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Chemical studies of anti-inflammatory secondary metabolites from marine sponges Yang, Lu 2006

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CHEMICAL STUDIES OF ANTI-INFLAMMATORY SECONDARY METABOLITES FROM MARINE SPONGES by LU YANG M.Sc, China Pharmaceutical University, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Chemistry THE UNIVERSITY OF BRITISH COLUMBIA November 2005 © Lu Yang, 2005 Abstract n SHIP is a 145 kDa SH2-domain-containing inositol 5-phosphatase. It has been proposed that upregulation of SHIP activity with small molecule agonists of SHIP could control inflammation. At the outset of the research there were no known modulators of SHIP activity. A library of marine invertebrate extracts was screened for SHIP activators. A previously described meroterpenoid, pelorol, that represents the first known SHIP activator was isolated from a tropical sponge Dactylospongia elegans (Thiele, 1899). In vitro studies of pelorol have shown that it is able to activate SHIP 'S enzyme activity in intact cells and inhibits activation of macrophages, an essential component of the innate and acquired immune response in inflammatory diseases. A total synthesis of pelorol and its analogs was undertaken in order to flesh out the structure activity of this family of anti-inflammatory compounds. One of these analogs, A Q X - 1 6 A , showed a 3-fold higher activation of SHIP than pelorol at the same molar concentration. It represents a novel class of drugs which activates a physiologically important negative regulator of the PI3K pathway in hemopoietic cells. Stereoselectivity is an important impact factor in drug action. Several stereo- and regio- isomers of pelorol and its analogs have been synthesized to answer the questions of how Ill the regiochemistry of the aromatic ring of pelorol affects its activity and whether the stereochemistry of the C-ring is important for the SHIP activating properties of pelorol. Contignasterol was isolated by our group in 1992 from the sponge Petrosia contignata collected in Papua New Guinea. It was the first example of an emerging family of sponge steroids that have a number of unprecedented structural features. Contignasterol was found to inhibit the release of histamine from sensitized rat mast cells stimulated with anti-lge. IZP576-092, an antiasthma drug developed from the contignasterol lead structure, is now in Phase II human clinical trials. The structure of contignasterol was initially solved by interpretation of spectroscopic data. At the time, the absolute configuration of the side chain chiral centers was not determined. A reinvestigation of the structure of contignasterol using a combination of spectroscopic analysis and chemical degradation has now resulted in the determination of the complete absolute configuration of the molecule. OH 4.1 Contignasterol iv TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations xvii Acknowledgements xxi Chapter 1: General Introduction 1 1.1. Introduction 1 1.2. Plant and Marine Secondary Metabolites 3 1.3. Microbial Secondary Metabolites 4 1.4. Drugs from the S e a 5 1.5. Anti-inflammatory Marine Natural Products 10 1.6. References 16 Chapter 2: Synthesis of Pelorol and Analogs 19 2.1. A Brief Review of the SHIP A s s a y 19 2.2. Introduction 23 2.3. Isolation and Characterization of Pelorol (2.1) 30 2.3.1. Isolation 30 V 2.3.2. Characterization 36 2.4. Total Synthesis of Pelorol 39 2.4.1. Introduction 39 2.4.2. Proposed Biogenesis of Pelorol 40 2.4.3. Proposed Synthesis of Pelorol 42 2.4.4. Synthetic results 45 2.5. Biological Activities of Pelorol and its Analog AQX-16A (2.63) 68 2.6. Experimental 78 2.7. References 103 Chapter 3: Structure Activity Relationship Study of Pelorol 108 3.1. Introduction 108 3.2. Synthesis of C-8 and C-9 Stereoisomers of Pelorol 112 3.3. Synthesis of Stereoisomers of 2.63 (AQX-16A) 124 3.4. Synthesis Desmethyl-2.63 and its Stereoisomers 127 3.5. Synthesis of a Regioisomer of Pelorol 130 vi 3.6. Synthesis of Akaol 133 3.7. Biological Activities of Pelorol analogs and Discussion of S A R 139 3.7.1. In vitro activation of SHIP 139 3.7.2. Inhibition of LPS-stimulated T N F a production from J774 141 macrophage : 3.7.3. Discussion of S A R 143 3.8. Experimental 145 3.9. References 187 Chapter 4: Absolute Configuration of Contignasterol 188 4.1. Introduction 188 4.2. Absolute Configuration of Moiety A 191 4.3. Absolute Configuration of Moiety C 194 4.4. Absolute Configuration of Moiety B 209 4.5. Experimental 212 4.8. References 222 VII Summary 224 Appendix 226 VIII LIST OF T A B L E S Table 1.1. Marine-Derived Natural Products in Clinical and Preclinical Trials 8 Table 2.1. N M R Data for 2.1 and 2.4 recorded in C D C I , 35 Table 4.1. Selected N M R Data for (R), (S ) -MPA esters (4.16)and (4.17), (R), 198 ( S ) - M T P A esters (4.18)and (4.19) recorded at 4 0 0 M H z in C D 2 C I 2 a t 300K Table 4.2. Selected N M R Data for Dithioacetal (4.15) Recorded at 400MHz in 201 C D 2 C I 2 a t 300K Table 4.3. Selected Chemical Shifts of (R) -MPA ester recorded in C D 2 C I 2 at 207 different temperatures ix LIST OF FIGURES Figure 2.1. Enzymatic synthesis and degradation of PI- 3.4.5-P3 20 Figure 2.2. SHIP assay-guided isolation of Sesquiterpenes from D.elegans 23 Figure 2.3. Summary of sesquiterpene-hydroquinone (-quinone) substructures isolated from D.elegans 25 Figure 2.4. Proposed biogenesis of pelerol related compounds from D.elegans 26 Figure 2.5. Sesquiterpenes from D.elegans with various biological activities 28 Figure 2.6. Sesquiterpenes from D.elegans with various biological activities (continued) 29 Figure 2.7. Image of the sponge Dactylospongia elegans (Thiele, 1899) 30 Figure 2.8. 1 H N M R of pelorol (2.1) isolated from sponge D. elegans recorded in C D C I 3 at 4 0 0 M H z 33 Figure 2.9. 1 3 C N M R of pelorol (2.1) isolated from sponge D. elegans recorded in C D C I 3 at 100MHz 34 Figure 2.10. Proposed biogenesis of Pelorol (2.1) 41 Figure 2.11. Overman's study on polyene tetracyclization 42 Figure 2.12. Retrosynthetic analysis of pelorol (2.1) 43 Figure 2.13. Synthetic route to 2.18 45 Figure 2.14. Synthetic route to 2.28 47 Figure 2.15. Synthetic route to 2.33 48 X Figure 2.16. Synthetic route to 2.34 49 Figure 2.17. Lewis acid catalyzed cyclization of 2.34 50 Figure 2.18. Proposed introduction of carboxylate at C-20 of 2.36 51 Figure 2.19. Synthetic route to 2.38 52 Figure 2.20. Synthetic route to 2.41 53 Figure 2.21. Synthetic route to 2.42 and 2.43 54 Figure 2.22. Synthetic route to 2.45 54 Figure 2.23. Proposed synthetic route to 2.42 via 2.19 55 Figure 2.24. Chackalamannil's approach to 2.19 from (+)-sclareolide 56 Figure 2.25. Synthetic route to 2.52 from 2.20 57 Figure 2.26. Synthetic route to 2.19 59 Figure 2.27. Synthetic route to pelorol analog 2.57 60 Figure 2.28. Synthetic route to 2.58 61 Figure 2.29. Synthetic route to 2.63 61 Figure 2.30. Synthetic route to 2.67 with proposed mechanism 63 xi Figure 2.31. Synthetic route to 2.70 with proposed mechanism 64 Figure 2.32. Synthetic route to pelorol (2.1) 65 Figure 2.33. Comparison of 1 H N M R of natural and synthetic pelorol (2.1) recorded in C D C I 3 at 4 0 0 M H z 66 Figure 2.34. Comparison of 1 3 C N M R of natural and synthetic pelorol (2.1) recorded in C D C I 3 at 100MHz 67 Figure 2.35. Pelorol and A Q X - 0 1 6 A increase in vitro SHIP enzyme activity.. 68 Figure 2.36. A Q X - 0 1 6 A stimulates SHIP 'S enzyme activity in intact cells 69 Figure 2.37a, b A Q X - 0 1 6 A inhibits activation of S H I P + / + but not SHIP"7" macrophages and mast cells 70 Figure 2.37c. A Q X - 0 1 6 A inhibits activation of S H I P + / + but not SHIP"'" macrophages and mast cells 71 Figure 2.37d. A Q X - 0 1 6 A inhibits activation of S H I P + / + but not SHIP"7" macrophages and mast cells 72 Figure 2.38. A Q X - 0 1 6 A inhibits PI3K-dependent signaling in S H I P + / + but not SHIP"7" macrophages and mast cells 73 Figure 2.39. A Q X - 0 1 6 A is protective in mouse models of septicemia and acute cutaneous anaphylaxis 74 Figure 2.40. A Q X - 0 1 6 A potentiates the cytostatic effect of Gleevec on B C R - a b l expressing cells 76 Figure 3.1. Evaluation of the differentiation-inducing activities of sesquiterpene quinones by the induction of hemoglobin production in K562 cells 109 Figure 3.2. Preliminary S A R study of pelorol by evaluation of their ability to supress T N F a production in murine mast cells stimulated wi th lgE. 110 Figure 3.3. Synthetic route to 3.2 113 XII Figure 3.4. Synthetic route to 2.54 114 Figure 3.5. Synthetic route to 3.3a and 3.3b 114 Figure 3.6. Synthetic route to 3.4 and 3.5 115 Figure 3.7. Expansion of the N O E S Y spectrum for 3.4 117 Figure 3.8. Expansion of the N O E S Y spectrum for 3.5. 118 Figure 3.9. Synthetic route to 3.6 and 3.7 119 Figure 3.10. Expansion of the N O E S Y spectrum for 3.6 120 Figure 3.11. Expansion of the N O E S Y spectrum for 3.7 121 Figure 3.12. Synthetic route to 9epi-pelorol (3.10) and 8epi-pelorol (3.13)... 122 Figure 3.13. Synthetic route to 3.14a and 3.14b 124 Figure 3.14. Synthetic route to 3.19 and 3.20 125 Figure 3.15. Synthetic route to 3.25 127 Figure 3.16. Synthetic route to 3.26a and 3.26b 128 Figure 3.17. Synthetic route to 3.31 and 3.32 128 Figure 3.18. Model study in synthesis of pelorol regioisomer 130 Figure 3.19. Model study in synthesis of pelorol regioisomer 131 XIII Figure 3.20. Model study in synthesis of pelorol regioisomer 131 Figure 3.21. Proposed synthetic route to akaol (3.43) 133 Figure 3.22. Synthetic route to 3.48 134 Figure 3.23. Model study of 3.48 under various cyclization conditions 135 Figure 3.24. Model study of 3.26a and 3.26b under various cyclization conditions 136 Figure 3.25. Synthetic route to 3.50 136 Figure 3.26. Synthetic route to 3.51 137 Figure 3.27. Synthetic route to akaol (3.43) 137 Figure 3.28. In vitro SHIP activation activities of pelorol and its analogs 139 Figure 3.29. Inhibition of LPS-stimulated T N F a production from J774 macrophage by pelorol and its analogs 141 Figure 3.30. Structure activity relationship of Pelorol 144 Figure 4.1. Separate coupling systems A , B, and C of Contignasterol 190 Figure 4.2. Synthesis of contignasterol 3,6,7-tri-p-methoxybenzoate 191 Figure 4.3. C D spectra of congansterol and contignasterol 3,6,7-tri-p-methoxybenzoate in E t O H . 191 Figure 4.4. Negative chirality between the three p-methoxybenzoate x iv c h r o m o p h o r e s o n m o i e t y A 192 F i g u r e 4 . 5 . D e r i v a t i z a t i o n of c o n t i g n a s t e r o l h e m i a c e t a l a l c o h o l to ( R ) - a n d ( S ) - M T P A e s t e r s (4.13 a n d 4.14) 194 F i g u r e 4 . 6 . S e l e c t e d A8 R S 1 H N M R v a l u e s in p p m fo r M T P A e s t e r s 4.13 a n d 4.14. (A6RS= 6 {R).MPA - 6 (S)-MPA) 1 9 5 F i g u r e 4 . 7 . C o n t i g n a s t e r o l s i d e c h a i n e x i s t s in e q u i l i b r i u m b e t w e e n c y c l i c h e m i a c e t a l a n d the o p e n c h a i n 2 2 - h y d r o x y - 2 9 - a l d e h y d e f o r m . . 1 9 6 F i g u r e 4 . 8 . C h e m i c a l c o n v e r s i o n o f t e t r a a c e t a t e 4.12 into t he M P A e s t e r s 4.16 a n d 4.17 a n d the M T P A e s t e r s 4.18 a n d 4.19 1 9 7 F i g u r e 4 . 9 . 1 H N M R c o m p a r i s o n of ( R ) - a n d ( S ) - M P A e s t e r r e c o r d e d at 3 0 0 K i n C D 2 C I 2 1 9 9 F i g u r e 4 . 1 0 . 1 H N M R c o m p a r i s o n of ( R ) - a n d ( S ) - M T P A e s t e r r e c o r d e d at 3 0 0 K in C D 2 C I 2 2 0 0 F i g u r e 4 . 1 1 . M o d e l s p r o p o s e d b y t he M o s h e r w i th e x p e c t e d s i g n of A5 S R a n d A5 R S 201 F i g u r e 4 . 1 2 . S e l e c t e d A5 1 H N M R v a l u e s in p p m fo r t h e M P A e s t e r s 4.16 a n d 4.17 a n d t he M T P A e s t e r s 4.18 a n d 4.19 2 0 3 F i g u r e 4 . 1 3 . C o m p a r i s o n of s i d e c h a i n c h e m i c a l sh i f ts o f t he c o n t i g n a s t e r o l de r i va t i ve 4.22 a n d I z zo ' s s y n t h e t i c p r o d u c t 4.23 2 0 4 F i g u r e 4 . 1 4 . ( R ) - M P A e s t e r of ch i ra l s e c o n d a r y a l c o h o l e x i s t s a s two m a i n c o n f o r m e r s ( sp a n d a p ) 2 0 5 F i g u r e 4 . 1 5 . 1 H N M R c o m p a r i s o n of ( R ) - M P A e s t e r r e c o r d e d at 3 0 0 K , 2 7 3 K , 2 5 3 K , 2 2 3 K , a n d 2 0 3 K in C D 2 C I 2 2 0 8 F i g u r e 4 . 1 6 . N e w m a n p ro jec t i ons for the C - 2 0 / C - 2 2 b o n d in the d i t h i ane 5 in w h i c h C - 2 2 h a s t he R con f i gu ra t i on a n d H - 2 0 a n d H - 2 2 a r e anti 2 0 9 F i g u r e 4 . 1 7 . T h e f r a g m e n t s t ruc tu re o f 4 . 1 5 w i th s e l e c t e d N O E S Y co r re l a t i on 211 F i g u r e 4 . 1 8 . E x p a n s i o n of t he N O E S Y s p e c t r u m fo r 4.15 211 F i g u r e A . 1 . 1 H N M R of 2.36 r e c o r d e d in C D C I 3 at 4 0 0 M H z 2 2 7 XV Figure A .2 . 1 3 C N M R of 2.36 recorded in C D C I 3 at 100MHz 228 Figure A .3 . 1 H N M R of 2.42 recorded in C D C I 3 at 4 0 0 M H z 229 Figure A.4 . 1 3 C N M R of 2.42 recorded in C D C I 3 at 100MHz 230 Figure A .5 . 1 H N M R of 2.57 recorded in C D C I 3 at 4 0 0 M H z 231 Figure A.6 . 1 3 C N M R of 2.57 recorded in C D C I 3 at 100MHz 232 Figure A . 7 . 1 H N M R of 2.61 recorded in C D C I 3 a t 4 0 0 M H z . . 233 Figure A . 8 . 1 3 C N M R of 2.61 recorded in C D C I 3 a t 100MHz 234 Figure A .9 . 1 H N M R of 2.62 recorded in C D C I 3 at 4 0 0 M H z 235 Figure A.10. 1 3 C N M R of 2.62 recorded in C D C I 3 at 100MHz 236 F igureA.11 . 1 H N M R of 2.63 recorded in C D C I 3 at 4 0 0 M H z 237 Figure A.12. 1 3 C N M R of 2.63 recorded in C D C I 3 at -100MHz 238 Figure A.13. 1 H N M R of 3.19 recorded in C D C I 3 at 4 0 0 M H z 239 Figure A.14. 1 3 C N M R of 3.19 recorded in C D C I 3 at 100MHz 240 Figure A . 15. 1 H N M R of 3.20 recorded in C D C I 3 at 4 0 0 M H z 241 Figure A.16. 1 3 C N M R of 3.20 recorded in C D C I 3 at 100MHz 242 Figure A . 17. 1 H N M R of 3.25 recorded in C D C I 3 at 4 0 0 M H z 243 Figure A.18. 1 3 C N M R of 3.25 recorded in C D C I 3 at 100MHz 244 Figure A.19 . 1 H N M R of 3.31 recorded in C D C I 3 at 4 0 0 M H z 245 xvi Figure A.20. 1 3 C N M R of 3.31 recorded in C D C I 3 at 100MHz 246 Figure A.21. 1 H N M R of 3.32 recorded in C D C I 3 at 4 0 0 M H z 247 Figure A.22. 1 3 C N M R of 3.32 recorded in C D C I 3 at 100MHz 248 xvii LIST OF ABBREVIATIONS 0 -degree(s) 1D -one-dimensional 2D -two-dimensional N 2 5D -specific rotation at wavelength of sodium D line at 2 5 ° C A c -acetate A c O H -acetic acid A c 2 0 -acetic anhydride AIDS -aquired immune deficiency syndrome AICI3 -aluminum chloride A M L -acute myelogenous leukemia Ar -argon B B r 3 -boron bromide B F 3 . E t 0 2 -boron fluoride etherate B H 3 -boron hydride B l 3 -boron iodide B M M C s -bone-marrow-derived mast cells B M m s O s -bone-marrow-derived macrophages br -broad B r 2 -bromine brs -broad singlet C -concentration ° C -degrees celcius calcd -calculated C D -cyclodextrin or circular dichroism CDCI3 -deuterated chloroform C D 3 C N -deuterated acetonitrile C 6 D 6 -deuterated benzene ( C F 3 C O ) 2 0 -trifluoroacetic anhydride C H -methine C H 2 -methylene C H 3 -methyl C H 2 C I 2 -dichloromethane cm"1 -wavenumbers C M L -chronic myelogenous leukemia C O S Y - g r -gradient selected two-dimensional correlation spectroscopy 5 -chemical shift in parts per million d -doublet D A H P -3-deoxy-D-arabino-heptulosonic acid 7-phosphate D C C -dicyclohexylcarbodiimide dd -doublet of doublets D I B A L - H -diisobutylaluminum hydride D M A P -4-(dimethylamino)pyridine D M F -A/,A/-dimethylformamide D N A -deoxyribonucleic acid D N F B -dinitrofluorobenzene X V I I I D 2 0 -deuterium oxide dt -doublet of triplets D M S O -dimethyl sulphoxide D M S O - d 6 -deuterated dimethyl sulphoxide Av -chemical shift difference at T c € -extinction coefficient E D T A -ethylenediaminetetraacetic acid E L I S A -enzyme linked immuno sorbent assay E t 3 N -triethyl amine E t 2 0 -diethyl ether E t O A c -ethyl acetate F D A -Food and Drug Adminstration F P P -farnesyl diphophate g -gram(s) H 2 -hydrogen HCI -hydrochloric acid H 2 0 -water H 2 0 2 -hydrogen peroxide HIV -human immunodeficiency virus H M B C -two-dimensional heteronuclear multiple bond correlation spectroscopy H M Q C -two-dimensional heteronuclear multiple quantum coherence spectroscopy H P L C -high-performance liquid chromatography H R E I M S -high-resolution electron impact mass spectrometry H R E S I M S -high-resolution electrospray ionisation mass spectrometry H R F A B M S -high-resolution fast atom bombardment mass spectrometry H S A -human serum albumin H S Q C -two-dimensional heteronuclear single quantum coherence spectroscopy H T S -high-throughput screening Hz -hertz IBX -o-iodoxybenzoic acid igE -immunoglobulin E IL-3 -interleukin 3 IP 4 -inositol-1,3,4,5-tetrakiphosphate IPs -inositol-1,3,4-trisphosphate J -coupling constant in hertz K -degrees Kelvin K 2 C 0 3 -potassium carbonate L D A -lithium diisopropylamine L P S -lipopolysaccharide ^max -wavelength at maximum intensity m -multiplet M + -molecular ion m C P B A -m-chloroperbenzoic acid M - C S F -macrophage colony stimulating factor MeAICI 2 -methyl aluminum dichloride xix M e C N -acetonitrile Mel -methyl iodide M e O D - d 4 -deuterated methanol M e O H -methanol mg -milligram(s) M g S 0 4 -magnesium sulphate M H z -megahertz mL -millilitre(s) mmol -millimol(s) M o O P H - oxodiperoxomolybdenum (pyridine)-(hexamethylphosphoric triamide) mp -melting point M P A -methoxy phenyl acetic acid M S -mass spectrometry M T G -monothioglycolate m/z -mass to charge ratio N -normal N 2 -nitrogen Na -sodium N a B H 4 -sodium borohydride N a C N B H 3 -sodium cyanoborohydride N a H C 0 3 -sodium bicarbonate N a O H -sodium hydroxide NHPI -N-hydroxyphthalimide nBuLi -n-butyl lithium nm -nanometre(s) N M R -nuclear magnetic resonance N O -nitric oxide N O E S Y -Nuclear Overhauser enhancement spectroscopy O R D -optical rotatory dispersion P C C -pyridinium chlorochromate Pd on C -palladium on charcoal P E P -phosphoenolpyruvate P G E 2 -prostaglandin E 2 P H -pleckstrin homology PI-3,4,5-P 3 -phosphatidylinositol-3,4,5-triphosphate PI-4,5-P 2 -phosphatidylinositol-4,5-bisphosphate PI3K -phosphatidylinositol-3 kinase P L A 2 -phospholipase A 2 P M A -phorbol myristate acetate P P A -polyphosphoric acid n P r O H -1-propanol p.s.i. -pounds per square inch P T B -phophotyrosine binding P T E N -phosphatase and tensin homologue deleted on chromosome ten P t 0 2 -platium oxide q -quartet X X R h / C -rhodium on charcoal (R) -MPA -(R)- a-Methoxy-a-phenylacetic acid (R)-MTPACI - ^-(-J-a-Methoxy-a-^rifluoromethyOphenylacetyl chloride R O E S Y -rotating frame N O E S Y R T -room temperature R T X -resiniferatoxin s -singlet S A M -S-Adenosylmethionine S A R -structure-activity relationship Sc (OTf) 3 -scandium triflate S C U B A -self-contained underwater breathing apparatus S E M -standard error in the mean S e 0 2 -selenium dioxide SHIP -Src Homology 2-containing Inositol 5'-Phosphatase (S) -MPA -(S)- a-Methoxy-a-phenylacetic acid (S) -MTPACI - fS)-(-)-a-Methoxy-a-(trifluoromethyl)phenylacetyl chloride S n C U -tin tetrachloride - s p 2 hybrid orbital sp 3 - sp 3 hybrid orbital t -triplet tBuLi -tert-butyl lithium T F A -trifluoroacetic acid T H F -tetrahydrofuran T L C -thin-layer chromatography T M E D A -N,N,N',N'-tetramethylethylenediamine T M S -tetramethylsilane T N F a -tumor necrosis factor a T s O H -p-toluenesulfonic acid (j,M -micromolar -microgram(s) ML -microlitre(s) U V -ultraviolet V T -varied temperature w -watt(s) Z n l 2 -zinc iodide xxi ACKNOWLEDGEMENTS For his selfless devotion of time and energy to my education I offer my most sincere thanks to my supervisor, Professor Raymond J . Andersen. My respect and admiration are immeasurable. T o Professors Alice F. Mui, Michel Roberge I extend my genuine thanks for their long time collaboration that makes this dissertation possible. T o Dr. Dave E . Williams and Mr. Mike Leblanc I am indebted for their invaluable helps and suggestions over past few years. T o Dr. Christopher Gray I give my appreciation for his helpful proof-reading of this dissertation. T o other members of the Andersen lab I owe my most heartfelt gratitude. I am certain that I will never again have the opportunity to be surrounded by such a nice and brilliant group of people. The privilege of calling them my colleagues has been the greatest reward of my graduate education. T o my wife and parents I owe whatever measure of success I've managed to achieve. My accomplishments are theirs. Chapter 1: General Introduction. 1 Chapter 1: General Introduction. 1.1 Introduction The prevalence of fatal diseases such as cancer and AIDS, for which no effective cures are available, has stimulated tremendous interest among researchers in natural products chemistry, pharmacology, and biochemistry in discovering new chemotherapies. Nature has been the prime source of such discoveries for centuries. Terrestrial plants and other materials are still used for the preparation of galenicals in many countries where traditional medicine is important, such as China and India. A s chemical techniques have improved, the active constituents of traditional medicines have been isolated from the original source, structurally characterized, and synthesized in the laboratory. Drugs of natural origin have been classified into three groups: the original active natural products, semi-synthetic analogues produced by chemical modification of natural products to give more potent and better tolerated drugs, and total synthetic analogues based on natural product pharmacophores. 1 Natural products make a core contribution on a global basis to the maintenance and enhancement of human health by providing drugs for treating diseases and nutritional supplements to increase the efficacy of foods. This is particularly evident in the areas of cancer and infectious diseases, where over 60% and 75% of current drugs used to treat these diseases, respectively, are of natural origin. 2 Chapter 1: General Introduction. With new natural products consistently being discovered in substantial numbers, this field has been broadened and diversified to include more scientific directions. These include research in the areas of chemical ecology, biosynthesis, total synthesis, enzymology, spectroscopy, and genetics. Natural products are often referred to as 'secondary metabolites', a term invented by plant physiologists and brought into general use for microbial products by J . D. Bu'Lock in 1961. 3 This terminology is often used in reference to small chemical substances found in nature that have distinct pharmacological effects, such as the antibiotic penicillin. 4 Secondary metabolites are not necessarily produced under all conditions. Unlike the intermediates and cofactors that take part in cell-structure syntheses and energy transduction, these substances are not essential for growth and reproductive metabolism. From this perspective, the majority of secondary metabolites are considered to show no benefit to the producer. 4 However, the opposite view, now widely held, is that every secondary metabolite is present because it displays a biological activity at some stage in evolution that endows the producer with increased fitness. 5 Even though in the vast majority of cases, the function of these compounds and their benefit to the organism is still not yet known, some are undoubtedly produced for easily appreciated reasons, e.g. as toxic materials providing defense against predators, as volatile attractants towards the same or other species, or as colouring agents. Chapter 1: General Introduction. ^ 1.2 Plant and marine secondary metabolites Natural products researchers first focused their attention on plants due to the abundance of the source materials and relatively easy methods of collection. Plants are major sources of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides. 6 It has been reported that up to 80% of the population in developing countries are totally dependent on plants for their primary health care . 7 Despite the remarkable progress in the synthetic organic chemistry of the twentieth century, it is still the case that over 25% of prescribed medicines in U S are derived from plants, directly or indirectly. 7 A high profile example of a plant secondary metabolite is paclitaxel (1.1), which is a complex diterpene isolated from the bark of the Taxus brevifolia. It is a widely used anticancer agent due to its unique mode of action as a stabilizer of microtubules. 8 The discovery that marine organisms constitute a rich source of previously undescribed secondary metabolites is not entirely surprising, considering that many algal and invertebrate phyla reside exclusively in the s e a . 9 Today, both academic and industrial interest in marine organisms is on the rise, given the fact that the findings of unique, biologically active marine secondary metabolites are increasing. Many of these compounds have shown significant pharmacological Chapter 1: General Introduction. 4 activities and some have reached advanced stages of clinical trials. A prominent example is didemnin B (1.2),10 a member of the family of cytotoxic cyclic depsipeptides isolated from the Caribbean tunicate Trididemnum solidum, which progressed to phase II clinical trials as an anticancer drug. 1.2 Didemnin B 1.2 Microbial secondary metabolites Even though natural drugs derived from microorganisms have a much shorter history than from plants, the investigation into secondary metabolites isolated from microorganisms has dramatically increased the number of novel bioactive compounds. Microbially produced antibiotics account for a very high portion of the drugs commonly prescribed nowadays. Microbial secondary metabolites have been found to have a variety of other biological activities as wel l . 