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Title:
Antarctic tunicates and endophytic fungi : chemical investigation and synthesis
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English
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Lebar, Matthew
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University of South Florida
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Subjects / Keywords:
Polyketide
Polyol
Alkaloid
Indole
Pyrimidine
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb's 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.
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Dissertation (PHD)--University of South Florida, 2010.
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by Matthew Lebar.
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ABSTRACT: Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb's 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.
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Antarctic Tunicates and Endophytic F ungi: Chemical I nvestigation and S ynthesis by Matthew D. Lebar A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Bill J. Baker, Ph.D. Abdul Malik, Ph.D. Roman Manetsch, Ph.D. Edward Turos, Ph.D. Date of Approval: November 5, 2010 Keywords: polyketide, polyol, alkaloid, indole, pyrimidi ne Copyright 2010, Matthew D. Lebar

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Dedication I dedicate this dissertation to my family, particularly my mother and father who have supported me in all ways possible. I appreciate their encouragement and assistance when it was time to leave Colorado and continue my education in Hawaii. I am grateful for the monetary and, especially, the emotional support provided in times of need. I am thankful for the possibilities afforded me by my parents love, support and encouragement. I dedicate this dissertation to the past and to the future: my grandparents, who brightened my childhood and the newest member of our family, Grace Kelli (b. 2010.09.08) who will, no doubt, kiddo! I thank my girlfriend, Lauren, for all the gr eat moments that kept me sane the past few years. Be it a trip to the grocery store or a trip through Europe, I enjoy and relish every moment we spend together. Lastly, I would like to dedicate this d issertation to my friends past and present for sharing Dr. Hunter S. Thompson (19 37 2005)

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Acknowledgement s I am indebted to Bill Baker for his ideas, guidance, and inspiration. He taught me that nothing is impossible. scientist, presenter and writer. I would also like to thank the other members of my committee. Edward Turos taught me a great deal of advanced organic chemistry in a thoughtful and organized way in classes as well as during our weekly journal meetings. Roman Manetsch was always around t o give pointers and warnings about any (dangerous!) reaction s myself or undergrad contemplated attempting ; he also oversaw many beneficial late night journal meetin g s I thank Abdul Malik for his thoughtful and kind advice and willingness to help in anyw ay necessary. I greatly appreciate the efforts of Dennis Kyle and his lab for biological screening. The projects in this dissertation would not have been as exciting and in some cases im possible without his knowledge and facilities I also enjoyed working with Chad Dickey and would like to thank him for our collaboration, which will hopefully lead to many more fruitful endeavors A special thanks to Janice G. Smith, who piqued my interest in organic chemistry through her excellent lectures and instruction I would also like to thank Randy Larsen for welcoming me to USF with generous aloha spirit. Last but not least, I offer my gratitude to the undergraduates that helped me throughout the years, particularly Kris Hahn, Lisha Luttenton, and Ryan Bake r

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i Table of c ontents List of t ables ................................ ................................ ................................ ................................ ... v List of f igures ................................ ................................ ................................ ................................ vi List of schemes ................................ ................................ ................................ ............................. vii List of abbreviations ................................ ................................ ................................ ...................... ix Abstract ................................ ................................ ................................ ................................ ........... xi Chapter 1 Drugs, n ature, and s ynthesi s ................................ ................................ .................... 1 1.1 Natural products as a source of drugs ................................ ................................ ... 1 1.2 Drugs from the sea ................................ ................................ ................................ 1 1.3 Drugs from microorganisms ................................ ................................ ................... 4 1. 4 Relevance of synthesis in natural products chemistry ................................ ........... 7 1. 4 .1 Structure verification via degradati on studies ................................ ........... 8 1. 4 .2 Structure verification via total synthesis ................................ .................. 10 1. 4 .3 Drug supply by total synthesis and semisynthesis ................................ 14 1. 4 .4 Synthetic derivatives of natural products as drugs ................................ 18 1.5 Research o bjectives ................................ ................................ ............................. 20 1 6 R e f e r e n c e s C i t e d ................................ ................................ ................................ 2 0 Chapter 2 Synthetic studies of palmerolide A ................................ ................................ ........ 2 4 2.1 Isolation, structure elucidation, and bioactivity of palmerolide A ......................... 2 4 2.2 Palmerolide A as a synthetic target ................................ ................................ ..... 26 2.2.1 Total syntheses ................................ ................................ ....................... 2 6 2.2.1.1 Total synthesis by De Brabander ................................ ............... 2 6 2.2.1.2 Total synthesis by Nicolaou ................................ ....................... 2 8 2.2.1.3 Total synthesis by Hall ................................ ............................... 3 1 2.2.2 Partial syntheses of palmerolide A ................................ ......................... 3 3 2.2.3 Formal total synthesis of palmerolide A ................................ .................. 3 5 2.3 Degradation of palmerolide A to confirm absolute configuration ......................... 3 5 2.3.1 Ozonolysis of palmerolide A ................................ ................................ ... 36 2.3.2 Synthesis of hexane 1,2,6 triol ................................ ............................... 3 6 2.3.3 Synthesis of hexane 1,2,3,6 tetraol ................................ ........................ 3 8 2.3.4 Re evaluation of absolute configuration ................................ ................. 3 9 2.4 Synthesis of the C3 14 fragment of palmerolide A ................................ .............. 40 2.4.1 Julia Kocienski olefination ................................ ................................ ....... 40 2.4.2 cross metathesis ................................ .............................. 4 2 2 5 R e f e r e n c e s C i t e d ................................ ................................ ................................ 4 3 Chapter 3 Meridianin A and psammopemmin A: structure investigation ........................... 4 6 3.1 Indole and pyrimidine containing natural products ................................ .............. 46

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ii 3.1.1 Indoles and pyrimidines from Southern cold water sponges .................. 4 8 3.1.2 Indoles and pyrimidines from Southern cold water tunicates ................. 4 9 3. 2 Review of meridianin syntheses ................................ ................................ .......... 5 1 3.2.1 S ynthesis of natural meridianins ................................ ............................. 51 3.2.2 S ynthesis and biological activity of merdianin analogs ........................... 5 3 3.2.3 Synthesis and biological activity of meriolins ................................ .......... 5 9 3.3 Isolation of meridianins A, B, C, and E from Antarctic tunicate Synoicum sp. ................................ ................................ ................................ ..... 61 3. 4 Synthesis and bio logical activity of 3 pyrimidylindoles ................................ ........ 6 3 3. 4 .1 Synthesis of meridianin A, 4 methoxymeridianin A, and 5 bromomeridianin E ................................ ................................ ........... 6 3 bromomeridianin E 3. 4 .2 Synthesis of psammopemmin A chloropsammopemmin A ......... 6 4 3. 4 3 Structural reassessment of psammopemmin A ................................ ...... 6 6 3.4.4 Synthesis of meridoquin ................................ ................................ .......... 6 8 3.4.5 Biological activity of 3 pyrimidylindoles ................................ ................... 69 3 5 R e f e r e n c e s C i t e d ................................ ................................ ................................ 7 4 Chapter 4 Antimalarial natural products ................................ ................................ ................. 7 7 4.1 The malaria dilemma ................................ ................................ ........................... 7 7 4.2 Current m alaria treatment ................................ ................................ .................... 78 4.2.1 Quinolines, antifolates, and artemisinins ................................ ................ 78 4.2.2 Drug resistant parasites ................................ ................................ .......... 80 4. 3 Natural products as antimalarial leads ................................ ................................ 81 4. 4 Medicines for Malaria Venture project ................................ ................................ 83 4. 4 .1 MMV project overview ................................ ................................ ............. 83 4. 4 .2 MMV project methodology ................................ ................................ ...... 84 4.4.2.1 Antimalaria and cytotoxicity assays, prioritization of active extracts ................................ ................................ ................... 84 4.4.2. 2 Antarctic microbe cultivation extraction, and processing .......... 85 4.4.2. 3 Fungi extraction and processing ................................ ................ 86 4.4.2. 4 Scale up fermentation and fractionation ................................ .... 87 4.4.2. 5 Active fractions to pure compounds ................................ ........... 87 4.4.2.6 Protocol validation ................................ ................................ ...... 88 4.4.3 Antimalarial compounds from endophytic mangrove fungi ..................... 88 4.4.3.1 Cytochalasins ................................ ................................ ............. 88 4.4.3.2 Trichothecenes ................................ ................................ .......... 90 4.4.4 MMV project outlook ................................ ................................ ............... 93 4 5 R e f e r e n c e s C i t e d ................................ ................................ ................................ 9 4 Chapter 5 Experimental ................................ ................................ ................................ ............. 9 7 5.1 General ................................ ................................ ................................ ................ 97 5.2 Experimental supporting Chapter 2 ................................ ................................ ..... 98 5.2.1 Ozonolysis of palmerolide A forming 1,2,3 trihydroxyhexane ( 2. 51 ) and 1,2,3,6 tetrahydroxyhexane ( 2. 52 ) ................................ .. 98 5.2.2 ( R ) hexane 1,2,6 triol ( R 2. 53 ) and ( S ) hexane 1,2,6 triol ( S 2. 53 ) ................................ ................................ ................................ 98 5.2.3 (R) (2,2 dimethyl 1,3 dioxolan 4 yl)methanol ( 2. 54 ) ............................... 99 5.2. 4 ( R ) 4 (3 (1,3 dioxolan 2 yl)prop 1 enyl) 2,2 dimethyl 1,3 dioxolane ( 2. 55 ) ................................ ................................ ................ 99

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iii 5.2. 5 (R) 2 (2,2 dimethyl 1,3 dioxolan 4 yl)ethanol ( 2. 56 ) ............................ 100 5.2.6 ethyl 4 (2,2 dimethyl 1,3 dioxolan 4 yl)but 2 enoate ( 2. 57 ) ................. 101 5.2.6.1 Synthesis of R 2.57 ................................ ................................ .. 101 5.2.6.2 Synthesis of S 2.57 ................................ ................................ .. 102 5.2. 6 .3 Physical data common to R 2.57 and S 2.57 .......................... 102 5.2.7 4 (2,2 dimethyl 1,3 dioxolan 4 yl)butan 1 ol ( 2. 58 ) .............................. 102 5.2.8 hexane 1,2,3,6 tetraol ( 2. 59 ) ................................ ................................ 103 5.2.8.1 Synthesis of (2 R ,3 R ) hexane 1,2,3,6 tetraol ( S S 2.59 ) .......... 103 5.2.8. 2 Synthesis of (2 S ,3 S ) hexane 1,2,3,6 tetraol ( S S 2.59 ) .......... 103 5.2.8.3 Physical data common to R R 2.59 and S S 2.59 ................... 104 5.2.9 ((4R,5R) 5 (2 (1,3 dioxolan 2 yl)vinyl) 2,2 dimethyl 1,3 dioxolan 4 yl)methyl acetate ( 2. 61 ) ................................ ................ 104 5.2.10 ethyl 3 ((4S,5S) 5 (benzyloxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)acrylate ( 2. 62 ) ................................ ................................ .......... 105 5.2.11 ethyl 3 ((4S,5S) 5 (hydroxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)propanoate ( 2. 63 ) ................................ ................................ ....... 106 5.2.12 (S) 5 (2 (tert butyldimethylsilyloxy)hept 6 enylsulfonyl) 1 phenyl 1H tetrazole ( 2. 65 ) ................................ ................................ .......... 107 5.2.13 ethyl 3 ((4S,5R) 5 formyl 2,2 dimethyl 1,3 dioxolan 4 yl) propano ate ( 2. 66 ) ................................ ................................ ........... 108 5.2.14 (S) hept 6 ene 1,2 diol ( 2. 67 ) ................................ ............................... 109 5.2.15 (S) 2 (tert butyldimethylsilyloxy)hept 6 enyl 4 methylbenzenesulfonate ( 2. 68 ) ................................ ...................... 110 5.2.16 (S) 7 (benzyloxy)hept 1 en 3 yl acetate ( 2. 69 ) ................................ ..... 111 5.2.17 (S) 6 (benzyloxy)hexane 1,2 diol ( 2. 70 ) ................................ ............... 112 5.2.18 (S) 6 (benzyloxy) 1 hydroxyhexan 2 yl acetate ( 2. 71 ) ......................... 113 5.2. 19 ethyl 3 ((4S,5S) 2,2 dimethyl 5 vinyl 1,3 dioxolan 4 yl) propanoate ( 2. 72 ) ................................ ................................ ........... 113 5.2.2 0 ethyl 3 ((4S,5S) 5 ((S,E) 3 acetoxy 7 (benzyloxy)hept 1 enyl) 2,2 dimethyl 1,3 dioxolan 4 yl)propanoate ( 2. 73 ) .......................... 114 5.3 Experimental supporting Chapter 3 ................................ ................................ ... 115 5.3.1 Isolation of meridianins A ( 3. 24 ), B ( 3. 25 ), C ( 3. 26 ), and E ( 3. 28 ) from Antarctic tunicate Synoicum sp. ................................ ............. 115 5.3. 2 synthetic meridianin A ( 24 ) ................................ ................................ ... 116 5.3. 3 4 indolol ( 3. 86 ) ................................ ................................ ...................... 117 5.3. 4 4 (tert butyldimethylsilyloxy) 1 H indole ( 4 (2,2 dimethyl 1,3 dioxolan 4 yl)butanal 3. 87 ) ................................ ............................. 117 5.3. 5 3 bromo 4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indole ( 3. 88 ) ................................ ................................ .................... 118 5.3. 6 4 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1H indol 3 yl)pyrimidin 2 amine ( 3. 91 ) ................................ ............................. 119 5.3.7 4 methoxy 1 H indole ( 3. 92 ) ................................ ................................ .. 120 5.3.8 3 bromo 4 methoxy 1 (triisopropylsilyl) 1 H indole ( 3. 93 ) ..................... 120 5.3.9 4 (4 methoxy 1 (triisopropylsilyl) 1 H indol 3 yl)pyrimidin 2 amine ( 3. 95 ) ................................ ................................ .............................. 121 5.3.10 4 methoxymeridianin A ( 3. 96 ) ................................ ............................... 122 5.3.11 5,7 dibromomeridianin A ( 3. 97 ) ................................ ............................ 123 5.3.12 4 amino 2 chloro 5 iodopyrimidine ( 3. 99 ) ................................ ............. 123 5.3.13 4 amino 2 bromo 5 iodopyrimidine ( 3. 100 ) ................................ .......... 124

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iv 5.3.14 2 bromo 5 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indol 3 yl)pyrimidin 4 amine ( 3.101 ) ................................ ............... 124 amine ( 3. 101 ) 5.3.15 synthetic psammopemmin A ( 3. 102 ) ................................ .................... 126 5.3.16 synthetic psammopemmin A HCl ( 3. 103 ) ................................ .............. 126 5.3.17 5 (5 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indol 3 yl) 2 chloropyrimidin 4 amine ( 3.104 ) ................................ ............ 127 amine ( 3. 104 ) 5.3.18 chloropsammopemmin A ( 3.105 ) ................................ ...................... 128 5.3.19 meridoquin ( 3. 106 ) ................................ ................................ ................ 12 9 5.3.20 2 chloro 4 N N diethylaminopyrimidine ( 3.107 ) and 4 chloro 2 N N diethylamino pyrimidine ( 3.108 ) ................................ .............. 129 5.3.21 3 bromo 6 chloro 1 (triisopropylsilyl) 1 H indole ( 3. 110 ) ....................... 130 5.3.2 2 4 (6 chloro 1 (triisopropylsilyl) 1H indol 3 yl) N,N diethylpyrimidin 2 amine ( 3.112 ) ................................ .................... 131 5.4 Experimental supporting Chapter 4 ................................ ................................ ... 132 5.4.1 Isolation of cytochalasin D ( 4.17 ) ................................ .......................... 132 5.4.2 Isolation of roridin E ( 4.18 ) and 12,13 deoxyroridin E ( 4.19 ) ................ 13 3 Appendices ................................ ................................ ................................ ................................ 1 3 4 Appendix A : NMR data supporting Chapter 2 ................................ ................................ 13 5 Appendix B : NMR data supporting Chapter 3 ................................ ................................ 15 9 Appendix C: NMR data supporting Chapter 4 ................................ ............................... 19 2 About the author ................................ ................................ ................................ ............... end page

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v List of tables Table 3. 1 1 H NMR and 13 C shifts of meridianin A ( 3.24 ) and psammopemmin A ( 3.16 ). .......... 6 2 Table 3.2 1 H and 13 C NMR shifts of synthetic psammopemmin A ( 3.102 ) and synthetic psammopemmin AHCl ( 3.103 ) ................................ ................................ .......... 6 7 Table 3.3 Primary screening of 3 pyrimidylindoles for 5 HT binding inhibition . ........................ 7 0 Table 4.1. Antimalarial and cytotoxic activity of microbial extracts ................................ ............. 85 Table 4.2 1 H and 13 C NMR shift comparison of isolated cytochalasin D ( 4.17 ) with literature values. ................................ ................................ ................................ ... 90 Table 4.3 1 H and 13 C NMR shift comparison of isolated roridin E ( 4.18 ) with literature values ................................ ................................ ................................ ................... 92 Table 4.4 1 H and 13 C NMR shift comparison of isolated 12,13 deoxyroridin E ( 4.19 ) with literature values. ................................ ................................ ................................ ... 93

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vi List of figures Fig 3. 1. Meriolin, a structural hybrid of meridianin and variolin ................................ ............... 59 Fig 3.2 Meridoquin, a structural hybrid of meridianin and chloroquine ................................ ... 6 8 Fig. 3.3. Meridianin A inhibits radioligand binding to 5 HT 2B ................................ .................... 71 Fig. 3.4. 4 M ethoxymeridianin A inhibits radioligand binding to 5 HT 2B ................................ ... 71 Fig. 3.5. 4 M ethoxymeridianin A inhibits radioligand binding to 5 HT 5A ................................ ... 7 1 Fig. 3.6. 4 M ethoxymeridianin A inhibits radioligand binding to 5 HT 7 ................................ ..... 7 1 Fig. 3.7. M eridianin A inhibits radioligand binding to DAT ................................ ........................ 72 Fig. 3.8. C hloropsammopemmin A inhibits radioligand binding to M 5 ................................ .. 72 Fig. 3.9. Summary of CNS, antimalarial, and cytotoxic activity of 3 primidylindoles ................ 73

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vii List of schemes Scheme 1.1. Degradation of calicheamicin 1 I via acidic methanolysis and methanolysis via strong cation exchange resin yielded carbohydrate and hexasubstituted benzene fragments ................................ ................................ ...... 9 Scheme 1.2 Triphenylphosphine initiated Michael addition prompting biradical cycloaromatazation to form 1.26 evidence for endiyne structure 1.25b present in the natrual product calicheamicin 1 I ................................ ................... 10 Scheme 1.3 Synthesis of orignially proposed diazonamide A led to structural reassessment and ultimately the correct structure. ................................ ............. 13 Scheme 1.4. Schreiber's synthesis of discodermolide starting with Roche ester. .................... 15 Scheme 1.5. ET 743 could be dervived from the antibotic cyanosafracin B. ........................... 16 Scheme 1.6. Pharma Mar's semisynthesis of ET 743 from cyanosafracin B. .......................... 17 Scheme 2.1. De Brabander's retrosynthetic strategy for formation of palmerolide A. .............. 26 Scheme 2.2 De Brabander's formation of palmerolide fragments 2.4 2.5 and 2.6 ............... 27 Scheme 2.3. Grignard attack on isocyanate forms enamine 2.14 ................................ ........... 27 Scheme 2.4. Nicolaou's most efficient strategies for coupling 2.15 2.16 and 2.17 ................. 29 Scheme 2.5. Formation of fragments 2.15 2.16 and 2.17 ................................ ....................... 30 Scheme 2.6. Nicolaou's revised palmerolide A ( 2.1b ) endgame. ................................ ............. 31 Scheme 2.7. Hall's convergent synthesis of 2.1b from 2.30 and 2.31 ................................ ..... 32 Scheme 2.8 Hall's synthesis of fragment 2.30 ................................ ................................ ........ 32 Scheme 2.9. Hall's stereoselective route to fragment 2.31 ................................ ...................... 33 Scheme 2.10. Partial syntheses of palmerolide A ( 2.1 ) ................................ ............................. 34 Scheme 2.11. Formal total synthesis of palmerolide A by Maier and Jagel. .............................. 35 Scheme 2.12. Reductive ozonolysis of palmerolide A isolated from Synoicum adareanum. ................................ ................................ ................................ .......... 36 Scheme 2.13. Initial synthesis of hexane 1,2,6 triol. ................................ ................................ .. 37

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viii Scheme 2.14. Synthesis of ( R ) and ( S ) hexane 1,2,6 triol. ................................ ....................... 38 Scheme 2.15. Synthesis of ( R R ) hexane 1,2,3,6 tetraol. ................................ .......................... 39 Scheme 2.16. Synthesis of ( S S ) hexane 1,2,3,6 tetraol. ................................ ........................... 39 Scheme 2.17. Julia Kocienski path to polyol coupling. ................................ ............................... 41 Scheme 2.18. Synthesis of fragments for Julia Kocienski coupling and OCM coupling. ............ 4 1 Scheme 2.19. Synthesis of fragment C3 8 for OCM coupling. ................................ ................... 4 2 Scheme 2.20. OCM forming fragment C3 14 ( 2.73 ) of palmerolide A ( 2.1b ). ............................ 4 3 Scheme 3.1. Suzuki coupling to form meridianins D and G ................................ ..................... 51 Scheme 3.2. Enaminone condensation to form meridianins A, C, D, and E. ........................... 52 Scheme 3.3. Sonagashira carbonylation to form meridianins C, D, and G. ............................. 52 Scheme 3.4. Formation of merdianin G from 3 cyanoacetylindole. ................................ .......... 53 Scheme 3.5. Synthesis of 3 (5' (2'amino)pyrimidyl)indoles pre dating meridianin isolation. 54 Scheme 3.6. Fischer indole synthesis of iso meridianins C and G. ................................ .......... 55 Scheme 3.7. Synthesis of a meridianin related trisubstituted pyrimidines. ............................... 55 Scheme 3.8. Synthesis of 6' alkyl subst ituted meridianin analogs. ................................ .......... 56 Scheme 3.9. Uracil meridianin derivatives. ................................ ................................ ............... 56 Scheme 3.10. Direct metal free alkynylation of indoles to form meridianin analogs. .................. 58 Scheme 3.11. Indolization of nitroarenes to form meridianin analogs. ................................ ....... 58 Scheme 3.12. Condensation with guanidine to form meriolin 1. ................................ ................. 59 Scheme 3.13. Isolation of Meridianins A, B, C, and E ................................ ................................ 61 Scheme 3.14. Synthesi s of meridianin A and analogs. ................................ ............................... 6 4 Scheme 3.15. Synthesis of psammopemmin A and 2' chloro analog. ................................ ........ 6 6 Scheme 3.16 Synthesis of meridoquin. ................................ ................................ ...................... 69

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ix List of abbreviations 5 HT 5 hydroxytryptamine (serotonin) Ac acetyl Ac 2 O acetic anhydride AcOH acetic acid ACT artemisinin combination therapy ATP adenosine triphosphate BIA biochemical induction assay Bn benzyl Boc t ert b utyloxycarbonyl BuLi butyllithium cGMP current Good Manufacturing Practice CBS Corey Bakshi Shibata CD circular dichroism CDK cyclin dependent kinase CNS central nervous system COSY Correlation spectroscopy CQ chloroquine CY City University of Hong Kong cytD cytochalasin D DAT dopamine active transporter DCM dichloromethane DIBALH diisobutylaluminum hydride DMAP 4 dimetylaminopyridine DME 1,2 dimethoxyethane (glyme) DMF dimethylformamide DMF DMA dimethylformamide dimethylacetal DMP Dess Martin periodinane DMSO dimethylsulfoxide EDC 1 ethyl 3 (3 dimethylaminopropyl) carbodiimide E S I electrospray ionization ent enantiomer Et ethyl ET 743 ecteinascidin 743 EtOAc ethyl acetate EtOH ethanol FABMS fast atom bombardment mass spectrometry FDA Food and Drug Administration Fm 9 fluorenylmethyl GC gas chromatography GSK glycogen synthase kinase h hour(s) HIF 1 Hypoxia Induction Factor 1 HMBC Heteronuclear Multiple Bond Correlation HPLC high pressure (performance) liquid chromatograpy HRFABMS high resolution fast atom bombardment mass spectrometry HRMS high resolution mass spectrometry HSQC Heteronuclear Single Quantum Coherence HTS high throughput screen ing

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x HWE Horner Wadswort h Emmons i Pr isopropyl i PrOH isopropanol ITDL in vitro drug luminescence KHMDS Potassium bis(trimethylsilyl)amide LAH lithium aluminum hydride LC MS liquid chromatograpy mass spectrometry LRMS low resolution mass spectrometry LDA lithium d iisopropylamide Me methyl MeOH methanol MMV Medicines for Malaria Venture MOM methoxymethyl MPLC medium pressure liquid chromatography MQ mefloquine MSA microtubule stabilizing agent MSX Mycosynthetix MTPA methoxytrifluoro methylphenylacetoyl NBS N bromosuccinimide NER n ucleotide e xcision r epair NIS N iodosuccinimide NMR nuclear magnetic resonance NOE Nuclear Overhauser Effect NTOU National Taiwan Ocean University OCM olefin cross metathesis Ph phenyl p in pinacol PKS polyketide synthase PMB para methoxybenzyl PPh 3 triphenylphosphine Pr propyl pyr pyridine RCM ring closing metathesis ROESY Rotating frame Overhause r Effect SpectroscopY RBC red blood cell rt room temperature SAR structure activity relationship SP sulfadoxine/pyrimethamine SPR structure property relationship TBAF tetra n butylammonium fluoride TBDPS tert butyldiphenyl silyl TBS tert butyldimethylsilyl t Bu tert butyl TEA triethylamine TES triethylsilyl Tf trifluoromethylsulfonyl (trifyl) TFA trifluoroacetic acid THF tetrahydrofuran TIPS triisopropylsilyl TLC thin layer chromatograpy TMS trimethylsilyl Troc Trichloroethyloxycarbonyl Ts 4 toluenesulfonyl (tosyl) USF University of South Florida V ATPase vacuolar ATPase WHO World Health Organization

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xi Abstract Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endo phytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum and subsequent synthetic preparation of the resulting polyols (1,2,6 hexanetriol and 1,2 ,3,6 hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product ( 7 R 10 R 11R to 7S 10S 11S ) A partial synthesis of palmerolide A (C3 14) was completed using nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synth etic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4 indolol nucleophile to the apposite pyrimidine electrophile. S everal synthetic 3 pyrimidylindole analogs were also prepared and investigated for central nervous system antimalarial, and cytotoxic activity. Chemi cal investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds : cytochalasin D, roridin E, and 12,13 deoxyroridin E.

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1 Chapter 1 Drugs, nature, and synthesis 1.1 Natural p roducts as s ources for d rugs Compounds derived from natural sources are essential for new drug discovery. According to analysis of all drugs approved world wide from 1981 2006, seventy percent of the new chemical entities reported are natural products, derived from natural products, or inspired by natural products. 1 It is not surprising that organisms produce a multitude of bioactive molecules. Organisms survive based on their ability to generate and retain chemical diversity at low cost. 2 The inherent diversity, selectivity, and potency of n atural products ensure their utility in the drug discovery and development process Both marine 3 and microbial 4 environments are sources for a wealth of natural products At the interface of macro and microenvironment are m icrobial symbion ts thought to be responsible for producing a number of compounds isolated from marine macroorganisms 5 The unfathomable diversity intrinsic to marine and microbial environments ensures unlimited drug discovery potential. 1.2 Drugs from the sea T he antecedent search for biologically active compounds from marine sources can be traced, in part, to the laborator y of Paul S c heuer who methodically investigated the chemistry of marine 6 Since those initial explorations, b ioactive d rugs derived from marine organisms have proven their therapeutic utility 7 The potential for marine natural products as pharmaceuticals is impressive; many compounds isolated from the marine environment have progressed to clinical trials. 8 To date, one non pe p tide drug originating from the marine environment has made it to the commercial sector.

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2 Ecteinascidin 743 (= ET 743, trabectedin, Yondelis 1.1 ) a tris(tetrahydroisoquinoline), was the first marine natural product anticancer drug to be sold comme rcially 9 Originally describe d by the Rinehart group 10 11 with simultaneous publication by Wright et al. 12 from a n antiproliferative extract of the Caribbean tunicate Ecteinascidia turbinat a ET 743 was isolated in low yields (0.0001%) along with several other analogs (ET 729: 1.2 ; E T 745: 1.3 ; ET 759A: 1.4 ; ET 759B: 1.5 ; ET 770: 1.6 ). ET 743 was found to target DNA transcription by a complex mechanism of action involving DNA binding 13 with an unusual requirement : the cell must possess a proficient n ucleotide e xcision r epair (NER) system. 14 This differs from all other known DNA interacting agents which require deficient NER systems to exert cytotoxic effects. S upply problem s of ET 743 were addressed initially with large scale aquaculture then later alleviated using an efficient semisyn thetic procedure (see section 1.4.3). Under the trade name Yondelis, ET 743 is marketed by PharmaM ar in partnership with Jo h nson and Johnson in the European Union as treatment for advanced soft tissue sarcoma and is currently in development for ovarian, prosta te, lung, breast and pediatric cancers. Discodermolide ( 1.7 ), a polypropionate derived polyhydroxy lactone isolated from the rare Bahaman deep water (300 m) sponge Discodermia dissoluta is a potent microtubule stabilizing agent (MSA) 15 16 impede the cellular cycle by stabilizing microtubules in conditions which would normally be destabilizing, effectively disrupting microtubule dynamics. 17 Discodermolide

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3 was found to stabilize microtubules more potently than taxol 18 generating immense interest in the compound. Problems with obtaining quantities suitable for preclinical evaluation led to the development of efficient gram scale syntheses of discodermolide (s ee section 1.4.3). Although an extremely promising lead, Phase 1 clinical trials were halted on discodermolide due to lack of efficacy at tolerated doses. Initial investigation of the shallow water Japanese sponge Halichond r ia okadai revealed a cytotoxic polyether C 38 fatty acid derivative designated okadaic acid ( 1.8 ) 19 Due to remarkable antitumor activity demonstrated by H. o k adai extracts in vivo subsequent reinvestigation of sponge revealed small quantities of several cytotoxic polyether macrolides including halichondrin B ( 1.9 ) which w as shown to potently (nanomolar concentrations) inhibit cell growth 20 The low yield s of halichondrins ( halichondrin B: 20 g/kg ; total halichondrins: ~0.14 mg/kg ) from H. o k adai presented a barrier for pre clinical trials Fortuitously, a new species of deep water sponge, Lissodendoryx n. sp. 1 from the South Island of New Zealand was discovered to contain the compounds in higher concentrations ( halichondrin B: ~0.4 mg /kg; total halichondrins: ~ 1.5 mg/kg) 21 One metric ton of Lissodendoryx n. sp. 1 was harvested via trawling (80 100 m) which resulted in isolation of halichondrin B (310 mg), quantities sufficient for preclinical trials. As aquaculture techniques 22 were in development to provide the gram scale quantities of halichondrin B necessary for clinical trials, truncated synthetic halichondrin analogs were found to Even though halichondrin D had advanced to Phase I clinical trials in 2002 focus was directed to the most promising of the truncated synthetic analogs (see section 1.4.4).

