40,41, 85 The 2 O CH 3 glyceric ac id liberated from the acid hydrolysate of symplostatin 5 ( 1 ) is proposed to have an R

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54 configuration, based on comparison with authentic standards of 2 O CH 3 glyceric acid synthesized from D and L Ser by a mo dified diazotization procedure. 86 The other analogs of symplostatin 5 ( 1 ) are proposed to have the same configuration of the Ahp and 2 O CH 3 glyceric acid moieties based on similar 1 H and 13 C NMR chemical shifts. Enzyme Inhibition We tested the antiproteolyt ic activity of symplostatins 5 10 ( 1 6 ) against porcine pancreatic elastase. Compounds 1 6 potently inhibited porcine pancreatic elastase with IC 50 s of 37 89 nM (Table 3 4 ), which was comparable to the activity of the related compounds lyngbyastatins 4 and 7. Symplostatins 8 10 ( 4 6 ), containing N Me Tyr, are slightly more potent than their N Me Phe congeners ( 1 3 ) in inhibiting elastase. In contrast, Ile to Val substitution in the macrocycle and pendant side chain did not affect activity. To demonstrate th at these compounds also inhibit the disease relevant human neutrophil elastase, we determined the antiproteolytic activity against this enzyme. Symplostatins 8 10 ( 4 6 ) and lyngbyastatins 4 and 7 potently inhibited human neutrophil elastase, while symplost atins 5 7 ( 1 3 ) gave higher IC 50 s (Table 3 4 ). Compounds 4 6 and lyngbyastatins 4 and 7 showed higher potency than the drug sivelestat, a selective human neutrophil elastase inhibitor, while 1 3 had similar activity as sivelestat. Symplostatins 5 10 ( 1 6 ) and lyngbyastatins 4 and 7 were analogously tested for antiproteolytic activity against human and bovine pancreatic chymotrypsin. All the compounds tested were less potent inhibitors of chymotrypsin than elastase (Table 3 4 ). To determine the selectivity of the cyanobacterial elastase inhibitors, we screened the most potent inhibitor lyngbyastatin 7 at a single concentration against a panel of 68 proteases (Figure 3 2A ). Lyngbyastatin 7 showed preferential inhibition for

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55 serine proteases at 10 M, comple tely inhibiting the serine proteases elastase, chymotrypsin and proteinase K. The serine proteases cathepsin G, kallikrein 8, kallikrein 12 and plasma kallikrein, the dipeptidyl peptidase cathepsin C and the cysteine proteases caspases 1, 9 and 11 were par tially inhibited. Lyngbyastatin 7 did not inhibit any member of the cysteine carboxypeptidases, metalloproteases or aspartic family of proteases. To validate the serine protease selectivity profile for this class of inhibitors, a dose response study agains t the same panel of 26 serine proteases was undertaken for the most abundant compound, symplostatin 5 ( 1 ) (Figure 3 2B ). Symplostatin 5 ( 1 ), like lyngbyastatin 7, preferentially inhibited elastase over chymotrypsin. Aside from these enzymes, the majority o f the serine proteases including proteinase K was less potently inhibited by 1 than by lyngbyastatin 7, with IC 50 s of 10 M or higher. Molecular Basis for Elastase Inhibition by Lyngbyastatins and Symplostatins In order to understand the potent and select ive inhibitory activity of the Abu bearing cyclic depsipeptides against elastase, we cocrystallized the most potent inhibitor, lyngbyastatin 7, with porcine pancreatic elastase using the hanging drop vapor diffusion method. The structure of the lyngbyastat in 7 porcine pancreatic elastase complex was solved at a resolution of 1.55 the best reported for an elastase cyclic depsipeptide inhibitor complex. The elastase complexes with the natural products scyptolin (no Abu) and FR901277 (bicyclic) were previou sly cocrystallized and analyzed at resolutions of 2. 8 and 1.6 respectively. 74, 87 Porcine pancreatic elastase, despite sharing only 40% amino acid sequence homology to human neutrophil elas tase, is an accepted model system to understand key enzyme inhibito r interactions. 88 They are structurally comparable and share analogous residues that compose the enzyme active

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56 site. The improved resolution of the lyngbyastatin 7 elastase complex provided better insights into key molecular interactions with the enzyme. The cocrystal structure of porcine pancreatic elastase and lyngbyastatin 7 indicated that these compounds act as substrate mimics, with the Abu moie ty and the N terminal residues occupying subsites S1 to S4. They exploit the same binding sites occupied by FR901277 and scyptolin, and the orientation of the macrocyle of these three compounds is also comparable (Figure 3 3A C ). The ethylidene moiety of t he Abu unit in subsite S1 contributes a non bonded interaction with Ser203 and within distances f 3D, E ), as previou sly hypothesized for FR901277. 87 It also forms hydrogen bonds with Gly201 and Ser222, and an indirect hydrogen bond with Thr44 via a water molecule. The cocrystal st ructure did not show covalent bond formation between the Abu moiety of lyngbyastat in 7 and elastase or hydrolytic cleavage of the macrocyle. Lyngbyastatin 7 showed extensive hydrogen bonding and van der Waals interactions with elastase and several water mo lecules in the active site (Figure 3 3 D). The difference in antiproteolytic activity between the N Me Phe containing symplostatins 5 7 ( 1 3 ) with their corresponding N Me Tyr congeners ( 4 6 ) was evaluated in the context of the cocrystal structure. The OH g roup of N Me Tyr forms hydrogen bonds with three water molecules. Val to Ile substitution in the macrocycle did not cause a significant difference in antiproteolytic activity and, based on the cocrystal structure, this moiety is indeed not close to any ami no acid residu es of elastase for interaction. Comparison of the antiproteolytic activity of symplostatin 9 ( 5 ), lyngbyastatins 4 and 7 9, which all bear exactly the same macrocycle, indicated the contribution of the pendant side chain in modulating the ant iproteolytic activity of these elastas e inhibitors. Lyngbyastatins 8 and 9 are less

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57 potent, with IC 50 s of 120 210 nM, 42 suggesting that the presence of mainly hydrophobic residues in the pendant side chain is unfa vorable. The preference for a polar group in the pendant side chain is supported by the cocrystal structure, wherein the Gln moiety of lyngbyastatin 7 participates in indirect hydrogen bonding with Gln200 and Ser225, via a water molecule; a molecular inter action is not possible with nonpolar moieties in the pendant side chain. The side chain carbonyl of the Gln moiety of lyngbyastatin 7 also participates in a network of inter and intramolecular hydrogen bonding interaction involving an active site water mo lecule, C=O (Thr) and C=O (Ahp) (Figure 3 3 F). This interaction has not been previously demonstrated and suggests the novelty of having a Gln or related moiety at this position. A linear terminal unit in the pendant side chain appears to be preferable, as the hexanoic acid of lyngbyastatin 7 displays a perfect fit to the elastase binding pocket and also participates in nonbonded interactions with Val103 and Arg226. Biological Activity Evaluation Cytoprotective Effects of Symplostatin 5 (1) Against Elastase Induced Antiproliferation and Apoptosis We utilized the bronchial epithelial cell line BEAS 2B, a SV 40 transformed cell line that maintains epithelial cell characteristics, as a model system. 89 We challenged th ese cells with disease relevant concentrations of exogenous elastase and tested if compound 1 was able to prevent the toxicity by elastase, which showed both a dose and time dependent antiproliferative effect based on MTT assay, with an IC 50 value of 77.5 4.9 nM at 24 h (Figure 3 4A). Symplostatin 5 ( 1 ) dose dependently protected the cells, causing a shift in the IC 50 of elastase (Figure 3 4B). The ordinarily toxic concentrations of elastase had little effect on cell viability when 1 was coadministered.

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58 At concentrations of 1 or 10 M symplostatin 5 ( 1 ), cell viability in elastase co treated cells was >75%. Sivelestat was also protective but required higher concentration (100 M) to completely negate elastase induced cytotoxicity (Figure 3 4C). In additio n, symplostatin 5 ( 1 ) did not significantly affect the proliferation of BEAS 2B cells even up to a concentration of 100 M (Figure 3 4D) when co administered with either the solvent control or 100 nM elastase, thus providing a wide therapeutic window at le ast in culture d cells. To determine the possible role of apoptosis in the observed antiproliferative effect of elastase, we assessed caspase 3/7 activity of BEAS 2B cells. Increased pro apoptotic activity was observed upon 12 24 h incubation with 100 nM el astase (Figure 3 4E), paralleling with the onset of cell viability changes associated with elastase (Figure 3 4A). Addition of a caspase 3 inhibitor abrogated the observed increase in caspase 3/7 activity from elastase treatment (Figure 3 4E). Furthermore, treatment of BEAS 2B cells with the caspase 3 inhibitor also caused significant protection from elastase induced antiproliferation. However, protection was incomplete, which suggests that elastase also reduces cell viability through mechanisms other than apoptosis (Figure 3 4C) 1 ) lowered the caspase 3/7 activity in elastase treated cells and shifted the EC 50 of elastase in activating caspases (Figure 3 4F). Thus, symplostatin 5 ( 1 ) counteracted both pro apoptotic and antiproliferative effect s of elastase. Recent reports demonstrated that elastase can activate apoptosis through a proteinase activated receptor 1 (PAR 1) dependent pathway that culminates in the upregulation of NF B and p53 and subsequent changes in mitochondrial permeability an d caspase activation. 90, 91 PARs are seven transmembrane G protein coupled receptors that are activated by proteases following cleavage of the extracellular

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59 N terminus, which triggers a chang e in conformation and coupling to the G protein. 92 Thrombin is a canonical activator of PARs, while elastase has been reported to have varied effects and PAR substrates, depending on the cell type. 93 It is unclear whether elastase directly or indirectly activates PARs. Furthermore, the antiproliferative effects of elastase may be mediated by other pathways as well as its cytostatic effect based on the partial cytoprotectio n using the caspase inhibitor when compared to 1 and sivelestat. It is then evident that the key to maximum abrogation of elastase mediated antiproliferative effect is disarming its proteolytic activity. Cytoprotective Effects of Symplostatin 5 (1) Against Elastase Induced Cell Detachment and Morphological Change A morphological change of BEAS 2B cells from an epithelial to a rounded and retracted appearance was the most obvious and immediate cellular event that occurred following elastase treatment (Figure 3 5A). This effect of elastase was observed within 2 3 h, and the early onset suggests that this was independent of cell death. Cells incubated with elastase remained viable after 3 and 6 h as assessed by MTT and trypan blue staining, despite the obvious change in cell morphology. Furthermore, caspase 3 inhibitor pre treated cells showed the same rounded appearance ( Appendices A D ). Symplostatin 5 ( 1 ) and sivelestat both dose dependently prevented elastase induced cell morphology change (Figure 3 5A), alt hough sivelestat required a higher concentration during longer incubation periods (12 and 24 h) (Appendi ces C and D ), consistent with results from cell viability assays (Figure 3 4C). Elastase caused a three fold increase in cell detachment from the collag en base matrix and neighboring cells after 12 h, which was dose dependently prevented by 1 (Figure 3 5B). At a concentration of 10 M of symplostatin 5 ( 1 ), elastase was unable to cause

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60 desquamation. Sivelestat also showed the same cytoprotective effect bu t again only at higher concentration (100 M). The ability of elastase to induce cell detachment and morphology change reflects its canonical role in degrading components of the extracellular matrix such as collagen, fibronectin, and elastin and also impli cates its effects on cell adhesion molecules. This role of elastase is also dependent on its proteolytic activity as evidenced by abrogation via small molecule inhibition using symplostatin 5 ( 1 ) and sivelestat, but not with the caspase inhibitor. Adhesion molecules such as the immunoglobulin like cell adhesion molecules (ICAM 1, 2, 3, VCAM, PECAM), integrins, selectins and cadherins are located on the cell surface, are involved in cell and extracellular matrix attachment and also function to modulate leu kocyte adhesion and migration, a process essential to progression of inflammation. 94 ICAM 1 is a key regulator of cell cell adhesion and exists as a membrane bound protein (mICAM 1) that can be cleaved to generate solu ble ICAM 1 (sICAM 1) which is liber ated into the medium. 95 sICAM 1 is increased with inflammation and cardiovascular disease and serves as a biomarker. 96, 97 To determine the possible effects of elastase on total ICAM 1 levels in bronchial epithelial cells, culture medium and whole cell l ysates were collected after 6 h mICAM 1 in whole cell lysates was assessed by immunoblotting (Figure 3 5C) and provides a snapshot of the remaining membrane bound form at the specific timepoint. sICAM 1 in culture supernatants was quantified by AlphaLisa and reflects accumulated amount over time (Figure 3 5D). Media from elastase challenged cells contained significantly increased sICAM 1 level, which was dose 1 ) (Figure 3 5 D). Inhibition of the proteolytic activity of elastase by cotreatment with

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61 symplostatin 5 ( 1 ) caused retention of mICAM 1, t hus confirming the role of elastase activity on this cellular event (Figure 3 5C). Sivelestat also showed a similar effect on sICAM 1 and mICAM 1 levels in response to elastase. This inverse relationship is consistent with sICAM 1 levels in the culture med ium and provided internal validation of the direct effects of elastase with the proteolytic cleavage of ICAM 1. Conversely, ICAM1 transcript levels were not significantly modulated in this cell type as assessed by reverse transcription followed by real tim e quantitative polymerase chain reaction (RT qPCR) ( Appendix E ). Taken together, this data further supported the role of elastase as a sheddase, which posttranslationally modifies the membrane bound form by proteolytic processing to the soluble form. Whil e elastase mediated activation of caspases has been related to cell surface receptors, we additionally demonstrated that it can proteolytically process ICAM 1, a critical cell surface receptor that controls cell cell adhesion, known to be affected by elast ase at both the transcript and protein levels in endothelial cells. 98 Purified elastase and sputum samples from cystic fibrosis patients with significant proteolytic activity were shown to induce cleavage of IC AM 1, independent of cell surface expression. 99, 100 Aside from controlling cell cell adhesion, mICAM 1 also binds to leukocytes via the LFA 1 receptor, and its normal expression is required for immune defense. 95 Shedding of mICAM 1 is proposed to serve as a rapid mechanism to regulate leukocyte adhesion and/or promote signal transduction, although it has not been fully elucidated. 101 The observed cellular effects of elastase on cell detachment and cell death are important clinical hallmarks of asthma and COPD. 102 Neutrophils mainly cause cell detachment, with e lastase and cathepsin G degr ading a variety of substrates. 103 In a

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62 cellular model system, TNF may function t ogether with serine proteases. 104 Furthermore, lung biopsies of patients indicated that detachment and apoptosis may be related, with the initial sites of cell detachment show ing increased apoptotic cells 104 Cell death has b een linked, in addition to persistent inflammation, to contr ibute to the severity of COPD. 105 Excessive apoptosis is proposed to exacerbate lung disease by preventing re epithelialisation, development of apoptotic resistance leading to fibrosis and ineffective removal of apoptotic cells, resulting in a p ersistent inflammatory state. 106 Attenuation of Global Transcript Changes Induced by Elastase Elastase has been dem onstrated to induce changes in transcript levels of pro inflammatory cytokines, adhesion molecules and chemokines in v itro, mostly mediated by an NF B dependent pathway. 98,107, 108 The expression of NF B inducible genes is precede d by degradation of cytosolic I B and nuclear translocation of p65. 109 To determine the possible changes in transcript levels in elastase an d elastase+symplostatin 5 ( 1 ) treatm ents, the amount of cytosolic I B and nuclear p65 was assessed by immunoblotting and ELISA, respectively. Elastas e caused a strong decrease in I B level, which was prevented by 1 (Figure 3 6A). In accord, a significant i ncrease in nuclear translocation was observed 3 h after elastase treatment and attenuated by cotreatment of 1 This data is indicative of possible transcript changes associated with elastase treatment that may also be modulated by 1 Microarray profiling u sing the Affymetrix GeneChip Human Genome U133 plus 2.0 arrays was performed to comprehensively determine global changes in transcript levels in bronchial epithelial cells following elastase treatment. Elastase caused a significant change in

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63 expression ( P < 0.05, fold change > 1.5) of 364 transcripts corresponding to 348 genes (Figures 3 6B, Table s 3 5, 3 6 ). Elastase affected the expression of signaling molecules including chemokines, cytokines, and receptors, as well as components of the spliceosome, tra nscription machinery, cell cycle and ubiquitin mediated proteolysis. In addition, 13% of elastase inducible genes currently have no annotation of identity and function, suggesting that our analysis may have identified novel target genes of elastase signali ng (Table 3 5) Also, of the other 87% of genes with known identity, 30% do not have a clear function in cellular signaling. Aside from the members of the NOD and MAPK signaling pathways (Table 3 6) the contribution of other elastase inducible genes to i nflammation or downstream cellular effects of elastase has not been clearly established. Upregulation of kinases (e.g., PTK2 MAP2K5 SIK2 CSNK1A ) and transcription factors (e.g., ARID1B NFIB RBM14 ) may suggest that elastase is promoting cellular signal ing by affecting signaling mol ecules and/or their activation. The contribution of caspase independent pathways to elastase mediated cell death may also be discerned, as several positive modulators of the cell cycle were also upregulated by elastase (e.g., GAS1 DAP3 DDIT4 ). Importantly, the transcriptional response to elastase was attenuated by co administration of 10 M symplostatin 5 ( 1 ). Comparison of the heat map of significantly modulated transcripts indicated that 1 potently prevented the global effe cts of elastase (Figure 3 6B). Symplostatin 5 ( 1 ) caused a 20 68 % reduction in transcript levels of elastase inducible genes including those involved in NOD and MAPK signaling pathways which are relevant to inflammation ( Table 3 6 ). Microarray results w ere validated by measuring expression levels of important pro inflammatory cytokines IL1A IL1B and IL8 using RT qPCR. IL1B

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64 showed the greatest increase in transcript levels at 3 h, which was strongly abrogated by cotreatment with 1 (Figure 3 6 C ). Similar results were obtained for IL1A and IL8 The effects of elastase on mRNA levels of these three pro inflammatory cytokines were also assessed at 6 h (Figure 3 6 C ). The same trend was observed for IL1B and IL8 while IL1A was not significantly affected at thi s time point. Transcriptome profiling of BEAS 2B cells treated with symplostatin 5 ( 1 ) alone enabled us to characterize possible off target genes of 1 that are independent of elastase (Table 3 7 ). This analysis identified only nine significantly upregulate d transcripts corresponding to nine genes, suggesting high specificity of symplostatin 5 ( 1 ) for elastase also in cells. Our profiling of the transcriptome of bronchial epithelial cells in response to elastase, with or without 1 and vehicle control treatme nts, indicated that this enzyme upregulates the expression of specific genes. Comprehensive profiling enabled us to identify IL1B as the major pro inflammatory cytokine induced by elastase. Although IL8 has been reported to be upregulated by elastase in vi tro, 107,110, 111 our microarray analysis indicated that this gene is less inducible compared to IL1B IL inflammatory cytokine and has increase d activity in both COPD and asthma, causing significant airway remodeling and pulmonary inflammation in animal models, and thus serves as an important biomarker for elastase mediated cellular effects. 112, 113 The expression of IL treated animals has been demonstrated to occur via an IL1R1/MyD88 pathway, thus further implicating the role of this enzyme in receptor activation. 112 We also demonstrated t hat elastase has a broad effect on the transcriptome, and our identification of other elastase target genes may open up new

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65 avenues towards the understanding of the physiological and pathological roles of this enzyme. Conclusion We have demonstrated that n ovel cyanobacterial cyclodepsipeptides can potently inhibit the proteolytic activity of elastase, thereby preventing the downstream cellular effects of this serine protease in a bronchial epithelial model system. Symplostatin 5 ( 1 ) alleviated elastase indu ced changes in cell viability, apoptosis, cell detachment and alterations in levels of the adhesion molecule ICAM 1, activation of transcription factor NF B and global transcriptome changes. At the same time, 1 did not show any cytotoxic effects on bronch ial epithelial cells, offering a remarkable therapeutic window. Symplostatin 5 ( 1 ) showed equipotent activity as sivelestat in enzyme inhibition and short term cellular assays. However, 1 showed higher potency in longer term assays and successfully allevia ted several clinical hallmarks of chronic inflammatory diseases such as excessive sICAM 1 production, expression of pro inflammatory cytokines IL1A IL1B and IL8 and increased cell death and desquamation. Establishment of the molecular basis and biomarkers for elastase inhibition can aid in the design of second generation inhibitors that are potent, selective and cytoprotective against both short and long term effects of elastase. Experimental Methods G eneral Experimental Procedures Optical rotations were measured on a Perkin Elmer 341 polarimeter. UV spectra were recorded on SpectraMax M5 (Molecular Devices). 1 H and 2D NMR spectra were recorded in DMSO d 6 on a Bruker Avance II 600 MHz spectrometer equipped with a 5 mm TXI cryogenic probe using residual sol vent signals [(DMSO d 6 : H 2.50; C 39.5)]

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66 as internal standards. HSQC and HMBC experiments were optimized for 1 J CH = 145 and n J CH = 7 Hz, respectively. TOCSY experiments were done using a mixing time of 100 ms. HRESIMS data was obtained using an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector. LRESIMS measurements based on HPLC. Biological Material The red Symploca sp. cyanobacterium was collected by hand from Ce tti Bay, Guam. Samples were kept frozen at 20 C after collection. A voucher specimen preserved in formaldehyde is deposited in the University of Guam Herbarium and at the Smithsonian Marine Station, Fort Pierce, FL. Frozen cyanobacterium samples were lyo philized prior to extraction. Extraction and Isolation The freeze dried cyanobacterium was extracted with EtOAc MeOH (1:1) to yield the nonpolar extract. This was partitioned between hexanes and 80% aqueous MeOH, the latter concentrated under reduced press ure and further partitioned between n BuOH and H 2 O. The n BuOH fraction was concentrated to dryness and chromatographed on Si gel eluting first with CH 2 Cl 2 followed by increasing concentrations of i PrOH, while after 100% i PrOH, increasing gradients of M eOH were used. The fraction collected from 50% i PrOH elution (Si column) was purified by C 18 column chromatography eluting with 25%, 50%, 75% and 100% MeOH in H 2 O. The fraction from 50% MeOH was further purified using semipreparative reversed phase HPLC (Phenomenex Synergi gradient of MeCN H 2 O (25% 100% Me CN in 30 min and then 100% MeCN for 10 min)