1 1 The discovery of penicillin (1.3), a /?-lactam compound first found to be produced by a mold called Penicillium notatum by a British scientist Alexander Fleming in 1928, 1 2 has become recognized as one of the greatest advances in medical Chapter 1: General Introduction. 5 history. However, the major impact goes back to the mass production and clinical utilization of penicillin produced by Penicillium chrysogenum, which is presently used in producing penicillin commercially, during the second half of the last century. 1 3 Since then, more and more compounds with microbial origins are used due to their effect on the health, nutrition and economics of our society. 1.3 Drugs from the sea With approximately 70% of our earth's surface covered with marine ecosystems, which also represent 95% of the biosphere, we are living on a planet of oceans . 1 4 Until the 1960s, our understanding of marine natural products chemistry was quite limited, largely due to technical barriers to collecting the source organisms. The situation changed with the advent of S C U B A which first found its scientific utilization in marine biology 60 years ago. In the mid-1960s, natural products chemists also started moving their eyes from terrestrial organisms to their marine counterparts. 1 5 Inspired by the pioneering work of the late Dr. John Faulkner, Dr. Paul J . Scheuer, etc. on bioactive marine natural products, over the past three decades, we have witnessed well over 14000 new natural products being isolated from marine organisms. 1 6 Most of these compounds have unique structures without direct precedent from terrestrial organisms. Moreover, at least H O' 1.3 Penicillin COOH Chapter 1: General Introduction. ° 300 patents have been issued between 1969 and 1999 covering mainly potential anticancer agents from the s e a . 1 5 Although the successful commercialization of the sponge-derived cytosine arabinoside ( A r a - C ) , 1 7 as a potent antileukemic agent, and its analog adenine arabinoside (Ara-A), as an antiviral drug, has validate the potential in drug discovery from the sea, it could not be regarded as the milestone in this field, since the real break-through in marine drug discovery has not yet happened. Since the early 1980s, a significant number of marine-derived compounds entered into preclinical and clinical antitumor trials. Prialt, generically called ziconotide, 1 8 is the synthetic form of the cone snail peptide w-cenotoxin M-VII-A and the first in a new class of painkillers approved by the F D A in December 2004. There are 20 (Table 1.1, see page 8) marine natural products currently in different stages of clinical trials, mostly targeted towards cancer, pain, and inflammatory diseases, and there is an encouraging and still increasing list of marine natural products that are in preclinical trials. It seems possible that more marine natural products will be licensed for clinical use in the near future. The most promising candidates are ecteinascidin 743 (ET-743) and Neovastat. Ecteinascidin 743 (1.4), a tetrahydroisoquinoline alkaloid isolated from the colonial tunicate Ecteinascidia turbinate,1920 is the only known chemotherapy agent that binds to the minor groove of D N A , bending the D N A towards the major groove. It exerts its therapeutic effect through several pathways, including interference with cellular transcription-coupled nucleotide excision repair ( T C -Chapter 1: General Introduction. ' N E R ) to induce cell death, causing slowing and arrest of tumour cell division and subsequent p53-independent apoptosis (programmed cell death), and inhibiting transcriptional activation of inducible genes . 2 1 Early efforts at drug development of ET-743 were very difficult because 1 tonne (wet weight) of E. turbinata yielded only 1 g of the promising anti-tumor agent ET-743. This example illustrates a serious problem associated with development of drugs from marine natural products, namely the 'supply problem'. Close inspection of Table 1.1 reveals that since the majority of promising compounds have complex structures, it would be a formidable task in most cases to provide enough material for drug development by total synthesis. The first total synthesis of ET-743 was accomplished by Corey and co-workers using a multistep enantiocontrolled process . 2 2 However, the 'supply problem' was solved by Pharma Mar chemists through a semisynthesis from cyanosafracin B (1.5), 2 3 a metabolite of the marine microbe Pseudomonas fluorescens. Their 21-step synthetic route provided the large scale preparation of material required for HO' 1.4 Ecteinascidin 743 1.5 Cyanosafracin B Chapter 1: General Introduction. ° clinical trials. Although this approach can not be generalized, in certain instances it could be expected to be a satisfactory solution to the supply problem. Another approach to solving the supply problem is mariculture. A notable example of this approach is the aquaculture of the bryozoan Bugula neritina (the source of the bryostatins), 2 4 which can provide 100-200g of bryostatin 1 per year for each aquaculture unit at a cost of $700,000. This approach can be extremely useful when mated with chemical synthesis to find new analogs with enhanced potencies or lower toxicities. T h e last but not the least important approach to solve the supply problem is to synthesize a basic core structure by aiming to mimic some properties of natural compounds, then using this as a template for combinatorial chemistry to generate a pool of chemical entities that is substantially more diverse and has greater biological relevance than a pure combinatorial chemistry library. W e have come a long way in our understanding of marine natural products chemistry since the identification of the Caribbean sponge Cryptotheca crypta derived antiviral compounds in the 1950s. 1 5 Given the enormous bio-diversity of the oceans, more novel marine natural products with various bioactivities will be identified and this vast repository of esoteric structures will always complement terrestrial natural products chemistry. Table 1.1 Marine-derived natural products in clinical and preclinical trials1 5 , 1 Coumpound name Compound Type Source Discovering laboratory Clinical Trial Ara-A Nucleoside Gorgonian Glaxo Smith-Kline In Clinical Use Ara-C Nucleoside Sponge Pharmacia In Clinical Use Chapter 1: General Introduction. 9 Ziconotide NRP Mollusc Olivera-Pfizer In Clinical Use Didemnin B PK-NRPS Tunicate Rinehart Phase II (dropped 90s) Dolastatin 10 NRP Sea Hare Pettit Phase II (dropped 90s) Girolline Imidazole Sponge' Potier Phase I (dropped 90s) deriv. Manoalide Terpene Sponge Scheuer-Allergan Phase II (dropped 90s) Bengamide derivative PK-NRPS Sponge Crews Phase I (dropped 2002) Cryptophycins NRP Algea Merck Phase I (dropped 2002) CGX-1007 NRP Mollusc Olivera Phase I (discontinued) Ecteinascidin 743 Alkaloid Tunicate Rinehart-PharmaMar; Phase III Ortho Biotech (J&J) Neovastat Mixture Shark Sorbera Phase III Bryostatin 1 PK-NRPS Bryozoan Pettit Phase II Cematodin NRP Synthetic deriv. BASF Pharma Phase II of dolastatin15 Dehydrodidemnin B PK-NRPS Tunicate Rinehart-PharmaMar Phase II (Aplidin) Hemiasterlins A&B NRP Sponge Andersen-Wyeth Phase II IPL-576092 (synthetic) Terpene Sponge Andersen-Aventis Phase II IPL-512602 Terpene Deriv. of 576092 Andersen-Aventis Phase II Kahalalide F NRP Mollusc Scheuer-PharmaMar Phase II Squalamine Aminosteroid Dogfish Shark Zasloff Phase II Synthadotin NRP Synthetic deriv. BASF Pharma Phase II of dolastatin 15 TZT-1027 NRP Synthetic deriv. Teikoku Hormine Phase II of dolastatin 15 Agelasphin derivative Glycosphingo Sponge Kirin Phase I CGX-1160 NRP Mollusc Cognetix Phase I Discodermolide PK Sponge Pomponi-Norvatis Phase I E7389 PK Synthetic Umeru, Kishi-Eisai Phase I halichondrin B deriv. GTS-21 Alkaloid Synthetic Kem Phase I IPL-550260 Terpene Deriv. of 576092 Andersen-Aventis Phase I NVP-LAQ824 Synthetic Schmitz & Crews Phase I Spisulosine Alkylamino Mollusc PharmaMar Phase I Alpkinidine Alkaloid Sponge Crews-Valeriote Preclinical Aplyronine PK Sea Hare Yamada Preclinical Ascididemnin Alkaloid Sponge Delfourne Preclinical Curacin PK-NRP Cyanophyte Gerwick-Wipf Preclinical Diazonamide NRP Tunicate Fenical Preclinical Dictyodendrins Alkaloid Sponge Fusetani Preclinical Eleutherobin Terp-Alkaloid Soft Coral Fenical Preclinical Fascaplysins Alkaloid Sponge Crews-Valeriote Preclinical Isogranulatamide Alkaloid Tunicate Andersen Preclinical Isohomohalichondrin B PK Sponge Munro-Blunt Preclinical Laulimalide/Fijianolide PK Sponge Mooberry-Crews Preclinical 5-Methoxyamphimdeine Alkaloid Sponge Crews-Valeriote Preclinical Motupramines Alkaloid Sponge Andersen Preclinical Peloruside A PK Sponge Battershill Preclinical Pelorol Terpene Sponge Andersen Preclinical Salicyclihalimides A and R PK Sponge Boyd Preclinical D Salinosporamide PK-NRP Actinomycete Fenical-Nereus Preclincal Sacordictin Terpene Sponge Andersen Preclinical Thiocoraline NRP Actinomycete Canedo-PharmaMar Preclinical Variolin B Alkaloid Sponge Munro-PharmaMar Preclinical Vitilevuamide NRP Sponge Ireland Preclinical CGX-1063 NRP Synthetic deriv. Cognetix Preclinical of 1007 AMM336 Mollusc Preclinical X-conotoxin Mollusc Preclinical CGX-1063 NRP Synthetic deriv. Cognetix Preclinical ACV1 Mollusc Univ. of Melbourne Preclinical Chapter 1: General Introduction. 10 1.5 Antiinflammatory marine natural products Inflammation is caused by trauma of some kind which causes tissue injury. Our understanding of inflammation can be traced back to the first century when Celsus described the inflammatory response in terms of cardinal signs of redness, oedema, heat, pain, and loss of function. Since then, innumerable individuals in inflammation research have described many of the mechanisms associated with tissue injury. These include Lewis (1881-1945), who described the Triple Response; Cohnheim and Samuel (late 1880s), who reported on leukocyte emigration and vascular permeability; and Metchnikoff (1845-1916), who defined phagocytosis . 2 6 A s colorful as the history of inflammation research is, the treatment of inflammatory diseases with natural products has also come a long way since the great physician Hippocrates used salicylates in the form of preparations of willow bark in the treatment of pain, inflammation, and fevers. Aspirin, derived from salicylic acid, is probably the most well-known drug in the world today. It is still in use for treatment of inflammatory diseases. It is note-worthy that natural products and their derivatives from terrestrial origins have been the major source of antiinflammatory drugs currently in clinical u s e . 2 7 T o date, there is no marine natural product that can be found on the shelves of pharmacies for treating inflammatory diseases. T h e reason for this situation can be partly attributed to the coincident situation at present in the field of marine drug development. However, compared with the number of antitumor marine Chapter 1: General Introduction. ' 1 natural products in clinial trials and preclinical research, the number of antiinflammatory marine natural products under evaluation is insignificant. The deeper reason may be that pharmaceutical companies have given top priority to focusing on developing therapies for the fatal diseases, such as cancer. The first antiinflammatory marine natural product emerged in the investigation of the marine sponge Halichondria moorei by a group of Roche scientists. They found fluorine was the major constituent of this sponge and it occurred as K 2 S i F 6 , which was a potent antiinflammatory agent . 2 8 This was followed by another report from the same group on the isolation of three antiinflammatory compounds 6-n-tridecylsalicylic acid, flexibilide, and dendalone 3-hydroxybutyrate from a brown alga, a soft coral, and a dictyoceratid sponge, respectively. 2 9 Since then, various classes of novel marine natural products with different antiinflammatory activities have been reported. A n updated review by Keyzers and Davies-Coleman provided the first holistic overview of the sponge derived antiinflammatory metabolites. 3 0 It is not surprising that antiinflammatory marine natural products are dominated by sponge-derived metabolites, since marine sponges are the cornucopia of novel marine natural products. The forerunners of antiinflammatory sponge metabolites in terms of drug development are manoalide and contignasterol. Manoalide (1 .6) , 3 1 , 3 2 a sesterterpenoid isolated from the Palauan sponge luffariella variabilis, is the first inhibitor of phospholipase A 2 (PLA 2 ) . It was found to irreversibly inhibit the Chapter 1: General Introduction. 12 release of arachidonic acid (the precursor of the acute inflammatory response mediator eicosanoids) from membrane phospholipids by the enzyme P L A 2 , thus terminating the inflammatory response. The chemical basis of manoalide's inhibitory activity is the formation of a Schiff base between the manoalide hemiacetals and a lysine residue of the P L A 2 protein, which prevents the enzyme from binding to membranes. Manoalide was licensed to Allergan Pharmaceuticals, and placed into clinical trials for treatment of psoriasis. It advanced to phase II, but was discontinued due to formulation problems. Contignasterol (1.7) is the first naturally occurring sponge steroid with the 'unnatural' H-14 (5 configuration. 3 3 , 3 4 Shortly after its discovery, a pharmacological evaluation revealed that contignasterol could inhibit the anti-immunoglobulin E stimulated release of histamine from sensitized rat mast cells. Several synthetic OH Chapter 1: General Introduction. 13 derivatives (IPL.576,092 (1.8) and two others) showing the same activity have been advanced to various stages of clinical trials as antiasthma agents by Inflazyme in conjunction with Aventiis Pharma. Izzo et al. recently reported the synthsis of hybrid structures of manoalide and IPL576.092 (eg. 1.9).35 This approach was inspired by the perspective of 'natural product hybrids', that combination of parts of structurally different naturally occurring bioactive products to yield hybrid structures can in principle, exceed the activities of their parent compounds, These analogs inhibited human synovial sPLA2-IIA by 63% at 100u.M and also reduced nitric oxide and P G E 2 (two important mediators of the inflammatory process) by 64 and 72%, respectively, at 10u.M on lipopolysaccharide stimulated human monocytes. However, in consideration of the different mechanisms that manoalide and IPL.576,092 have utilized to inhibit the inflammatory response, Izzo's approach is questionable.35 OH Antiinflammatory marine natural products from many other resources have also been discovered. Some of them have been shown to possess interesting chemistry and remarkable activity. The pseudopterosins (1.10a-d),36 a family of diterpene glycosides from the gorgonian Pseudopterogorgia elisabethea, Chapter 1: General Introduction. 14 exhibited potent antiinflammatory and analgesic activities by inhibiting pancreatic PLA 2 and affecting both the cyclooxygenase and lipoxygenase pathways. 1.10a Pseudopterosin A R1=R2=R3=H 1.10b Pseudopterosin B R-t=Ac, R2=R3=H 1.10c Pseudopterosin C R2=Ac, R1=R3=H 1.1 Od Pseudopterosin D R3=Ac, Ri=R2=H Cyclomarin A (1.11),37 a cyclic peptide produced by a marine bacterium Streptomyces sp., displayed significant antiinflammatory activity in a PMA-induced (PMA-phorbol myristate acetate) mouse ear edema assay and in an in vitro assay. N ° ,0 HN O o i HN' H o MeO1 O 1.11 Cyclomarin A Oxepinamide A (1.12),38 the first of a family of alkaloids isolated from a marine fungal extract, exhibited good antiinflammatory activity in a topical RTX-induced mouse ear edema assay, to name but a few. Chapter 1: General Introduction. 1.12 Oxepinamide A n important requirement for a compound to be approved as a drug is that it is safe to use. However, the antiinflammatory drugs currently on the market all have side-effects. This includes the best selling antiinflammatory drug V I O X X , which increases the relative risk for confirmed cardiovascular events, such as heart attack and stroke. This imposes serious limitations on the use of these drugs. Developing new antiinflammatory drugs with higher potency and lower or no side-effects is the desired outcome for the workers in this field. Chapter 1: General Introduction. 16 1.6 References 1. Cragg, G. M.; Newman, D. J . ; Snader, K. M. J. Nat. Prod. 1997, 60, 52-60. 2. Newman, D.J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66, 1022-1037. 3. Bu'Lock, J. D. Adv. Appl. Microbiol. 1961, 3, 293-342. 4. Vining, L. C. Annu. Rev. Microbiol. 1990, 44, 395-427. 5. Firn, R. D.; Jones, C. G. Molecular Microbiology 2000, 37, 989-994. 6. Vanisree, M.; Lee, C.-Y.; Tsay, H.-S.; Bot. Bull. Acad. Sin. 2004, 45, 1-22. 7. Newman, D.J.; Cragg, G.M.; Snader, K.M. Nat. Prod. Rep. 2000, 7 7, 215-234. 8. Kusama, H.; Hara, R.; Kawahara, S.; Nishimori, T.; Kashima, H.; Nakamura, N.; Morihira, K.; Kuwajima, I. J. Am. Chem. Soc. 2000, 722, 3811-3820. 9. Jensen, P. R.; and Fenical, W. Annu. Rev. Microbiol. 1994, 48, 559-84. 10. Rinehart, K.; Gloer, J. B.; Cook, J. C. J. Am. Chem. Soc. 1981, 103, 1857-1859. 11. Demain, A. L., International Microbiology, 1998, 1,259-264. 12. Newall, C. E.; Hallam, P. D. Comprehensive Medicinal Chemistry (Vol 2), Pergamon Press, Oxford,1990, 609-653. 13. William, M and Bulger, R. J. , Annual Review of Medicine, 1964, 15, 393-412. 14. Ellis, R. Aquagenesis. The origin and evolution of life in the sea; Viking Eds.: New York, 2001. 15. Newman, D. J. ; Cragg, G. M. J. Nat. Prod. 2004, 67, 1216-1238. 16. MarineLit, Version September 2003. A marine literature database produced and maintained by the Department of Chemistry, University of Canterbury, New Zealand. 17. Wolf, S. N.; Marion, J. ; Stein, R. S. Blood. 1985, 65, 1407-1411. 18. http://www.bookofjoe.eom/2004/12/behindthemedspe_30.html 19. Rinehart, K.; Holt, T. G.; Fregeau, N. L; Stroh, J . G.; Kiefer, P. A.; Sun, F.; Chapter 1: General Introduction. 17 Li, L. H. ; Martin, D. G . J. Org. Chem. 1990, 55, 4512-4515. 20. Wright, A . E . ; Forleo, D. A. ; Gunawardana, G . P.; Gunasekera , S. P.; Koehn, F. E . ; McConnel l , O . J . J. Org. Chem. 1990, 55, 4508-4512. 21. van Kesteren, C ; de Vooght, M . M . M . ; Lopez-Lazaro, L . ; Mathot, R. A . A.; Schellens, J . H . M . ; Jimeno, J . M . ; Beijnen, J . H . Anti-Cancer Drugs 2003, 14, 487-502. 22. Corey, E . J . ; Gin, D. Y. ; Kania, R. S. J. Am. Chem. Soc. 1996, 118, 9202-9203. 23. Cuevas , C ; Perez, M . ; Martin, M . J . ; Chicharro, J . L . ; Fernandez-Rivas, C ; Flores, M . ; Francesch, A . ; Gallego, P.; Zarzuelo, M . ; de laCalle, F.; Garcia, J . ; Polanco, C ; Rodruiguez, I.; Manzanares, I. Org. Lett. 2000, 2, 993-996. 24. Mendola, D. Drugs from the sea, Fusetani, N . , E d . ; Karger: Basel , 2000, pp 74-85. 25. Dr.Philip Crews website at http://chemistry.ucsc.edu/mnpr/research.htm 26. Florey, H . W . General Pathology (Lloyd-Luke Ltd, London, 1970) 27. Lewis, D. A . Anti-Inflammatory drugs from plant and marine sources (Birkhauser Verlag, Basel , 1989) 28. Gregson, R. P.; Baldo, B. A . ; Thomas , P. G . ; Quinn, R. J . ; Bergquist, P. R.; Stephens, J . F.; Home, A . R. Science. 1979, 206, 1108-9. 29. Buckle, P. J . ; Baldo, B. A. ; Taylor, K. M . Agents and Actions. 1980, 10, 361-7. 30. Keyzers, R. A . ; Davies-Coleman, M . T. Chem. Soc. Rev. 2005, 34, 355-365. 31. de Silva, E . D.; Scheuer, P. Tetrahedron Lett. 1980, 21, 1611-1614. 32. Kernan, M . R.; Faulkner, D. J . ; Jacobs, R. S. J. Org. Chem. 1987, 52, 3081-3083. 33. Burgoyne, D. L. ; Andersen, R. J . ; Allen, T . M . J. Org. Chem. 1992, 57, 525-528. 34. Takei , M . ; Burgoyne, D. L ; Andersen, R. J . J. Pharm. Sci. 1994, 83, 1234-1235. 35. Izzo, I.; Avallone, E . ; Delia, M . C ; Casapullo, A . ; Amigo, M . ; De Riccardis, F. Tetrahedron. 2004, 60, 5587-5593. 36. Look, S .A.; Fenical, W. ; Jacobs, R.S . ; Clardy, J . J. Org. Chem. 1986, 51, Chapter 1: General Introduction. 18 5140-5145. 37. Renner, M . K.; Shen, Y . C ; Cheng , X . C ; Jensen, P. R.; Frankmoelle, W. ; Kauffman, C . A. ; Fenical, W. ; Lobkovsky, E . ; Clardy, J . J. Am. Chem. Soc. 1999, 727, 11273-11276. 38. Belofsky, G . N.; Anguera, M . ; Jensen, P. R.; Fenical, W. ; Kock, M . Chem.-A Euro. J. 2000, 6, 1355-1360. Chapter 2: Synthesis of Pelorol and Analogs. 19 Chapter 2: Synthesis of Pelorol and Analogs' 2.1 A brief review of the SHIP assay T h e phosphatidylinositol-3 kinase (PI3K) signaling pathway plays an important role in regulating various cellular activities. These include, depending on the cell type, survival, adhesion, movement, proliferation, differentiation and end cell activation. A key second messenger in this pathway is the membrane-associated phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3) (Figure 2.1), which is present in low concentration in resting cells, but is transiently synthesized from PI-4,5-P2 by P I 3 K in response to a diverse array of extracellular stimuli, and attracts pleckstrin homology (PH) domain containing proteins to the plasma membrane to mediate its effects.1"4 T o ensure the activation of this signaling pathway is properly restrained, the ubiquitously expressed tumor suppressor P T E N (phosphatase and tensin homologue deleted on chromosome ten) reverses P I 3 K activity by catalyzing the hydrolysis of PI-3 ,4,5-P3 back to PI-4,5-P 2 . Around 50% of human cancers contain biallelic inactivating mutations of P T E N and familial cancer predisposition disorders can be mimicked in P T E N knock-out mice, which illustrates the importance of these phospholipid phosphatases in preventing uncontrolled cell growth. 5 Aversion of this chapter has been published. Yang, Lu; Williams, David E.; Mui, Alice; Ong, Christopher; Krystal, Gerald; van Soest, Rob; Andersen, Raymond J . (2005) Synthesis of Pelorol and Analogs: Activators of the Inositol 5-Phosphatase SHIP. Organic Letters, 7(6), 1073-1076. Chapter 2: Synthesis of Pelorol and Analogs. 20 Figure 2.1 Enzymatic synthesis and degradation of PI- 3,4,5-P3 Similar to P T E N , the hemopoietic-restricted Src homology 2-containing inositol 5-phosphatases SHIP, sSHIP, and SHIP2 hydrolyze PI-3,4,5-P 3 to PI-3,4-P 2 . SHIP, also known as SHIP1, is a 145kDa protein that is widely expressed in hemopoietic cells. 4 It contains an amino-terminal src homology 2 (SH2) domain, a centrally located 5'-phosphatase domain that selectively hydrolyses PI-3,4,5-P3 and inositol-1,3,4,5-tetrakiphosphate (IP 4) in vitro, two phosphotyrosine binding (PTB) consensus sequences, and a proline-rich region at the carboxyl tail. Chapter 2: Synthesis of Pelorol and Analogs. ^ 1 sSHIP is a 104kDa protein that is only expressed in murine embryonic stem cells and hemopoietic stem cells. The function of sSHIP is to regulate PI-3,4,5-P 3 levels in stem cells in response to extracellular stimuli. Besides SHIP and sSHIP, there is a more widely expressed 150kDa SHIP2 protein, which is encoded by a separate gene and has a structure similar to SHIP. Like SHIP and sSHIP, SHIP2 specifically hydrolyzes the 5'-phosphate from PI-3,4,5-P 3 and IP 4 in vitro. In 1998, Krystal and co-workers generated mice containing a homozygous deletion of SHIP ( S H I P - / - mice) by replacing SHIP 'S first exon with the neomycin resistance gene in an antisense orientation. 6 Although these animals are viable and fertile, they have a shortened lifespan and normally do not survive beyond 14 weeks. They overproduce granulocytes and macrophages, suffer from progressive splenomegaly, extramedullary hemopoiesis, massive myeloid infiltration of the lung, and osteoporosis. Experiments with S H I P - / - mice and with S H I P - / - bone-marrow-derived mast cells ( B M M C s ) and macrophages (BMm(j)S) obtained from these mice, have demonstrated that SHIP is a negative regulator of immunoglobulin E (IgE) or Steel Factor induced mast cell activation, 7 a negative regulator of lipopolysaccharide (LPS) induced macrophage activation, and a negative regulator of osteoclast formation and resorptive function. There is also evidence that SHIP acts as a tumor suppressor in both acute myelogenous leukemia ( A M L ) 8 and in chronic myelogenous leukemia ( C M L ) . 9 Macrophages Chapter 2: Synthesis of Pelorol and Analogs. 22 from older SHIP-/- mice display an impaired LPS-induced inflammatory cytokine production, which suggests that regulation of the PI3K signaling pathway may play an important role in programming the macrophage innate immune response.10 It has been suggested that therapeutic manipulation of the PI3K pathway might be beneficial in treating osteoporosis,: inflammatory disorders, and cancers. Current atempts to develop drugs based on intervention in signaling pathways are overwhelmingly biased toward finding selective kinase inhibitors. There has been some recent interest in examining the therapeutic potential of phosphatase inhibitors,1 but there has been virtualy no effort to explore the usefulness of smal molecule phosphatase activators. The important role of SHIP as a negative regulator of mast cell and macrophage activation, osteoclast formation and resorptive function, as wel as in AML and CML, combined with its occurrence only in hemopoietic cells, makes it an attractive drug target. Krystal hypothesized that selective activators of SHIP would be useful experimental tools and potential drug candidates to provide proof of principle validation for a new approach to the treatment of inflammation, osteoporosis, and leukemia. Chapter 2: Synthesis of Pelorol and Analogs. 