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4 The abovementioned compounds represent a small fraction of marine derived chemistry exhibiting intriguing chemical structure and exciting bioactivity, a majority of which are isolated from temperate and tropical waters. Because m arine natural product discovery programs have traditionally focused on organisms in tropical and temperate waters, a wealth of cold water organisms remain largely unexplored. 23 Although understudied, marine invertebrates residing in cold water have elaborated many interesting and biologically relevant compounds. Chapters 2 and 3 of this disserta tion examine some remarkable chemistry isolated from cold water marine invertebrates. 1.3 Drugs from microorganisms Microbe derived drug discovery began in 1928 in the laboratory of Alexander Fle ming 24 when a Penicillium mold was observed to kill Staphylococcus aureus leading to the discovery of the antibiotic lactam, penicillin ( 1.10 ) An abundance of useful antibiotics derived from microbes have been isolated since that initial discovery including streptomycin ( 1.11 ) an aminoglycoside from Streptomyces griseus, the dichloroacetamide chloramphenicol ( 1.12 ) from Streptomyces

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5 venezuelae and the tetracyclines from Streptomyces spp ., the first of which was named chlortetracycline ( 1.1 3 ). As well as antibiotic activity, microbial secondary metabolites display a broad range of biological utility including, but not limited to, immunosuppressant, antitumor, insecticidal, heribicidal, and antiparasitic properties 4 Calicheamicins s alino s poramide s and epothilones are notable microb ial natural products currently in clinical development possess ing both interesting structur al features in addition to compelling antitumor properties. Calicheamicin s, extraordinarily potent antitumor compound s were isolated from the bacterium Micromonaspora echinospora 25 Calicheamicin 1 Br ( 1.14 ) was initially isolated from culture but upon refermentation in the presence of sodium iodide, a similar compound differing only in halogenation o f the benz e ne ring was discovered ( calicheamicin 1 I 1.15 ). 26 The remarkabl e and complex structure of calicheamicin 1 I including four carbohydrate moieties, a hexasubstituted benzene ring, an N O glycosidic bond, a tri sulfide, and a bicyclo enediyne system, was determined via spectroscopic techniques in conjunction with noteworthy chemical degradation studies (see section 1.4.1) Antitumor properties of calicheamicin 1 I arise from its ability to cleave double stranded DNA. 27 Although calicheamicin 1 I is generally toxic, a prodrug was developed by attaching an antibody (anti CD33 monoclonal ) which directs the molecule selectively to certain cancer cells types The resulting drug,

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6 gemtuzumab ozogamicin, is approved by the Food and Drug Administration (FDA) for the treatment of relapsing patients suffering from acute myeloid lymphoma. 28 Exploration of microbial constituents isolated from ocean sediment resulted in the i dentification of a new genus of acti n omycetes ( Salinospora ) which was found to yield a potent proteosome inhibitor, s alinosporamide A ( from S. tropica CNB476, shake flask: 4 mg/L culture media 1.1 6 ). 29 Nereus Pharmaceuticals licensed the compound and began industrial scale fermentation after modifying the culture media to meet current Good Manufacturing Practice (cGMP) guidelines 30 The company isolated an individual colony of S. tropica CNB476 from the origin al strain that produced more salinoporamide A concomitant with less undesirable analogs Researchers at Nereus discovered that a ddition of solid resin (XAD 16) to the culture considerably increased quantities of salinosporamide A due to the inherent instability of the lactone in aqueous solution. The fermentation was ultimately optimized to obtain quantities of material suitable for clinical trials resulting in about a hundred fold increase in production of salinosporamide A (fermentor: 360 mg/L shake flask: 450 mg/L ). Salinosporamide A completed Phase 1 clinical trials for th e treatment of multiple myeloma in 2010, just seven years since its discovery

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7 Epothilones, microtublule stabilizing polyketide macrolides, were isolated from myxobacterium Sorangium cellulosum 31 Epothilones A ( 1.1 7 ) and B ( 1.1 8 ) were the first non taxane based 32 33 Both compounds are at least as potent as taxol in stabilizing microtubules. Gram scale isolation (4.8 g 1.1 7 ; 2.1 g 1.1 8 from 230 L culture broth) and c rystal structure of epothilones A and B were subsequently reported 34 Due to the excellent potency and efficacy of the naturally occurring epothilones many promising clinically significant synthetic analogs have been also been prepared (see section 1.4.4) N atural epothilone B as well as five synthetic epothilones have completed P hase I clinical trials leading to investigat ion of several (e pothilone B and two synthetic derivatives) in Phase II studies to assess efficacy in a variety of tumor types 35 Phase III clinical trials of epothilone B are currently underway for treatment of patients with taxane and platinum resistant disease. 36 1.4 Relevance of synthesis in natural product chemistry The essential role of synthesis in natural product drug discovery is undeniable. 37 The structural complexity present in many naturally occurring compounds necessitates advanced spectroscopic techniques often times in conjunction with wet chemical methods to fully elucidate absolute configuration. Derivatization an d degradation are common synthetic methods utilized to elucidate intricate chemical structure s T otal synthesis considered to be the ultimate verification of structure is especially useful for validating complex natural products isolated in scarce amounts Because many natural products are found in low abundance, total and semisynthes e s have been utilized to alleviate supply problems of clinically promising drugs. Inspired chemists have constructed analogs of biologically active natural products to probe and tailor bioactivity, often

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8 times resulting in more potent and / or less toxic deri vatives. The symbiotic relationship between natural product chemistry and organic synthesis cannot be overstated The value of chemical synthesis to identify, supply, and modify compounds originating from natural sources will be emphasized in the remaind er of this chapter 1. 4 .1 Structure elucidation and v erif ication via d egradati on studies Degradati on studies of naturally occurring compounds have proved instrumental in the structure elucidation and verification of complex molecules Degradation studies to determine chemical structure were commonplace before the advent of X ray and nuclear magnetic resonance ( NMR ) 38 Although not as common as it once was, chemical degradation for structure eluc idation and verification is still an important part of natural products chemistry, evidenced by an extraordinary study in which the structure of calicheamicin 1 I was determined 26 Spectroscopic analysis of calicheamicin 1 I ( 1.15 ) a compound displaying activity in biochemical induction assay s (BIA ; used to identify DNA damaging antitumor compounds ), 39 revealed four glycosides and one aglycone. D ifficulty further interpreting NMR data due to signal overlap prompted degradation studies (Scheme 1.1) N acetylated calicheamicin 1 I ( 1.19 ) was subjected to acidic methanolysis yielding among other products, the methyl glycoside of a 6 deoxyhexopyranose ( 1.20 ; both and anomers), the methyl glycoside of a n N acetyl N ethylaminoxylopyranose ( 1.21 ; 7/3 mixture of anomers confirmed 40 by synthesis ) and hexasubstituted benzene 1.22 To find degradation products retaining BIA activity, methanolysis was performed on a strong cation exchange resin (Dowex) column resulting in thioesters 1.23 and 1.24 (as well as their anomers) along with the BIA active calicheamicin pseudoaglycon e 1.25 Structures of 1.23 and 1.24 were determined with NMR analysis. Mass data indicated that fragment 1.25 was the natural product minus ring D and E. Based on careful fast atom bombardment mass spectroscopy ( FABMS ) and NMR examination of 1.25 ring A and B were determined to be linked through a n unusual N O glycosidic bond.

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9 The arduous task of determining the aglycon e structure of 1.25 a was completed in part, with thoughtful X ray, NMR, and FABMS analysis of reaction product 1.26 and deuterated 1.26 d 2 which were recovered from mixtures of PPh 3 /CH 2 C l 2 /CH 3 OH and PPh 3 /CD 2 Cl 2 /CD 3 OD respectively (Scheme 1.2) The deuterated benzene product ( 1.26 d 2 ) could only be explained by a enediyne bi radical cycloaromati zation (Bergman cyclization) initiated by the destabilizing effect of thiol adding in Michael fashion to the un satura ted ketone ( 1.2 5b 1.2 7 ) Because only natural and degradation product s retaining the endiyne system displayed BIA activity, the endiyne moiety was determined to be responsible for the observed DNA damaging effects. The degradation study revealed that n ature has found an elegant way to retain and discriminately liberate the quite reactive endiyne functionality. Nicolaou et. al 41 synthesized calicheamicin 1 I

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10 and confirmed the proposed structure further validating the effectiveness of degradation to elucidate complex natural products. 1.4.2 Structure v erification via total synthesis Spectroscopic techniques [ NMR, high resolution mass spectroscopy ( HRMS) ] and chemical degradation are quite powerful and allow for structure elucidation of elaborate molecules. Structural misassignment in the literature is not uncommon, however, due to investigator error by data misinterpretation Simple bookkeeping errors 42 are often responsible for misassignment, although structural data from exceptionally complex molecules can lead the most careful scientist to the wrong conclusion. Total synthesis provides a n unambiguous means to verify proposed structures. Chemical lit erature abounds with structures of natural product s that have been

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11 corrected through total synthesis. 43 44 T he total synthesis of diazonamide A highlight s the structure reassignment of an exceedingly complex and bioactive marine natural product In 1991, d iazonamide A ( proposed: 1.28 ; revised: 1.29 ), an unusual halogenated cyclic peptide with potent antitumor properties, was isolated by Clardy and Fenical 45 from the colonial ascidian Diazona anngulata collected in the Philippines. compelling bioactivity captured the interest of dozens of synthetic research groups which culminated after ten years of investigation and frustration in structural reassignment. When first isolated, the structure of diazonamide A could not be elucidated with NMR and HRFABMS alone due to a large number of unprotonated carbons and heteroatoms. Several subunits were revealed but their connectivity remained elusive. Because the compound would not crystalli ze X ray analysis of diazonamide A was not possible. Due to similar NMR, ultraviolet ( UV ) and infrared ( IR ) data between diazonamide A and diazonamide B, X ray diffraction of the p bromobenzamide derivat i ve of diazonamide B ( 1.30 initially assigned ) afforded connectivity of The conversion of proposed hemiacetal 1.28 to the acetal found in 1.30 was postulated to occur during the acylation reaction. The proposed structure for diazonamide A ( 1.28 ) was synthesized in 2001 by Harran et. al. 46

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12 Synthesis of the proposed structure of diazonamide A ( 1.28 ) began with formation of modified dipeptide 1.30 from 1.31 1.32 and 1.33 (Scheme 1.3) A pivotal Heck cyclization of 1.30 formed 1.34 which after phenol protection was dihydroxylated to 1.35 The diazonamide core ( 1.36 ) was formed from 1.35 through an acid catalyzed pinacol rearrangement in a near perfect stereoselective manner. After reduction, protecting group mani pulation, and selective bromination, 1.36 was converted to lactone 1.37 upon treatment with acid. Lactone opening with N dimethylaluminumtryptamine followed by oxidation furnished 1.38 which was briefly photolyzed (350 nm) to afford 1.39 after acetylation Oxidation/cyclodehydration of 1.39 formed bis(oxazoyl)indole 1.40 Surprisingly, exposing 1.40 to UV (300 nm) light resulted in the formation of 1.41 with the loss of HBr. With the critical framework synthesized, 1.41 was converted to the proposed str ucture of diazonamide A ( 1.28 ) with facile dichlorination and removal of protecting groups Unfortunately, however, the synthesized compound was quite unstable and differed in physical properties with the natural product.

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13

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14 Harran et al re that the natural product did not release valine upon acid digestion nor did spectroscopic data support the valine residue. They theorized the C37 substituen t was actually and OH and not an NH 2 This change necessitated an alteration elsewhere in the molecule to rectify the molecular mass observed R e examination of mass data of natural diazonamide A and crystal structure of the p bromobenzamide derivatve of diazonamide B ( 1.30 ) indicated the acetal proposed for 1.30 was likely retained in natural diazonamide A as opposed to the proposed hemiacetal. X ray and HRMS suggested the acetal structure was actually a hemiaminal ether in which O3 should be an NH leading Harran et al to propose revised structure 1.2 9 for diazonamide A. 47 A year later, Nicolaou et al. completed the total synthesis of 1.2 9 using strategies developed by Harran as well as another method differing in order of macrocycle formation fina lly verifying the revised structure as that of natural diazonamide A 48 1.4.3 Drug s upply by total synthesis and semisynthesis Culturable microbes and cultivatable plants are excellent sources for drugs. However, reliable drug supply fro m natural sources is often hampered by rarity or inaccessibility of the producing organism Particularly susceptible are drugs from the sea. Although marine organisms afford a wealth of interesting bioactive chemistry, sustainable supply of marine derived drugs is limited. Not only are most marine macroorganisms and their microbial fauna largely uncultu r able but m any marine natural products are found in low natural abundance C onsequently large scale aquaculture would likely be too inefficient as a viable sou rce of drugs Pharmacological investigation of discodermolide and ect e inascidin, two promising marine derived antitumor agents, was hindered by lack of adequate supply Supply demands were alleviated through total synthesis and semisynthesis respectively. Several highly efficient syntheses of discodermolide w ere developed allowing preparation of gram quantities sufficient for initial clinical testing. 49 Schr ei ber et al completed the total synthesis of ent di s codermolide ( ent 1.7 ) establishing the absolute configuration of the molecule. 50

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15 Surprisingly, ent discodermolide was found to have similar cytotoxic effects as the natural product. Schr ei ber et al used the same methodology to synthesize the natural ena n tiomer. 51 The synthesis relied upon the absolute configuration of Roche ester starting material ( 1.43 ) which was converted to two diastereomers via Roush crotylation ( 1.44 1.45 ) further elaborated to three fragments of discodermolide ( 1.46 1.47 1.48 ) t hat were then stitched together to form the natural product effectively completing the divergent/convergent synthesis (Scheme 1.4) Key reactions include Stille Gennari HWE olefination in the formation of 1.46 Negishi coupling in the formation of 1.48 a Nozaki Kishi coupling of 1.46 and 1.47 and finally enolate alkylations to join 1.48 and 1.49 The synthesis was completed in a n overall yield of 4.3% ( longest linear sequence : 24 steps). Smith et al. reported the synthesis of discodermolide in 6% overall yield (24 linear steps) which resulted in an impressive 1.0 g ram of the natural product using a Ro che ester derived aldehyde to form fragments which were coupled to generate the natural pr synthesis 52 Pa terson et al employed two synthetic methods to produce discodermolide. The relatively high yielding (10.3% over 23 steps longest linear sequence ) first generation synthesis employed chiral auxiliary groups and reagents to direct stereochemistry 53 A second generation

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16 synthesis was lower yielding (5.1% over 24 linear steps ) but required fewer total steps (35) and no chiral auxiliary groups or reagents depending instead on substrate to control stereochemistr y 54 Novartis Pharma AG utilizing key steps from syntheses reported by Smith and Paterson completed large scale synthesis of discodermolide in apposite quantities (60 g!) for Phase I clinical trials. 55 A quaculture provided suitable quantities of ecteinascidin 743 (ET 743, 1.1 ) for preclinical and early Phase clinical trials but yields were variable. S uppl y issues plagued development until scientists at Pharma Mar developed a semisynthesis of ET 743 from cyanosafracin B ( 1.50 Scheme 1.5 ), a readily available (kilogram scale!) antibiotic from the optimized fermentation broth of Pseudomonas fluorescens 56 Cyanosafracin B ( 1.50 ), after protecting group manipulation, was converted to 1.51 though an unstable hydoquinone intermediate (Scheme 1.6) More protecting group manipulation and cleavage of the amide by Edman degradation formed amine 1.52 A key step in the semisynthesis was the conversion of amine 1.52 to alcohol 1.53 upon treatment with sodium nitrit e/acetic acid ( NaNO 2 /AcOH) effectively completing a formal total synthesis by generating an intermediate in the total synthesis of E T 743 previously reported by Corey et al. 57 The remaining conversion of 1.53 to ET 1.53 deallylation, and subsequent oxidation of the phenol with (PhSeO) 2 O resulted in

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17 hydroxylated product 1.54 Q uinone methide formation via Swern protocol then liberation of the thioether to generate thiolate ion resulted in nucleophilic attack of quinone forming 1.55 after acetylation. Removal of the Troc group, oxidation of the resulting amino lactone to the keto lactone then treatment with 2 [3 hydroxy 4 methoxyphenyl]ethylamine ( 1.56 ) afforded stereospecific formation of the spiro tetrahydroisoquinoline 1.57 Protecting group removal and conversion of cyano group to alcohol with silver nitrate ( AgNO 3 ) resulted in the semisynthesis of ET 743 ( 1.1 ) from cyano sa fracin B ( 1.50 ). The initial synthetic work of Corey et al. in combination with the ingenuity of Pharma Mar scientists led to the develop ment of gram scale semisynthesis of a rare marine natural product enabling production of commercial quantities.

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18 1.4.4 Synthetic derivatives of natural products as drugs The first examples of marine natural product inspired, clinically useful synthetic derivatives were antiviral a ra A (vidarabine, 1.58 ) and chemotherapy agent a ra C (cytarabine, 1.59 ). Both compounds owe their structure to sponge derived bioactive arab i nose containing nucleosides isolated by Bergmann in 58 More recently it was found that twenty seven percent of the 1184 drugs approved worldwide from 1981 2006 are derived from natural product s including semisynthetic derivatives and totally synthetic compounds containing a pharmacophore that is natural in origin 1 The number jumps to thirty seven per cent if synthetic natural product mimics such as pe p tidic isoster e s are included in the an a l y sis. Medicinal chemists, through synthetic procedures, tweak already biologically active natural products to identify pharmacophores responsible for desired bioactivity, eliminate unneeded or detrimental structural features and, ultimately, optimize pharmacological properties. 59 Eribulin mesylate and several epothlilone analogs are examples of clinically successful synthetic derivatives of natural origin Using synthetic methods developed by Kishi, 60 61 s everal truncated analogs of halichondrin B ( 1.9 ) were found to be equipotent to the natural product in antitumor assays 62 Because obtaining halichondrin B from natural sources proved problematic, the truncated analogs, with about 70% the molecular mass of the natural compound were more easily synthesized and thus, an attractive alternative Further screening by Eisai Company reveal ed that the truncated halichondrin B ketone analog E7389 ( ER 086526 ) later developed as e ribulin mesylate ( 1. 60 ), possessed highly potent in vitro and in vivo anticancer activities with a wide therapeutic window in vivo 63 Eribulin mesylate completed Phase II studies as a monotherapy for refractory breast cancer 64 and showed promising results in patients with advanced breast cancer. 65 Currently

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19 eribulin mesylate is undergoing Phase III trials as a late stage treatment for locally recurrent or metastatic breast cancer. 66 After comple ting total synthesis of epothil ones A ( 1.17 ) 67 and B ( 1.18 ), 68 Danishefsky et al proceeded to assemble close to fifty epothilone analogs. Danishefsky discovered that synthetic desoxyepothilone B, ( KOS 862, epothilone D, 1.61 ) displayed low in vivo toxicity in contrast with epothilone B (highly toxic in mice) while retaining antitumor effects. 69 Desoxyepothilone B has completed Phase II clinical trials for colorectal, gastric, ovary, renal and other cancers 3 5 The production and evaluation of over 300 semisynthetic epothilone analogs by researchers at Bristol Meyers Squibb led to i xabepilone (BMS 247550, aza epothilone B 1.62 ) the l actam derivative of epothilone B which displayed antitumor properties toward a wide range of cancers in Phase II trials. 70 Recently, P hase III clinical trials of combination ixabepilone therapy in the treatment of patients resistant to taxol and anthracy c lines showed a statistically significant improvement in progression free survival although toxicity was a concern. 36

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20 1.5 Research objectives The interconnectivity and interdependence of natural product chemistry and chemical synthesis in dru g discovery is demonstrated through the c hemical exploration reported herein. Investigation of l ess accessible and therefore less studied environments, particularly Antarctica and endophytic microbial communities, ha s afforded ex citing chemistry and promising biologically active leads. Synthetic analogs inspired by Antarctic derived natural products have displayed promising bioactivity. The c hemical investigation, degradation, and synthetic studies presented herein dislcose a number of remarkable compounds derived from natural sources as well as synthetic operations utilized to study their interesting structure and biological activity. 1.6 References Cited 1 Newman, D. J.; Cragg, G. M. J. Nat. Prod 2007 70 461 477. 2 Firn, R. D.; Jones, C. G. Nat. Prod. Rep 2003 20 382 391. 3 Saleem, M.; Ali, M S. ; Hussain, S.; Jabbar, A.; Ashraf, M.; Lee Y, S. Nat. Prod. Rep 2007 24 1142 1152. 4 Demain, A.; Sanchez, S J. Antibiol 2009 62 5 16. 5 Salomon, C. E.; Magarvy, N. A., Sherman, D. H. Nat. Prod. Rep 2004 21 105 121. 6 Okuda, R. J. Nat. Prod 2004 67 1201 1203. 7 Molinski, T. F.; Dalisay D. S.; Lievens, S. L.; Saludes, J. P. Nat. Rev. Drug Discovery 2009 8 69 85. 8 Newman, D. J.; Cragg, G. M. J. Nat. Prod 2004 67 1216 1238. 9 Cuevas, C.; Francesch, A. Nat. Prod. Rep 2009 26 322 337. 10 Rinehart, K. L.; Holt, T. G.; Frege au, N. L.; Stroh, J. G.; Keifer, P. A.; Sun, F.; Li, H. L.; Martin, D. G J. Org.Chem. 1990 55 4512 4515. 11 Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.; Stroh, J. G.; Keifer, P. A.; Sun, F.; Li, H. L.; Martin, D. G. J. Org.Chem. 1991 56 1676. 12 Wright, A. E.; Forleo, D. A.; Gunawardana, G. P.; Gunasekera, S. P.; Koehn, F. E.; McConnell, O. J. J. Org.Chem. 1990 55 4508 4512. 13 Fayette, J.; Coquard, I. R.; Alberti, L.; Boyle, H.; Meeus, P.; Decouvelaere, A. V.; Thiesse, P.; Sunyach, M. P. ; Ranchere, D.; Blay, J. Y. Curr. Opin. Oncol. 2006 18 347 353.

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21 14 Takebayashi, Y.; Zimonji, D. B.; Nakayama, K.; Emmert, S.; Ueda, T.; Urasaki, Y.; Kanzaki, A.; Akiyama, S. I.; Popescu, N.; Kraemer, K. H.; Pommier, Y. Nat. Med. 2001 7 961 966. 15 Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1990 55 4912 4915. 16 Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1991 56 1346. 17 Jordan, M. A.; Wilson, L. Nat. Rev. Canc. 2004 4 253 265. 18 Haar, E.; Kowalski, R.; Hamel, E.; Lin, C.; Longley, R.; Gunasekara, S.; Rosenkranz, H.; Day, B. Biochemistry 1996 35 243 250. 19 Tachibana, K.; Scheuer, P. J.; Tsukitani, Y.; Kikuchi, H.; Engen, D. V.; Clardy, J. J. Am. Chem. Soc. 1981 103 2469 2471. 20 Hirata, Y.; Uemura, D Pure Appl. Chem 1986 58 701 710. 21 Munro, M. H. G.; Blunt, J. W.; Dumdei, E. J.; Hickford, S. J. H.; Lill, R. E.; Li, S.; Battershill, C. N.; Duckworth, A. R J. Biotechnol 1999 70 15 25. 22 Battershill, C. N.; Page, M .J. Aquac. Update 1996 Spring 5 6. 23 Lebar, M. D.; Heimbegner, J. L.; Baker, B. J. Nat. Prod. Rep. 2007 24 774 797. 24 Fleming, A. Br. J. Exp. Pathol 1929 10 226 236. 25 Lee, M. D.; Manning, J. K.; Williams, D. R.; Kuck, N. A,; Testa, R. T.; Borders, D. B. J. Antibiot. 1989 42 1070 1087. 26 Lee, M. D.; Dunne, T. S.; Chang, C. C.; Siegel, M. M.; Morton, G. O.; Ellestad, G. A.; McGahren, W. J.; Borders, D. B. J. Am. Chem. Soc 1992 114 985 997. 27 Walker, S.; Landovitz, R.; Ding, W. D.; Ellestad, G. A.; Kahne, D. Proc. Natl. Acad. Sci. USA 1992 89 4608 4612. 28 Bross, P. F.; Beitz, J.; Chen, G.; Chen, X. H.; Duffy, E.; Kieffer, L.; Roy, S.; Sridhara, R., Rahman, A.; Williams, G.; Pazdur, R. Clin. Cancer Res 2001 7, 1490 1496. 29 Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Angew. Chem. Int. Ed. 2003 42 355 357. 30 Potts, B. C.; Lam, K. S. Mar. Drugs 2010 8 835 880. 31 Gerth, K.; Bedorf, N.; Hofle, G.; Irschik, H.; Reichenbach, H. J. Antibiot. 1996 49 560 564. 32 Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995 55 2325 2333. 33 Kowalski, R. J.; Giannakakou, P.; Hammel, E. J. Biol. Chem. 1997 272 2534 2541.

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22 34 Hofle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H. Angew. Chem. Int. Ed 1996 35 1567 1569. 35 Larkin, J. M. G.; Kaye, S. B. Ann. Oncol 2007 18 v28 v34. 36 Sabbatini, P.; Spriggs, D. R. J. Clin. Oncol 2009 27 3079 3081. 37 Nicolaou, K. C.; Chen, J. S.; Dalby, S. M. Bioorg. Med. Chem. Lett 2009 17 2290 2303. 38 Hudlick y, T.; Reed, J. W. The Way of Synthesis; Wiley VCH: Weinheim, 2007. 39 Elespuru, R. K.; White, R J. Cancer Res. 1983, 43 2819 2830. 40 Kahne, D.; Yang, D.; Lee, M. D. Tetrahedron Lett. 1990, 31, 21 22. 41 Nicolaou, K. C.; Hummel, C. W.; Pitsinos, E. N.; Nakada, M.; Smith, A. L.; Shibayama, K.; Saimoto, H J. Am. Chem. Soc. 1992 114 10082 10084. 42 Erickson, K. L.; Beutler, J. A.; Cardellina II, J. H.; Boyd, M. R. J. Org. Chem. 2001, 66 1532. 43 Nicolaou, K. C.; Snyder, S. A. Angew. Chem Int. Ed 2005 44 1012 1044. 44 Usami Y. Mar. Drugs 2009 7 314 330. 45 Lindquist, N.; Fenical, W.; Van Duyne, G. D.; Clardy, J. J. Am. Chem. Soc 1991 113 2303 2304. 46 Li, J.; Jeong, S.; Esser, L.; Harran, P. G. Angew. Chem. Int. Ed 2001 40 4765 4769. 47 Li, J. ; Burgett, W. G.; Esser, L.; Amezcua, C.; Harran, P. G. Angew. Chem. Int. Ed 2001 40 4770 4773. 48 Nicolaou, K. C.; Chen, D. Y. K.; Huang, X.; Ling, T.; Bella, M.; Snyder, S. A. J. Am. Chem. Soc. 2004 126 12888 12896. 49 Paterson, I.; Florence, G. J. Eur. J. Org. Chem 2003 12 2193 2280. 50 Nerenberg, J. B.; Hung, D. T.; Somers, P. K.; Schreiber, S. L. J. Am. Chem. Soc. 1993 115 12621 12622. 51 Nerenberg, J. B.; Hung, D. T.; Schreiber, S. L. J. Am. Chem. Soc. 1996 118 11054 11080. 52 Sm ith III, A. B.; Beauchamp, T. J.; LaMarche, M. J.; Kaufman, M. D.; Qiu, Y. P.; Arimoto, H.; Jones, D. R.; Kobayashi, K. J. Am. Chem. Soc. 2000 122 8654 8664. 53 Paterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P.; Sereinig, N. J. Am. Chem. Soc 2001 123 9535 9544. 54 Paterson, I.; Delgado, O.; Florence, G. J.; Lyothier, I.; Scott, J.; Sereinig, P. N. Org. Lett. 2003 5 35 38.

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23 55 Mickel, S. J.; Niederer, D.; Daeffler, R.; Osmani, A.; Kuesters, E.; Schmid, E.; Schaer, K.; Gamboni R.; Chen, W.; Loeser, E.; Kinder Jr., F. R.; Konigsberger, K.; Prasad, K.; Ramsey, O. R.; Wang, R. M. Org. Process Res.Dev. 2004 8 122 130. 56 Cuevas, C. Perez, M.; Martin, M. J.; Chicharro, J. L.; Fernandez Rivas, C.; Flores, M.; Francesch, A.; Ga llego, P.; Zarzuelo, M.; de la Calle, F.; Garcia, J.; Polanco, C.; Rodrigueuz, I.; Manzanares, I. Org. Lett. 2000 2 2545 2548. 57 Corey, E. J.; Gin, D. Y.; Kania, R. J. Am. Chem. Soc. 1996 118 9202 9203. 58 Bergmann, W.; Burke, D. C. J. Org. Chem 1956 21 226 228. 59 Wilson, R. M.; Danishefsky, S. J. Angew. Chem. Int. Ed 2010 49 6032 6056. 60 Aicher, T. D.; Buszek, K. R.; Fang, F. G.; Forsyth, C. J.; Jung, S. H.; Kishi, Y.; Matelich, M. C.; Scola, P. M.; Spero, D. M.; Yoon, S. K. J. Am. Chem. Soc 1992 114 3162 3164. 61 Stamos, D. P.; Chen, S. S.; Kishi, Y. J. Org. Chem 1997 62 7552 7553. 62 Wang, Y.; Habgood, G. J.; Christ, W. J.; Kishi, Y.; Littlefield, B. A.; Yu, M. Bioorg. Med. Chem. Lett. 2000 10 1029 1032. 63 Towle, M. J.; Salvato, K. A.; Budrow, J.; Wels, B. E.; Kuznetsov, G.; Aalfs, K. K.; Welsh, S.; Zheng, W.; Seletsky, B. M. ; Palme, M. H.; Habgoed, G. J.; Singer, L. A.; Dipietro, L. V.; Wang, Y.; Chen, J. J.; Quincy, D. A.; Davis, A.; Yoshimatsu, K. ; Kishi, Y.; Y u, M. J.; Littlefield, B. A. Cancer Res 2001 61 1013 1021. 64 Blum, J.; Forero, L.; Heiskala, M. K.; Meneses, N.; Chandrawansa, K.; Fang, F.; Shapiro, G.; Fields, S. Z.; Silberman, S.; Vahdat, L. J. Clin. Oncol 2006 24 (Suppl. 18) 653. 65 Blum J. L.; Pruitt, B.; Fabian, C. J.; Rivera, R. R.; Shuster, D. E.; Meneses, N. L.; Chandrawansa, K.; Fang, F.; Fields, S. Z.; Vahdat, L. J. Clin. Oncol 2007 25 (Suppl. 18) 1034. 66 Twelves, C.; Cortes, J. ; Vahdat, L. T.; Wanders, J.; Akerele, C.; Ka ufman, P. A. Clin. Breast Cancer 2010 10 160 163. 67 Balog, A.; Meng, D.; Kamenecka, T.; Bertinato, P.; Su, D. S.; Sorensen, E. J.; Danishefsky, S. J. Angew. Chem. Int. Ed 1996, 35 2801 2803. 68 Su, D. S.; Meng, D.; Bertinato, P.; Balog A.; Sorensen, E. J.; Danishefsky, S. J.; Zheng, Y. H.; Chou, T. C.; He, L.; Horwitz, S. B. Angew. Chem. Int. Ed. 1997, 36 757 759. 69 Chou, T. C.; Zhang, X. G.; Balog, A.; Su, D. S.; Meng, D.; Savin, K.; Bertino, J. R.; Danishefsky, S. J. Proc. Na tl. Acad. Sci. USA 1998 95 9642 9647. 70 Lee, F. Y. F.; Borzilleri, R.; Fairchild, C. R.; Kim, S. H.; Long, B. H.; Reventos Suarez, C.; Vite, G. D.; Rose, W. C.; Kramer, R. A. Clin. Cancer. Res 2001 7 1429 1437.

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24 Chapter 2 Synthetic studies of palmerolide A 2.1 Isolation, structure elucidation, and bioactivity of palmerolide A The palmerolides are a family of macrocylic polyketides found in the abundant Antarctic tunicate Synoicum adareanum Antarctic Peninsula 1 The major metabolite, palmerolide A ( 2 1 ), displays 18 nM inhibition of UACC 66 melanoma and includes among its biochemical targets the pH regulatory vacuolar ATPase (V ATPase), 2 for which it is a potent inhibitor (IC 50 = 2 nM). V ATPases are largely responsible for cellular and organellular pH regulation but h ave been implicated in cancer treatment 3 due in part to the low pH requirement and concomitant overexpression of V ATPases of some cancer cell types. 4 In ongoing studies at the National Cancer Institute at Frederick 5 palmerolide A induced markers of autophagy and the transcription factor Hypoxia Induction Factor 1 (HIF 1 ), but the mechanism underlying palmerolide A induced cell death in human tumor cells remains unclear. Palmerolide A remains of interest for development due, in contrast to other V ATPase inhibitors such as bafilomycin ( 2. 2 ) 6 7 to its lack of neurotoxicity at therapeutic levels. The microbial community of S adareanum has been investigated for genes codin g for polyketide synthase s (PKS) in an ongoing effort to characterize bacteria responsib le for producing polyketide der ived palmerolide A. 8

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25 The planar structure of palmerolide A was established on the basis of extensive 2D NMR experimentation while the stereochemical assignment required a combination of spectroscopic and derivatization techniques 1 The two secondary alcohols, C7 and C 10, were amenable to stereochemical analys e s by Mosher s method. 9 The remaining stereocenter s were determined relative to C 10 using through space NMR techniques such as Rotating frame Overhauser Effect SpectroscopY ( ROESY ) along with n J CH based analysis. 10 Configuration of C19 and 20 relative to C7, 10 and 11 was proposed based on NOE data suggesting the macrolide conformed to a teardrop like structure. The originally proposed structure ( 1a ) was determined to have an absolute configuration of 7R 10R 11R 19R 20R The unique and potent bioactivity as well as the intriguing structure of pal m erolide A has generated considerable interest in developing synthetic access to the compound (see section 2.2 for a review) prompted us to perform degradative studies using a chiral pool based strategy 11 to verify the initial stereochemical assignments (see section 2.3 ) which then led us to synthesize the C3 14 fragment 12 (see section 2.4 ).