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67 to yield compounds 5 ( t R 17.8 min, 1.0 mg), 4 ( t R 19.0 min, 1.0 mg) and 6 ( t R 36.6 min, 0.3 mg). The 75% MeOH fraction was purified using the same HPLC conditions to yield compounds 2 ( t R 23.8 min, 3.5 mg), 1 ( t R 24.0 min, 7.0 mg) and 3 ( t R 25.3 min, 1.0 mg). Enant ioselective Analysis Portions of 1 6 h), 2 O. The absolute configurations of the amino acids (Ile, Val, N Me Tyr, N Me Phe, Phe, Thr) were determined by enantioselective HPLC MS [column, Chirobiotic TAG (250 4.6 mm), Supelco; solvent, M eOH 10 mM NH 4 OAc (40:60, pH 5.30); flow rate, 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times ( t R min; MRM ion pair) of the authentic amino acids were as follows: L D Val (13.7); N Me L Phe (22.7; 18 N Me D Phe (40.4); L D Phe (17.5); N Me L N Me D Tyr (35.4); L L allo Thr (7.2), D Thr (8.0), D allo Thr (10.2). In order to separate Ile isomers, the mobile phase was modified to MeOH 10 mM NH 4 OAc (90:10, pH 5.65) while keeping the same chromatographic conditions. The retention times of authentic standards were as follows: L Ile (10.4; L allo Ile (11.2), D allo Ile (20.1), D Ile (22.2). The acid hydrolysates of 1 6 showed retentio n times at 6.8 and 12.1 min corresponding to L Thr and L Phe, respectively. L Val ( t R 7.8min) was detected in the acid hydrolysates of 1 2 4 and 5 The acid hydrolysates of 1 3 4 and 6 showed a peak corresponding to L allo Ile ( t R 11.2 min). 3 and 6 h ad an additional peak corresponding to L Ile ( t R 10.4 min). N Me L Phe ( t R 22.7 min) was detected in the acid

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68 hydrolysate of 1 3 while N Me L Tyr ( t R 18.8 min) was present in the acid hydrolysate of 4 6 The modified (2 S ) 2 O Me glyceric acid residue was prepared using L Ser (50 3 4 (60 mg) and anhydrous MeOH (1 mL). The mixture was heated at 110 C for 4 h, cooled down to room temperature and filtered. The filtrate was evaporated to dryness unde r N 2 to yield (2 S ) 2 O Me glyceric acid. The same procedure was employed to prepare (2 R ) 2 O Me glyceric acid from D Ser. LRESIMS and 13 C NMR spectrum for (2 S ) 2 O Me glyceric acid and (2 R ) 2 O Me glyceric acid were in agreement with rep orted literature va lues. 86 The absolute configuration of Glu and 2 O Me glyceric acid was also determined using HPLC MS [column, Chirobiotic TAG (250 4.6 mm), Supelco; solvent, MeOH 10 mM NH 4 OAc (40:60, pH 5.30); flow rate, 0. 5 mL/min] with detection in the negative ion mode (MRM). The retention times of the authentic standards ( t R min; MRM pair): L Glu D Glu (6.1), (2 S ) 2 O R ) 2 O Me glyceric acid (6.6). Compound 1 was oxidize d using CrO 3 and hydrolyzed using 6 N HCl peak at 5.1 min corresponding to L Glu. The acid hydrolysate of 1 yielded a peak for (2 R ) 2 O Me glyceric acid ( t R 6.6 min). Symplostatin 5 ( 1 ): colorless, amorphous solid; [ ] 20 D 3.6 ( c 0.14, MeOH); UV (MeOH); max (log ) 210 (4.49); 1 H NMR, 13 C NMR, COSY, and HMBC data, see Table 3 1; HRESIMS m/z 1044.3981 [M + Na] + (calcd for C 47 H 64 N 7 O 15 SNa, 1044.3971).

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69 Symplostatin 6 ( 2 ): colorless, amorphous solid; 20 D 5.2 ( c 0.2 6, MeOH); UV (MeOH); max (log ) 204 (4.49); 1 H NMR and 13 C NMR data, see Table 3 2 ; HRESIMS m/z 1030.3815 [M + Na] + (calcd for C 46 H 62 N 7 O 15 SNa, 1030.3815). Symplostatin 7 ( 3 ): colorless, amorphous solid; 20 D 14 ( c 0.15, MeOH); UV (MeOH); max (log ) 202 (4.48); 1 H NMR and 13 C NMR data, see Table 3 3 ; HRESIMS m/z 1058.4104 [M + Na] + (calcd for C 48 H 66 N 7 O 15 SNa, 1058.4128). Symplostatin 8 ( 4 ): colorless, amorphous solid; 20 D 6.7 ( c 0.09, MeOH); UV (MeOH); max (log ) 210 (4.13); 1 H NMR and 13 C NM R data, see T able 3 1 ; HRESIMS m/z 1060.3941 [M + Na] + (calcd for C 4 7 H 64 N 7 O 16 SNa, 1060.3920). Symplostatin 9 ( 5 ): colorless, amorphous solid; 20 D 3.5 ( c 0.10, MeOH); UV (MeOH); max (log ) 204 (3.94); 1 H NM R and 13 C NMR data, see Table 3 2 ; HRESIMS m/z 1046.3747 [M + Na] + (calcd for C 46 H 6 2 N 7 O 16 SNa, 1046.3764). Symplostatin 10 ( 6 ): colorless, amorphous solid; 20 D 3.3 ( c 0.03, MeOH); UV (MeOH); max (log ) 200 (5.24); 1 H NM R and 13 C NMR data, see Table 3 3 ; HRESIMS m/z 1074.4060 [M + Na] + (calcd for C 48 H 66 N 7 O 16 SNa, 1074.4077). In Vitro Protease Assay Porcine pancreatic elastase (Elastin Products Company, Owensville, MO) was dissolved in Tris HCl (pH 8.0) to give a concentration of 75 g/mL. Test compounds (1 L, DMSO), 5 L elastase solution and 7 9 L Tris HCl (pH 8.0) were pre incubated at room temperature for 15 min in a 96 well microtiter plate. At the end of the incubation, 15 L substrate solution were added [2 mM N succinyl Ala Ala Ala p nitroanilide (Sigma Aldrich, St. Louis, MO) in Tris HCl pH 8.0] to each well, and the reaction was monitored by recording the absorbance at 405 nm every 30 s. The inhibitory activity against human neutrophil elastase was also determined using the same procedure with

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70 minor modifications, using 100 g/mL human neutrophil elastase (Elastin Products Company) and 2 mM N (OMe succinyl) Ala Ala Pro Val p nitroanilide (Sigma Aldrich), both prepared in 0.1 M Tris NaCl buffer (pH 7.5). Enzyme activity was determined by calculating the initial slope of each progress curv e, expressed as a percentage of the Aldrich) were assessed using the substrates N succinyl Gly Gly Phe p nitroanilide (Sigma Aldrich) and N succinyl Ala Ala Pro Phe p nitroanilide (Sigma Aldrich), respectively. In brief, the reaction buffer (39 The absorbance was monitored at 405 nm. Enzyme activity in each well was calculated based on the slope of the reaction curve compared to that of the solvent control. For high throughput screening, enzyme and inhibitor [symplostatin 5 ( 1 ) or lyngbyastati n 7] were incubated for 15 min in the reaction buffer before the addition of the substrate. The reaction was monitored for 2 h and the initial linear portion of the slope was analyzed. Detailed information on the enzymes, substrates, reaction buffers and d etect ion conditions are given in Table 3 8 High th roughput protease screening was carried out by Reaction Biology Inc. Cocrystallization of Lyngbyastatin 7 wi th Porcine Pancreatic Elastase A 10 L aliquot of high purity porcine pancreatic elastase:lyngby astatin 7 solution (3:1) was incubated in a hanging drop setup equilibrated against a 0.42 M sodium sulfate solution. Diffraction data was collected on beamline X6A at the National Synchrotron Light Source (Upton, NY). Diffraction data was processed using HKL200051 and the structure was solved by molecular replacement using 2V0B as

PAGE 71

71 search model in MOLREP (CCP4). 114, 115 The model was re fined using REFMAC and COOT. 116, 117 Detailed information on the refinement statistics are provided in Table 3 9 Coordinates are deposited in the Protein Databank with accession number 4GVU. Cocrystallization experiments and data analysis were c arried out by Ms. Kanchan Taori, Dr. Jean Jakoncic and Dr. David A. Ostrov, In V itro Cellular Assays General cell culture procedure Bronchial epithelial cells (BEAS 2B, ATCC) were grown in bronchial epithelial basal media (BEBM ) (Lonza, Walkersville, MD) supplemented with bronchial epithelial growth factors (Lonza) under a humidified environment with 5% CO 2 at 37 C. All culture plates and flasks were coated with collagen before use. Cell viability assay BEAS 2B (5,000/well) cells were seeded in collagen c oated 96 well plates and treated with varying concentrations of elastase or vehicle (40% sodium acetate in BEBM ) after 24 h of seeding. These were co treated with varying doses of either symplostatin 5 ( 1 ) or sivelestat (Sigma Aldrich) or with DMSO. The ce lls were incubated for an additional 24 h before the addition of the MTT reagent. Cell viability was 50 calculations were done by GraphPad Prism 5.03 based on duplicate experi ments. Cell d e tachment and morphology c hange BEAS 2B cells were seeded in 6 cm dishes. The cells were treated with vehicle+DMSO, elastase+DMSO and elastase+inhibitor. Brightfield photographs were taken at 3 h, 6 h, 12 h and 24 h using a Nikon Eclipse T i U microscope (10 magnification). Media were collected after 12 h and the detached cells were pelleted by

PAGE 72

72 centrifugation. Adherent cells were collected by trypsinization and pelleted afterwards. Cell pellets were resuspended in fresh culture medium containi ng 0.04% trypan blue. A 10 L aliquot was utilized for cell counting using a hemacytometer. Percent detachment was calculated based on the ratio of the detached cells and total number of cells. Graphs and data analysis were performed using the Prism softw are and analyzed test. Caspase activation measurement BEAS 2B cells were prepared similar to the cell viability assay. Cells were treated 24 h post seeding with vehicle+DMSO, elastase+DMSO, elastase+symplostatin 5 ( 1 ). In addition, BEAS 2B cells were pre incubated with 10 M Z D(OMe)E(OMe)VD(OMe) FMK, a caspase 3 inhibitor (Calbiochem, Billerica, MA), for 1 h prior to addition of varying concentrations of elastase. At the end of the 24 h incubation period, the medium was replaced with fresh BEBM and incubated for 10 min at room temperature. The caspase reagent was prepared according to the min to ensure complete cell lysis. Luminescence was measured and the relative caspase 3/7 activity of elastase and elastase+symplostatin 5 ( 1 ) treated cells were compared to the control. Measurement of sICAM 1 l evels BEAS 2B cells (60,000/well) were seeded in collagen coated 24 well plates. After overni ght incubation, the medium was replaced with supplement free media and the cells were further incubated for 24 h. At the end of the incubation period, cells were replenished with new supplement free medium prior to treatment. Cells were treated with elasta se together with DMSO or varying concentrations of symplostatin 5 ( 1 )

PAGE 73

73 dissolved in DMSO. Control cells were treated with DMSO (1%) and sodium acetate in supplement free medium (4%). The cells were incubated and culture supernatants were collected after 6 h sICAM 1 levels were determined using ICAM 1 AlphaLisa Kit data analysis were performed using the Prism software and analyzed using ANOVA test. Im muno blot a nalysis of mICAM 1 l evels BEAS 2B cells (150,000/well) were grown in collagen coated 6 cm tissue culture dishes. The supplemented medium was replaced with BEBM after overnight incubation and further left to acclimatize for 24 h in supplement fre e medium. Cells were replenished with fresh BEBM and treated with elastase together with DMSO or varying concentrations of symplostatin 5 ( 1 ) or sivelestat Cells were harvested and lysed with PhosphoSafe lysis buffer (Novagen, Madison, WI) after 6 h. Th e protein concentration of whole cell lysates was measured with the BCA Protein Assay kit (Pierce Chemical, Rockford, IL). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (4 12%), transferred to polyvinylidene difluoride ( PVDF) membranes, probed with anti ICAM 1 antibody (Abcam, Cambridge, MA) and detected with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). The immunoblots were stripped by heating i n a water bath (90 C) and reprobed with anti actin antibody (Cell Signaling, Danvers, MA) to confirm equal protein loading. Isolation of n uclear and c ytoplasmic p roteins Cells (150,000/well) were seeded in collagen coated 10 cm dishes. Culture media wer e replaced prior to treatment with elastase and/or elastase+symplostatin 5 ( 1 ). After 3 h the culture supernatant was collected and phosphate buffered saline

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74 supplemented with 1.5 protease inhibitor cocktail (Roche, Indianapolis, IN) was added to each di sh. The cells were lifted from the culture dish using a cell scraper and pelleted by centrifugation (300 g ) at 4 C for 5 min. Cytoplasmic proteins were collected using the NE PER Cytoplasmic Extraction Reagent ( Pierce inst ruction. After collection of the cytoplasmic fraction, the insoluble pellet was washed with PBS, centrifuged for 1 min and the supernatant was discarded. Nuclear proteins were isolated from the insoluble pellet. All extracts were incubated on ice, and prot ein concentration was determined using the BCA reagent (Pierce). Measurement of d egradat ion and NF B p65 t ranslocation I proteins. Equal amounts of the cytoplasmic fraction was loaded and separated in a 4 12% Bis Tris HCl gel, transferred on a PVDF membra ne and probed with an anti I antibody (Cell Signaling) and detected with SuperSignal Femto Max reagent (Pierce). The blots were stripped after detection by incubating at 90 C and subsequently probed with anti tubulin (Cell Signaling) to assess prote in loading. NF B p65 translocation was measured using the TransAM NF B Chemi p65 brief, equal amounts of the nuclear protein were prepared in the TransAM complete lysis buffer. The nuclear extracts were added to oligonucleotide coated plates containing complete binding buffer. This was allowed to incubate at room temperature with mild agitation for 1 h, washed and incubated with NF B p65 primary antibody for 1 h. The wells were washed and subsequently incubated for 1 h with anti rabbit horseradish peroxidase conjugated antibody. The chemiluminescent reagent was

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75 added after the incubation period and luminescence was measured. The relative NF B p65 translocation of elastase and elastase+symplostatin 5 ( 1 ) treated cells were compared to the control. To ascertain the specificity of the measured activity, elastase treatments were also incubated with wild type oligonucleotide AM20 which prevented NF B p65 binding to the oligonucleotide probe immobilized on the plate. Each experiment was performed in triplicate. Graphs and data analysis were performed using the Prism test. RNA i sol ation and reverse t ranscription A total of 1.2 10 6 BEAS 2B cells were seeded in 10 cm dishes and incubated further for 24 h in supplement free medium prior to treatment. RNA was isolated at 3 and 6 h post treatment using RNeasy mini kit (QIAGEN, Valencia, CA). Total RNA was quantified by UV absorbance. From 2 g total RNA, cDNA synthesis was done using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT) 12 18 (Invitrogen). Real time quantitative polymerase chain r eaction (qPCR) qPCR after reverse transcripti on (RT qPCR) was performed on a 25 L reaction solution containing a 1.5 L aliquot of cDNA, 12.5 L TaqMan gene expression master mix, 1.25 L of 20 TaqMan gene expression assay mix and 9.25 L RNase free water. qPCR was carried out on an ABI 7300 sequ ence detection system using the thermocycler program: 2 min at 50 C, 10 min at 95 C, and 15 s at 95 C (40 cycles) and 1 min at 60 C. Each experiment was performed in triplicate. IL1A (Hs00174092_m1), IL1B (Hs01555410_m1), and IL8 (Hs00174103_m1) were u sed as target genes, while GAPDH (Hs02758991_g1) was used as endogenous control.

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76 Graphs and data analysis were performed using the Prism software and analyzed test. Transcriptome p rofiling RNA was analyzed using a NanoD rop Spectrophotometer and Agilent 2100 Bioanalyzer to determine the RNA concentration and quality, respectively. RNA samples were processed using the GeneChip brief, 250 ng RNA were used for cDNA synthesis by reverse transcription and the cDNA was utilized as a template for the biotin labeled RNA prepared by in vitro transcription reaction. The labeled RNA was further purified, fragmented and hybridized with ro tation at 45 C for 16 h to the Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. The arrays were washed and stained using the GeneChip Hybridization Wash and Stain kit on an Affymetrix Fluidics Station 450. The chips were scanned using a GeneChip 7G Scanner. Analysis of the microarray data was done acc ording to the reported method. 35 Raw data was normalized using the Robust Multichip Analysis approach and statistical analysis was done using the Bioconductor st detection call was estimated using the Wilcoxon signed rank based algorithm. Probe sets that are absent in all of the study samples were removed from further analyses. Differential expression analysis was p erformed using a linear modeling approach and the empirical Bayes statistics as implemented in the limma package of the R software. The P values obtained were controlled for multiple testing (false discovery rate) using the Benjamini Hochberg method. P va lue and fold induction were calculated. Differentially expressed transcripts were ranked by P values, and P < 0.05 and fold induction >1.5 were considered at a statistically significant level. Hierarchical clustering

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77 of the data was computed on log transfo rmed and normalized data by using complete linkage and Pearson correlation distances. Computation and visualization were done with R packages. Gene ontology was performed using the DAVID Bi oinformatics Resources 6.7. 118, 119 Omnibus with accession number GSE41600.

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78 Figure 3 1. Elastase inhibitors from marine cyanobacteria and the clinically approved human neutrophil elastase inhibitor sivelestat.

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79 Figure 3 2. Selectivity profile of Abu containing cyclic depsipeptides from marine cyanobacteria. ( A ) Screening of lyngbyastatin 7 (10 M) against a panel of 68 proteases. ( B ) Selectivity profiling for symplostatin 5 ( 1 ) on a panel of 26 s erine proteases. Assays were performed by Reaction Biology, Inc.

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80 Figure 3 3. Cocrystal structures of natural cyclic depsipeptide elastase inhibitors. (A) ( F o F c) plot for lyngbyastatin 7. (B) Comparison of lyngbyastatin 7 (yellow, PDB ID 4GVU) and scyp tolin (white, PDB ID 1OKX) binding to elastase. (C) Comparison of lyngbyastatin 7 (yellow) and FR901277 (green, PDB ID 1QR3) binding to elastase. (D) Ligplot of the lyngbyastatin 7 porcine pancreatic elastase complex. The Abu moiety serves as the key resid ue for elastase inhibition. Chain designations are (A) elastase, (B) lyngbyastatin 7, (C) H 2 O. (E) Proposed CH moiety (F) Network of inter and intramolecular hydrogen bonding interaction in lyngbyasta tin 7 mediated by a water molecule Data courtesy of Ms. Kanchan Taori, Dr. Jean Jakoncic and Dr. David A. Ostrov.

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81 Figure 3 4. Changes in cell viability and caspase activation mediated by elastase and effects of inhibitors. (A) Elastase displayed both time and dose dependent decrease in cell viability, with substantial changes at 12 24 h. (B) Symplostatin 5 ( 1 ) attenuated the antiproliferative effects of elastase. (C) Sivelestat and the caspase 3 inhibitor Z D(OMe)E(OMe)VD(OMe) FMK also partially prote cted against the antiproliferative effects of elastase. (D) Symplostatin 5 ( 1 ) did not show any significant antiproliferative effect on BEAS 2B cells at 24 h. (E) Treatment with 100 nM elastase caused a time dependent increase in caspase activation which w as abrogated by the caspase 3 inhibitor. (F) Incubation of BEAS 2B cells with elastase for 24 h caused a dose dependent increase in caspase 3/7 activity. Symplostatin 5 ( 1 ) attenuated the potency and efficacy of elastase to activate distal caspases. Data a re presented as mean SEM (n = 2)

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82 Figure 3 5. Elastase acts as a sheddase and promotes cell morphology change and desquamation. (A) Elastase caused cell rounding after incubation for 3 h. Cotreatment with 10 M symplostatin 5 ( 1 ) or sivelestat preven ted this effect of elastase (10 magnification). (B) Significant increase in cell detachment was observed after 12 h of incubation with elastase, which was abrogated by both symplostatin 5 ( 1 ) and sivelestat. (C) Levels of mICAM 1 in whole cell lysates in elastase tr eated and elastase inhibitor co treated cells as assessed by immunoblotting (D) sICAM 1 in culture supernatants of elastase tr eated and elastase inhibitor co treated cells.Data are presented as mean + SEM, P < 0.05, ** P < 0.01, *** P < 0.001 co mpared to HNE treated control cells using test (n = 3).

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83 Figure 3 6. Elastase caused a global change in transc ript levels via, in part, an NF B dependent pathway. (A) Symplostatin 5 ( 1 ) dose dependently inhibited elastase induced I B degradation and p65 nuclear translocation at 3 h of cotreatment. (B) Heat map of differentially regulated transcripts by elastase with or without symplostatin 5 ( 1 ) cotreatment. Global transcriptome profiling (Affymetrix GeneChip Human Genome U133 plus 2 .0 arrays) was carried out using duplicate biological samples. (C) Validation of the microarray analysis using RT qPCR. Data are presented as mean + SEM for A and mean + SD for C P < 0.05, ** P < 0.01, *** P < 0.001 compared to HNE treated control cells test (n = 3).