23 2.2 Introduction A s part of the ongoing efforts in the Andersen lab to search for bioactive natural products from marine sources through bioassay-guided separation, crude extracts of marine invertebrates were screened for in vitro activation of the SHIP-catalyzed conversion of inositol-1,3,4,5-tetrakisphosphate (IP 4) to inositol-1,3,4-trisphosphate (IP3). Out of approximately 2000 extracts tested, 10 exhibiting SHIP activation were identified. A M e O H extract of the sponge Dactylospongia elegans (Thiele, 1899), collected in Papua New Guinea, exhibited promising activity in the assay. Bioassay-guided fractionation of the extract led to the identification of pelorol (2.1) as the sole SHIP-activating component (Figure 2.2). OMe Figure 2.2 Chapter 2: Synthesis of Pelorol and Analogs. ^ Three related sesquiterpenes, illimaquinone (2.2), 1 2 mamanuthaquinone (2.3), 1 3 and dactyloquinone A (2.4), 1 4 were also isolated from the D. elegans extract, but were not active in the assay. Pelorol (2.1) and dactyloquinone A (2.4) were undescribed when first isolated in our laboratory, but while further biological studies were in progress, pelorol (2.1) was isolated by Konig's g r o u p , 1 5 also from D. elegans, and by Schmitz's group from Petrosaspongia metachromia.16a Dactyloquinone A (2.4) was subsequently isolated from the Okinawan sponge D.elegans by Yamada's group in 2001.14 A review of the literature reveals that 36 sesquiterpene hydroquinones and quinones have been isolated from the species D. elegans to date and half of them were new compounds when they were initially i d e n t i f i e d . 1 5 , 1 7 - 2 2 All of these compounds have a 4,9-friedodrimane (A,B) or drimane (C) skeleton, which couples with a hydroquinone, quinone, or lactone moiety. A n early paper from C r e w s 1 7 classified quinone (hydroquinone) sesquiterpenoids from sponges and algae in accordance with their structure type and the corresponding marine resource. A s he indicated, quinone (hydroquinone) sesquiterpenoids from D. elegans had the largest variety of structures among the sponge families Dysideidae, Thorectidae, Spongiidae, and Haploscleridae. Subsequent work on this species enriched the sesquiterpene quinone and hydroquninone family which comprises fourterpene skeletons (A,B,C,D) and four different benzenoid-derived substructures (W, X,Y,Z) (Figure 2.3). The benzenoid-derived substructures W-Z Chapter 2: Synthesis of Pelorol and Analogs. 25 A B C W a b c h i Figure 2.3 Summary of sesquiterpene-hydroquinone (-quinone) substructures isolated from D.elegans. could be further categorized into different subunits (a-i) as a function of the heteroatom substitution, presence or absence of an additional carbon and oxidative transformations. Of interest, ilimaquinone (2.2) that we have isolated in our current work, is the only component present in all investigations on this species by six research groups (Andersen, Crews, Kobayashi , Konig, Riguera, Chapter 2: Synthesis of Pelorol and Analogs. 26 Dactylospongenones A (2.8) (16R, 20S) Dactylospongenones B (2.9) (16S, 20R) Dactylospongenones C (2.10) (16R, 20R) Dactylospongenones D (2.11) (16S, 20S) Figure 2.4 Proposed biogenesis of pelerol related compounds from D.elegans Yamada) sampled from seven different geographical sites (Papua New Guinea, Fiji & Thailand, Indonesia, Australia, Malaysia, Japan), which exemplifies its chemotaxonomical importance to the species D. elegans. More interesting is that only three groups have reported the isolation of pelorol (2.1 ) 1 6 , 2 0 from D. elegans. Recently, the other groups isolated either (-)smenodiol (2 .6) , 1 7 , 2 3 which was believed to be the formal biogenetic precursor of pelorol, or smenospondiol (2 .7) , 1 7 , 2 4 the rearrangement product. Riguera's examination of this species led to the isolation of some ring-contracted derivatives of ilimaquinone, Chapter 2: Synthesis of Pelorol and Analogs. ^ ' dactylospongenones A - D (2.8-2.11) 1 7, which were originally discovered by Faulkner's group from the Palauan sponge Dactylospongia sp. together with smenospondiol (2.7). 2 5 Faulkner refuted the hypothesis that dactylospongenones might be artifacts formed from ilimaquinone during the isolation and separation. O n the basis of this evidence, we proposed that pelorol (2.1), smenodiol (2.6) and smenospondiol (2.7) may have a common precusor (Figure 2.4), which is the bicyclic cationic cyclization intermediate (2.5). This intermediate could undergo elimination to form smenodiol (2.6), cyclization to give pelorol (2.1), and the Wagner Meerwein rearrangement of the drimane terpene skeleton to give smenospondiol (2.7), which could be further oxidized, decarboxylated and cyclized to yield the four diastereomers dactylospongenone A - D (2.8-2.11). D.elegans is a rich source of sesquiterpene quinones / hydroquinones that possess interesting biological and pharmacological properties. Similar structural motifs in the sesquiterpene quinone and hydroquinone family members often translates to a variety of exciting biological properties unique to individual compounds. For example, ilimaquinone (2.2) was found to exhibit anti-HIV, 2 6 antimicrobial, antimitotic, and antiinflammatory activities, 2 5 in addition to inhibiting the cytotoxicity of ricin and diphtheria toxin, 2 7 selectively fragmenting the Golgi apparatus , 1 2 and interacting with methylation e n z y m e s , 2 8 as well as inhibiting the lyase activity of D N A polymerase p . 2 9 Smenospongine (2.12) (Figure 2.5), an aminoquinone sesquiterpene, was reported to have antimicrobial, and Chapter 2: Synthesis of Pelorol and Analogs. 28 Figure 2.5 antileukemic act ivit ies 1 7 , 2 4 and was found to induce the differentiation of human chronic myelogenous Leukemia (CML) K562 cells into erythroblasts along with cell-cycle arrest at the G1 phase. It also exhibits strong inhibition of D N A synthesis in L1210 leukemia cells and inhibition of the proliferative response of mitogens in murine splenocytes and human peripheral lymphocytes . 2 2 , 3 0 The akylated aminoquinone sesquiterpene smenospongiarine (2.13), which has a similar structure to smenospongine (2.12), is less toxic to L1210 leukemia cells and shows moderate toxicity to tumor c e l l s . 1 7 , 2 4 , 3 0 Another aminoquinone sesquiterpene, smenospongidine (2.14), was found to have antimicrobial activity and differentiation-inducing activity for the conversion of C M L K562 cells into erythroblasts, but was not cytotoxic to L1210 leukemia c e l l s . 1 7 , 2 2 , 2 4 This is consistent with the activity of (2.13), that a free amino group is essential to the cytotoxicity. 5-Epi-smenospongiarine (2.15) (Figure 2.6) was reported to show strong in vitro antileukemic activity and potent in vitro toxicity to solid tumor models. 5-Epi-smenospongidine (2.16) was found to have moderate toxicity to leukemia cells (P388) and solid tumor cells, in addition to exhibiting differentiation inducing activity in K562 leukemia c e l l s . 1 7 , 2 2 Dactylospongenone B (2.9) is the only non-quinone containing derivative that exhibits in vitro activity against Chapter 2: Synthesis of Pelorol and Analogs. 29 Figure 2.6 leukemia cells (P388). 1 7 Neodactyloquinone (2.17) 2 1 expresses moderate cytotoxic activity toward Hela cells and mamanuthaquinone (2.3) 1 3 is toxic to human colon tumor cells with an I C 5 0 of 2/vg/mL. A s we noted above, while the interesting structure of pelorol (2.1) was reported by the Konig and Schmitz g r o u p , 1 5 , 1 6 and Konig et al described anti-protozoan activity of this compound, neither group appreciated the ability of pelorol to activate SHIP or modify the biology of mammalian cells. Chapter 2: Synthesis of Pelorol and Analogs. ^ u 2.3 Isolation and Characterization of Pelorol (1)' 2.3.1 Isolation Specimens of the brownish sheet sponge Dactylospongia elegans (Thiele, 1899) (order Dictyoceratida, family Spongiidae) (Figure 2.7) were collected by hand using S C U B A at a depth of 5-10 m from a protected overhang in Rasch Passage on the outer reef of Madang Lagoon, Papua New Guinea, in January 1995. Freshly collected sponge was frozen on site and transported to Vancouver over dry ice. The sponge was identified by Professor Rob van Soest, University of Amsterdam, and a voucher sample has been deposited at the Zoological Museum of Amsterdam (ZMA P O R . 15986). Figure 2.7 Image of the sponge Dactylospongia elegans (Thiele, 1899) ' Isolation and characterization work for pelorol, ilimaquinone, mamanuthaquinone, and actyloquinone A was performed by Dr. D.E. Williams, Andersen research group, University of British Columbia. Chapter 2: Synthesis of Pelorol and Analogs. 3' The frozen sponge (120 g) was cut into small pieces, immersed in and subsequently extracted with M e O H (3 x 250 mL). The combined methanolic extracts were concentrated in vacuo, and partitioned between E t O A c (4 x 100 mL) and H 2 0 (300 mL). The combined E t O A c extract was evaporated to dryness in vacuo to yield 490 mg of a brown purply oil that exhibited significant activity at 2 uM in a SHIP assay. The E t O A c soluble material was chromatographed on Sephadex LH-20 with 4:1 M e O H / C H 2 C I 2 as eluent to give two SHIP active fractions. O n e fraction (14.9 mg) consisted primarily of the novel metabolite 2.1 and lipid material. T h e second active strongly coloured fraction (30.5 mg) (appearing orange under basic and blue/purple under acidic conditions) contained a mixture of ilimaquinone (2.2), mamanuthaquinone (2.3), dactyloquinone A (2.4) and fats. A pure sample of 2.1 (8.9 mg) was obtained by semi-preparative reversed-phase H P L C , using a Whatman Magnum-9 Partisil 10 O D S - 3 column, with 13:7 M e C N / 0 . 0 5 % aqueous T F A ) as eluent. The mixture of ilimaquinone (2.2) and mamanuthaquinone (2.3), dactyloquinone A (2.4) was further fractionated by semi-preparative reversed-phase H P L C , using the same conditions as for the purification of 2.1, to give pure dactyloquinone A (2.4) (1.9 mg), pure ilimaquinone (2.2) (17.0 mg) and mamanuthaquinone (2.3) contaminated with a small quantity of 2.2. Pure 2.3 (0.8 mg) was obtained after an additional step of H P L C purification using the same conditions. T h e structures of 2.2 and 2.3 were solved by spectroscopic analysis and the data obtained were then compared to those found in the l i terature. 1 2 , 1 3 Chapter 2: Synthesis of Pelorol and Analogs. 3A It is important to note that the same products were isolated in a subsequent workup in which M e O H was substituted for E t O H throughout. Hence, compounds 2.1, 2.2, 2.3 and 2.4 can all be considered to be natural products and not artifacts of isolation. Pelorol (2.1): Isolated as a pale oil; [af5D - 3 7 . 1 ° (c 0.275, C H 2 C I 2 ) ; U V ( C H 2 C I 2 ) Imax 227 (e 7436), 253 (e 6557), 289 (e 2519) nm; 1 H N M R , see Table 2.1; 1 3 C N M R , see Table 2.1; positive ion H R F A B M S [M+H] + m/z 373.23808 (calcd for C 2 3 H 3 3 0 4 , 373.23799). Dactyloquinone A (2.4): Isolated as a pale oil; [af5D - 4 6 . 3 ° (c 0.095, C H 2 C I 2 ) ; U V ( C H 2 C I 2 ) Imax 290 (e 15469), 406 (e 394) nm ; 1 H N M R , see Table 2.1; 1 3 C N M R , see Table 2.1; negative ion H R F A B M S [M]- m/z 356.19950 (calcd for C 2 2 H 2 8 0 4 , 356.19884). Figure 2.8 1 H N M R of pelorol (2.1) isolated from sponge D. elegans CDCI3 at 400MHz recorded in Chapter 2: Synthesis of Pelorol and Analogs. 34 99ZZ91 8i.9em ES986I. 6890'IZ ZQSZv-Z 86wee weeee 6 ^ 9 8 1391Z8 frOOZOfr 0 8 6 ^ 3 * o C\l o co o 0fr89'8t-0969' 1.9 o IT) 8 Z U Z 9 3£9l- '99 o CD O 91899Z 0000'ZZ 9818ZZ o co o E a CL o — o 1 4 9 8 ' H I . 69C1--8U o CN 299663I-1 .609'En 01.91.'891. o CO o o o CD o o co Figure 2.9 1 3 C N M R of pelorol (2.1) isolated from sponge D. elegans recorded in CDCI 3 at 100MHz Chapter 2: Synthesis of Pelorol and Analogs. 35 Table 2.1 NMR Data for 2.1 and 2.4 recorded in C D C I 3 1 2.1 2.4 Atom # 1 a 5 H 13 b 5 C Atom # 1 a 5 H 13 b 5 C 1 ax 0.98 td (13.2. 3.4), 1H 40.2 1 ax 1.92 m, 1H 29.1 1 eq 1.54 m 1H 1 eq 1.87 m, 1H 2 ax 1.40 m, 1H 18.4 2 1.90 m, 1H 23.2 2 eq 1.64-1.67 m, 1H 1.71 m, 1H 3 ax 1.14 td (13.7,4.1), 1H 42.5 3 ax 2.18 ddm, (14.4, 4.0), 1H 30.0 3 eq 1.40 m, 1H 3 eq 2.57 m, 1H 4 33.1 4 153.0 5 ax 0.91 dd (12.3, 2.4), 1H 57.1 5 44.5 6 ax 1.64-1.67 m, 1H 19.5 6 ax 1.95 m, 1H 32.6 6 eq 1.54 m, 1H 6 eq 1.77 m, 1H 7 ax 1.38-1.40 m, 1H 36.5 7 ax 1.61 qm (15.4), 1H 26.9 7 eq 2.51 m, 1H 7 eq 1.27 m, 1H 8 48.5 8 ax 1.41 m, 1H 33.8 9 ax 1.64-1.67 m, 1H 65.2 9 38.9 10 37.2 10 88.1 11 Me ax 0.84 s, 3H 21.1 11 proE 11 proZ 4.78 s, 1H 4.88 s, 1H 107.5 12Me eq 0.83 s, 3H 33.3 12Me ax 1.36 s, 3H 27.6 13Me ax 1.21 s, 3H 19.9 13Me eq 0.74 d (6.2), 3H 16.2 14Me ax 1.04 s, 3H 16.3 14Me ax 1.11 s, 3H 19.9 15 ax 2.48 m, 1H 24.3 15 2.09 ABd(19.1), 1H 28.1 15 eq 2.60 dd (14.3, 6.2), 1H 2.62 ABd (19.1), 1H 16 130.0 16 114.1 17. 143.5 17 150.91 18 140.5 18 181.6 19 7.08 s, 1H 114.9 19 5.71 s, 1H 104.8 20 118.1 20 159.41 21 149.5 21 181.1 22 168.2 22 3.78 s, 3H 56.3 23 3.81 s, 3H 51.7 a 500 MHz. b 100 MHz. 1 Assignments within a column are interchangeable. Assignments based on H M Q C , HMBC, C O S Y , R O E S Y and 1D N O E S Y data. Chapter 2: Synthesis of Pelorol and Analogs. 36 2.3.2 Characterization Pelorol (2.1) was obtained as an optically active amorphous solid that gave a [M+H] + ion in H R F A B M S at m/z 373.2381 appropriate for a molecular formula of C 2 3 H 3 2 0 4 (calculated for C 2 3 H 3 2 0 4 373.2380), requiring eight sites of unsaturation. The 1 3 C N M R spectrum (CDCI 3 ) obtained for 2.1 contained resonances accounting for 23 carbon atoms in agreement with the H R F A B M S data. H M Q C data showed that only 30 hydrogen atoms were attached to carbons ( 5 x C H 3 , 6 x C H 2 , 3 x C H , 9xC), requiring the presence of 2 O H s . Broad singlets at 8 5.17 and 8 5.37 in the 1 H N M R spectrum, that were not correlated to carbon resonances in the H M Q C spectrum, were assigned to the O H protons. Preliminary analysis of the 1 H and 1 3 C N M R spectra indicated that pelorol (2.1) had both benzenoid and sesquiterpenoid fragments. Four aliphatic methyl singlets (8 0.83, Me-12; 0.84, Me-11; 1.04, Me-14; 1.21, Me-13), one methoxyl singlet (8 3.81, Me-23), a benzylic methylene [8 2.48 (m, H-15) and 8 2.60 (dd, J= 14.3, 6.2 Hz, H-15')] coupled to a methine (8 1.64-1.67, H-9), and an aromatic hydrogen (8 7.08, H-19) were present in the 1 H N M R spectrum of 2.1. Resonances that could be assigned to a pentasubstituted benzene ring [8 114.9 (C-19), 118.1 (C-20), 130.0 (C-16), 140.5 (C-18), 143.5 (C-17), 149.5 (C-21)] and an ester carbonyl (8 168.2, C-22) were observed in the 1 3 C N M R spectrum. The aromatic ring and ester functionalities accounted for 5 of the 8 Chapter 2: Synthesis of Pelorol and Analogs. 0 5 ' required sites of unsaturation and the remaining 3 sites of unsaturation had to be rings. Structural features of 2.1 identified from the N M R data were similar to the structural components of (-)smenodiol (2.6),17 previously isolated from the same species D. elegans., suggesting that the compounds were related and the 1 3 C N M R data of 2.1 fit well for the decalin ring system. C O S Y correlations identified the spin system extending from the methylene protons at C-1 (8 0.98 and 1.54) through to the methylene protons at C-3 (8 1.14 and 1.40), and starting from H-5 (8 0.99) through to H-7 (8 1.39 and 2.51), from H-9 (8 1.64-1.67) to H-15 (8 2.48 and 2.60). This information suggested that 2.1 had the A / B rings of a drimane skeleton (Type C terpene skeleton in Figure 2.3). H M B C correlations observed between Me-11 (8 0.84), Me-12 (8 0.83) and C-5 (8 57.1), C-3 (8 42.5), C-4 (8 33.1) ; between Me-14 (8 1.04) and C-1 (8 40.2), C-5 (8 57.1) , C-9 (8 65.2), C-10 (8 37.2); between Me-13 (8 1.21) and C-7 (8 36.5), C -8 (8 48.5), C-9 (8 65.2); between H-9 (8 1.64-1.67) and C-8 (8 48.5), C-10 (8 37.2) , C-15 (8 24.3), C-13 (8 19.9), C-14 (8 16.3) confirmed the presence of the A / B ring system in 2.1. The proton resonance at 8 7.08 (H-19) showed strong H M B C correlations to C -17 (8 143.5), C-18 (8 140.5), C-21 (8 149.5) and C-22 (8 168.2). This set of H M B C correlations confirmed the presence of a pentasubstituted benzene ring in 2.1. T h e Me-13 (8 1.21) showed a strong H M B C correlation to C-21 (8 149.5) indicating that the benzene ring C-21 was bonded to C-8 . T h e benzylic methylene proton resonances at 8 2.48 (H-15ax) and 2.60 (H-15eq) showed Chapter 2: Synthesis of Pelorol and Analogs. correlations to C-16 (8 130.0) and C-21 (8 149.5) establishing the connection of the drimane skeleton and the aromatic ring through the C-15 and C-16 bond to give the final ring required by the molecular formula of pelorol (2.1) and generating a constitution that was consistent with all of the spectroscopic data. 1D N O E S Y data provided evidence for the regiochemistry of the aromatic ring and the relative stereochemistry of . pelorol (2.1). Correlations observed between Me-23 (8 3.81) and H - 7 a x (8 1.38-1.40, m) and H-19 (8 7.08) placed the methyl ester at C-20 and the single aromatic proton at C-19, leaving the two hydroxyls to be situated at C-17 and C-18. N O E correlations between Me-14 (8 1.04), Me-13 (8 1.21), Me-12 (8 0.83), H - 6 a x (8 1.64-1.67, m), and H - 1 5 a x (8 2.48) are only possible if the decalin system of pelorol (2.1) adopts the fairly typical trans fused double-chair conformation and the drimane skeleton and the aromatic ring are connected by a trans fused five membered ring. Chapter 2: Synthesis of Pelorol and Analogs. ^ y 2.4. Total synthesis of pelorol 2.4.1 Introduction Pelorol (2.1) is the first known SHIP activator. In vitro biological studies treating macrophages with lipopolysaccharide (LPS) in the presence or absence of pelorol showed that pelorol inhibited the release of the pro-inflammatory mediator T F N a production as efficiently as interleukin-10, a potent, physiological inhibitor of macrophage activation. These exciting bioassay results prompted us to carry out further biological studies on this compound, but the limited quantity (8.9 mg) of pelorol (2.1) available from the source sponge D. elegans was inadequate to support a detailed in vitro and in vivo evaluation of its ability to activate SHIP. In order to satisfy the need for additional material, confirm the absolute configuration of the natural product, and generate analogs to flesh out the structure activity relationship for this family of anti-inflammatory compounds, the total synthesis of pelorol (2.1) and analogs, where the methyl ester at C-20 was replaced by methyl and ethyl residues, was undertaken. Chapter 2: Synthesis of Pelorol and Analogs. 40 2.4.2 Proposed biogenesis of pelorol A s mentioned above, pelorol (2.1) and its biogenetically related compounds smenodiol (2.6), smenospondiol (2.7), etc. might be derived from a common precursor, which is the bicyclic cationic intermediate 2.5. It is fairly common for nature to synthesize 2.5 from the linear polyene derivative, which could be the coupling product of the farnesyl diphosphate (FPP) and the shikimate un i t . 3 1 , 3 2 O n the basis of this evidence, a biosynthetic pathway for pelorol was proposed as shown in Figure 2.10. In this proposal, the shikimate building block of the molecule, protocatchuic acid, was synthesized beginning with the coupling of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), followed by elimination of phosphoric acid and an intramolecular aldol reaction to form the six-membered ring intermediate, 3-dehydroquinic acid. Dehydration of 3-dehydroquinic acid leads to 3-dehydroshikimic acid, which could lose another molecule of H2O and undergo enolization to give the protocatechuic a c i d . 3 2 This simple phenolic acid could be alkylated at the position ortho to the hydroxyl group by a suitable alkylating agent, in this case farnesyl diphosphate. T h e newly introduced farnesyl group can then cyclize upon protonation of the terminal double bond to give the tertiary carbocation intermediate 2.5, which might undergo enzyme-catalysed Friedel-Crafts alkylation to give the meroterpenoid phenolic acid. O-methylation using S A M would lead to the natural product pelorol (2.1), though suspending the Chapter 2: Synthesis of Pelorol and Analogs. 41 phenolic acid in M e O H may give the same product. However, there was no evidence that pelorol (2.1) was an artifact formed during isolation and separation. Pelorol (2.1) (2.5) Figure 2.10 Proposed biogenesis of pelorol (2.1) Chapter 2: Synthesis of Pelorol and Analogs. 42 2.4.3 Proposed synthesis of pelorol From the proposal for the biosynthesis of pelorol, it was natural to consider a biomimetic construction of this compound, although the question as to whether nature employed the polyene cyclization cascade in the biosynthesis of pelorol had not yet been experimentally answered. In spite of its productive and aesthetic appeal, the polyene cyclization approach would be difficult to execute in the laboratory. O n e of the anticipated difficulties was the nonenzymatic capture of the cationic intermediate 2.5 with a nucleophilic arene shown in Figure 2.10. A recent publication by the Overman group (Figure 2.11) describing the enantioselective total synthesis of adociasulfate I was the only precedent that used an arene to terminate an epoxide-initiated polyene cyclization to afford a tetracyclic fragment similar to pelorol . 3 3 In their early scouting studies, they found that treatment of A with a variety of Lewis acids under different conditions delivered only bi-, tri-, and tetracyclic products, not the desired pentacycle B . Although the transformation was eventually accomplished by addition of a OMe OMe OMe \ , / 3' HO' OH B Pelorol OBn A OBn Figure 2.11 Overman's study on polyene tetracyclization Chapter 2: Synthesis of Pelorol and Analogs. 4o removable activating substituent at the 3'-position of the arene, the highest yield they could achieve was 15% after screening a range of Lewis acids and cyclization conditions. It seemed the polyene cyclization approach to pelorol was not suitable for large scale synthesis of the product and generating a variety of analogs with different functionalities on the arene ring, which would be highly important for a S A R study. A more succinct and conservative pathway for a synthesis of pelorol was, therefore, sought. A s shown in Figure 2.12, pelorol could be assembled in a convergent manner from two building blocks, the sesquiterpene and the arene. If sesquiterpene building block III (2.18 or 2.19) had a tertiary alcohol at C-8 , which would act as the initiator of cyclization in the intermediate II upon treatment with Lewis acid, then the chirality of this alcohol was not important to the resulting carbocation I. O n the basis of sound biogenetic arguments, Schmitz predicted that the absolute Pelorol (1) •OMe OR R Figure 2.12 Retrosynthetic analysis of pelorol (2.1) Chapter 2: Synthesis of Pelorol and Analogs. 4 4 configuration of pelorol was 5 S , 8 R , 9 R , 1 0 S . 1 6 Therefore, a logical starting material for intermediate III would be (+)-sclareolide (2.20), which has the same absolute configurations at C-5 , C-9 , and C-10 as those predicted for pelorol. Sclareolide is commercially available in gram quantities at an acceptable price. Vanillin was selected as the starting material for the arene unit. T h e key step in the convergent synthesis is the biomimetic carbocation-initiated cyclization of the intermediate I to generate the C-8/C-21 bond. Steric bulk associated with the C -14 axial methyl was expected to cause preferential approach of the phenyl ring from the bottom face of C - 8 , 3 4 resulting in the formation of the required trans B / C ring fusion. Synthetic routes to both C-8 epimers of III starting from sclareolide have been reported by Quideau et al. in the synthesis of (+)-puupehenone 3 5 and Chackalamannil et al in the preparation of wiedendiol . 3 6 T h e plan was to examine both as intermediates in the preparation of direct precursors to the carbocation I and ultimately cyclized products. Chapter 2: Synthesis of Pelorol and Analogs. 45 2.4.4 Synthetic results A s presented in the retrosynthetic analysis of pelorol (Figure 2.12), the key intermediates were the drimane aldehydes 2.18 or 2.19. A s already mentioned, aldehyde 2.18 has been prepared by Quideau et al. as a precursor in the synthesis of the antituberculosis marine sponge metabolite (+)-puupehenone. 3 5 | KOH, MeOH f 95% Figure 2.13 Synthetic route to 2.18. Following Quideau's procedure, commercially available (+)-sclareolide (2.20) was dissolved in a mixture of sulfuric acid and formic acid at room temperature for seven hours. These conditions brought about near quantitative inversion of the configuration at C-8. Th e ease of this reaction was due to the release of the 1,3-diaxial interaction between the 13- and 16-methyls in 2.20. The subsequent transformation required introduction of a hydroxyl functionality a to C-12 of 2.21. Chapter 2: Synthesis of Pelorol and Analogs. 46 This was accomplished by using Vedejs' M o 0 5 » p y r i d i n e » H M P A (MoOPH)-based protocol 3 7 and the use of magnesium bis (dissopropylamide) [(DA) 2Mg] as a chelating agent . 