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26 2.2 Palmerolide A as a synthetic target To date three total syntheses and one structure activity relationship study of palmerolide A have been published Several partial syntheses as well as a formal total synthesis have also been reported. 2.2.1 Total synthes e s of palmerolide A 2.2.1.1 Total synthesis by De Brabander De Brabander et al. reported the first total synthesis of ent palmerolide A in 2007. 13 His group began by synthesizing the originally proposed structure 2. 1a De Brabander envisioned constructing the C1 24 portion ( 2. 3 ) of palmerolide A with a convergent approach (Scheme 2. 1) utilizing three main fragments : 2. 4 (C9 15) 2. 5 (C16 24) and 2. 6 (C1 8). Vinyl pinacol borate ester 2. 4 was formed from D arabitol ( 2. 7 ) through intermediate 2. 8 (Scheme 2. 2). A key step in the formation of fragment 2. 5 was the diastereoselective Muk a iyama aldol condensation of dieno l silyl ether 2. 9 a and vinyl iodide 2. 10 to form 2. 11 Phosphonate ester 2. 6 was synthesized from valerolactone ( 2. 12 ) C oupling fragments 2. 4 and 2. 5 via Suzuki methodology yielded an intermediate that was e sterifi ed with 2. 6 se t t ing up a ring closing Horner Wadsworth Emmons (HWE) olefination to form macrolactone 2. 3

PAGE 41

27 Ster e oselective reduction of 2. 3 gave the preferred C7 epimer Curtius rearrangement followed by trapping the isocyanate intermediate ( 2. 1 2a ) with Grignard reagent 2. 1 3 completed the enam id e pendant 2. 1 4 (Scheme 2. 3) Installing the carbamate at C11 follow ed by protecting group removal afforded initially proposed palmerolide A ( 2. 1a ) Differences in the NMR data of synthesized 2. 1a and that of natural palmerolide A led De Brabander et al. to reevaluate the assignments The group was confident in their stereochemical assignment of synthetic 2. 1a Mosher analysis of C7 absolute configuration. Stereochemist r y at C10 and 11 w as derived from D arabitol. X ray analysis of a

PAGE 42

28 crystal intermediate containing C19 and 20 corroborat ed the assignments at those centers. De Brabander then examined the natural product data and found assignments for C7, 10, and 11 were made on sound evidence. He also felt that the relative configuration between C19 and 20 w as justifiable but the relative configuration of C19 and 20 to C7, 10, and 11 (f ounded on ROESY analysis) was suspect. The researchers decided to repeat the synthesis with 2. 4 ent 2. 5 and 2. 6. The resulting compound ( ent 2. 1b ) possessed the same absolute configuration at C7, 10, and 11 (all R ) but opposite configuration at C19 and 20 (now both S ) when compared to 2. 1a NMR data, as well as thin layer chromatography ( TLC ) and high performance liquid chromatography ( HPLC ) behavior of e nt 2. 1b coincided with that of natural palmerolide A. Circular dichroism ( CD ) spectra of the two compounds were mirror images, however, suggesting that De Brabander et al. had synthesized the enantiomer of the natural product T he absolute configuration of naturally occurring palmerolide A was revised to 7S 10S 11S 19R 20R ( 2. 1b ). 2.2.1.2 Total synthesis by Nicolaou Shortly after De Brabander reported synthesis of revised palmerolide A ( ent 2. 1b ), Nicolaou et al. reported a total synthesis of the natural ena n tiomer ( 2. 1b ) along with several stereoisomers including the originally proposed structure 14 15 Nicolaou also used a convergent, three fragment approach to achieve synthesis of the originally proposed structure which could be easily amended to synthesize other stereoisomers of palmerolide A Using this methodology the group was first to synthesi ze the revised structure 2. 1b from fragments 2. 15 2. 16 and 2. 17 (Scheme 2. 4) By varying the order in which the fragments were combined, Nicolaou was able to determine the most efficient method to f orm the macrocy c le was via olefin ring closing metathesis (RCM) from 2. 18 or Yamaguchi macrolactonization from 2. 19

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29 Analogous 2. 5 via stereoselective Muk a iyama aldol methodology Nicolaou synthesized 2. 15 using 2. 9b and 2.10 (Scheme 2. 2) resulting in comparable yields and better d iastereomeric r atio s Configuration at C10 and 11 was selectively formed by crotylation of 2. 20 with diisopinocamphe n ylborane 2. 21 resulting in 2. 22 which was further elaborated to afford vinyl stannane 2. 16 (Scheme 2. 5) Epoxidation of 2. 2 3 then hydrolytic 2. 2 4 ) resulted in diol 2. 2 5 and epoxide 2. 2 6 en route to 2. 17 and ent 2. 17 respectively.

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30 With 2. 15 2. 16 and 2. 17 in hand, Nicolaou et al. proceeded to assemble the fragments with particular focus on efficiency (Scheme 2.4) Stille coupling of vinyl iodide 2. 15 and vinyl stannane 2. 16 then Yamaguchi type esterification with acid 2. 17 furnished 2. 18 Alternatively, e sterif ying 2. 15 with 2. 17 followed by coupling to 2. 16 improved yield of 2. 18 Protecting group removal and oxidation of 2. 18 set up chain elongation via Takai olefination resulting in vinyl iodide 2. 26 (Scheme 2. 6). RCM with Grubbs II catalyst ( 2. 2 7 ) proved to be an efficient method to form the macrolide which was then coupled to primary enamide 2. 2 8 using Buchwald methodology affording the revised structure of palmerolide A ( 2. 1b )

PAGE 45

31 Nicolaou et al. found that Yamaguchi macrolactonization was also an effective method to form the macrolide portion of palmerolide A (Scheme 2. 4) Less efficient ring closing methods were also investigated includ ing Mitsunobu cylization forming the macrolide at C1 19 in ter molecular HWE olefination at C2 3, and int er molecular Stille coupling at C15 16 In 2008, Nicolaou and co workers reported the synthesis and bioactivity of a multitude of palmerolide A stereoisomers and analogs 16 using strategies developed en route to palmerolide A 14.15 Along with ent 2. 1b nine diastereomers of palmerolide A, were prepared as were six analogs differing only in their amide linkages at C24. Several deoxygenated derivatives of palmerolide A were also synthesized. From this structure activity relationship ( SAR ) study, Nicolaou inferred the anticancer properti es of palmerolide A are most dependent on the enamide moiety as changes to the substituent s on the ring had less effect on activity It was also noted that the benzoylam ide analog was more potent than 2. 1b while removing the C7 hydroxyl group had no effe ct on potency. Less complex analogs, either equipotent or more so, would be more easily accessible and thus more attractive drug leads. 2.2.1.3 Total synthesis by Hall In 2009, Hall et al. 17 reported a third total synthesis of palmerolide A ( 2. 1b ) utilizing catalytic asymmetric organoboron methodologies developed in their lab. Using a less modular but still

PAGE 46

32 convergent approach compared to those employed by De Brabander and Nicolaou, Hall envisioned constructing macrolide 2. 29 (Scheme 2 7) comprising C14 24 ( 2. 30 (C1 13 2. 31 ). Hall began synthesis of 2. 30 with an enantioselective E crotylboration of aldehyde 2. 10 with 2. 32 using catalyst 2. 3 3 resulting in alcohol 2. 34 in excellent yield, diastereoselectiv ity and enantiomeric excess (Scheme 2 8). Successive Wittig olefinations furnished 2. 35 which underwent Sonogashira coupling and alkyne hydrozirconation yielding fragment 2. 30 portion of the macrolide. Synthesis of fragment 2. 31 commenced (Scheme 2 9) with an enantioselective hetero [4 + 2] cycloaddition/allylboration of 2. 36 and 2. 37 by way of Jacobsen designed ( Schiff base ) chromium

PAGE 47

33 (III) catalyst ( 2. 38 ) Intermediate 2. 3 9 reacted with another equivalent of 2. 36 resulting in alcohol 2. 40 a which upon a cylation gave 2. 4 0b An unprecedented Claisen Ireland [3,3] rearrangement followed (through transition state 2. 4 1 ) giving 2. 4 2 a and finally, after oxidation, 2. 4 2 b With configuration at C7, 10, and 11 set, 2. 42b was then elaborated to fragment 2. 31 portion of the macrolide. Hall et al. then coupled fragments 2. 30 and 2. 31 with a n sp 2 sp 3 B alkyl Suzuki coupling. Yamaguchi macrolactonization completed 2. 29 (Scheme 2 3) converting 2. 29 to isocyanate 2. 1 2 b and finally enamide 2. 14b Hall then installed the carbamate at C11. Protecting group removal afforded palmerolide A ( 2. 1b ). 2.2.2 Partial syntheses of palmerolide A Several approaches to various fragments of p almerolide A have been reported 18 22 ( Scheme 2. 10 ) Shortly before DeBrabander published the revised structure of palmerolide A, Kaliappan and Gowrisankar reported synthesizing the of originally proposed 2. 1a comprising C1 9 and C15 21 ( 2. 43 ) 18 Notable transformations include using an Evans chiral auxiliary to set configuration at C 7 a palladium (II) ( Pd II ) catalyzed allylic rearrangement to furnish E 16 selectively, and olefin cross metathesis ( O CM) nd gen eration catalyst to form E 2. Maier et al. constructed the linear fragment C3 23 ( 2. 44 ) en route to 2. 1a 19 Key steps

PAGE 48

34 include forming C10 and 11 stereocenters via Sharpless dihydroxylation, Red Al mediated reduction of an alkyne to form E 8, and Stille coupling at C15 16. Chandrasekar et al. synthesized the C1 14 fragment ( 2. 45 a ) of revised palmerolide A ( 2. 1b ). 20 The researchers relied on deoxygenative rearrangement of an alkynol, an asymmetric dihydroxylation of the resulting diene ester which installed C11 and 12 hydroxyls and CBS ( Corey Bakshi Shibata ) reduction to selectively afford the C7 hydroxyl Cantagrel et al. reported the C3 15 ( 2. 46 ) and C16 23 ( 2. 47 ) fragment s of 2. 1b 21 Significant transformations included condensation forming C19 and 20 centers an enatioselective set C7 configuration and a diastereoselective acetylenic Grignard addition forming C10 configuration. Dudley et al. 22 synthesized the C1 15 region ( 2.45b ) of revised palmerolide A using an optimized Claisen type conden sation of vinylogous acyl triflates to form the C1 8 subunit and appending it to C9 15 via a convergent HWE olefination. Ster e ocenters C10 and 11 were formed via asymmetric dihydroxylation ( AD mix Configuration at C7 was installed using a C BS reduction

PAGE 49

35 2.2.3 Formal total synthesis of palmerolide A Formation of an a dvanced intermediate from 14,15 ( 2. 47 Scheme 2. 11 ) by Maier and Jagel 23 was reported culminating in the formal total synthesis of palmerolide A ( 2. 1b ) Using methods they had previously reported 19 t he researchers were able to synthesize 2. 48 which could then be coupled to 2. 49 via HWE olefination yielding 2. 50 The macrolide was formed via a highly stereospecific Heck cyclization. Takai olefination then cleavage of silyl protecting groups yielded 2. 47 completing the formal total synthesis. 2.3 Degradation of palmerolide A to confirm absolute configuration Naturally occurring palmerolide A was subjected to degradative studies 11 to verify the stereochemical assignments determined by spectroscopic and derivatization techniques. 1 Reductive ozon olysis of palmerolide A could be used to cleave the molecule into s everal polyol

PAGE 50

36 fragments. Comparison of the naturally derived fragments to those synthesized from chiral pool starting material would verify the proposed structure. 2.3.1 Ozonolysis of palmerolide A Subjecting palmerolide A to ozonolysis followed by reduction with sodium borohydride (Scheme 2. 12) resulted in quantitative yields of hexane 1,2,6 tri ol ( 2. 51 ) and hexane 1,2,3,6 tetra ol ( 2. 52 ) Specific rotations of 9.0 and 8.1 were recorded for triol 2. 51 and tetraol 2. 52 respectively. D egradation of 2. 1a should afford triol R 2. 51 as well as ( 2 R 3 R ) 2. 52 H exane 1,2,6 triol is available commercially but only in racemic form. ( S ) H exane 1,2,6 triol has been reported, 24 having a specific rotation of 3.4 This was at odds with the rotation of degradation product 2. 51 (i.e. R 2. 51 should bear a rotation of +3.4) in sign as well as magnitude prompting us to develop chiral pool based synthese s of polyols hexane 1,2,6 triol and hexane 1,2,3,6 tetraol 2.3.2 Synthesis of hexane 1,2,6 triol ( R ) H exane 1,2,6 triol ( R 2. 53 ) was prepared from the ( R ) acetonide of glycerol ( 2. 54 Scheme 13 ). Acetonide 2. 54 was oxidized via Swern 25 protocol. The resulting aldehyde underwent Wittig olefination for ming 2. 55 26 In the course of this preparation the Witti g product 2. 55 was reductively ozonolyzed back to 2. 54 to confirm that epimerization had not occurred. Recovered 2. 54 however, was found to have significantly epimerized (ee 56% of original).

PAGE 51

37 A homologue of 2. 54 the acetonide of ( R ) butane 1,2,4 triol ( R 2. 56 ), after oxidation, could be subjected to Wittig olefination to R 2. 57 while retaining its optical purity based on a similar reductive ozonolysis back to R 2. 5 6 (Scheme 2. 14) A 1,4 reduction of the conjugated ester with lithium aluminium hydride ( LAH ) produced the terminal alcohol R 2. 58 in moderate yields. H ydrol ysis afforded the desired triol R 2. 53 under mild ly acid ic conditions. Spectral and chromatographic data ( 1 H NMR, 13 C NMR, GC/MS, ESI MS ) of ( R ) hexane 1,2,6 triol ( R 2. 53 ) matched that of commercially available () hexane 1,2,6 triol T he specific rotation of synthetic ( R ) hexane 1,2,6 triol ( R 2. 53 +11.1) was then compared to degradation product ( 2. 5 1 9.0). We were satisfied to find the magnitude of the specific rotation from the synthetic product more closely matched the degradation product, but were disappointed to find the sign of the rotation to be opposite t hat of the degradation product. Final verification of the C 7 configuration was achieved by preparatio n of ( S ) hexane 1,2,6 triol ( S 2. 53 ), starting from the acetonide of ( S ) butane 1,2,4 tri ol ( S 2. 56 Scheme 2. 14 ). Oxidation of S 2.56 to the requisite aldehyde was achieved via Swern protocol, avoiding use of Dess Martin periodinane, a costly reagent. Horner Wadsworth Emmons olefination was utilized to form S 2.57 in a more efficient manner than the Wittig reaction used to form the R ena n tiomer. As an alternative to the modest yielding reduction of R 2. 57 with LAH a two step reduction with diisobutylaluminum hydride ( DIBALH ) to the allylic alcohol the n catalytic hydrogenation afforded S 2. 58 in better overall yield. The specific rotation of the ( S ) triol ( S 2. 53 11.6) matched the degradation product ( 2. 5 1 9.0)

PAGE 52

38 obtained from ozonolysis of palmerolide, suggesting the con 7 stereocenter as bearing the S configuration, rather than the originally published 7 R 2.3.3 Synthesis of hexane 1,2,3,6 tetraol Stereocenters at C10 and 11 of palmerolide A were analyzed by comparing the degradation product tetraol 2. 5 2 to analogs synthesized from the chiral pool. The configuration at C10 and C11 could be explored by synthesis of both ( 2R,3R ) and ( 2S,3S ) hexane 1,2,3,6 tetraol ( R R 2. 59 and S S 2. 59 respectively) S ynthesis from either L(+) or D 2,3 O Isopropylidene threitol ( 2. 60 ) yielded the desired tetraol in six steps. Preparation of ( 2R,3R ) hexane 1,2,3,6 tetraol ( Scheme 2. 15 ) began with the monoacetylation of diol ( R,R ) 2. 60 followed by oxidation of the unprotected al cohol via Dess Martin periodinan e. Immediate W ittig olefination of the subsequent aldehyde produced an inseparable mixture of E / Z isomers ( 2. 61 ) The desired tetraol ( R,R ) 2. 59 was obtained from 2. 6 1 after catalytic hydrogenation, acid hydrolysis, then reduction. The specific rotation of ( R,R ) 2. 59 (+9.9) was at odds with the corresponding ozonolysis product 2. 5 2 ( 8.1).

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39 Preparation of tetraol ( S,S ) 2. 59 was achieved using a slightly different synthetic route starting with commercially available ( S,S ) 2. 6 0 (Scheme 2. 16) Selective benzylation [silver oxide ( Ag 2 O ) and benzyl bromide ] 27 of ( S,S ) 2. 6 0 produced the mono benzylated alchohol which was subjected to Dess Martin oxidation and Wittig olefination to afford alkene 2. 6 2 as a mixture of E / Z isomers E 2. 6 2 could be obtained exclusively using HWE conditions. Catalytic h ydrogenation of 2. 6 2 yielded the unsaturated and deprotected alcohol 2. 6 3 Tetraol ( S,S ) 2. 59 was realized by r eduction with LAH then acid hydrolysis of 2. 63 ( S,S ) 2. 59 display ed a specific rotation of 1 0 .0 which agreed with th at of the degradation product obtained from ozonolysis ( 8.1) in sign and magnitude inferring the natural product was actually 10S 11S 2.3.4 Re evaluation of absolute configuration The results of the degradation study compelled us to re examine the previously reported 1, 28 data from the Mosher s analysis to establish whether our procedures were in error or the method itself failed Lab notebooks indicated the correct conversion of the acid chloride stereochemistry to the corresponding ester (ie. ( R ) acid chloride to ( S ) ester) T he esterification of palmerolide A with

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40 ( R ) methoxytrifluoromethylphenylacetoyl chloride was repeated forming the ( S ) MTPA ester which w as found to bear 1 H NMR shifts that matched the data originally assigned to the ( R ) MTPA ester. 2 8 Thus, t he samples or data sets derived from the original MTPA esters were found to be mislabelled necessitating a revision to the absolute configuration of palmerolide A at C7 from R to S The C1 0 configurational assignment was made from the same MTPA products, necessitating that we re evaluate the C7/ C 10 MTPA diester. We found that C 10 had been subject to the sam e transposition. Because the C 11 configuration is based on 2 J CH and 3 J CH c onformational analysis of the C10/C 11 spin system, both C 10 and 1 1 must be revised to the S configuration. In summary, the absolute configuration of palmerolide A should be revised to 7S 10S 11S based on this degradation study and re evaluation of the Moshers analysis. 2.4 Synthesis of the C3 14 fragment of palmerolide A With the correct absolute configuration at C7, 10, and 11 determined, efforts were directed to the reconstruction 12 of palmerolide A using similar synthetic routes as the ones described above employed to generate the chiral polyols. The syntheses of the fragments 2. 5 3 and 2. 5 9 were modified for coupling using one of the many olefination reactions described in literature 2.4.1 Julia Kocienski olefination T o form the C3 14 segment of the macrolide we decided that E 8 alkene could be formed from an aldehyde derived from triol 2. 51 coupled to a sulfone derived from tetraol 2. 52 based on Julia Kocienski protocol. 29 Compostella et al. 30 demonstrate d that Julia Kocienski olefination is useful in constructing trans olefins with an alkoxy aldehyde and an aliphatic sulfone or a alkoxy sulfone (no elimination observed) and aliphatic aldehyde, but to our knowledge no examples exist in which both coupling components contain alkoxy substituents. Using this methodology, we envisioned fragment C3 14 ( 2. 64 ) could be formed from sulfone 2. 65 derived from triol 2. 5 3 coupled with aldehyde 2. 66 derived from tetraol 2. 5 9 (Scheme 2. 17)

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41 The synthesis of triol 2. 53 was modified to generate sulfone 2. 65 (C3 8) from alcohol S 2. 58 (Scheme 2. 18 ). Anticipating an olefin ring closing metathesis reaction to form the macrolide portion of palmerolide A, intermediate terminal alkene 2. 67 was required. Dess Martin oxidation of S 2. 58 followed by Wittig olefination and hydrolysis yielded the desired al kene 2. 67 Monotosylation followed by silylation of the free secondary alcohol led to sulfone precursor 2. 68 Treating tosylate 2. 68 with 1 phenyl 1 H tetrazole 5 thiol and potassium carbonate under refluxing conditions 31 yielded a thioether intermediate, which was then oxidized with catalytic amounts of sodium tungstate, phenylphosphonic acid, methyltrioctylammonium hydrogen sulfate and an excess of 30% hydrogen peroxide 32 to generate sulfone 2. 65 Aldehyde 2. 66 was realized by Dess Martin o xidation of 2. 63 (Scheme 2. 18), an intermediate in the synthesis of ( 2S,3S ) hexane 1,2,3,6 tetraol [(S,S) 2. 59 ]

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42 Attempts at coupling 2. 65 and 2. 66 using Julia Kocienski methodology were not successful in our hands, yielding low recoveries of unreacted starting material with no evidence of elimination of the tert butyldimethylsilyl ( TBS ) ether in 2. 65 2.4.2 A new route to join fragments derived from 2. 53 and 2. 59 was devised utilizing olefin cross metathesis (OCM) 33 We chose Grubbs second generation catalyst because Type II/Type III cross couplings are predicted to produce moderate to high yields with little to no homodimerization. 34 The new route required that each fragment terminate in an olefin. The synthesis of a fragment bearing the palmerolide A ( 2. 1 b ) C3 8 centers (e.g., 2. 65 ) was modified to produce olefin metathesis substrate 2. 69 (Scheme 2. 19 ). Benzylation followed by acid hydrolysis of intermediate S 2. 58 resulted in the formation of diol 2. 70 A one pot protecting group manipulation produced secondary acetate 2. 71 Dess Martin oxidation and Wittig olefination resulted in the formation of terminal olefin 2. 69 The fragment bearing the palmerolide A ( 2. 1b ) C9 14 segment, 2. 7 2 (Scheme 2. 18 ), was derived from aldehyde 2. 66 by a Wittig reaction. Combination of olefins 2. 69 and 2. 72 (Scheme 2. 20 ) using Grubbs second generation catalyst ( 2. 27 ) proceeded smoothly to generate the desired E i s omer, as predicted, in moderate yields ( 2. 7 3 C3 14 of palmerolide A) with no recovery of homodimers nor unreacted starting material. Steric bulk at both allylic positions in the product may explain why Z 2. 7 3 was not observed. One cannot rule out the possibility of E / Z isomeri z ation via secondary metathesis of 2. 7 3 which could also explain selective E isomer

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43 formation. The fact that no homodimers were found and only the E isomer of 2. 7 3 was isolated suggests 2. 69 and 2. 7 2 may be reacting in a selective type II/type III fashion as postulated by Grubbs et al. 3 4 However, due to the moderate yield of 2. 7 3 and no evidence of either homodimer, it is unclear of which olefin type (II or III) 2. 69 and 2. 72 should be considered. In summary, 2. 73 the C3 14 portion of palmerolide A, was constructed from commercially available chiral building blocks S 2. 56 and ( S S ) 2. 60 The total synthesis of palmerolide A based on this chiral pool approach is ongoing and when completed should offer a facile route to a multitude of derivatives for use in structure activity relationship ( SAR ) and structure property relationship ( SPR ) studies. 2.5 References Cited 1 Diyabalanage, T.; Amsler, C. D.; McClintock, J. B.; Baker, B. J. J. Am. Chem. Soc. 2006, 128 5630 5631 2 Beutler, J. A.; McKee, T. C. Curr. Med. Chem 2003 10 787 796. 3 Fais, S.; De Milito, A.; You, H.; Qin, W. Cancer Res 2007 67 10627 10630. 4 Sennoune, S. R.; Bakunts, K.; Martnez, G. M.; Chua Tuan, J. L.; Kebir, Y.; Attaya, M. N.; Martnez Zaguiln R. Am. J. Physiol. Cell Physiol. 2004 286 C1443 C1452. 5 Anne Monks, personal communication. 6 Shacka, J. J.; Klocke, B. J.; Roth, K. A. Autophagy 2006 2 228 230. 7 Xie, X. S.; Padron, D.; Liao, X.; Wang, J.; Roth, M. G.; De Brabander, J. K. J. Biol. Chem 2004 279 19755 19768. 8 Riesenfeld C. S.; Murray, A. E.; Baker, B. J. J. Nat. Prod 2008 71 1812 1818.

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44 9 Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991 113 4092 4096. 10 Murata, M.; Matsuoka, S.; Matsumori, N ; Paul, G. K.; Tachibana, K. J. Am. Chem. Soc. 1999 121 870 871. 11 Lebar, M. D.; Baker, B. J. Tet rahedron Lett. 2007 48 8009 8010. 12 Lebar, M. D.; Baker, B. J. Tetrahedron 2010 66 1557 1562. 13 J iang, X.; Liu, B.; Lebreton, S.; DeBrabander, J. K. J. Am. Chem. Soc. 2007 129, 6386 6387. 14 Nicolaou, K. C.; Guduru, R.; Sun, Y.; Banerji, B.; Chen, D. Y. K. Angew. Chem. Int. Ed. Engl. 2007 46, 5896 5900. 15 Nicolaou, K. C.; Sun, Y.; Guduru, R.; Banerji, B.; Chen, D. Y. K. J. Am. Chem. Soc. 2008 130, 3633 3644. 16 Nicolaou K. C.; Leung, G.; Dethe, D. H.; Gurudu, R.; Sun, Y. P.; Lim, C. S.; Chen, D. Y. K. J. Am. Chem. Soc. 2008 130, 10019 10023. 17 Penner, M.; Rauniyar, V.; Kaspar, L. T.; Hall, D. G. J. Am. Chem. Soc. 2009 131 14216 14217. 18 Kaliappan, K. P.; Gowrisankar, P. SynLett 2007 10, 1537 1540. 19 Jagel, J.; Schmauder, A.; Binanzer, M.; Maier, M. E. Tetrahedron 2007 63, 13006 13017. 20 Chandrasekhar, S. Vijeender, K., Chandrashekar, G., Reddy, C. R. Tetrahedron: Asymmetry 2007 18 2473 2478. 21 Cantagrel, G.; Meyer, C.; Cossy, J. SynLett 2007 19, 2983 2986. 22 Jones, D. M.; Dudley G. B. Synlett 2010 2 0223 0226. 23 Jagel, J.; Maier, M. E. Synthesis 2009 17 2881 2892. 24 Regeling H.; Chittenden G. J. Carbohydr. Res 1991 216 79 91. 25 Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem 1978 43 2480 2482. 26 Servi, S. J. Org. Chem 1985 50 5865 5867. 27 Bouzide, A.; Sauve, G Tetrahedron Lett 1997 38, 5945 5948. 28 Diyabalanage, T. Ph.D. Dissertation, University of South Florida, 2006. 29 Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morely, A. Synlett 1998 26 28. 30 Compostella, F.; Franchini, L.; Panza, L.; Prosperi, D.; Ronchetti, F. Tetrahedron 2002 58 4425 4428.

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45 31 Toschi, G.; Baird, M. S. Tetrahedron 2006 62, 3221 3227. 32 Sato, K.; Hyodo, M.; Aoki, M.; Zheng, X.; Noyori, R. Tetrahedron 2001 57 2469 2476. 33 Krishna, P. R.; Dayaker, G. Tetrahedron Lett 2007 48 7279 7282. 34 Chatterjee, A. K.; Choi T. L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem. Soc 2003 125 11360 11370.

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46 Chapter 3 Meridianin A and psammopemmin A: structure investigation 3.1 Indole and pyrimidine containing natural products Nitrogen containing heterocyclic compounds derived from natural sources are abundant in structu ral complexity and bioactivity Of particular interes t to this study are indole and pyrimidine containing metabolites derived from marine invertebrates. Sponges (Porifera) and tunicates (Urochordata ) have afforded a diverse assortment of biologically promising indole 1 and pyrimidine 2 c ontaining alkaloids I ndole alkaloids that bear a nitrogen containing heterocycle at the 3 position have displayed a rich array of bioactivity. Dragmacidin ( 3. 1 ), a piperazine linked bisindole alkaloid with cytotoxic properties, was isolated from the deep water marine sponge Dragmacido n sp. 3 The compound display ed low in vitro IC 50 values toward P 388 (leukemia), A 549 (human lung), HCT 8 (human colon), and MDAMB (human mammary) cancer cell lines. Dragmacidin D ( 3. 2 ), a nother piperazine linked bis indole was isolated from the sponge Spongosorites sp 4 Dragmacidin D exhibited antiviral properties versus feline leukemia virus (FeLV) antitumor properties versus P 388 and A 549 cancer cells, and also inhibit ed the growth of the fungal pathogens Candida albicans and C ryptococcus neoformans

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47 Topsentin ( 3. 3 ), 5 bromotopsentin ( 3. 4 ) 5 and nortopsentins A C ( 3. 5 3. 7 ), 6 bis (indolyl)imidazole alkaloids isolated from the deep sea Caribbean sponge Spongosorites ruetzleri exhibit antitumor and antiviral activity toward several important targets. Topsentin display ed activity toward P 338, HCT 8, A 549, and T47D (breast) cancer cells in vitro as well as in vivo activity toward P 388 and B16 (melanoma) cell lines. Topsent in and bromotopsentin exhibit ed in vitro antiviral activit y toward herpes simplex ( HSV 1 ) Vesivular stomati tis virus (VSV), and corona virus A 59. Nortopsentins A C ( 3. 5 3. 7 ) display ed inhibitory activity toward P 388 cancer cells. S emisynthetic nortopsentins, dimethylated at the imidazole nitrogens show ed a significant increase in activity toward P 388. A multitude of a plysinopsins tryptophan derivatives b ear ing an imidazoline moiety, have been isolated from sponge genera worldwide 7 Aplysinopsins have also been found in corals (predominately from the genus Tubastraea ) as well as a Tubastraea predator, the mollus k Phestilla melanobrachia Aplysinopsin ( 3. 8 ) 8 initially found in eight Indo Pacific sponge species has shown anticancer 9 and antimicrobial properties 10 Several aplysinopsins and synthetic analogs have also show n activity in serotonin (5 HT) receptor assays. 7

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48 3.1. 1 Indoles and pyrimidines from Southern cold water sponges A plethora of d iscorhabdins cytotoxic pyrroloiminoquinone alkaloids, have been isolated from both temperate and tropical sponges of the family Latrunculiidae. 11 The cold wa ter sponge Latrunculia apicalis found in McMurdo Sound, Antarctica, has afforded discorhabdin G ( 3. 9 ) 12 as well as discorhabdin C ( 3. 10 previously reported from New Zealand collected Latrunculia sp.). 13 Discorhabdin C is a particularly potent deterrent, defending L. apicalis from predation by the spongivorous sea star Perknaster fuscus Discorhabdin R ( 3. 11 ) found to contain a sulfide as well as an epoxide, has been isolated from two Antarctic sponges in the Latrunc u li i dae family ( Negombata and Latrunculia sp.) 14 The bright red Antarctic sponge Kirkpatrickia variolosa elaborates colorful pigments named variolins A ( 3. 12 ) and B ( 3. 13 ) 15 16 aminopyrimidine containing fused tricyclic heteroaromatic alkaloids. 17 N (3 ) methyl tetrahydrovariolin B ( 3. 14 ) was also isolated from K. variolosa Variolin B was shown to be a very potent inhibitor of P 388 cancer cells. Variolin B was later found to inhibit cyclin dependent kinase 2 (CDK2) and CDK3 selectively over CDK4. 18 Variolin B and a synthetic derivative, deoxyvariolin B ( 3. 15 ), were found to inhibit phosphorylation of histone H1 mediated by several CDKs effectively interrupting the normal progression of the cell cycle. 19 These promising findings have prompted PharmaMar a company specializing in marketing drugs derived from marine sources, to assess the variolins in preclinical trials.

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49 Psammopemmin s A C ( 3. 16 3. 18 ), 4 hydroxyindole alkaloids were isolated from the Antarctic sponge Psammopemma sp. 20 The 5 bromo 4 aminopyrimidine) moiety reported in psammopemmins is unprecedented in both terrestrial and marine natural products. No bioactivity data was reported for psammopemmins A C. 3.1. 2 Indoles and pyr imidines from Southern cold water tunicates Aplicyanins A F ( 3. 19 3. 2 4 ), tetrahydropyrimidine substituted indoles isolated from the Antarctic tunicate Aplidium cyaneum were shown to be cytotoxic and antimitotic 21 The absolute configuration of the aplicyanins is not known. Aplicyanin E, when subjected to chiral HPLC, was shown to be enantiomerically pure. Aplicyanins C F bear a rather uncommon 1N methoxyindole subunit. Aplicyanins B, D, and F displayed GI 50 values in the submicromolar range versus HT 29 (colon) A 549, and MDAMB cancer cells lines while aplicy anins A, C, and E were far less active The N acetyl group is clearly crucial for the biological activity observed.