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84 Table 3 1. NMR data of s ymplostatin 5 ( 1 ) and s ymplostatin 8 ( 4 ) in DMSO d 6 Symplostatin 5 Symplostatin 8 unit C/H no C a H ( J in Hz) b COSY b HMBC b C a H ( J in Hz) b Ile 1 170.0 ,C e 2 54.0, CH 4.89, br NH 1 54.0, CH 4.87, d (11.0) 3 37.5, CH 1.86, m H 3 6 37.0, CH 1.86, m 4a 25.8, CH 2 1.30, m H 4b, H 3 5 2, 3 26.0, CH 2 1.29, m 4b 1.11, m H 4a, H 3 5 2, 3 1.12, m 5 11.2, CH 3 0.92, t (7.2) H 4a, H 4b 3 11.4, CH 3 0.91, t (7.3) 6 14.1, CH 3 0.71, d (7.0) H 3 2, 3 14.3, CH 3 0.70, d (6.8) NH 7.40, br H 2 7.40, br N Me Phe c / 1 172.7, C e N Me Tyr d 2 60.2, CH 5.00, br H 3a, H 3b 1 60.7, CH 4.90, d (10.6) 3a 33.4, CH 2 3.23, brd ( 13.5) H 2, H 3b 4,5/9 32.7, CH 2 3.11, d ( 14.2) 3b 2.84, m H 2, H 3a 5/9 2.70, dd ( 14.2, 10.6) 4 137.9, C e 5/9 129.4, CH 7.23, d (7.5) H 6 130.3, CH 6.99, d (7.8) 6/8 128.4, CH 7.39, m H 5, H 7 4 115.2, CH 6.77, d (7.8) 7 126.5, CH 7.30, m H 6 e OH 8.13, br s N Me 30.1, CH 3 2.77, s 2, 1 (Phe) 30.3, CH 3 2.75, s Phe 1 170.3, C 2 49.6, CH 4.70, dd (11.4,4.7) H 3a, H 3b 1, 2 (Ahp) 50.0, CH 4.73, m 3a 34.8, CH 2 2.84, dd ( 14.7,11.4) H 2, H 3b 4 34.6, CH 2 2.87, dd ( 14.2, 11.3) 3b 1.68, m H 2, H 3a 4 1.81, m 4 136.5, C e

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85 Table 3 1. Continued Symplostatin 5 Symplostatin 8 unit C/H no C a H ( J in Hz) b COSY b HMBC b C a H ( J in Hz) b 5/9 129.2, CH 6.77, d (7.5) H 6 7 129.3, CH 6.84, d (7.3) 6 127.6, CH 7.18, m H 5, H 7 4 127.7, CH 7.19, m 7 126.1, CH 7.15, m H 6 126.2, CH 7.15, m Ahp 2 168.7, C e 3 47.8, CH 3.75, m H 4a, H 4b, NH 2 48.0, CH 3.79, m 4a 21.7, CH 2 2.38, m H 3, H 4b, H 5a 21.9, CH 2 2.41, m 4b 1.56, m H 3, H 4a 1.58, m 5a 29.0, CH 2 1.68, m H 4a, H 5b, H 6 29.2, CH 2 1.71, m 5b 1.50, m H 5a, H 6 1.56, m 6 73.4, CH 5.03, br s H 5a, H 5b, OH 2 73.5, CH 5.07, m OH 6.05, s H 6 6.07, br s NH 7.34, br H 3 7.33, br Abu 1 162.9, C e 2 130.0, C e 3 131.7 CH 6.50, q (7.2) H 3 4 1, 4 131.5, CH 6.49, q (7.2) 4 12.8, CH 3 1.47, d (7.2) H 3 1, 2 13.0, CH 3 1.47, q (7.2) NH 9.24, br s 9.21, brs Thr 1 f e 2 55.1, CH 4.67, br NH 55.2, CH 4.67, m 3 71.5, CH 5.52, br s H 3 4 e 5.53, brs 4 17.5, CH 3 1.22, d (6.5) H 3 2 17.7, CH 3 1.22, d (6.2) NH 8.18, br s H 2 7.70, br s Val 1 172.2, C e 2 56.4, CH 4.47, t (7.2) NH 1 56.7, CH 4.47, t (7.3) 3 30.7, CH 2.09, m H 3 4, H 3 5 30.6, CH 2.09, m

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86 Table 3 1. Conti nued Symplostatin 5 Symplostatin 8 unit C/H no C a H ( J in Hz) b COSY b HMBC b C a H ( J in Hz) b 4 18.9, CH 3 0.88, d (6.7) H 3 1 19.1, CH 3 0.88, d (7.0) 5 17.5, CH 3 0.83, d (6.7) H 3 1 17.5, CH 3 0.83, d (7.0) NH 7.71, br s H 2 7.72, br s 2 O C H 3 1 168.9, C e Glyceric Acid 2 79.9, CH 3.98, dd (7.4,3.4) H 3a, H 3b 80.2, CH 3.97, dd (7.3, 3.4) 3a 66.1, CH 2 3.90, dd ( 10.8,3.4) H 2, H 3b 66.2, CH 2 3.89, dd ( 10.7, 3.3) 3b 3.73, m H 2, H 3a 3.72, m OCH 3 57.1, CH 3 3.33 g 2 57.3, C H 3 3.32 g a Deduced from HSQC and HMBC, 600 MHz. b 600 MHz. c Refers to symplostatin 5 ( 1 ). d Refers to symplostatin 8 ( 4 ). e Not determined, predicted to have comparable chemical shifts based on highly homologous structures. f No correlation observed from HMBC. g Overlapping with residual water.

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87 Table 3 2. N MR data of s ymplostatin 6 ( 2 ) and s ymplostatin 9 ( 5 ) in DMSO d 6 Symplostatin 6 Symplostatin 9 u nit C/H no C a H ( J in Hz) b C a H ( J in Hz) b Val 1 170.2, C e 2 56.0, CH 4.68, m 55.7, CH 4.70, m 3 30.7, CH 2.00, m 30.3, CH 2.08, m 4 19.0, CH 3 0.89, d (6.8) 18.8, CH 3 0.88, d (6.8) 5 17.1, CH 3 0.76, d (6.8) 17.0, CH 3 0.75, d (6.8) NH 7.51, d (8.8) 7.48, d (8.1) N Me Phe c / 1 169.3, C e N Me Tyr d 2 60.3, CH 5.01, d (11.3) 60.5, CH 4.89, d (10.9) 3a 33.4, CH 2 3.23, m 32.3, CH 2 3.10, d ( 14.2) 3b 2.85, m 2.71, m 4 137.9, C e 5/9 129.4, CH 7.23, d (7.9) 130.1, CH 6.99, d (8.4) 6/8 128.4, CH 7.39, m 114.8, CH 6.77, d (8.4) 7 126.5, CH 7.30, m N Me 30.2, CH 3 2.79, s 29.9, C H 3 2.76, s OH Phe 1 170.3, C e 2 49.7, CH 4.71, dd (11.8,4.4) 49.8, CH 4.73, dd (11.4, 3.8) 3a 34.9, CH 2 2.85, m 34.9, CH 2 2.87, dd ( 14.4, 11.4) 3b 1.69, m 1.81, dd ( 14.4, 3.8) 4 136.5, C e 5/9 129.1, CH 6.77, d (7.5) 129.1, CH 6.8 4, d (7.6) 6 127.6, CH 7.18, m 127.4, CH 7.19, m 7 126.0, CH 7.14, m 126.0, CH 7.14, m Ahp 2 168.7, C e 3 47.9, CH 3.76, m 47.7, CH 3.78, m 4a 21.6, CH 2 2.42, m 21.5, CH 2 2.42, m 4b 1.56, m 1.57, m

PAGE 88

88 Table 3 2. Continued Symplostatin 6 Symplostatin 9 unit C/H no C a H ( J in Hz) b C a H ( J in Hz) b 5a 29.1, CH 2 1.70, m 29.0, CH 2 1.71, m 5b 1.51, m 1.56, m 6 73.5, CH 5.04, s 73.3, CH 5.07, s OH 6.10, br s NH 7.23, br s 7.23, br s Abu 1 163.0, C e 2 129.9, C e 3 131.6,CH 6.51, q (7.0) 131.6, CH 6.51, q (7.1) 4 12.9, CH 3 1.49, d (7.0) 12.9, CH 3 1.49, d (7.1) NH 9.20, br s Thr 1 e e 2 55.2, CH 4.65, m 54.8, CH 4.65, m 3 71.4, CH 5.54, br s 71.2, CH 5.54, br s 4 17.6, CH 3 1. 23, d (6.3) 17.4, CH3 1.23, d (6.4) NH 7.80, br 2 7.70, br s Val 1 171.7, C e 2 56.5, CH 4.47, m 56.3, CH 4.46, m 3 30.4, CH 2.09, m 30.3, CH 2.09, m 4 19.0, CH 3 0.89, d (6.3) 18.8, CH 3 0.88, d (6.8) 5 17.5,CH 3 0.82, d (6.7) 17.3, CH 3 0.82, d (6.8) NH 8.13 br s 8.18, br s 2 O CH 3 1 168.9, C e Glyceric Acid 2 80.0, CH 3.98, dd (7.3,3.4) 79.8, CH 3.98, dd (7.3, 3.4) 3a 66.0, CH 2 3.90, dd ( 11.1, 3.4) 65.8, CH 2 3.89, dd ( 10.9, 3.4) 3b 3.74, dd ( 11.1, 7.3) 3.73, dd ( 10.9,7.3) O CH 3 57.4, CH 3 3.32 f 57.0, CH 3 3.33 f a Deduced from HSQC, 600 MHz. b 600 MHz. c Refers to symplostatin 6 ( 2 ). d Refers to symplostatin 9 ( 5 ). e Not determined, predicted to have comparable chemical shifts based on highly homologous structures f Overlapping with residual water.

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89 Table 3 3. NMR data of s ymplostatin 7 ( 3 ) and s ymplostatin 10 ( 6 ) in DMSO d 6 Symplostatin 7 Symplostatin 10 unit C/H no C a H ( J in Hz) b C a H ( J in Hz) b Ile 1 e e 2 54.1, CH 4.88, br d 53.9, CH 4.88, m 3 37.0, CH 1.88, m 3 6.9, CH 1.86, m 4a 26.0, CH 2 1.30, m 25.8, CH 2 1.29, m 4b 1.12, m 1.12, m 5 11.3, CH 3 0.92, t (7.2) 11.9, CH 3 0.91, t (7.4) 6 14.4, CH 3 0.71, d (6.7) 14.2, CH 3 0.70, d (6.8) NH 7.44, br s 7.38, br s N Me Phe c / 1 e e N Me Tyr d 2 60.3, CH 5 .01, br d 60.6, CH 4.90, m 3a 33.6,CH 2 3.24, br d ( 13.4) 32.5, CH 2 3.11, d ( 14.2) 3b 2.85, m 2.70, dd ( 14.2,11.8) 4 e e 5/9 129.5, CH 7.24, d (7.6) 130.2, CH 6.99, d (8.3) 6/8 128.4, CH 7.40, m 115.1, CH 6.76, d (8.3) 7 126.6, CH 7.31, m N Me 30.2, CH 3 2.79, s 30.0, CH 3 2.74, s OH 9.31, br s Phe 1 e e 2 49.9, CH 4.72,m 49.9, CH 4.72, dd (11.4,4.8) 3a 35.0, CH 2 2.85, m 35.0, CH 2 2.87, ( 14.3,12.3) 3b 1.69, m 1.79, m 4 e e 5/9 129.2, CH 6.83, d (7.4) 129.2, CH 6.83 d (7.2) 6 127.6, CH 7.19, m 127.6, CH 7.19, m 7 126.1, CH 7.16, m 126.0, CH 7.14, m Ahp 2 e e 3 47.9, CH 3.77, m 47.9, CH 3.77, m 4a 21.8, CH 2 2.39, m 21.8, CH 2 2.39, m

PAGE 90

90 Table 3 3. Continued Symplostatin 7 Sym plostatin 10 unit C/H no C a H ( J in Hz) b C a H ( J in Hz) b 4b 1.57, m 1.57, m 5a 29.1, CH 2 1.71, m 29.1, CH 2 1.71, m 5b 1.55, m 1.55, m 6 73.5, CH 5.06, s 73.5, CH 5.06, br s OH 6.04, s 6.03, s NH 7.35, br s 7.35, br s Abu 1 e e 2 e e 3 131.4, CH 6.48, q (7.1) 131.4, CH 6.48, q (7.2) 4 12.8, CH 3 1.46, d (7.1) 12.8, CH 3 1.46, d (7.2) NH 9.22, br s 9.22, br s Thr 1 e e 2 55.0, CH 4.68, m 55.0, CH 4.68, m 3 71.6, CH 5.52, br s 71.6, CH 5.52, br s 4 17.5, CH 3 1.21, d (6.5) 17.5, CH 3 1.21, d (6.3) NH 8.24, br s 8.19, br s Ile 1 e e 2 55.8, CH 4.48, m 55.8, CH 4.48, m 3 36.9, CH 1.86, m 36.9, CH 1.85, m 4a 23.7, CH 2 1.43, m 23.7, CH 2 1.43, m 4b 1.06, m 1.06, m 5 10.6, CH 3 0.80, t (7.4) 10.6, CH 3 0.8 0, t (7.4) 6 15.0, CH 3 0.85, d (6.5) 15.0, CH 3 0.85, d (6.8) NH 7.75, br s 7.73, br s 2 O CH 3 1 e e Glyceric Acid 2 79.9, CH 3.96, dd (7.5,3.4) 79.9, CH 3.96, dd (7.3,3.2) 3a 66.1, CH 2 3.88, dd ( 10.8,3.4) 66.1, CH 2 3.88, dd ( 10.7,3.2) 3b 3 .72, dd ( 10.8,7.5) 3.72, dd ( 10.7,7.3)

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91 Table 3 3. Continued Symplostatin 7 Symplostatin 10 unit C/H no C a H ( J in Hz) b C a H ( J in Hz) b OCH 3 57.1, CH 3 3.31 f 57.1, CH 3 3.31 f a Deduced from HSQC, 600 MHz. b 600 MHz. c Refers to symplostatin 7 ( 3 ). d Refers to symplostatin 10 ( 6 ). e Not determined, predicted to have comparable chemical shifts based on highly homologous structures f Overlapping with residual water.

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92 Table 3 4 Antiproteolytic activity of Abu containing c yclic d epsipep tides from m arine c yanobacteria a Compound Porcine Pancreatic Elastase b IC 50 (nM) Human Neutrophil Elastase c IC 50 (nM) Bovine Pancreatic Chymotrypsin d IC 50 (nM) Human Pancreatic Chymotrypsin e IC 50 (nM) (% Activity at 10 M) Symplostatin 5 68 9.7 144 2.9 322 3.2 > 10000 (53.4 3.2) Symplostatin 6 89 11 121 12 503 65 > 10000 (90.6 7.6) Symplostatin 7 77 5.4 195 28 515 43 > 10000 (70.7 3.8) Symplostatin 8 43 3.2 41 9.0 268 11 > 10000 (69.0 2.0) Symplostatin 9 37 3.1 28 5.8 324 27 > 10000 (73.9 1.0) Symplostatin 10 44 1.5 21 2.9 222 5.1 > 10000 (79.4 3.2) Lyngbyastatin 4 4 1 2.0 49 1.4 614 6.3 > 10000 (72.2 3.3) Lyngbyastatin 7 30 6.8 23 1.1 314 37 2000 Sivelestat 2810 95 136 18 4084 37 > 10000 (55.1 3.3) a Data are presented as mean SD (n = 3). b e Substrates. b N succinyl Ala Ala Ala p nitroanil ide. c N (methoxysuccinyl) Ala Ala Pro Val p nitroanilide. d N succinyl Gly Gly Phe p nitroanilide. e N succinyl Ala Ala Pro Phe p nitroanilide.

PAGE 93

93 Table 3 5 Non inflammatory e lastase inducible g enes Probe ID Symbol Annotation Fold induction a % Reduction b Transcription Factors 203394_s_at HES1 H airy and enhancer of split 1 (Drosophila) 2.98 58 c 215898_at TTLL5 T ubulin tyrosine ligase like family, member 5 2.96 62 c 215191_at KDM2A L ysine (K) specific demethylase 2A 2.50 54 215470_at GTF2H2 G eneral tr anscription factor IIH, polypeptide 2, 44kDa 2.32 47 243561_at YAF2 YY1 associated factor 2 2.18 47 230791_at NFIB N uclear factor I/B 2.12 50 232865_at AFF4 AF4/FMR2 family, member 4 2.10 49 c 1556462_a_at KLF12 Kruppel like factor 12 1.93 47 c 23243 1_at NR3C1 N uclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) 1.91 53 c 240008_at ARID1B AT rich interactive domain 1B (SWI1 like) 1.89 54 c Other Targets 232528_at NA NA 3.67 59 c 238774_at KIAA1267 KIAA1267 3.62 64 219995_s_a t ZNF750 Z inc finger protein 750 3.62 68 c 230332_at ZCCHC7 Z inc finger, CCHC domain containing 7 3.30 67 c 235847_at ZFAND3 Z inc finger, AN1 type domain 3 3.29 60 c 23149_at EIF4G3 E ukaryotic translation initiation factor 4 gamma 3 3.22 59 1564378_a_a t EXT1 E xostoses (multiple) 1 3.20 67 c 234989_at NEAT1 N on protein coding RNA 84 3.08 48 c 241457_at FBXL7 F box and leucine rich repeat protein 7 3.06 64 c 242476_at NA NA 2.95 64 c 207746_at POLQ P olymerase (DNA directed), theta 2.46 62 c 1559360_at EFNA5 E phrin A5 2.43 63 c 1554638_at ZFYVE16 Z inc finger, FYVE domain containing 16 2.24 60 c 1563075_s_at NA NA 2.21 59 c 224917_at MIR21 M icroRNA 21 2.08 43 c a Relative to control, P < 0.05. b In response to inhibitor cotreatment. c Significant difference with inhibitor treatment, P < 0.05.

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94 Table 3 6. Relevant g enes i nvolved in NOD and MAPK s ignaling p athways s ignificantly m odulated by e lastase Probe ID Symbol Annotation Fold Induction a % Reduction b 39402_at IL1B Interleukin 1B 2.91 58 c 241786_at PPP 3R1 Protein phosphatase 3, regulatory subunit B 2.42 56 c 205207_at IL6 Interleukin 6 2.26 22 1569540_at NLK Nemo like kinase 2.23 49 239409_at RAP1A RAP1A, member of RAS oncogene family 2.21 53 c 230337_at SOS1 Son of sevenless homolog 1 1.89 46 c 21011 8_at IL1A Interleukin 1A 1.85 34 1565889_at TAB2 Mitogen activated kinase kinase 7 interacting protein 1.83 42 211506_at IL8 Interleukin 8 1.53 22 a Relative to control, P < 0.05 b In response to inhibitor cotreatment. c Significant difference with inhibi tor treatment, P < 0.05.

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95 Table 3 7. Symplostatin 5 ( 1 ) inducible g enes p otentially i ndependent of e lastase a Probe ID Symbol Annotation P value Fold I nduction b 243598_at GPD2 G lycerol 3 phosphate dehydrogenase 2 (mitochondrial) 0.03 2.15 242824_at NFIA N uclear factor I/A 0.03 2.02 229728_at NA NA 0.03 2.01 236545_at PPP3CA P rotein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform 0.04 1.97 233037_at NA NA 0.03 1.96 242696_at NUDCD3 NudC domain containing 3 0.05 1.94 226840_at H2AFY H2A h istone family, member Y 0.04 1.88 236685_at NA NA 0.04 1.79 1553145_at FLJ39653 H ypothetical FLJ39653 0.05 1.67 a These genes were not significantly affected by elastase treatment. b Relative to control

PAGE 96

96 Table 3 8 Reaction c onditions for p rotease a ssay s Protease Substrate [Sub] Ex/Em max Buffer a ACE1 MCA RPPGFSAFK(Dnp) 10 320/405 A Activated Protein C (H) in 50% gly Boc DVLR ANSNH C4H9 50 355/460 C ADAM9 MCA PLAQAV Dpa RSSSR NH3 10 320/405 I ADAM10 MCA PLAQAV Dpa RSSSR NH3 10 320/405 I BACE1 MCA SEVNLDAEFRK(Dnp) RR NH2 10 320/405 J Calpain 1 Biomol, N Succinyl Leu Tyr AMC 10 355/460 K Caspase 1 Ac LEHD AMC 5 355/460 G Caspase 2 Ac LEHD AMC 5 355/460 G Caspase 3 Ac DEVD AMC 5 355/460 F Caspase 4 Ac LEHD AMC 5 355/460 G Caspase 5 Ac LEHD AMC 5 355/460 G Caspase 6 A c LEHD AMC 5 355/460 G Caspase 7 Ac DEVD AMC 5 355/460 F Caspase 8 Ac LEHD AMC 5 355/460 G Caspase 9 Ac LEHD AMC 5 355/460 G Caspase 10 Ac LEHD AMC 5 355/460 G Caspase 11 Ac LEHD AMC 5 355/460 G Caspase 14 Ac LEHD AMC 5 355/460 G Cathepsin B Z FR AM C 5 355/460 L Cathepsin C Z FR AMC 5 355/460 L Cathepsin D MCA KPILFFRLK(Dnp) D R NH2 10 320/405 P Cathepsin E MCA KPILFFRLK(Dnp) D R NH2 10 320/405 P Cathepsin G Suc AAPF AMC 10 355/460 C Cathepsin H R AMC 10 355/460 M Cathepsin K Z GPR AMC 10 355/4 60 C Cathepsin S Z FR AMC 10 355/460 M Cathepsin V Z FR AMC 10 355/460 E Cathepsin X/Z MCA RPPGFSAFK(Dnp) 10 320/405 D Chymase Suc AAPF AMC 10 355/460 C Chymotrypsin (Human Pancreatic) Suc AAPF p Na 3 405 U Chymotrypsin (Bovine Pancreatic) Suc GGF p Na 1.5 405 T Complement Component C1s (CCC1s) Dabcyl SLGRKIQI EDANS 10 340/490 A DPP IV H GP AMC 10 355/460 H DPP VIII H GP AMC 10 355/460 H DPP IX H GP AMC 10 355/460 H