3 8 A s described by Quideau er al.,35 along with the required ot-hydroxylated product, a crystalline side-product 2.23 was also recovered after the standard workup conditions. The intermediate 2.22 was subjected to reduction by D I B A L - H at room temperature for 48 hours. After quenching the reaction slowly with dilute HCI at 0 ° C and workup, a mixture of lactol 2.25 and the desired triol 2.24 was obtained. T h e final step to generate the (3-hydroxy drimane aldehyde 2.18 was realized by adding N a l 0 4 to the triol in T H F / H 2 0 at 0 ° C for half an hour, which gives the oxidative cleavage product 2.18 in excellent yield. Of particular note is the oxidation of the side-product lactol 2.25 by Nal04 in the last step, which led to the formate 2.26. The formate was subsequently hydrolyzed by K O H to give the desired product 2.18. With the sesquiterpenoid unit of pelorol in hand, the next step was to synthesize the shikimate unit, which was elaborated from commercially available vanillin (2.27) (Figure 2.14). A s we have noted above, Overman had shown in his synthesis of adociasulfate that a highly nucleophilic arene was required to trap a carbocation such as I (Figure 2.12) in preference to proton elimination to give an uncyclized olefin. Therefore, 1-bromo-2,3,5-trimethoxybenzene (2.28) was chosen as the appropriate shikimate unit to couple with the aldehyde 2.18. Synthesis of the trimethoxyphenyl bromide 2.28 followed a known procedure by Furtsener et a / . , 3 9 which started from the commercial product vanillin (2.27). Chapter 2: Synthesis of Pelorol and Analogs. 47 MeO. CHO MeO. Mel, K 2C0 3, DMF 95% MeO 2.29 CHO 2.30 MeO MeO' i) mCPBA, CH2CI2, 16 h ii) aq.HCI, aq.MeOH, 30 min OMe MeO Mel, K 2C0 3, DMF MeO 70% 95% 2.28 Figure 2.14 Synthetic route to 2.28. Vanillin was brominated regioselectively at the 5-postion in methanol at 0 ° C by adding bromine slowly to the stirred solution to give compound 2.29. O -methylation of 2.29 by Mel in D M F gave quantitatively 6-bromoveratraldehyde (2.30), which was available commercially but is very expensive. Bayer-Villiger oxidation of 2.30 was carried out with m C P B A by refluxing in CH2CI2 for 16 hours. Subsequent acid hydrolysis in aqueous M e O H for 30 minutes gave phenol 2.31. T h e phenol was O-methylated to give the trimethoxyphenyl bromide 2.28. With both the sesquiterpenoid and aromatic units in hand, the way was cleared for the coupling reaction. nBuLi was slowly added to the trimethoxyphenyl bromide 2.28 in T H F at - 7 8 ° C . T h e mixture became a slurry after half an hour, which indicated completion of the halogen-metal exchange and formation of aggregates of trimethoxyphenyllithium 2.32. T h e drimane aldehyde 2.18 (0.5 equiv) was added to the slurry which was further stirred for 2 hours. After quenching with ice water, standard workup conditions followed by flash silica gel column chromatography afforded the diol 2.33. There are many methods Chapter 2: Synthesis of Pelorol and Analogs. 48 Figure 2.15 Synthetic route to 2.33 available for reducing benzylic alcohols to methylene groups. These fall into three general categories: i) dissolving metal reductions, 4 0 ii) a mixture of Lewis acid (or protic acid) and a strong reducing a g e n t 4 1 or iii) catalytic hydrogenat ion . 4 2 , 4 3 Dissolving metal reduction was not suitable for selectively removing the benzylic alcohol of the diol 2.33, because the tertiary alcohol did not survive the reduction. The diol 2.33 was dissolved in triethylsilane, a hydride donor, and treated with T F A at - 7 8 ° C for five minutes. T L C indicated that a less polar product formed, but subsequent N M R analysis revealed that instead of generating the desired reduction product 2.34, a retroaldol reaction product 2.35 was the only product. Changing the hydride donor to B H 3 or using a Lewis acid gave the same result. Sodium cyanoborohydride together with zinc iodide was reported to be an effective reducing combination for benzylic alcohols . 4 4 However, when 2.33 was refluxed in dichloroethane containing sodium cyanoborohydride for 24 hours no trace of 2.34 was detected. W h e n all the other choices were exhausted, catalytic hydrogenation was explored as a method to obtain 2.34. Quideau et al. 3 5 successfully utilized Chapter 2: Synthesis of Pelorol and Analogs. 49 OMe Figure 2.16 Synthetic route to 2.34 hydrogenolysis in removal of both benzyl protective groups and the benzylic hydroxyl group in their synthesis of puupehenone, providing a precedent that catalytic hydrogenation should be a feasible approach to 2.34. Diol 2.33 was dissolved in ethyl acetate containing 10% Pd on activated charcoal and hydrogenated at 45 psi in a Parr hydrogenator with agitation for 24 hours. T L C monitoring showed that all the starting material had disappeared. Subsequent N M R analysis confirmed that 2.34 was the only product. W h e n the reaction was scaled up to 200mg, no reaction was observed. Elevating the pressure to 600 psi and extending the reaction time to 72 hours only led to the generation of a small amount (less than 5%) of the required product 2.34. Simply changing the catalyst from 10% Pd on activated carbon to 10% Pd on carbon successfully solved the problem. This result suggests that a trace amount of acidic impurity in the Pd on Chapter 2: Synthesis of Pelorol and Analogs. ^ carbon catalyst complexes with the benzylic hydroxyl group facilitating the breaking of the C - 0 bond and uptake of hydrogen. This was confirmed by carefully adding of microliter scale of cone. HCI to the hydrogenation reaction with 10% Pd on activated carbon as the catalyst, which gave the same hydrogenolysis product 2.34. It was found that 45 psi was the optimal pressure for the reaction. Higher pressures may lead to C-8 tertiary alcohol removal. Figure 2.17 Lewis acid catalyzed cyclization of 2.34 Treatment of 2.34 with S n C I 4 in C H 2 C I 2 at - 2 0 ° C for 30 minutes afforded the cyclization product 2.36 in good yield (78%). W e anticipated that the steric repulsion of the C-14 angular methyl would lead to the phenyl ring attacking from the bottom of the C-8 tertiary cation to give a trans fused five-membered ring. The 1 4 - C H 3 peak at 51.06 (s, 3H) in 1 H N M R spectrum of the product suggested the frans-annulation nature of the B / C rings in 2.36 and the corresponding methyl in the c/s-annulation product was supposed to shift noticeably upfield to around 5 0.40 due to the shielding effect of the axial phenyl r i n g . 1 6 b This result meant that construction of a trans fused five-membered ring containing two aromatic carbons via the route we adopted was possible and high yielding. Comparing compound 2.36 with our target molecule pelorol (2.1), it was not difficult to see Chapter 2: Synthesis of Pelorol and Analogs. 51 OMe OMe 2.36 OH OMe OMe OTf Figure 2.18 Proposed introduction of carboxylate at C-20 of 2.36 that if the C-20 methoxy group could be replaced by a carbonyl group, the synthesis of pelorol would not be far away. T h e complicating factor in this approach was that there were 3 methoxyls on the phenyl ring and selectively deprotecting the C-20 methoxyl followed by triflation and Stille coupl ing 4 5 to introduce the carbonyl group would be a real challenge. Unfortunately, all our attempts to selectively demethylate the C-20 methoxyl failed. In order to overcome this problem, the dimethoxyethylphenyl bromide 2.38 was chosen as the aromatic unit to couple with the drimane aldehyde 2.18. There were two reasons to choose 2.38 as the candidate: 1) the ethyl group could act as an electron donating group along with the methoxyls on the ring, though its electron donating potential was weaker compared with the methoxyl group; 2) the Chapter 2: Synthesis of Pelorol and Analogs. ^ ethyl could be oxidized to a carboxylic acid which differentiates it from the methoxyls on the phenyl ring. Synthesis of 2 .38 4 6 started from the 6-bromoveratraldehyde (2.30), which was refluxed in T H F with M e P P h 3 B r under basic conditions for two hours until T L C analysis indicated that all of the starting material had been converted to a less polar product with a strong U V absorbance. T h e Wittig reaction gave high yield of styrene 2.37, that was light sensitive. In our experience, avoiding exposure to light during the synthesis and following workup steps was essential. Numerous heterogeneous catalysts, such as P t 0 2 , Raney Ni, R h / C and P d / C , can catalyze the hydrogenation of olefins. However these catalysts generally do not effect hydrogenation of olefins with a high degree of chemical selectivity (e.g., other sensitive groups such as nitro, ketone, arylhalide, benzyloxy.etc. also are reduced). In the initial trial, we found that hydrogenation of the styrene derivative 2.37 using a P d / C catalyst provided mainly the debrominated product with only a small amount of the desired product 2.38. Facile and highly selective reduction of the olefinic functionality occurred with no overreduction of the aromatic bromide when the hydrogenation catalyst was changed to 5% R h / C . Thus , styrene 2.37 Figure 2.19 Synthetic route to 2.38 was smoothly and completely hydrogenated with R h / C in CH2CI2 to afford the Chapter 2: Synthesis of Pelorol and Analogs. 53 ethyl benzene 2.38, which could be used for the next step without further purification. Under the same protocol used to generate 2.34, dimethoxyethylphenyllithium 2.39 was coupled with the drimane aldehyde 2.18 to give the diol 2.40, which was further reduced to the tertiary alcohol 2.41 with P d / C . OMe OMe Figure 2.20 Synthetic route to 2.41 Initial reactions of 2.41 with S n C U gave variable yields of the desired product 2.42 and the undesired elimination product 2.43 (Figure 2.21). In order to prompt the cyclization of the side product 2.43 to give 2.42, the alkene was dissolved in benzene with T s O H and refluxed overnight. W e anticipated that electrophilic addition of the hydrogen to the double bond of 2.43 could regenerate the tertiary carbocation 2.44, which would readily cyclyze to afford the expected product 2.42 under the forcing conditions. However, contrary to our expectation the reaction gave only the unanticipated cyclization product 2.45. Treatment of the tertiary alcohol 2.41 under the same conditions or the protic acid P P A afforded the identical product 2.45 (Figure 2.22). Chapter 2: Synthesis of Pelorol and Analogs. 54 Figure 2.21 Synthetic route to 2.42 and 2.43 OMe OMe Figure 2.22 Synthetic route to 2.45 Chapter 2: Synthesis of Pelorol and Analogs. 55 W e envisaged that the formation of 2.43 and 2.45 resulted from reduction of the nucleophilicity of the arene (R= Et vs R= O M e ) making the elimination reaction leading to 2.43 and the Wagner Meerwein rearrangements leading to 2.45 competitive with direct trapping of the C-8 carbocation by the arene to give the desired product 2.42. After optimization of the reaction conditions by testing different Lewis acids ( B F 3 . E t 0 2 > Sc(OTf) 3 , S n C I 4 , MeAICI 2 ) at various temperatures ( - 7 8 ° C , - 2 0 ° C , 0 ° C , r.t.), it was found that the SnCI 4 -catalyzed cyclization ( - 2 0 ° C ) gave consistently high yields (-76%) for the conversion of 2.41 to 2.42. Although it was possible to obtain 2.42 via the drimane aldehyde 2.18, the preparation of 2.18 from sclareolide was cumbersome. It not only required multiple chromatographic separations, but also preparation of the oxidizing agent OMe 2 .19 2 . 4 7 MeO ,OMe SnCI4 H 2 . 4 4 Figure 2.23 Proposed synthetic route to 2.42 via 2.19. Chapter 2: Synthesis of Pelorol and Analogs. 56 M o O P H ( M o 0 5 . P y . H M P A ) that was not commercially available, because it was not stable at room temperature to light or moisture. Therefore, we turned our attention to the aldehyde 2.19 (Figure 2.23), having an equatorial O H group at C -8, since it could couple with the shikimate unit 2.39, and after hydrogenolysis give a tertiary alcohol 2.47, which was the diastereomer of 2.41. Brief exposure of 2.47 to S n C I 4 in C H 2 C I 2 would lead to the same tertiary carbocation intermediate 2.44 as that generated from 2.41, which we expected would undergo cyclization to give the desired product 2.42. There were several synthetic routes in the literatue for the synthesis of 2.19 starting from (+)-sclareolide. Chackalamannil et al. reported a three-step procedure to synthesize 2.19 with an overall yield around 64%. 3 6 Their procedure, which included using (+)-(10-camphorsulfonyl)oxaziridine as the a-hydroxylation agent, was not reproducible in our hands. Figure 2.24 Chackalamannil's approach to 2.19 from (+)-sclareolide. Recently, Hua et al. reported a route similar to Chackalamanni l ' s . 4 7 They simply changed the oxidizing agent from (+)-(10-camphorsulfonyl)oxaziridine to M o O P H , which led to very good yield of 2.19, but they did not circumvent the arduous task Chapter 2: Synthesis of Pelorol and Analogs. 57 Figure 2.25 Synthetic route to 2.52 from 2.20. of making M o O P H . A search of the literature revealed that Kuchkova et al. had described a short efficient synthesis of drimane diol 2.52 from (+)-sclareolide. 4 8 W e felt this could be exploited to generate the aldehyde 2.19. Following Kuchkova's procedure, the reaction of sclareolide (2.20) with methyllithium in a molar ratio 1:2 afforded the desired hydroxy ketone 2.48 in 40% yield. The diol 2.49 was produced as the major product in 51% yield, which was much higher than Kuchkova's report of 18% yield for this compound. Decreasing the amount of methyllithium to 1 equivalent effectively suppressed the dialkylation product 2.49, but also increased the amount of unreacted sclareolide (2.20). In order to Chapter 2: Synthesis of Pelorol and Analogs. 3 ° find the optimal ratio between sclareolide (2.20) and methyllithium, various amounts of methyllithium were added to the substrate on different time scales. Eventually it was found that 1:1.2 was the best ratio for sclareolide and methyllithium, giving a 91% of 2.48 and only a trace amount of 2.49. The reaction time was optimized to 30 minutes to assure the adequate transformation of 2.20. Ketone 2.48 existed in equilibrium with its hemiacetal form 2.50. With a rather good yield of 2.48 in hand, purification was not necessary and the methylation product could be subjected to the next step after a simple workup which removed the base. The oxidation of 2.48 to afford 2.51 in quantitative yield was accomplished with an excess of trifluoroperacetic acid, prepared in situ from trifluoroacetic anhydride and 50% hydrogen peroxide, in dichloromethane in the presence of sodium hydrogen carbonate. The successful Baeyer-Villiger transformation of 2.48 to 2.51 was largely dependent on the addition of N a H C 0 3 to the reaction mixture. A s Kuchkova et al. have mentioned, 4 8 the ratio of sodium hydrogen carbonate and trifluoroacetic anhydride [molar ratio of N a H C 0 3 and ( C F 3 C O ) 2 0 , ca. 1:1] was critical to assure a high yield of hydroxyl ester 2.51. If this ratio is more than one, the yield of 2.51 is lowered because of the simultaneous formation of hydroperoxide 2.53. In practice, adding NaHCC>3 to the reaction mixture at a lower rate and vigorous stirring was found to result in higher yields; ineffective stirring led to a sharp increase in the side product 2.53. Finally, the hydroxyl ester 2.51 was hydrolyzed in basic M e O H at 0 ° C for 30 minutes. Standard workup conditions followed by flash silica gel column chromatography Chapter 2: Synthesis of Pelorol and Analogs. 59 (hexane: E t O A c = 70:30) gave the drimane diol 2.52. Thus , sclareolide (2.20) was converted to the diol 2.52 in excellent yield (90%) using a three-step sequence modification of the literature procedure. The subsequent transformation required oxidation of the resulting diol 2.52 to the aldehyde 2.19. This was accomplished by three different reactions: i) Corey's P C C (pyridinium chlorochromate) oxidation, 4 9 ii) Dess-Martin periodinane oxidation, 5 0 and iii) Swern oxidation. 5 1 Of particular note was that all these oxidation reactions gave the elimination products 2.54 and 2.55 in various yields. P C C oxidation of 2.52 was quite clean in the reaction vessel, but after the reaction was quenched with ether and passed through a pad of Silica gel during workup, nearly 50% of the required product 2.19 lost H 2 0 to give the elimination side products. Changing the oxidant to Dess-Martin periodinane didn't improve 2.52 + 2.19 2.54 2.55 PCC, CH2CI2, r.t, 2 h 38% 35% 17% Triacetoxyperiodinane, CH2CI2, 0°C, 2 h 35% 42% 10% (COCI)2, DMSO, CH2CI2, -78°C, 0.5 h, then Et3N — r.t. 70% 10% 10% Figure 2.26 Synthetic route to 2.19. Chapter 2: Synthesis of Pelorol and Analogs. 60 2.19 2.56 JL > - ^ 2 0 2.57 BBr3, CH2CI2 90% 2.47 SnCI4, -20°C 80% .OMe 2.42 Figure 2.27 Synthetic route to pelorol analog 2.57 the yield of 2.19. Initial trials of the Swern oxidation gave a mixture of 2.19 and the other three elimination products in varied ratios. Monitoring the reaction by T L C and 1 H N M R showed that the ratio of 2.19 and the side products was directly related to the volume of triethylamine added in quenching the reaction and the time required to warm the mixture from - 7 8 ° C to room temperature. It was found that a high yield of the reaction product 2.19 could be produced by addition of 5 equivalents of triethylamine to quench the reaction and reducing the time for warming to 20 minutes. Either increasing the volume of triethylamine or extending the time resulted in more elimination products. With the aldehyde 2.19 in hand, it could be coupled with the dimethoxyethylphenyllithium 2.39 as shown in Figure 2.27 to give the epimeric Chapter 2: Synthesis of Pelorol and Analogs. 61 benzyl alcohols 2.56 in good yield. Hydrogenolysis of 2.56 cleanly removed the benzylic alcohols to afford 2.47, which underwent S n C I 4 catalyzed cyclization to give the desired tetracyclic intermediate 2.42 in high yield, without any trace of the elimination product 2.43. Dimethyl ether 2.42 was converted to the catechol 2.57, which differed from pelorol (2.1) in the C-20 substituent. T o provide more pelorol (2.1) analogs for preliminary S A R study, 2.19 was also coupled with dimethoxymethylphenylbromide 2.58 produced from reduction of 6-bromovertraldehyde (2.30) with N a C N B H 3 and Z n l 2 , a powerful deoxygenation combination described a b o v e . 4 4 T h e entire sequence was repeated with Figure 2.28 Synthetic route to 2.58 OMe OMe CHO THF, -78°C 60% -OMe 2.59 2.60 2.61 2.19 'H SnCI4, CH2CI2 -20°C, 90% MeO ,OMe BBfg, CH2CI2 90% Fiqure 2.29 Synthetic route to 2.63 Chapter 2: Synthesis of Pelorol and Analogs. D ^ phenyllithium 2.59. After hydrogenolysis, cyclization, and demethylation, the methyl analog 2.63 was obtained. Both compounds 2.57 and 2.63 could be regarded as simplified versions of pelorol (2.1) and the short and efficient syntheses of these compounds made them attractive tools for further biological studies and ideal candidates for future drug development. The next critical transformation in the pelorol synthesis was the regioselective introduction of oxygen at the C-22 methylene of 2.42. Conventional regioselective oxidation of C - H bonds requires neighboring activating groups and the oxidation of benzylic methylenes is common in synthesis. The most successful reagents for this transformation are derivatives of selenium (IV) 5 2 and chromium (VI). 5 3 The recent development of I B X 5 4 by K.C.Nicolaou as an oxidant for benzylic carbons and Ishii's aerobic oxidation m e t h o d 5 5 which uses NHPI analogs as key catalysts, have provided new tools for our arsenal. Both 2.42 and 2.62 were subjected to the standard IBX-induced oxidation condition [2.5 equiv IBX, f luorobenzene/DMSO (2:1) at 6 5 ° C ] and more forcing conditions, but unfortunately no reaction occurred. Changing to Ishii's mild aerobic oxidation condition also failed to yield the desired product. Compound 2.42 was then refluxed in ethanol overnight in presence of SeC>2, and again only starting material was recovered. Gentle refluxing of 2.42 with P C C 5 6 in C H 2 C I 2 for 20 hours and standard workup and purification of the reaction mixture with column chromatography gave methyl ketone 2.67. T h e disappearance of the C-22 methylene signal and the appearance of a methyl ketone signal at 52.49 ppm in Chapter 2: Synthesis of Pelorol and Analogs. 63 / 2.65 Figure 2.30 Synthetic route to 2.67 with proposed mechanism the 1 H N M R of 2.67 indicated the reaction had proceeded as desired. The mechanism for this reaction might be as shown in Figure 2.30. Chromium trioxide might first add to the arene in an "ene" reaction to yield the allylchromic acid 2.65, which can rearrange to the benzyl chromoxylate 2.66. Compound 2.66 can then cleave to give the oxidation product 2.67. Repeating the same reaction on the methyl analog 2.62 didn't afford the expected C-22 aldehyde. T o complete the synthesis of pelorol, it was now necessary to convert the methyl ketone into a carboxylic acid. Chapter 2: Synthesis of Pelorol and Analogs. Treatment of methyl ketone 2.67 with l 2 in aqueous N a O H , 5 7 in an attempt to effect a haloform reaction to give benzoic acid 2.72, unexpectedly resulted in the near quantitative formation of the a-ketoacid 2.70. This anomalous result might Figure 2.31 Synthetic route to 2.70 with proposed mechanism be caused by the steric bulk of the C-8 carbon ortho to the C-22 ketone preventing the formation of a triiodinated methyl. If a diiodinated methyl ketone 2.68 was attacked by hydroxide to give a tetrahedral intermediate, the diiodomethyl may not be a good enough leaving group to depart in the normal fashion to give a carboxylic acid. Instead, an intramolecular S N 2 displacement of iodide can form an epoxide, which after fragmentation as shown in Figure 2.31 would lead to the a-ketoaldehyde 2.69. Oxidation of 2.69 via iodination of the aldehyde hydrate can generate the final a-ketoacid 2.70. Simply changing the Chapter 2: Synthesis of Pelorol and Analogs. 65 Figure 2.32 synthetic route to pelorol (2.1) halogen to B r 2 led to a clean transformation of methyl ketone 2.66 to the desired benzoic acid 2.72 (Figure 2.32). The synthesis of pelorol (2.1) was completed by esterification of 2.72 with Mel followed by selective cleavage of the phenyl methyl ethers with B l 3 at - 7 8 ° C . Efforts in selective demethylation of 2.73 with BBr3 led to the free carboxylic acid 2.74. Synthetic pelorol (2.1) was identical by N M R and M S comparison with the natural product. The [a]o of the synthetic material was - 6 4 ° compared with values o f - 6 9 ° reported by Konig and - 7 1 ° reported by Schmitz, confirming that the absolute configuration is 5S ,8R,9R,10S as predicted by S c h m i t z . 1 6 3 Chapter 2: Synthesis of Pelorol and Analogs. 6 6 E Q. Figure 2.33 Comparison of 1 H N M R of natural and synthetic pelorol (2.1) recorded in CDCI 3 at 400MHz. Figure 2.34 Comparison of 1 3 C N M R of natural and synthetic pelorol (2.1) recorded in CDCI3 at 100MHz. Chapter 2: Synthesis of Pelorol and Analogs. 0 0 2.5 Biological acitivities of pelorol and its analog AQX-16A (2.63) ' Because of the promising biological properties of pelorol, we undertook its total chemical synthesis and successfully developed an efficient, high yielding, 12-step protocol for the synthesis of pelorol from the commercially available terpenoid sclareolide. This allowed the production of sufficient quantities for testing in biological assays. Additionally, certain intermediate compounds derived during the synthesis, as well as structural analogs of pelorol, were tested Figure 2.35. Pelorol and AQX-016A increase in vitro SHIP enzyme activity. 40 u.g/mL of purified Pelorol or AQX-016A were tested for their ability to enhance SHIP'S enzyme activity (expressed as u.moles phosphate released/min). The error bars indicate the standard error in the mean (SEM) of triplicate determinations and the data are representative of at least 3 experiments. for their ability to activate SHIP 'S enzymatic activity. O n e of these, designated AQX-16A (compound 2.63 generated in 9 chemical steps from sclareolide in section 2.4 of this chapter) showed a 3-fold higher activation of SHIP than pelorol at the same molar concentration (Figure 2.35). Due to the relative ease of synthesis of AQX-16A and its enhanced potency, most of the subsequent studies were performed with AQX-16A. o CO 0 E N c 0 Q. X w 6.0 c E a> 15 4.0 .c a. CO o -c a. in 0 2.0 0.0 No drug Pelorol AQX-016A AQX-16A was the designation give to compound 2.63 by our biological collaborators, G. Krystal, A.Mui and C.Ong. The biological data presented here was generated by Krystal, Mui and Ong. Chapter 2: Synthesis of Pelorol and Analogs. 69 a 2500 2000 " 1500 CO Q_ i IT) < 1000 co i Q_ 500 Q. O b 5000 . 4000. 