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50 The green tunicate Aplidium meridianum collected in the South Georgia Islands, elaborates seven aminopyrimidine containing indole alkaloids, meridianins A G ( 3. 2 4 3. 30 ). 22 23 Meridianins A E were initially screened against murine mamarian adenocarcinoma cells (LMM3 ). M eridianin s B E displayed micromolar IC 50 values against LMM3 cells Further biological investigation revealed that meridianins A F were low micromolar inhibitors of various cyclin dependent kinases ( CDKs ) glycogen synthase kinase 3 (GSK 3), protein kinase A (PKA), as well as other protein kinases 24 The most potent inh ibitors, meridianins B and E, are 4 hydroxyindole indole alkaloids differing in bromination pattern. Meridianins B F exhibited cytotoxic activity at low micromolar ranges. Meridianin A, however, displayed no cytotoxicity toward HEp 2 ( laryngeal carcinoma ), HT 29, and LMM3 cells at the highest concentration examined

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51 3.2 R eview of meridianin syntheses The meridianins have attracted significant synthetic interest due to their biological activity as well as their unique and relatively simple structure. Many of the naturally occurring meridianins have been synthesized. Particular attention has been focused on meridiani n analogs as well as methods to efficiently construct a variety of derivatives for further biological examination. 3.2.1 Synthesis of natural meridianins Meridianin D ( 3. 27 ) and its debromo analog meridianin G ( 3. 30 ) were synthesized via coupling of 2 amino 4 chloropyrimidine ( 3. 31 ) and the appropriate protected indole boronic acid ( 3. 32 ) using a Suzuki Miyaura protocol (Scheme 3. 1) 25 Along with the tricyclic core of variolin, the synthes i s of meridianins C ( 3. 26 ), D ( 3. 27 ), and E ( 3. 28 ) w as reported. 26 A follow up pu blication described the synthesis of meridianin A ( 3. 24 ) using the same methods. 27 Heating the suitably functionalized N tosyl 3 acylindole ( 3. 33 ) with dimethylformamide dimethylacetal (DMF DMA) afforded enaminone 3. 34 ( Bredereck protocol, 28 Scheme 3. 2) Condensation of 3. 34 with guanidine chlorohydrate in the presence of sodium carbonate resulted in concomitant detosylation forming meridianins C, D, and intermediate 3. 35 Debenzylation and debromination of 3. 35 with H 2 and palladium ( Pd ) yielded meridianin A Meridian E was form ed via selective debenzylation of 3. 35 with trifluoroacetic acid ( TFA ) and thioanisole. Hydrogenation in different solvents formed either meridianin A or E In the initial report (ref. 2 6 ) meridianin E was produced from intermediate 3. 35 via hydrogenation (H 2 Pd/C, EtOH, 88%). I n the follow up report (ref. 2 7 ) meridianin E was formed from 3. 35 by treatment

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52 with TFA and thioanisole (65%) while hydrogenation of 3. 35 (H 2 Pd/C, EtOAc) resulted in the debrominated compound, meridianin A in 83% yield. Meridianins C, D, and G were synthesized efficiently using a carbonylative alkynylation followed by subsequent cyclocondensation 29 Carbonylative Sonogashira coupling of trimethylsilylacetylene and appropriately functionalized 3 iodoindole 3. 3 6 using Pd(PPh 3 ) 2 Cl 2 catalyst resulted in trimethylsilylalkynone 3. 3 7 (Scheme 3.3) Condensation of 3. 3 7 with guanidine in the presence of sodium carbonate with concurrent de protection resu lted in meridanins C, D, and G. Absent from the report is any discussion of the reactivity of 4 hydroxyindoles (or protected forms thereof) toward carbonylative alkynylation Meridianin G ( 3. 30 ), referred to in this report as 6 debromomeridianin D, was produced from 3 cyanoacetylindole ( 3. 38 ) in four steps (Scheme 3. 4) 30 Treatment of 3. 38 with DMF DMA

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53 afforded enaminonitrile 3. 39 which was then condensed with guanidine cyanomeridianin G ( 3. 40 ). Base hydrolysis then decarboxylation yielded the desired meridianin G. 3.2.2 Synthesis and biological activity of meridianin analogs Several 3 alkaloids were synthesized prior to the discovery of the meridianins in 1998 The first report, in 1967, describes the synthesis of two compounds very similar in structure to that of the meridians. Alkaloids 3. 41a and 3. 41b were formed by reacting 3 me thylthio 3 (3 indolyl)acrylic acid derivatives ( 3. 42a,b ) with guanidine (Scheme 3. 5). 31 In 1993 the synthesis of over 200 pyrimidine derivatives and their utility in asthma therapy was reported 32 Enaminone 3. 4 3 3 methylaniline ( 3. 4 4 ) and cyanamide were condensed under acidic conditions to form 3. 4 5 in effect, methylaniline substituted meridianin G (Scheme 3. 5 ) Pyrimidylindole 3. 4 5 was found to inhibit histam ine release from basophil cells Compounds that b lock immunological release of mediators like histamine could be developed into antiasthma agents

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54 Several synthetic ( bis ) indole and pyrazine meridianin analogs constructed using Suzuki methodology were found to inhibit growth of a variety of cancer cell lines. 33 (Bis)indole alkaloid 3. 4 6 displayed strong activity against IGROV1 (ovarian carcinoma). The pyrazine substituted indole alkaloids displayed varying amounts of cytotoxicity and selectivity. N tosyl indole 3. 4 7 showed across the board cytotoxicity, inhibiting the growth of every cell line tested. Alternatively, methoxy substituted pyrazine 3. 4 8 specifically and effectively inhibited HOP 92 (non small lung cancer) cells. Iso meridianins C ( 3. 49 ) and G ( 3. 50 ) were prepared by microwave assisted Fischer indole synthesis (Scheme 3. 6 ) 34 Suitably substituted phenylhydrazine 3. 51 (from pyrimidine 3. 52 ) was

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55 heated in a conventional microwave oven with zinc chloride ( ZnCl 2 ) in a small amount of dimethylformamide ( DMF ) resulting in N Boc deprotection and indole formation The resulting iso meridianins C and G were devoid of biological activity. An environmentally benign method to construct the piperidinyl meridianin analog 3. 53 a and related trisubstituted pyrimidines h a s been described using basic alumina as a solid support as well as a catalyst. 35 Chalcone 3. 54 S benzylthiuronium chloride ( 3. 55 ) and piperidine dissolved in ethanol concentrated onto basic aluminum and heated intermittently with microwave resulted in 3. 53 a (Scheme 3. 7). B iological activity of 3. 53 a and other trisubstituted pyrimidines synthesized in this study was not reported. A reaction of the apposite chalcone ( 3. 54 ) with guanidine derivative 56 afforded several similar trisubstituted pyrimidines ( 3. 53b d Scheme 3. 7) that displayed antimalarial properties, inhibiting Plasmodium falciparum in vitro. 36

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56 Reaction of suitably substituted chalcone 3. 57 with guanidine afforded alkyl substituted meridianin analogs 3. 58a c 37 When screened for in vivo pregnancy interceptive activity in hamsters, 3. 58c was 50% effective, whereas 3. 58a and 3. 58b were devoid of activity. Indoyluracils 3. 59 resembling meridians have been synthesized from condensation of ureidopropenoate 3. 60 (Scheme 3.9 ). 38 Using DMF DMA and a substituted guanidine, 3. 60 was formed in similar fashion as 3. 3 4 (Scheme 3. 2) and 3. 39 (Scheme 3. 4). Perplexingly, n o mention of biological activity or lack thereof was reported for these compounds. The authors note that the pyrimidyl N1 could be coupled to a sugar to form novel nucleosides. Using Bredereck methods described in Scheme 3 2, 2 6 ,2 7 meridianins C ( 3. 26 ) G ( 3. 30 ) and seven monohalogenated meridianin analogs ( 3. 61 a g ) were synthesized from the suitably

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57 substituted enaminone 3. 34 39 In a similar fashion numerous monohalogenated N 1 alkyl substituted meridianin derivatives were constructed from alkylated enaminone 3. 62 The c rystal structure of N1 methylmeridianin G was obtained to assist in molecular modeling of the interaction between merdi anin analogs and the adenonsine triphosphate ( ATP ) pocket of kinases. Structure activity relationship studies were ongoing at the time of publicat ion. N o bioactivity data is av ailable to date. Following a Bredereck protocol and a Suzuki coupling was aryl substituted meridianin G derivatives ( 3. 63a g ) 40 The N 1 methyl derivatives of 3. 63a g were also synthesized. The anticancer utility of 3. 63a g was reported in a follow up publication. 41 Screening the compounds against various kinases as well as MCF 7 (breast) and PA 1 (ovarian) cancer cell lines revealed that N 1 methyl 3. 63c N 1 methyl 3. 63b 3. 63f and 3. 63g were selectively cytotoxic toward PA 1 cells Interestingly, none of the active compound s displayed any significant kinase inhibition. substituted meridianin C and G analogs were synthesized (Scheme 3.10) via metal free alk y ny lation of indole 3. 64 with various oxo ketene dithioacetals ( 3. 65 ) 42 Refluxing 3. 64 and 3. 65 in trifluoroacetic acid:dichloromethane ( TFA:DCM ) resulted in the formation of functionalized indole 3. 66 Condensation with guanidine nitrate yielded N 1 alkylated substituted meridianin derivatives 3. 67a g O xidative debenzylation of 3. 67a d substituted meridianins ( 3. 6 8 a d ). Biological activity was not reported.

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58 Meridi a n in analogs 3. 69a c chloromeridianin derivatives were constructed by thermal annulation of nitrosoarenes with 4 ethynylpyrimidines. 43 Heating a 1:1 ratio of substituted nitrosoarene 3. 70 and 2 amino 4 ethynylpyrimidine ( 3. 71 ) in toluene resulted in the formation of meridianin derivatives 3. 69a c (Scheme 3. 11) At time of publication investigation of the mechanism of the reaction as well as the bioactivity of the compounds synthesized was ongoing.

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59 3.2.3 Synthesis and biological activity of meriolins The remarkable biological activity of the meridianins and the variolins as well as the structural commonalities between the two alkaloids prompted intere st in synthesizing 3 ( amino pyrimidin 4 yl) 7 azaindole, a structural hybrid later dubbed meriolin 1 ( 3. 70 Fig. 3. 1 ). The first synthesis of 3 aminopyrimidin 4 yl) 7 azaindole ( 3. 70 ) was reported along with the synthesis of meridianins A, C, D, and E and similarly relied on Bredereck methodology 2 7 Analogous to the synthesis of meridianins in Scheme 3. 2, m eriolin 1 ( 3. 70 ) was formed through condensation of guanidine and a zaindolylenaminone 3. 71 (Scheme 3. 12) Meriolin 1 was also synthesized via condensation of guanidine and trimethylsilylalkynone 3. 72 (Scheme 3. 12) reported in conjunction with the synthesis of meridianins C, D, and G (see Scheme 3. 3) 2 9 Initial screening demonstrated that meriolin 1 inhibi ts protein kinases hSGK1 and Tie 2 at low micromolar levels

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60 The synthesis and biological characterization of fourteen meriolin analogs ( meriolins 1 14, 3. 70 3. 83 ) revealed the compounds to be potent CDK inhibitors. 44 45 When screened for selectivity against thirty two kinases, meriolins displayed enhanced specificity toward CDKs over variolins especially CDK2 and CDK9 Upon crystallization of variolin B and meriolin 3 with pCDK2/cyclin A, both compounds were found to bi nd in the ATP binding site of the kinase but in different than the control, roscovitine (a purine analog, 3. 84 ) in xenograft assays. Meriolin 3 also showed nanomolar IC 50 values against spheroid and monolayer HCT 116 (colon) cancer cell lines. SAR methylthiomeriolin (meriolin 12, 3. 81 ) H meriolin (mer iolin 13, 3. 82 ) showed a significant loss of activity. Changing the substituent at C4 ( R 1 ) to a more aliphatic group led to increased activity, theorized to be due to favorable interactions with the glycine rich loop of the kinase. Bulky substitutes at C 6 (R 2 ) led to less inhibition, perhaps by shielding hydrogen bonding interactions between N2, N7 and the kinase. M eriolins are potent CDK inhibitors as well as strong antiproliferatives and warrant further investigation

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61 3.3 Isolation of meridianins A, B, C, and E from Antarctic tunicate Synoicum sp. In our ongoing search for bioactive metabolites, we investigated t he chemical composition of the yellow top tunicate, Synoicum sp., 46 collected in the benthos surrounding Palmer Station, Antarctica. The lyophilized tunicate was extracted with polar and nonpolar solvents. The nonpolar extract was subjected to successive medium and high performance liquid (Scheme 3. 13) to yield meridianins A ( 3. 24 ) B ( 3. 25 ) C ( 3. 26 ) and E ( 3. 28 ) NMR data of meridianins isolated from Synoicum sp. were compared to th at of the previously reported meridianins isolated from Aplidium meridianum 2 2 and found to be congruous.

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62 In the process of verifying the structure of the meridianins, it was noted that the physical data reported for meridianin A, B, and E bore a striking resemblance to psammopemmin A ( 3. 16 ), C ( 3. 18 ), and B ( 3. 17 ) respectively indole alkaloids isolated from the Antarctic sponge Psammopemma sp. 20 Little difference exists between the reported NMR data of meridianin A and psammopemmin A (Table 3. 1). To verify the structure originally proposed for psammopemmin A as well as investigate b iological activity of the meridianins and analogs thereof synthetic studies were initiated. Table 3. 1 1 H NMR and 13 C shifts a of meridianin A ( 3. 24 ) and psammopemmin A ( 3. 16 ) Position 3. 24 H b 3. 24 H c 3. 24 C b 3. 24 C c Position 3. 16 H d 3. 16 C d 4 OH 13.55 (s) 13.57 (s) 4 OH 13.55 (s) N1 H 11.71 (brs) 11.76 (brs) N1 H 11.75 (brs) 2 8.20 (d) 8.22 (d) 128.5 128.5 2 8.22 (d) 128.3 3 113.8 114.0 3 113.7 3a 114.5 114.7 3a 114.3 4 152.1 152.0 4 152.0 5 6.36 (dd) 6.36 (dd) 105.6 105.4 5 6.38 (dd) 105.4 6 6.96 (dd) 6.98 (dd) 124.4 124.4 6 6.98 (dd) 124.3 7 6.78 (dd) 6.79 (dd) 102.4 102.3 7 6.81 (dd) 102.3 7a 139.4 139.4 7a 139.2 161.9 161.7 161.7 e 160.6 160.5 160.7 e 7.09 (d) 7.10 (d) 104.5 104.4 7.12 (d) 104.3 8.10 (d) 8.10 (d) 158.5 158.4 H 8.12 (brd) 158.3 e NH 2 6.69 (s, 2H) 6.72 (s, 2H) NH 2 6.68 (brs, 2H) a in ppm in d 6 DMSO. b isolated from Aplidium meridianum 22 c isolated from Synoicum sp 46 d isolated from Psammopemma sp. 20 e assignments may be interchanged. 20

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63 3.4 Synthesis and bio logical activity of 3 pyrimidylindoles A strategy to couple indoles to amino substituted pyrimidines was developed using Suzuki Miyaura protocol to synthesize both meridian in A and psammopemmin A, 47 as well as 3 pyrimidyindole analogs. The compounds were evaluated for biological activity. 3.4.1 Synthesis of meridianin A 4 methoxymeridianin A and 5 bromomeridinanin E Meridianin A ( 3. 24 ) was formed in six steps from indolone 3. 85 in 17 % overall yield (Scheme 3. 14) The 4 indolol moiety of meridinain A was synthesized by the dehydrogenation of commercially available tetrahydroindolone 3. 85 using Pd/C in refluxing diisobutyl ketone, a modest yielding yet economic reaction. 48 The phenol of 4 indolol ( 3. 86 ) was then masked as a silyl ether ( 3. 87 ). A two step, one pot N1 silylation and bromination 49 reaction resulted in the suitably substituted indole 3. 88 Transmetallation with t butyllithium then addition of borate 3. 89 generated vinyl borate 3. 90 which could be coupled to 2 amino 4 chloropyrimidine using a Suzuki protocol result ing in an intermediate ( 3. 91 ) bearing the 4 hydroxymeridianin skeleton. 25 Desilylation with tetra n butylammonium fluoride ( TBAF ) afforded meridianin A ( 3. 24 ) in good yields. The 4 methoxy analog of meridianin A was synthesized using a route analogous to that producing meridianin A (Scheme 3. 14) 4 I ndolol ( 3. 86 ) was refluxed with methy l iodide under basic conditions to form 4 methoxyindole ( 3. 92 ). After silylation and bromination resulting in the formation of 3. 93 transmetallation followed by addition of 3. 89 gave vinyl borate 3. 94 Coupling 3.94 to 2 amino 4 chloropyrimidine with Pd 0 resulte d in 3 pyrimidylindole 3.95 Upon treatment with TBAF, 4 methoxymeridianin A ( 3. 96 ) was obtained. Dibromination of meridianin A ( 3. 24 ) with pyridinium tribromide 50 resulted in the formation of 5 bromomeridianin E ( 3. 97 Scheme 14).

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64 3.4.2 chloropsammopemmin A Psammopemmin A ( 3. 16 ) differs from meridianin A ( 3. 24 ) in connectivity and substitution of the 3 pyrimidyl moiety. Using the Suzuki methodology developed to synthesize meridianin A, we needed only to change the pyrimidine coupling partner to afford psam m opemmin A. Synthesis of the pyrimidine moiety (Scheme 3. 15 ) began with the amination of 2,4 dich lo ropyrimidine ( 3. 9 8 ) followed by iodination to yield 4 amino 2 chloro 5 iodopyrimidine ( 3. 9 9 ). 51 Substitution of the chloro group using hydrogen bromide in acetic acid ( HBr/AcOH ) 52 resulted in 4 amino 2 bromo 5

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65 iodopyrimidine ( 3. 10 0 ). As a general rule, oxidative addition of Pd 0 to halopyrimidines takes place at C4>C2>>C5. 53 We expected compound 3. 100 to couple at C5 rather than C4, however, due to the more reactive iodo substituent. V inyl borate 3. 90 was then coupled to pyrimidine 3. 1 0 0 with tetrakistriphenylphospine palladium result ing in an intermediate ( 3. 101 ) bearing the psammopemmin skeleton. Desilylation of the resulting protected 3 pyrimidyl) indole using TBAF resulted in very low yields of the psammopemmin A free base, 3. 102 which was quite unstable unde r basic conditions. However, deprotection of 3. 101 with hydrogen fluoride/pyridine ( HFpyridine ) 54 produced the psammopemmin A free base cleanly as a white precipitate from the reaction solution. Forming the reported 20 HX salt of psammopemmin A was problematic and led to decomposition. P sammopemmin A hydrochloride ( 3. 10 3 ) could be prepared by bubbling HCl gas through a d 6 DMSO solution of 3. 102 Immediately analyzing the sample via NMR was necessary as the compound began to decompose after a few hours. Vinylborate 3. 90 was coupled to 4 amino 2 chloro 5 iodopyrimidine ( 3. 99 ) to form 3. 10 4 which, upon deprotection with TBAF in low yields or HF pyr in higher yields chloropsammopemmin A ( 3. 10 5 Scheme 3. 15 ).

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66 3.4.3 Structural reassessment of psammopemmin A NMR data (Table 3 2) for synthetic psammopemmin A HCl ( 3. 10 3 ) and its free base ( 3. 102 ) w ere then compared to the natural product psammopemmin A ( 3. 16 Table 3. 1 ). The phenolic 4 OH signals differed significantly ( 3. 16 : 13.55, s; 3. 102 : 9.30, s; 3. 10 3 : 9.47, vbrs) suggesting these protons were in dissimilar electronic environments. However, the 4 OH signal in meridianin A ( 3. 24 ) was identical to that reported for psammopemmin A ( 3. 16 Table 3. 1). The signal for H2 also differs between reported ( 3. 16 : 8.22, d) and synthetic ( 3. 102 : 7.27 d; 3. 103 : 7.33) psammopemmin A. Both meridianin A ( 3. 24 ) and natural psammopemmin A ( 3. 16 ) show coupling between protons on the pyrimidine ring ( 3. 24 3. 16 there is no obvious evidence protonation in synthetic psammopemmin A hydrochloride ( 3. 103 ). It is not clear from the 1 H NMR spectrum of 3. 103 which pryimidyl nitrogen accepts a proton NH 2 signal of 3. 102 exists as a very broad signal from 6.2 7.6 (centered around

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67 NH 2 signal of 3. 103 appears even more broad ( 6.1 8.5, centered around 7.45). The rather sharp 4 OH singlet in the 1 H NMR spectrum of 3. 102 ( 9.30) significantly broadens as to be almost imperceptible in 3. 1 03 ( 3.16 It has been demonstrated that monoprotonation of simple 4 aminopyrimidines occurs almost completely on N1, para to the amino group. 55 The 13 C NMR data for 3. 103 however, lacks etween N1 and N3 of pyridine. This phenomenon has been reported to occur in 2,4 diaminopyrimidine resulting in two carbon signals for C2, 4, 5, and 6. 56 Unfortunately, 3. 103 slowly degrades in d 6 DMSO so the doubling of those carbons due to proton exchange, if occurring, could not be detected. Table 3. 2. 1 H and 13 C NMR shifts a of synthetic psammopemmin A ( 3. 102 ) and synthetic psammopemmin AHCl ( 3. 103 ) Position 3. 102 H 3. 102 C 3. 102 HMBC 3. 103 H 3. 103 C 4 OH 9.30 (s) 9.30 (s) N1 H 11.29 ( brs) 11.29 (brs) 2 7.27 (d) 123.6 7.33 124.2 3 105.6 not observed 3a 115.4 H2, 5, 7, N1H, 4 OH 115.2 4 151.2 H5, 6, 4 OH 151.1 5 6.38 (d) 103.8 H7, 4 OH 6.48 104.0 6 6.93 (dd) 122.6 6.90 b 122.8 7 6.88 (d) 103.2 H5 6.90 b 103.2 7a 138.6 H2, 6 138.6 148.9 not observed 163.6 163.9 113.0 113.0 7.79 (s) 156.4 7.88 not observed NH 2 7.00 (vbrs) 7.45 H not observed a in ppm in d 6 DMSO. b signals overlap. From comparison of the 1 H and 13 C NMR data of 3. 16 3. 24 3. 102 and 3. 103 we infer that the correct structure of psammopemmin A isolated from Psammopemma sp. is the same as that of meridianin A ( 3. 24 ). The structure of psammopemmin C ( 3. 18 ) should also be revised to that of

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68 meridianin B ( 3. 25 ) due to the nearly identical 1 H and 13 C NMR signals reported. 20,22 It is also likely that the structure of psammopemmin B ( 3. 17 ) is that of meridianin E ( 3. 28 ), although the NMR data for the two were obtained in different solvents so co mparison is difficult. 3.4.4 Synthesis of meridoquin Inspired by the meriolins ( 3. 70 3. 83 ) synthetic hybrids of meridianins and variolins (Fig. 3. 1) we envisioned a meridianin chloroquine hybrid dubbed meridoquin ( 3. 106 Fig. 3. 2). By c ombining the bioactive meridianin skeleton with characteristics from the widely used antimalarial chloroquine (see section 4.2) we hoped to build a new bioactive scaffold as a starting point for future studies. M eridoquin ( 3. 106 ) was achieved using Suzuki methodology to couple a suitably substituted indole moiety to the desired pyrimidine (Scheme 3.1 6) A low yielding but quick reaction of neat diethylamine and 2,4 dichloropyrimidine ( 3. 98 ) resulted in the formation of 2 chloro 4 N N diethylaminopyrimidine ( 3. 10 7 ) as well as the desired 4 chloro 2 N N diethylaminopyrimidine ( 3. 10 8 ). Construction of the indole coupling partner began with the one pot N1 silylation 3 bromination of commercially available 6 chloroindole ( 3. 1 09 ) resulting in dihaloindole 3. 1 1 0 T ransmetallation of 3. 11 0 with t butyllithium then addition of 3. 89 resulted in intermediate 3. 11 1 which was immediately coupled to pyrimidine 3. 10 8 using the previously developed Suzuki protocol. Due to the electron withdrawing effects of the ring nitrogens, 4 halopyrimidines are exceptionally good electrophiles for use in Suzuki coupling reactions, 5 3 explaining the absence of

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69 any bisindoles in the reaction mixture. The indole pyrimidine coupling product ( 3. 112 ) was observed exclusively. Desilylation of 3. 11 2 with TBAF afforded meridoquin ( 3. 106 ). 3.4.5 Biological evaluation of 3 pyrimidylindoles Serotonin ( 3.113 5 hydroxytryptamine, 5 HT) transmission is thought to play a role in central nervous system (CNS) disorders. Compounds that bind to specific serotonin receptor subtypes could lead to treatment of CNS diseases. 57 Selective antagonists of 5 HT 2C helped to establish Neuropsychiatric disorders such as major depression, anxiety, and migraine are currently being treated with 5 HT selective receptor ligands while drugs that target 5 HT 1A 5 HT 2 A and 5 HT 2C receptors are under clinical investigation for the treatment of depression, schizophrenia, and anxiety, respectively. We were prompted to examine the binding affinity of several of our 3 pyrimidylindoles to various serotonin

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70 receptors d ue to the structural features (amin e containing indole) common to our compounds and serotonin. The binding affinity of meridianin A ( 3. 24 ), 4 methoxymeridianin A ( 3. 96 ), synthetic psammopemmin A ( 3. 102 ), and chloropsammopemmin A ( 3. 105 ) toward eleven 5 HT receptor subtypes was evaluated. 58 Primary screening was conducted in vitro by measuring the percent inhibition of radioligand bound to the receptor in question (% inhibition = 100% % radioactively bound) Primary assay results are summarized in Table 3. 3. Secondary screening was performed in vitro on compounds showing >50% inhibition (highlighted in Table 3. 3). Table 3. 3 Primary screening of 3 pyrimidylind oles for 5 HT binding inhibition. a 5 HT 1 A 5 HT 1 B 5 HT 1 D 5 HT 1 E 5 HT 2 A 5 HT 2 B 5 HT 2 C 5 HT 3 5 HT 5 A 5 HT 6 5 HT 7 3. 24 81.3 0.6 57.1 5.1 11.7 103.7 84.9 12.5 21.1 36 6.9 3. 96 83.7 43.8 76 1.6 8 100.3 91.4 7.5 79.9 12.7 69.8 3. 102 20.3 41.7 58.2 2.2 11.4 1.4 13.3 5.2 0.8 10.8 10 3. 105 10.6 44.1 27.9 2 4.1 3.7 7.2 5.7 8.7 17 12.7 a % inhibition = 100% % radioactively bound Meridianin A significantly inhibited binding of the radioligand to 5 HT 1 A 5 HT 1 D 5 HT 2 B and 5 HT 2 C in primary screening To date, secondary screening using radioligand competition binding assays has been carried out on 5 HT 1D 5 HT 2B and 5 HT 2C Secondary screening revealed meridianin A did not inhibit binding of 5 HT 1D and 5 HT 2C with [ 3 H]5 carboximidotryptamine and [ 3 H]mesulergine, respectively. Meridainin A did, however, inhibit the binding of radioligand [ 3 H] lysergic acid diethylamide ( [ 3 H]LSD ) with 5 HT 2B ( K i = 150 nM Fig. 3. 3)

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71 Primary screening of 4 methoxymerididanin A showed the compound inhibited radioligand binding to 5 HT 1A 5 HT 2B 5 HT 2C and 5 HT 5A and 5 HT 7 Secondary screening toward 5 HT 1D and 5 HT 2C showed 4 methoxymeridianin A did not inhibit binding of the radiolabled compounds. Like meridianin A, the 4 methoxy analog significantly in hibited binding of radioligand [ 3 H]LSD with 5 HT 2B ( K i = 88 nM, Fig. 3. 4) 4 M ethoxymeridianin A also inhibited, to a lesser extent, binding of [ 3 H]LSD with 5 HT 5A and 5 HT 7 ( K i = 1525 nM 1985 nM ; Fig. 3. 5 and Fig. 3. 6, respectively) Secondary screening showed psammopemmin A did not inhibit binding of [ 3 H]LSD to 5 HT 1D These initial findings show merid ianin A is a more selective inhibitor than other compounds tested while m ethylation of the 4 OH group leads to more potent inhibition of radioligand binding to 5 HT 2B as well as broader but less potent activity versus other receptor subtypes

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72 In addition to 5 HT receptor s the binding effects of 3 pyrimidylindoles on other CNS receptors and transporters w ere investigated The dopamine active transporter (DAT) was screened for radioligand ([ 3 H]WIN35428, a synthetic tropane analogous to cocaine) inhibition. Meridianin A was the only compound to display activity in prim ary and secondary DAT screens ( K i = 2.35 M Figure 3.7 ) One compound was also found to inhibit radioligand binding ([ 3 H]3 quinuclidinyl benzilate [ 3 H]Q N B ) of the muscarinic acetylcholine receptor (M 5 chloropsammopemmin A ( K i = 7.04 M, Figure 3.8). Malaria is a devastating disease affecting disadvantaged populations world wide (see Chapter 4). Because many current treatments for malaria are losing efficacy due to drug resistant parasites, new drugs are required to overcome resistance Toward this end, t he potential antimalarial activity 59 and cytoxicity 60 of meridianin A ( 3. 24 ), 4 methoxymeridianin A ( 3. 96 ), synthetic psammopemmin A ( 3. 102 chloropsammopemmin A ( 3. 105 ) and meridoquin ( 3. 106 ) w ere investigated. Merid i anin A, 4 methoxymeridianin A, and meridoquin were a ctive against the malaria parasite Plasmodium falciparum in initial screening Secondary screening was conducted to determine IC 50 values toward P. falciparum Meridianin A was the most potent (IC 50 = 12 M) but 4 methoxymeridianin A (IC 50 = 40 M ) and meridoquin (IC 50 = 200 M ) also displayed some antimalarial activity. Meridianin A was cytotoxic toward A 549 lung cancer cells (IC 50 = 15 M) but, fortuitously both 4 methoxymeridianin A and meridoquin display ed no toxic effects at the highest concentration examined (IC 50 > 4 20 M and > 333 M, respectively) Psammopemmin A

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73 chloro analog showed no activity against P. falciparum at the highest concentration tested The results of this screen are interesting in that 4 OH methylation of meridianin A significantly decreases the cytotoxicity of the compound while retaining antiparasitic activity. In summary, several meridianin and psammopemmin analogs were synthesized and examined for biological activity (Figure 3. 9 ) Meridianin A inhbited binding of [ 3 H]LSD to 5 HT 2B inhibited radioligand binding to DAT, and inhibited growth of P. falciparum but was, unfortunately found to be cytotoxic. 4 M ethoxymeridianin A inhibited binding of [ 3 H]LSD with 5 HT 2B 5 HT 5A and 5 HT 7, inhibited growth of P. falciparum and displayed no cytotoxic effects. Meridoquin had moderate antimalarial activity and was found to be non toxic. Psammopemmin A was devoid of activity but its chloro analog inhibited radioligand binding of M 5

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74 3.5 References Cited 1 Gul, W.; Hamann, M. T.; Life Sciences 2005 78 442 453. 2 Lagoja I. M. Chem. Biodiversity 2005 2 1 50. 3 Kohmoto, S.; Kashman, Y.; McConnell, O. J.; Rinehart, K. L.; Wright, A.; Koehn, F. J. Org. Chem 1998 5 3, 3116 3118. 4 Wright, A. E.; Pomponi, S. A.; Cross, S. S.; McCarthy, P. J. Org. Chem 1992 57 4772 4775. 5 Tsujii, S.; Rinehart, K. L.; Gunasekera, S.P.; Kashman, Y.; Cross, S. S.; Lui, M. S.; Pomponi, S. A.; Diaz, M. C. J. Org. Chem 1988 53 5446 5453. 6 Sakemi, S.; Sun, H. H. J. Org. Chem 1991 56 4304 4307. 7 Bialonska, D.; Zjawiony J. K. Mar. Drugs 2009 7 166 183. 8 Kazlauskas, R.; Murphy, P. T.; Quinn, R. J.; Wells, R. J. Tetrahedron Lett. 1977 1 61 64. 9 Hollenbeak, K. H.; Schmitz, F. J. Lloydia 1977 40 479 481. 10 Segraves, N. L.; Crews, P. J. Nat. Prod. 2005 68 1484 1488. 11 Atunes, E. M.; Copp, B. R., Davies Coleman, M. T.; Samaal, T. Nat. Prod. Rep 2005 22 62 72. 12 Yang, A.; Baker, B. J.; Grimwade, J.; Leonard, A.; McClintock, J. B. J. Nat. Prod 1995 58 1596 1599. 13 Perry, N. B.; Blunt, J. W.; McC ombs, J. D.; Munro, M. H. G. J. Org. Chem 1986 51 5476 5478. 14 Ford, J.; Capon, R. J. J. Nat. Prod 2000 63 1527 1528. 15 Perry, N. B.; Ettouati, L.; Litaudon, M.; Blunt, J. W.; Munro, M. H. G. Tetrahedron 1994 50 3987 3992. 16 Trimurtulu G.; Faulkner, D. J.; Perry, N. B.; Ettouati, L.; Litaudon, M.; Blunt, J. W.; Munro, M. H. G.; Jameson, G. B. Tetrahedron 1994 50 3993 4000. 17 Walker, S. R.; Carter, E. J.; Huff, B. C; Morris, J. C. Chem. Rev 2009 109 3080 3098. 18 Fresneda, P. M.; Delgado, S.; Francesch, A.; Manzanares, I.; Cuevas, C.; Molina, P. J. Med. Chem 2006 49 1217 1221. 19 Simone, M.; Erba, E.; Damia, G.; Vikhanskaya, F.; Di Francesco, A. M.; Riccardi, R.; Ba illy, C.; Cuevas, C.; Sousa Faro, J. M. F. Eur. J. Cancer 2005 41 2366 2377. 20 Butler, M. S.; Capon, R. J.; Lu, C. C. Aust. J. Chem 1992 45 1871 1877.

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75 21 Reyes, F.; Fernandez, R.; Rodriguez, A.; Francesch, A.; Taboada, S.; Avila, C.; Cuevas, C. Tetrahedron 2008, 64 5119 5123. 22 Franco L. H.; Joffe, E.; Puricelli, L.; Tatian, M.; Seides, A. M.; Palermo, J. A. J. Nat. Prod 1998 61 1130 1132. 23 Seldes, A. M.; Brasco, M. F. R.; Franco, L. H.; Palermo, J. A. Nat. Prod. Res 2007 21 555 563. 24 Gompel, M.; Leost, M.; De Kier Joffe, E. B.; Puricelli, L.; Hernandez Franco, L.; Palermo, J. ; Meijer, L. Bioorg. Med. Chem. Lett 2004 14 1703 1707. 25 Jiang, B.; Yang, C. G. Heterocycles 2000 53 1489 1498. 26 Fresneda, P. M.; Molina, P.; Delgado, S.; Bleda, J. A. Tetrahedron Lett 2000 41 4777 4780. 27 Fresneda, P. M.; Molina, P.; Bleda, J. A. Tetrahedron 2001 57 2355 2363. 28 Bredereck, H.; Effenberger, F.; Botsch, H.; Rehn, H. Chem. Ber 1965 98 1081 1086. 29 Karpov, A. S.; Merkul, E.; Rominger F.; Muller, T. J. J. Angew. Chem. Int. Ed 2005 44 6951 6956. 30 Radwan, M. A. A.; El Sherbiny, M. Bioorg. Med. Chem 2007 15 1206 1211. 31 Kobayashi, G.; Furukawa, S.; Matsuda, Y.; Washida, Y. Yakugaku Zasshi 1967 87 857 860. 32 Paul, R.; Hallett, W. A.; Hanifin, J. W.; Reich, M. F.; Johnson, B. D.; Lenhard, R. H.; Dusza, J. P.; Kerwar, S. S.; Lin, Y.; Pickett, W. C.; Seifert, C. M.; Torley, L. W.; Tarrant, M. E.; Wrenn, S J. Med. Chem 1993 36 2716 2725. 33 Jiang, B.; Yang, C. G.; Xio ng, W. N.; Wang, J. Bioorg. Med. Chem 2001 9 1149 1154. 34 Franco, L. H.; Palermo, J. A. Chem. Pharm. Bull 2003 51 975 977. 35 Kidwai, M.; Rastogi, S.; Saxena, S. Bull. Korean Chem. Soc 2003 24 1575 1578. 36 Agarwal, A.; Srivastava, K.; Puri K. K.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett 2005 15 3133 3136. 37 Agarwal, A.; Kumarr, B.; Mehrotra, P. K.; Chauhan, P. M. S Bioorg. Med. Chem 2005 13 1893 1899. 38 Casar, Z.; Bevk, D.; Svete, J.; Stanovik, B Tetrahedron 2005 61 7508 7 519. 39 Simon, G.; Couthon Gourves, H.; Haelters, J. P.; Corbel, B.; Kervarec, N.; Michaud, F.; Meijer, L J. Heterocyclic Chem. 2007 44 793 799. 40 Rossignol, E.; Youssef, A.; Moreau, P. ; Prudhomme, M.; Anizon, F. Tetrahedron 2007 63 10169 10176. 41 Rossignol, E.; Debiton, E.; Fabbro, D.; Moreau, P.; Prudhomme, M.; Anizon, F. Anti Cancer Drugs 2008 19 789 792.