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97 Table 3 8 Continued Protease Substrate [Sub] Ex/Em max Buffer a Elasta se (Human Neutrophil) (OMeSuc) AAPV p Na 2 405 V Elastase (Porcine Pancreatic) Suc AAA p Na 2 405 S Factor VIIa Z VVR AMC 10 355/460 A Factor Xa CH 3 SO 2 D CHA Gly Arg AMC AcOH 10 355/460 N Factor XIa (Boc Glu(OBzl) Ala Arg) MCA 10 355/460 A Granzyme B Ac IEPD AMC 10 355/460 A Hepatitis C virus NS3/4A protease Anaspec EnzoLyte (Hylite Biosciences, Catalogue: 22991) 10 340/490 R Kallikrein 1 Z GPR AMC 10 355/460 A Kallikrein 5 Z VVR AMC 10 355/460 A Kallikrein 8 VPR AMC 10 380/460 E Kallikrein 12 VPR A MC 10 380/460 B Kallikrein 13 VPR AMC 10 380/460 A Kallikrein 14 VPR AMC 10 380/460 A MMP1 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP2 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP3 (5 FAM/QXLTM) FRET peptide 5 485/520 H 3 7 MMP7 (5 FAM/QXLTM) FRET pepti de 5 485/520 H MMP8 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP9 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP10 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP11 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP12 (5 FAM/QXLTM) FRET peptide 5 485/520 H MMP13 (5 FAM /QXLTM) FRET peptide 5 485/520 H MMP14 (5 FAM/QXLTM) FRET peptide 5 485/520 H Papain Z FR AMC 10 355/460 M Plasma Kallikrein Z FR AMC 10 380/460 A Plasmin H D CHA Ala Arg AMC.2AcOH 10 355/460 A Proteinase K H D CHA Ala Arg AMC.2AcOH 10 355/460 A TA CE MCA PLAQAV Dpa RSSSR NH 2 10 320/405 I Thrombin alpha H D CHA Ala Arg AMC.2AcOH 10 355/460 O Ti ssue Plasminogen Activator Z GPR AMC 10 355/460 Q Trypsin H D CHA Ala Arg AMC.2AcOH 10 355/460 A Tryptase beta 2 Z GPR AMC 10 355/460 A Tryptase gamma 1 Z GPR AMC 10 355/460 A Urokinase Bz b Ala Gly Arg AMC.AcOH 10 355/460 A

PAGE 98

98 Table 3 8 Continued a Buffers A 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35 B 50 mM Tris pH 7.5, 10 mM CaCl 2 150 mM NaCl, 0.05% Brij35 C 25 mM Tris pH 9, 150 mM NaCl D 25 mM Sodium Acetate pH 3.5, 5 mM DTT E 25 mM Sodium Acetate pH 5.5, 0.1 M NaCl, 5 mM DTT F 50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% CHAPS, 0.1 mM EDTA, 10 mM DTT G 50 mM HEPES pH 7.4, 1 M sodium citrate, 100 mM NaCl, 0.01% CHAPS, 0.1 mM EDTA, 10 mM DTT H 5 0 mM HEPES pH 7.5, 100 mM CaCl 2 0.01% Brij35, store at 4C, add 0.1 mg/mL BSA before use I 2 0.005% Brij J 0.1 M Sodium acetate, pH 4.0 K 75 mM Tris pH 7.0, 0.005% Brij35, 3 mM DTT, 0.5 mM CaCl 2 L 25 mM MES pH 6.0, 50 mM NaCl, 0.005% Brij35, 5 mM DTT M 75 mM Tris pH 7.0, 1 mM EDTA, 0.005% Brij35, 3 mM DTT N 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 0.25 mg/mL BSA O 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 2.5 mM CaCl 2 1.0 mg/mL BSA P 0.1 mM Sodium Acetate pH 3.5, 0.1 M NaCl Q 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 1.0% BSA R Assay kit Buffer S 1.0 M Tris pH 8.0 T 50 mM Tris pH 7.8, 100 mM NaCl, 1 mM CaCl 2 U 0.1 M Tris pH 8.3, 25 mM CaCl 2 V 0.1 M Tris pH 7.5, 0.5 M NaCl

PAGE 99

99 Table 3 9 Crystallogra phy d ata and r efinement s tatistics PDB Title Elastase/Lyngbyastatin 7 complex PDB ID 4GVU Data collection Space group P2 1 2 1 2 1 Cell dimensions A,b c () ( = 90) 53.40 57.42 74.33 Resolution () 30.00 1.55 (1.56 1.55) Total reflect ions 257532 Unique reflections 33573 (699) R merge 0.044 (0.55) I 41.1 (2.0) Completeness (%) 99.6 (84.0) Redundancy 7.7 (4.9) Mean Mosaicity ( ) 0.47 Wilson B factor (A 2 ) 20.6 Matthews coefficient (A 3 Da 1 ) 2.18 Solvent content (%) 43.7 Refinem ent Resolution () 28.71 1.55 (1.60 1.55) No. reflections 33518 R work/ R free (%) 17.6 / 20.6 (27.2 / 33.0) No. atoms : All 2139 Protein 1822 SO 4 / Ca / Lyngbyastatin7 5 / 1 / 68 Water 243 B factors ( 2 ) : All 24.4 Protein : all / main / si de 23.2 / 21.9 / 24.6 SO 4 / Ca / Lyngbyastatin7 29.1 / 21.1 / 21.6 Water 34.0 RMSD Bond lengths () 0.013 Bond angles () 1.709 Ramachandran plot % residues Favored 85.4 Additional 14.6 Generously allowed 0 Disallowed 0 Numbers in parenthes es refer to the highest resolution shell.

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100 CHAPTER 4 VERAGUAMIDES A G: CYTOTOXIC CYCLIC HEXADEPSIPEPTIDES WITH A C 8 P OLYKETIDE DERIVED HYDROXY ACID MOIETY FROM CETTI BAY G UAM Introduction Marine cyanobacteria have provided both structurally diverse an d potent antiproliferative compounds with varying mechanisms of action as lead structures for drug discovery. 20, 21 Most of these are products of nonribosomal peptide synthetases or mixed nonri bosomal peptide synthetases and polyketide synthases to yield cyclic and linear modified peptides and depsipeptides. Cyanobacteria utilize mainly nonpolar and neutral proteinogenic amino acids as building blocks, commonly Val, Ala, Phe, Tyr, Pro, and Ile. 20 These proteinogenic amino acids may be further modified by N or O methylation, halogenation, or epimerization to yield the unnatural D amino acids Amino aci ds such as Cys or Ser can undergo cycloaddition with o ther amino acids to yield heterocyclic moieties such as thiaz oline/thiazole or oxazoline/oxazole rings. 120, 121 Marine cyanobac hydroxy acids amino acids as building blocks of the peptide polyketide hybrid compounds In addition, fatty acid type moieties consisting of four to twelve carbons in length are also a signature polyketid e derived r esidue in marine cyanobacteria, and oftentimes bear a mono carbon position and decorated by a terminal alkyne, alke ne, or halogenated alkyne functionality. 20, 122 125 A recent survey of cyanobacteria metabolites containing a 2,2 dimethyl 3 hydroxy 7 octynoic acid (Dhoya) or a 3 hydroxy 2 methyl 7 octynoic acid suggested that the se lipopeptides are widely Reproduced in part with permission from Salvador, L. A.; Biggs, J. S.; Paul, V. J.; Luesch, H. J. Nat. Prod 2011 74 917 927. Copyright 2011 American Chemical Society.

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101 distributed and may be products of an ancient biosynthetic pathway common across different cyanobacteria genera. 126 Thus, this class of secondary metabolites from marine cyanobacteria cle Incorporation of unnatural amino acids as well as polyketide derived units aside from increasing the structural diversity, is also postulated to contribute to the stability of this class of compound against hydrolytic cleavage. 127 From the screening profile of several cyanobacteria collection, we prioritized a Symploca cf. hydnoides from Cetti Bay, Guam for discovery of new antiproliferative agents. This col lection showed antiproliferative activity, with the active principle not related to the known compounds dolastatin 10, symplostatin 1 or largazole. Presented herein is the cytotoxicity directed fractionation of this S cf. hydnoides collection from Cetti B ay, Guam, which afforded the known compound dolastatin 16, 128 together with seven new cyclic depsipeptides, given the trivial names veraguamides A G ( 7 13 ). The trivial names were assigned to conform with the na ming by W. Gerwick and co workers, who concurrently isolated members of this compound class. 129 Initial structure activity relationship studies and effects on cancer cell populations of the veraguamides are also discus sed Isolation and Structure Elucidation The freeze dried Symploca sp. cyanobacterium from Cetti Bay, Guam was extracted with EtOAc MeOH (1:1). This extract showed antiproliferative activity at a concentration from initial profiling data This extract was further solvent partitioned into hexanes n BuOH, and H 2 O soluble fractions, with the n BuOH soluble fraction being the most cytotoxic This fraction was further purified by silica column chromatography, the

PAGE 102

102 fraction eluting with 20% i PrOH in CH 2 Cl 2 showed characteristic 1 H NMR resonances for peptides and modified peptides and potent antiproliferative activity. Reversed phase HPLC purif ication of this silica fraction yielded veraguamides A G ( 7 1 3 ) (Figure 4 1 ). HRESIMS of the major compound in this ser ies, veraguamide A ( 7 ) (Figure 4 1 ), showed the distinctive 1:1 isotopic cluster for a Br containing compound for the [M + H] + peak at m /z 767.3675/769.3660, suggesting a molecular formula of C 37 H 59 BrN 4 O 8 The 1 H NMR spectrum of 7 displayed characteristic peptide resonances for a secondary amide proton ( H 6.25), two tertiary amide N CH 3 H 3.00, H protons ( H 3.85 4.95). 2D NMR analysis (Table 4 1) in CDCl 3 using HSQC, COSY, TOCSY, and HMBC established the presence of four amino acids (Pro, Val, 2 N Me hydroxy acid [(2 hydroxy 3 methylpentanoic acid (Hmpa)]. The last spin system consisted of a CH 3 do ublet ( H 1.25) that showed a COSY correlation to a methine ( H 3.11) and HMBC correlations to a carbonyl ( C 170.8) and an oxymethine ( C 76.4). Further extension of this unit using HMBC and COSY established the presence of a 8 bromo 3 hydroxy 2 methyl 7 octynoic acid (Br Hmoya) moiety in 7 This was supported by HMBC correlations of the methylene ( C 6 19.2/ H 6 2.23) with two quaternary carbons at C 38.4 and C 79.3 and by the large difference in chemical shifts between these quaternary carbons charact e ristic for an alkynyl bromide. 130 The linear sequence of N Me Val 1 Pro Hmpa N Me Val 2 Val Br Hmoya was established prot ons and carbonyl groups (Table 4 1 ) and was verifie d by MS/MS fragmentation (Figure 4 2 ). The deshielded C 3 methine of the Br Hmoya unit suggested acylation with the carbonyl of N Me Val 1 to form a cyclic

PAGE 103

103 hexadepsipeptide, corroborated by HMBC and consistent with the molecular formula requirements based on HRESIMS. Veraguamide B ( 8 ) showed a 1:1 isotopic pattern for the pseudomolecular ion [M + H] + at m/z 753.3517/755.3508, suggesting the presence of a Br as in 7 with a negative difference of 14 amu corresponding to one less CH 2 unit and thus a molecula r formula of C 36 H 57 BrN 4 O 8 Comparison of the 1 H NMR spectrum of 7 and 8 showed differences in the splitting pattern in the CH 3 region at H 0.93 ppm and the chemical proton ( H hydroxy acid (Table 4 2). The vicinal methine ( H hydroxy acid showed COSY correlations to two methyl groups ( H 0.93, H 1.02) instead of COSY correlations to methylene and methyl protons in 7 Therefore, 8 possesses a 2 hydroxyisovaleric acid (Hiva) instead of the Hmpa unit as in 7 (Figu re 4 1 ). The HRESIMS spectrum of veraguamide C ( 9 ) showed a negative deviation of 79 amu compared with 7 which indicated the lack of Br and a molecular formula of C 37 H 60 N 4 O 8 This was supported by the absence of the 1:1 isotopic pattern for the [M + H] + p eak when compared to 7 The 1 H NMR spectrum of 9 showed an additional triplet at H 1.93 with J H,H = 2.5 Hz (Table 4 2); otherwise it was virtually identical to that of 7 This proton correlated to a methine at C 68.8 and a quaternary C ( C 83.6) in the HSQC and HMBC spectra, respectively. These signals are indicative of a terminal al kyne; hence 9 had to bear a 3 hydroxy 2 methyl 7 octynoic acid (Hmoya) moiety in lieu of Br Hmoya present in 7 and 8 (Figure 4 1 ). Veraguami de D ( 10 ) appeared closely related to 9 as its 1 H NMR spectrum showed the acetylenic proton at H 1.93 (Table 4 3). In comparison to 9 the HRESIMS

PAGE 104

104 of 10 showed a positive difference of 14 amu, corresponding to an additional CH 2 unit and in agreement with a molecular formula of C 38 H 62 N 4 O 8 The 1 H NMR and HSQC spectra of 10 showed a high field CH 3 at C 11.4/ H 0.73, ch aracteristic of an Ile or Ile derived moiety. COSY ( H / H 0.93/1.46, 1.46/2.02, 2.02/4.01) and HMBC correlations ( C / H 15.8/4.01, 28.7/4.01) correlations (Table 4 3) established that the N Me Val 1 residue is replaced by an N Me Ile in veraguamide D ( 10 ) (Figure 4 1 ). Compound 11 (C 39 H 64 N 4 O 8 ) exhibite d a close relationship to both 9 and 10 showing a positive deviation of 28 amu and 14 amu, respectively, and also having the Hmoya moiety. The NMR data of 11 (Table 4 3) indicated the presence of two high fi eld methyl ( C 11.7/ H 0.96, C 11.5/ H 0.85) and additional methylene ( C 26.6/ H 1.57, 1.06; C 23.9/ H 1.47, 1.05) groups in comparison with 9 which suggested that two isopropyl groups in the latter are replaced with sec buty l groups in the former (Figure 4 1 ). Th is is further corroborated by HMBC and COSY correlations (Table 4 3), which established the replacement of Val and N Me Val 2 moieties with Ile and N Me Ile, respectively, in veraguamide E ( 11 ). The N Me Val residue that was replaced by N Me Ile was loc ate d at different positions in 10 and 11 ; with N Me Val 1 replaced in the former and N Me Val 2 in the latter. This NMR result was verified by MS/MS fragmentation of both 10 and 11 (Figure 4 2 ). The 1 H NMR spectrum of veraguamide F ( 10 ) (Table 4 4) showed add itional resonances for aromatic protons at H 7.2 7.4 ppm, upfield shifted N Me protons to H 2.60 ppm presumably due to the shielding by the aromatic ring, and a low proton of the hydroxy acid ( H 5.47), with the acetylenic proton still present ( H 1.93). COSY correlations of H 5.47 to diastereotopic CH 2 protons at H 3.17/ H 2.91, together with

PAGE 105

105 HMBC correlations of the latter to aromatic carbons at C 136.2/ C 129.3 (Table 4 4) established the presence of phenyllactic acid (Pla) as t hydrox y acid in 12 (Figure 4 1 ). These NMR derived conclusions fulfilled the molecular formula requirements for C 40 H 58 N 4 O 8 based on HRESIMS of 12 Veraguamide G ( 13 ) lacked the acetylenic signal ( C 68.8/ H 1.93) observed for 9 12 and instead showed downfield re sonances of a terminal methylene ( C 114.9/ H 4.97) and a methine ( C 138.2/ H 5.74) (Table 4 5) These signals indicated that the terminal alkyne group of the C 8 polyketide derived moiety is replaced by a terminal vinyl group (Figure 4 1 ). This conclusion was further supported by the positive deviation of 2 amu compared to 9 and a molecular formula of C 37 H 62 N 4 O 8 Hence, the Hmoya unit present in 9 12 was replaced by 3 hydroxy 2 methyl 7 octenoic acid (Hmoea) in 13 Enantioselective HPLC analysis coupled w ith mass spectrometry or UV detection of the acid hydrolysates of 7 12 allowed us to assign the absolute configuration of all hydroxy acid components as L and S respectively. To determine the absolute configuration at C 2 and C 3 of the Br Hmoya unit, veraguamide A ( 7 ) was subjected to methanolysi s to y ield the linear fragment 1 5 (Figure 4 3 ). The observed coupling constant of 3.2 Hz was characteristic for a syn configuration, whereas a coupling constant near 6.3 Hz would have been expected for the anti configuration. 131 The absolute configuration at C 3 and consequently f or C 2 of the Br Hmoya unit of 15 values (Figure 4 3 ) predicted an R configuration at C 3 and hence from the relative configuration, C 2 should have an S configuration. Of note, comparison of the 3 J H,H values of H 2 and H 3 with a model system to assign the relative configuration could only be applied when C 3 bears a free

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106 hydroxy group. This moiety is involved in intramolecular hydrogen bonding with the adjacent carbonyl group, thus hindering free bond rotation across C 2 and C 3. 132, 133 Accordingly, the corresponding MTPA esters ( 1 6 17 ) did not show the same 3 J H,H values for H 2 and H 3 as that of 15 The same absolute configuration at C 2 and C 3 for Hmoya, Hmoea and 3 hydroxy 2 methyl octanoic acid (Hmoaa) is expecte d based on virtually identical 13 C NMR shifts and specific optical rotations observed for 7 13 Biological Activity Studies To gain insight into structure activity relationships, veraguamide A ( 7 ) was partially (Lindlar catalyst, H 2 ) and fully (Pd/C, H 2 ) h ydrogenated to yield the semisynthetic veraguamide G ( 13 ) and tetrahydroveraguamide A ( 14 ) (Figure 4 1 ), respectively. The cytotoxic activities of 7 1 4 and semisynthetic veraguamide G ( 13 ) were evaluated for effects on viability of HT29 colorectal and HeLa cervical adenocarcinoma cells (Table 3 6). The IC 50 values of the natural and semisynthetic veraguamide G ( 13 ) were comparable suggesting the activities of these compounds were not likely due to traces of highly biologically active impurities. The most ac tive in this series of compounds are veraguamides D ( 10 ) and E ( 11 ), with IC 50 values more than 5 fold lower than those for their related congener veraguamide C ( 9 ). This suggested that increased hydrophobicity of specific units (II, IV, V, VI) increased t he cytotoxicity of this compound class, with the position having minimal effect on the bioactivity as 10 and 11 showed comparable IC 50 s. However, modification with bulkier groups is detrimental to the activity, as exemplified by a close to 10 fold decrease in the cytotoxic activity of 12 (Table 4 6) compared to 9 where a phenyllactic acid (Pla) moiety is introduced at position IV of the former instead of the Hmpa unit. The C 8 polyketide derived moiety also plays a role in the cytotoxicity of these compound s. Comparing the

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107 biological activities of related compounds 7 9 13 and 14 weaker cytotoxicity was observed for compounds with a Br Hmoya or Hmoaa unit, while compounds with Hmoya or Hmoea were about equally potent. This then suggests the importance of a system combined with the presence of acetylenic or vinylic protons in this moiety for cytotoxic activity. In order to gain insights on the possible mode of cell death mediated by veraguamides, cell cycle analysis by flow cytometry was performed. HeLa an d HT29 cells were treated with 1.0, 3.2, and ( 1 0 ) for 24 h, permeabilized with EtOH and stained with propidium iodide. A dose dependent increase in cell populations at sub G1 and G2 were observed with veraguamide D ( 1 0 ) (Figure 4 4 ). Th e observed change in cell populations was however, incremental. This further suggested that the veragu am ides are not likely to act as antimitotics. Antimitotic agents such as the dolastatins, paclitaxel and vinca alkaloids cause a dramatic increase in cel ls at G2/M. 134 The result of the cell cycle analysis also corroborates the moderate antiproliferative activity observed using the MTT assay. The veraguamides are reminiscent of other cyanobacterial compounds such as hantupeptins, 133, 135 antanapeptins, 124 and trungapeptins. 125 These compounds are also cyclic hexadepsipeptides with the characteristic C 8 polyketide derived units as Hmoya, Hmoea, or Hmoaa. Veraguamide F ( 1 2 ) is a constitutional isomer of antanapeptin D, 124 where an N Me Phe and Hiva are prese nt in the latter instead of Pla and N Me Val as in 12 It is interesting that subtle changes in structure of these compounds have a profound effect on the cytotoxicity. Antanapeptins A D (brine shrimp) as well as trungapeptin A (KB and LoVo cells) did not display cytotoxicity at the reported

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108 concentr 124, 125 while hantupeptins A C were cytotoxic against MOLT 4 leukemia and MCF7 breast cancer cells, with hantupeptin A being the most active in this series with IC 5 0 133, 135 Trungapeptins B and C wer e not tested for cytotoxicity 125 Conclusion Cytotoxicity d irected purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cycli c depsipeptides, veraguamides A G ( 7 13 ), together with the known compound dolastatin 16. The planar structures of 7 13 were elucidated using NMR and MS e xperiments, while analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of th e stereocenters. Veraguamides A G ( 7 13 ) are characterized by the presence of an invariant p roline residue, multiple N methylated amino acids, an R hydroxy acid, and a C 8 polyketide hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue ( 14 ) s howed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound cl ass. Additional studies are required to elucidate the mechanism of action of the veraguamides. Experimental Methods Biological Material The Symploca cf. hydnoides cyanobacterium was collected by hand while snorkeling in the shallow wate rs of the southern fore reef (1 3 m) of Cetti Bay, Guam,

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109 on April 17, 2009. A voucher specimen, which is preserved in 100% EtOH, is deposited in the University of Guam Herbarium (accession no. GUAM GH11446). A voucher specimen is also r etained at the Smithsonian Marine Station, Fort Pierce, FL. Extraction and Isolation The freeze dried cyanobacterium (14 2.0 g) was extracted with EtOAc MeOH (1:1) to yield the nonpolar extract (11.6 g). This was partitioned between hexanes and 20% aqueous MeOH, the latter concentrated under reduced pressure and further partitioned between n BuOH and H 2 O. The n BuOH fraction w as concentrated to dryness (2.8 g) and chromatographed on Si gel eluting first with CH 2 Cl 2 followed by increasing concentrations of i PrOH; after 100% i PrOH, increasing gradients of MeOH were used. The 20% i PrOH fraction was subjected to a C18 SPE eluting with 25%, 50%, 75%, and 100% MeOH in H 2 O. The 100% MeOH fraction was purified by semipreparative reversed phase HPLC (Phenomenex S ynergi rate, 2.0 mL/min) using a linear gradient of MeOH H 2 O (70% 100% MeOH in 60 min and then 100% MeOH for 10 min) to yield dolastatin 16 ( t R 32.6 min, 21.6 mg), semipure veraguamide C ( t R 36.4 min, 15.1 mg), semipure veraguamide F ( t R 37.4 min, 10.0 mg), a mixture of veraguamides B and D ( t R 40.0 min, 25.0 mg), veraguamide A ( 7 ) ( t R 42.6 min, 25.9 mg), and a mixture of veraguamides E and G ( t R 43.7 min, 9.0 mg). The final purification of the semipure veraguamide C ( 9 ) was achieved u sing semipreparative HPLC (Phenomenex Phenyl a linear gradient of MeOH H 2 O (85% 100% MeOH in 40 min and then 100% MeOH for 5 min) to yield veraguamide C ( 9 ) ( t R 19.9 min, 10.7 mg). Using the same chromatographic co nditions, purification of the semipure veraguamide F yielded veraguamide F ( 12 ) ( t R 21.7 min, 6.8 mg). The mixture of veraguamides B and D was