3000 °r 2000 co" 5 1 1000 mm LPS AQX-016A LY294002 LPS AQX-016A LY294002 Figure 2.36 AQX-016A stimulates SHIP'S enzyme activity in intact cells. J16 cells were treated for 30 min with 5 ng/mL AQX-016A, 25 \iM LY294002 or carrier prior to stimulation with 50 ng/mL of LPS with or without 100 ng/mL IL-10, as indicated, for 15 min. Cellular lipids were extracted and analyzed for PIP 3 (panel a) and PI-3,4-P2 (panel b) levels as described in Methods. The error bars indicate the SEM of three independent replicates for each stimulation condition and the experiment was repeated twice. To be an effective drug candidate, AQX-16A needs to be cell permeable and able to activate SHIP in intact cells. To directly test whether AQX-16A was indeed able to activate SHIP'S enzyme activity in intact cells the inositol phospholipid content of macrophages stimulated with lipopolysaccharide (LPS) in the presence and absence of AQX-16A was measured. As shown in Figure 2.36a, LPS stimulated a 3-5 fold increase in PIP3 levels, in keeping with the ability of LPS to activate the PI3K pathway.58"60 The addition of AQX-16A abolished this increase (Figure 2.36a) and resulted in a corresponding increase in the SHIP hydrolysis product PI-3,4-P2 (Figure 2.36b). For comparison, LPS-stimulated cells were also treated with the PI3K inhibitor LY294002 and, as expected, P I P 3 levels were diminished without a corresponding increase in PI-Chapter 2: Synthesis of Pelorol and Analogs. ' u 3,4-P 2 levels. Thus , both AQX-16A and LY294002 inhibit the PI3K-mediated increase in intracellular P I P 3 levels, but through different mechanisms. A n important property of any potential drug is its specificity: it must act via its designated target and alter biological responses in a predictable manner. A rigorous way to test the specificity of AQX-16A for its target SHIP is to compare its effects on SHIP-regulated processes in wild-type S H I P + / + vs SHIP"7" cells. In this regard, it has been previously shown that activation of macrophages by L P S 5 8 , 6 0 , and mast cells by IgE receptor (FcsRI) cross-l inking 6 1 " 6 3 is negatively regulated by SHIP. Macrophages are an essential component of the innate and acquired immune response, and stimulation with L P S is associated with a PI Pa-Figure 2.37a,2.37b AQX-016A inhibits activation of SHIP*'* but not SHIP"'" macrophages and mast cells. In panel a, SHIP*'* and SHIP "'" (§) BMrn^s were pretreated with the indicated concentration of AQX-016A or carrier 30 min prior to stimulation with 10 ng/mL of LPS at 37°C. Supernatants were collected 1 hr later for TNFa determination by ELISA. Values were plotted as a percentage of maximum TNFa production for SHIP*'* and SHIP"'" cells (623 and 812 pg/ml, respectively). This experiment was repeated 3 times, the error bars indicate the SEM of triplicates. In panel b, SHIP*'* (|) and SHIP"'" (|) BMm<(>s were pretreated with the indicated concentration of LY294002 or carrier for 30 min prior to stimulation with 10 ng/mL of LPS at 37°C. Supernatants were collected 1 hour later for determination by ELISA and values were plotted as a percentage of maximum TNFa production for SHIP*'* and SHIP''" cells (693 and 921 pg/mL, respectively). Error bars indicate SEM of triplicate determinations, this experiment was repeated twice. AQX-016A AQX-016A AQX-016A 5ng/mL 1ng/mL LY294002 LY294002 LY294002 50|iM 25nM Chapter 2: Synthesis of Pelorol and Analogs. 71 c Vehicle AQX-016 S H I P + / + £2 0 100 300 Time (s) 500 0 100 300 Time (s) 500 S H I P -I- S2 2 2 0 100 300 Time (s) 500 0 100 300 Time (s) 500 Figure 2.37c SHIP + / + (top panels) and SHIP"'" (bottom panels) BMMCs were pre-loaded with IgE and incubated with Fura-2 as described in Methods, and pretreated for 30 min with 0.5 /yg/ml AQX-016A (right panels) or carrier (left panels). Cells were then stimulated (as indicated by the arrow) with 0 (••••), 1 (—) , 5 (—) or 10 (_) ng/mL DNP-HSA and intracellular calcium levels monitored over time by spectroflourometry61. dependent release of pro-inflammatory mediators such as TNFa. The action of AQX-16A on wild-type vs SHIP"7" bone marrow derived macrophages (BMm<j>s) was examined, and as shown in Figure 2.37a, AQX-16A was far more effective at inhibiting LPS-stimulated TNFa production in wild-type than in SHIP"7" cells, while as expected, LY294002 inhibited TNFa production equally well in both wild-type and SHIP"7" BMm<j)S (Figure 2.37b). Mast cells also play a key role in triggering inflammation and in initiating allergic responses. Activation of mast cells results in the release of intracellular granule contents (i.e., degranulation) and this is preceded by an influx of extracellular calcium that plays an essential Chapter 2: Synthesis of Pelorol and Analogs. 72 Figure 2.37d SHIP ' (top panel) and SHIP"7" (bottom panel) BMMCs were pre-loaded with IgE and incubated with Fura-2 as above, and pretreated for 30 min with vehicle (_), 5 (—), or 10 (—) JJM LY294002. Cells were then stimulated 5 ng/mL DNP-HSA or 0 ng/mL of DNP- HSA (••••) and intracellular levels monitored as in c. These experiments were repeated at least twice. role in triggering the degranulation process. A s shown in Figure 2.37c, AQX-16A inhibited the IgE + antigen-induced calcium influx to a substantially greater degree in wild-type than in SHIP"7" bone marrow derived mast cells ( B M M C s ) . T o rule out that this difference was not simply due to SHIP"7" B M M C s being less sensitive to PI3K inhibition, the effect of LY294002 on IgE + antigen-induced calcium influxes was tested in these two cell types and it was found that S H I P + / + and SHIP"7" B M M C s were inhibited to a similar extent by this PI3K inhibitor (Figure 2.37d). These data indicate AQX-16A inhibits both macrophage and mast cell activation in a SHIP-dependent manner. The downstream signaling proteins that are activated by PIP 3 -dependent pathways in m a c r o p h a g e s 5 8 , 6 0 and mast cells 6 3 " 6 5 have been well characterized. (1 LY294002 0 100 300 500 Time (s) 8, SHIP -I-Time (s) Chapter 2: Synthesis of Pelorol and Analogs. 73 a SHIP+/+ - 5.0 2.5 + + + + SHIP+/+ + + SHIP-/-- 5.0 2.5 AQX-016A (mg/mL) + + + LPS + - . Vehicle mm —• phospho-PKB mm mm mm P h o s P h ° - p 3 8 M A p K SHIP SHIP-/-+ + + + + AQX-016A DNP Vehicle Phospho-PKB Phospho-P38 MAPK Phospho-ERK1/2 SHIP Grb2 Figure 2.38 AQX-016A inhibits PI3K-dependent signaling in SHIP+/+ but not SHIP"'" macrophages and mast cells. In panel a, SHIP + / + and * BMmtjjs were pretreated for 30 min with AQX-016A or carrier prior to stimulation with 10 ng/mL of LPS for 15 min. In panel b, SHIP + / + and "'" BMMCs were pre-loaded overnight with 0.2 //g/ml anti-DNP-lgE and then treated for 30 min with 10 /vg/ml AQX-016A or carrier prior to stimulation with 20 ng/ml DNP-HSA for 5 min. Total cell lysates were analyzed for the indicated phospho-proteins or proteins by immunoblot analysis. These experiments were repeated three times. Therefore, the ability of AQX-16A to inhibit the activation (as assessed by site specific phosphorylation) of these proteins in wild-type vs SHIP"'" cells was compared. L P S stimulation of macrophages results in the activation of the P I P 3 -dependent protein kinase P K B (also known as Akt) and the downstream kinase P38MAPK A S S N O W N I N F I G U R E 2.38a, AQX-16A inhibited the LPS-stimulated phosphorylation of both P K B and p 3 8 M A P K in wild-type but not in SHIP"7" BMm<|>s. FcsRI cross-linking in mast cells results in the PIP3-dependent activation of the kinases P K B , p 3 8 M A P K , and M A P K (Erk1/2) 6 3 . A s shown in Figure 2.38b, AQX-16A inhibited the activation of all of these proteins in wild-type but not in SHIP"7" Chapter 2: Synthesis of Pelorol and Analogs. 74 B M M C s . Thus , in both cell types, AQX-16A inhibits PIP 3-regulated intracellular signal transduction events in a SHIP-dependent manner. The litmus test for the clinical utility of any potential therapeutic is the ability to exert effects in animal models. Therefore, the in vivo efficacy of AQX-16A was evaluated in mouse models of septicemia and acute cutaneous anaphylaxis. In humans, septic s h o c k 6 6 is a condition in which uncontrolled bacterial infection leads to the over-enthusiastic production of T N F a and nitric oxide (NO). Death often ensues due to hypotension. T h e mouse model of this condition involves intraperitoneal injection of bacterial L P S (also known as endotoxin) and measurement of serum T N F a levels 2 hrs later. 6 7 AQX-16A or the steroidal drug dexamethasone were orally administered to mice 30 min prior to the L P S challenge. A s predicted for an activator of SHIP and an inhibitor of macrophage z 600 * « 400 • « 200 • « t 1 X 0 Vehicle Dexamethasone AQX-016A 1.2 c 0.8 0.4 0.0 Vehicle AQX-016A No DNFB Figure 2.39 AQX-016A is protective in mouse models of septicemia and acute cutaneous anaphylaxis. In panel a, mice were administered 20 mg/kg AQX-016A or 0.4 mg/kg dexamethasone orally 30 min prior to an intraperitoneal injection of 20 mg/kg LPS. Blood was collected 2 hrs later for TNFa determination by ELISA. In panel b, *\0 \xg of AQX-016A or vehicle was applied to the right ears of DNP-sensitized mice 30 min prior to application of DNFB to both ears. The no DNFB group had DNFB applied only to the left ear. Each symbol indicates one mouse and data are representative of three independent experiments. Chapter 2: Synthesis of Pelorol and Analogs. 75 activation, AQX-16A reduced the level of serum TNFa and did so to the same extent as dexamethasone (Figure 2.39a). Anaphylactic or alergic responses on the other hand are mediated by alergen-induced degranulation of pre-sensitized mast cells23. The mouse ear edema/cutaneous anaphylaxis model24 reflects this process and involves pre-sensitization of mice with the alergen dinitrofluorobenzene (DNFB) one week prior to elicitation of the alergic reaction by painting DNFB onto the ears of the mice. The efficacy of potential anti-inflammatory compounds is tested by topical application of the test substance to one ear and comparing the resulting ear edema or inflammation of the two ears. As shown in Figure 2.39b, AQX-16A dramaticaly inhibited alergen-induced anaphylaxis in this model. Thus AQX-16A is protective in both septicemia and alergy models, and is oraly bioavailable. The data presented above suggest that AQX-16A might be a useful therapeutic agent for the treatment of various inflammatory disorders. However, AQX-16A may also be useful for the treatment of hematologic malignancies. Loss of SHIP protein has been implicated in human chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML). The transforming oncogene in CML is the tyrosine kinase BCR-abl and expression of BCR-abl has been shown to mediate its oncogenic effects via activation of the PI3K pathway63 and intriguingly has been shown to directly downregulate SHIP levels. The BCR-abl inhibitor Gleevec Chapter 2: Synthesis of Pelorol and Analogs. 76 Parental Ba/F3 4000 0 1000 2000 3000 4000 [Gleevec] nM Fig 2.40: AQX-016A potentiates the cytostatic effect of Gleevec on BCR-abl expressing cells Parental (panel a) or BCR-abl (panel b)-expressing Ba/F3 cells were grown for 18 hrs in RPMI + 1 //g/ml IL-3 + 10% FCS in the presence of 1 jug/ml AQX-016A (B) or vehicle (p) and the indicated concentrations of Gleevec. [3H]-Tdr was then added for 2 hrs and the cells harvested for liquid scintillation counting. Error bars indicate the SEM and data are representative of three independent experiments. very efficiently inhibits B C R - a b l kinase activity and achieves astonishing remission rates when administered to C M L patients. 7 0 However, there are side effects 7 1 associated with this drug and Gleevec resistant ce l l s 7 2 eventually appear in the treated patients. Thus , a great deal of effort is currently focused on discovering new B C R - a b l inhibitors which inhibit using different mechanisms or developing combination therapies with other drugs to circumvent the problem of Gleevec side effects and resistance. 7 3 Drugs inhibiting the PI3K pathway have attracted particular attention for use in combination with B C R - a b l inhibitors 7 3 so Chapter 2: Synthesis of Pelorol and Analogs. ' ' AQX-16A was tested for the ability to synergize with Gleevec in inhibiting B C R -Abl transformed cells. A s predicted, it was found that addition of AQX-16A substantially enhanced the sensitivity of B C R - a b l expressing Ba/F3 (Figure 2.40b) cells to Gleevec while having no activity on control Ba/F3 cells (Figure 2.40a). In summary, pelorol (2.1) and analogs such as AQX-16A represent a new class of anti-inflammatory and anti-leukemic drugs which target and activate the negative regulator, SHIP. This work marks the first time that a small molecule activator of a phosphatase has been described. T h e restricted expression of SHIP to immune/hematopoietic cells limits the action of these compounds to these cells. The novel mechanism of action of these compounds creates new treatment options as well as provides potential synergies with existing therapeutics. Chapter 2: Synthesis of Pelorol and Analogs. 78 2.6 Experimental General: All starting materials and reagents were obtained from commercial sources and were used without further purification. Diethylether (Et 2 0) and tetrahydrofuran (THF) were purified by distillation from sodium/benzophenone under argon immediately before use. Dichloromethane was distilled from C a H 2 under argon. Moisture and oxygen sensitive reactions were carried out in flame-dried glassware under N 2 or argon. Evaporations were conducted under reduced pressure at room temperature. Analytical thin layer chromatography was performed on silica gel 60 F-254 pre-coated aluminum plates (Merck). Column chromatography was carried out on 70-230 mesh silica gel and 10g normal phase Sepaks with the indicated solvent. U V spectra were recorded on a Waters 2487 spectrophotometer. Optical rotations were determined with a J A S C O J -1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm micro cell. N O E S Y , H M Q C , and H M B C spectra were recorded on a Bruker Avance 400 N M R spectrometer. 1 3 C spectra and 1 H spectra were recorded on Bruker A M 4 0 0 , Bruker A M X 5 0 0 , and Bruker Avance 400 N M R spectrometers. Chemical shifts were referenced to solvent peaks (8H 7.24, 8c 77.0 for C D C I 3 ) . Low resolution ESI mass spectra were recorded on a Bruker Esquire L C mass spectrometer. High resolution ESI mass spectra were recorded on a Macromass L C T mass spectrometer. Both low and high resolution F A B M S were recorded on a Kratos Concept II H Q mass spectrometer. H P L C separations were achieved using a Waters 600 pump and a Waters 486 tuneable absorbance detector. Chapter 2: Synthesis of Pelorol and Analogs. ' y Solvents were all H P L C grade (Fisher) and those used for H P L C were filtered prior to use. Preparation of 1-bromo-2,3,5-trimethoxybezene (2.28) OMe 2.28 was synthesized according to the reported procedure. Preparation of 1-bromo-2,3-dimethoxy-5-ethyl benzene (2.38) OMe The precursor of 2.38, 1-bromo-2,3-dimethoxy-5-ethenyl benzene, was synthesized according to the literature procedure . 4 6 T h e hydrogenation of 1-bromo-2,3-dimethoxy-5-ethenyl benzene (5.0 g, 20.4 mmol) was carried out overnight in C H 2 C I 2 (15 mL) under H 2 (3 bars) in the presence of 5% R h / C (800mg). T h e mixture was filtered and then washed with acetone, and the filtrate and washings were evaporated and lyophilized to afford 4.84 g of 2.38 as a colorless oil (96%) which was not purified and could be used in the next step. Chapter 2: Synthesis of Pelorol and Analogs. 80 1 H N M R (CDCI3) 5 1.20 (t, J= 7.6 Hz, 3H), 2.55 (q, J= 7.6 Hz, 2H), 3.81 (s, 3H), 3.83 (s, 3H), 6.66 (d, J= 1.83 Hz, 1H), 6.95 (d, J= 1.83 Hz, 1H); 1 3C N M R (CDCI3) 5 15.4, 28.5, 56.0, 60.5, 111.5, 117.3, 123.7, 141.4, 144.4, 153.4; H R E S I M S calcd for C i 0 H 1 4 O 2 8 1 B r 247.0157, found 247.0157. Preparation of 1-bromo-2,3-dimethoxy-5-methyl benzene (2.58) OMe T o a stirred solution of 5-bromoveratraldehyde (2.0 g, 8.7 mmol) in 1,2-dichloroethane (45.0 mL) at ambient temperature were added zinc iodide (3.9 g, 12.2 mmol) and sodium cyanoborohydride (3.8 g, 61.2 mmol). The reaction mixture was heated under gentle reflux for 20 hrs and then filtered through celite. The filtrate was evaporated to dryness to afford 1.5 g of 2.58 as a light yellow oil (80%). 1 H N M R (CDCI3) 5 2.26 (s, 3H), 3.80 (s, 3H), 3.82 (s, 3H), 6.63 (d, J= 1.4 Hz, 1H), 6.92 (d, J= 1.4 Hz, 1H); Chapter 2: Synthesis of Pelorol and Analogs. 81 1 3 C N M R (CDCI3) 5 20.9 ,56.0 ,60.4 , 112.6, 117.2, 124.9, 135.0, 144.2, 153.3; H R E S I M S calcd for C 9 H n 0 2 N a 8 1 B r 254.9820, found 254.9830. Preparation of tertiary alcohol 2.41 OMe I I X)H ^"""o 2.41 A 1.7 M solution of t-BuLi in pentane (1.74 mL, 2.79 mmol) was added slowly to a stirred solution of 2.38 (627.4 mg, 2.54 mmol) in dry T H F (20.0 mL) at -78°C. After stirring for 30 minutes, a solution of 2.18 (300mg, 1.26mmol) in dry T H F (5.0 mL) was added. The mixture was further stirred at -78°C for 2hrs. Then H 2 0 (10.0 mL) was added and the mixture was extracted with E t 2 0 (120mL twice). T h e combined E t 2 0 extracts were washed with saturated brine, dried (MgSCv) and concentrated to give a residue, which was chromatographed on a normal phase Seppak (hexane:ethyl acetate 9:1) to give 306 mg (60%) of 2.40 as a white solid. 1 H N M R (CDCI3) 5 0.87 (s, 3H), 0.88 (s, 3H), 1.03 (s, 3H), 1.21 (t, J= 7.6 Hz, 3H), 1.36 (s, 3H), 1.92 (brd, J= 12.5Hz, 1H), 2.59 (q, J= 7.7 Hz, 2H), 3.77 (s, 3H), 3.84 (s, 3H), 5.49 (s, 1H), 6.63 (d, J= 1.7Hz, 1H), 7.03 (d, J= 1.7 Hz, 1H); Chapter 2: Synthesis of Pelorol and Analogs. 82 1 3 C N M R (CDCI3) 8 15.7, 16.3, 18.4, 18.8, 21.8, 29.0, 32.8, 33.5, 33.8, 39.3, 39.9, 41.9, 44.1, 55.6, 55.7, 59.1, 60.0, 69.5, 75.4, 76.7, 77.0, 77.3, 110.0, 118.9, 139.2, 139.8, 143.3, 151.9; H R E S I M S calcd for C25H 4 o0 4 Na 427.2824, found 427.2828. Hydrogenolysis of 2.40 (202 mg, 0.5 mmol) was carried out in E t O A c (8.0 mL) under H 2 (3 bars) in the presence of 10 % P d / C (150 mg) overnight. The reaction mixture was filtered and concentrated to afford a brown oil, which was further purified by column chromatography to give 2.41 (165 mg, 85%) as a colorless oil. 1 H N M R ( C D C I 3 ) 8 0.85 (s, 3H), 0.87 (s, 3H), 0.90 (s, 3H), 1.06 (s, 3H), 1.21 (t, J= 7.6 Hz, 3H), 1.73 (dt, J= 13.1, 2.7 Hz, 1H), 1.79 (brd, J= 12.3 Hz, 1H), 2.57 (q, J= 7.5 Hz, 2H), 2.60 (dd, J= 15.7, 2.9 Hz, 1H), 2.91 (dd, J= 15.7, 7.3 Hz, 1H), 3.78 (s, 3H), 3.82 (s, 3H), 6.54 (d, J= 1.8 Hz, 1H), 6.63 (d, J= 1.8 Hz, 1H); 1 3 C N M R ( C D C I 3 ) 8 15.2, 15.8, 18.5, 18.5, 21.9, 23.4, 28.9, 31.6, 33.4, 33.6, 39.0, 40.0, 42.0, 42.9, 55.6, 56.0, 58.9, 60.6, 73.3, 109.0, 120.5, 137.9, 139.7, 144.5, 152.4; H R E S I M S calcd for C 2 5 H 4 o 0 3 N a 411.2875, found 411.2880. Chapter 2: Synthesis of Pelorol and Analogs. 83 Preparation of tetracycle 2.36 OMe OMe The procedure for the synthesis of the tertiary alcohol 2.34 from the diol 2.33 was the same as the synthesis of 2.41 from 2.40 and afforded a colorless oil (20mg, 60%). It was dissolved in 5ml_ of C H 2 C I 2 and S n C I 4 (0.05 mL) was added slowly during stirring at - 2 0 ° C under argon for 2 min. T h e resulting mixture was further stirred for 20min and then diluted with C H 2 C I 2 (10mL) and poured into ice. T h e aqueous phase was extracted with C H 2 C I 2 twice (10mL) and the combined extracts were washed with sat. NaHCG -3 , sat. brine and dried over M g S 0 4 . Evaporation afforded 2.36 (14 mg, 70%) as a colorless oil. 1 H N M R ( C D C I 3 ) 8 0.84 (s, 3H), 1.01 (s, 3H), 1.06 (s, 3H), 1.23 (s, 3H), 1.71 (dd, J= 12.8, 6.1 Hz, 1H), 2.40 (m, 1H), 2.50 (dd, J= 14.7, 12.9 Hz, 1H), 2.69 (dd, J= 14.7, 6.2 Hz, 1H), 3.74 (s, 3H), 3.76 (s, 3H), 3.82 (s, 3H), 6.28 (s, 1H); 1 3 C N M R (CDCI3) 8 16.2, 18.4, 19.5, 20.7, 21.1, 25.9, 33.1, 33.4, 37.0, 38.4, 40.2, 42.6, 46.8, 55.9, 56.3, 57.5, 60.6, 64.5, 96.3, 134.3, 136.8, 139.8, 150.9, 150.9; H R E S I M S calcd for C 2 4 H 3 6 0 3 N a 395.2562, found 395.2564. Chapter 2: Synthesis of Pelorol and Analogs. 84 Preparation of tetracycle 2.45 OMe Tertiary alcohol 2.41 (100 mg, 0.26 mmol) was dissolved in 3.0 mL of polyphosphoric acid (PPA) and the mixture was heated under argon at 6 0 ° C for 30 minutes. The resulting orange mixture was diluted with ice water and extracted with ether. The combined extracts were washed with brine repeatedly and concentrated to give a brown oil, which was further purified by column chromatography to give 76.3 mg (80%) of 2.45. 1 H N M R (CDCI 3 ) 8 0.73 (s, 3H), 0.75 (s, 3H), 0.82 (s, 3H), 0.99 (d, J= 7.2 Hz, 3H), 1.21 (t, J= 7.5 Hz, 3H), 2.13 (d, J= 18.4 Hz, 1H), 2.61 (m, 2H), 3.08 (m, 1H), 3.20 (d, J= 18.4 Hz, 1H), 3.76 (s, 3H), 3.82 (s, 3H), 6.59 (s, 1H); 1 3 C N M R (CDCI3) 8 14.8, 15.9, 20.6, 21.6, 24.4, 26.3, 27.3, 28.8, 30.5, 32.1, 32.9, 33.7, 34.7, 37.6, 38.2, 38.9, 42.1, 55.5, 59.8, 111.4, 130.2, 132.0, 138.1, 144.4, 149.4; H R E S I M S calcd for CasHssOaNa 393.2770, found 393.2768. Chapter 2: Synthesis of Pelorol and Analogs. 85 Preparation of diol 2.56 OMe The procedure for the synthesis of diol 2.56 from the aldehyde 2.19 and the dimethoxyethyllithium 2.39 was the same as the synthesis of 2.40 from the aldehyde 2.18 and the dimethoxyethyllithium 2.39 and afforded a white solid (63%). 1 H N M R (CDCI 3 ) 6 0.28 (td, J= 13.1, 3.4 Hz, 1H), 0.76 (s, 3H), 0.81 (s, 3H), 1.03 (s, 3H), 1.21 (t, J= 7.6 Hz, 3H), 1.54 (s, 3H), 1.85 (dt, J= 12.2, 3.2 Hz, 1H), 2.11 (d, J= 8.4 Hz, 1H), 2.60 (q, J= 7.6 Hz, 2H), 3.84 (s, 3H), 3.86 (s, 3H), 5.25 (d, J= 8.4 Hz, 1H), 6.65 (d, J= 1.7 Hz, 1H), 6.90 (d, J=1.7 Hz, 1H); 1 3 C N M R ( C D C I 3 ) 6 15.7, 15.9, 18.4, 20.0, 21.7, 25.8, 28.9, 33.3, 33.6, 38.9, 40.0, 41.5, 44.7, 55.7, 56.0, 61.3, 62.7, 69.5, 74.7, 111.0, 119.6, 140.5, 140.6, 143.5,152.3; H R E S I M S calcd for C 2 5H4o0 4 Na 427.2824, found 427.2828. Chapter 2: Synthesis of Pelorol and Analogs. 86 Preparation of tertiary alcohol 2.47 OMe The procedure for the synthesis of tertiary alcohol 2.47 from the diol 2.56 was the same as the synthesis of tertiary alcohol 2.41 from the diol 2.40 and afforded colorless oil (87%). 1 H N M R (CDCI 3 ) 5 0.78 (s, 3H), 0.83 (s, 3H), 0.89 (s, 3H), 1.08 (td, J= 13.3, 3.8Hz, 1H), 1.19 (t, J= 7.6Hz, 3H), 1.26 (s, 3H), 1.82 (dt, J= 12.5, 3.2Hz, 1H), 2.54 (q, J= 7.6Hz, 2H), 2.81 (dd, J= 14.5, 5.5Hz, 1H), 3.81 (s, 6H), 6.54 (d, J= 1.8Hz, 1H), 6.64 (d ,J= 1.8Hz, 1H); 1 3 C N M R ( C D C I 3 ) 5 15.4, 15.7, 18.6, 20.3, 21.6, 24.3, 25.3, 28.8, 33.3, 33.5, 39.4, 40.3, 41.8, 43.7, 55.6, 56.1, 60.6, 62.8, 73.7, 109.6, 122.0, 137.6, 140.0, 144.3, 152.3; H R E S I M S calcd for C 2 5 H4o0 3 Na 411.2875, found 411.2871. Chapter 2: Synthesis of Pelorol and Analogs. 87 Preparation of diol 2.60 OMe The procedure for the synthesis of diol 2.60 from the aldehyde 2.19 and the dimethoxymethyllithium 2.59 was the same as the synthesis of 2.40 from the aldehyde 2.18 and the dimethoxyethyllithium 2.39 and afforded a white solid (61%). 1 H N M R (CDCI 3 ) 5 0.27 (td, J= 12.8, 3.1Hz, 1H), 0.75 (s, 3H), 0.80 (s, 3H), 1.02 (s, 3H), 1.53 (s, 3H), 1.84 (dt, J= 12.2, 2.9 Hz, 1H), 2.10 (d, J= 8.5 Hz, 1H), 2.31 (s, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 5.26 (d, J= 8.5 Hz, 1H), 6.61 (d, J= 1.5 Hz, 1H), 6.89 (d, J= 1.5 Hz, 1H); 1 3 C N M R ( C D C I 3 ) 5 15.8, 18.4, 20.0, 21.5, 21.6, 25.9, 33.2, 33.6, 38.8, 39.8, 41.5, 44.6, 55.6, 55.9, 61.4, 62.6, 69.0, 74.7, 112.1, 120.5, 134.1, 140.7, 143.3, 152.2; H R E S I M S calcd for C 2 4 H 3 8 0 4 N a 413.2668, found 413.2664 Chapter 2: Synthesis of Pelorol and Analogs. 88 Preparation of tertiary alcohol 2.61 OMe T h e procedure for the synthesis of tertiary alcohol 2.61 from the diol 2.60 was the same as the synthesis of tertiary alcohol 2.41 from the diol 2.40 and afforded a colorless oil (89%). 1 H N M R (CDCI 3 ) 5 0.78 (s, 3H), 0.83 (s, 3H), 0.88 (s, 3H), 1.27 (s, 3H), 1.76 (brd, J= 12.6 Hz, 1H), 1.82 (dt, J= 12.5, 3.2 Hz, 1H), 2.25 (s, 3H), 2.54 (dd, J= 14.5, 4.6 Hz, 1H), 2.78 (dd, J= 14.5, 5.6 Hz, 1H), 3.79 (s, 3H), 3.80 (s, 3H), 6.52 (d, J= 1.7 Hz, 1H), 6.62 (d, J= 1.7 Hz, 1H); 1 3 C N M R (CDCI3) 8 15.4, 18.6, 20.3, 21.4, 21.6, 24.3, 25.0, 33.3, 33.5, 39.4, 40.3, 41.8, 43.7, 55.6, 56.1, 60.6, 62.8, 73.7, 110.8, 123.1, 129.6, 133.6, 137.7, 152.3; H R E S I M S calcd for C 2 4 H 3 8 0 3 N a 397.2719, found 397.2713. Chapter 2: Synthesis of Pelorol and Analogs. 89 Preparation of tetracycle 2.42 MeO OMe T o a stirred solution of 2.41 or 2.47 (38.8 mg, 0.1 mmol) in C H 2 C I 2 (10 mL), S n C I 4 (0.1 mL) was added slowly at - 2 0 ° C under argon for 2 min. T h e resulting mixture was further stirred for 20min and then diluted with C H 2 C I 2 (20 mL) and poured into ice. The aqueous phase was extracted with C H 2 C I 2 twice (20 mL) and the combined extracts were washed with sat. N a H C 0 3 , sat. brine and dried over MgSCu. Evaporation afforded 29.5 mg (76%) of 2.42 as a colorless oil. 1 H N M R (CDCI 3 ) 8 0.85 (s, 6H), 1.02 (s, 3H), 1.08 (s, 3H), 1.22 (t, J= 7.6 Hz, 3H), 2.36 (dt, J= 12.0, 3.4 Hz, 1H), 2.49 (dd, J= 14.5, 13.0 Hz, 1H), 2.56 (dd, J= 14.5, 7.5 Hz, 1H), 2.69 (m, 2H), 3.80 (s, 3H), 3.81 (s, 3H), 6.49 (s, 1H); 1 3 C N M R ( C D C I 3 ) 6 16.0, 16.1, 18.3, 19.7, 21.1, 21.3, 24.7, 25.2, 33.1, 33.4, 37.1, 39.3, 40.2, 42.5, 47.9, 55.9, 57.1, 60.4, 64.3, 111.1, 133.7, 135.8, 143.6, 144.8, 150.3; H R E S I M S calcd for C 2 5 H380 2 Na 393.2770, found 393.2768. Chapter 2: Synthesis of Pelorol and Analogs. 90 Preparation of tetracycle 2.62 MeO OMe The procedure for the synthesis of tetracycle 2.62 from the tertiary alcohol 2.61 was the same as the synthesis of tetracycle 2.42 from the tertiary alcohol 2.41 or 2.47 and afforded a colorless oil (85%). 1 H N M R (CDCI 3 ) 8 0.85 (s, 6H), 1.02 (s, 3H), 1.06 (s, 3H), 2.24 (s, 3H), 2.32 (dt, J= 11.7, 3.1 Hz, 1H), 2.49 (dd, J= 14.8, 13.0 Hz, 1H), 2.69 (dd, J= 14.8, 6.1 Hz, 1H), 3.47 (s, 6H), 6.41 (s, 1H); 1 3 C N M R (CDCI3) 8 16.1, 18.3, 18.6, 19.6, 20.5, 21.1, 25.3, 33.1, 33.4, 37.1, 38.8, 40.1, 42.5, 47.6, 55.9, 57.1, 60.4, 64.4, 113.0, 127.1, 135.8, 143.7, 145.2, 150.0; H R E S I M S calcd for C^HaeC^Na 379.2613, found 379.2614 Chapter 2: Synthesis of Pelorol and Analogs. 