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76 42 Yu, H.; Yu, Z. Angew. Chem. Int. Ed 2009 48 2929 2933. 43 Tibiletti, F.; Simonetti, M.; Nicholas, K. M.; Palmisano, G.; Parravicini, M.; Imbesi, F. Tollari, S.; Penoni, A. Tetrahedron 2010 1280 1288. 44 Bettayeb, K.; Tirado, O. M.; Marionneau Lambot, S.; Ferandin, Y.; Lozach, O.; Morris, J. C.; Mateo Lozana, S.; Drueckes, P.; Schachtele, C.; Kubbuatat, M. H. G, Liger, F. ; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J. A.; Notario, V.; Meijer, L. Cancer Res 2007 67 8325 8334. 45 Echalier, A.; Bettayeb, K.; Ferandin, Y.; Lozach, O.; Clement, M.; Valette, A.; Liger, F.; Marquet, B.; Morris, J. C.; Endicott, J. A.; J oseph, B.; Meijer, L. J. Med. Chem 2008 51 737 751. 46 The tunicate was identified as Synoicum (family Polyclinidae) by Dr. Linda Cole, Smithsonian Institution, Washington, D.C. A voucher specimen is held at USF (PSC08 17). 47 Lebar, M. D.; Baker, B. J. Aust. J. Chem 2010 63 862 866. 48 Hirotaka, F.; Kenji, N.; Tomoaki, N.; Kiyoshi, W. Japan Patent 60 146870 1985 49 Amat, M.; Seffar, F.; Lior, N.; Bosch, J. Synthesis 2001 267 275. 50 Yamada, F.; Tamura, M.; Hasegawa, A.; Somei, M. Chem. Pharm. Bull 2002 50 92 99. 51 Minakawa, N.; Kojima, N.; Hikishima, S.; Sasaki, T.; Kiyosue, A.; Atsumi, N.; Ueno, Y.; Matsuda, A. J. Am. Chem. Soc 2003 125 9970 9982. 52 Schlosser, H.; Wingen, R. U. S. Patent 5,371, 224 1994 53 Palucki, M. Palladium in Heterocyclic Chemistry (Ed. J. J. Li, G. W. Gribble) 2007, Vol 26. Ch. 11. pp. 475 509 (Elsevier, Oxford, UK). 54 Jung, M. E.; Salehi Rad, R. Angew. Chem. Int. Ed. 2009 48 8766 8769. 55 JRiand, J.; Chenon, M. T.; Lumbroso Bader, N. J. Am. Chem. Soc 1977 99 6838 6845. 56 Griffiths, D. V.; Swetnam, S. P.; J. Chem. Soc. Chem. Comm 1981 1224 1225. 57 Barnes, N. M.; Sharp, T. Neuropharmacol 1999 38 1083 1152 58 K i determinations were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN 271 2008 00025 C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA. 59 IC 50 determinations toward Plasmodium falciparum were provided by Dennis E. Kyle at the Department of Global Health, College of Public Health, Tampa FL, USA. 60 IC 50 determinations toward A 549 (human lung cancer) were pr ovided by Alberto van Olphen at the Department of Global Health, College of Public Health, Tampa FL, USA.

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77 Chapter 4 Antimalarial natural products 4.1 The m alaria dilemma Malaria, a devastating infectious disease, was responsible for almost a million deaths and 247 million reported clinical episodes in 2008 1 Malaria is caused by Plasmodium parasites transmitted through infected Anopheles mosquitoes. Many Plasmodium species exist worldwide and can infect a variety of vertebrates P. falciparum is the most deadly to humans, accounting for the majority of malaria related deaths. P. vivax causes the most morbidity of the human parasites. P. ovale and P. mal a riae are also human pathogens of concern. The malaria parasite life cycle is complex, involving discreet stages in two hosts. 2 The bite from a malaria infected female Anopheles mosquito transmit s sporozoites into a human host which infect hepatocytes (liver cells). The spor o zoites mature and release merozoites into the blood circulation, invading erythrocyt e s ( red blood cells RBC). After deve loping through ring, trophozoit e and schizont stages and asexu s, mature schizonts again rupture releasing more merozoites Anopheles mosquito during blood meal. Sexual reproduction in the mosquito gut forms non motile zygote s which develop into mot ile ookinetes that move to the midgut wall. Ookinetes transform into oocysts which rupture releasing sporozoites that tra verse to the sal ivary gland of the mosquito. The parasite life cycle repeats in a new host when the mosquito takes a human blood meal Malaria persists in 108 countries and territories particularly affecting disadvantaged populations in Africa and, to a lesser extent, South East Asia and Latin America 3 The most at risk are y oung children residing in Africa where m alaria is responsible for 20% of childhood deaths. Other at risk

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78 groups include non immune pregnant women, individuals with human immunodeficiency virus/acquired immune deficiency syndrome ( HIV/AIDS ) international travelers and families returning to end emic areas after prolonged absence International funding for the treatment prevention and eradication of malaria increased to $1.7 billion in 2009 from $300 million in 2003 fueling noticeable reductions in the malaria burden However, c urrent funding falls short of the $5 billion required annually to fully combat the disease globally Although devastating, malaria is treatable and preventable. 4.2 Current malaria treatment 4.2.1 Quinolines, antifolates, and artemisinins Europeans learned of the antim alarial properties of South American C incho n a bark (Family: Rubiaceae) i n the seventeenth century 4 The antimalarial constituent of the bark, a quinoline containing alkaloid named quinine ( 4.1 ) is thought to prevent the parasite from polymerizing heme. 5 As the parasite develops in the RBC it cannibalizes hemoglobin subsequently releasing heme which the parasite convert s to h emozoin, a non toxic crystalline heme polymer. Inhibition of heme polymerization results in toxic leve ls of heme and death of the parasite. The mechanism s by which the parasite polymerize s heme and quinoline based drugs inhibit t he polymerization process is not fully understood It is clear, though, that quinoline based therapies have been quite effective in the t reatment of malaria evidenced by the success of chloroquine ( CQ, 4.2 ), a synthetic 4 aminoquinoline derivative. 6 C Q cure billions of clinical cases of malaria. Excellent clinical efficacy, economical synthes e s ease of use and prophylactic properties contributed to the success of CQ and other synthetic quinoline derivatives including amodiaquin e ( 4.3 ) and mefloquine ( MQ, 4.4 ) overused, however, leading to the emergence of chlor oquine resistant parasites. 7 Other drugs targeting different metabolic processes in the parasite were developed to combat resistant strains

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79 Drugs that target folate metabolism eradicate Plasmodium parasites by inhibit ing ability to produce folic acid and thus its ability to biosynthesize pyrimidine, purine, and amino acids A combination of the antifolate pyrimidine containing drugs sulfadoxine ( 4.5 ) and pyrimethamine ( 4.6 ) was found to be efficacious i n treating uncomplicated CQ resistant malaria. 8 While both compounds show mode st antiparasitic properties individually, a synergistic effect is observed when combination therapy is administered. Unfortunately, the switch from CQ to sulfadoxine/pyrimetham ine (SP) to treat CQ resistant strains while initially effective, quickly led to the appearance of SP resistan t parasites 9 Artemisinin ( 4.7 ), dubbed qinghaosu by the Chinese, is the powerful antimalarial component of the Chinese medicinal herb Artemisia annua 7 Artemisinin, a unique sequiterpene 1,2,4 trioxane lactone rapidly clears all blood stages of Plasmodium parasitemia through a highly contested

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80 mechanism of action which could involve the peroxide moiety forming radical or ionic intermediates 10 Poor solubility of the natural product led to semisynthetic derivatives including more lipophilic artemether ( 4.8 ) and more hydrophilic artesunate ( 4.9 ). In an intriguing e ffort to utilize microbes as an inexpensive producer of artemisinin, the yeast Saccharomyces cerevisiae was genetically modified, using genes from A. annua to produce the artemisinin precursor artemisinic acid ( 4.10 ). 11 The precursor can be easily converted to artemisinin and derivatives using through economical, well studied synthetic procedures. Although artemisinin and its derivatives are effective single drug treatments, artemisinin combination therapy (ACT) is reco mmended by the World Health Orgaization (WHO) to delay development of parasite drug resistance. ACT has been reported as 95% effective in curing malaria. 12 Most malaria endemic countries now use ACT as a first line treatment for uncomplicated P. falciparu m infection. Unfortunately, parasites displaying artemisinin resistance ha ve been recently documented on the Cambodia Thailand border. 13 4.2.2 Drug resistant parasites Widespread drug resistant malaria is of enormous concern. 14 Parasites resistant to quinoline based drugs and sulfadoxine/pyrimethamine combination therapy ha ve been extensively observed 15 The more recent emergence of artemisinin combination therapy resistance underscores an urgent need for the developm ent of a new class of compounds. Success of future a ntimalarial drug discovery depends on the ability to identify parasites displaying drug resistance 16 and subsequently screen diverse chemotypes for the ability to inhibit drug resistant parasitemia Gratifyingly, worldwide awareness of the malaria burden has led to collaborations between academic institutions, non profit organizations and the pharmaceutical industry 17 W ith

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81 the support of international funding from public and private sources these collaborators are working to combat the global malaria burden 4.3 Natural products as antimalarial leads Recent s creening of vast synthetic chemical libraries provided by Glaxo Smith Kline has afforded hundreds of potential antimalarial leads. 18 19 Particularly exciting was the identification of nineteen new inhibitors of four validated drug targets as well as fifteen novel malarial protein binders. Researchers hunting for new antimalarial chemotypes commonly depend on synthetic combinatorial librar ies to generate large numbers of compounds which supply high throughput screening (H TS ) operations. 20 S ynthetic chemical librar ies however, simply cannot match the Given that the most widely used and most efficacious malaria therapies were derived from natural products further chemical investigation of natural sources for antimalarial consti tuents is fully warranted. Plants have proven to be an excellent source of antimalarial alkaloids and terpenes. 21 Flora used in traditional medicine to treat malaria like symptoms, like Cinchona and Artemisina are particularly well studied. Less studied but no less promising sources for potential antimalrial chemistry are microorganisms and marine macroorganisms A number of antimalarial agents have been recently identified from marine macroorganisms 22 Crambe s cidin 800 ( 4.1 1 ), previously isolated from the Mediterranean red encrusting sponge Crambe crambe 23 displayed nanomolar activity against CQ resistant P. falciparum FCR3 in vitro. 24 Crambescidin s bear a complex molecular structure comprised of a pentacyclic guanidine linked by a linear hydroxy fatty acid to a hydroxyspermidine moiety. Several crambescidin analogs have been prepared that show even more promising antimal a rial activity.

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82 M anzamine A ( 4.1 2 ) initially isolated from an Okinawan sponge Haliclona sp., is a n antimalarial carboline alkaloid containing a complicated array of 5 6 8 and 13 membered rings 25 More than sixty related compounds have been isolated from various sponge species. 26 A single intraperitoneal injection of manzamine A was found to clear parasitemina in mice infected with P. berghei 27 N atural manzamines display toxicity but the semisynthetic derivative, per acylated 8 hydroxymanzami n e A ( 4.1 3 ) has no cyto toxicity at the highest dose tested while retaining antiparasitic properties 28 Manzamines are purported to have been isolated from a symbiotic bacteri um suggesting the molecules produced in the sponge are of microbial origin. 29 If large scale fermentation of this microbe is feasible, ample quantities of manzami n es would be available for further experimentation. Bacteria and fungi produce a wide range of biologically active compounds that posse ss great diversity in chemical structure. 30 31 Screening microbial extracts has resulted in the discovery of many compounds possessing antimalarial activity S everal microbe derived natural products

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83 display potent and promising antiparasitic activity Nostocarboline ( 4.1 4 ), isolated from the cyanobacterium Nostoc sp., and synthetic dimers thereof inhibited P. falciparum in nanomolar concent rations. 32 The compounds are only weakly cytotoxic the most encouraging possessing a selectivity index (SI) of > 2,500. The screening of eighty crude marine derived bacterial extracts led to the identification of s alino s poramide A ( 4.1 5 see also section 1.3 ) as a nanomolar in vitro inhib itor of human Plasmodium parasites 33 The compound also significantly reduced P. yoelii parasitemia in mice. Isolated from the yeast Candida lipolytica 34 the indoloquinazoline tryptanthrin ( 4.1 6 ) along with several synthetic analogs displayed potent in vitro activity against CQ and MQ resistant Plasmodium strains. 35 Microbes are an ideal source for novel antimalarial compounds. Not only do microbes produce profound chemical diversity, they are also amendable to large scale fermentation resulting in facile access to compounds with complicated structures Complex chemicals are un desirable mostly because the malaria burden in underdeveloped nations requires inexpensive treatment. However, an abundantly producing microbe could yield complex natural products very cost effectively Ou r lab, with international collaboration, has examine d a multitude of microorganisms for antimalarial constituents with the goal of identifying lead compounds possessing potent activity against Plasmodium falciparum 4.4 Medicines for Malaria Venture proje ct 4.4.1 MMV project overview With funding from Medicines for Malaria Venture (MMV), 36 we endeavored to examine niche environment microbiota for new antimalarial chemotypes. The planned project would seek to

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84 analyze ~70,000 microbes from niche environments worldwide including our own microbial libraries at the University of South Florida (USF) as well as the libraries of our academic and commercial collaborators. Our lab supplied 1 ,000 + Antarctic marine Florida ma rine, and Florida mangrove microbial endophytes Bacterial and fungal endophytes were isolated from samples collected on various expeditions to McMurdo Station and Palmer Station, Antarctica as well as collecting trips to several locations in Florida wate rs, including the Florida Keys, the Gulf of Mexico, and the Florida Everglades Eubacteria (~5,000) isolated from Caribbean marine cave dwelling invertebrates were supplied by Magellan BioScience (Todd Daviau and John Cronin) City University of Hong Kon g ( CY, Lilian Vrijmoed) and National Taiwan Ocean University ( NTOU, Ka Lai Pang) provided 5,000 + isolates of mangrove endophytes from the co a stal region of China. The majority (~55,000) of the microbes analyzed were filamentous fungi isolated from various sources largely plant in origin, collected around the world and housed at Mycosynthetix ( MSX, Cedric Pearce). Biological activity screening (section 4.4.2.1) was performed at USF, College of Public Health (Dennis Kyle). Processes involving preparation of extracts, fractionation of active samples, as well as analyzing and identifying active components were carried out in our lab using the procedures described in section 4.4.2.2. 4.4.2 MMV project methodology 4.4.2.1 Antimalaria and cytotoxicity assays prioritization of active extracts Primary screening employed a novel luciferase reporter assay that utilizes transgenic blood stage parasites to rapidly identify antimal ar ial extracts from a 96 well plate format The in vitro drug luminescence (ITDL) assa y 37 uses Plasmodium falciparum (3D7) transfected with a luciferase construct, allowing for an assay with signal to noise ratio better than many currently in use (Sybr Green, DAPI, Pico Green). (50 g crude extract/mL) Extracts that displayed >67% parasite growth inhibition at both high and low concentration s were designated

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85 In vitro cyto toxicity of the active extracts toward A 549 human cancer cells allowed for further prioritization of antimalarial extracts. concentrations previously described. Both high and low concentration s were divided into three categories : cytotoxic (< 7 5% cell viability), moderately cytotoxi c ( 7 5 95% cell viability), and non toxic (>95% cell viability) Current hit rates are summarized in Table 4.1. Table 4.1. Antim al arial and cytotoxic activity of microbial extracts Origin Extracts screened Active extracts (% of screened) Partially active extracts (% of screened) non toxic active extracts (% of actives) Non toxic partially active extracts (% of p.a.) MSX 24,780 a 278 ( 0.9 %) 2214 (9%) 108 (39%) 872 (39%) CY /NTOU 5192 35 (0.7%) 269 (5%) 10 (29%) 186 (36%) USF 1056 3 ( 0.3 %) 40 (4%) 2 (67%) 14 (35%) a includes only extracts for which both antimalarial and cytotoxicity data was available 4.4.2. 2 Antarctic microbe cultivation, extraction, and processing Tissue from sessile marine invertebrates (i.e. sponges, tunicates, soft corals, etc.) collected in the waters surrounding McMurdo Station and Palmer Station, Antarctica were preserved in glycerol solution and kept at 80 o C during shipment from Antarctica to USF. Using sterile technique, the warmed tissue (5 o C) was sectioned into fractions and immediately plated on a Difco marine agar dish which was then sealed with parafilm. The microbes were a llowed to grow at 5 o C for about two weeks. Sterile loops were used to isolate various microbes growing from the tissue. The agar plates streaked with the new isolates were allowed to grow for an additional two weeks. This process was repeated until a pure isolate was obtained. Each isolate was archived in glycerol stock and kept in our microbe library at 80 o C. The i solate s w ere cultured on a small scale to obtain enough biomass for biological analyses. To limit error and speed processing time, all procedures were conducted in a 96 well format. A

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86 60 mL capacity each in a 12 column X 8 row grid was us ed to culture 88 isolates concurrently (the last column was reserved for controls). Difco marine broth ( 100 mL) was added to each well. After sterilization, each isolate, either directly from the isolation plate or from re grown stock, was transferred via sterile loop to a designated well in the shaker rack. The culture media was allowed to ferment under aeration at 5 o C for three weeks. Acrylic ester resin (XAD 7, 3 g) was then added to each well to collect small non polar and semi polar organic metaobolites C ells were lysed with three successive freeze thaw cycles T he resin was then filtered along with any cell mass and washed with water. The filtrate was sterilized and discarded. The resin and cell mass w ere then extracted with methanol (10 mL) for 24 h and removed via filtration from the methanolic extract. Concentration of the methonolic extract yielded a crude extract for each of the 88 samples cultured. The extract was redissolved in dimethylsulfoxide (DMSO) at 30 mg/mL. A portion of this solution (150 L) was transferred to a 96 well plate and submitted for antimalarial screening. The remain in g DMSO solution was transferred to a deep well plate and kept frozen for further testing. 4.4.2. 3 Fungi extraction and processing Lyophilized fungal cultures were received from City University of Hong Kong (CY) and National Taiwan Ocean University (NTOU) in 30 mL falcon tubes. Each culture was extracted with methanol (~15 mL) for at least 24 h. The methanolic extract was carefully transferred to a clean 20 mL scintillation vial which was place d in a compartmentalized tray keep ing the extractions in a 96 well format (88 per tray) Concentration of the methanol extracts under a stream of air afforded the dried extract. Mycosynthetix (MSX) samples were received as solid culture material (~5 mL) in 20 mL scintillation vials. After each sample was extracted with methanol ( 24 h, 15 mL), the methanolic extract was subjected to the same concentration protocol used for the CY and NTOU samp les. All dried extracts were processed and submitted (30 mg/ mL, DMSO) in 96 wel l plates analogous to the procedure developed for the Antarctic microb ial extracts.

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87 4.4.2. 4 Scale up f ermentation and fractionation Antarctic microbe extracts found active in the antimalarial assay were regrown in 6X1 L cultures (Difco marine broth). After 3 weeks of aerated fermentation, XAD 7 (30 g/L) resin was added to extract the small organic meta bolites from the media. Cells were then lysed in three successive freeze thaw cycles. The resin and cell mass was separated from the broth by filtration, rinsed with water, and extracted with methanol. The methanolic extract was concentrated in vac uo to afford the crude scale u p extract. Scaled up fermentations (100X 1000X) were received from CY, NTOU, and MSX and extracted exhaustively with methanol. The meth ano lic extracts were concentrated to afford crude scale up extracts. Fractionation of the scale up extracts was achiev ed with normal phase silica gel medium pressure liquid chromatography (MPLC). A portion (400 mg or 2 g) of the extract was fractionated through a silica g el cartridge bout ten fractions of increasing polarity. After concentration, a portion of each fraction was redissolved in DMSO (2 mg/ mL) and submitted for biological evaluation. 4.4.2. 5 Active fractions to pure compounds Fractions that remained active in the antima larial assay were further investigated via nuclear magnetic resonance (NMR) spectroscopy and liquid chromatograpy mass spectrometry (LC MS) Proton ( 1 H ) NMR spectra were obtained for each active fraction. Those fractions with interesting spectra (i.e. those with downfield signals indicative of heteroatoms and consequently, functional groups) were further analyze d by thin layer chromatography (TLC) and f inally purified with high performance liquid chromatography (HPLC) Pure compounds were identified by obtaining physical data ( 1 H and 13 C NMR, MS) and searching databases such as AntiMarin for compounds with similar or identical data. Alternatively, part ial structures could be elucidated via various 2 D NMR techniques [ Correlation spectroscopy ( COSY ) Heteronuclear Single Quantum Coherence

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88 ( HSQC ) Heteronuclear Multiple Bond Correlation ( HMBC ), etc.] which would be used to narrow down potential structures. 4.4.2.6 Protocol validation To validate our sample preparation procedures as well as the antimalarial assay a crude Artemisina annua extract 38 was subjected to our fractionation protocol. The concentrated f ractio ns were then added to a plate containing MSX fractions and submitted for biological analysis. The Artemisina extract fractions were analyzed in an identical manner alongside the fungal fractions B ioassay results indicated one active Artemisina fraction which, upon NMR and LC MS ana ly s e s, was revealed to contain artemisinin. 4.4.3 Antimalarial compounds from endophytic mangrove fungi Preliminary results led to isolation of several fungi derived antimal a rial natural products. Extracts from end ophytic fungi isolated from mangroves inhabiting coastal China were the first to be analyzed. Scale up fermentation and extraction followed by fractionation of several fungal samples afforded two known and, unfortunately, cytotoxic and cytostatic classes of metabolites, cytochalasins and trichothecenes. 4.4.3.1 Cytochalasins Scale up fermentation of CY 4202 was found to contain cytochalasin D ( cytD, 4.17 ), a polyketide 39 mycotoxin originally isolated 40 from cultures of Metarrhizium anisopliae and Hypoxylon terricola Cyt ochalasin D is a reversible potent inhibitor of actin polymerization and thus is used to probe the role of actin in various cell processes. 41 Physical data ( Table 4.2. 1 H and 13 C NMR data ) of cytD isolated from CY 4202 was identical to that previously reported. 42 An LC MS method was developed to quickly screen fractions for the presence of cytD leading to the identifi cation in another cytD producing coastal China mangrove endopyte, CY 4204 the pre sence of which was further confirmed with 1 H NMR data. Several other CY and MSX samples

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89 appear to contain cytochalasin s as their 1 H NMR spectra are similar to cytD but LC MS traces differ from cytD. Cyt ochalasin D was previously observed to a ffect development of P. berghei 43 44 but have had no inhibitory effect on P. yoelli 45 We found that many of the cytD containing fractions inhibit P. falciparum ( in vitro ) perhaps enhanced by synergistic effects from other compounds in the mixture Pure cytD also displayed in vitro inhibition of P. falciparum with activity at nanomolar concentrations (IC 50 = 26 nM) However, because the compound inhibits actin poly merization resulting in cytost asis, cytD is a poor candidate for malaria therapy.

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90 Table 4.2 1 H and 13 C NMR shift a comparison of isolated cytochalasin D ( 4.17 ) with literature values. Position ref. 4 2 H 4.17 H ref. 4 2 C 4.17 C 1 173.6 173.7 3 3.22, m 3.24, m 53.5 53.5 4 2.84, m 2.84, m 47.0 47.0 5 2.14, t 2.15, t 50.0 49.9 6 147.5 147.6 7 3.80, d 3.80, d 69.8 69.8 8 2.83, m 2.83, m 32.6 32.6 9 53.2 53.3 10 2.65, dd; 2.83, m 2.76, m (2H) 45.3 45.3 11 0.95, d 0.94, d 13.6 13.6 12 5.09, s, 5.29, s 5.08, s, 5.29, s 114.5 114.4 13 5.35, m 5.33, m 134.1 134.1 14 5.65, dd 5.69, dd 130.6 136.0 15 2.02, dd, 2.51, dd 2.02, dd, 2.51, dd 37.7 37.7 16 2.73, m 2.73, m 42.3 42.3 17 210.2 210.2 18 77.7 77.7 19 5.15, dd 5.14, d 127.1 127.0 20 6.11, dd 6.11, dd 132.3 132.3 21 5.63, dd 5.62, m 77.3 77.1 22 1.20, d, 3H 1.20, d, 3H 19.4 19.4 23 1.51, s, 3H 1.51, s, 3H 24.2 24.2 137.2 137.2 7.25, m, 2H 7.13, d 2H 129.1 129.1 7.25, m, 2H 7.28, m, 2H 128.9 128.9 7.25, m 7.28, m 127.6 127.6 O C OCH 3 169.7 169.6 O C O CH 3 2.26, s, 3H 2.26, s, 3H 20.8 20.8 NH 5.53, brs 4.64, brs a in ppm in CDCl 3 4.4.3.2 Trichothecenes Investigation of scale up fermentation CY 3923 revealed two known tricothecene mycotoxins that were in vitro inhibitor s of the malaria parasite Trichothecenes are tetracyclic sesquiterpenes usually containing an epoxide ring. We found that roridin E ( 4.1 8 ) potently inhibited P. falciparum (IC 50 < 190 nM) while its deoxy analog, 12,13 deoxyroridin E ( 4. 19 ), displayed less but still significant inhibition (IC 50 = 765 nM) The compounds were cytotoxic, however, in our initial toxicity screen at the lowest dose examined (5 g/mL). The structures of 4.18 and 4.19 were

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91 confirmed upon comparison of 1 H and 13 C data with literature values ( 4.18 : Table 4.3, 4.19 : Table 4.4) 46 The antimalarial properties of roridin E and related trichothecenes ha ve been previously reported. 47 Roridin E showed extremely potent activity against P. falciparum (K1, EC 50 = 0.3 nM) but unfortunately, similar cyto to xicity (KB, BC1, vero cells: EC 50 = 0.8 to1.4 nM). Interestingly, roridin E acetate ( 4.20 ) more potently inhibited P. falciparum (K1, EC 50 = 0.1 nM) while possessing ten to twenty fold less cytotoxicity (KB, BC1, vero cells: EC 50 = 8.0 to 28.0 nM) than roridin E. This simple addition of an acetate moiety had a profound effect on activity. Although most tricothecenes are cytotstatic or cytotoxic, it is possible that a derivative will be unc overed or semisynthetically derived that possesses the excellent antiparasitic properties currently observed as well as low or no cytotoxicity.

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92 Table 4. 3 1 H and 13 C NMR shift a comparison of isolated roridin E ( 4.18 ) with literature values. Position ref. 4 6 H 4.1 8 H ref. 4 6 C 4.1 8 C 2 3.85, d 3.85, d 79.2 79.2 3 2.09, m; 2.56, m 2.06, m; 2.53, m 35.8 35.8 4 6.22, m 6.21, dd 74.2 74.2 5 48.4 48.4 6 42.7 42.7 7 1.67, m; 2.06. m 1.67, m; 2.06. m 21.6 21.6 8 2.02, m; 2.00, m 2.02, m; 2.00, m 27.7 27.6 9 140.2 140.2 10 5.48, br d 5.48, d 118.9 118.8 11 3.92, d 3.90 d 67.2 67.2 12 65.6 65.6 13 2.83, d; 3.15, d 2.82, d; 3.14, d 48.1 48.1 14 0.8, s 0.79, s, 3H 6.7 6.7 15 3.95, d; 4.32, d 3.93, d; 4.31, d 63.7 63.7 16 1.72, s 1.71, s, 3H 23.2 23.2 166.4 166.5 5.96, s 5.96, s 117.2 117.1 159.2 159.2 2.53, m 2.53, m 41.2 41.2 3.59, m; 3.71, m 3.59, m; 3.70, m 69.6 69.8 83.9 83.8 5.91, dd 5.90, dd 138.0 137.9 7.54, dd 7.52, dd 126.6 126.6 6.58, dd 6.57, dd 143.6 143.6 5.75, d 5.75, d 117.8 117.8 165.8 165.8 2.28, d 2.26, d, 3H 20.3 20.3 3.65, m 3.65, m 70.7 70.7 1.21, d 1.20, d, 3H 18.3 18.2 a in ppm in CDCl 3

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93 Table 4. 4 1 H and 13 C NMR shift a comparison of isolated 12,13 deoxyroridin E ( 4.19 ) with literature values. Position ref. 45 H 4.1 9 H ref. 45 C 4.1 9 C 2 4.45, d 4.48, d 79.0 79.8 3 1.75, m; 2.56, m 1.77, m; 2.55, m 37.2 37.2 4 6.26, dd 6.27, m 74.4 74.4 5 51.0 51.0 6 42.5 42.5 7 1.50, m; 1.97, m 1.51, m; 1.96, m 20.9 20.9 8 2.00, m, 2H 2.00, m, 2H 27.7 27.7 9 139.8 140.0 10 5.44, br d 5.45, d 119.1 118.8 11 3.98, d 4.01, d 67.0 67.1 12 152.7 152.5 13 4.70, s; 5.14, s 4.72, s; 5.16, s 105.3 105.5 14 1.02, s, 3H 1.03, s, 3H 11.3 11.3 15 3.96, d; 4.28, d 3.96, d; 4.30, d 64.2 64.3 16 1.68, s, 3H 1.69, s, 3H 23.2 23.3 166.5 166.6 6.02, br s 6.03, s 117.5 117.5 158.7 158.8 2.53, m, 2H 2.53, m, 2H 41.3 41.3 3.55, m; 3.65, m 3.56, m; 3.65, m 69.8 69.8 3.70, m 3.70, m 83.9 83.7 5.90, dd 5.90, d 137.8 137.7 7.56, dd 7.56, dd 126.6 126.7 6.57, dd 6.58, dd 143.5 143.5 5.66, d 5.68, d 117.7 117.8 165.6 165.7 2.25, d, 3H 2.25, s, 3H 20.0 20.0 3.64, m 3.64, m 70.6 70.8 1.20, d, 3H 1.23, d, 3H 18.3 18.2 a in ppm in CDCl 3 4.4.4 MMV project outlook With preliminary extract screening nearing completion 48 and fractionation of scale up fermentation extracts well underway, we expect to discover many more antimalarial constituents in our 70,000 strong microbial extract library. The identification of artemisinin combined with the isolation of cytochalsin D and roridin E contributes to the validity of both the isolation procedure as well as the antimalarial assay. Cytotoxicity data for those extracts displaying activity in the malaria assay should steer us away from active but toxic extracts resembling the ones discussed herein. Further development of LC MS techniques to identify common toxic compounds li ke

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94 cytochalasins and roridins would speed up identification of compounds we do not want. The time saved would allow us to analyze a greater quantity of promising active extracts increasing the chance of drug lead discovery. A microbe derived lead compoun d w ould then be investigated synthetically to identify the pharmaco phore, as well as to tailor bioactivity and physical properties, analogous to operations that led to the development of clinically successful quinoline and artemisinin derivatives. 4.5 Ref erences Cited 1 World Health Organization. Fact sheet no. 94 April, 2010 (Geneva, Switzerland) 2 Tuteja, R. FEBS J 2007 274 4670 4679 3 World Health Organization. World Malaria Report 2009 ; WC 765 NTIS ( Geneva, Switzerl and) 4 Rocco F. Quinine: Malaria and the Quest for the Cure that Changed the World ; Harper Perennial : San Francisco, 2004 5 Rathore, D.; Dewal, J.; Nagarkatti, R.; Kumar, S. Drug Discovery Today: Ther. Strategi es 2006 3 153 158. 6 Sullivan, D. J.; Gluzman, I. Y.; Russels, D. G.; Goldberg, D. E Proc. Natl. Acad. Sci. USA 1996 93 11865 11870. 7 Curr. Top. Mol. Immun 2005 295 3 38. 8 Bell, D. J.; Nyirongo, S. K.; Mukak a, M.; Zijlstra, E. E.; Plowe, V. P.; Molyneus, M. E.; Ward, S. A.; Winstanly, P. A. PLoS ONE 2008 3 e1578. 9 Gatton, M. L.; Martin, L. B.; Cheng, Q. Antimicrob. Agents Chemother 2004 48 2116 2123. 10 . ; Barton, V. E.; Ward, S. A. Molecules 2010 15 1705 1721. 11 Ro, D. K.; Paradise, E. M.; Ouellet, M.; Fisher, K. J.; Newman, K. L.; Ndungu, J. M.; Ho, K. A.; Eachus, R. A.; Ham, T. S.; Kirby, J.; Chang, M. C. Y.; Withers, S. T.; Shiba, Y.; Sarpong, R.; Keasling, J. D. Nature 2006 440 940 943 12 Kar, S.; Kar, S. Nat. Rev. Drug Discovery 2010 9 511 512. 13 Dondorp, A. M. Nature Rev. Microbiol. 2010 8 272 280. 14 Hayton, K.; Su, X. Z Curr. Drug Targets Infect. Disord 2004 4 1 10. 15 Hyde, J. FEBS J. 2007 274 4688 4698. 16 Bacon, D. J.; Jambou, R.; Fandeur, T.; Le B ras, J.; Wongrichanalai, C.; Fukuda, M. M.; Ringwald, P.; Sibley, C. H.; Kyle, D. E. Malaria Journal 2007 6, 120.