PAGE 110

110 resolved using the same chromatographic condition with a different linear gradient (70% 100% MeOH in 45 min and t hen 100% MeOH for 10 min) to yield veraguamide D ( 10 ) ( t R 36.6 min, 4.0 mg) and veraguamide B ( 8 ) ( t R 37.3 min, 11.5 mg). The mixture of veraguamides E and G was further purified using the same chromatographic conditions to yield veraguamide G ( 13 ) ( t R 39. 9 min, 4.4 mg) and veraguamide E ( 11 ) ( t R 40.5 min, 3.6 mg). Hydrogenation of 7 A catalytic amount of 10% Pd/C was added to a methanolic solution of 7 (1.8 mg/mL). The reaction was left to stir for 6 h under a hydrogen balloon. The catalyst was filtered t hrough a Celite pad, and the filtrate, upon concentration, was purified by semipreparative HPLC (Phenomenex Phenyl a linear gradient of MeOH H 2 O (70% 100% MeOH in 45 min and then 100% MeOH for 10 min) to yield 14 ( t R 40.9 min, 1.2 mg). Partial hydrogenation of 7 was carried out with Lindlar catalyst, using the same reaction and chromatographic conditions stated above. This afforded the semisynthetic veraguamide G ( t R 39.9 min, 1.7 mg). The LRESIMS and 1 H NMR spectr a of the semisynthetic veraguamide G were in good agreement with the spectra for the natural product ( 13 ). Veraguamide A ( 7 ): colorless, amorphous solid; [ ] 20 D 44 ( c 0.44, MeOH); UV (MeOH); max (log ) 202 (6.29); 1 H NMR 13 C NMR, COSY, and HMBC data, see Table 4 1; HRESIMS m/z 767.3675 [M + H] + (calcd for C 37 H 60 79 BrN 4 O 8 767.3594), m/z [M + H] + 769.3660 (calcd for C 37 H 60 81 BrN 4 O 8 769.3574) (100:100 [M + H] + ion cluster). Veraguamide B ( 8 ): colorless, amorphous solid; 20 D 40 ( c 0.16, MeOH); UV (MeOH); max (log ) 202 (4.30); 1 H NMR and 13 C NMR data, see Table 4 2; HRESIMS

PAGE 111

111 m/z 753.3517 [M + H] + (calcd for C 36 H 58 79 BrN 4 O 8 753.3438), m/z [M + H] + 755.350 8 (calcd for C 36 H 58 81 BrN 4 O 8 755.3418) (100:100 [M + H] + ion cluster). Veraguamide C ( 9 ): colorless, amorphous solid; 20 D 44 ( c 0.31, MeOH); UV (MeOH); max (log ) 202 (4.17); 1 H NMR and 13 C NMR data, see Table 4 2; HRESIMS m/z 689.4486 [M + H] + (calc d for C 37 H 61 N 4 O 8 689.4490). Veraguamide D ( 10 ): colorless, amorphous solid; 20 D 57 ( c 0.11, MeOH); UV (MeOH); max (log ) 202 (4.30); 1 H NMR and 13 C NMR data, see Table 4 3; HRESIMS m/z 703.4639 [M + H] + (calcd for C 38 H 63 N 4 O 8 703.4646). Veraguamide E ( 11 ): colorless, amorphous solid; 20 D 56 ( c 0.22, MeOH); UV (MeOH); max (log ) 202 (4.30); 1 H NMR and 13 C NMR data, see Table 4 3; HRESIMS m/z 717.4799 [M + H] + (calcd for C 39 H 65 N 4 O 8 717.4802). Veraguamide F ( 12 ): colorless, amorphous solid; 20 D 41 ( c 0.13, MeOH); UV (MeOH); max (log ) 206 (4.33); 1 H NMR and 13 C NMR data, see Table 4 4; HRESIMS m/z 723.4411 [M + H] + (calcd for C 40 H 59 N 4 O 8 723.4333). Veraguamide G ( 13 ): colorless, amorphous solid; 20 D 48 ( c 0.17, MeOH); UV (MeOH); max (lo g ) 202 (4.26); 1 H NMR and 13 C NMR data, see Table 4 5; HRESIMS m/z 691.4649 [M + H] + (calcd for C 37 H 63 N 4 O 8 691.4646). Tetrahydroveraguamide A ( 14 ): colorless, amorphous solid; 20 D 43 ( c 0.05, MeOH); UV (MeOH); max (log ) 202 (4.33); 1 H NMR and 13 C NMR data, see Table 4 5; HRESIMS m/z 693.4791 [M + H] + (calcd for C 37 H 65 N 4 O 8 693.4802). Acid Hydrolysis of Veraguamides and Enantioselective Analysis Portions of 7 13 f 6 N HCl, 110 C, 20 h), and the product mixtu 2 O, and analyzed by enantioselective HPLC UV and enantioselective HPLC MS. The absolute configurations

PAGE 112

112 of the amino acids N Me Ile, Ile, N Me Val, Val, and Pro were determined by enantioselective HPLC MS [column, Chir obiotic TAG (250 4.6 mm), Supelco; solvent, MeOH 10 mM NH 4 OAc (40:60, pH 5.30); flow rate, 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The acid hydrolysates of 7 1 0 12 and 13 showed retention times at 7.8, 11.6, and 13.6 min corres ponding to L Val, N Me L Val, and L Pro, respectively. The acid hydrolysate of 1 0 in addition showed a retention time at 12.4 min, corresponding to N Me L Ile. The acid hydrolysate of 11 showed retention times at 8.4, 11.6, 12.4, and 13.6 min, correspondin g to L Ile, N Me L Val, N Me L Ile, and L Pro, respectively. The retention times ( t R min; MRM ion pair) of the authentic amino acids were as follows: N Me L 86), N Me D Val (34.3), L Val (7.8; 118 72), D Val (13.7), N Me L Ile (12.4; 146 10 0), N Me L allo Ile (15.0), N Me D Ile (49.0), N Me D allo Ile (51.0), L Ile (8.4; 132 86), L allo Ile (8.6), D allo Ile (17.6), D Ile (20.2), L Pro (13.6; 116 70), D Pro (36.0). Compound dependent parameters used were as follows: N Me Val: DP 29.4, EP 4.2 CE 17.4, CXP 2.7, CEP 10.6; Val: DP 5.7, EP 9.0, CE 40.0, CXP 8.0, CEP 10.0; N Me Ile: DP 35.0, EP 7.0, CE 17.0, CXP 2.0, CEP 10.0; Ile: DP 40.0, EP 9.0, CE 15.0, CXP 3.0, CEP 8.0; Pro: DP 35.0, EP 7.7, CE 22.7, CXP 5.0, CEP 10.3. Source gas parameters u sed were as follows: CUR 40, CAD Medium, IS 4500, TEM 750, GS1 65, GS2 65. The absolute configurations of the R hydroxy acids [2 hydroxyisovaleric acid (Hiva), 2 hydroxy 3 methylpentanoic acid (Hmpa), and phenyllactic acid (Pla)] were determined using enan tioselective HPLC [column, CHIRALPAK MA ( + ) (50 4.6 mm); solvent, CH 3 CN 2 mM CuSO 4 (10:90); flow rate, 1.0 mL/min; detection by UV (254 nm)]. The acid hydrolysates of 7 9 11 and 13 each showed peaks at 33.0 min, corresponding to (2 S ,3 S ) Hmpa. The acid hydrolysate of 8

PAGE 113

113 contained a component that had a retention time at 10.0 min, corresponding to (2 S ) Hiva, while 12 gave a peak at 51.0 min, corresponding to (2 S ) Pla. The retention times of the authentic standards were as follows: (2 R ) Hiva (6.0), (2 S ) Hiv a (10.0), (2 R ,3 S ) Hmpa (16.0), (2 R ,3 R ) Hmpa (19.0), (2 S ,3 R ) Hmpa (26.0), (2 S ,3 S ) Hmpa (33.0), (2 R ) Pla (33.5), (2 S ) Pla (51.0). All other amino acid units eluted within less than 5.0 min using this chromatographic condition. Methanolysis of 7 Compound 7 (5 .0 mg) was dissolved in 2.0 mL of 5% (w/w) methanolic KOH solution and stirred for 24 h at room temperature. The solvent was evaporated and the residue was partitioned between CH 2 Cl 2 and H 2 O. The organic layer was collected, dried over anhydrous MgSO 4 and concentrated to dryness under nitrogen. The crude methanolysis product was further purified by semipreparative reversed phase HPLC (Phenomenex Synergi MeOH H 2 O (70% 100% MeOH in 60 min and t hen 100% MeOH for 10 min) to yield 15 ( t R 19.3 min, 1.4 mg). 15 : colorless, amorphous solid; 1 H NMR (CDCl 3 ) 6.30 (d, J = 8.1 Hz, 1H), 4.92 (d, J = 10.5 Hz, 1H), 4.76 (dd, J = 9.1, 6.6 Hz, 1H), 3.78 (ddd, J = 8.6, 4.4, 3.2 Hz, 1H), 3.49 (s, 3H), 3.08 (s, 3H), 2.42 (qd, J = 6.8, 3.2 Hz 1H), 2.23 (m, 2H), 2.03 (m, 1H), 1.74 (m, 2H), 1.56 (m, 1H), 1.46 (m, 2H), 1.01 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); HRESIMS m/z 497.1640 [M + Na] + (calcd for C 21 H 35 79 BrN 2 O 5 Na, 497.1627), m / z [M + Na] + 499.1616 (calcd for C 21 H 35 81 Br N 2 O 5 Na, 499.1607) (100:100 [M + H] + ion cluster).

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114 Preparation of MTPA Esters of 15 The methanolysis product 15 3 and was divided into two equal portions and to each was added 0.75 mL triethylamine. To one portion was R ) MTPA S ) MTPA Cl to give the ( S ) MTPA ester ( 1 6 ) and ( R ) MTPA est er ( 1 7 ), respectively. Each reaction was N N dimethylaminopropylamine was added to quench the reactions. The reaction products were dried under N 2 and applied ont o silica SPE eluting with EtOAc hexanes (1:1). The semip ure product was further purified by semipreparative HPLC (Phenomenex Phenyl a linear gradient of MeOH H 2 O (70% 100% MeOH in 45 min and then 100% MeOH for 10 min) to yield 1 6 ( t R 38.0 min, 0.1 mg) or 1 7 ( t R 37.8 min 0.1 mg). 1 6 : colorless, amorphous solid; 1 H NMR (CDCl 3 ) 7.57 (dd, J = 6.4, 2.7 Hz, 2H), 7.41 (m, 3H), 6.14 (d, J = 9.2 Hz, 1H), 5.28 (q, J = 6.9 Hz, 1H), 4.93 (d, J = 10.6 Hz, 1H), 4.68 (dd, J = 9.1, 7.6 Hz, 1H), 3.69 (s, 3H), 3.58 (s, 3H), 3.06 (s, 3H ), 2.46 (quintet, J = 7.1 Hz 1H), 2.21 (m, 1H), 2.15 (t, J = 6.8 Hz, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.47 (m, 2H), 1.09 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H); HRESIMS m/z 729.1746 [M + K] + (calcd for C 31 H 42 79 BrF 3 N 2 O 7 K, 729.1759), m / z [M + K] + 731.1747 (calcd for C 31 H 42 81 BrF 3 N 2 O 7 K, 731.1755) (100:100 [M + K] + ion cluster); LRESIMS m/z 691/693 (100:100 [M + H] + ion cluster), 713/715 (100:100 [M + Na] + ion cluster). 1 7 : co lorless, amorphous solid; 1 H NMR (CDCl 3 ) 7.56 (dd, J = 4.3, 3.6 Hz, 2H), 7.41 (m, 3H), 6.27 (d, J = 8.3 Hz, 1H), 5.27 (q, J = 6.0 Hz, 1H), 4.94 (d, J = 10.4 Hz, 1H), 4.73 (dd, J = 9.1, 7.5 Hz, 1H), 3.69 (s, 3H), 3.57 (s, 3H), 3.07 (s, 3H), 2.54 (quintet,

PAGE 115

115 J = 6.7 Hz, 1H), 2.22 (m, 1H), 2.08 (td, J = 7.2, 2.9 Hz, 2H), 2.00 (m, 1H), 1.63 (m, 1H), 1.31 (m, 2H), 1.19 (d, J = 6.7 Hz, 3H), 1.01 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 7.0 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H); HRESI/APCIMS m/z 691 .2202 [M + H] + (calcd for C 31 H 43 79 BrF 3 N 2 O 7 691.2206), m / z [M + H] + 693.2186 (calcd for C 31 H 43 81 BrF 3 N 2 O 7 693.2186) (100:100 [M + H] + ion cluster). Biological Activity Assays Cell viability assay HT29 colorectal adenocarcinoma and HeLa cervical carcinoma c ells were 10% fetal bovine serum (FBS, Hyclone) under a humidified environment with 5% CO 2 at 37 C. HeLa (3,000) and HT29 (12,500) cells were seeded in 96 well plates and t reated with varying concentrations of test samples and solvent control (DMSO) after 24 h of seeding. The cells were incubated for an additional 48 h before the addition of the MTT tions (Promega). IC 50 calculations were done by GraphPad Prism 5.03 based on duplicate experiments. Paclitaxel was used as positive control. Cell cycle analysis by flow c ytometry HeLa (75,000) and HT29 (200, 000) cells were seeded 24 h prior to treatment i n 6 well dishes and kept under a humidified environment with 5% CO 2 at 37 C. At the end of the incubation time, the growth medium was replaced prior to sample treatment. Cells were incubated with incre asing concentrations of 10 for 24 h, with DMSO and pa clitaxel as the solvent and positive control, respectively. Cells were harvested at the end of the 24 h incubation by trypsinization, followed by centrifugation at 4 C. The supernatant was discarded and the cell pellet was recovered for further use. Singl e cell

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116 suspensions were prepared in PBS and subsequently permeabilized by dropwise addition of EtOH. Cells were centrifuged, resu spended in PBS, containing 1.0 mM EDTA and 1 00 g/mL RNAse A and stained with propidium iodide. Cells were sorted using a FACS can (Becton Dickson) based on the fluorescence of the propidium iodide DNA complex.

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117 Figure 4 1 Structures of veraguamides A G ( 7 13 ) and the semisynthetic tetrahydroveraguamide A ( 14 )

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118 Figure 4 2 MS/MS fragmentation of veraguamide A ( 7 ), veragu amide D ( 10 ), and veraguamide E ( 11 ).

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119 Figure 4 3 Assignment of absolute configuration of veraguamide A ( 7 ) using = (S MTPA ester) (R MTPA ester).

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120 Figure 4 4 Cell cycle analysis of HT29 and HeLa cells treated with varying concentrations of veraguamide D ( 10 ) Dose dependent increase in sub G1 and G2 cell populations was observed.

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121 Table 4 1. NMR d ata for v eraguamide A ( 7 ) in CDCl 3 unit C/H no C a H ( J in Hz) b COSY b HMBC b Br Hmoya 1 170.8, C 2 42.4, CH 3.11, br q (7.4) H 3,H 3 9 1, 3, 4, 9 3 76.4, CH 4.85, dt (10.2, 2.5) H 2, H 4a, H 4b 1, 1 ( N Me Val 1) 4a 27.4, CH 2 2.06, m H 3, H 4b, H 5a, H 5b 5, 6 4b 1.59, m H 3, H 4a, H 5a, H 5b 5a 25.0, CH 2 1.60, m H 4a, H 4b, H 5b, H 2 6 7, 8 5b 1.41, m H 4a, H 4b, H 5a, H 2 6 6, 8 6 19.2, CH 2 2.23, m H 5a, H 5b 7, 8 7 38.4, C 8 79.3, C 9 14.1, CH 3 1.25, d (7.4) H 2 1, 2, 3 N Me Va l 1 1 170.6, C 2 65.0, CH 3.93, d (10.3) H 3 1, 3, 4, N Me, 1 (Pro) 3 28.3, CH 2.30, m H 2, H 3 4, H 3 5 2, 4, 5 4 19.56, CH 3 0.98, d (6.6) H 3 2 5 19.51, CH 3 0.91, d (6.6) H 3 2 N Me 28.6, CH 3 3.00, s 2, 1 (Pro) Pro 1 172.1, C 2 57.3, C H 4.94 dd (8.9, 4.5) H 3a, H 3b 1, 3, 4, 1 (Hmpa) 3a 29.5, CH 2 2.30, m H 2, H 3b, H 4a 1, 2, 5 3b 1.79, m H 2, H 3a, H 4a 1, 2, 5 4a 24.9, CH 2 2.03, m H 3a, H 3b, H 4b, H 5a, H 5b 2, 3, 5 4b 1.98, m H 4a, H 5a, H 5b 2, 3, 5 5a 47.3, CH 2 3.84, d t ( 17.0, 7.1) H 4a, H 4b, H 5b 2, 3, 4 5b 3.61, dt ( 17.0, 7.1) H 4a, H 4b, H 5a 3, 4

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122 Table 4 1 Continued unit C/H no C a H ( J in Hz) b COSY b HMBC b Hmpa 1 165.9, C 2 76.1, CH 4.90, d (9.1) H 3 1, 3, 4, 5, 1 ( N Me Val 2) 3 35.7, CH 1.97, m H 2, H 3 6 4a 24.81, CH 2 1.54, m H 4b, H 3 5 4b 1.13, m H 4a, H 3 5 5 10.5, CH 3 0.87, t (7.3) H 4a, H 4b 3, 4 6 13.8, CH 3 1.01, d (6.8) H 3 2, 3, 4 N Me Val 2 1 169.6, C 2 66.0, CH 4.15, d (9.5) H 3 1, 3, 5, N Me, 1 (Val) 3 28.5, CH 2.27, m H 2, H 3 4, H 3 5 1 4 20.4, CH 3 1.00, d (7.0) H 3 2 5 20.2, CH 3 1.11, d (7.0) H 3 2, 3 N Me 30.0, CH 3 2.94, s 2, 1 (Val) Val 1 173.4, C 2 52.8, CH 4.70, dd (8.7, 6.5) H 3, NH 1, 3, 4, 5, 1 (Br Hmoya) 3 32.1, CH 1.96, m H 2, H 3 4, H 3 5 5 4 20.3, CH 3 0.95, d (6.7) H 3 2, 3, 5 5 17.5, CH 3 0.88, d (6.7) H 3 2, 3 NH 6.25, d (8.7) H 2 1, 1 (Br Hmoya) a 10 0 MHz b 600 MHz

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123 Table 4 2. NMR d ata for v eraguamide B ( 8 ) and v eraguamide C ( 9 ) in CDCl 3 Veraguamide B Veraguamide C unit C/H no. C a H ( J in Hz) b C a H ( J in Hz) b Br Hmoya c / 1 170.8, C 170.8, C Hmoya d 2 42.3, CH 3.13, br q (7.4) 42.4, CH 3.10, b r q (7.2) 3 76.4, CH 4.85, d (8.7) 76.4, CH 4.86, dt (10.4, 2.5) 4a 27.5, CH 2 2.07, m 27.4, CH 2 2.07, m 4b 1.60, m 1.62, m 5a 24.93, CH 2 1.61, m 25.2, CH 2 1.62, m 5b 1.42, m 1.44, m 6 19.2, CH 2 2.21, m 18.0, CH 2 2.19, m 7 38.4, C 83.6, C 8 79.4, C 68.8, CH 1.93 t (2.5) 9 14.6, CH 3 1.25, d (7.4) 14.5, CH 3 1.25, d (7.2) N Me Val 1 1 170.7, C 170.7, C 2 65.0, CH 3.94, d (10.4) 65.0, CH 3.93, d (11.0) 3 28.26, CH 2.29, m 28.3, CH 2.28, m 4 19.57, CH 3 0.98, d (6.5) 19.58, CH 3 0. 98, d (6.8) 5 19.55, CH 3 0.92, d (6.5) 19.56, CH 3 0.91, d (6.8) N Me 28.7, CH 3 3.00, s 28.7, CH 3 3.00, s Pro 1 172.1, C 172.2, C 2 57.2, CH 4.95 dd (8.6, 4.8) 57.3, CH 4.94 dd (8.4, 5.0) 3a 29.4, CH 2 2.28, m 29.5, CH 2 2.28, m 3b 1.79, m 1.79 m 4a 24.99, CH 2 2.04, m 24.89, CH 2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH 2 3.80, dt ( 16.4, 6.9) 47.3, CH 2 3.84, dt ( 16.8, 7.1) 5b 3.60, dt ( 16.4, 6.9) 3.60, dt ( 16.8, 7.1) Hiva c /Hmpa d 1 165.8, C 165.9, C 2 77.2, CH 4.85, d (8.7) 76.0 CH 4.89, d (9.4) 3 29.6, CH 2.17, m 35.7, CH 1.98, m