91 Preparation of catechol 2.57 HO OH T o a stirred solution of 2.42 (38.6mg, 0.1 mmol) in CH2CI2 (1 mL) under argon at 0 ° C , B B r 3 in CH2CI2 (2.0 mL 1M) was added, and stirring was continued for 1.5 h at room temperature. The mixture was then poured into H 2 0 and extracted with CH2CI2 (50 mL). The combined extracts were then dried over MgSC>4, filtered and concentrated. The residue was purified by flash chromatography (hexane:EtOAc=7:3) to afford 32mg (90%) of 2.57 as a white solid. 1 H N M R ( C D C I 3 ) 5 0.85 (s, 6H), 1.02 (s, 3H), 1.20 (s, 3H), 2.35 (dt, J= 12.2, 3.4 Hz, 1H), 2.43 (dd, J= 13.9, 13.6 Hz, 1H), 2.57 (m, 2H), 2.61 (dd, J= 14.5, 7.3 Hz, 1H), 6.47 (s, 1H); 1 3 C N M R (CDCI3) 5 15.9, 16.1, 18.3, 19.6, 21.0, 21.3, 24.1, 24.4, 33.0, 33.3, 37.0, 40 .1 ,42 .4 ,48 .0 , 57.0, 64.5, 113.5, 128.4, 130.8, 137.8, 141.3, 144.7; H R E S I M S calcd for C 2 3 H340 2 Na 365.2457, found 365.2456 Chapter 2: Synthesis of Pelorol and Analogs. 92 Preparation of catechol 2.63 HO OH T he procedure for the synthesis of catechol 2.63 from the tetracycle 2.62 was the same as the synthesis of catechol 2.57 from the tetracycle 2.42 and afforded a white solid (85%). 1 H N M R (CDCI 3 ) 5 0.85 (s, 6H), 1.02 (s, 3H), 1.04 (s, 3H), 1.74 (dd, J= 12.6, 6.4 Hz, 1H), 2.17 (s, 3H), 2.31 (dt, J= 11.6, 3.0 Hz, 1H), 2.43 (dd, J= 14.0, 12.5 Hz, 1H), 2.57 (dd, J= 14.3, 6.4 Hz, 1H), 6.40 (s, 1H); 1 3 C N M R ( C D C I 3 ) 8 16.1, 18.2, 18.3, 19.5, 20.5, 21.1, 24.5, 33.0, 33.4, 37.0, 38.7,40.0, 42.5,47.8, 57.0, 64.6, 115.7, 124.1, 128.7, 138.0, 141.0, 145.2; H R E S I M S calcd for C 2 2H 3 20 2 Na 351.2300, found 351.2303. Chapter 2: Synthesis of Pelorol and Analogs. 93 Preparation of methyl ketone 2.67 MeO OMe Pyridium chlorochromate (416mg, 1.92mmol) was added to 2.42 (74mg, 0.2mmol) dissolved in 20mL of C H 2 C I 2 . T h e mixture was stirred at gentle reflux for 20 hours under argon. T h e reaction was diluted with E t 2 0 (80mL) and the resulting dark solution was filtered through a pad of silica gel. Concentration of the filtrates and further purification afforded 40.7 mg (53 %) of 2.67 as colorless oil. 1 H N M R (CDCI 3 ) 5 0.82 (s, 3H), 0.84 (s, 3H), 1.03 (s, 3H), 1.22 (s, 3H), 2.31 (dt, J= 12.5, 3.7Hz, 1H), 2.49 (s, 3H), 2.54 (dd, J= 14.9, 12.5Hz, 1H), 2.69 (dd, J= 14.9, 6.7Hz, 1H), 3.84 (s, 3H), 3.86 (s, 3H), 6.79 (s, 1H); 1 3 C N M R ( C D C I 3 ) 5 16.2, 18.3, 19.4, 20.5, 21.1, 25.2, 30.2, 33.1, 33.4, 36.4, 37.1, 40.2, 42.5, 48.0, 56.2, 57.2, 60.4, 64.9, 110.1, 130.5, 137.4, 147.6, 147.9, 149.6, 201.8; H R E S I M S calcd for C 25H360 3Na 407.2562, found 407.2563. Chapter 2: Synthesis of Pelorol and Analogs. 94 Preparation of ct-Keto ester 2.71 MeO OMe OMe O 2.67 (15.0 mg, 0.04 mmol) was dissolved and stirred in 20.0 mL of N a O H (10%) solution (contain 5.0 mL T H F ) . 50 mg of iodine was added subsequently and the mixture was further stirred for 20 min and acidified by adding 30.0 mL of 10% H 2 S 0 4 . T h e solution was extracted with 150.0 mL of E t 2 0 , washed with sat. brine and concentrated to afford a residue, which was dissolved in 10.0 mL of D M F . Potassium carbonate (0.5 g) and Mel (1.0 mL) were added to the mixture and stirring was continued overnight. The reaction was quenched by adding 10.0 mL of brine and the resulting mixture was extracted with E t 2 0 , washed with brine, dried with M g S 0 4 and evaporated to dryness. The residue was further purified by column chromatography to give 15.0 mg of 2.71 (95%). 1 H N M R ( C D C I 3 ) 8 0.84 (s, 3H), 0.87 (s, 3H), 1.19 (s, 3H), 1.41 (s, 3H), 1.79 (dd, J= 11.9, 7.8 Hz, 1H), 2.41 (brd, J= 11.9 Hz, 1H), 2.74 (dd, J= 15.7, 12.5 Hz, 1H), 2.93 (dd, J= 15.7, 8.1 Hz, 1H), 3.81 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.93 (s, 1H); Chapter 2: Synthesis of Pelorol and Analogs. ^ 1 3 C N M R (CDCI3) 5 18.4, 18.5, 21.9, 24.2, 24.5, 30.1, 32.9, 33.5, 36.6, 36.7, 38.4, 42.4, 47.9, 48.1, 52.8, 53.4, 56.2, 60.4, 63.2, 114.1, 118.2, 125.3, 136.4, 149.4, 150.6, 164.6, 187.4; H R E S I M S calcd for CseHseOsNa 451.2460, found 451.2457. Preparation of methyl ester 2.73 MeO OMe A solution of 0.21 g of sodium hydroxide in 2.0 mL of water was added 0.15 mL of bromine at 0 ° C over 10 minutes and 38.0 mg (0.1 mmol) of 2.67 in 1.0 mL of dioxane was added subsequently. After being stirred at 4 0 ° C for 30 minutes, the yellow mixture was quenched by sodium bisulfite. After acidification with 10% H 2 S O 4 , the mixture was extracted with E t 2 0 , washed with brine and dried to furnish a residue, which was dissolved in 10.0 mL of D M F . Potassium carbonate (1.0 g) and Mel (2.0 mL) were added to the mixture and stirring was continued overnight. T h e reaction was quenched by adding 20.0 mL of brine and the resulting mixture was extracted with ET.2O, washed with brine, dried with MgSO-4 Chapter 2: Synthesis of Pelorol and Analogs. 96 and evaporated to dryness. The residue was further purified by column chromatography to give 32.0 mg of 2.73 (80%). 1 H N M R (CDCI 3 ) 6 0.83 (s, 3H), 0.84 (s, 3H), 1.04 (s, 3H), 1.22 (s, 3H), 2.44 (dt, J= 12.3, 3.4Hz, 1H), 2.54 (dd, J= 14.3, 12.9Hz, 1H), 2.69 (dd, J= 14.3, 6.3Hz, 1H), 3.82 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 7.00 (s, 1H); 1 3 C N M R (CDCI 3 ) 8 16.3, 18.3, 19.5, 19.9, 21.1, 25.2, 33.1, 33.3, 36.5, 37.1, 40.2, 42.5, 48.1, 51.7, 56.1, 57.1, 60.4, 64.8, 111.6, 121.2, 137.0, 148.3, 148.7, 149.8, 168.3; H R E S I M S calcd for CssHseCuNa 423.2511, found 423.2513. Preparation of pelorol (2.1) HO OH T o a solution of 2.73 (5.0 mg, 0.013 mmol) in 2.0 mL of C H 2 C I 2 at -78 U C under argon was added B l 3 (60.0 mg) in 2.0 mL of C H 2 C I 2 slowly and the stirring was continued for 0.5 h. T he mixture was then poured into H 2 0 and extracted Chapter 2: Synthesis of Pelorol and Analogs. 97 with CH2CI2 (50.0 mL). T he combined extracts were then dried over MgSC>4, filtered and concentrated. The residue was purified by flash chromatography to furnish 1 (2.3 mg, 50%). [a] D 2 2 8 = -63.5 (0.06, CHCI3); 1 H N M R ( C D C I 3 ) 5 0.83 (s, 3H), 0.84 (s, 3H), 0.97 (td, J= 12.8, 3.7 Hz, 1H), 1.04 (s, 3H), 1.21 (s, 3H), 1.66 (dd, J= 12.5, 6.4 Hz, 1H), 2.48 (dd, J= 14.6, 12.6 Hz, 1H), 2.51 (m, 1H), 2.60 (dd, J= 14.3, 6.4 Hz, 1H), 3.81 (s, 3H), 7.07 (s, 1H); 1 3 C N M R (CDCI3) 5 16.3, 18.4, 19.5, 19.8, 21.1, 24.3, 33.0, 33.3, 36.5, 37.1, 40.2, 42.5, 48.5, 51.7, 57.1, 65.2, 114.8, 118.2, 130.0, 140.5, 143.4, 149.5, 168.0; H R E S I M S calcd for C 2 3H320 4 Na 395.2198, found 395.2199. Chapter 2: Synthesis of Pelorol and Analogs. y o lln vitro SHIP enzyme assay. The SHIP enzyme assay was performed in 96-well microtitre plates with 10 ng of enzyme/well in a total volume of 25 u,L of 20 m M Tris HCI, pH 7.5 and 10 m M M g C ^ . SHIP enzyme was incubated with extract or vehicle for 15 min at 2 3 ° C before the addition of 200 m M inositol-1,3,4,5-tetrakisphosphate (Echelon Biosciences Inc, Salt Lake City, Utah). The reaction was allowed to proceed for 20 min at 3 7 ° C and the amount of inorganic phosphate released assessed by the addition of Malachite Green reagent followed by an absorbance measurement at 650 nm. Inositol phospholipid analysis. J16 cells, a macrophage cell line immortalized from C57BI6 mice, were grown in 10% F C S in IMDM supplemented with 10 u.M 2-mercaptoethanol, 150 u,M monothioglycolate ( M T G ) and 1 m M glutamine. 5x10 6 cells were plated the night before in 10 cm tissue culture dishes. The next day, cells were washed three times with phosphate-free medium before being starved in phosphate free RPMI (MP Biomedicals, Irvine, C A ) supplemented with 10% dialyzed F C S (Invitrogen, Burlington, Ont) and 1% RPMI for 2 hrs. Cells were then labeled with 1.0 mCi of orthophosphate (MP Biomedicals, Irvine, CA)/ml for 2 hrs at 3 7 ° C . Cells were pretreated for 30 min with 2.63, LY294004 or vehicle prior to stimulation with L P S (50 ng/ml) for 15 min, or directly treated with L P S and IL-10 (100 ng/ml) for 15 min. Extraction of inositol phospholipids and H P L C analysis of deacylated lipids were performed as previously descr ibed 7 4 . The amount of radioactivity contained in the elution peak for each lipid (two to five 'The biological experiments presented here were carried out by our collaborators, G. Krystal, A.Mui and C.Ong. Chapter 2: Synthesis of Pelorol and Analogs. y y fractions) was summed to give the total counts for each lipid, and data were normalized to the first 60 fractions to adjust for fluctuations in total lipid labeling and recovery between samples. Production of SHIP+/+ and SHIP"'" bone marrow derived mast cells (BMMCs) and macrophages (BMm<|>s). T o obtain B M M C s , bone marrow cells were aspirated from 4 to 8 week old C57BI6 x 129Sv mixed background mice and S H I P + / + and SHIP"7" B M M C s prepared as described previously 6 1 . After 8 weeks in IMDM + 15% F C S (StemCell Technologies, Vancouver, Canada) + 150 uM M T G + 30 ng/ml IL-3 ( B M M C medium) more than 99% of the cells were c-kit and FcsR1 positive as determined by flow cytometry with FITC-labeled anti-c-kit (BD Pharmingen, Mississauga, Canada) and FITC-labeled IgE (anti-Epo 26), respectively. BMrri(j)S from S H I P + / + and SHIP^mice were obtained as described previously 6 0 and maintained in IMDM supplemented with 10% F C S , 150 u.M M T G , 2% C127 cell conditioned medium as a source of macrophage colony stimulating factor ( M - C S F ) (BMm<j) medium) LPS stimulation of BMm<|)S. For the analysis of LPS-stimulated T N F a production, 2 x10 5 cells were plated the night before in 24 well plates in BMm<|) medium. The next day, the medium was changed and 2.63 or carrier was added to cells at the indicated concentrations for 30 min prior to the addition of 10 ng/mL L P S . Supernatants were collected for T N F a determination by E L I S A (BD Biosciences, Mississauga, O N , Canada) . For analysis of intracellular signaling, 2 Chapter 2: Synthesis of Pelorol and Analogs. 100 x10 6 cells were plated the night before in 6 cm tissue culture plates. The next day, the cells were cultured in BMrncj) medium without M - C S F for 1 hr at 3 7 ° C and then pretreated with 2.63 or carrier for 30 min prior to the addition of 10 ng/mL of L P S for 15 min. Cell lysates were rinsed with P B S and collected into lysis buffer (50 m M Hepes, 2 m M E D T A , 1mM N a V 0 4 , 100 m M N a F , 50 m M NaPPj and 1%NP40) supplemented with Complete Protease Inhibitor Cocktail (Roche, Montreal, Canada) . Lysates were rocked at 4 ° C for 30 min and clarified by centrifuging 20 min at 12000 x g. Lysates were then made 2 x in Laemmli's buffer, boiled 2 min and loaded onto 7.5% S D S polyacrylamide cells. Immunoblot analysis for phospho P K B , phospho p 3 8 M A P K (Cell Signalling, Mississauga, Ont), SHIP 6 0 and actin (Santa Cruz, Santa Cruz, C A ) were carried out as described previously. 6 0 Stimulation of BMMCs by FceRI crosslinking. S H I P + / + and S H I P ' - B M M C s were pre-loaded overnight in B M M C medium lacking IL-3 in the presence of 0.1 //g/ml anti-DNP IgE (Sigma, Oakville, Ont). For calcium flux measurements, the cells were incubated with 2 JJM fura 2-acetoxymethyl ester (Molecular Probes, Eugene, O R ) in Tyrode's buffer 5 9 at 2 3 ° C for 45 min.. T h e cells were then washed and incubated in the presence of vehicle control, LY294002 or 2.63 30 min prior to stimulation with the indicated concentration of DNP-human serum albumin ( D N P - H S A ) . Calcium influx was then monitored by spectrofluorometry as described previously 5 9 . For analysis of intracellular signaling, cells were pre-loaded with anti-DNP IgE as above, pre-treated with 2.63 or buffer control for 30 min at 3 7 C ° and stimulated with 20 ng/ml D N P - H S A for 5 min. Total cell lysates Chapter 2: Synthesis of Pelorol and Analogs. 1 u 1 were then prepared and analyzed for phospho-PKB, p h o s p h o - p 3 8 M A P K , phospho-M A P K , Grb-2 (Cell Signalling, Mississauga, Ont) and S H I P 6 0 by immunoblot analysis as descr ibed . 6 3 Mouse septicemia model. 6-8 week old C57BI6 mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, B C ) were orally administered 20 mg/kg 2.63, 0.4 mg/kg dexamethasone or carrier 30 min prior to an intra-peritoneal injection of 20 mg/kg of L P S (E. Coli serotype 0111:B4, Sigma, Oakville, Ont). Blood was drawn 2 hr later for determination of plasma T N F p b y E L I S A . Mouse acute cutaneous anaphylaxis model. 6-8 week old CD1 mice (University of British Columbia Animal Facility, Vancouver, B C ) were sensitized to the hapten D N P by cutaneous application of 25 uL of 0.5% dinitroflourobenzene (DNFB) (Sigma, Oakville, Ont) in acetone to the shaved abdomen of mice for two consecutive days. One week after the first application, 20 uCi of tritiated thymidine ([ 3H]-Tdr ( G E Healthcare, Piscataway, NJ) was injected intra-peritoneally. 24 hr later, test substances (dissolved in 10 uL of 1:2 D M S O : M e O H ) were painted on the left ear while the right ear received vehicle control. 30 min after drug application, D N F B was applied to both ears to induce mast cell degranulation. The resulting neutrophil infiltration was quantitated by taking a 6mm diameter punch from the ear 1 hr later for dissolution in Solvable (Perkin Elmer-Packard, Woodbridge, Ont) and scintillation counting. The ability of test substances to inhibit mast cell degranulation was then determined by Chapter 2: Synthesis of Pelorol and Analogs. ' u ^ calculating the ratio of [ 3H]-Tdr in the test (right) ear vs the control (left) ear. One group of mice had D N F B applied only to the left ear leaving the right ear non-inflamed, in order to control for basal [ 3H]-Tdr incorporation into ear parenchymal cells. Effect of 48 on the proliferation of BCR-abl-expressing Ba/F3 cells. The hematopoietic cell line Ba /F3 (control cells) or Ba /F3 cells expressing the oncogene B C R - A b l (BCR-abl cel ls) 7 5 were maintained in RPMI supplemented with 10% F C S and 1 /yg/mL IL-3. T o test the effect of 2.63 on cell proliferation, 2 x 10 4 cells/well were plated into 96 well round bottom microtitre plates with 1 u.g/mL of 2.63 or carrier, the indicated concentrations of Gleevec in RPMI + 1 jt/g/mL IL-3 + 10% F C S . 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Blood, 2005, 7,191. 59. Fang, H . et al. J. Immunol. 2004, 773, 360-366. 60. Sly, L. M . ; Rauh, M . J . ; Kalesnikoff, J . ; Song, C . H. ; Krystal, G . Immunity. 2004, 27, 227-239. 61. Huber, M . e ta l . Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11330-11335. 62. Huber, M . ; Kalesnikoff, J . ; Reth, M . ; Krystal, G . Immunol. Lett. 2002, 82, 17-21. 63. Kalesnikoff, J . et al. J. Immunol. 2002, 768, 4737-4746. 64. Djouder, N. et al. J. Immunol. 2001, 766, 1627-1634. 65. Kitaura, J . et al. J. Exp. Med. 2000, 792, 729-740. 66. Annane, D., Bellissant, E . ; Cavaillon, J . M . Lancet 2005, 365, 63-78. 67. Galanos, C ; Freudenberg, M . A . Immunobiology 1993, 787, 346-356. 68. Kemp, S. F.; Lockey, R. F. J. Allergy Clin. Immunol. 2002, 770, 341-348. 69. Young, J . M . et al. J. Invest. Dermatol. 1984, 82, 367-371. 70. Peggs, K. Clin. Exp. Med. 2004, 4, 1-9. Chapter 2: Synthesis of Pelorol and Analogs. 107 71. Hensley, M . L ; Ford, J . M . Semin. Hematol. 2003, 40, 21-5. 72. Gambacorti-Passerini, C . B. et al. Lancet Oncol. 2003, 4, 75-85. 73. Kharas, M . G . ; Fruman, D. A . Cancer Res 2005, 65, 2047-2053. 74. Krahn, A . K.; M a , K.; Hou, S.; Duronio, V . ; Marshall, A . J . J. Immunol. 2004, 7 72, 331-339. 75. Weisberg, E . ; Griffin, J . D. Blood. 2000, 95, 3498-3505. Chapter 3 : Structure Activity Relationship Study of Pelorol. 108 Chapter 3: Structure Activity Relationship Study of Pelorol. 3.1 Introduction A typical natural product drug discovery process starts from the identification of the bioactive sample with H T S (high-throughput screening), then the natural product is extracted from the source material, concentrated, fractionated and purified to yield essentially a single biologically active component. In those cases in which the biological activity profile of this component meets criteria for potency and selectivity, preliminary S A R studies are conducted. W e have described our discovery and synthesis of the sponge meroterpenoid pelorol and its two analogs, A Q X - 1 6 A and A Q X - 1 8 A , which represent a new class of anti-inflammatory and anti-leukemic drugs that specifically target and activate the negative regulator, SHIP. The promising biological study results of A Q X - 1 6 A , which produced a 3-fold higher activation of SHIP than the natural product pelorol at the same molar concentration, provides sound reasons to carry out further S A R research on pelorol. The goal of the study is to determine which functionality is essential for the activation of the SHIP enzyme and which part of the molecule is not important for the biological activity, thus simplifying the pharmacophore. More importantly, the rational generation of chemical diversity by synthesis of a library of pelorol analogs may lead to the discovery of more promising drug development candidates having improved potency and drug-like properties, essentially optimizing the pelorol drug lead. Chapter 3: Structure Activity Relationship Study of Pelorol. 1 u y A s Koehn er a/. 1 mentioned in their paper, one of the distinguishing features of natural products is their frequent occurrence as suites or complexes of structurally related analogues. It might be the consequence of an organism's need to generate its own chemical diversity to optimize the activity of its secondary metabolites, essentially performing its own S A R optimization. There are two recent reports concerning the structure-activity relationship studies of marine sesquiterpenoid quinones based on the compound pool generated by marine organisms. 2 , 3 Aoki et al. evaluated the differentiation-inducing activity to R= OCH 3 R= OCHj Figure 3.1 Evaluation of the differentiation-inducing activities of sesquiterpene quinones by the induction of hemoglobin production in K562 cells.2 ATRA-nonrespons ive chronic myelogenous leukemia cells (K562 cells) into erythroblasts often sesquiterpene quinones isolated from D.elegans (Figure 3.1). The structure-activity relationship study of these compounds clarified that the quinone skeleton (a to h) is indispensable and the configuration at C-5 in the sesquiterpene fragment is not important for their differentiation-inducing activity to K562 cells. 2 A similar investigation by Prokof'eva et al. on the S A R of some sesquiterpene quinones for cytotoxic and hemolytic activities, using developing Chapter 3: Structure Activity Relationship Study of Pelorol. 110 sea urchin eggs, Ehrlich carcinoma cells, and mouse red blood cells has shown that the quinone ring is essential for both cytotoxic and hemolytic activities and the structure of the terpenoid portions of the compounds have no significant influence on biological activity. 3 Although the S A R studies of these sesquiterpene quinone/hydroquinone families on different cell lines can not be directly compared, there are some common structural features that are required for the biological activity in both of these series. First, the quinone or hydroquinone moiety is essential for activity and modifying the substitution pattern on this moiety has a major input on its activity. Second, the sesquiterpene skeleton is not important to activity. In our synthesis of pelorol (Chapter 2), pelorol (2.1), dimethylpelorol 2.73, the analogs 2.57 and 2.63, the corresponding methyl ethers 2.42 and 2.62, the R=COOMe pelorol (2.1) Me AQX-16A (2.63) Et AQX-18A (2.57) R= COOMe dimethylpelorol (2.73) Me dimethylAQX-16A (2.62) Et dimethylAQX-18A(2.42) OMe trimethoxy analog (2.36) Figure 3.2 Preliminary SAR study of pelorol by evaluation of their ability to supress T N F a production in murine mast cells stimulated with IgE. Chapter 3 : Structure Activity Relationship Study of Pelorol. 1 1 1 trimethoxy pelorol analog 2.36, and the uncyclized precursor 2.61 were tested for in vitro activation of SHIP and the ability to suppress degranualation and T N F a production in murine mast cells stimulated with IgE (Figure 3.2). Synthetic pelorol 2.1, the ethyl analog 2.57, and the methyl analog 2.63 showed significant activity in all three assays, but the methyl ethers 2.36, 2.42, 2.61, 2.62 and 2.73 were inactive. T h e relative effectiveness of the active compounds in the SHIP activation assay was 2.63 >2.57 « 2.1, showing that replacement of the methyl ester at C-20 in pelorol with a methyl group gives enhanced activity. Lack of activity in the dimethyl ethers 2.62, 2.42, and 2.73 demonstrates that at least one phenol is required for activity. Based on these preliminary S A R study results in combination with the previous S A R results on sesquiterpene quinones, we proposed that: i) the aromatic ring of pelorol would be important for its activity and changing the substitutions on the ring may significantly alter its activity, ii) the five-membered C ring should also play a significant role in its activity, opening of this ring would result in loss of activity, and iii) the A , B ring of pelorol would not have remarkable impact on its activity. This chapter will be focused on answering the questions of how the functionality on the aromatic ring of pelorol affects its activity and whether the stereochemistry of the C-ring is important for the SHIP activating properties of pelorol. Chapter 3: Structure Activity Relationship Study of Pelorol. > > 3.2 Synthesis of C-8 and C-9 stereoisomers of pelorol Biological systems are largely constructed from chiral molecules such as L-amino acids or D-sugars. It is then understandable that some drugs, which have a chiral center or centers exhibit a high degree of stereoselectivity in their interactions with biological macromolecules. Stereoselectivity in drug action at specific receptors with enzymes or ion channels, is well known. For example, the antihypertensive agent labetol is both an a- and p-adrenoceptor antagonist. The drug possesses two chiral centers and is used as a mixture of four diastereomers. T h e R, R form provides most of the (3-blocking activity while the S, R diastereomer is an a-receptor antagonist. The S, S and R, S forms do not contribute significantly to the pharmacology of the drug . 4 Although the mechanism of how pelorol activates the SHIP enzyme is still not clear, it seems reasonable to predict that the stereochemistry of pelorol may be an important factor in the interaction of pelorol with the enzyme. Pelorol has four stereogenic centers at C-5 , C-8, C-9 , and C-10 on the sesquiterpene skeleton. A s we proposed above, varying the A , B ring stereochemistry of pelorol may not lead to a significant change in its activity and it is the C-8 , C-9 stereochemistry that is likely most critical for its activity. Our interest was in how the stereochemistry of the C ring fusion affected pelorol's activity. T o examine this Labetol Chapter 3: Structure Activity Relationship Study of Pelorol. 113 S A R issue, four stereoisomers, pelorol, 8-ep/'-pelorol, 9-ep/-pelorol, and 8-ep/,9-ep/-pelorol, had to be synthesized, and their activity in the SHIP assay compared. In our synthesis of pelorol, we synthesized an intermediate 3.1, which underwent a Nazarov cyclization reaction under acidic conditions at - 7 8 ° C as shown in Figure 3.3, to give the tetracycle 3.2 and trace amount of the C-8 epimer 3.2a. This result led us to postulate that varying the cyclization conditions, especially the reaction temperature, might increase the yield of the 3.2a epimer. Furthermore, we anticipated that the A 9 double bond of 3.2 and its epimer could be reduced to generate two of the four possible stereoisomers at C-8 and C-9 . More importantly, 8-ep/-pelorol, 9-ep/-pelorol, and 8-ep/',9-ep/'-pelorol could be synthesized in this approach by simply replacing the C-20 methoxyl with an ethyl group. Figure 3.3 Synthetic route to 3.2 Chapter 3: Structure Activity Relationship Study of Pelorol. 