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95 17 Gelb, M. H. Cur. Opin. Chem. Biol 2007 11 440 445 18 Fransisco Javier, G.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alverez, E.; Lavandera J. L.; Vanderwall, D. E.; Green, D. V. S.; Hasan, S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia Bustos, J. F. Nature 2010 465 305 310 19 Guiguemde, W. A.; Shelat A. A.; Bouck, D.; Duffy, S.; Crowther, G. J.; Davis, P. H.; Smithson, D. C.; Connelly, M.; Clark, J.; Zhu, F.; Jimenez Diaz, M. B.; Martinez, M. S.; Wilson, E. B.; Tripathi, A. K.; Gut, J.; Sharlow, E. R.; Bathurst, I.; Mazouni, F. E.; Fowble, J. W.; Forquer, I.; McGinley, P. L.; Castro, S.; Angulo Barturen, Ferrer, S.; Rosenthal, P. J.; DeRisi, J. L.; Sullivan, D. J.; Lazo, J. S.; Roos, D. S.; Riscoe, M. K.; Phillips, M. A.; Rathod, P. K.; Van Voorhis, W. C. ; Avery, V. M., ; Guy, R. K. Nature 2010 465 311 315. 20 Olliaro, P. L.; Yuthavong, Y. Pharmacol. Ther 1999 81 91 110. 21 Saxena, S.; Pant, N.; Jain, D. C.; Bhakuni, R. S. Curr. Science 2003 85 1314 1329. 22 Gademann, K.; Kobylinska, J. Chem. Rec 2009 9 187 198. 23 Jares Erijman, E. A.; Sakai, R.; Rinehart, K. L J. Org. Chem 1991 56 5712 5715. 24 Lazaro J. E. H. ; Nitcheu, J.; Mahmoudi, N.; Ibana, J. A.; Mangalindan, G. C. ; Black, G. P. ; Howard Jones, A. G.; Moore, C. G. ; Thomas, D. A. ; Mazier, D. ; Ireland, C. M. ; Concepcion, G. P. ; Murphy, P. J. ; Diquet, B. J. Antibiot. 2006 59 583 590. 25 Sakai, R.; Higa, T.; Jefford, C. W.; Bernardinelli, G. J. Am. Chem. S oc 1986 108, 6404 6405 26 Hu, J. F.; Hamann, M. T.; Hill, R. T.; Kelly, M. The Manzamine Alkaloids. In T he Alkaloids: Chemistry and Biology; Elsevier; 2003 Vol. 60 ; pp 207 285. 27 Ang, K. K. H.; Holems, M. J.; Higa, T.; Hamann, M. T.; Kara, U. A. K. Antimicrob. Agents Chemother. 2000 44 1645 1649. 28 Shilabin, A. G.; Kasanah N.; Tekwani, B. L.; Hamann, M. T. J. Nat. Prod. 2008 71 1218 221. 29 Hill, R.; Hamann, M.T.; Peraud, O.; Kasanah, N. Manzamine producing actinomycetes. US Patent. WO2003US2423820030801[WO2004013297] 2006 30 Demain, A.; Sanchez, S J. Antibiol 2009 62 5 16. 31 Brase, S.; Encinas, A.; Keck, J.; Nising, C. F. Chem. Rev 2010 109 3903 3990. 32 Barbaras, D.; Kaiser, M.; Brun, R.; Gademann, K. Bioorg. Med. Chem. Lett 2008 18 4413 4415. 33 Prudhomme, J.; McDaniel, E.; Ponts, N.; Bertani S.; Fenical, W.; Jensen, P.; Le Roch, K. PLoS ONE 2008 3 e2335 34 Schindler, F.; Zahner, H. Arch. Mikrobiol 1971 79 187 203.

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96 35 Bhattacharjee, A. K.; Hartell, M. G.; Nichols, D. A.; Hicks, R. P.; Stanton, B.; van Hamont, J.E.; Milhous, W.K. E ur. J. Med. Chem 2004 39 59 67. 36 Medicines for Malaria Ventures g rant: MMV08/0105 37 Franke Fayard, B.; Djokovic, D.; Dooren, M. W.; Ramesar, J. ; Waters, A. P.; Falade, M. O.; Kranendonk, M.; Martinelli, A.; Cravo, P.; Janse, C. J. Int. J. Parasitol 2008 38 1651 1662. 38 Artemisina annua extract was generously provided by Jim McChesney, Chromadex, Inc. 39 Schumann, J.; Hertweck, C. J. Am. Chem. Soc 2007 129 9564 9565 40 Aldridge. D. C., Armstrong, J. J.; Speake, R. N.; Turner, W. B. J. Chem. Soc: C 1967 1661 1676. 41 Hoppe, H. C.; Joiner, K. A.; Smythe, W. A. Cell Microbiol 2008 10 452 464. 42 Xu, H.; Fang, W. S.; Chen, X. G.; He, W. Y.; Cheng, K. D. J. Asian Nat. Prod. Res 2001 3 151 155. 43 Carrolo M.; Giordano, S.; Cabrita Santos, L.; Corso, S.; Vigario, A. M.; Silva, S.; Leiriao, P.; Carapau, D.; Armas Portela, R.; Comoglio, P. M.; Rodriguez, A.; Mota, M. M. Nat. Med 2003 9 1363 1369 44 Silvie, O.; Franetich, J. F.; Renia, L.; Mazier, D. TRENDS Mol. Med 2004 10 101. 45 Silvie, O.; Franetich, J. F.; Renia, L.; Mazier, D. TRENDS Mol. Med 2004 10 97 100. 46 Namikoshi, M.; Akano, K.; Meguro, S.; Kasuga, I.; Mine, Y.; Takahashi, T.; Kobayashi, H. J. Nat. Prod 2001 64 396 398 ( 1 H NMR shifts for roridin E are listed in the supporting information). 47 Isaka, M.; Punya, J.; Lertwerawat, Y.; Tanticharoen, M.; Thebtaranonth, Y. J. Nat. Prod 1999 62 329 331 48 We are indebted to numerous researchers (> 50!) who contributed to processing the seemingly never ending multitude of samples in both the labs of Bill Baker and Dennis Kyle.

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97 Chapter 5 Experimental 5.1 General Procedures Unless otherwise stated, all experiments were performed under inert atmosphere (nitrogen or argon) in oven or flame dried glassware equipped with a magneti c stir bar and a rubber septum. All solvents used were reagent grade. Anhydrous dichloromethane ( DCM ) was obtained by distillation from calcium hydride ( CaH ) Anhydrous tetrahydrofuran ( THF ) was obtained by distillation from sodium/benzophenone. Dry methanol (MeOH) was obtained by distillation from magnesium and iodine ( Mg 0 I 2 ) All other chemicals were purchased from Sigma Aldrich and were used as received. Low temperature baths of 78 C, 60 C, and 40 C were obtained with an immersion cooler bath using aceto ne, chloroform, or acetonitrile, respectively, with dry ice (CO 2 ). Thin layer chromatography (TLC) was carried out using Whatman normal phase Silica gel 60 Par t isil. TLC plates were visualized with 5% phosphomolybdic acid in ethanol ( EtOH ) and heating and / or UV (254 nm). Products were chromatographed on a Teledyne Isco Combiflash Companion medium pressure liquid chromatography ( MPLC ) instrument using normal phase silica gel cartridges purchased from Teledyne Isco. Melting points were reco rded on an Electrothermal Mel Temp 3.0 instrument. Specific rotations were measured on an Autopol IV automatic polarimeter using Na lamp corrected to 20C. IR spectra were recorded on a Nicolet Avatar 320 spectrometer with a Smart Miracle accessory. Low resolution mass spectrometry ( LRMS ) data w ere recorded on a n A gil ent LC/MSD VL electrospray ionization mass spectrometer. High resolution mass spectrometry ( HRMS ) data w ere obtained on an Agilent LC/MSD TOF electrospray ionization mass spectrometer. 1 H and 13 C NMR spectra were recorded on a Varian 400 MHz or 500 MHz instrument using residual protonated solvent as 1 H internal standard or 13 C absorption lines of solvents for 13 C internal standard. NMR data were obtained in CDCl 3 (Sigma Aldrich), CD 3 OD, or d 6 DMSO ( both from Cambridge Isotope Labs).

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98 5.2 Experimental supporting Chapter 2 5.2.1 Ozonolysis of palmerolide A forming hexane 1,2,3 t riol ( 2 51 ) and hexane 1,2,3,6 tetra ol ( 2 52 ) To a solution of palmerolide A ( 2. 1 8.5 mg, 0.0145 mmol, 1 eq) dissolved in dry methanol, ozone was bubbled through at 78 o C under stirring. A solution of methanol (1.5 mL) and sodium borohydride (10 mg, 0.264 mmol, 18.2 eq.) was added to the solution. The mixture was warmed to room temp erature ( rt ) and stirred for 25 min. After solvent removal under reduced pressure, the resulting residue was chromatographed by silica gel MPLC to yield fragment 2. 51 (eluting at 15% MeOH in chloroform ~2 mg, 0.0149 mmol, quantitative) and fragment 2. 5 2 (eluting at 20% MeOH in chloroform ~2.5 mg, 0.0165 mmol, quantitative) as colorless oils. 2. 51 :. [ ] D 20 9.0 ( c 0.100, MeOH); 1 H NMR (500 MHz, CD 3 OD) (multiplicity, integration) : 1.41 (m, 2H); 1.56 (m, 4H), 3.47 ( m 2H), 3.58 ( m 2H), 3.60 (m); 13 C NMR (125 MHz, CD 3 OD) 2 3.1 3 3.8 3 4.3 6 3.0 6 7.5 7 3.3 ; ESI MS ( m/z ) [M+ H ] = 135.1. 2. 52 : [ ] D 20 8 1 ( c 0. 2 00, MeOH) 1 H NMR ( 5 00 MHz, CD 3 OD ) (multiplicity, integration): 1. 62 ( m 4H), 3.53 (m, 4H), 3.64 (m, 2H) ; 13 C NMR (1 25 MHz, CD 3 OD ) : 30.3, 30.9, 63.2, 64.7, 72.7, 75.8 ; ESI MS ( m/z ) [M+ H ] = 151.1 5.2.2 Synthes i s hexane 1,2,6 triol ( 2. 53 ) R 2. 5 8 (19 mg, 0.109 mmol) w as dissolved in 50% acetic acid:H 2 O (3 mL). The solution stirred for 20 min at rt after which the aqueous acid was evaporated under a stream of air. The resulting residue was chromatographed by silica gel MPLC (eluting at 15% MeOH in chloroform) to yield R 2. 53 as a viscous colorless oil (13.6 mg, 0.101 mmol, 93%). A similar procedure applied to 40

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99 mg (0.230 mmol) of S 2. 5 8 yielded ( 27.5 mg, 0.205 mmol, 89%) of ( S ) hexane 1,2,6 triol ( S 2. 53 ). R 2. 53 : [ ] D 20 +11.1 ( c 0.100, MeOH) ; S 2. 53 : [ ] D 20 11.6 ( c 0.100, MeOH); 1 H NMR (500 MHz, CD 3 OD) 1.41 (m, 2H); 1.56 (m, 4H), 3.47 ( m 2H), 3.58 ( m 2H), 3.60 (m, 1H); 13 C NMR (125 MHz, CD 3 OD) 2 3.1 3 3 8 3 4 3 6 3.0 6 7.5 7 3.3 ; ESI MS ( m/z ) [M+ H ] = 135.1. 5.2.3 Synthesis of ( R ) (2,2 dimethyl 1,3 dioxolan 4 yl)methanol ( 2. 54 ) A stream of ozone was bubbled through a stirring solution of 2. 55 (24 mg, 0.112 mmol) in dry methanol (3 mL) at 78 o C. A solution of methanol (1.5 mL) and sodium borohydride (20 mg, 0.518 mmol) was added to the solution. The mixture was warmed to rt and stirred for 25 min. After solvent removal under reduced pressure, the resulting residue was chromatographed by sili ca gel MPLC to yield 2. 54 (eluting at 38% ethylacetate ( EtOAc ) :Hexane, 6.5 mg, 0.492 mmol, 44% yield, 58% ee). (R) (2,2 dimethyl 1,3 dioxolan 4 yl)methanol purchased from Sigma Aldrich: [ ] D 20 13.7, ( c =0.1, MeOH); 2. 54 : [ ] D 20 7.0, ( c =0.1, MeOH); 1 H NMR ( 4 00 MHz, CD Cl 3 ) 1. 39 (s 3 H) 1.46 (s, 3H), 3.60 ( m ), 3.75 ( m ), 3.81 ( m ), 4.05 ( m ), 4.25 (m) ; 13 C NMR (1 00 MHz, CD Cl 3 ) 25.3 26.7, 63.0, 65.7, 76.1, 109.4; ESI MS ( m/z ) [M+ H ] = 13 3 .1. 5.2.4 Synthesis of ( R ) 4 (3 (1,3 dioxolan 2 yl)prop 1 enyl) 2,2 dimethyl 1,3 dioxolane ( 2. 55 ) To 2 0 mL dry DCM at 60 o C was added oxalyl chloride ( 540 mg, 4.16 mmol, 0. 36 mL ). After stirring for 5 min, DMSO ( 592 mg, 7.57 mmol, 0. 54 mL ) was added. After stirring 2 min, R 2.56 (500 mg, 3. 78 mmol ) dissolved in 3 mL dry DCM was added over a 5 min period. After stirring for

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100 an additional 10 min at 60 o C, triethylamine ( TEA 1.91 g, 18.9 mmol, 2. 7 mL) was added. The mixture was then warmed to rt and partitioned between EtOAc and water. The organic layer was collected. The aqueous layer was washed 2X with aliquots of EtOAc. The organic layers were combined, dried over anhydrous magnesium sulfae (anh yd MgSO 4 ) and concentrated to afford ( S ) 2,2 dimethyl 1,3 dioxolane 4 carbaldehyde ( 285 mg, 2.19 mmol, 58 %) To a solution of 2 (1,3 Dioxolan 2 yl)ethyltriphenylphosphonium bromide ( 26 mg, 0.15 mmol, dried with heat under vacuum) in dry THF (7 mL) at 78 o C was added n BuLi dropwise until the solution turned a dark yellow (1. 4 M, 0.22 mL, 0.30 mmol) After stirring for 15 min., ( S ) 2,2 dimethyl 1,3 dioxolane 4 carbaldehyde (dissolved in 1.5 mL dry THF) was added to the stirring solution. The solution stirred at 78 o C for 10 additional minut es then was allowed to warm to rt The mixture was partitioned between diethyl ether ( Et 2 O ) and water. The aqueous layer was washed 2X with aliquots of Et 2 O. The combined organic layers were dried over anhyd. MgSO 4 concentrated and chromatographed on silica gel MPLC affording 2. 55 as a mixture of E / Z isomers as a colerless oil (eluting at 15% EtOAc:hexanes 24 mg, 0. 11 mmol, 75 % ). 5.2.5 Synthes e s of 2 (2,2 dimethyl 1,3 dioxolan 4 yl)ethanol ( 2. 56 ) To a stirring solution of R 2. 57 (47 mg, 0.219 mmol) in dry MeOH (5 mL) was added ozone at 78 o C. Sodiu m borohydride (40 mg, 1.06 mmol ), dissolved in 1 mL MeOH was added to the solution. The mixture was warmed to rt and stirred for 25 min. After solvent removal under reduced pressure, the resulting residue was chromatographed by silica gel MPLC (eluting at 45% EtOAc in hexanes) to yield R 2. 56 as a colorless oil (17.5 mg, 0.120 mmol, 55%). A similar procedure applied to 55 mg of S 2. 57 (0.258 mmo l) yielded 22.6 mg (0.155 mmol, 60%) of S 2. 56 [ ] D 20 R 2. 5 6 : 2.4 o 89% ee; S 2. 5 6 : +2.4 o 97% ee ( c 0.100, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 )

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101 (multiplicity, J (Hz) integration): 1.3 6 (s, 3H), 1. 42 (s, 3H), 1. 82 (dt, 5.8, 5. 4 2H), 2. 31 (brs), 3.5 9 (dd 7. 8.0 4, 7. 4 ), 3. 80 (t, 5.4, 2H), 4.0 9 (dd 8.1, 6.0 ), 4.2 7 ( m ) ; 13 C NMR (1 00 MHz, CD Cl 3 ) 25.7, 26.9 35.6, 60.6, 69.4, 75.1, 109.1; ESI MS ( m/z ) [M+ H ] = 1 4 7 .1. 5.2.6 Synthesis of ( E ) ethyl 4 (2,2 dimethyl 1,3 dioxolan 4 yl)but 2 enoate ( 2 57 ) To 10 0 mL dry DCM at 60 o C was added oxalyl chloride ( 866 mg, 6. 82 mmol, 0. 6 mL ). After stirring for 5 min, DMSO (6 68 mg, 8.55 mmol, 0. 6 mL ) was added. After stirring 2 min, R 2.5 6 (5 00 mg, 3. 42 mmol ) dissolved in 3 mL dry DCM was added over a 5 min period. After stirring for an additional 10 min at 60 o C, TEA ( 2.08 g, 20.5 mmol, 2. 9 mL) was added. The mixture was then warmed to rt and partitioned between EtOAc and water. The organic layer was collected. The aqueous layer was washed 2X with aliquots of EtOAc. The organic layers were combined, dried over anhydrous MgSO 4 and concentrated to afford the aldehyde ( R ) 2 (2,2 dimethyl 1,3 dioxolan 4 yl)acetaldehyde (4 58 mg, 3.17 mmol, 93 %) as a colorless oil. A similar procedure was applied to S 2.56 ( 500 mg, 3.42 mmol) resulting in ( S ) 2 (2,2 dimethyl 1,3 dioxolan 4 yl)acetaldehyde ( 480 mg, 3.38 mmol 99 %). 5.2.6.1 Synthesis of R 2.57 To a mixture of (ethoxycarbonylmethyl)triphenylphosphonium bro mide (4.855 g, 11.31 mmol ) in dry DCM (100 mL) was added triethylamine (TEA, 1.71 6 g, 2.354 mL, 16.96 mmol ) at rt After mixture stirred under nitrogen for 10 min, ( R ) 2 (2, 2 dimethyl 1,3 dioxolan 4 yl)acetaldehyde (815 mg, 5.65 mmol ) dissolved in 2.5 mL dry DCM was added. The mixture stirred for 17 h at rt then was partitioned between 250 mL diethyl ether: 250 mL H 2 O. The organic layer was collected. The aqueous layer was extracted with 250 mL diethyl ether. The organic layers were combined and dried over anhydrous MgSO 4 After solvent removal under reduced pressure, the resulting

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102 residue was chromatographed by silica gel MPLC (eluting at 20 25% EtOAc in hexanes) to yiel d R 2.5 7 as a colorless oil (544 mg, 2.54 mmol, 45%). 5.2.6.2 Synthesis of S 2.57 To a slurry of sodium hydride ( NaH 196 mg 60% in mineral oil, 4.91 mmol) in dry THF (12 mL) at 0 o C and inert atmosphere was added triethyl phosphonoacetate (1.1 g, 4.91 mmol, 0.98 mL). After stirring 10 min., ( S ) 2 (2,2 dimethyl 1,3 dioxolan 4 yl)acetaldehyde (544 mg, 3.77 mmol) dissolved in 2.5 mL dry THF was added. The mixture stirred an additional 10 min. and was partitioned between EtOAc:H 2 O. The aqueous layer was washed 2X with aliquots of EtOAc. The combined organic layers were dried over anhyd. MgSO4, concentrated and purified via MPLC (silica, elution at 25% EtOAc:hexane) to afford 5 7 3 mg of S 2.5 7 (2. 6 8 mmol, 71 %). 5.2. 6 .3 Physical data common to R 2.57 and S 2.57 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration) 1.29 (t, 7.1, 3H), 1.36 (s, 3H), 2.48 (m, 2H), 3.60 (dd 8.0, 6.5 ), 4.06 (dd 8.0, 6.2 ), 4.19 (q, 7.1, 2H), 4.20 ( m ), 5.91 (d t, 15.6, 1.4 ), 6.93 (dt 15.6, 7.1 ); 13 C NMR (1 00 MHz, CD Cl 3 ) 14.2, 25.5, 26.8, 36.4, 60.3, 68.8, 74.2, 109.3, 123.9, 143.7, 166.2 ; ESI MS ( m/z ) [M+ H ] = 215 .1. 5.2.7 Synthesis of 4 (2,2 dimethyl 1,3 dioxolan 4 yl)butan 1 ol ( 2. 58 ) To a mixture of R 2. 5 7 (66 mg, 0.308 mmol) in anhydrous THF (7 mL) was added LiAlH 4 (35 mg, 0.924 mmol) under an atmosphere of nitrogen. The mixture stirred at rt for 2 h. Potassium hydroxide solution (0.1 mL, 1 M) was carefully added to quench the reaction. After stirring for 10 min the mixture was partitioned between DCM:H 2 O. The organic layer was collected. The aqueous layer was extracted 2X with DCM. The organic layers were combined and dried over

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103 anhydrous MgSO 4 After solvent removal under reduced pressure, the resulting residue was chromatographed by silica gel MPLC (eluting at 38 42% EtOAc in hexanes) to yield R 2. 58 as a colorless oil (24.1 mg, 0.13 8 mmol, 45%). A similar procedure applied to 465 mg (2.17 mmol) of S 2. 57 yielded 140 mg (0.803 mmol, 37%) of S 2. 58 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, integration): 1.3 6 (s, 3H), 1.4 2 (s, 3H), 1.49 (m, 2H), 1.53 (m, 2H), 1.62 (m, 2H), 3. 52 (t 7.3 ), 3. 67 ( t 6.4, 2H), 4.06 ( m ), 4.12 (m ); 13 C NMR (1 00 MHz, CD Cl 3 ) 22.0, 25.7, 26.9, 32.6 33.2, 62.7, 69.4, 76.0, 108.7; ESI MS ( m/z ) [M+ H ] = 175.1 5.2.8 Synthes i s of hexane 1,2,3,6 tetraol ( 2. 59 ) 5.2.8.1 Synthesis of (2 R ,3 R ) hexane 1,2,3,6 tetraol ( R R 2. 59 ) To a flask containing activated 10% Pd/C (10 mg) and a stir bar was added 2. 61 (40 mg, 0.147 mmol) dissolved in 5 mL EtOH. A balloon containing H 2 gas was affixed to the flask. The mixture stirred for 4 h at rt then was filtered through Ce Lite. The filtrate was concentrated to afford the intermediate saturate d acetal (38 mg, 0.138 mmol, 94 %). The intermediate saturated acetal (37 mg, 0.135 mmol) was stirred in a solution of 50% acetic acid in water for 2h a t 60 o C. The solvent was then evaporated under a stream of air to yield the diolal (24 mg, 0.126 mmol, 94% ). To a solution of the diolal (24 mg, 0.126 mmol, 1.0 eq.) in 4 mL MeOH was added sodium borohydride (7.6 mg, 0.202 mmol, 1.6 eq.). The solution st irred for 2h then was concentrated onto silica and chromatographed on MPLC to afford ( R,R ) 2. 59 as a colorless oil (11 mg, 0.073 mmol, 58% yield, eluting at 20% MeOH in cholorform). [ ] D 20 + 9.9 ( c 0.100, MeOH) 5.2.8. 2 Synthesis of (2 S ,3 S ) hexane 1,2,3,6 tetraol ( S S 2. 59 ) To a mixture of lithium aluminum hydride (LAH, 8 mg, 0.187 mmol) in dry THF (10 mL) at rt was added 2. 63 (58 mg, 0.250 mmo l) dissolved in 1 mL dry THF. The mixture stirred for 2h. TLC showed some starting material remained so additional LAH (6 mg, 0.158 mmol) was added. After

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104 20 min., no more starting material remained. KOH solution (1 M, 0.5 mL) was cautiously added. The mixture was diluted with EtOAc, dried over anhydrous MgSO 4 and concentrated on to silica. After MPLC purification the i ntermediate diol (eluting at 100% EtOAc, 40 mg, 0.210 mmol, 84% yield) was obtained. The intermediate diol was added to a 50% acetic aci d in water solution. The mixture stirred for 3h. The solvent was removed under a stream of air to afford ( S,S ) 2. 59 (11 mg, 0.073 mmol, 41% ) as a viscous colorless oil. [ ] D 20 10.0 ( c 0.100, MeOH) 5.2.8.3 Physical data common to R R 2. 59 and S S 2. 59 1 H NMR (400 MHz, CD 3 OD ) (multiplicity, J (Hz), integration): 1. 62 ( m 4 H), 3.5 3 (m, 4 H), 3.64 (dd, 11.1, 4.9, 2H) ; 13 C NMR (100 MHz, CD 3 OD ) : 30.3, 30.9, 63.2, 64.7, 72.7, 75.8 ; ESI MS ( m/z ) [M+H] = 151.1. 5.2.9 Synthesis of ((4 R ,5 R ) 5 (2 (1,3 dioxolan 2 yl)vinyl) 2,2 dimethyl 1,3 dioxolan 4 yl)methyl acetate ( 2. 61 ) 2,3 O Isopropylidene D threitol ( R,R 2. 60, 255 mg, 1.54 mmol) in 10 mL dry DCM at rt under inert atmosphere was added 4 dimethylaminopyridine (DMAP, 18 mg, 0.15 mmol), TEA (155mg, 0.214 mL, 1.54 mmol), and acetic anhydride (158 mg, 0.147 mL, 1.54 mmol). The solution stirred for 30 min then was concentrated and subjected to chromatographic purification on silica MPLC. The monoacetat e (176 mg, 0.862 mmol, 57% ) eluted at 25 30% EtOAc:Hex. To a stirring slurry of Dess Martin periodinane (287 mg, 0.676 mmol) in 10 mL dry DCM was added t he monoacetate (92 mg, 0.451 mmol) dissolved in 2 mL dry DCM followed by pyridine (180 mg, 2.28 mmol). After 2h at rt the mixture was quenched by addition of saturated sodium bicarbonate (sat. NaHCO 3 5 mL) and 1 M sodium thiosulfate ( Na 2 S 2 O 3 ) solution (5 mL).

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105 This mixture was allowed to stir until both layers were clear. The mixture was partitioned between EtOAc and water. The organic layer was collected. The aqueous layer was extracted X2 with aliquots of EtOAc. The combined organic layers were dried over anhydrous MgSO 4 and concentrated to yield cr ude aldehyde (84 mg, 0.415 mmol, 92% yield). A slurry of (1,3 Dioxolan 2 ylmethyl)triphenylphosphonium bromide (308 mg, 0.714 mmol,) and potassium tert butoxide (80 mg, 0.714 mmol) in dry THF stirred at rt for 25 min under inert atmosphere. To the result ing dark yellow mixture was added the intermediate aldehyde (84 mg, 0.446 mmol) dissolved in 2 mL dry THF. After stirring for 2.5 h, the dark orange mixture was concentrated then partitioned between Et 2 O and H 2 O. The organic layer was collected. The aqu eous layer was extracted 2X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 concentrated, and chromatographed on silica MPLC (eluting at 30% EtOAc:Hex) to yield a mixture of E / Z 2. 6 1 (51 mg, 0.187 mmol, 42% ) as a colorless oil. 5.2.10 Synthesis of ethyl 3 ((4 S ,5 S ) 5 (benzyloxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)acrylate ( 2. 62 ) A mixture of (+) 2,3 O isopropylidene L threitol [( S S ) 2. 60 500 mg, 3.08 mmol], benzyl bromide (580 mg, 3.39 mmol, 1.1 equiv), and silver oxide (Ag2O, 1.07 g, 4.62 mmol) in dry toluene was stirred at rt for 8 h. The mixture was filtered through a plug of silica and concentrated. The resulting residue was chroma tographed on silica (eluting at 35 42% EtOAc in hexanes) to yiel d ((4S,5S) 5 (benzyloxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)methanol (598 mg, 2.38 mmol, 77%) as a colorless oil. To a stirring slurry of Dess Martin periodinane (630 mg, 1.49 mmol) in 100 mL dry DCM was added ((4S,5S) 5 (benzyloxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)methanol (342 mg, 1.35

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106 mmol) dissolved in 5 mL dry DCM followed by pyridine (534 mg, 0.54 mL, 6.75 mmol). After 30 min at rt, the mixture was quenched by addition of satd NaHCO 3 (50 mL) and 1 M Na 2 S 2 O 3 solution (50 mL). This mixture was allowed to stir until both layers were clear and was then partitioned between EtOAc and water. The aqueous layer wa s extracted 2 X with aliquots of EtOAc. The combined organic layers were dried over anhydrous MgSO 4 and concentrated to yield the crude aldehyde (360 mg). A slurry of ( e thoxycarbonylmethyl)triphenyl phosphonium bromide (683 mg, 1.59 mmol, 1.1 equiv) and N aH (38 mg, 1.59 mmol, 1.1 equiv) in 50 mL dry THF at rt stirred for 4 h. The crude aldehyde (360 mg, ~ 1.44 mmol) dissolved in 5 mL dry THF was then added. The mixture stirred for 6 h and was then partitioned with Et 2 O/H 2 O. The organic layer was collecte d. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 and concentrated under reduced pressure. The concentrate was chromatographed by silica gel MPLC (eluting at 15% EtOAc : hexane) to afford a mixture of E / Z isomers of conjugate ester 2. 62 (298 mg, 0.93 mmol, 69%, two steps) as a colorless oil. The E isomer could be formed exclusively by substituting ( e thoxy carbonylmethyl)triphenyl phosphonium bromide with triethylphosphonoacetate (60 %, two steps). E 2. 62 : [ ] D 20 23.4 ( c 1.0, CHCl 3 ); IR (neat) (cm 1 ): 2988, 1722, 1654, 1090; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.29 (t, 6.9, 3H), 1.44 (s, 3H), 1.45 (s, 3H), 3.63 (d, 4.7, 2H), 3.96 (dt, 8.4, 4.7, 1H), 4.20 (q, 6.9, 2H), 4.43 (ddd, 8.4, 5.6, 1.7, 1H), 4.60 (s, 2H), 6.09 (dd, 15.7, 1.7, 1H), 6.89 (dd, 15.7, 5.6, 1H), 7.34 (m, 5H). 13 C NMR (100 M Hz, CDCl 3 ) : 14.3, 26.8, 27.1, 60.7, 69.5, 73.8, 77.6, 79.7, 110.3, 122.7, 127.8 (2C), 127.9, 128.6 (2C), 137.9, 144.2, 166.1. ESI HRMS [M + Na] + calcd for [C 18 H 24 O 5 Na] + : 343.1516, found 343.1510. 5.2.11 ethyl 3 ((4 S ,5 S ) 5 (hydroxymethyl) 2,2 dimethyl 1,3 dioxolan 4 yl)propanoate ( 2. 63 )

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107 To activated 10% Pd/C (50 mg) was added 2. 62 (278 mg, 0.87 mmol) dissolved in 10 mL EtOH. A balloon containing H 2 gas was affixed to the flask. The mixture stirred for 12 h at rt, was diluted with EtOAc, and filtered through Celite. The filtrate was concentrated to afford 2. 63 as a colorless oil (194 mg, 0.84 mmol, 96%). 1 H NMR (400 MHz, CDCl3) (multiplicity, J (Hz), integration): 1.23 (t, 7.2, 3H), 1.37 (s, 3 H), 1.38 (s, 3H), 1.60 (br s), 1.83, (m), 1.94 (m), 2.46 (m, 2H), 3.61 (m), 3.75 (m), 3.78 (m), 3.89 (dt, 7.7, 3.6 ), 4.12 (q, 7.2, 2H); 13 C NMR (100 MHz, CDCl 3 ) : 14.4, 27.2, 27.5, 28.2, 30.9, 60.6, 62.0, 76.3, 81.2, 109.2, 173.4; ESI MS ( m/z ) [M+H] = 233.1. 5.2.12 ( S ) 5 (2 (tert butyldimethylsilyloxy)hept 6 enylsulfonyl) 1 phenyl 1H tetrazole ( 2. 65 ) To 1 phenyl 1H tetrazole 5 thiol (288 mg, 1.62 mmol) and potassium carbonate (K 2 CO 3 372 mg, 2.60 mmol) was added 2. 68 (215 mg, 0.54 mmol) dissolved in 5 mL dry acetone. The stirring mixture refluxed for 20 h and was cooled to rt, and partitioned between Et 2 O/H 2 O. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic extracts were dried over anhyd rous MgSO 4 concentrated under reduced pressure, and subjected to silica gel MPLC to afford the thioether intermediate as white needles (mp 35 36 o C, 174 mg, 0.430 mmol, 80%). To the thioether intermediate (103 mg, 0.254 mmol) in 2 mL EtOAc was added H 2 O 2 (86 L 30% solution, 26 mg H 2 O 2 0.762 mmol), sodium tungstate (Na 2 WO 4 2H 2 O, 170 L of a 5 mg/mL solution in EtOAc, 0.85 mg, 0.00254 mmol), phenylphosphonic acid (80 L of a 5 mg/mL solution in EtOAc, 0.4 mg, 0.00254 mmol), and methyltrioctylammonium hydrogensulfate (Oct 3 MeNHSO 4 240 L of a 5 mg/mL solution, 1.2 mg, 0.00254 mmol). After 40 h the reaction was not yet complete via TLC so another aliquot of sodium tungstate ( 0.00254 mmol ), phenylphosphonic acid ( 0.00254 mmol ), Oct 3 MeNHSO 4 ( 0.00254 mmol ), and ( 0.762 mmol ) was added. This mixture stirred another 60 h and was partitioned between EtOAc / H 2 O. The organic layer was dried over

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108 anhydrous MgSO 4 concentrated and chromatographed via silica gel MPLC afford a mixture of diastereomers of the partially oxidized sulfoxide (20 mg, 0.05 mmol, 20%) as well as desired sulfone 2. 65 as a white solid (mp 62 64 o C, 48 mg, 0.110 mmol, 43%). [ ] D 20 + 15.6 ( c 0.4 CHCl 3 ); IR (neat) (cm 1 ): 3073, 2950, 2934, 2858, 1345, 1254, 1157; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 0.03 (s, 3H), 0.06 (s, 3H), 0.84 (s, 9H), 1.48 (m, 2H), 1.67 (m, 2H), 2.10 (dt, 7.0, 7.0, 2H), 3.86 (dd, 14.9, 4.6, 1H), 4.00 (dd, 14.9, 6.6z, 1H), 4.48 (m, 1H), 5.00 (m, 2H), 5.77 (ddt, 16.9, 6.9, 3.9, 1H), 7.64 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) : 4.7, 4.1, 18.1, 23.6, 25.8 (3C), 33.6, 37.1, 62.1, 66.6, 115.4, 125.3 (2C), 129.9 (2C), 131.6, 133.3, 138.1, 154.4; ESI HRMS [M + H] + calcd for [C 20 H 33 N 4 O 3 SSi] + : 437.2037, found 437.2024. 5.2.13 ethyl 3 ((4 S ,5 R ) 5 formyl 2,2 dimethyl 1,3 dioxolan 4 yl)propanoate ( 2. 66 ) To a stirring solution of Dess Martin periodinane (424 mg, 1.02 mmol) in 10 mL dry DCM at rt was added 2. 63 (198 mg, 0.85 mmol) then pyridine (336 mg, 0.35 mL). T he solution stirred for 1 h and was then quenched with 5 mL 1 M Na 2 S 2 O 3 and 5 mL sat. NaHCO 3 solution. The mixture stirred until both layers were no longer cloudy. The organic layer was concentrated then repartitioned in EtOAc/H 2 O. The organic layer was collected, dried over anhydrous MgSO 4 and concentrated to yield 2. 66 as a colorless oil (150 mg, 0.65 mmol, 76%). [ ] D 20 7.4 ( c 1.0, CHCl 3 ); IR (neat) (cm 1 ): 2985, 2938, 1073 1731; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.19 (t, 7.3, 3H), 1.34 (s, 6H), 1.95 (m, 2H), 2.42 (m, 2H), 3.91 (dt), 4.04 (m), 4.07 (q, 7.3, 2H), 9.67 (s); 13 C NMR (100 MHz, CDCl 3 ) : 14.3, 26.4, 27.2, 28.7, 30.4, 60.7, 76.1, 84.7, 110.1, 172.9, 201.1; ESI HRMS [M + H] + calcd for [ C 11 H 19 O 5 ] + : 231.1227, found 231.1221.