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124 Table 4 2. Continued Veraguamide B Veraguamide C unit C/H no. C a H ( J in Hz) b C a H ( J in Hz) b 4 18.1, CH 3 1.02, t (6.6) 24.81, CH 2 1.54, m 1.13, m 5 18.5, CH 3 0.93, d (6.6) 10.5, CH 3 0.86, t (7.3) 6 13.8, CH 3 1.01, d (6.7) N Me Val 2 1 169.6, C 169.6, C 2 66.1, CH 4.15, d (10.2) 66.0, CH 4.13, d (10.0) 3 28.34, CH 2.28, m 28.5, CH 2.28, m 4 20.3, CH 3 0.99, d (6.8) 20.4, CH 3 0.99, d (6.4) 5 20.1, CH 3 1.11, d (6.8) 20.2, CH 3 1.10, d (6.4) N Me 30.0, CH 3 2.94, s 30.0, CH 3 2.93, s Val 1 173.5, C 173.4, C 2 52.8, CH 4.71, dd (8.6, 6.4) 52.8, CH 4.70, dd (8.6, 6.2) 3 32.2, CH 1.98, m 32.1, CH 1.96, m 4 20.3, CH 3 0.94, d (6.4) 20.3, C H 3 0.94, d (6.7) 5 17.5, CH 3 0.88, d (6.4) 17.6, CH 3 0.87, d (6.7) NH 6.26, d (8.6) 6.26, d (8.6) a 100 MHz b 600 MHz c Refers to v eraguamide B d Refers to v eraguamide C

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125 Table 4 3. NMR d ata for v eraguamide D ( 10 ) and veraguamide E ( 11 ) in CDCl 3 Ver aguamide D Veraguamide E unit C/H no. C a H ( J in Hz) b C a H ( J in Hz) b Hmoya 1 170.8, C 170.71, C 2 42.4, CH 3.13, br q (7.2) 42.4, CH 3.08, br q (7.4) 3 76.4, CH 4.86, dt (10.8, 2.6) 76.5, CH 4.85, d (9.0) 4a 27.5, CH 2 2.07, m 27.5, CH 2 2.06, m 4b 1.63, m 1.62, m 5a 25.2, CH 2 1.63, m 25.2, CH 2 1.61, m 5b 1.47, m 1.43, m 6 17.5, CH 2 2.18, m 18.0, CH 2 2.18, m 7 83.6, C 83.6, C 8 68.8, CH 1.93, t (2.5) 68.8, CH 1.93, t (2.3) 9 14.4, CH 3 1.24, d (7.2) 14.5, CH 3 1.23, d (7.4) N Me Ile c / 1 170.7, C 170.69, C N Me Val d 2 64.0, CH 4.01, d (10.6) 64.9, CH 3.93, d (10.0) 3 34.6, CH 2.02, m 28.3, CH 2.29, m 4 25.7, CH 2 1.46, m 19.59, CH 3 0.91, d (6.4) 5 11.4, CH 3 0.93, t (6.5) 19.56, CH 3 0.98, d (6.4) 6 15.8, CH 3 0.94, d (6.8) N Me 28.7, CH 3 2.99, s 28.6, CH 3 3.00, s Pro 1 172.7, C 172.2, C 2 57.2, CH 4.94 dd (8.9, 4.8) 57.3, CH 4.94 dd (9.0, 5.3) 3a 28.8, CH 2 2.26, m 29.5, CH 2 2.29, m 3b 1.78, m 1.78, m 4a 24.9, CH 2 2.03, m 24.89, CH 2 2.01, m 4b 1.97, m 1.99, m 5a 47.2, CH 2 3.82, dt ( 17.0 7.3) 47.3, CH 2 3.86, dt ( 17.0, 7.0) 5b 3.60, dt ( 17.0, 7.3) 3.60, dt ( 17.0, 7.0) Hmpa 1 165.9, C 166.0, C 2 76.0, CH 4.90, d (9.2) 76.1, CH 4.85, d (9.0)

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126 Table 4 3. Continued Veraguamide D Veraguamide E uni t C/H no. C a H ( J in Hz) b C a H ( J in Hz) b 3 35.7, CH 1.98, m 35.1, CH 1.98, m 4 24.8, CH 2 1.53, m 24.86, CH 2 1.54, m 1.12, m 1.13, m 5 10.5, CH 3 0.86, t (7.6) 10.5, CH 3 0.86, t (7.0) 6 13.9, CH 3 0.99, d (6.9) 13.8, CH 3 1.01, d (6.8) N Me Val c / 1 169.6, C 169.7, C N Me Ile d 2 66.0, CH 4.15, d (9.4) 65.2, CH 4.22, d (9.6) 3 28.4, CH 2.28, m 35.7, CH 1.98, m 4 20.3, CH 3 1.10, d (6.8) 26.6, CH 2 1.54, m 1.06, m 5 20.2, CH 3 0.99, d (6.8) 11.7, CH 3 0.96, t (7.2) 6 16.5, CH 3 1.04, d (6.9) N Me 30.0, CH 3 2.92, s 30.1, CH 3 2.93, s Val c /Ile d 1 173.4, C 173.5, C 2 52.8, CH 4.70, dd (8.6, 6.6) 52.4, CH 4.70, dd (8.4, 6.7) 3 32.1, CH 1.98, m 38.6, CH 1.69, m 4 19.0, CH 3 0.94, d (6.6) 23.9, CH 2 1.47, m 5 17.6, CH 3 0. 87, d (6.6) 1.05, m 11.5, CH 3 0.85, d (6.6) 6 16.3, CH 3 0.91, d (6.6) NH 6.24, d (8.6) 6.26, d (8.4) a 125 MHz b 600 MHz c Refers to v eraguamide D d Refers to v eraguamide E

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127 Table 4 4. NMR d ata for v eraguamide F ( 12 ) in CDCl 3 unit C/H no. C a H ( J in Hz) b Hmoya 1 170.9, C 2 42.2, CH 3.24 br q (7.3) 3 76.6, CH 4.89, dt (10.6, 2.4) 4a 27.5, CH 2 2.08, m 4b 1.64, m 5a 25.2, CH 2 1.65, m 5b 1.45, m 6 18.0, CH 2 2.20, m 7 83.5, C 8 68.9, CH 1.93, t (2.5) 9 14.6, CH 3 1.29, d ( 7.3) N Me Val 1 1 170.7, C 2 65.3, CH 3.95, d (10.4) 3 28.3, CH 2.32, m 4 19.6, CH 3 1.00, d (6.5) 5 19.7, CH 3 0.93, d (6.5) N Me 28.7, CH 3 3.06, s Pro 1 172.3, C 2 57.1, CH 4.97, dd (9.0, 4.5) 3a 29.1, CH 2 2.27, m 3b 1.83, m 4a 25.0, CH 2 2.07, m 4b 1.97, m 5a 47.0, CH 2 3.62, m 5b 3.56, m Pla 1 165.4, C 2 72.8, CH 5.47, dd (9.8, 4.0) 3 36.7, CH 2 3.17, m

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128 Table 4 4. Continued unit C/H no. C a H ( J in Hz) b 2.91, m 4 136.2, C 5//9 129.3, CH 7.18, d (8.0) 6/8 128.5, CH 7.28, m 7 126.7, CH 7.20, m N Me Val 2 1 168.9, C 2 65.8, CH 4.05, d (10.5) 3 27.4, CH 2.08, m 4 19.8, CH 3 0.89, d (6.8) 5 20.0, CH 3 0.91, d (6.8) N Me 29.0, CH 3 2.60, s Val 1 173.4, C 2 52.7, CH 4.76, dd (8.5, 6.1) 3 32.3, CH 1.98, m 4 20.3, CH 3 0.91, d (6.6) 5 17.7, CH 3 0.88, d (6.6) NH 6.28, d (8.5) a 100 MHz b 600 MHz

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129 Table 4 5. NMR d ata for v eraguamide G ( 13 ) and t etrahydroveraguamide A ( 14 ) in CDCl 3 Veraguamide G Tetrahydroveraguamide A unit C/H no. C a H ( J in Hz) b C b,c H ( J in Hz) b Hmoea d / 1 170.9, C 170.7, C Hmoaa e 2 42.4, CH 3.10, br q (7.4) 42.1, CH 3.08, br q (7.4) 3 76.8, CH 4.85, dt (10.6, 2.4) 76.8, CH 4.86, dt (10.1, 2.1) 4a 27.9, CH 2 1.98, m 31.0, CH 2 1.21, m 4b 1.45, m 1.26, m 5a 25.5, CH 2 1.48, m 28.2, CH 2 1.39, m 5b 1.30, m 6a 33.2, CH 2 2.05, m 25.8, CH 2 1.39, m 6b 1.20, m 7 138.2, CH 5.74, m 22.2, CH 2 1.26, m 8 114.9, CH 2 4.97, m 13.6, CH 3 0.85, t (6.9) 9 14.4, CH 3 1.22, d (7.4) 14.0, CH 3 1.23, d (7.4) N Me Val 1 1 170.7, C 170.7, C 2 65.0, CH 3.93, d (9.8) 64.9, CH 3.93, d (10.7) 3 28.3, CH 2.28, m 28.3, CH 2.28, m 4 19.59, CH 3 0.98, d (6.4) 19.2, CH 3 0.98, d (6.5) 5 19.54, CH 3 0.92, d (6.4) 19.3, CH 3 0.91, d (6.5) N Me 28.6, CH 3 3.01, s 2 8.4, CH 3 3.00, s Pro 1 172.2, C 172.0, C 2 57.3, CH 4.95, dd (8.7, 5.0) 57.1, CH 4.94, dd (9.0, 5.0) 3a 29.4, CH 2 2.29, m 29.1, CH 2 2.28, m 3b 1.79, m 1.79, m 4a 24.9, CH 2 2.03, m 24.6, CH 2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH 2 3.84, d t ( 16.7, 7.1) 47.0, CH 2 3.84, dd ( 17.0, 7.3) 5b 3.61, dt ( 16.7, 7.1) 3.60, dd ( 17.0, 7.3) Hmpa 1 165.9, C 165.7, C

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130 Table 4 5. Continued Veraguamide G Tetrahydroveraguamide A unit C/H no. C a H ( J in Hz) b C b,c H ( J in Hz) b 2 76.0, CH 4.90, d (8.7) 76.6, CH 4.90, d (8.8) 3 35.7, CH 1.98, m 35.4, CH 1.98, m 4 24.8, CH 2 1.54, m 24.5, CH 2 1.54, m 1.13, m 1.13, m 5 10.5, CH 3 0.86, t (7.3) 10.2, CH 3 0.86, t (7.1) 6 13.8, CH 3 1.00, d (6.0) 13.5, CH 3 1.00, d (6.4) N Me Val 2 1 169.6, C 169.5, C 2 66.0, CH 4.15, d (10.2) 65.8 CH 4.14, d (9.6) 3 28.6, CH 2.28, m 28.1, CH 2.28, m 4 20.4, CH 3 1.00, d (6.1) 20.0, CH 3 0.99, d (6.6) 5 20.2, CH 3 1.10, d (6.1) 19.9, CH 3 1. 10, d (6.6) N Me 30.0, CH 3 2.93, s 29.7, CH 3 2.93, s Val 1 173.4, C 173.3, C 2 52.7, CH 4.70, dd (8.6, 6.7) 52.5, CH 4.70, dd (8.6, 6.2) 3 32.1, CH 1.98, m 31.7, CH 1.96, m 4 20.3, CH 3 0.93, d (6.8) 19.3, CH 3 0.93, d (6.3) 5 17.6, CH 3 0.86, d (6.8) 17.2, CH 3 0.87, d (6.3) NH 6.23, d (8.6) 6.26, d (8.6) a 125 MHz b 600 MHz c Based on HSQC and HMBC d Refers to v eraguamide G e Refers to t etrahydroveraguamide A

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131 Table 4 6. Antiproliferative a ctivity (IC 50 M) of n atural and semisynthetic v eragua mides a Compound HT29 HeLa Veraguamide A ( 7 ) 26 3.1 21 0.8 Veraguamide B ( 8 ) 30 2.4 17 1.0 Veraguamide C ( 9 ) 5.8 0.8 6.1 1.0 Veraguamide D ( 10 ) 0.84 0.09 0.54 0.01 Veraguamide E ( 11 ) 1.5 0.09 0.83 0.06 Veraguamide F ( 12 ) 49 12 4 9 1.4 Veraguamide G ( 13 ) 2.7 0.7 2.3 0.9 Tetrahydroveraguamide A ( 14 ) 33 0.2 48 2.5 a Data are presented as mean SD (n = 2).

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1 32 CHAPTER 5 CAYLOBOLIDE B AND AMANT E LIDES A AND B: ANTIPROLIFERATIVE POLYKETIDES FROM MARINE CYANOBACTERIA Introdu ction Secondary metabolites assembl ed solel y by polyketide synthases represent a minor fraction of isolated compounds from the phylum Cyanobacteria. These usually polyhydroxylated compounds are reminiscent of secondary metabolites from dinoflagellates 136 such as the cytotoxic amphidinolides, amphidinols, and luteophanols as well as bacteria de rived antibiotics desertomycins 137 and oasomycins. 138 Polyketides from marine and terrestrial cyanobacteria also possess interesting biological activities and may be decorated with unusual moieties. Tolytoxin and the related scytophycins, produced by terrestrial cyanob acteria are pote nt cytotoxins. 139 Tolytoxins are distinguished by an epoxide substituent in their back bone structure. Oscillariolide, 140 a polyketide isolated from the genus Oscillato ria inhibited the development of fertilized echinoderm eggs, suggestive of its effects on cell division. Phormidolide, 141 a compound related to oscillariolide, was isolated from the genus Phormidium and is a lso a potent cytotoxin. Both oscillariolide and phormidolide macrocycles contain a tetrahydrofuran ring and a terminal vinyl bromide appended to their ring stru cture. In addition, one hydroxy group in phormidolide is esterified with a C 16 carboxylic acid. The well studied marine cyanobacterium Lyngbya majuscula afforded the polyketide caylobolide A, which is characterized by its contiguous pentad of 1,5 diols. 142 Reproduced with permission from Salvador, L. A.; Paul V. J.; Luesch, H. J. Nat. Prod 2010 73 1606 1609. Copyright 2010 American Chemical Society. Reproduced with permission from J. Nat. Prod ., submitted for publication. Unpublishe d work copyright 2013 American Chemical Society.

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133 The structure elucidation of polyketides is part icularly challenging due to difficulty in establishing the relative and absolute configuration of the multiple stereocenters and substantial overlap in the methylene region. Their configurational assignment has greatly benefited from the development of Kis 143 145 as 146 and ex tensions of this method, 147 although applications still have certain limitations, particularly for those bearing 1, n diol ( n 5) moieties. Assignment of the configuration of 1, n diols has so far been demonstrated on model systems using exciton coupling CD after derivatization with arylcarboxylate chromophores within liposomes. 148 Here we report the isolation, structure elucidation, and antiproliferative ac tivity of three related polyketides characterized by a polyhydroxylated macro cyle bearing a pendant alkyl side chain, given the names caylobolide B ( 18 ) and amantelides A and B ( 19 20 ) from Floridian Phormidium spp. and a Guamanian gray cyanobacterium co llections, respectively. Isolation and Structure Elucidation Caylobolide B (18) A freeze dried sample of an assemblage of Phormidium cf. dimorph um and Phormidium inundatum from Key West, Florida was extracted with EtOAc MeOH (1:1). This extract was cytoto xic at a concentration of 100 ng/mL and contained symplostatin 1 based on the HPLC MS profiling. The nonpolar extract was solvent partitioned to yield the hexanes n BuOH and H 2 O soluble fractions. The n BuOH fraction was cytotoxic and was subjected to a bioactivity guided isolation using silica gel chromatography and reversed phase HPLC to yield caylobolide B ( 18 ) (Figure 5 1 ). The major cytotoxic activity was attributed to the known compound symplostatin 1 based on comparison of

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134 LRESIMS and 1 H NMR wit h literature values. Symplostatin 1 gave an IC 50 of ~1.5 nM against HT29 cells. However, because our cyanobacterial collection was largely a binary mixture of two different Phormid um species, it is unclear if caylobolide B ( 18 ) and the co isolated cytotoxi n symplostatin 1 were produced by the same or both species. Caylobolide B ( 18 ) was isolated as a colorless, amorphous solid with molecular formula of C 42 H 80 O 11 based on pseudomolecular ion peaks observed by HRESI/APCIMS at m/z 761.5767 [M + H] + and m/z 78 3.5594 [M + Na] + Fragmentation of the [M + H] + peak using positive ionization showed repetitive loss of 18 amu, corresponding to elimination of H 2 O typical for alcohols. The structure of 18 was determined by NMR analysis in DMSO d 6 The presence of exchan geable hydroxy protons was evident from the lack of HSQC correlations for nine protons which resonate at H 4.2 4.6 ppm. Detailed interpretation of HSQC, TOCSY, HSQC TOCSY and HMBC experiments with 18 (Table 5 1 Figure 5 1 ) established that the hydroxy groups are part of methine carbinols that form a highly oxygenated backbone structure consisting of a 1,3 d iol system (C 7, C 9), a 1,3,5 triol system (C 25, C 27, C 29) an d repeating 1,5 diol moieties. Degenerate 1 H and 13 C NMR chemical shifts were observed for three oxygenated methines at C 69.6 (C 13, C 17, C 21), seven methylenes at C 37.3 (C 12, C 14, C 16, C 18, C 20, C 22, C 24), and two methylenes at C 21.6 (C 15, C 19) that make up the contiguous chain of 1,5 diol. The 13 C NMR chemical shifts are in good agreement with reported values for 1,5 diol units of luteophanol. 1 49 These degenerate signals together with HSQC TOCSY correlations (Figure 5 2 ) between C 37.3/ H 4.20 and C 21.6/ H 4.20 supported the 1,5 diol substitution pattern. HSQC TOCSY correlations (Figure 5 2 ) between C 15/9

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135 OH, C 23/25 OH sug gested that the contiguous chain of 1,5 diol is flanked by the 1,3 diol and 1,3,5 triol units. HSQC TOCSY correlations between C 31/29 OH, C 32/29 OH, C 32/33 OH, C 33/H 35 enabled the extension of the polyhydroxylated chain which terminates to form an est er linkage with a carbonyl group at C 165.4 (C 1). The low field chemical shift of H 35 ( H 5.00) due to anisotropy from an unsaturated system and HMBC correlation between C 1/H 35 confirmed the presence of the ester linkage. From HMBC and TOCSY co rrelations of C 35/H 35 (Table 5 1 ), it was evident that C 35 was modified by an isohexyl side chain substitution. An additional unsaturation is present in 18 due to a carbon carbon double bond between C 2 and C 3. HMBC correlations (C 1/H 2, C 3/H 2) and the characteristic chemical shifts for C 2 ( C 116.5) and C 3 ( C 159.4) were suggestive of a polarized carbon carbon double bond, unsaturated ester functionality. HMBC correlations between C 2/H 3 42 and C 3/H 3 The structur e of 18 bears a close resemblance to the 36 membered macrolactone ring present in th e known compound caylobolide A 142 (Figure 5 1 ) and was therefore termed caylobolide B. The C 1 to C 9 portion of these compoun ds presents a major difference, where an additional carbon carbon double bond and a different hydroxylation pattern are present in 18 The isolated 1,3 diol system (C 7 to C 9) is a distinctive feature of 18 instead of a 1,5 diol unit from C 5 to C 9 chai n in caylobolide A. The structure of caylobolide B ( 18 ) was confirmed using ESIMS fragmentation in the negative ionization mode (Figure 5 3 ). It was evident that to the hydroxy groups, similar to fragmen tation pat terns observed for amphidinols. 137

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136 Amantelides A and B (19 20) A gr a y cyanobacterium collected at Amantes Point, Tumon Bay, Guam was extracted with CH 2 Cl 2 MeOH (1:1). The resulting nonpolar extract exhi bit ed antiproliferative activity against HT29 cells at a concentration contain largazole, symplosta tin 1 or dolastatin 10 based on the HPLC MS profiling Solvent partitioning of the nonp olar extract gave the hexanes n BuOH and H 2 O s oluble fractions. The antiproliferative n BuOH fraction was further purified by silica column chromatography, with the bioactivi ty concentrated in the fraction eluting from 70% i PrOH in CH 2 Cl 2 Reversed phase HPLC purification, afforded two related polyketide derived compounds amantelide s A ( 19 ) and B ( 20 ) as bioactiv e constituents The HRESIMS spectrum of amantelide A ( 19 ) suggested a molecular formula of C 44 H 84 O 11 based on the observed pseudomolecular [M + Na] + ion at m/z 811.5927. The three d unsaturated ester based on 1 H and 13 C NM R, HSQC, and HMBC spectra, suggesting the presence of one ring system to fulfill the molecular formula requirements. HMBC correlations with the sp 2 C ( C 16 0.3) were observed for the CH 3 singlet ( H 1.85) and a vinyl group ( H 5.62), with the latter also having long range correlations to a carbonyl group ( C 165.5), confirming unsaturated ester (Figure 5 4, Table 5 2 ). The presence of a n ester functionality was also corroborated by the presence of a low field methine ( C / H 76.6/4.93), which also showed HMBC correlations to C 1 ( C 165.5). In addition, two other spin system consisting of a 1,3 methine carbinol and a tert butyl moiety wer e also deduced. Using COSY, TOCSY and HMBC correlations, a partial structure (Figure 5 5 ) for amantelide A ( 19 ) was derived. This i s reminiscent of the C 1 to C 9 and C 33 to C

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137 40 moieties of caylobolide B ( 18 ) (Fi gures 5 1 5 5 ). However, instead of an is ohexyl pendant side chain, amantelide A ( 19 ) bears a tert butyl moiety (Figures 5 4, 5 5) The overlapping 1 H and 13 C NMR signals only allowed for partial assignment of the structure of 19 Comparison of the 1 H and 13 C chemical shifts of 18 and 19 indicate d that the latt er lacks the distinctive 1,3,5 triol system present in caylobolides A 142 and B ( 18 ) (Table s 5 1, 5 2) Based on the 1 H and 13 C NMR chemical shifts as well as the remaining C 27 H 52 O 6 to be accounte d for from the partial structure and molecular formula of 19 a contiguous chain of 1,5 diol is proposed to form the macrocyclic structure of amantelide A ( 19 ) The observed degenerate 13 C NMR shifts in amantelide A ( 19 ) (Table 5 2) are in accorda nce with literature values for 1 ,5 diols in luteophanols 149 and caylobolides A 142 and B ( 18 ) (Table 5 1) T o verify the proposed structure, MS/MS fragmentation of amantelide A ( 19 ) was done under negative ionization (Figure 5 6) Fragmentations were observed at positions to the methine carbinols (Figure 5 6) and confirmed that amantelide A ( 19 ) has a closely related structure to the caylobolides HRESIMS data for amantelide B ( 20 ) showed pseudomolecular ion [M + Na] + at m/z 853.6044, with a 42 amu mass d ifference with amantelide A ( 19 ), suggesting a molecular formula of C 46 H 86 O 12 1 H and 13 C NMR, HSQC and HM BC spectra of amantelide B ( 20 ) suggested that these compounds belong to the same structural class with an additional acetyl group in amantelide B ( 2 0 ). This was corroborated by a singlet CH 3 ( C / H 20.7/1.97 ) that showed an HMBC correlation to a carbonyl group ( C 170. 0 ) (Table 5 2) This acetyl group is proposed to modify a methine carbinol and is evident from the appearance of a downfield shifted methine ( C / H 73. 3 /4.73) that also showed