114 Figure 3.4 Synthetic route to 2.54 The synthesis of pelorol stereoisomers started with diol 2.52. A s mentioned in Chapter 2, all the oxidation conditions tried gave the elimination side products 2.54 and 2.55. T o increase the yield of the desired enal 2.54, the volume of Et3lM used to quench the reaction was doubled and the duration of the Swern oxidation was extended from 20 minutes to 3 hours. A s expected, the major product under these conditions was the desired enal 2.54 and the minor elimination product 2.55 could be converted to the thermodynamically favored product 2.54 by dissolving the alkene mixture in M e O H and treating with K O H at room temperature. Compared to the reported precedures for preparation of this key intermediate 2.54,5 the new synthetic route is superior in yield and succinctness. OMe Figure 3.5 Synthetic route to 3.3a and 3.3b Chapter 3: Structure Activity Relationship Study of Pelorol. ' ' 5 With the enal 2.54 in hand, it was added to one equivalent of phenyl lithium 24 in T H F at - 7 8 ° C . The mixture was stirred for 2 hours at - 7 8 ° C and then allowed to slowly warm to room temperature. T L C analysis indicated the disappearance of 2.54, which was characterised by its strong U V absorbance, and two polar spots with weak U V absorbance were generated. Standard workup followed by normal phase chromatography (hexane : E t O A c = 93:7) yielded two epimeric benzylic alcohols 3.3a and 3.3b in a ratio of 8.4:1 (Figure 3.5). Initial attempts to acquire N M R data on these epimers failed due to their instability. It was found that the benzylic alcohols cyclized readily at room temperature in protic or acidic N M R solvents such as M e O D , C D C I 3 , etc. C 6 D 6 was tried but some of the aliphatic signals from 3.3a/b were obscured by the solvent peak in the carbon spectrum. Finally, C D 3 C N was chosen as a suitable solvent, because it didn't cause the decomposition of 3.3a and 3.3b and all the signals were well resolved. OMe Figure 3.6 Synthetic route to 3.4 and 3.5 Chapter 3: Structure Activity Relationship Study of Pelorol. > >D Treatment of 3.3a and 3.3b with S n C I 4 in C H 2 C I 2 resulted in cyclization to give a mixture of the C-8 epimers 3.4 and 3.5 in high yield (Figure 3.6). Assuming that the driving force for this cyclization is the formation of the stable indene structure within the tetracycle product, then the nucleophilicity of the arene is not so important to a successful cyclization. This explains why this acid-catalyzed cyclization is much easier to achieve than the acid-catalyzed Friedel-Crafts alkylicyclization that we utilized in the synthesis of pelorol. It is worth noting that the ratio of the cyclization products varied with the temperature used in the cyclization step. At - 7 8 ° C , compound 3.4 was the major product, while at - 2 0 ° C , a 6:4 mixture of 3.4 and 3.5 was formed. W e envisioned that the steric bulk of the C-14 methyl played a major role in determining the ratio of isomers formed in the cyclization. At the lower temperature ( - 7 8 ° C ) , the steric strain between the C-14 axial methyl and the nucleophilic aromatic ring made attack of the arene from the bottom of C-8 predominant. T h e higher temperature ( - 2 0 ° C ) would provide sufficient energy for the arene to overcome the steric hinderance, facilitating attack from the top face of C-8 resulting in a higher yield of 3.5. After passing through a normal phase Seppak, the mixture formed at - 2 0 ° C was subjected to H P L C separation with a chiral column, eluted with hexane: E t O A c (97:3) to give pure samples of 3.4 and 3.5. T h e stereochemistries of 3.4 and 3.5 were confirmed by 2D N O E S Y experiments (Figure 3.7 and 3.8). Chapter 3: Structure Activity Relationship Study of Pelorol. 117 Figure 3.7 Expansion of the N O E S Y spectrum for 3.4 Chapter 3: Structure Activity Relationship Study of Pelorol. 118 Figure 3.8 Expansion of the N O E S Y spectrum of 3.5 Chapter 3: Structure Activity Relationship Study of Pelorol. 119 Figure 3.9 Synthetic route to 3.6 and 3.7 Catalytic hydrogenation of 3.4 over P d / C in ethanol gave exclusively 3.6 in excellent yield, and under the "same conditions compound 3.5 cleanly gave 3.7, also in high yield (Figure 3.9). The stereochemistry of catalytic hydrogenation of the cycloalkenes is strongly affected by the catalyst and slightly affected by the hydrogen pressure. A palladium catalyst was found to promote the generation of cis products in both reactions. Either low pressure (30 psi) or high pressure (600 psi) did not change the reaction outcome. Efforts to synthesize the C-9 epimer of 3.7, which was the precusor of 8-ep/',9-ep/'-pelorol, by homogeneous catalytic hydrogenation using chiral catalyst (Wilkinson's catalyst) did not afford the desired product. Reduction of 3.5 with sodium borohydride (NaBhU) in the presence of nickel dichloride was attempted, but no reaction was observed. The stereochemistry of compound 3.6 was confirmed by a 2 D - N O E S Y experiment (Figure 3.10), which showed strong correlations between the C-14 methyl (81.22) Chapter 3: Structure Activity Relationship Study of Pelorol. 120 Figure 3.10 Expansion of the N O E S Y spectrum for 3.6 Chapter 3: Structure Activity Relationship Study of Pelorol. 121 Figure 3.11 Expansion of the N O E S Y spectrum of 3.7 Chapter 3: Structure Activity Relationship Study of Pelorol. 122 OMe ,0Me PCC, 40°C 20h, 53% HQ OH ^ / 0 MeO 3.10 Bl3, CH2CI2; -78°C 38% 1) Br2, NaOH, 40°C 2) Mel, K 2C0 3 , DMF two steps from 3.8 64% OMe PCC, 40UC 20h, 47% 3.11 HQ OH BI3, CH2CI2 \ ^ MeO 42% 3.13 1) Br2, NaOH,40X 2) Mel, K 2C0 3, DMF two steps from 3.11 71% MeC OMe MeO 3.12 Figure 3.12 Synthetic routes to 9-ep/-pelorol (3.10) and 8-ep/-pelorol (3.13) Chapter 3: Structure Activity Relationship Study of Pelorol. 123 and C-15 methyl (8 1.61) resonances and was devoid of correlations between the C-11 methylene (8 2.66 and 2.90) and C-15, C-14 methyl resonances. The stereochemistry of 3.7 was also determined by a 2 D - N O E S Y experiment (Figure 3.11) and the characteristic C-14 methyl chemical shift at 8 0.44, which moved upfield due to the shielding effect of the aromatic ring. Following the same procedure used in the synthesis of pelorol, 3.6 and 3.7 were oxidized by P C C to give the corresponding ketones 3.8 and 3.11. A haloform reaction using B r 2 / N a O H followed by methylation of the resulting acid with Mel afforded the methyl ethers 3.9 and 3.12. A s in the synthesis of pelorol, the phenyl methyl ethers were selective removed by treating with BI3 in CH2CI2 at - 7 8 ° C to give the 9-ep/-pelorol (3.10) and 8-ep/-pelorol (3.13). Chapter 3: Structure Activity Relationship Study of Pelorol. 124 3.3 Synthesis of stereoisomers of 2.63 (AQX-16A) Our synthesis of pelorol 2.1, the ethyl analog 2.57, and the methyl analog 2.63 which had significantly improved potency, answered the question of whether a methyl ester on C-20 was essential for activity. Furthermore, the synthesis of 8ep/'-pelorol 3.13 and 9epi-pelorol 3.10 was expected to reveal how the stereochemistries of C-8 and C-9 in pelorol affect its activity. Another question arises at this point, would the stereochemical changes at C-8 or C-9 and the functionality replacement at C-20 have a synergistic effect in enhancing activity or cancel each other out? T o answer this question, both C-8 and C-9 stereoisomers of 2.63 were synthesized by adopting a similar synthetic route to the one used in the synthesis of 8-ep/and 9-ep/-pelorols. Coupling the dimethoxymethylphenyllithium 2.59 with enal 2.54 yielded two epimers 3.14a and 3.14b in high yield with an isolated ratio of 8:2 (Figure 3.13). A s with their ethyl anologs, 3.14a and 3.14b were quite unstable in protic OMe Li- -OMe HO, THF, -78°C tor.t.,2h 2.59 2.54 3.14a y. 67% 3.14b y.17% Figure 3.13 Synthetic route to 3 .14a and 3.14b Chapter 3: Structure Activity Relationship Study of Pelorol. 125 OMe Figure 3.14 Synthetic route to 3.19 and 3.20 solvents or under acidic conditions. Therefore, C D 3 C N was used as N M R solvent. Cyclization of 3.14a and 3.14b catalysed by S n C I 4 at - 2 0 ° C in C H 2 C I 2 afforded two epimers 3.15 and 3.16, in high yield, in a ratio of 1:1, which were separated by chiral H P L C . T h e same reaction run at - 7 8 ° C gave a mixture of Chapter 3: Structure Activity Relationship Study of Pelorol. ' ^ D 3.15 and 3.16 in a ratio of 8:2 (Figure 3.14). T h e stereochemistries of 3.15 and 3.16 were confirmed by 2D N O E S Y experiments. It was interesting to note that the proportion of the epimer 3.16 in the mixture was higher compared to its ethyl analog 3.5 under both conditions ( - 2 0 ° C and - 7 8 ° C ) , which was probably due to less steric strain between the C-10 angular methyl and the C-20 aromatic methyl than the bulky ethyl in 3.5. Catalytic hydrogenation of 3.15 and 3.16 over P d / C gave the corresponding cis products 3.17 and 3.18. Their stereochemistries were characterised by 1 H N M R and 2D N O E S Y experiments. Demethylation of 3.17 and 3.18 with B B r 3 at room temperature furnished the desired catechols 3.19 and 3.20 in excellent yield. Chapter 3: Structure Activity Relationship Study of Pelorol. 127 3.4 Synthesis of desmethyl-2.63 and its stereoisomers The preliminary S A R study of pelorol showed that at least one phenol was required for activity and replacement of the methyl ester with methyl gave enhanced activity. This led us to ask whether the C-20 functionality was required for activity. T o answer this question, desmethyl-2.63 (3.25) and its stereoisomers were synthesized. CHO "OH Li-THF, -78°C 2h 73% OMe 3.21 -OMe OMe H2, 45 psi, Pd/C, EtOH 2.19 3.22 3.23 iH SnCI4, -20°C i M e BBfg, CH2CI2 3.25 Three steps from 3.22 62% 3.24 Figure 3.15 Synthetic route to 3.25 One equivalent of aldehyde 2.19 was reacted with 2 equivalents of 3-lithioveratrole generated in situ by reaction of veratrole with butyllithium 6 in the Chapter 3: Structure Activity Relationship Study of Pelorol. 128 OMe 2 54 3.26a y. 65% 3.26b y.16% Figure 3.16 Synthetic route to 3.26a and 3.26b OMe Scheme 3.17 Synthetic route to 3.31 and 3.32 Chapter 3: Structure Activity Relationship Study of Pelorol. > ^ y presence of T M E D A , to give the epimeric diols 3.22, which on hydrogenolysis with P d / C in ethanol afforded 3.23 in 81% yield. Reaction of 3.23 with S n C I 4 in CH2CI2 at - 2 0 ° C furnished the tetracycle 3.24 nearly quantitatively. It was a surprise to find that one para methoxyl provided sufficient nucleophilicity in the arene to bring about the Friedel Crafts cyclization. This was in contrast to our initial expectation that both para and ortho activation were required for a successful cyclization. B B r 3 deprotected the methyl ether to give the desired catechol 3.25 (Figure 3.16). Following the procedure used in the synthesis of 3.19 and 3.20, one equivalent of enal 2.54 was coupled with one equivalent of the 3-lithioveratrole 3.21 to give the two epimers 3.26a and 3.26b (8:2), which underwent cyclization to afford the epimers 3.27 and 3.28 in a ratio of 6:4 at - 2 0 ° C . T h e two epimers were subsequently separated by chiral H P L C . Stereospecific hydrogenation of 3.27 and 3.28 led to the tetracycles 3.29 and 3.30, respectively, which after treatment with B B r 3 gave the desired catechols 3.31 and 3.32 in high yield. Chapter 3: Structure Activity Relationship Study of Pelorol. 130 3.5 Synthesis of a regioisomer of pelorol A s part of our efforts to synthesize a small library of pelorol analogs for S A R evaluation, we tried to synthesize regioisomers of pelorol that differed in the position of the C-20 methyl ester. In our model study to synthesize the regioisomer of 9-ep/-pelorol 3.38 as shown in Figure 3.18, enal 2.54 was coupled OMe 3.37 3.36 3.35a y. 6% HO OH ? J OMe 3.38 Figure 3.18 Mode l study in synthes is of pelorol reg io isomer 3.38 Chapter 3: Structure Activity Relationship Study of Pelorol. * ^ ' with dimethoxyethyllithium 3.33, the regioisomer of 2.42. The coupling products 3.34a and 3.34b (9:1) were reacted with SnCI 4 in CH 2 CI 2 at -20°C to give exclusively one epimer 3.35 as well as small amount of elimination side product 3.35a. This reaction outcome is different from all the cyclization results we obtained before, which may due to the decrease of nucleophilicity on C-21. Catalytic hydrogenation of 3.35 afforded the c/'s product 3.36 quantitatively. Unfortunately, 3.36 failed to undergo the desired oxidation reaction upon treatment with PCC and other oxidants to furnish 3.37. 3 .29 3 .39 3 . 4 0 Figure 3.19 Model study in the synthesis of a pelorol regioisomer To circumvent this problem, 3.29 was treated with BuLi in TMEDA at -78°C. After warming to room temperature for half an hour, the mixture was cooled down to 0°C and quenched with Br2. However, the envisioned directed ortho metalation 3.31 3.41 3 .42 Figure 3.20 Model study in synthesis of pelorol regioisomer Chapter 3: Structure Activity Relationship Study of Pelorol. > reaction proceeded with partial decomposition of the starting material 3.29 and no desired product 3.39 was recovered (Figure 3.19). Since direct bromination of 3.29 with Br 2 in AcOH gave the undesired C-20 brominated product, catechol 3.31 was subjected to bromination (Figure 3.20). Bromine was added dropwise to catechol 3.31 dissolved in MeOH at 0°C. The mixture was gradually warmed to room temperature and the reaction was monitored by TLC. After quenching the reaction mixture with H 2 0 and followed by standard workup, the 1 H NMR of the product showed the signal of the unreacted 3.31, characterised by the two doublets at 5 6.50 (d, J= 7.9 Hz, 1H), 6.67 (d, J= 7.9 Hz, 1H) and some singlets around 5 6.90, which might be the signals of 3.41 and 3.42. This reaction could be optimized to selectively forming 3.41 by using a literature procedure that utilizes Br 2 and t-butylamine. The synthesis of 3.41 would provide a practical approach to the desired pelorol isomer. Chapter 3: Structure Activity Relationship Study of Pelorol. 133 3.6 Synthesis of Akaol ,OH OMe Three years after the first identification of pelorol by Schmitz's group, another meroterpenoid, akaol (3.43),7 was isolated by the same group from a marine sponge of the genus Aka collected at Y a p Island, Federated States of Micronesia. Akaol was tested for activity in a CDK4/cyc l in D1 assay at a concentration of 10 ug/mL, but was found to be inactive. However, the 8a, 9a stereochemistry of akaol's B / C ring fusion drew our interest because it shared the same cis B / C ring fusion as the 8-ep/'-pelorol that we had synthesized. Akaol OBn .OMe 2.54 3.45a 3.45b HQ BnQ ,OBn OMe Figure 3.21 Proposed synthetic route to akaol (3.43) Chapter 3: Structure Activity Relationship Study of Pelorol. 134 differed from 8-ep/'-pelorol in the C-20 substituent, which was a benzyl methyl ether. Though it was possible to prepare akaol by transforming the C-20 methyl ester in 8epi-pelorol to the methyl ether, we thought there was a shorter route to akaol by manipulation of the protecting groups on the aromatic portion of the starting material. Figure 3.21 shows our proposed synthetic route to akaol, which was initiated from the same terpene portion as we used in the synthesis of stereoisomers of pelorol. The aromatic portion 3.44 was the benzyl methyl ether with the two phenols protected by benzyl groups and anion generation on the ring was conducted in situ by lithium-halogen exchange. As with the many successful precedents, the coupled products 3.45a and 3.45b could cyclize under acidic conditions to give 3.46, though we might face the problem of separating 3.46 from its epimer. The advantage of this route is the last step where low pressure catalytic hydrogenation could be used to reduce the double bond and remove the benzyl protecting groups in one step. Though this approach seemed simpler than our synthesis of 8-ep/'-pelorol, there were a number of potential problems underlying this synthetic plan. For example, it was not clear that all the protecting groups would survive the cyclization conditions and what conditions would be suitable for 3.45a and 3.45b to cyclize. To answer these questions, a model study was carried out on 3.48 and 3.26. MeO. XHO BnO. XHO BnO^ ^ \ MeO 2.30 ii) BnBr, K 2C0 3, Acetone BnO' two steps from 2.30 61% i) Pyridine, AICI3, CHCI3 ii) NaH, Mel, THF, r.t. BnO 3.47 two steps from 3.47 95% i) NaBH4, EtOH, r.t 3.48 "OMe Br Br Br Figure 3.22 Synthetic route to 3.48 Chapter 3: Structure Activity Relationship Study of Pelorol. 1 J 0 Compound 3.48 (Figure 3.22) was synthesized from 5-bromoveratraldehyde 2.30 by demethylation with pyridine and AICI3 in refluxing C H C I 3 to expose the phenols, 8 which were protected by benzyl groups under basic conditions to give 3.47. Reduction of 3.47 with N a B H 4 and methylation of the benzyl acohol give model compound 3.48. TFA, CH2CI2, »- No reaction -20°C, 2hr SnCI4, CH2CI2, Deprotection mixture -20°C, 2hr Figure 3.23 Model study of 3.48 under various cyclization conditions A s shown in Figure 3.23, 3.48 was exposed to various cyclization conditions. It was stable when treated with excess T s O H at 0°C or T F A at -20°C. At higher temperatures, treatment with T F A or with the Lewis acid S n C U was found to remove the protecting groups on the phenols. O n c e the cyclization conditions suitable for the protecting groups were chosen, it was necessary to optimize the specific conditions for cyclization. 3.26a and 3.26b were selected as the substrates for this purpose. W h e n 3.26a and 3.26b were treated with T s O H at 0°C in CH2CI2, some rearrangement products were formed but no cyclization product was observed in the reaction mixture. 3.26a and 3.26b reacted with T F A at -20°C cleanly to give the epimers 3.27a and 3.27b (6:4). It was obvious that Chapter 3 : Structure Activity Relationship Study of Pelorol. 136 T F A at - 2 0 ° C would be the optimal condition for the cyclization of 3.45a and 3.45b. OMe 3.27a 3.27b Figure 3.24 Model study of 3.26a and 3.26b under various cyclization conditions The enal 2.54 was reacted with phenyllithium 3.44 (Figure 3.21) in T H F at - 7 8 ° C for two hours and warmed to - 2 0 ° C over a period of two hours. T h e reaction was quenched with H2O followed by standard workup. However, 1 H N M R indicated that the envisioned coupling reaction proceeded with unacceptably low yield. The Figure 3.25 Synthetic route to 3.50 Chapter 3: Structure Activity Relationship Study of Pelorol. 137 OMe 2.54 3.51 Figure 3.26 Synthetic route to 3.51 OMe Scheme 3.27 Synthetic route to akaol (3.43) Chapter 3: Structure Activity Relationship Study of Pelorol. 138 discouraging result of the coupling reaction may be explained by the shielding effect of the bulky benzyl protecting group ortho to the anion, which made the coupling difficult. T o overcome this problem, substrate 3.50 was prepared in two steps from 2.30 (Figure 3.25). The coupling reaction between 3.50 and 2.54 proceeded smoothly to give a 41% yield of 3.51 (Figure 3.26). Cyclization was realized with T F A at - 2 0 ° C to give epimers 3.52 and 3.53, as well as around 20% of the elimination product 3.54, which was presumably the result of decreased nucleophilicity of the arene with a benzylic methoxyl at C-20. 3.52, 3.53, and 3.54 were separated on a chiral H P L C column. 3.52 and 3.53 were subjected to catalytic hydrogenation using 5% R h / C as catalyst to afford stereospecific products 3.55 and 3.56. The stereochemistries of 3.55 and 3.56 were confirmed by 2D N O E S Y experiments. Removing the methyls of 3.55 with B B r 3 at 0 ° C , followed by methylation at C-20 with T s O H in M e O H did not give the desired product akaol. Simply changing BBr3 to B l 3 and keeping the temperature a t - 7 8 ° C furnished the demethylation product, which after remethylation in M e O H with T s O H gave akaol. Chapter 3: Structure Activity Relationship Study of Pelorol. 139 3.7 Biological Activities of Pelorol analogs and Discussion of SAR 3.7.1 In vitro activation of SHIP. Pelorol (2.1), the stereoisomers 8-ep/'-peloroI (3.13) and 9-ep/'-pelorol (3.10), A Q X - 1 6 A (2.63), desmethylpelorol (2.74), A Q X - 1 8 A (2.57) were evaluated side by side for in vitro activation of SHIP (15 min, 30u.M) as shown in Figure 3.28. 0.130 0.120 If) U . I 117 CO Q ° 0.100 > •fj 0.090 TO | 0.080 0.070 0.060 cP <y Figure 3.28 In vitro SHIP activation activities of pelorol and its analogs Chapter 3 : Structure Activity Relationship Study of Pelorol. 140 9epi-pelorol (3.10) 8epi-pelorol (3.13) A Q X - 1 6 A (2.63) stands out as the most active compound in this bioassay. Pelorol (2.1), 9-ep/-pelorol (3.10), A Q X - 1 8 A (2.57), and desmethylpelorol (2.74) exhibited similar acitivity, which is weaker than that of 8-ep/'-pelorol (3.13). Chapter 3: Structure Activity Relationship Study of Pelorol. 141 3.7.2 Inhibition of LPS-stimulated TNFa production from J774 macrophage. Pelorol (2.1), and its analogs were also tested on L P S stimulated T N F a production from J774 macrophage cells. The J774 assay can be used to assess the ability of test compounds to cross the cell membrane and to activate SHIP in intact cells. Test compounds were dissolved in cyclodextrin (CD) and then tested in the J774 assay at 2 ug/mL. Cells were stimulated with 10 ng/mL L P S in the presence of the compounds or D M S O (the same volume of D M S O diluted into C D as in the compounds) for 4 hours. Supernatants were collected and assayed for T N F a by E L I S A . 3000 2500 2000 ! 1500 a s LL Z 1000 500 ML *> ^ J> J>.# S .J> n # ^ £ # <y e>° e>° & ^ > v ^ .<y +r J X?V .^OT -LT' Figure 3.29 Inhibition of LPS-stimulated T N F a production from J774 macrophage by pelorol and its analogs. Chapter 3: Structure Activity Relationship Study of Pelorol. 142 R=COOMe pelorol (2.1) R= Et dimethylAQX-18A (2.57) Me AQX-16A (2.63) OMe trimethoxy analog (2.36) Et AQX-18A(2.57) H desmethyl-2.63 (3.25) A s shown in Figure 3.29, A Q X - 1 6 A (2.63) has greater activitiy in inhibition of T N F a production than other analogs. Both pelorol (2.1) and A Q X - 1 8 A (2.57) exhibited comparable activity to A Q X - 1 6 A (2.63). It is worth noting that 9-ep/'-pelorol (3.10) shows similar activity to A Q X - 1 6 A (2.63), however, 8-ep /-pelorol (3.13) is inactive. This result is opposite to what we observed in the SHIP activation assay. The reciprocal biological activity of 9-ep/' (3.10) and 8 - e p / -pelorol (3.13) in two assays probably reflects 9-ep/-pelorol has better cell permeability. The same tendency could be observed when comparing the three stereoisomers desmethyl-2.63 (3.25), 9-ep/'-desmethyl-2.63 (3.31) and 8 - e p / -desmethyl-2.63 (3.32). T h e relative activities of these compounds in the bioassay are 3.25 « 3.31 > 3.32. The dimethylAQX-18A (2.57) didn't show inhibitory Chapter 3: Structure Activity Relationship Study of Pelorol. 143 activity, which is in good agreement with the previous conclusion that at least one phenol group is required for activity. O n e exception in this assay is that the trimethoxy analog (2.36) shows somewhat inhibitory activity when dissolved into C D . Further work is being carried out to confirm this result. 3.7.3 Discussion of SAR. O n the basis of the structure activity relationship studies, the following evidence was obtained (Figure 3.30). 1) At least one phenol is required for activity. This was. demonstrated by the prominent activity of pelorol (2.1), A Q X - 1 6 A (2.63), A Q X - 1 8 A (2.57) and lack of acitivity in the dimethyl ethers 2.73, 2.62, 2.42 (Figure 3.2). 2) The substituent at C-20 is important for activity. A distinct decrease of activity is observed when there is no substituent at C-20 of pelorol and 9epi-pelorol (Figure 3.29). The C-20 methyl analog A Q X - 1 6 A (2.63) is the most active compound in all assays. 3) The stereochemistry at C-9 has no drastic effect on in vitro SHIP activation (Figure 3.28) and inhibition of T N F a production in intact cells (Figure 3.29). 4) The stereochemistry at C-8 is critical for activity in intact cells. All C-8 epi stereoisomers are less active than the C-9 epi stereoisomers in the J774 assay (Figure 3.29). Chapter 3: Structure Activity Relationship Study of Pelorol. 144 5) The five-membered C-ring is required for activity. T h e open ring compound is inactive (Figure 3.2). The stereochemistry at C-9 has no drastic consequence on in vitro SHIP activation and inhibition of TNFa production in intact cells. At least one phenol is required for activity. Methylation or acetylation reduces activity significantly. HQ The substituent at C-20 is important for activity. The C-20 methyl analog AQX-16A (2.63) is the most active compound in all assays. The stereochemistry at C-8 is critical for activity in intact cells. All C-8 epi stereoisomers are less active than the C-9 epi stereoisomers in the J774 assay. Opening of the five-membered C-ring loses activity. Figure 3.30 Structure activity relationships of Pelorol (2.1) 3.8 Experimental General: See chapter 2. Chapter 3: Structure Activity Relationship Study of Pelorol. Preparation of enal (2.54) 2.54 T o a solution of oxalyl chloride (1.4 mL, 15.8 mmol) in CH2CI2 (50 mL) was added dropwise D M S O (1.3 mL, 18.9 mmol) at - 7 8 ° C . After the mixture was stirred for 20 minutes at the same temperature, a solution of alcohol 2.52 (1.51 g, 6.3 mmol) in C H 2 C I 2 (2 mL) was added. The resulting reaction mixture was stirred for 40 minutes at - 7 8 ° C and triethylamine (8.8 mL, 62.8 mmol) was added. The cold dewar was removed and the mixture was allowed to warm to room temperature and further stirred for 3 hours after which brine (50 mL) was added and the mixture was extracted with C H 2 C I 2 (200 mL, twice). T h e combined CH2CI2 extracts were washed with H 2 0 , dried ( M g S 0 4 ) , and concentrated to give a residue, which was dissolved in 20% K O H in M e O H at room temperature and stirred for another 20 minutes. After addition of H2O (80 mL), the mixture was extracted with E t 2 0 (200 mL, twice). The combined E t 2 0 extracts were washed with saturated brine, dried ( M g S 0 4 ) , and concentrated to give a brown residue, which was chromatographed on a normal phase silica gel column to give 1.18 g (85%) of 2.54 (light yellow oil). The spectral data of 2.54 were identical to reported values . 5 Chapter 3: Structure Activity Relationship Study of Pelorol. 146 Preparation of benzyl alcohol (3.3a) and (3.3b) OMe 3.3a 3.3b A solution of 1.7 M tBuLi in pentane (3.48 mL, 5.58 mmol) was added slowly to a stirred solution of 2.38 (1.25 g, 5.08 mmol) in dry T H F (40.0 mL) at -7 8 ° C . After stirring for 30 minutes, a solution of 2.54 (1.1g, 5 mmol) in dry T H F (10.0 mL) was added. The mixture was stirred at - 7 8 ° C for another 30 minutes and the cold dewar was removed to allow the mixture warm to room temperature. Stirring was continued for 2 hours after which H 2 0 (20 mL) was added and the mixture was extracted with E t 2 0 (120 mL, twice). T h e combined extracts were washed with brine, dried ( M g S 0 4 ) , and concentrated to yield a residue. Silica gel column chromatography give 1.63 g (84%) of 3.3a and 0.2 g (10%) of 3.3b as colorless oils. 3.3a 1 H N M R ( C D 3 C N ) 5 0.87 (s, 3H), 0.93 (s, 3H), 1.00 (s, 3H), 1.16 (t, J= 7.6 Hz, 3H), 1.33 (td, J= 13.0, 3.5 Hz, 1H), 1.43 (s, 3H), 1.64 (dt, J= 13.7, 3.4 Hz, 1H), 2.55 (q, J= 7.6 Hz, 2H), 3.65 (d, J= 3.7 Hz, 1H), 3.78 (s, 3H), 3.82 (s, 3H), 5.64 (d, J= 3.7 Hz, 1H), 6.73 (d, J= 1.8 Hz, 1H), 6.77 (d, J= 1.8 Hz, 1H) Chapter 3: Structure Activity Relationship Study of Pelorol. 