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109 5.2.14 ( S ) hept 6 ene 1,2 diol ( 2. 67 ) To 10 mL dry DCM at 60 o C was added oxalyl chloride (787 mg, 6.20 mmol, 0.525 mL). After stirring for 5 min, DMSO (605 mg, 7.75 mmol, 0.55 mL) was added. After stirring 2 min, S 2. 58 (540 mg, 3.10 mmol) dissolved in 3 mL dry DCM was added over a 5 min period. After stirring for an additional 10 min at 60 o C, TEA (1.88 g, 18.6 mmol, 2.6 mL) was added. The mixture was then warmed to rt and partitioned between EtOAc /H 2 O The organic layer was collected. The aqueous layer was washed 2 X with aliquots of EtOAc. The organic layers were combined, dried over anhydrous MgSO 4 and concentrated to afford the aldehyde (S) 4 (2,2 dimethyl 1,3 dioxola n 4 yl)butanal (488 mg, 2.83mmol, 91%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.28 (s, 3H),1.34 (s, 3H),1.58 (m, 4H), 2.44 (dt, 7.3, 1.6, 2H), 3.45 (t, 7.1), 3.97 (t, 7.1), 4.0 (m), 9.71 (t, 1.6); 13 C NMR (100 MHz, CDCl 3 ) : 18.4, 25.9, 27.1, 33.1, 43.8, 69.5, 76.2, 109.1, 202.4. To a stirring slurry of Ph 3 PCH 3 Br (1.95 g, 5.46 mmol) in dry THF (50 mL) at 0 o C was added potassium bis(trimethylsilyl)amide ( KHMDS 0.5 M in toluene, 10.92 mL, 5.46 mmol) over a 10 min period. The mixture was warmed to rt and stirred for 30 min. The mixture then was cooled to 0 o C and ( S ) 4 (2,2 dimethyl 1,3 dioxolan 4 yl)butanal (470 mg, 2.72 mmol, 1.0 equiv) dissolved in 10 mL dry THF was added over a 10 min period. The mixture stirred at 0 o C for 1.5 h. Methanol (0.6 mL) was added to quench the reaction. The mixture was diluted with Et 2 O and filtered through Celite. The filtrate was concentrated and chromatographed by silica gel MPLC (eluting at 10 12% EtOAc : hexanes) to afford ( S ) 2,2 dimethyl 4 (pent 4 enyl) 1,3 dioxolane as a colorless oil (260 mg, 1.53 mmol, 56%). 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.29 (s, 3H), 1.34 (s, 3H), 1.45 (m, 2H), 1.55 (m, 2H), 2.02 (dt, 6.8, 1.4, 2H), 3.44 (t,

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110 7.4), 3.96 (dt, 7.4, 5.8), 3.99 (m), 4.89 (m), 4.94 (m), 5.73 (ddt, 17.2, 6.8, 3.5); 13 C NMR (100 MHz, CDCl 3 ) : 25.1, 25.8, 27.1, 33.1, 33.7, 69.6, 76.2, 108.9, 115.0, 138.8. ( S ) 2,2 dimethyl 4 (pent 4 enyl) 1,3 dioxolane (233 mg, 1.37 mmol) w as stirred in 5 mL 50% AcOH:H 2 O in a flask open to air for 2 h at rt. The solvent was then removed under a stream of air to yield diol 2. 67 as a colorless oil (164 mg, 1.26 mmol, 92%). [ ] D 20 6.5 ( c 1.0, CHCl 3 ); IR (neat) (cm 1 ): 3365, 3079, 2935, 1070, 1039; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.44 (m, 4H), 1.68 (br s), 2.03 (m, 2H), 2.07 (br s), 3.37 (dd, 10.8, 7.7), 3.59 (dd, 10.8, 3.0), 3.65 (m), 4.93 (m, 2H), 5.74 (ddt, 17.0, 6.6, 3.5); 13 C NMR (1 00 MHz, CDCl 3 ) : 24.9, 32.7, 33.7, 67.0, 72.3, 115.0, 138.7; ESI HRMS [M + NH 4 ] + calcd for [C 7 H 18 O 2 N] + : 148.1332, found 148.1328. 5.2.15 ( S ) 2 (tert butyldimethylsilyloxy)hept 6 enyl 4 methylbenzenesulfonate ( 2. 68 ) To a solution of tosyl chloride (265 mg, 1.39 mmol, 1.1 eq uiv) and DMAP (15 mg, 0.13 mmol ) in 9 mL dry DCM and stirring at 0 o C was added 2. 67 (164 mg, 1.26 mmol) dissolved in 1 mL dry DCM. The solution stirred for 5 min, then TEA (141 mg, 0.2 mL, 1.39 mmol) w as added. The solution stirred at 0 o C for 4 h and then rt for 4 h. The solution was poured into a flask containing 20 mL ice, 20 mL H 2 O and 10 mL 2 N HCl. The resulting mixture was extracted 2 X with 50 mL aliquots of DCM. The combined organic layers w ere dried over anhydrous MgSO 4 and concentrated under reduced pressure. The crude concentrate was chromatographed by silica gel MPLC to afford the monotosylated alcohol as a colorless oil (315 mg, 1.11 mmol, 88%). To the monotosylated alcohol (150 mg, 0. 53 mmol) in 5 mL dry DCM under stirring at 0 o C was added 2,6 lutidine (169 mg, 0.18 mL, 1.58 mmol) then tert butyldimethylsilyl trifluoromethanesulfonate (TBS OTf, 348 mg, 0.30 mL, 1.32 mmol). The mixture stirred for 2 h and was partitioned between

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111 DCM/H 2 O. The organic layer was collected. The aqueous layer was extracted 2 X with aliquots of DCM. The combined organic extracts were dried over anhydrous MgSO 4 concentrated under reduced pressure, and subjected to silica gel MPLC to yield 2. 68 as a colorless oil (215 mg, 0.53 mmol, quantitative). [ ] D 20 6.0 ( c 0.4, CHCl 3 ); IR (neat) (cm 1 ): 3079, 2952, 2857, 1170; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 0.01 (s, 3H), 0.02 (s, 3H), 0.83 (s, 9H), 1.39 (m, 4H), 2.00 (dt, 6.6, 6.6, 2H), 2.45 (s, 3H), 3.85 (m, 2H), 3.85 (m), 4.96 (m), 5.74 (ddt, 17.0, 6.6, 3.2) 7.34 (d, 8.0, 2H), 7.79 (d, 8.0, 2H); 13 C NMR (100 MHz, CDCl 3 ) : 4.8, 4.6, 21.6, 24.0, 25.7 (3C), 26.9, 33.4, 33.6, 69.8, 73.1, 114.7, 127.9 (2C), 129.8 (2C), 133.0, 138.3, 144.7; ESI HRMS [M + H] + calcd for [C 20 H 35 O 4 SSi] + : 399.2020, found 399.2032. 5.2.16 ( S ) 7 (benzyloxy)hept 1 en 3 yl acetate ( 2. 69 ) To a stirring solution of 2. 71 (66 mg, 0.248 mmol) in 5mL dry DCM at 0 o C was added Dess Martin periodinane (158 mg, 0.372 mmol) in one portion. The mixture was warmed to rt and stirred for 45 min. Saturated NaHCO 3 (5 mL) and 1.0 M Na 2 S 2 O 3 (5 mL) was added. The mixture stirred for 5 min then was partitioned between Et 2 O/H 2 O. The organic layer was collected, dried over anhydrous MgSO 4 and concentrated under reduced pressure to yield the aldehyde inte rmediate as a colorless oil (65 mg, 0.248 mmol, quantitative ). To a mixture of ethyltriphenylphosphonium bromide (89 mg, 0.25 mmol) in 5 mL dry THF at 0 o C was added KHMDS (0.5 M in toluene, 0.5 mL, 0.25 mmol) dropwise. After 10 min stirring at 0 o C, the aldehyde intermediate (60 mg, 0.227 mmol) dissolved in 2 mL dry THF was added dropwise. After 5 min the reaction was partitioned between Et 2 O/H 2 O. The organic layer was collected. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 and concentrated. The crude residue was chromatographed by silica gel MPLC (eluting at 10 13% EtOAc : hexanes) to afford 2. 69 as a colorless oil (37 mg, 0.141 mmol,

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112 62%). [ ] D 20 2.9 ( c 0.6, CHCl 3 ); IR (neat) (cm 1 ): 3035, 2945, 2857, 1745, 1244, 1106; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.40 (m, 2H), 1.61 (m, 4H), 2.04 (s, 3H), 3.44 (t, 6.6, 2H), 4.48 (s, 2H), 5.14 (dd, 10.6, 1.0), 5.21 (m, 2H), 5.75 (ddd, 17.3, 10.5, 6.4), 7.27 (m, 1H), 7.31 (m, 4H); 13 C NMR (100 MHz, CDCl 3 ) : 21.4, 22.0, 29.7, 34.2, 70.3, 73.1, 74.9, 116.8, 127.7, 127.8 (2C), 128.5 (2C), 136.7, 133.8, 170.7; ESI HRMS [M + Na] + calcd for [C 16 H 22 O 3 Na] + : 285.1461, found 285.1463. 5.2.17 ( S ) 6 (benzyloxy)hexane 1,2 diol ( 2. 70 ) To a stirring solution of S 2. 58 (215 mg, 1.25 mmol) in 5 mL dry THF at rt was added NaH (33 mg, 1.37 mmol) in one portion. The mixture bubbled vigorously for 5 min. After gas evolution had subsided, benzyl bromide (257 mg, 1.5 mmol) was added. The mixtu re stirred for 20 h and was partitioned between Et 2 O/H 2 O. The organic layer was collected. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 and concentrated. The crude residue was chromatographed by silica gel MPLC (eluting at 12 14% EtOAc/hexane) to afford the benzylated intermediate as a colorless oil (142 mg, 0.54 mmol, 43%). The benzylated intermediate was stirred in 2 mL 50% AcOH:H 2 O in a flask opened to air for 3 h. The mixture was then concentrated to afford 2. 70 as a colorless oil (125 mg, 0.47 mmol, 94%). [ ] D 20 3.3 ( c 1.0, CHCl 3 ); IR (neat) (cm 1 ): 3382, 2938, 2867, 1456, 1096, 1029; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.44 (m, 2H), 1.59 (m, 4H), 1.91 (br s), 2.14 (br s), 3.41 (dd,11.1, 7.6), 3.47 (t, 6.4, 2H), 3.62 (dd, 11.1, 3.0), 3.69 (m), 4.49 (s, 2H), 7.27 (m), 7.32 (m, 4H); 13 C NMR (100 MHz, CDCl 3 ) : 22.4, 29.7, 33.0, 67.0, 70.3, 72.4, 73.1, 1 27.8, 127.9 (2C), 128.6 (2C), 138.8; ESI HRMS [M + H] + calcd for [C 13 H 21 O 3 ] + : 225.1485, found 225.1481.

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113 5.2.18 ( S ) 6 (benzyloxy) 1 hydroxyhexan 2 yl acetate ( 2. 71 ) To a stirring solution of 2. 70 (100 mg, 0.45 mmol) in 3 mL dry THF at rt was added TEA (54 mg, 71 mL, 0.54 mmol) followed by chlorotrimethylsilane (53 mg, 63 mL, 0.49 mmol) dropwise. After stirring 25 min, the mixture was diluted with 3 mL dry DCM. Additional TEA (162 mg, 213 mL, 0.1 6 mmol) followed by DMAP (5 mg, 0.045 mmol) was added. Acetic anhydride (137 mg, 1.34 mmol) was added to the stirring solution dropwise. The solution stirred for 1 h then was partitioned between Et 2 O/2 N HCl. The organic layer was collected. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 and concentrated. The crude residue was chromatographed by silica gel MPLC (eluting at 45 65% EtOAc : hexanes) to afford 2. 71 as a colorless oi l (66 mg, 0.249 mmol, 56%). Starting material (24%) was also recovered. 2. 71 : [ ] D 20 1.0 ( c 1.0, CHCl 3 ); IR (neat) (cm 1 ): 3452, 2941, 2864, 1735, 1241, 1096; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.42 (m, 2H), 1.60 (m, 4H), 2.07 (s, 3H), 3.45 (t 6.3, 2H), 3.61 (dd, 11.5, 6.3 ), 3.70 (dd, 11.5, 2.5 ), 4.48 (s, 2H), 4.89 (m ), 7.27 (m), 7.32 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) : 21.3, 22.3, 29.7, 30.4, 65.0, 70.2, 73.2, 75.7, 127.7, 127.8 (2C), 128.6 (2C), 138.7, 171.6; ESI HRMS [M + H] + calcd for [C 15 H 23 O 4 ] + : 267.1591, found 267.1594. 5.2. 19 ethyl 3 ((4 S ,5 S ) 2,2 dimethyl 5 vinyl 1,3 dioxolan 4 yl)propanoate ( 2. 72 ) To a stirring slurry of Ph 3 PMeBr (414 mg, 1.16 mmo) in 10 mL dry THF at 0 o C was added dropwise KHMDS (0.5 M in toluene, 2.32 mL, 1.16 mmol ). The mixture was warmed to rt and

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114 stirred for 30 min. The mixture was then cooled back down to 0 o C. Aldehyde 2. 66 (150 mg, 0.65 mmol) diss olved in 3 mL dry THF was then added dropwise. The reaction was then partitioned between Et 2 O / H 2 O. The aqueous layer was extracted 2 X with aliquots of Et 2 O. The combined organic layers were dried over anhydrous MgSO 4 and concentrated. The crude residue was chromatographed by silica gel MPLC (eluting at 12 16% EtOAc/hexanes) to afford 2. 72 as a colorless oil (91 mg, 0.40 mmol, 62%). [ ] D 20 2.3 ( c 0.7, CHCl 3 ); IR (neat) (cm 1 ): 3082, 2985, 1741, 1167, 1073; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.25 (t, 7.3, 3H), 1.39 (s, 3H), 1.40 (s, 3H), 1.83 (m),1.96 (m), 2.45 (m, 2H), 3.69 (dt, 8.3, 3.7), 4.00 (dd, 8.3, 1.4), 4.13 (q, 7.3, 2H), 5.26 (dd, 10.2, 1.4), 5.38 (dd, 17.4, 1.4), 5.80 (ddd, 17.4 10.2, 7.0); 13 C NMR (100 MHz, CDCl 3 ) : 14.4, 27.0, 27.1, 27.4, 30.9, 60.6, 79.8, 82.6, 109.0, 119.3, 135.2, 173.3; ESI HRMS [M + Na] + calcd for [C 12 H 20 O 4 Na] + : 251.1254, found 251.1256. 5.2.2 0 ethyl 3 ((4 S ,5 S ) 5 (( S E ) 3 acetoxy 7 (benzyloxy)hept 1 enyl) 2,2 dimethyl 1,3 dioxolan 4 yl) propanoate ( 2. 73 ) To a solution of 2. 69 (20 mg, 0.076 mmol) and 2. 72 (17 mg, 0.076 mmol) in dry DCM under stirring at rt was added Grubbs second generation catalyst (7 mg, 0.008 mmol). The solution stirred for 48 h then was concentrated and chromatographed by silica gel MPLC (eluting at 25 30% EtOAc : hexanes) to afford the E isomer 2. 73 as a colorless oil, which solidified when placed in freezer (14 mg, 0.03 mmol, 40%). [ ] D 20 15.6 ( c 0.36, CHCl 3 ); IR (neat) (cm 1 ): 3032, 2988, 2931, 2867, 1738, 1372, 1241, 1093, 1079, 1022; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz ), integration): 1.22 (t, 7.1, 3H), 1.36 (s, 6H), 1.38 (m, 2H), 1.59 (m, 4H), 1.84 (m, 2H), 2.02 (s, 3H), 2.41 (m, 2H), 3.43 (t, 6.5, 2H), 3.64 (dt, 7.7, 4.0), 3.97 (dt, 7.7, 7.2), 4.10 (q, 7.1, 2H), 4.47, (s, 2H), 5.24 (d, 6.7), 5.60 (dd, 15.6, 7.2), 5.68 (dd, 15.6, 6.7), 7.25 (m), 7.31 (m, 4H); 13 C NMR

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115 (100 MHz, CDCl 3 ) : 14.4, 21.4, 22.0, 27.0, 27.1, 27.4, 29.7, 30.8, 34.3, 60.6, 70.2, 73.1, 73.7, 79.9, 81.5, 109.1, 127.7, 127.8 (2C), 128.5 (2C), 129.7, 133.2, 138.7, 170.4, 173.3; ESI HRMS [M + Na] + calcd for [C 26 H 38 O 7 Na] + : 485.2510, found 485.2524. 5.3 Experimental supporting Chapter 3 5.3.1 Isolation of meridianins A ( 3 24 ) B ( 3 25 ) C ( 3 26 ) and E ( 3 28 ) from Antarctic tunicate Synoicum sp. Lyophilized Synoicum sp. (192 g), collected at Palmer Station, Antarctica, was extracted 3 x 24 h with 1:1 DCM:MeOH followed by extraction with 1:1 MeOH:H 2 O ( 3x24 h ) Concentration of the organic filtrate under reduced pressure yielded 21.0 g of lipophilic extract. A portion of this extract (4.6 g) was fractionated on MPLC with a gradient of hexane to EtOAc to MeOH. Fractions eluting at 20 50% MeOH:EtOAc were combined (25 mg) and chromatographed via analy tical reverse phase HPLC (55% MeOH:H 2 O) to yield meridianins A, B, C, and E. NMR data matched that of the previously reported meridianins (Chapter 3, ref. 22) Meridianin A ( 3. 24 ), 5 mg, 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration, position) : 6.36 (dd, 7.3, 0.5, H5), 6.27 (s, 2H, 2 NH 2 ), 6.79 (dd, 7.5, 0.5, H7), 6.96 (dd, 7.5, 7.3 H6), 7.10 (d, 5.4 H5 ), 8.10, (d, 5.4 H6 ), 8.22 (s, H2), 11.76, (brs, N1 H), 13.57 (s, 4 OH); 13 C NMR (100 MHz, d 6 DMSO) : 102.3, 104.4, 105.4, 114.0, 114.7, 124.4, 128.5, 139.4, 152.0, 158.4, 160.5, 161.7.

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116 Meridianin B ( 3. 2 5 ), 5 mg, 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration, position): 6.51 (d, 1.5 H5), 6.85 (s, 2H, 2 NH 2 ), 7.00 (d, 1.5 H7), 7.15 (d, 5.4 H5 ), 8.18 (d, 5.4 H6 ), 8.28 (s, H2), 11.90 (brs, N1 H), 14.21 (s, 4 OH). Meridianin C ( 3. 26 ), < 1 mg, 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration, position): 6.49 ( s, 2H, 2 NH 2 ), 7.00 (d, 5.3, H5 ), 7.29 (dd, 8.5, 1.9 H6), 7.39 (d, 8.4 H7), 8.10 (d, 5.3 H6 ), 8.24 (s, H2), 8.75 (d, 1.9, H4), 11.85 (brs, N1 H). Meridianin E ( 3. 28 ), 3 mg, 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration, position): 6.37 (d, 8.3 H5), 6.85 (2H, 2 NH 2 ), 7.19 (d, 8.3 H6), 7.24 (d, 5.4 H5 ), 8.18 (d, 5.4 H6 ), 8.31 (s, H2), 11.92 (brs, N1 H), 13.93 (s, 4 OH). 5.3. 2 synthetic meridianin A ( 3.24 ) To a stirring solution of 3.91 (75 mg, 0.15 mmol) in dry THF (2 mL) at rt was added dropwise tetra n butylammonium fluoride ( TBAF 1.0 M, 0.3 mL, 0.3 mmol). After 5 min. aqueous sodium carbonate ( Na 2 CO 3 2 M, 2 mL) was added. The mixture was then partitioned between sat. ammonium chloride ( NH 4 Cl ) / 10% MeOH in EtOAc. The aqueous layer was extracted with another aliquot of 10% MeOH in EtOAc. The combined organic layers were dried over anhyd. MgSO 4 concentrated, and purified via MPLC (silica, eluting at 10% MeOH:DCM) to afford meridianin A. The compound was recrystallized from MeOH / H 2 O to yield meridianin A as yellow needles ( 3.24 30 mg, 0.13 mmol, 87% yield). Mp = 1 68 o C ; 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration) : 6.36 (d, 7.6), 6.73 (br s, 2H), 6.79 (d, 7.9), 6.96 (dd, 7.6, 7.9),

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117 7.10 (d, 5.4), 8.10 (d, 5.4), 8.21 (d, 2.6), 11.73 (br s), 13.58 (s); 13 C NMR (100 MHz, d 6 DMSO) : 102.4, 104.4, 105.5, 113.8, 114.4, 124.4, 128.5, 139.3, 152.0, 158.5, 160.5, 161.8 ; ESI HRMS [M + H] + calculated for [C 12 H 11 N 4 O] + :227.0927, found: 227.0929. 5.3. 3 4 indolol ( 3. 86 ) A stirring mixture of 1,5,6,7 tetrahydro 4 H indol 4 one ( 3. 85 2.50 g, 18.4 mmol) and 10% Pd/C (625 mg) in 2,6 dimethyl 4 heptanone (25 mL) was refluxed (190 o C) under N 2 for 48 h. The mixture was cooled to rt and filtered through Celite. The filter cake was washed with DCM. The filtrate was concentrated in vacuo Kugel rohr distillation of the concentrate afforded discolored 4 indolol (150 o C, 4 torr) which was subject ed to MPLC (silica; eluting at 30 35% EtOAc:Hex) to yield 3. 86 (1.19 g, 9.0 mmol, 49% yield) as a white crystalline solid. 1 H NMR (400 MHz, d 6 DMSO, not stable in CDCl 3 ) (multiplicity, J (Hz), position): 6.31 (d, 6.1 H3), 6.43 (m, H5), 6.81 (m, H6), 6.82 (m, H7), 7.10 (d, 2.4 H2), 9.21 (s, OH), 10.86 (brs, NH); 13 C NMR (100 MHz, d 6 DMSO) : 98.5, 102.8 (2C), 117.8, 121.8, 122.9, 137.9, 150.5; ESI HRMS [M + H] + calculated for [C 8 H 8 NO] + : 134.0600, found: 134.0600. 5.3. 4 4 (tert butyldimethylsilyloxy) 1 H indole ( 3. 87 ) A mixture of 4 indolol ( 3. 86 1.05 g, 7.89 mmol), tert butyldimethylsilyl chloride (1.43 g, 9.47 mmol) and imidazole (1.74 g, 25.3 mmol) was stirred in 25 mL dry DMF at rt under N 2 for 1 h. The mixture was partitioned (EtOAc:water). The organic layer was washed with water then dried (anhyd. MgSO 4 ), filtered and concentrated under reduced pressure onto silica. Purification using

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118 MPLC (silica; eluting at 18% EtOAC:hexane) afforded 3.87 as a white solid (1.76 g, 7.14 mmol, 91% yield). Mp= 79 80 o C; IR (neat) (cm 1 ): 3399, 3056, 2961, 2928, 1583; 1 H NMR (400 MHz, d 6 DMSO, not stable in CDCl 3 ) (multiplicity, J (Hz), integration, position) : 0.19 (s, 6H, Si(CH 3 ) 2 ), 1.02 (s, 9H, SiC(CH 3 ) 3 ), 6.35 (m, H3), 6.41 (d, 7.5 H5), 6.93 (dd, 7.5, 8.0 H6), 7.02 (d, 8.0 H7), 7.21 (s, H2), 11.0 (brs, NH); 13 C N MR (100 MHz, d 6 DMSO) : 4.4 (2C), 18.0, 25.7 (3C), 98.3, 105.4, 107.6, 121.2, 121.5, 123.8, 137.9, 148.1; ESI HRMS [M + H] + calculated for [C 14 H 22 NOSi] + : 248.1465, found: 248.1468. 5.3. 5 3 bromo 4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indole ( 3. 88 ) To 3. 87 (1.77 g, 7.14 mmol) in 35 mL dry THF under stirring and N 2 was added dropwise n butyllithium ( 5.43 mL, 1.6 M in hexanes 8.76 mmol ) at 78 o C. The mixture was warmed to 10 o C and stirred for 10 min. before being cooled back to 78 o C. Triisopropylsilyl chloride (1.93 g, 10.0 mmol, 2.14 mL) was added dropwise. The mixture was then warmed to 0 o C and stirred until reaction was complete (via TLC). The mixture was cooled to 7 8 o C. N bromosuccinimide (1.72 g, 9.64 mmol) dissolved in dry THF (10 mL) was added to the cooled mixture. After stirring for 2 h at 78 o C, the mixture was warmed to rt diluted with 1% pyridine in hexane and filtered through Celite. Any 3 bromo 4 (ter t butyldimethylsilyloxy) 1H indole in the mixture will lead to decomposition of the desired product upon concentration. The filtrate was concentrated onto neutral alumina and chromatographed on neutral alumina (eluting in hexanes). Brown impurities were removed by further purification via MPLC (silica; eluting at 5% EtOAc:Hex) to afford white crystalline solid 3. 88 (2.69 g, 5.59 mmol, 78% yield). Mp = 107 109 o C; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration, position) : 0.35 (s, 6H, Si(CH 3 ) 2 ), 1.08 (s, 9H, SiC(CH 3 ) 3 ), 1.14 (d, 18H, SiCH(CH 3 ) 2 ) 1.65 ( sept. 3H, Si C H), 6.52 (d, 7.7 H5), 6.98 (dd, 7.7, 8.1 H6), 7.07 (d, 8.1 H7), 7.10 (s, H2); 13 C NMR (100 MHz, CDCl 3 ) : 3.7 (2C), 13.0 (3C), 18.2 (6C), 18.7, 26.2

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119 (3C), 90.6, 107.7, 109.1, 121.0, 122.8, 129.7, 142.7, 149.5; ESI HRMS [M + H] + calculated for [C 23 H 41 BrNOSi 2 ] + : 482.1905, found: 482.1905. 5.3. 6 4 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1H indol 3 yl)pyrimidin 2 amine ( 3. 91 ) To a stirring solution of 3. 88 ( 500 mg, 1.04 mmol) in dry THF (5 mL) at 78 o C under N 2 was added dropwise tert butyllithium (1.6 M in pentane) until the solution remained yellow then and additional aliquot (1. 4 mL, 2.29 mmol) was added dropwise. The solution stirred for 15 min. Neat 2 Isopropoxy 4,4,5,5 tetramethyl 1,3,2 dioxaborolane ( 3. 89 3 24 mg, 1. 74 mmol, 0. 35 mL) was added dropwise. The mixture stirred for 1 h at 78 o C and was quenched with sat. NH 4 Cl. The mixt ure warmed to rt and was partitioned between Et 2 O / sat. NH 4 Cl. The aqueous layer was extracted 2X with EtO 2 The combined organic layers were dried (anhyd. MgSO 4 ), concentrated to afford 4 (tert butyldimethylsilyloxy) 3 (4,4,5,5 tetramethyl 1,3,2 dioxabor olan 2 yl) 1 (triisopropylsilyl) 1H indole ( 3. 90 ) which was used immediately in the next reaction without further purification. A stirring mixture of crude 3. 90 ( 1.04 mmol), tetrakis(triphenylphosphine)palladium (10 0 mg, 0.09 mmol), and 2 amino 4 chloro p yrimidine ( 113 mg, 0. 87 mmol), benzene (25 mL, degassed by sparging with N 2 ), methanol (5 mL, degassed), and aqueous sodium carbonate (1.25 mL, 2 M, degassed) was refluxed under N 2 for 24 h. The mixture was allowed to cool to rt diluted with EtOAc and dried with anhyd. MgSO 4 The filtrate was concentrated onto silica and purified via MPLC (silica, eluting at 40 % EtOAc:hexane) to afford 3. 91 (2 00 mg, 0.4 0 mmol, 4 6 % yield). Mp= 83 85 o C ; 1 H NMR ( 5 00 MHz, CDCl 3 ) (multiplicity, J (Hz), integration, position) : 0.10 ( s, 6H, Si(CH 3 ) 2 ), 0.90 (s, 9H, SiC(CH 3 ) 3 ), 1.17 (d, 18H, 7.6, SiCH(C H 3 ) 2 ), 1.73 (sept., 7.6 3H, SiCH),

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120 7.8 H5), 7.05 (dd, 7.8, 8.1, H6), 7.15 (d, 8.1, H7), 7.22 (d, 5. 3 3 ; 13 C NMR (1 25 MHz, CDCl 3 ) : 0.4 (2C), 12.8 (3C), 18.2 (6C), 18.6, 26.0 (3C), 107.9, 110.9, 113.4, 118.4, 120.7, 122.5, 133.7, 144.2, 149.5, 156.6, 162.5, 163.1 ; ESI HRMS [M + H] + calculated for [ C 27 H 4 5 N 4 OSi 2 ] + : 497.3126, found: 497.3116. 5.3. 7 4 methoxy 1 H indole ( 3. 92 ) A stirring mixture of K 2 CO 3 (3.9g, 28.5 mmol), 3. 86 (380 mg, 2.85 mmol), and iodomethane (4.05g, 28.5 mmol, 1.8 mL) in dry acetone (10 mL) under N 2 was refluxed for 6 h. The mixture was then cooled to rt and partitioned between EtOAc / H 2 O. The aqueous layer was extracted 2X with aliquots of EtOAc. The combined organic layers were dried over anhyd. MgSO 4 concentrated, and purified via MPLC (silica eluting at 22% EtOAc:Hex) to afford 3. 92 (326 mg, 2.22 mmol, 78% yield). Mp= 66 o C ; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration) : 3.98 (s, 3H), 6.55 (d, 7.7, 1H) 6.68 (m, 1H), 7.05 (d, 8.2), 7.12 (m, 2H), 8.16 (br s ) ; 13 C NMR (100 MHz, CDCl 3 ) : 55.3, 99.5, 99.8, 104.4, 118.5, 122.61, 122.72, 137.18, 153.33 ; ESI HRMS [M + H] + calculated for [ C 9 H 10 NO ] + : 148.0757 found: 148.0757 5.3. 8 3 bromo 4 methoxy 1 (triisopropylsilyl) 1 H indole ( 3. 93 ) To 3. 92 (275 mg, 1.87 mmol) in 7.5 mL dry THF under stirring and N 2 was added dropwise n butyllithium ( 1.58 mL, 1.6 M in hexanes, 2.52 m mol ) at 78 o C. The mixture was warmed to 10 o C and stirred for 10 min. before being cooled back to 78 o C. Triisopropylsilyl chloride ( 0.5 g,