PAGE 138

138 an H MBC correlation to the carbonyl at C 170. 0 (Table 5 2) C 7 and C 9 were eliminated as possible sites of acetylation since the characteristic 1 H and 13 C NMR shifts of this moiety can still be clearly discerned (Table 5 2). A TOCSY correlation between H 4 .73 and H 3.2 1 suggested that C 33 bears the additional acetyl group in 20 Hence, amantelide B ( 20 ) is the C 33 monoacetylated analog of amantelide A ( 19 ) (Figure 5 4). Verification by MS/MS fragmentation was, however not possible due to the immediate l oss of the acetyl group upon ionization yielding a similar fragmentation pattern as amantelide A ( 19 ) Amantelides A and B ( 19 20 ) showed similarities to caylobolides A and B ( 18 ), with the presence of a polyhydroxylated macrola c tone ring that is modifie d by a pendant aliphatic side chain. The C 1 to C 21 portions of the macrolactone ring of 1 8 20 are similar with the characteristic 1,3 diol moiety (C 7 to C 9) flanked by a 1,5 diol moiety (C 10 to C unsaturated ester (C 1 to C 3). Compound s 19 and 20 are distinguished by their 1,5 di hydroxylation pattern (C 25 to C 39), as well as a larger 40 membered macrolactone ring instead of a 36 membered macrocycle in caylobolides. Aman t e lides also possess a tert butyl side chain instead of an isohexyl moiety as in the caylobolides. Tert butyl bearing natural prod u cts are rare and present only a small portion of secondary metabolites Among the cyanobacterial metabolites, the cytotoxins apratoxins, 60 64 laingolides, 150, 151 madangoli de 152 and bisebromoamide 153 and the Ca 2+ blocker palmyrolide A 154 bear a tert butyl moiety Configurational Analysis The relative con figuration of selected stereogenic centers of 1 8 was assigned by independently considering the 1,3 diol and 1,3,5 NMR Databas e (Database 2 ). 143 145 The 13 C NMR chemical shift of C 7/C 9 was in good

PAGE 139

139 agreement with syn arrangement of 1,3 diol model system ( Figure 5 7 ) The 1,3,5 triol system was assigned as either syn/anti or anti/syn between C 25/C 27, C 27/C 29 b ased on comparison of C at C 27 with the characteristic C of the central carbon of the 1,3,5 triol model system (Figure 5 7 ). This method cannot differentiate between syn/anti or anti/syn orientation. he absolute configuration and was limited by the low yield of 18 The lack of chemical shift dispersion in the contiguous chain of 1,5 diols in caylobolide B ( 18 ) limits the assignment of the absolute configuration of this moiety as well as those for 19 a nd 20 The absolute configuration of the stereocenters in amantelides A and B ( 19 20 ) was not determined. The relative configuration at C 7/C 9 of amantelides were assigned as syn based on comparison with caylobolide B ( 18 ) (Tables 5 1, 5 2) and also in agreement diols. 143 Biological Activity Studies Antiproliferative Activity Caylobolide B ( 18 ) exhibited moderate cytotoxic activity against HT29 colorectal ade nocarcinoma and HeLa cervical carcinoma cells with IC 50 respectively (Table 5 3) The cytotoxic activity of 18 is comparable to that of caylobolide A against the human colon carcinoma HCT116 cells (IC 50 142 Due to the limited amount o f caylobolide B ( 18 ) and its weak cytotoxic activity, it was not pursued for further biological studies. Amantelide A ( 19 ) showed superior antiproliferative activity in HT29 and HeLa cancer cell lines with submicromolar IC 50 s compared to the caylobolides (Table 5 3). Monoacetylation of a mantelide A ( 19 ) at C 33 however, caused more than 10 fold de crease in anti proliferative activity, as observed for

PAGE 140

140 amantelide B ( 20 ) (Table 5 3) T his the n suggested the role of acetylation and hydroxylation in modulating the an tiproliferative activity of cyanobacterial polyketides belonging to t he caylobolide class In order to gain insight into the role of acetylation in the antiproliferative activity of amantelides, a semisynthetic derivative of 19 was prepared using ac etic anhydride and pyridine to yield the peracetylated amantelide A ( 21 ). Antiproliferative activity testing on 21 indicated that peracetylation caused a dramatic decrease in potency, causing a 20 fold and 67 fold increase in IC 50 in HeLa and HT29 cells, r espectively (Table 5 3) In addition to acetylation of the hydroxy groups, the difference in antiproliferative activities of caylobolides and amantelides may suggest that the size of the macrolide ring, hydroxylation pattern and aliphatic side chain may co ntribute to the antiproliferative activity of these compounds. Elucidation of the Mechanism of Action of Cyanobacterial Polyketides The preliminary SAR for 18 21 suggested that the hydroxy groups of cyanobacterial polyketides are important to the biologic al activity. Time course cell viability analysis of HeLa and HT29 cells treated with amantelide A ( 19 ) indicated that the cellular effects of 19 are observed within 1 h post treatment (Figure 5 8) This then indicate d that t hese compounds may be acting as cell membrane disrupting agents based on the rapid cellular effects of 19 This mechanism of action is observed for amphotericin B, a natural product isolated from Streptomyces nodosus where changes in membrane permeability culminate in the leakage of mo no and divalent ions 155 Close inspection of the structure of amphotericin B and the cyanobacterial polyketides 18 20 indi cated several similarities. C 1 to C 11 of amphotericin B bears close resemblance to C 1 to C 13 of 18 20 The C 35 to C 37 moiety of amphote ricin B is homologous to the C 37 to C 39 of amantelides A and B ( 19 20 ) and C 33 to C 35 of caylobolide B ( 18 )

PAGE 141

141 (Figures 5 1, 5 4) In amphot ericin B, C 35 is a methine carbinol, while C 36 bears a meth yl group, and C 37 is derivatized to an ester which forms the macrocycle. Recent investigations on the mechanism of action of amphotericin B indicated that it binds to ergosterol in yeast cells through the mycosamine moiety and also forms ion channels via the polyhydroxylated portion of the molecule. 155 157 The formation of ion channels by amphotericin B i s postulated to be through the formation of both monom eric and dimeric structures, with C 1 to C 13 and C 35 to C 37 being criticial structural elements. 155 157 The hydroxy group at C 35 is in particular import ant ; suggested to bridge the amphotericin backbone to the lipid bilayer. 155 This critical structural element parallels the observation for amantelides, where the presence of an acetyl group at C 33 caused a decr eased in activity. In order to probe the mechanism of action of 18 20 we utilize d amantelide A ( 19 ) as the mo del compound since it gave the highest potency in the antiproliferative assay and also present in sufficient amounts To verify the proposed mech anism of action of amantelide A ( 19 ), t he antiproliferative activity and cellular phenotype of amphotericin B and amantelide A ( 19 ) treated cells were compared (Figure 5 8) Significant changes i n cell viability were observed for b oth amantelide A ( 19 ) an d amphotericin B tre a ted cells after 1 h (Figure 5 8). Amphotericin B however, induced cell death at a slower rate compared to amantelide A ( 19 ). This is in accordance with the close to 10 fold higher potency of 19 compared to amphotericin B (Table 5 3) in preventing the growth of HT29 and HeLa cancer cells. Based on visual inspection, a mantelide A ( 19 ) and amphotericin B also both induced rapid morphological changes

PAGE 142

142 in HeLa cells, within 1 h of treatment The morphology of amantelide A ( 19 ) and amphoteric in B treated cells we re distinct from control treatments. Conclusion Bioactivity guided p urification of two cyanobacteria colle c tions yielded the closely related polyketide macrolactones caylobolide B ( 18 ) and amantelides A and B ( 19 20 ). The structures of 18 20 were assigned based on 1 H and 13 C NMR HSQC, HMBC, TOCSY and COSY experiments. Compounds 18 20 are characterized by a polyhydroxylated macrocycle modified by an aliphatic pendant side chain. Caylobolide B ( 18 ) is characterized by a 36 membered mac rocycle consisting of 1,3 and 1,5 diol and a 1,3,5, triol systems and an isohexyl pendant side chain. Amantelides A and B ( 19 20 ) have a distinctive 40 membered macrocycle composed of 1,3 and 1,5 diol moieties and a tert butyl pendant side chain with c ompound 20 additionally being acetyl ated at C 33 Antiproliferative activity assays with 18 20 indicated the importance of the hydroxy groups for bioactivity and w ere verified by the loss of activity of the peracetylated derivative of 1 9 Compounds 18 20 be ar structural similarities with amphotericin B. This, together with the results of time course cell viability determination for amphotericin B and amantelide A ( 19 ) treated cells suggested that the latter may also affect the integrity of the cell membra n e, similar to amphotericin B Additional e xperiments to visualize the effects of 19 on the cell membrane may be carried out Experimental Methods General Experimental Procedures O ptical rotation was measured on a Perkin Elmer 341 polarimeter. The UV spectrum was recorded on SpectraMax M5 Molecular Devices. 1 H and 2D NMR spectra were recorded in DMSO d 6 on a Bruker Avance II 600 MHz spectrometer equipped with

PAGE 143

143 a 5 mm triple resonance high temperature su perconducting (HTS) cryogenic probe using residual solvent signals ( H 2.50; C 39.5) as internal standards. The 13 C NMR spectrum was recorded in DMSO d 6 on a Bruker 500 MHz spectrometer, operating at 125 MHz. HSQC and HMBC experiments were optimized for 1 J CH = 145 and n J CH = 7 Hz, respectively. TOCSY and HSQC TOCSY experi ments were done using a mixing time of 100 ms. HRMS data were obtained using an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector. ESIMS/MS data were obtained on a 3200 QTRAP (Applied Biosystems) by direct injection u sing a syringe driver. Biological Material The cyanobacteria, Phormidium spp., were hand collected on June 24, 2008, at the breakwater at Fort Zachary Taylor State Park (Key West), Florida, by snorkeling in shallow waters. The collection was later identifi ed to consist primaril y of P cf. dimorphum and P inundatum Voucher specimens (#VP_6_24_08_FZT1) are maintained at Smithsonian Marine Station, Fort Pierce, F L T he gr a y cyanobacteri um belonging to the Family Oscilliatoriales w as collected from Amantes Po int, Tumon Bay, Guam. Vo u c her specimens are maintained at Smithsonian Marine Station, Fort Pierce, F L Extraction and Isolation Caylobolide B (18) The freeze dried organism (54.2 g) was extracted with EtOAc MeOH (1:1) to yield 5.7 g of the nonpolar extract Subsequent extraction of the freeze dried material with EtOH H 2 O (1:1) gave 11.5 g of the polar extract. The nonpolar extract was further partitioned between hexanes and 20% aqueous MeOH. The latter was concentrated

PAGE 144

144 under reduced pressure and was further partitioned between n BuOH and H 2 O. The n BuOH (0.56 g) fraction was concentrated and subjected to Si gel column chromatography eluting first with CH 2 Cl 2 followed by increasing concentrations of i PrOH. After 100% i PrOH, increasing gradients of MeOH wer e used until 100% MeOH. The fraction that eluted with 25% MeOH was subjected to reversed phase HPLC (semipreparative, Phenomenex Synergi using a linear gradient of MeOH H 2 O (40 100% MeOH in 40 min and then 100% MeOH for 10 min) to yield cay lob olide B ( 18 ) ( t R 31.1 min, 2.1 mg). Purification of the fraction from 50% MeOH using the same conditions yielded symplostatin 1 ( t R 31.4 min, 1.5 mg). Caylobolide B ( 18 ): colorless, amorphous solid; [ ] 20 D 15 ( c 0.15, MeOH); UV (MeOH); max (log ) 215 (4.09); 1 H NMR, 13 C NMR, TOCSY, and HMBC data, see Table 5 1; HRESI/APCIMS m/z 761.5767 [M + H] + (calcd for C 42 H 81 O 11 761.5779); m/z 783.5594 [M + Na] + (calcd for C 42 H 80 O 11 Na, 783.5593). Symplostatin 1: colorless, amorphous solid; [ ] 20 D 98 ( c 0.03, MeO H) {lit. 28 [ ] D 45 ( c 1.6, MeOH)}; UV (MeOH); max (log ) 204 (3.52), 240 (2.94); 1 H NMR spectrum (Appendix F) is identical to that of an authentic sample, 28 ; L RESIMS m/z 799.3 [M + H] + Amantelides A (19) and B (20) The cyanobacteria collection ( 22.0 g) was extracted wit h CH 2 Cl 2 MeOH (1:1) to yield 3.4 g of the nonpolar extract. The lipophilic extract was further partitioned between hexanes:80% aque ous MeOH. The latter was concentrated and further partitioned between n BuOH :H 2 O. The n BuOH fraction ( 0.488 g) was further purified on a silica column, eluting with increasing gradients of i PrOH in CH 2 Cl 2 until 100% i PrOH followed by 100% MeOH. The fraction from 70 % i PrOH elution was subjected to reversed phase HPLC (semipreparative, Phenomenex Synergi

PAGE 145

145 a linear gradient of MeOH H 2 O (40% 100% MeOH in 30 min and then 100% MeOH for 10 min) to yield amantelide A ( 19 ) ( t R 29.4 min, 13.3 mg) and aman telide B ( 20 ) ( t R 30.8 min, 5.7 mg). Amantelide A ( 19 ): colorless, amorphous solid; [ ] 20 D 5.0 ( c 0.06, MeOH); UV (MeOH); max (log ) 220 (3.99); HRESI/APCIMS m/z 811.5927 [M + Na] + (calcd for C 44 H 84 O 11 Na, 811.5911) Amantelide B ( 20 ): colorless, amorpho us solid; [ ] 20 D 68 ( c 0.02, MeOH); UV (MeOH); max (log ) 218 (3.76); HRESI/APCIMS m/z 853.6044 [M + Na] + (calcd for C 46 H 86 O 12 Na, 853.6017). Acetylation of a mantelide A (19) Acetic anhydride (0.5 mL), pyridine (0.5 mL) and 19 (6.0 mg) was left to stir overnight. The reaction was terminated and dried under N 2 to yield peracetylated amantelide A ( 21 ) Peracetylated amantelide A ( 2 1 ) : oily liquid ; [ ] 20 D 5 8 ( c 0.02, MeOH ); UV (MeOH); max (log ) 21 4 ( 4.19 ); HRESI/APCIMS m/z 1169.6891 [M + Na] + (calcd for C 60 H 102 O 20 Na, 1169.6857 ). ESIMS/MS Fragmentation of Caylobolide B (18) and Amantelide A (19) Individual s olution s of 18 and 19 in MeOH were directly infused into the mass spectrometer using a syringe driver. MS fragmentation was obtained by positive and n egative ionization using the enhanced product ion (EPI) and MS2 scan. The [M + H] + and [M H] ions were fragmented by ramping the collision energy through the possible allowed range. Compound dependent and source gas param eters used were as follows: DP 65.0, EP 10.0, CUR 10.0, CAD High, IS 4500, TEM 0, GS1 10, GS2 0.

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146 Cell Viability Assay HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells were 10% fetal bovi ne serum (FBS, Hyclone) under a humidified environment with 5% CO 2 at 37 C. HeLa (3 ,000) and HT29 (12, 500) cell s were seeded in 96 well plates. V arying concentrations of 1 8 21 and amphotericin B were added to each well 24 h post seeding with treatments d one in duplicate. The cells were incubated for an additional 1, 3, 6, 12 and 48 h before the addition of the MTT reagent. Cell viability was measured IC 50 calculations were done by GraphPad Prism 5.0 3 based on duplicate experiments.

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147 Figure 5 1. Caylobolide B ( 18 ) and closely related compound caylobolide A. Absolute configuration for C 25, C 27 and C 29 is proposed by analogy to caylobolide A. Only relative configuration is shown for C 7 and C 9, which could not be related to C 25 C 29.

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148 Figure 5 2. Key HSQC TOCSY correlations for caylobolide B ( 18 ).

PAGE 149

149 Figure 5 3. ESI MS/MS of caylobolide B ( 18 ).

PAGE 150

150 Figure 5 4. Amantelides A and B ( 19 20 ) and the semisynthetic derivative peracetylated amant elide A ( 21 ). Only the r elative configurations for C 7 and C 9 are indicated.

PAGE 151

151 Figure 5 5 Partial structure of amantelide A ( 19 ) derived from NMR experiments in DMSO d 6 COSY correlations ar e indicated by solid double headed arrows. Protons showing HMB C correlations to indicated carbons are shown by single headed arrows.

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152 Figure 5 6. ESI MS/MS fragmentation of amantelide A ( 19 ).

PAGE 153

153 Figure 5 7 Assignment of relative configuration of caylobolide B ( 18 Universal NMR Database (Databas e 2). values between the model system and 18 are shown. The relative configuration shown is based on the best fit with the model system. The 1,3 diol is assigned as syn The values for the characteristic central carbon of the 1,3,5 triol system suggest eith er anti / syn or syn / anti arrangement.

PAGE 154

154 Figure 5 8. Time course antiproliferative activities of amantelide A ( 19 ) and amphoter icin B against cancer cells. (A) HT29 and (B) HeLa cell viability afte r 1, 3, 6, 12 and 48 h incubation with amantelide A ( 19 ) (C) HT29 and (D) HeLa cell viability afte r 1, 3, 6, 12 and 48 h incubation with amphotericin B.

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155 Table 5 1. NMR d ata of c aylobolide B ( 18 ) in DMSO d 6 Position C a H ( J in Hz) b HMBC b TOCSY b 1 165.4, C 2 116.5, CH 5.63, s 1,3,42 H 4a, H 4b, H 42 3 159.4, C 4a 4b 32.8, CH 2 2.64, m 2.42, m 2, 3 ,5, 42 2, 3, 5, 42 H 2,H 4b,H 5a,H 5b,H 7, 7 OH H 2,H 4a,H 5a,H 5b,H 7, 7 OH 5a 23.6, CH 2 1.52, m H 4a,H 4b,H 7,7 OH 5b 1.40, m 7 H 4a,H 4b 6 37.1, CH 2 1.37, m 7 OH 7 68.8, CH 3.58, m 9 H 4a, H 4b,H 5a,7 OH 7 OH 4.52, d (4.4) 6,7,8 H 4a, H 4b, H 5aH 6,H 7 8 44.15, CH 2 1.39, m 9 69.0, CH 3.54, m 10 9 OH,13 OH 9 OH 4.47, d (4.8) 8,9,10 H 9,H 13 10 37.6 CH 2 1.28, m 11 21.3, CH 2 1.21, m 12 37.3, CH 2 1.28, m 13 69.6, CH 3.35, m 9 OH, 13 OH 13 OH 4.20, m 12,13,14 H 9, H 13 14 37.3, CH 2 1.28, m 15 21.6, CH 2 1.21, m 16 37.3, CH 2 1.28, m 17 69.6, CH 3.37, m 17 OH 17 OH 4.20, m 16,17,1 8 H 17 18 37.3, CH 2 1.28, m 19 21.6, CH 2 1.21, m 20 37.3, CH 2 1.28, m 21 69.8, CH 3.36, m 21 OH,25 OH 21 OH 4.20, m 20,21,22 H 21,H 25 22 37.3, CH 2 1.28, m 23 20.9, CH 2 1.32, m

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156 Table 5 1. Continued Positi on C a H ( J in Hz) b HMBC b TOCSY b 24 37.3, CH 2 1.29, m 25 68.1, CH 3.54, m 23,27 21 OH,25 OH, H 27 25 OH 4.37, d (4.4) 24,25,26 H 21,H 25,H 27 26 44.4, CH 2 1.42, m 27 65.8, CH 3.79, dq (13.5, 6.4) 25,26,29 H 25,27 OH, H 29 27 OH 4.47, d (4.9) 27 H 27 28 44.08, CH 2 1.39, m 29 66.6, CH 3.61, m 28,31 H 27, 29 OH 29 OH 4.28, d (5.2) 28,30 H 27,H 29 30 37.5, CH 2 1.31, m 31a 21.0, CH 2 1.29, m 31b 1.22, m 32 38.1, CH 2 1.29, m 33 66.5, CH 3.31, m 32,34 33 OH, H 35 33 OH 4.29, d (6.03) 33,34 H 33, H 35 34 37.3, CH 2 1.49, m 35 H 35 35 73.6, CH 5.00, ddd (10.2, 4.3, 2.2) 1,33,41 H 33,OH 33,H 34,H 36,H 37b,H 3 40,H 3 41 36 36.0, CH 1.66, m 35 H 35,H 37b,H 41 37a 31.6, CH 2 1.31, m 41 H 37b,H 38a 37b 1.06, dd (17.7, 8.9) 38a 28.8, CH 2 1.31, m H 37a,H 37b 38b 1.23, m 39 22.3, CH 2 1.26, m 40 H 3 40 40 13.6, CH 3 0.86, t (7.0) 38,39 H 35, H 39 41 14.4, CH 3 0.80, d (6.9) 35,36,37 H 36 42 24.5, CH 3 1.85, s 2, 3, 4 H 2 a 125 MHz. b 600 MHz.