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121 2.62 mmol, 0.56 mL) was added dropwise. The mixture was then warmed to 0 o C and stirred until reaction was complete (via TLC). The mixture was cooled to 78 o C. N bromosuccinimide ( 0.45 g, 2.52 mmol) dissolved in dry T HF (10 mL) was added to the cooled mixture. After stirring for 2 h at 78 o C, the mixture was warmed to rt diluted with 1% pyridine in hexane and filtered through Celite. Any 3 bromo 4 methoxy 1 H indole in the mixture will lead to decomposition of the desired product upon concentration. The filtrate was concentrated onto neutral alumina and chromatographed on neutral alumina (eluting in hexanes). Brown impurities were removed by further purifica tion via MPLC (silica; eluting at 5% EtOAc:Hex) to afford white crystalline solid 3. 93 ( 610 mg 1.60 mmol, 85 % yield). Mp = 65 o C ; 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration) : 1.06 (d, 7.4, 18H), 1.71 (sept., 7.6, 3H) 3.84 (s, 3H), 6.61 (d, 7.6, ), 7.10 (d, 8.2,), 7.13 (dd, 8.24, 7.6), 7.26 (s ); 13 C NMR (100 MHz, d 6 DMSO ) : 11.9 (3C) 17.76 (6C) 55.24, 89.5, 101.1, 107.3, 118.0, 123.4, 129.4, 141.4, 153.0 ; ESI HRMS [M + H] + calculated for [ C 18 H 28 BrNOSiNa ] + : 404.1016 found: 404.1025 5.3. 9 4 (4 methoxy 1 (triisopropylsilyl) 1 H indol 3 yl)pyrimidin 2 amine ( 3. 95 ) To a stirring solution of 3. 93 (350 mg, 0.91 mmol) in dry THF (4.5 mL) at 78 o C under N 2 was added dropwise tert butyllithium (1.6 M in pentane) until the solution remained yellow then and additional aliquot (1.2 mL, 2.0 mmol) was added dropwise. The solution stirred for 15 min. Neat 2 Isopropoxy 4,4,5,5 tetramethyl 1,3,2 dioxaborolane ( 3. 89 340 mg, 1.82 mmol, 0.37 mL) was added d ropwise. The mixture stirred for 1 h at 78 o C and was quenched with sat. NH 4 Cl. The mixture warmed to rt and was partitioned between Et 2 O / sat. NH 4 Cl. The aqueous layer was extracted 2X with EtO 2 The combined organic layers were dried (anhyd. MgSO 4 ), c oncentrated

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122 to afford 4 methoxy 3 (4,4,5,5 tetramethyl 1,3,2 dioxaborolan 2 yl) 1 (triisopropylsilyl) 1 H indole ( 3. 94 ) which was used immediately in the next reaction without further purification. A stirring mixture of crude 3. 94 (0.91 mmol), tetrakis(triphenylphosphine)palladium (104 mg, 0.09 mmol), and 2 amino 4 chloropyrimidine (118 mg, 0.91 mmol), benzene (25 mL, degassed by sparging with N 2 ), methanol (5 mL, degassed), and aqueous sodium carbonate (1.25 mL, 2 M, degassed) was refluxed under N 2 for 24 h. The mixture was allowed to cool to rt diluted with EtOAc and dried with anhyd. MgSO 4 The filtrate was concentrated onto silica and purified via MPLC (silica, eluting at 40% EtOAc:hexane) to afford 3. 95 (205 mg, 0.52 mmol, 5 7 % yield). Mp = 106 o C ; 1 H (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration) : 1.11 (d, 7.5, 18H), 1.74 (sept., 7.5, 3H), 3.86 (s, 3H), 6 .33 (br s, 2H), 6.71 (d, 7.7 ), 7.1 6 ( m 3H ), 7.77 (s), 8.16 (d, 5.2 ); 13 C NMR (100 MHz, d 6 DMSO ) : 12.0 (3C), 17.9 (6C), 55.0, 102.0, 107.5, 110.2, 117.4, 118.5, 123.1, 133.0, 143.1, 153.3, 157.0, 161.14, 163.2 ; ESI HRMS [M + H] + calculated for [ C 22 H 33 N 4 OSi ] + : 397.2418 found: 397.2418 5.3.1 0 4 methoxymeridianin A ( 3. 96 ) To a stirring solution of 3. 9 5 ( 80 mg, 0. 2 mmol) in dry THF (2 mL) at rt was added dropwise TBAF (1.0 M, 0. 2 mL, 0 2 mmol). After 5 min. aqueous Na 2 CO 3 (2 M, 2 mL) was added. The mixture was then partitioned between sat. NH 4 Cl and EtOAc. The aqueous layer was extracted 2X with aliquot s of EtOAc. The combined organic layers were dried over anhyd. MgSO 4 concentrated, and purified via MPLC (silica, eluting at 10% MeOH:DCM) to afford 4 methoxymeridianin A ( 3. 96 30 mg, 0.13 mm ol, 87% yield). Mp = 213 215 o C ; 1 H NMR (400 MHz, d 6 DMSO) (multiplicity, J (Hz), integration) : 3.87 (s, 3H), 6.27 (brs, 2H), 6.64 (d, 7. 2 ), 7.08 ( m ), 7.09 ( m ), 7.25 (d, 5.3), 7.84

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123 (d, 2.6 ), 8.15 ( d, 5.3), 11.64 (brs ) ; 13 C NMR (100 MHz, d 6 DMSO) : 55.0, 101.2, 105.6, 109.6, 114.5, 115.4, 122. 7, 127.5, 138.8, 153.3, 157.0, 161.8, 163.1. M ; ESI HRMS [M + H] + calculated for [ C 13 H 13 N 4 O ] + : 241.1084 found: 241.1084 5.3.1 1 5,7 dibromomeridianin A ( 3. 97 ) To a stirring solution of meridianin A ( 3. 24 5 mg, 0.022 mmol) in 2.0 mL dry DCM was added AcOH (0.2 mL) and pyridinium tribromide (14 mg, 0.044 mmol) The mixture stirred for 5.5 h. Na 2 S 2 O 3 (5% aqueous, 10 mL) and 10 mL EtOAc was added. The aqueous layer was washed 2X with aliquots of EtOAc. The combined organic layers were dried over anhyd. MgSO 4 concentrated to afford crude 3. 96 ( 5 mg, 0.013 mmol, 58 % yield). 1 H NMR (400 MHz, d 6 DMSO ) (multiplicity, J (Hz)) : 6.97 (s, 2H) 7.27 (d, 5.4), 7.46 (s), 8.21 (d, 5.4), 8 .38 (s), 11.24 (brs), 15.10 (s); ESI MS ( m/z ) [M+H] = 382.9 (50%), 384.9 (100%), 386.9 (45%). 5.3.1 2 4 amino 2 chloro 5 iodopyrimidine ( 3. 99 ) In a flask sealed with a rubber stopper 2,4 dichloropyrimidine ( 3. 98 1 g, 6.76 mmol) was stirred in 29% NH 4 OH (20 mL) for 12 h. The solvent was removed in vacuo The crude product was chromatographed via MPLC ( silica ) to afford 2 amino 4 chloropyrimidine (400 mg, 3.0 mmol, eluting at 6% MeOH:CHCl 3 44% yield) and 4 amino 2 chloropyrimidine (454 mg, 3.5 mmol, eluting at 10% MeOH:CHCl 3 52% yield).

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124 N iodosuccinimide (2.7 g, 12.0 mmol) and 4 amino 2 chloropyrimidine (1.3 g, 10.0 mmol) were stirred in AcOH (30 mL) at 60 75 o C for 3 h. After solvent removal in vacuo the residue was partitioned between 5% Na 2 S 2 O 3 and CHCl 3 The organic layer was washed with sat. NH 4 Cl, dried over anhyd. MgSO4 and chromatographed via MPLC (silica, eluting at 45% EtOAc:hexane) to afford 3. 99 (1.45 g 5.67 mmol, 57% yield). 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, integration) : 8.38 (s, ), 5.65 (br s, 2 H); 13 C NMR ( 100 MHz, CDCl 3 ) : 74.4, 160.5, 163.1, 163.9. The NMR data are consistent with literature values (Chapter 3 r ef. 51). 5.3.1 3 4 amino 2 bromo 5 iodopyrimidine ( 3. 100 ) 4 amino 2 chloro 5 iodopyrimidine ( 3. 99 0.73 g, 2.86 mmol) in 33% HBr:AcOH was stirred for 1 h then refluxed for an additional hour. The mixture cooled to rt Solvent was removed in vacuo Water was added to the concentrate. The solid was collected via filtration and dried under vacuum aff ording 3. 100 (0.74 g, 2.46 mmol, 86% yield). Mp= 189 190 o C; IR (neat) (cm 1 ): 3330, 3168, 3009, 1633, 1549; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, integration) : 5.78 (2H, brs, NH 2 ), 8.31 (s, H6); 13 C NMR (100 MHz, CDCl 3 ) : 75.4, 152.1, 162.8, 163.9; ESI HRMS [M+H] + calculated for [C 4 H 4 BrIN 3 ] + : 299.8629, found: 299.8626. 5.3.14 2 bromo 5 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indol 3 yl)pyrimidin 4 amine ( 3. 101 )

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125 To a stirring solution of 3. 88 (470 mg, 0.98 mmol) in dry THF (5 mL) at 78 o C under N 2 was added dropwise tert butyllithium (1.6 M in pentane) until the solution remained yellow then and additional aliquot (1.3 mL, 2.15 mmol) was added dropwise. The solution stirred for 15 min. Neat 2 Isopropoxy 4,4,5,5 tetramethyl 1,3,2 dioxaborolane ( 3. 89 364 mg, 1.96 mmol, 0.4 mL) was added dropwise. The mixture stirred for 1 h at 78 o C and was quenched with sat. NH 4 Cl. The mixture warmed to rt and was partitio ned between Et 2 O:sat. NH 4 Cl. The aqueous layer was extracted 2X with EtO 2 The combined organic layers were dried (anhyd. MgSO 4 ), concentrated to afford crude 3. 90 which was used immediately in the next reaction without further purification. A stirring mixture of crude 3. 90 (0.98 mmol), tetrakis(triphenylphosphine)palladium (104 mg, 0.09 mmol), and 4 amino 2 bromo 5 iodopyrimidine ( 3. 100 283 mg, 0.98 mmol), benzene (25 mL, degassed by sparging with N 2 ), methanol (5 mL, degassed), and aqueous sodium carbonate (1.25 mL, 2 M, degassed) was refluxed under N 2 for 24 h. The mixture was allowed to cool to rt diluted with EtOAc and dried with anhyd. MgSO 4 The filtrate was concentrated onto silica and purified via MPLC (silica, eluting at 18% EtOAc :hexane) to afford 3. 101 (252 mg, 0.44 mmol, 45% yield from 3. 88 ). Mp = 131 133 o C; IR (neat) (cm 1 ): 3351, 3130, 2951; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration, position) : 0.09 (s, 6H, Si(CH 3 ) 2 ), 0.83 (s, 9H, SiC(CH 3 ) 3 ), 1.16 (d, 18H, S i CH(CH 3 ) 2 ), 1.69 (sept, 3H, SiCH), 5.25 (brs, 2H, NH 2 ), 6.54 (d, 7.1 H5), 7.04 (dd, 7.9, 8.1, H6), 7.07 (s, H2), 7.15, (d, 8.1 13 C NMR (100 MHz, CDCl 3 ) : 4.1 (2C), 13.0 (3C), 18.3 (6C), 18.4, 25.8 (3C), 107.9, 109.0, 109.3, 114.1, 121.7, 123.2, 130.3, 143.9, 149.6, 150.3, 156.2, 163.9; ESI HRMS [M + H] + calculated for [C 27 H 44 N 4 OSi 2 ] + : 575.2232, found: 575.2248.

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126 5.3.15 synthetic psammopemmin A ( 3. 102 ) To 3. 101 (50 mg, 0.087 mmol) in 1.0 mL acetonitrile (dried over 4 molecular sieves) and 0.1 mL pyridine (dried over 4 molecular sieves) was added 50 L hydrogen fluoride pyridine (70% as HF, 30% as pyridine). After 30 min. the starting material had completely dissolved. After 1 h, a precipitate had formed. The mixture stirred for 11 additional h at rt The precipitate was collected via filtration and washed with acetonitrile. Drying the precipitate under high vacuum resulted in 3. 1 02 as a tan powder (15 mg, 0.049 mmol, 56%). IR (neat) (cm 1 ): 3429, 3313, 3163, 3015; 1 H NMR (400 MHz, d 6 DMSO ) (multiplicity, J (Hz), integration, position) : 6.38 (d, 7.1 H5), 6.88 ( d 8.3, H7), 6.93, (dd, 8.3, 7.1 13 C NMR (100 MHz, d 6 DMSO ) : 103.2, 103.8, 105.6, 113.0, 115.4, 122.6, 123.6, 138.6, 148.9, 151.2, 156.4, 163.6; ESI HRMS [M + H] + calculated for [ C 12 H 10 BrN 4 O ] + : 305.0033, found: 305.0033. 5.3.16 synthetic psammopemmin A HCl ( 3. 103 ) In a 3mm NMR tube, HCl gas was bubble through 3. 102 (5 mg) in d 6 DMSO (200 L) for 1 min to form 3. 103 Because 3. 103 is not stable, 1 H and 13 C NMR data w ere obtai ned immediately (s ee Table 3. 2 for NMR data )

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127 5.3.17 5 ( 5 (4 (tert butyldimethylsilyloxy) 1 (triisopropylsilyl) 1 H indol 3 yl) 2 chloropyrimidin 4 amine ( 3. 104 ) To a stirring solution of 3. 88 ( 200 mg, 0. 42 mmol) in dry THF (5 mL) at 78 o C under N 2 was added dropwise tert butyllithium (1.6 M in pentane) until the solution remained yellow then and additional aliquot ( 0.57 mL, 0 92 mmol) was added dropwise. The solution stirred for 15 min. Neat 2 Isopropoxy 4,4,5,5 tetramethyl 1,3,2 dioxaborolane ( 3. 89 155 mg, 0.83 mmol, 0. 17 mL) was added dropwise. The mixture stirred for 1 h at 78 o C and was quenched with sat. NH 4 Cl. The mixt ure warmed to rt and was partitioned between Et 2 O / sat. NH 4 Cl. The aqueous layer was extracted 2X with EtO 2 The combined organic layers were dried (anhyd. MgSO 4 ), concentrated to afford crude 3. 90 which was used immediately in the next reaction without f urther purification. A stirring mixture of crude 3. 90 (0. 42 mmol), tetrakis(triphenylphosphine)palladium ( 49 mg, 0.0 4 mmol), and 4 amino 2 chloro 5 iodopyrimidine ( 3. 99 106 mg, 0. 42 mmol), benzene ( 10 mL, degassed by sparging with N 2 ), methanol ( 2.0 mL, degassed), and aqueous sodium carbonate ( 0.5 mL, 2 M, degassed) was refluxed under N 2 for 24 h. The mixture was allowed to cool to rt diluted with EtOAc and dried with anhyd. MgSO 4 The filtrate was concentrated onto silica and purified via MPLC (silica, eluting at 18% EtOAc:hexane) to afford 3. 10 4 ( 123 mg, 0. 23 mmol, 56 % yield). Mp = 83 85 o C; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration, position) : 0.10 (s, 6H, S i(CH 3 ) 2 ), 0.83 (s, 9H, SiC(CH 3 ) 3 ), 1.16 (d, 7.3, 18H, SCH(CH 3 ) 2 ), 1. 71 (sept, 7.3, 3H, SiCH), 5. 40 (brs, 2H, H 2 ), 6.5 5 (d, 7. 8 H5), 7.0 5 (dd, 7. 8 8. 2 H6), 7.0 9 (s, H2), 7.1 6 (d, 8. 2 H7), 8.02 13 C N MR (100 MHz, CDCl 3 ) : 4. 3 (2C), 12.8 (3C), 18. 1 (6C), 18. 2 25. 6 (3C), 107. 7 10 8 9 109. 1 11 3 5 121.6, 12 3 0 130. 2 143. 7 149. 4 15 6 1 15 8 6 16 4 1 ; ESI HRMS [M + H] + calculated for [ C 27 H 43 Cl N 4 O Si 2 Na ] + : 553.2556 found: 553.2540

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128 5.3.18 chloropsammopemmin A ( 3.105 ) T o a stirring solution of 3. 104 (35 mg, 0.066 mmol) in dry THF (1 mL) was added dropwise TBAF (123 L, 1.0 M, 0.132 mmol). After 8 min. the solution turned brown and 2 M Na 2 CO 3 (1 mL) was added. The mixture was then partitioned between EtOAc:H 2 O The organic layer was collected, dried over andhyd. MgSO 4 and chromatographed on MPLC (silica, eluting at 10% MeOH:DCM) to afford 3. 105 (5 mg, 0.019 mmol, 29% yield). Alternative pro cedure: To 3. 104 ( 80 mg, 0. 15 mmol) in 1.0 mL acetonitrile (dried over 4 molecular sieves) and 0.1 mL pyridine (dried over 4 molecular sieves) was added 125 L hydrogen fluoride pyridine (70% as HF, 30% as pyridine). After 30 min. the starting material had completely dissolved. After 1 h, a precipitate had formed. The mixture stirred for 11 additional h at rt The precipitate was collected via filtration and washed with aceton itrile. Drying the precipitate under high vacuum resulted in 3.10 5 as a tan powder ( 20 mg, 0.04 8 mmol, 32 %) 1 H NMR (400 MHz, d 6 DMSO ) (multiplicity, J (Hz), integration position ): 6.3 9 (d, 7. 3 H5), 6.8 9 ( d 8.3, H7), 2 ), 6.9 2 (dd, 7 .3, 7. 8 H6), 7.27 (s, H2), 7. 87 8 (brs, NH); 13 C NMR (100 MHz, d 6 DMSO ) : 103. 1 103.8, 105.6, 11 2.6 115. 5 122.6, 123. 7 138.6, 1 51.2 15 6.4, 15 7 0 16 4.0 ; ESI HRMS [M + H] + calculated for [C 12 H 10 ClN 4 O] + : 261.0538 found: 261.0536

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129 5.3.19 meridoquin ( 3. 106 ) To 3. 112 (62 mg, 0.136 mmol) in 1 mL dry THF under Ar and stirring was added TBAF (0.14 mL, 0.14 mmol, 1.0 M in THF). After 5 min., the reaction was complete. Sodium carbonate (1 mL, 2.0 M) was added and the mixture was partitioned between EtOAc / H 2 O. The aqueous layer was washed 2X with EtOAc aliquots. The combined EtOAc extracts were dried over anhyd. MgSO 4 and purified via MPLC (silica, eluting at 35% EtOAc:hexane) to afford 3. 106 (35 mg, 0.116 mmol, 86%). Mp = 166 o C ; 1 H NMR (500 MHz, CDCl 3 ) (multi plicity, J (Hz), integration): 1.29 (t, 6.9, 6H), 3.75 (q, 6.9, 4H), 6.78 (dd, 5.3, 2.1), 7.24 (d, 8.6), 7.43 (dd, 2.1, 2.1), 7.83 (s), 8.27 (dd, 5.3, 2.1), 8.45 (d, 8.6), 8.60 (br s ). 13 C NMR (125 MHz, CDCl 3 ) : 13.2 (2C), 42.1 (2C), 104.3, 111 2, 116.5, 121.9, 123.1, 124.2, 126.5, 12 8.7, 137.2, 156.9, 161.1, 161.7; ESI HRMS [M + H] + calculated for [ C 16 H 18 ClN 4 ] + : 301.1215 found: 310.1220 5.3.20 2 chloro 4 N N diethylaminopyrimidine ( 3. 107 ) and 4 chloro 2 N N diethylamino pyrimidine ( 3. 108 ) At rt, neat diethylamine (2 mL) was slowly added t o 2,4 dichloropyrimidine ( 3. 98 500 mg, 3.38 mmol) The mixture stirred for 1 min., was diluted with EtOAc and concentrated onto silica. Purification via MPLC (silica, gradient from 0 to 35% EtOAc:hexane) afforded 4 chloro 2 N N diethylaminopyrimidine ( 3. 108 eluting at 12% EtOAc:Hex, 54 mg, 0. 44 mmol, 13 % yield) and 2 chloro 4 N N diethylaminopyrimidine ( 3. 107 eluting at 26% EtOAc:hexane, 372 mg, 2.0 mmol, 59 % yield).

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130 4 ( N N diethyl ) 2 chloro aminopyrimidine ( 3.107 ): viscous colorless liquid, solidifies in freezer; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.18 (t, 7.0, 6H), 3 .48 (br s, 4H), 6.26 (d, 5.9), 7.96 (d, 5.9 ); 13 C NMR (125 MHz, CDCl 3 ) : 12.5, 42.4, 100.9, 156.5, 160.7, 161.7; ESI HRMS [M + H] + calculated for [C 8 H 13 ClN 3 ] + : 186.0793, found: 186.0794. 2 ( N N diethyl ) amino 4 chloro pyrimidine ( 3. 108 ): viscous colorless liquid; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.19 (t, 7. 1 6H), 3.6 1 (q, 7. 1 4H), 6.44 (d, 4.7), 8.14 (d, 4.7); 13 C NMR (1 00 MHz, CDCl 3 ) : 12.9, 42. 0 108. 0 158. 7, 160.9, 161.0 ; ESI HRMS [M + H] + calculated for [ C 8 H 13 ClN 3 ] + : 186.0793 found: 186.0791 5.3.21 3 bromo 6 chloro 1 (triisopropylsilyl) 1 H indole ( 3. 110 ) To 6 chloroindole ( 3. 109, 1.0 g, 6.6 mmol) in 20 mL dry THF under stirring and N 2 was added dropwise n butyllithium (5.6 mL, 1.6 M in hexanes, 8.91 mmol) at 78 o C. The mixture was warmed to 10 o C and stirred for 10 min. before being cooled back to 78 o C. Triisopropylsilyl chloride (1.78 g, 9.24 mmol, 1.96 mL) was added dropwise. The mixture was then warmed to 0 o C and stirred until reaction was complete (via TLC). The mixture was cooled to 78 o C. N bromosuccinimide (1.59 g, 8.91 mmol) was added to the cooled mixture in one portion After stirring for 2 h at 78 o C, the mixture was warmed to rt diluted with 1% pyridine in hexane and filtered through Celite The concentrate filtrate was purifi ed via MPLC (silica; eluting in hexane ) to afford white crystalline solid 3. 110 ( 2.26 g, 5.87 mmol, 8 9 % yield). Mp = 52 o C ; 1 H NMR ( 4 00 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.15 (d, 7.6, 18H), 1.67 (q, 7.6, 3H), 7.18 (dd, 8.3, 1.4 ), 7.22 (s), 7.46 (d, 1.4), 7.48 (d, 8.3 ). 13 C NMR (1 00 MHz, CDCl 3 ) : 12.7 (3C), 18.0 (6C), 93.6, 113.8, 119.9, 121.3, 128.6, 128.7 130.4, 140.4 ; ESI HRMS [M + H] + calculated for [ C 17 H 26 BrClNSi ] + : 386.0701 found: 386.0698

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131 5.3.2 2 4 (6 chloro 1 (triisopropylsilyl) 1H indol 3 yl) N,N diethylpyrimidin 2 amine ( 3. 112 ) To a stirring solution of 3. 110 (200 mg, 0.52 mmol) in dry THF (5 mL) at 78 o C under inert atmosphere was added dropwise tert butyllithium (1.6 M in pentane) until the solution remained yellow then and additional aliquot (0.350 mL, 0.57 mmol) was added dropwise. The solution stirred f or 15 min. Neat 2 Isopropoxy 4,4,5,5 tetramethyl 1,3,2 dioxaborolane (194 mg, 1.04 mmol, 0.21 mL) was added dropwise. The mixture stirred for 1 h at 78 o C and was quenched with sat. NH 4 Cl. The mixture warmed to rt and was partitioned between Et 2 O:sat. NH 4 Cl. The aqueous layer was extracted 2X with EtO 2 The combined organic layers were dried (anhyd. MgSO 4 ), concentrated to afford crude 6 chloro 3 (4,4,5,5 tetramethyl 1,3,2 dioxaborolan 2 yl) 1 (triisopropylsilyl) 1 H indole which was used immediately i n the next reaction without further purification. A stirring mixture of crude 6 chloro 3 (4,4,5,5 tetramethyl 1,3,2 dioxaborolan 2 yl) 1 (triisopropylsilyl) 1 H indole (0.52 mmol), tetrakis(triphenylphosphine)palladium (35 mg, 0.03 mmol), and 2 ( N N diethy l)amino 4 chloropyrimidine (283 mg, 0.29 mmol), benzene (5 mL, degassed by sparging with N 2 ), methanol (1 mL, degassed), and aqueous sodium carbonate (0.25 mL, 2 M, degassed) was refluxed under N 2 for 2 h. The mixture was allowed to cool to rt diluted with EtOAc and dried with anhyd. MgSO 4 The filtrate was concentrated onto silica and purified via MPLC (silica, eluting at 18% EtOAc:hexane) to afford 3. 112 (82 mg, 0.179 mmol, 62%). Mp = 126 o C ; 1 H NMR (400 MHz, CDCl 3 ) (multiplicity, J (Hz), integration): 1.18 (d, 7. 4 18H), 1.29 (t, 7, 6H), 1.72 (sept., 7. 3 3H), 3.76 (q, 7.3, 4H), 6. 80 (d, 5. 5 ), 7.2 2 ( d d, 8. 7 1.8 ), 7.4 9 (d, 1.8 ), 7.8 8 (s), 8.2 6 (dd, 5. 4 ), 8.4 6 (d, 8. 7 ); 13 C NMR (100 MHz, CDCl 3 ) : 12.7 (3C), 13.3

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132 (2C), 18.06 (6C), 42.04 (2C), 104.3, 113.8, 118.0, 121.7, 122.8, 127.4, 128.1, 13 3.8, 142.4, 157.1, 161.2, 161.6; ESI HRMS [M + H] + calculated for [ C 25 H 38 ClN 4 Si ] + : 457.2549 found: 457.2569. 5.4 Experimental supporting Chapter 4 5.4.1 Isolation of cytochalasin D ( 4.17 ) Lyophilized 2L scale up fermentation of CY 4202 was exhaustively extracted with methanol (3 X 24h). The combined, filtered methanolic extracts were concentrated under reduced pressure. A portion of this extract (2 g) was fractionated on silica MPLC (40g column, gradient: to afford fractions A F, eluting in increasing polarity. A portion (50 mg) of a ntimalarial fraction C (250 mg) was further purified on HPLC (Sunfire Prep OBO, 10X250 mm) with an isocratic solvent system (1% MeOH:CHCl 3 ) A large UV active peak eluting at 4.4 min. was found to be cytochalasin D (18 mg). ESI MS ( m/z ) [M+ H ] = 508.3; 1 H and 13 C NMR data are presented in comparison with literature values in Ta ble 4. 2

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133 5.4.2 Isolation of roridin E ( 4.18 ) and 12,13 deoxyroridin E ( 4.19 ). Lyophilized 2L scale up fermentation of CY 3923 was exhaustively extracted with methanol (3 X 24h). The combined, filtered methanolic extracts were concentrated under reduced pressure. A ions A F, eluting in increasing polarity. The antimalarial fraction D ( 52 mg) was further purified on HPLC (Sunfire Prep C 18 10X250 mm) with an isocratic solvent system ( 30% water:MeOH, 0.1% TFA ). A UV (254 nm) active peak eluting at 30 min was found to 12,13 deoxyroridin E ( 4.19 3.3 mg) but the major component of the mixture was not isolated and was thought to have degraded in the acidic solution. Another portion of the original extract (2g) was carefully fractionated on silica MPLC (40g column) with a gradual gradient ( 50% EtOAc: 100%EtOAc). Fractions of a strong UV active (254 nm) peak that eluted at 90% EtOAc:hexane were analyzed via 1 H NM R which revealed pure roridin E ( 4.18 40 mg). 1 H and 13 C NMR data are presented in comparison with literature values in Table 4.3 ( 4.18 ) and Table 4.4 ( 4.19 ).

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134 Appendices

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135 Appendix A: NMR data supporting Chapter 2 1 H and 13 C NMR spectr a ( CD 3 OD) of 2.51 ................................ ................................ ................... 13 6 1 H and 13 C NMR spectr a ( CD 3 OD) of 2.5 2 ................................ ................................ ................... 137 1 H and 13 C NMR spectr a (CD 3 OD) of R 2.5 3 ................................ ................................ ............... 13 8 1 H and 13 C NMR spectr a (CD 3 OD) of S 2.5 3 ................................ ................................ ............... 139 Comparison of 2.51 and 2.53 1 H and 13 C NMR spectr a ( CD 3 OD) ................................ ............... 140 1 H and 13 C NMR spectr a ( CD Cl 3 ) of 2.5 4 ................................ ................................ ..................... 141 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2.5 6 ................................ ................................ ..................... 142 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2.5 7 ................................ ................................ ..................... 143 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2.5 8 ................................ ................................ ..................... 144 1 H and 13 C NMR spectr a (CD 3 OD) of R,R 2.5 9 ................................ ................................ ........... 145 1 H and 13 C NMR spectr a (CD 3 OD) of S,S 2.5 9 ................................ ................................ ........... 146 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 62 ................................ ................................ ..................... 147 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 63 ................................ ................................ ..................... 148 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 65 ................................ ................................ ..................... 149 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 66 ................................ ................................ ..................... 150 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 67 ................................ ................................ ..................... 151 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 68 ................................ ................................ ..................... 152 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 69 ................................ ................................ ..................... 153 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 70 ................................ ................................ ..................... 154 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 71 ................................ ................................ ..................... 155 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2 .72 ................................ ................................ ..................... 156 1 H and 13 C NMR spectr a (CD Cl 3 ) of 2. 73 ................................ ................................ ..................... 157 1 H NMR spectrum ( C DCl 3 expanded) of 2.73 ; H8 9 coupling simulation ................................ ... 158

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159 Appendix B : NMR data supporting Chapter 3 1 H and 13 C NMR spectr a ( d 6 DMSO ) of meridianin A from Synoicum sp. ( 3.24 ) ......................... 1 60 gHSQC spectrum ( d 6 DMSO ) of meridianin A from Synoicum sp. ( 3.24 ) ................................ .... 161 gHMBC spectrum ( d 6 DMSO ) of meridianin A from Synoicum sp. ( 3.24 ) ................................ ... 162 1 H NMR spectr a ( d 6 DMSO ) of meridianin B ( 3.25 ) and meridianin C ( 3.26 ) .............................. 163 1 H NMR spectr um ( d 6 DMSO ) of meridianin E ( 3.2 8 ) ................................ ................................ ... 164 1 H and 13 C NMR spectr a ( d 6 DMSO ) of synthetic meridianin A ( 3.24 ) ................................ ........ 1 6 5 1 H and 13 C NMR spectr a ( d 6 DMSO ) of synthetic and natural 3.24 ................................ ............. 16 6 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 86 ................................ ................................ ............... 16 7 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 87 ................................ ................................ ............... 16 8 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 88 ................................ ................................ ..................... 16 9 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 91 ................................ ................................ ..................... 170 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 92 ................................ ................................ ..................... 171 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 93 ................................ ................................ ............... 172 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 95 ................................ ................................ ............... 173 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 4 methoxymeridianin A 3. 96 ................................ ......... 174 1 H NMR spectr um ( d 6 DMSO ) of 5 bromomeridianin E 3. 97 ................................ ....................... 175 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 100 ................................ ................................ ................... 176 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 101 ................................ ................................ ................... 177 1 H and 13 C NMR spectr a ( d 6 DMSO ) of synthetic psammopemmin A ( 3. 102 ) ........................... 178 gHSQC spectrum ( d 6 DMSO) of synthetic psammopemmin A ( 3.102 ) ................................ ....... 179 gHMBC spectrum ( d 6 DMSO) of synthetic psammopemmin A ( 3.102 ) ................................ ....... 180 1 H NMR spectr a ( d 6 DMSO ) of 3. 102 and 3 103 ................................ ................................ ......... 1 81 13 C NMR spectr a ( d 6 DMSO ) of 3. 102 and 3 103 ................................ ................................ ........ 182 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 24 and 3 10 2 ................................ .............................. 183 1 H and 13 C NMR spectr a ( d 6 DMSO ) of 3. 24 and 3 1 16 ................................ .............................. 184 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 104 ................................ ................................ ................... 185 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 105 ................................ ................................ ................... 186 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 106 ................................ ................................ ................... 187 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 107 ................................ ................................ ................... 188 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 108 ................................ ................................ ................... 189 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 110 ................................ ................................ ................... 190 1 H and 13 C NMR spectr a ( CDCl 3 ) of 3. 112 ................................ ................................ ................... 191

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192 Appendix C : NMR data supporting Chapter 4 1 H and 13 C NMR spectr a ( CD Cl 3 ) of cytochalasin D ( 4.17 ) ................................ .......................... 1 9 3 1 H and 13 C NMR spectr a ( CD Cl 3 ) of roridin E ( 4.1 8 ) ................................ ................................ .... 1 9 4 1 H and 13 C NMR spectr a ( CD Cl 3 ) of 12,13 deoxy roridin E ( 4.1 9 ) ................................ ................ 1 9 5

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About the author Matthew Dennis Lebar, raised in Lakewood, C olorado briefly attended the University of Northern Colorado before deciding it was time to get out of the state. A eye opening vacation with family in high school to Hawaii combined with the Western Undergraduate Exchange program was all it took for Matt to re alize he would continue his studies at the University of Hawaii, Manoa. With inspiration and a newly minted Bachelor of Science in Chemistry from UH Matt decided it was again time to move on. Friends wanted to move to Florida and Matt had acclimated to warmer climates. Luckily, the University of South Florida Chemistry Department gave Matt a warm welcome and accepted him into the graduate program. The rest is history. Matt conducted all research characterized in Chapters 2.3, 2.4, 3.3, and 3.4 of this dissertation with the exception of the biological activity screening. He was also instrumental in developing extraction, fractionation and isolation methods and protocols described in Chapter 4 and was responsible for all work described in section 4.4 3. All experiments described in the experiment al section (Chapter 5) were carried out by Matt. Outside the lab, Matt enjoys (in no particular order) baseball, hiking, family, snowboarding, his girlfriend, traveling, swimming, diving, cacti, reading, computers, islands (tropical), video games, friendships, brewery tours, and gin + tonics. He also fosters two cats presently