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157 Table 5 2. NMR d ata of a mantelid e A ( 19 ) and a mantelide B ( 20 ) in DMSO d 6 Amantelide A Amantelide B Position C a H ( J in Hz) b C a H ( J in Hz) b 1 165.5, C 165.8, C 2 116.2, CH 5.62, s 116.1, CH 5.67, s 3 160.3, C 160.0, C 4 32.9, CH 2 2.54, m 32.4, CH 2 2.56, m 5a 23.6, CH 2 1.54, m 23.4, CH 2 1.52, m 5b 1.40, m 1.40, m 6 37.0, CH 2 1.27, m 37.0, CH 2 1.2 3, m 7 68.7, CH 3.58, m 68.6, CH 3.58, m 7 OH 4.54, br 4.50, br 8 44.1, CH 2 1.37, m 44.2, CH 2 1.37, m 9 68.5, CH 3.55, m 68.8, CH 3.55, m 9 OH 4.52, br 4.55, br 10 37.0, CH 2 1.22, m 37.0, CH 2 1.23, m 11 21.3, CH 2 1.21, m 21.2, CH 2 1.22, m 12 37 .0, CH 2 1.31, m 36.9, CH 2 1.30, m 13 69.4, CH 3.34, m 69.4, CH 3.34, m 13 OH 4.23, m 4.23, m 14 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 15 20.8, CH 2 1.31, m 20.8, CH 2 1.32, m 16 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 17 69.4, CH 3.34, m 69.4, CH 3.34, m 17 OH 4.23, m 4.23, m 18 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 19 20.8, CH 2 1.31, m 20.8, CH 2 1.32, m 20 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 21 69.4, CH 3.34, m 69.4, CH 3.34, m 21 OH 4.23, m 4.23, m 22 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 23 20.8, CH 2 1.31, m 20.8, CH 2 1.32, m

PAGE 158

158 Table 5 2. Continued Amantelide A Amantelide B Position C a H ( J in Hz) b C a H ( J in Hz) b 24 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 25 69.4, CH 3.34, m 69.4, CH 3.34, m 25 OH 4.23, m 4.23, m 26 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 27 20.8, CH 2 1.31, m 20.8, CH 2 1.32, m 28 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 29 69.4, CH 3.34, m 69.4, CH 3.34, m 29 OH 4.23, m 4.23, m 30 37.0, CH 2 1.31, m 36.9, CH 2 1.30, m 31 20.8, CH 2 1.31, m c c 32 37.0, CH 2 1.31, m c c 33 69.4, CH 3.34, m 73.3, CH 4.73, m 33 OH 4.23, m 34 37.0, CH 2 1.31, m c c 35 20.8, CH 2 1. 31, m c c 36 37.0, CH 2 1.31, m c c 37 66.4, CH 3.21, m 66.4, CH 3.21, m 37 OH 4.26, m 4.27, br 38 37.1, CH 2 1.43, m 37.1, CH 2 1.41, m 39 76.6, CH 4.93, br d 76.2, CH 4.93, br d 40 34.1, C 34.2, C 41 43 26.3, CH 3 0.82, s 25.7, CH 3 0.83, s 44 24 .6, CH 3 1.85, s 24.3, CH 3 1.86, s 45 170.0, C 46 20.7, CH 3 1.97, s a 125 MHz. b 600 MHz. c Cannot be assigned due to significant overlap of signals

PAGE 159

159 Table 5 3 Cytotoxic a ctivity (IC 50 M) of the isolated c yanobacterial p olyketides ( 18 2 1 ) a a Data are presented as mean SD (n = 2). Compound HT29 HeLa Caylobolide B ( 18 ) 4.5 1.2 12.2 1.0 Amantelide A ( 19 ) 0.87 0.02 0.87 0.07 Amantelide B ( 20 ) 12 1.6 9.9 0.05 Peracetylated amantelide A ( 21 ) 58 6.7 18 1.6 Amphotericin B 10 2.7 10 4.4

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160 CHAPTER 6 GENERAL CONCLUSION In the last 30 years, marine cyanobacteria have been utilized as a source of small molecule therapeutics In this study, we aimed to exploit the diverse secondary metabolites from th e marine cyanobacte ria belonging mainly to the genera of Phormidium and Symploca for drug discovery. Cyanobacteria collections from Guam, Florida, and the US Virgin Island were extracted and profiled in an antiproliferative assay using the HT29 human colorectal adenocarcinom a cell line and an HPLC MS based dereplication as a preliminary screening of bioactivity and chemical space, respectively. Based on the profilin g results, four cyanobacteria collections were prioritized for further purifica tion of secondary metabolites. B i oactivity and 1 H NMR directed approach es for the prioritized cyanobacteria collections yielded symplostatins 5 10 ( 1 6 ), veraguamides A G ( 7 13 ), caylobolide B ( 18 ) and amantelides A and B ( 19 20 ). The planar structure s of purified compounds were establi shed using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. Absolute configurations of stereocenters were assigned by enantioselective HPLC MS and/or HPLC UV analysis by comparison with authentic standards as well as derivatization with c hiral reagents and J based analysis. A Guamanian Symploca sp. collection yielded the cyclic depsipeptides symplostatins 5 10 ( 1 6 ), bearing the modified amino acid residue 3 amino 6 hydroxy 2 piperidone (Ahp) and 2 amino 2 butenoic acid (Abu). The Ahp bear ing cyclodepsipeptides from cyanobacteria constitutes a predominant class of metabolites, with more than 100 members isolated to date from terrestrial, marine and freshwater origins. These cyanobacterial metabolites are serine protease inhibitors with the Abu

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161 bearing compounds such as symplostatins 5 10 ( 1 6 ) and the related lyngbyastatins 4 10 being potent elastase inhibitors. Using the structural diversity of these agents, the molecular basis for potent elastase inhibition was established using structure activity relationship (SAR) and X ray cocrystallization studies. Aside from the Ahp and Abu moieties, an N Me Tyr residue in the macrocyle and a polar functionality in the pendant si de chain are contributors to potent elastase inhibition. This was verifie d from the X ray cocrystal structure of lyngbyastatin 7 porcine pancreat ic elastase, where the hydroxy group of the N Me Tyr and the terminal amid e group of Gl n in lyngbyastatin 7 co ntribute critical hydrogen bonding interactions with the enzyme and active site water molecules The involvement of the pendant side chain, which is highly variable among members of serine protease profiling for symplostatin 5 ( 1 ) and lyngbyastat in 7 demonstrated preferential inhibition of elastase by these agents. The cellular effects of symplostatin 5 ( 1 ) against the downstream cellular effects of elastase in bronchial epithelial cells were also interrogated. Symplostatin 5 ( 1 ) attenuated the ef fects of elastase on cell death, detachment, genome wide transcript changes as well as proteolytic processing of adhesion molecules. Compound 1 alleviated key pro inflammatory mediators stim ulated by elastase, such as NF B activation and upregulation of i nterleukins IL1A IL1B and IL8 Compared to the clinically approved elastase inhibitor sivelestat, symplostatin 5 ( 1 ) has a long lasting effect against the cellular effects of elastase to bronchial epithelial cells, while having no cytotoxic effects. There fore, key aspects in protease inhibitor development selectivity, potency and cellular activity have been addressed in this study. Additional investigations are warranted to determine the in vivo cellular effects of

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162 this class of compounds in a COPD ani mal model system, as well as S AR studies to further probe the effects of the highly divergent pendant side chain to selectivity and pote ncy of this class of compounds. Antiproliferative agents constitute the majority of the purified seco ndary metabolites i n this study. A Guamanian Symploca cf. hydnoides collection yielded the modified cyclic depsipeptides veraguamides A G ( 7 13 ), characterized by a C 8 polyketide derived hydroxy acid, multiple N methylated amino acid s hydroxy acid. Compounds 7 13 and a semisynthetic derivative tetrahydroveraguamide A ( 14 ) showed moderate to weak antiproliferative activity against HT29 human colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure activity relationship studies on verag uamides indicated that t hydroxy acid and the terminal functionality of the C 8 polyketide derived hydroxy acid moieties are major contributors to the antiproliferative activity of this class of compounds. Veraguamide D ( 10 ) caused an incremental change in cell populations a t sub G1 and G2. Additional studies are needed to determine the mechanism of cell death induced by the veraguami des. Three polyketide compounds, caylobolide B ( 18 ) and amantelides A and B ( 19 20 ) were isolated from Floridian Phormidium spp. assemblage an d a G uamanian gray cyanobacterium collection s respectively. These compounds bear a polyhydroxylated macrolactone ring with an alkyl pendant side chain. Caylobolide B ( 18 ) bears a contiguous chain of 1,3 and 1,5 diol and 1,3,5 triol moieties and an isohex yl side chain. Amantelides A ( 19 ) and B ( 20 ) bear a contiguous chain of 1,3 diol and 1,5 diol systems and a tert butyl side chain. The C 33 of 20 bears an acetyl group instead of a hydroxy group, which differentiates 19 and 20 Among the purified p olyketid es amantelide A

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163 ( 19 ) displayed the most potent antiproliferative activity, with sub nanomolar IC 50 against HT29 and HeLa cells, indicating that acetylation of the hydroxy groups of this class of compound is detrimental to the activity. This was corroborat ed by the weak antiproliferative activity of the semisynthetic derivative of 19 peracetylated amantelide A ( 21 ). Preliminary studies on the mechanism of action of amantelide A ( 19 ) indicated that this class of cyanobacterial metabolites may target the cel l membrane, leading to cytotoxicity. This study demonstrated that marine cyan obacteria are validated source organisms of novel bioactive secondary metabolites yielding both structurally and pharmacol ogically diverse compounds that have potential applicat ions as small molecule therapeutics in malignancies and elastase mediated pathologies

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164 APPENDIX A CELL MO RPHOLOGY AT 3 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR)

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165 APPENDIX B CELL MORPHOLOGY AT 6 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR)

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166 APPEN DIX C CELL MORPHOLOGY AT 12 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR)

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167 APPENDIX D CELL MORPHOLOGY AT 24 h POST TREATMENT WITH ELASTASE (+/ INHIBITOR)

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168 APPENDIX E ICAM1 TRANSCRIPT LEV ELS AT 3 h AND 6 h a a Data are presented as mean + SD (n = 3)

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169 APPENDIX F NMR SPECTRA OF ISOLATE D COMPOUNDS On the following pages are the NMR spectra of isolated compounds in this study, which includes the known compounds largazole, dolastatin 10, symplostatin 1 and dolastatin 16, as well as the new secondary metabolites symplostatins 5 10 ( 1 6 ), veraguamides A G ( 7 13 ), caylobolide B ( 18 ) and amantelides A, B ( 19 20 ) and the semisynthetic analogs tetrahydroveraguamide ( 14 ) and peracetylated amantelide A ( 21 ).

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170 1 H NMR SPECTRUM OF LARGAZOLE IN CDC l 3 (600 MH z )

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171 1 H NMR SPECTRUM OF DOLASTATIN 10 IN CD 2 C l 2 (600 MH z )

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172 1 H NMR SPECTRUM OF SYMPLOSTATIN 1 IN CD 2 C l 2 (600 MH z )

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173 1 H NMR SPECTRUM OF DO LASTATIN 16 IN CDC l 3 (400 MH z )

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174 1 3 C NMR SPECTRUM OF DO LASTATIN 16 IN CDC l 3 (100 MH z )

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175 1 H NMR SPECTRUM OF SY MPLOSTA TIN 5 ( 1 ) IN DMSO d 6 (600 MH z )

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176 HSQC SPECTRUM OF SYMPLOST ATIN 5 ( 1 ) IN DMSO d 6 (600 MH z )

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177 COSY SPECTRUM OF SYMPLOST ATIN 5 ( 1 ) IN DMSO d 6 (600 MH z )

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178 HMBC SPECTRUM OF SYMPLOST ATIN 5 ( 1 ) IN DMSO d 6 (600 MH z )

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179 NOESY SPECTRUM OF SYMPLOST ATIN 5 ( 1 ) IN DM SO d 6 (600 MH z )

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180 1 H NMR SPECTRUM OF SY MPLOSTATIN 6 ( 2 ) IN DMSO d 6 (600 MH z )

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181 HSQC SPECTRUM OF SYMPLOST ATIN 6 ( 2 ) IN DMSO d 6 (600 MH z )

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182 COSY SPECTRUM OF SYMPLOST ATIN 6 ( 2 ) IN DMSO d 6 (600 MH z )

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183 1 H NMR SPECTRUM OF SYMP LOSTATIN 7 ( 3 ) IN DMSO d 6 (600 MH z )

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184 HSQC SPECTRUM OF SYMPLOST ATIN 7 ( 3 ) IN DMSO d 6 (600 MH z )

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185 CO SY SPECTRUM OF SYMPLOST ATIN 7 ( 3 ) IN DMSO d 6 (600 MH z )

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186 1 H NMR SPECTRUM OF SYMP LOSTATIN 8 ( 4 ) IN DMSO d 6 (600 MH z )

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187 H SQC SPECTRUM OF SYMPLOST ATIN 8 ( 4 ) IN DMSO d 6 (600 MH z )

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188 COSY SPECTRUM OF SYMPLOST ATIN 8 ( 4 ) IN DMSO d 6 (600 MH z )

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189 1 H NMR SPECTRUM OF SYMP LOSTATIN 9 ( 5 ) IN DMSO d 6 (600 MH z )

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190 HSQC SPECTRUM OF SYMPLOST ATIN 9 ( 5 ) IN DMSO d 6 (600 MH z )

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191 COSY SPECTRUM OF SYMPLOST ATIN 9 ( 5 ) IN DMSO d 6 (600 MH z )

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192 1 H NMR SPECTRUM OF SYMP LOSTATIN 10 ( 6 ) IN DMSO d 6 (600 MH z )

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193 HSQC SPECTRUM OF SYMPLOST ATIN 10 ( 6 ) IN DMSO d 6 (600 MH z )

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194 COSY SPECTRUM OF SYMPLOST ATIN 10 ( 6 ) IN DMSO d 6 (600 MH z )

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195 1 H NMR SPECTRUM OF VERA GUAMIDE A ( 7 ) IN CDC l 3 (600 MH z )

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196 1 3 C NMR SPE CTRUM OF VERAGUAMIDE A ( 7 ) IN CDC l 3 (100 MH z )

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197 HSQC SPECTRUM OF VERAGUAM IDE A ( 7 ) IN CDC l 3 (600 MH z )

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198 COSY SPECTRUM OF VERAGUAM IDE A ( 7 ) IN CDC l 3 (600 MH z )

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199 HMBC SPECTRUM OF VERAGUAM IDE A ( 7 ) IN CDC l 3 (600 MH z )

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200 1 H NMR SPECTRUM OF VERAGU AMIDE B ( 8 ) IN CDC l 3 (600 MH z )

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201 1 3 C NMR SPECTRUM OF VE RAGUAMIDE B ( 8 ) IN CDC l 3 (100 MH z )

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202 HSQC SPECTRUM OF VER AGUAMIDE B ( 8 ) IN CDC l 3 (600 MH z )

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203 COSY SPECTRUM OF VER AGUAMIDE B ( 8 ) IN CDC l 3 (600 MH z )

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204 HMBC SPECTRUM OF VER AGUAMIDE B ( 8 ) IN CDC l 3 (600 MH z )

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205 1 H NMR SPECTRUM OF VERA GUAMIDE C ( 9 ) IN CDC l 3 (600 MH z )

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206 1 3 C NMR SPECTRUM OF VE RAGUAMIDE C ( 9 ) IN CDC l 3 (100 MH z )

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207 HSQC SPECTRUM OF VER AGUAMIDE C ( 9 ) IN CDC l 3 (600 MH z )

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208 COSY SPECTRUM OF VER AGUAMIDE C ( 9 ) IN CDC l 3 (600 MH z )

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209 HMBC SPECTRUM OF VER AGUAM IDE C ( 9 ) IN CDC l 3 (600 MH z )

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210 1 H NMR SPECTRUM OF VE RAGUAMIDE D ( 10 ) IN CDC l 3 (600 MH z )

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211 1 3 C NMR SPECTRUM OF VE RAGUAMIDE D ( 10 ) IN CDC l 3 (125 MH z )

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212 HSQC SPECTRUM OF VER AGUAMIDE D ( 10 ) IN CDC l 3 (600 MH z )

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213 COSY SPECTRUM OF VER AGUAMIDE D ( 10 ) IN CDC l 3 (600 MH z )

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214 HMBC SPECTRUM OF VER AGUAMIDE D ( 10 ) IN CDC l 3 (600 MH z )

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215 1 H NMR SPECTRUM OF VE RAGUAMIDE E ( 11 ) IN CDC l 3 (600 MH z )

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216 1 3 C NMR SPECTRUM OF VE RAGUAMIDE E ( 11 ) IN CDC l 3 (125 MH z )

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21 7 HSQC SPECTRUM OF VER AGUAMIDE E ( 11 ) IN CDC l 3 (600 MH z )

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218 COSY S PECTRUM OF VERAGUAMI DE E ( 11 ) IN CDC l 3 (600 MH z )

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219 HMBC SPECTRUM OF VER AGUAMIDE E ( 11 ) IN CDC l 3 (600 MH z )

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220 1 H NMR SPECTRUM OF VE RAGUAMIDE F ( 12 ) IN CDC l 3 (600 MH z )

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221 1 3 C NMR SPECTRUM OF VE RAGUAMIDE F ( 12 ) IN CDC l 3 (100 MH z )

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222 HSQC SPECTRUM OF VER AGUAM IDE F ( 12 ) IN CDC l 3 (600 MH z )

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223 COSY SPECTRUM OF VER AGUAMIDE F ( 12 ) IN CDC l 3 (600 MH z )

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224 HMBC SPECTRUM OF VER AGUAMIDE F ( 12 ) IN CDC l 3 (600 MH z )

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225 1 H NMR SPECTRUM OF VE RAGUAMIDE G ( 13 ) IN CDC l 3 (600 MH z )

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226 1 3 C NMR SPECTRUM OF VE RAGUAMIDE G ( 13 ) IN CDC l 3 (100 MH z )

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227 HSQC SPECTRUM OF VER AGUAMIDE G ( 13 ) IN CDC l 3 (600 MH z )

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228 COSY SPECTRUM OF VER AGUAMIDE G ( 13 ) IN CDC l 3 (600 MH z )

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229 HMBC SPECTRUM OF VER AGUAMIDE G ( 13 ) IN CDC l 3 (600 MH z )

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230 1 H NMR SPECTRUM OF TE TRAHYDROVERAGUAMIDE A ( 14 ) IN CDC l 3 (600 MH z )

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231 HSQC SPECTRUM OF TET RAHYDROVERAGUAMIDE A ( 14 ) IN CDC l 3 (600 MH z )

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232 1 H NMR SPECTRUM OF LI NEAR FRAGMENT 15 IN CDC l 3 (500 MH z )

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233 COSY SPECTRUM OF LIN EAR FRAGMENT 15 IN CDC l 3 (500 MH z )

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234 1 H NMR SPECTRUM OF R MTPA ESTER 1 6 IN CDC l 3 (600 MH z )

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235 COSY SPECT RUM OF R MTPA ESTER 1 6 IN CDC l 3 (600 MH z )

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236 1 H NMR SPECTRUM OF S MTPA ESTER 1 7 IN CDC l 3 (600 MH z )

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237 COSY SPECTRUM OF S MTPA ESTER 1 7 IN CDC l 3 (600 MH z )

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238 1 H NMR SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (600 MH z )

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239 1 3 C NMR SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (125 MH z )

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240 HSQC SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (600 MH z )

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241 COSY SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (600 MH z )

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242 HMBC SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (600 MH z )

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243 HSQC TOCSY SPECTRUM OF CAYLOBOLIDE B ( 1 8 ) IN DMSO d 6 (600 MH z )

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244 1 H NMR SPECTRUM OF AMANTELIDE A ( 1 9 ) IN DMSO d 6 (600 MH z )

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245 1 3 C NMR SPECTRUM OF AM ANTELIDE A ( 1 9 ) IN DMSO d 6 (125 MH z)

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246 HSQC SPECTRUM OF AMA NTELIDE A ( 1 9 ) IN DMSO d 6 (600 MH z)

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247 COSY SPECTRUM OF AMA NTELIDE A ( 1 9 ) IN DMSO d 6 (600 MH z)

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248 HMBC SPECTRUM OF AMA NTELIDE A ( 1 9 ) IN DMSO d 6 (600 MH z)

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249 1 H NMR SPECTRUM OF AM ANTELIDE B ( 20 ) IN DMSO d 6 (600 MH z)

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250 1 3 C NMR SPECTRUM OF AM ANTELIDE B ( 20 ) IN DMSO d 6 (125 MH z)

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251 HSQC SPECTRUM OF AMA NTELIDE B ( 20 ) IN DMSO d 6 (600 MH z)

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252 COSY SPECTRUM OF AMANTELIDE B ( 20 ) IN DMSO d 6 (600 MH z)

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253 HMBC SPECTRUM OF AMA NTELIDE B ( 20 ) IN DMSO d 6 (600 MH z)

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254 TOCSY SPECTRUM OF AM ANTELIDE B ( 20 ) IN DMSO d 6 (600 MH z)

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255 1 H NMR SPECTRUM OF PE RACETYLATED AMANTELI DE A ( 21 ) IN CDC l 3 (600 MH z)

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256 HSQC SPECTRUM OF P ERACETYLATED AMANTEL IDE A ( 21 ) IN CDC l 3 (600 MH z)

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266 BIOGRAPHICAL SKETCH Lilibeth Apo Salvador was born in Quezon City, Phi lippines. She received her B achelor of S cience in chemistry at the Un iversity of the Philippines Diliman in 2000. On the same year, she became a qualified chemist and joined the Marine Science Institute at the University of Philippines Diliman as s cience r esearch s pecialist, under the supervision of Professor Gise la P. Concepcion and Professor Amelia P. Guevara. She worked with the Antibody and Molecular Onc ology Research (AMOR) p rogram and the National Cooperative Drug Discovery Group (NCDDG) on the discovery of anticancer therapeutics from Philippine plants and marine sponges During this time, she developed a strong interest on natural products intiated drug discovery. Lilibeth fin ished her M aster of S cience in chemistry in 2006 and subsequently served as r esearch and d evelopment c onsultant for Euro Med Laboratories Inc. and TEDA Pha rmaceuticals Inc. She joined the Department of Medicinal Chemistry, College of Pharmacy at the University of Florida in 2008, under the mentorship of Professor Hendrik Luesch. Lilibeth worked on the purification and structure determination of novel seconda ry metabolites from marine cyanobacteria as well as elucidation of the mechanism s of action and pharmacokinetics of cyanobacterial derived compounds She received her Ph.D. in p harma ceutical sciences medicinal c hemistry from the University of Florida in the spring of 2013.