147 1 J C N M R ( C D 3 C N ) 8 16.3, 19.9 (Cx2), 21.0, 22.2, 22.3, 29.6, 33.9, 34.2, 35.6, 37.9, 40.2, 42.5, 53.4, 56.4, 60.7, 68.7, 112.2, 120.6, 132.9, 138.8, 140.2, 141.0, 146.0,153.4 H R E S I M S calcd for CasHa&OaNa 409.2719, found 409.2715 3.3b 1 H N M R ( C D 3 C N ) 5 0.76 (td, J= 13.6, 4.0 Hz, 1H), 0.86 (s, 3H), 0.91 (s, 3H), 1.07 (td, J= 13.3, 3.7 Hz, 1H), 1.13 (s, 3H), 1.17 (t, J= 7.6 Hz, 3H), 1.34 (brd, J= 12.9 Hz, 1H), 1.65 (s, 3H), 1.73 (dd, J= 13.1, 7.2 Hz, 1H), 2.55 (q, J= 7.6 Hz, 2H), 3.28 (d, J= 3.5 Hz, 1H), 3.83 (s, 3H), 3.84 (s, 3H), 5.56 (d, J= 3.5 Hz, 1H), 6.70 (d, J= 1.7 Hz, 1H), 6.80 (d, J= 1.7 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 8 16.3, 19.6, 19.8, 20.3, 22.1, 22.9, 29.6, 33.7, 34.0, 35.4, 37.0, 39.8, 42.4, 52.8, 56.5, 61.1, 67.9, 112.9, 121.2, 132.8, 137.7, 140.3, 141.0, 146.5, 153.6 H R E S I M S calcd for C 2 5H380 3 Na 409.2719, found 409.2726 Chapter 3: Structure Activity Relationship Study of Pelorol. 148 Preparation of indenes 3.4 and 3.5 T o a stirred solution of 3.3a or 3.3b (98 mg, 0.25 mmol) in C H 2 C I 2 (25 mL), S n C I 4 (0.25 mL) was added slowly at - 2 0 ° C under argon for 2 minutes. The mixuture was further stirred for 5 minutes, and diluted with CH2CI2 (40 mL) and then poured into ice water. The aqueous phase was extracted with C H 2 C I 2 twice (40 mL) and the combined extracts were washed with saturated N a H C 0 3 , H 2 0 , and dried ( M g S 0 4 ) . Concentration of the C H 2 C I 2 gave a brown oil, which was passed through a normal phase Seppak eluted with Hexane : E t O A c (93:7) to give an yellow oil. The mixture was subjected to H P L C seperation with a chiral column to afford 46 mg of 3.4 and 30 mg of 3.5 as colorless oils. 3.4 1 H N M R (CDCI3) 5 0.83 (s, 3H), 0.91 (s, 3H), 1.21 (t, J= 7.6 Hz, 3H), 1.24 (s, 3H), 1.41 (s, 3H), 1.86 (brd, J= 11.7 Hz, 1H), 2.43 (dt, J= 12.8, 3.0 Hz, 1H), 2.70 (m, 2H), 3.83 (s, 3H), 3.85 (s, 3H), 6.33 (s, 1H), 6.45 (s, 1H) 1 3 C N M R (CDCI3) 5 16.3, 18.6, 19.2, 19.5, 21.6, 23.3, 24.5, 33.5, 33.7, 37.8, 38.5, 39.3, 42.2, 52.6, 56.0, 56.1, 61.2, 108.9, 114.1, 133.9, 135.9, 140.0, 144.3, 151.1,168.3 Chapter 3: Structure Activity Relationship Study of Pelorol. 149 H R E S I M S calcd for C25H37O2 [M+H] + 369.2794, found 369.2794 3 .5 3.5 1 H N M R (CDCI3) 5 0.90 (s, 3H), 0.92 (s, 3H), 1.15 (s, 3H), 1.22 (t, J=7.5 Hz, 3H), 1.49 (s, 3H), 1.71 (dt, J= 13.5, 3.3 Hz, 1H), 2.01 (dd, J= 12.6, 5.8 Hz, 1H), 2.36 (m, 1H), 2.70 (m, 2H), 3.83 (s, 3H), 3.84 (s, 3H), 6.43 (s, 1H), 6.47 (s, 1H) 1 3 C N M R (CDCI3) 8 16.1, 17.6, 19.5, 21.5, 24.7, 25.5, 26.1, 28.0, 33.2, 33.7, 38.9, 40.3, 42.4, 45.2, 52.3, 56.0, 61.2, 108.5, 117.8, 133.6, 136.7, 139.7, 144.8, 151.2,170.4 H R E S I M S calcd for C 2 5H 3 60 2 Na 391.2613, found 391.2613 Chapter 3: Structure Activity Relationship Study of Pelorol. 150 Preparation of tetracycle 3.6 Catalytic hydrogenation of 3.4 (25 mg, mmol) was carried out in EtOH (3.0 mL) under H 2 (45 psi) in the presence of 10% Pd/C ( 50 mg) overnight. The reaction mixture was filtered and concentrated to afford an oil, which was purified with normal phase Seppak to give 23 mg of 3.6 (90%) as a colorless oil. 1 H NMR (CDCI 3 ) 5 0.83 (s, 3H), 0.85 (s, 3H), 1.22 (t, J= 7.5 Hz, 3H), 1.22 (s, 3H), 1.61 (s, 3H), 1.68 (brd, J= 13.7 Hz, 1H), 1,81 (dd, J= 12.3, 8.2 Hz, 1H), 1.97 (brd, J= 11.1 Hz, 1H), 2.66 (dd, J= 15.7, 12.3 Hz, 1H), 2.54-2.77 (m, 2H), 2.90 (dd, J= 15.7, 8.1 Hz), 3.80 (s, 3H), 3.81 (s, 3H), 6.52 (s, 1H) 1 3 C NMR (CDCI3) 5 16.3, 18.4, 18.5, 21.9, 24.5, 25.2, 26.9, 30.1, 32.9, 33.5, 36.6, 37.6, 38.3, 42.4, 47.6, 47.9, 55.9, 60.3, 62.4, 112.3, 134.3, 135.0, 142.8, 144.2, 150.4 HRESIMS calcd for C 25H380 2Na 393.2770, found 393.2772 Chapter 3: Structure Activity Relationship Study of Pelorol. 151 Preparation of tetracycle 3.7 T h e procedure for the preparation of tetracycle 3.7 from the indene 3.5 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and afforded 21 mg of 3.7 (87%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.44 (s, 3H), 0.77 (s, 3H), 0.86 (s, 3H), 0.96 (dd, J= 11.3, 4.1 Hz, 1H), 1.19 (s, 3H), 1.22 (t, J= 7.6 Hz, 3H), 2.49 (dt, J= 14.2, 5.3 Hz, 1H), 2.56-2.74 (m, 2H), 2.75 (brd, J= 16.9 Hz, 1H), 2.86 (dd, J= 16.9, 7.6 Hz, 1H), 3.78 (s, 3H), 3.81 (s, 3H), 6.47 (s, 1H) 1 3 C N M R (CDCI3) 5 15.5, 16.0, 18.5, 19.8, 21.9, 25.4, 28.3, 32.1, 33.2, 33.4, 35.3, 37.3, 41.0, 42.1, 48.8, 52.7, 55.8, 59.9, 62.7, 111.8, 134.2, 137.3, 141.4, 142.4, 150.5 H R E S I M S calcd for C25H 3 80 2 Na 393.2770, found 393.2773 Chapter 3: Structure Activity Relationship Study of Pelorol. 152 Preparation of methylketone 3.8 T he procedure for the preparation of methylketone 3.8 from the tetracycle 3.6 was the same as that used in the preparation of methylketone 2.67 from the tetracycle 2.42, and it afforded 11 mg of 3.8 (53%) as a colorless oil. 1 H N M R (CDCI 3 ) 8 0.83 (s, 3H), 0.87 (s, 3H), 1.18 (s, 3H), 1.35 (s, 3H), 1.75 (dd, J= 11.9, 8.2 Hz, 1H), 2.28 (m, 1H), 2.53 (s, 3H), 2.74 (dd, J= 15.5, 12.3 Hz, 1H), 2.90 (dd, J= 15.5, 7.9 Hz, 1H), 3.83 (s, 3H), 3.86 (s, 3H), 6.75 (s, 1H) 1 3 C N M R (CDCI3) 6 18.4, 18.6, 21.9, 24.2, 24.6, 29.9, 30.9, 32.9, 33.5, 36.6, 37.7, 38.5, 42.4, 47.6, 47.9, 56.3, 60.3, 63.3, 110.7, 132.9, 136.0, 146.3, 147.0, 149.6, 203.6 H R E S I M S calcd for CasHseOsNa 407.2562, found 407.2569 Chapter 3: Structure Activity Relationship Study of Pelorol. 153 Preparation of methylketone 3.11 T he procedure for the preparation of methyketone 3.11 from the tetracycle 3.7 was the same as that used in the preparation of methylketone 2.67 from the tetracycle 2.42, and it afforded 7 mg of 3.11 (47%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.28 (s, 3H), 0.71 (s, 3H), 0.84 (s, 3H), 1.29 (s, 3H), 1.65 (d, J= 7.0 Hz, 1H), 1.71 (brd, J= 12.9 Hz, 1H), 2.27 (m, 1H), 2.54 (s, 3H), 2.76 (d, J= 16.7 Hz, 1H), 2.90 (dd, J= 16.7, 7.0 Hz, 1H), 3.82 (s, 3H), 3.84 (s, 3H), 6.66 (s, 1H) 1 3 C N M R (CDCI3) 5 15.3, 18.4, 19.6, 22.1, 28.3, 31.1, 32.6, 33.0, 33.6, 34.2, 37.2, 40.7, 42.0, 48.4, 53.4, 56.1, 60.0, 63.3, 109.3, 132.3, 139.1, 142.3, 146.3, 150.0, 204.2 H R E S I M S calcd for C 2 5 H 3 6 0 3 N a 407.2562, found 407.2570 Chapter 3: Structure Activity Relationship Study of Pelorol. 154 Preparation of methylester 3.9 T he procedure for the preparation of methylester 3.9 from the methylketone 3.8 was the same as that used in the preparation of methylester 2.73 from the methylketone 2.67, and it afforded 5 mg of 3.9 (64%) as a colorless oil. 1 H N M R (CDCI 3 ) 8 0.84 (s, 3H), 0.87 (s, 3H), 1.19 (s, 3H), 1.42 (s, 3H), 1.77 (dd, J= 11.9, 7.6 Hz, 1H), 2.36 (m, 1H), 2.73 (dd, J= 15.5, 12.2 Hz, 1H), 2.90 (dd, J= 15.5, 8.1 Hz, 1H), 3.82 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 6.98 (s, 1H) 1 3 C N M R (CDCI3) 5 18.4, 18.6, 21.9, 24.2, 24.7, 29.8, 32.9, 33.5, 36.6, 36.6, 38.4, 42.4, 47.5, 47.9, 51.9, 56.1, 60.3, 63.4, 112.5, 123.0, 135.6, 147.4, 147.4, 149.9,169.3 H R E S I M S calcd for C 2 5H360 4 Na 423.2511, found 423.2513 Chapter 3: Structure Activity Relationship Study of Pelorol. 155 Preparation of methylester 3.12 T he procedure for the preparation of methylester 3.12 from the methylketone 3.11 was the same as that used in the preparation of methylester 2.73 from the methylketone 2.67, and it afforded 3 mg of 3.12 (71%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.27 (s, 3H), 0.70 (s, 3H), 0.84 (s, 3H), 1.31 (s, 3H), 1.67 (d, J= 7.2 Hz, 1H), 1.71 (brd, J= 12.8 Hz, 1H), 2.37 (dt, J= 14.5, 4.0 Hz, 1H), 2.77 (d, J= 16.6 Hz, 1H), 2.89 (dd, J= 16.6, 7.2 Hz, 1H), 3.82 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 6.89 (s, 1H) 1 3 C N M R (CDCI3) 5 15.3, 18.4, 19.7, 22.0, 28.3, 31.7, 33.0, 33.6, 34.2, 37.2, 40.8, 42.0, 48.4, 51.9, 53.3, 56.0, 60.0, 63.4, 111.4, 122.5, 138.8, 143.8, 146.8, 150.2, 169.6 H R E S I M S calcd for C^HseCUNa 423.2511, found 423.2505 Chapter 3: Structure Activity Relationship Study of Pelorol. 156 Preparation of 9-ep/-pelorol (3.10) The procedure for the preparation of 9-ep/'-pelorol (3.10) from the methylester 3.9 was the same as that used in the preparation of pelorol (2.1) from the methylester 2.73, and it afforded 1 mg of 3.10 (38%) as a colorless oil. 1 H NMR (CDCI 3) 5 0.84 (s, 3H), 0.87 (s, 3H), 1.19 (s, 3H), 1.43 (s, 3H), 1.81 (dd, J= 12.0, 8.5 Hz, 1H), 2.42 (brd, J= 11.7 Hz, 1H), 2.70 (dd, J= 15.4, 12.2 Hz, 1H), 2.82 (dd, J= 15.4, 8.4 Hz, 1H), 3.82 (s, 3H), 7.03 (s, 3H) HRESIMS calcd for C 23H320 4Na 395.2198, found 395.2202 Chapter 3: Structure Activity Relationship Study of Pelorol. 157 Preparation of 8-ep/-pelorol (3.13) 3.13 The procedure for the preparation of 8-ep/-pelorol 3.13 from the methylester 3.12 was the same as that used in the preparation of pelorol (2.1) from the methylester 2.73, and it afforded 0.8 mg of 3.13 (42%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.31 (s, 3H), 0.72 (s, 3H), 0.84 (s, 3H), 1.32 (s, 3H), 2.39 (dt, J= 14.3, 4.3 Hz, 1H), 2.67 (d, J= 16.0 Hz, 1H), 2.84 (dd, J= 16.0, 7.4 Hz, 1H), 3.83 (s, 3H), 6.97 (s, 3H) H R E S I M S calcd for C 23H320 4Na 395.2198, found 395.2205 Chapter 3: Structure Activity Relationship Study of Pelorol. 158 Preparation of benzyl alcohol 3.14a and 3.14b OMe 3.14a 3.14b A solution of 1.7 M tBuLi in pentane (1.16 mL, 1.86 mmol) was added slowly to a stirred solution of 2.38 (0.42 g, 1.70 mmol) in dry T H F (17.0 mL) at -7 8 ° C . After stirring for 30 minutes, a solution of 2.54 (367 mg, 1.67 mmol) in dry T H F (4.0 mL) was added. The mixture was stirred at - 7 8 ° C for another 30 minutes and the cold dewar was removed to allow the mixture warm to room temperature. Stirring was continued for 2 hours after which H 2 0 (10.0 mL) was added and the mixture was extracted with E t 2 0 (40 mL, twice). T h e combined extracts were washed with brine, dried ( M g S 0 4 ) , and concentrated to yield a residue. Silica gel column chromatography give 417 mg of 3.14a (67%) and 104 mg of 3.14b (17%) as colorless oils. 3.14a 1 H N M R ( C D 3 C N ) 5 0.87 (s, 3H), 0.93 (s, 3H), 1.01 (s, 3H), 1.42 (s, 3H), 2.25 (s, 3H), 3.61 (d, J= 3.7 Hz, 1H), 3.76 (s, 3H), 3.80 (s, 3H), 5.61 (d, J= 3.7 Hz, 1H), 6.71 (d, J= 1.2 Hz, 1H), 6.74 (d, J= 1.2 Hz, 1H) Chapter 3: Structure Activity Relationship Study of Pelorol. 159 1 J C N M R ( C D 3 C N ) 8 19.9, 20.0, 20.9, 21.6, 22.2, 22.3, 33.9, 34.2, 35.7, 37.9, 40.2, 42.4, 53.4, 56.4, 60.7, 68.5, 113.2, 121.7, 132.9, 133.7, 138.9, 141.0, 145.8, 153.3 H R E S I M S calcd for C s ^ s e O s N a 395.2562, found 395.2560 3.14b 1 H N M R ( C D 3 C N ) 8 0.77 (td, J= 12.9, 3.4 Hz, 1H), 0.86 (s, 3H), 0.91 (s, 3H), 1.13 (s, 3H), 1.22 (dd, J= 12.3, 1.5 Hz, 1H), 1.65 (s, 3H), 1.73 (dd, J= 12.9, 6.9 Hz, 1H), 2.25 (s, 3H), 3.27 (d, J= 3.5 Hz, 1H), 3.81 (s, 3H), 3.83 (s, 3H), 5.55 (d, J= 3.5 Hz, 1H), 6.66 (d, J= 1.7 Hz, 1H), 6.78 (d, J= 1.7 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 8 19.6, 19.8, 20.3, 21.6, 22.1, 22.9, 33.7, 33.9, 35.3, 36.9, 39.7, 42.3, 52.6, 56.4, 61.1, 67.8, 113.9, 122.3, 132.7, 133.8, 137.7, 140.9, 146.4, 153.5 H R E S I M S calcd for C s ^ e O a N a 395.2562, found 395.2565 Chapter 3: Structure Activity Relationship Study of Pelorol. 160 Preparat ion of indenes 3.15 a n d 3.16 T h e procedure for the preparation of indenes 3.15 and 3.16 from the benzyl alcohols 3.14a and 3.14b was the same as that used in the preparation of indenes 3.4 and 3.5 from the benzyl alcohols 3.3a and 3.3b, and it afforded 21 mg of 3.15 (43%) and 20 mg of 3.16 (42%) as colorless oils. 3.15 1 H N M R (CDCI 3 ) 5 0.83 (s, 3H), 0.91 (s, 3H), 1.23 (s, 3H), 1.41 (s, 3H), 1.86 (brd, J= 11.7 Hz, 1H), 2.34 (s, 3H), 2.48 (dt, J= 12.6, 3.1 Hz, 1H), 3.82 (s, 3H), 3.85 (s, 3H), 6.32 (s, 1H), 6.38 (s, 1H) 1 3 C N M R (CDCI3) 5 18.5, 18.6, 19.1, 19.4, 21.6, 22.0, 33.5, 33.7, 36.7, 38.5, 39.3, 42.2, 52.5, 56.0, 56.1, 61.3, 110.8, 114.0, 127.2, 136.0, 140.1, 144.9, 150.7, 168.4 H R E S I M S calcd for C24H35O2 [M+H] + 355.2637, found 355.2634 3.15 Chapter 3: Structure Activity Relationship Study of Pelorol. 161 3.16 3.16 1 H NMR (CDCI3) 5 0.90 (s, 3H), 0.92 (s, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 2.34 (s, 3H), 3.81 (s, 3H), 3.83 (s, 3H), 6.36 (s, 1H), 6.46 (s, 1H) 1 3 C NMR (CDCI3) 5 17.5, 18.6, 19.5, 21.5, 24.4, 26.0, 26.8, 33.2, 33.7, 39.0, 40.3, 42.4, 45.2, 52.2, 56.0, 61.3, 110.4, 117.8, 127.0, 136.8, 139.8, 145.3, 150.9,170.5 HRESIMS c a l c d for C24H35O2 [M+H]+355.2637, f o u n d 355.2638 Chapter 3: Structure Activity Relationship Study of Pelorol. 162 Preparation of tetracycle 3.17 T he procedure for the preparation of tetracycle 3.17 from the indene 3.15 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and it afforded 15 mg of 3.17 (88%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.85 (s, 3H), 0.86 (s, 3H), 1.23 (s, 3H), 1.60 (s, 3H), 1.81 (dd, J= 12.0, 8.4 Hz, 1H), 1.98 (brd, J= 12.2 Hz, 1H), 2.32 (s, 3H), 2.68 (dd, J= 15.7, 12.2 Hz, 1H), 2.90 (dd, J= 15.8, 8.2 Hz, 1H), 3.80 (s, 6H), 6.46 (s, 1H) 1 3 C N M R (CDCI3) 5 18.4, 18.4, 19.9, 21.9, 24.5, 26.0, 30.1, 32.9, 33.5, 36.6, 36.7, 38.3, 42.4, 47.4, 47.9, 56.0, 60.3, 62.3, 114.3, 128.2, 134.4, 143.0, 144.7, 150.0 H R E S I M S calcd for C24H37O2 [M+H] + 357.2794, found 357.2790 Chapter 3: Structure Activity Relationship Study of Pelorol. 163 Preparation of tetracycle 3.18 3.18 T he procedure for the preparation of tetracycle 3.18 from the indene 3.16 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and it afforded 14 mg of 3.18 (83%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.43 (s, 3H), 0.77 (s, 3H), 0.86 (s, 3H), 0.96 (dd, J= 11.4, 4.3 Hz, 1H), 1.17 (s, 3H), 1.48 (tt, J= 13.6, 3.2 Hz, 1H), 1.72 (brd, J= 12.6 Hz, 1H), 2.31 (s, 3H), 2.53 (dt, J= 13.9, 5.8 Hz, 1H), 2.75 (d, J= 16.8 Hz, 1H), 2.88 (dd, J= 16.9, 7.8 Hz, 1H), 3.78 (s, 3H), 3.79 (s, 3H), 6.41 (s, 1H) 1 3 C N M R (CDCI3) 5 15.5, 18.5, 19.5, 19.7, 21.9, 28.4, 31.0, 33.2, 33.4, 34.7, 37.3, 41.1, 42.1, 48.6, 52.6, 55.9, 59.9, 62.6, 113.7, 127.4, 137.2, 142.0, 142.7, 150.2 H R E S I M S calcd for C24H37O2 [M+H] + 357.2794, found 357.2789 Chapter 3: Structure Activity Relationship Study of Pelorol. 164 Preparation of catechol 3.19 ,OH 3.19 T h e procedure for the synthesis of catechol 3.19 from the tetracycle 3.17 is the same as the synthesis of catechol 2 . 5 7 from the tetracycle 2 . 4 2 and it afforded a white solid 9.1 mg ( 85%) . 1 H N M R ( C D C I 3 ) 5 0 . 8 3 (s, 3 H ) , 0 . 8 5 (s, 3 H ) , 1.22 (s, 3 H ) , 1.59 (s, 3 H ) , 1.67 (tt, J= 1 4 . 5 , 3.1 Hz, 1 H ) , 1.85 (dd, J= 1 1 . 3 , 8 .4 Hz, 1 H ) , 1.97 (brd, J = 1 2 . 3 Hz, 1H) , 2 . 2 4 (s, 3 H ) , 2 . 64 (dd, J= 1 7 . 1 , 11 .9 Hz, 1 H ) , 2 . 7 9 (dd, J = 1 5 . 2 , 8 .2 Hz, 1H ) , 4 . 7 0 (brs, 1 H ) , 4 . 7 8 (brs, 1 H ) , 6 . 4 2 (s, 1 H ) 1 3 C N M R (CDCI3) 5 18 .4 ( 2 x C ) , 1 9 . 5 , 2 1 . 9 , 2 4 . 5 , 2 6 . 0 , 2 9 . 4 , 3 2 . 9 , 3 3 . 5 , 3 6 . 6 , 3 6 . 8 , 3 8 . 3 , 4 2 . 4 , 4 7 . 7 , 4 8 . 0 , 6 2 . 5 , 1 1 6 . 8 , 1 2 5 . 0 , 1 2 7 . 2 , 1 3 7 . 5 , 1 4 0 . 9 , 1 4 4 . 7 H R E S I M S calcd for C22H33O2 [M+H] + 3 2 9 . 2 4 8 1 , found 3 2 9 . 2 4 6 7 Chapter 3: Structure Activity Relationship Study of Pelorol. 165 Preparation of catechol 3.20 .OH 3.20 T h e procedure for the synthesis of catechol 3.20 from the tetracycle 3.18 is the same as the synthesis of catechol 2.57 from the tetracycle 2.42 and it afforded a white solid 7 mg (83%). 1 H N M R (CDCI 3 ) 5 0.44 (s, 3H), 0.78 (s, 3H), 0.86 (s, 3H), 0.97 (dd, J= 11.3, 4.6 Hz, 1H), 1.15 (s, 3H), 1.48 (tt, J= 13.6, 3.4 Hz, 1H), 2.23 (s, 3H), 2.48 (dt, J= 13.9, 5.8 Hz, 1H), 2.62 (d, J= 16.3 Hz, 1H), 2.81 (dd, J= 16.5, 7.9 Hz, 1H), 4.78 (brs, 1H), 4.91 (brs, 1H), 6.40 (s, 1H) 1 3 C N M R (CDCI3) 8 15.4, 18.5, 19.2, 19.4, 21.8, 27.7, 31.0, 33.2, 33.3, 34.4, 37.3, 41.1, 42.1, 48.8, 52.4, 62.8, 116.5, 124.4, 129.9, 137.2, 141.0, 142.2 H R E S I M S calcd for C22H 320 2 Na 351.2300, found 351.2304 Chapter 3: Structure Activity Relationship Study of Pelorol. 166 Preparation of diol 3.22 OMe 3.22 T o a stirred solution of veratrol (552 mg, 4 mmol), 6 mmol of T M E D A (0.91 mL) and 6 mL T H F at 0 ° C was added 5 mmol BuLi (3.125 mL, 1.6M). The reaction mixture was stirred at room temperature for 1 hour, then cooled to -7 8 ° C , and 380 mg of 2.19 in 5 mL of T H F was added dropwise. After stirring at -7 8 ° C for another hour, the reaction was quenched with water and partitioned with E t 2 0 . The ether layer was washed with sat. brine successively and evaporated to dryness. The residue was subjected to silica gel column chromatography and eluted with Hexane: E t O A c (100:0-100:7) to give 438 mg solid of 3.22 (73%). 1 H N M R (CDCI 3 ) 6 0.27 (td, J= 13.4, 3.8 Hz, 1H), 0.75 (s, 3H), 0.80 (s, 3H), 0.86 (dd, J= 12.2, 2.1 Hz, 1H), 0.94 (dd, J= 13.6, 4.0 Hz, 1H), 1.04 (s, 3H), 1.55 (s, 3H), 1.63 (brd, J= 14.0 Hz, 1H), 1.85 (dt, J= 12.8, 3.5 Hz, 1H), 2.12 (d, J= 8.4 Hz, 1H), 3.85 (s, 3H), 3.89 (s, 3H), 5.28 (d, J= 8.4 Hz, 1H), 6.81 (dd, J= 8.1, 1.5 Hz, 1H), 7.05 (t, J= 7.9 Hz, 1H), 7.11 (dd, J= 7.9, 1.5 Hz, 1H) 1 3 C N M R (CDCI3) 5 15.9, 18.4, 20.0, 21.6, 25.9, 33.3, 33.6, 38.9, 39.9, 41.5, 44.8, 55.7, 56.0, 61.3, 62.8, 69.2, 74.8, 111.2, 120.6, 124.4, 141.3, 145.6, 152.6 Chapter 3: Structure Activity Relationship Study of Pelorol. 167 HRESIMS calcd for C 23H360 4Na 399.2511, found 399.2514 Preparation of catechol 3.25 .OH T h e procedure for the synthesis of catechol 3.25 from the tetracycle 3.24 is the same as the synthesis of catechol 2.57 from the tetracycle 2.42 and it afforded a white solid 15 mg (81%). 1 H N M R ( C D C I 3 ) 5 0.85 (s, 6H), 0.95 (dd, J= 10.5,3.7 Hz, 1H), 1.00 (s ,3H), 1.02 (s, 3H), 1.17 (m, 1H), 2.07 (m, 1H), 2.49 (dd, J= 14.0, 12.5 Hz, 1H), 2.59(dd, J= 14.3, 6.4 Hz, 1H), 4.82 (s, 1H), 4.93 (s, 1H), 6.46 (d, J= 7.9 Hz, 1H), 6.64 (d, J= 7.8 Hz, 1H) 1 3 C N M R (CDCI 3 ) 5 16.2, 18.3, 19.5, 21.1, 23.3, 24.6, 33.2, 33.5, 37.0, 37.4, 40.2, 42.6, 46.0, 57.5, 64.9, 112.0, 113.3, 128.1, 140.2, 141.3, 149.6 HRESIMS calcd for C 2 i H 3 i 0 2 [M+H]+ 315.2324, found 315.2326 Chapter 3: Structure Activity Relationship Study of Pelorol. Preparation of benzyl alcohols 3.26a and 3.26b OMe 3.26a 3.26b T o a stirred solution of veratrol (414 mg, 3 mmol), 4.5 mmol of T M E D A (0.68 mL) and 4.5 mL T H F at 0 ° C was added 3.75 mmol BuLi (2.34 mL, 1.6M). The reaction mixture was stirred at room temperature for 1 hour, then cooled to -7 8 ° C , and 260 mg of 2.54 in 4 mL of T H F was added dropwise. After stirring at -7 8 ° C for another hour, the mixture was allowed to warm to room temperature over one hour. The reaction was quenched with water and partitioned with ether. The ether layer was washed with saturated brine successively and evaporated to dryness. The residue was subjected to silica gel column chromatography and eluted with Hexane: E t O A c (100:0-100:7) to give 260 mg of 3.26a (65%) and 63mg 3.26b (16%). 3.26a 1 H N M R ( C D 3 C N ) § 0.86 (s, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.30 (td, J= 12.9, 3.7 Hz, 1H), 1.38 (s, 3H), 1.59 (dt, J= 13.7, 3.5 Hz, 1H), 3.58 (d, J= 3.7 Hz, 1H), 3.76 (s, 3H), 3.79 (s, 3H), 5.61 (d, J= 3.7 Hz, 1H), 6.90 (m, 3H) Chapter 3: Structure Activity Relationship Study of Pelorol. 169 1 J C N M R ( C D 3 C N ) 5 19.9, 19.9, 20.9, 22.2, 22.3, 33.9, 34.1, 35.7, 37.9, 40.2, 42.4, 53.4, 56.4, 60.7, 68.5, 112.6, 121.5, 124.1, 132.9, 139.4, 141.0, 148.1, 153.6 H R E S I M S calcd for C s s H a ^ a N a 381.2406, found 381.2404 3.26b 1 H N M R ( C D 3 C N ) 5 0.73 (td, J= 13.0, 3.5 Hz, 1H), 0.85 (s, 3H), 0.90 (s, 3H), 1.05 (td, J= 13.6, 4.0 Hz, 1H), 1.33 (brd, J= 13.1 Hz, 1H), 1.64 (s, 3H), 1.72 (dd, J= 13.1, 6.7 Hz, 1H), 3.29 (d, J= 3.7 Hz, 1H), 3.83 (s, 3H), 3.87 (s, 3H), 5.58 (d, J= 3.5 Hz, 1H), 6.85 (dd, J= 6.7, 2.6 Hz, 1H), 6.95 (m, 2H) 1 3 C N M R ( C D 3 C N ) 5 19.6, 19.8, 20.3, 22.1, 22.9, 33.7, 33.9, 35.3, 36.9, 39.7, 42.3, 52.7, 56.5, 61.2, 67.8, 113.2, 122.0, 124.2, 132.8, 138.2, 141.0, 148.7, 153.8 H R E S I M S calcd for C s s H a ^ N a 381.2406, found 381.2407 Chapter 3: Structure Activity Relationship Study of Pelorol. 170 Preparat ion of indenes 3.27 a n d 3.28 T he procedure for the preparation of indenes 3.27 and 3.28 from the benzyl alcohols 3.26a and 3.26b was the same as that used in the preparation of indenes 3.4 and 3.5 from the benzyl alcohols 3.3a and 3.3b, and it afforded 39.5 mg of 3.27 (47%) and 25.5 mg of 3.28 (31%) as colorless oils. (3.27) 1 H N M R (CDCI 3 ) 5 0.84 (s, 3H), 0.92 (s, 3H), 1.23 (s, 3H), 1.36 (s, 3H), 1.42 (brd, 1H), 1.55 (dt, J= 13.3, 3.5 Hz, 1H), 1.85 (brd, 1H), 2.16 (dt, J= 12.6, 3.1 Hz, 1H), 3.84 (s, 3H), 3.91 (s, 3H), 6.37 (s, 1H), 6.66 (d, J= 7.9 Hz, 1H), 6.86 (d, J= 7.9 Hz, 1H) 1 3 C N M R (CDCI3) 5 18.6, 19.2, 19.3, 21.6, 25.0, 33.6, 33.8, 38.2, 39.1, 39.4, 42.3, 51.0, 56.2, 56.7, 61.2, 108.3, 113.8, 115.8, 135.2, 142.0, 148.8, 151.0, 168.1 3.27 H R E S I M S calcd for C23H33O2 [M+H] + 341.2481, found 341.2478 Chapter 3: Structure Activity Relationship Study of Pelorol. 171 (3.28) 1 H N M R (CDCI 3 ) 5 0.90 (s, 3H), 0.91 (s, 3H), 1.14 (s, 3H), 1.23 (td, J= 13.4, 3.7 Hz, 1H), 1.42 (s, 3H), 1.69 (tt, J= 13.6, 3.4 Hz, 1H), 2.04 (dd, J= 12.6, 6.1 Hz, 1H), 2.11 (ddd, J= 12.3, 9.9, 1.8 Hz, 1H), 3.83 (s, 3H), 3.89 (s, 3H), 6.51 (s, 1H), 6.63 (d, J= 8.1 Hz, 1H), 6.83 (d, J= 8.1 Hz, 1H) 1 3 C N M R (CDCI 3 ) 5 17.1, 19.5, 21.5, 26.4, 27.0, 28.2, 33.3, 33.7, 38.9, 40.2, 42.5, 45.2, 51.2, 56.2, 61.2, 108.1, 116.0, 117.9, 136.2, 141.8, 148.7, 151.1, 170.5 H R E S I M S calcd for C23H33O2 [M+H] + 341.2481, found 341.2479 Chapter 3: Structure Activity Relationship Study of Pelorol. 172 Preparation of tetracycle 3.29 3.29 T he procedure for the preparation of tetracycle 3.29 from the indene 3.27 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and it afforded 29 mg of 3.29 (89%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.85 (s, 3H), 0.87 (s, 3H), 1.18 (s, 3H), 1.81 (dd, J= 11.6, 8.2 Hz, 1H), 2.78 (dd, J= 15.8, 11.7 Hz, 1H), 2.90 (dd, J= 15.7, 8.1 Hz, 1H), 3.81 (s, 3H), 3.85 (s, 3H), 6.72 (t, J= 9.0 Hz, 2H) 1 3 C N M R (CDCI3) 8 18.2, 18.8, 22.0, 23.8, 25.8, 29.8, 33.0, 33.5, 36.5, 38.5, 39.2, 42.5, 45.1, 47.8, 56.1, 60.3, 62.9, 110.8, 116.2, 133.5, 145.3, 148.5, 150.7 H R E S I M S calcd for C23H35O2 [M+H] + 343.2637, found 343.2640 Chapter 3: Structure Activity Relationship Study of Pelorol. 173 Preparation of tetracycle 3.30 3.30 T he procedure for the preparation of tetracycle 3.30 from the indene 3.28 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and it afforded 18 mg of 3.30 (88%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.32 (s, 3H), 0.72 (s, 3H), 0.87 (s, 3H), 1.05 (s, 3H), 1.14 (td, J= 12.3, 3.8 Hz, 1H), 1.70 (brd, J= 6.7 Hz, 1H), 2.28 (dt, J= 13.9, 3.5 Hz, 1H), 2.76 (d, J= 16.6 Hz, 1H), 2.91 (dd, J= 16.6, 6.9 Hz, 1H), 3.81 (s, 3H), 3.83 (s, 3H), 6.69 (s, 2H) 1 3 C N M R (CDCI3) 5 15.2, 18.4, 19.4, 22.1, 28.6, 33.0, 33.7, 34.0, 35.1, 37.0, 41.3, 42.1,45.9, 53.0,56.0, 59.9, 62.3, 110.3, 114.9, 136.3, 145.0, 145.8, 150.8 H R E S I M S calcd for C 2 3 H 3 5 0 2 [M+H] + 343.2637, found 343.2643 Chapter 3: Structure Activity Relationship Study of Pelorol. 174 Preparation of catechol 3.31 T h e procedure for the synthesis of catechol 3.31 from the tetracycle 3.29 was the same as the synthesis of catechol 2.57 from the tetracycle 2.42 and it afforded a white solid 3.0 mg (87%). 1 H N M R (CDCI 3 ) 5 0.84 (s, 3H), 0.85 (s, 3H), 1.18 (s, 3H), 1.42 (s, 3H), 1.56 (brd, J= 13.4 Hz, 1H), 1.84 (dd, J= 10.4, 8.4 Hz, 1H), 2.71 (dd, J= 15.1, 11.4 Hz, 1H), 2.80 (dd, J= 15.4, 8.2 Hz, 1H), 4.89 (s, 1H), 4.99 (s, 1H), 6.50 (d, J= 7.9 Hz, 1H), 6.67 (d, J=7 .9 Hz, 1H) 1 3 C N M R ( C D C I 3 ) 5 18.4, 18.8, 22.0, 23.7, 25.8, 29.0, 33.0, 33.5, 36.4, 38.5, 39.3, 42.5, 45.4,47.9, 63.1, 113.2, 113.5, 126.5, 139.8, 141.4, 148.6 H R E S I M S calcd for C21H31O2 [M+H] + 315.2324, found 315.2326 Chapter 3: Structure Activity Relationship Study of Pelorol. 175 Preparation of catechol 3.32 ,OH T h e procedure for the synthesis of catechol 3.32 from the tetracycle 3.30 was the same as the synthesis of catechol 2.57 from the tetracycle 2.42 and it afforded a white solid 2.6 mg (84%). 1 H N M R (CDCI 3 ) 5 0.32 (s, 3H), 0.72 (s, 3H), 0.86 (s, 3H), 1.74 (brd, J= 6.9 Hz, 1H), 2.25 (m, 1H), 2.63 (d, J= 16.4 Hz, 1H), 2.83 (dd, J= 16.0, 6.9 Hz, 1H), 4.86 (s, 1H), 4.95 (s, 1H), 6.47 (d, J= 8.0 Hz, 1H), 6.65 (d, J= 8.0 Hz, 1H) 1 3 C N M R (CDCI3) 5 15.0, 18.4, 19.4, 22.1, 27.7, 33.1, 33.6, 33.9, 34.9, 37.0, 41.3, 42.1, 46.2, 52.8, 62.6, 112.1, 113.3, 129.2, 139.6, 141.6, 146.1 H R E S I M S calcd for C 2 i H 3 o 0 2 N a 337.2144, found 337.2147 Chapter 3: Structure Activity Relationship Study of Pelorol. 176 Preparation of benzyl alcohols 3.34a and 3.34b 3.34a 3.34b The procedure for the preparation of benzyl alcohols 3.34a and 3.34b was the same as that used in the preparation of benzyl alcohols 3.14a and 3.14b, and it afforded 102 mg of 3.34a (74%) and 11 mg of 3.34b (8%) as colorless oils. 3.34a 1 H N M R ( C D 3 C N ) 8 0.87 (s, 3H), 0.93 (s, 3H), 0.99 (s, 3H), 1.17 (t, J= 7.6 Hz, 3H), 1.34 (td, J= 13.1, 3.8 Hz, 1H), 1.43 (s, 3H), 2.61 (q, J= 7.6 Hz, 2H), 3.56 (d, J= 3.8 Hz, 1H), 3.79 (s, 3H), 3.85 (s, 3H), 5.64 (d, J= 3.7 Hz, 1H), 6.85 (d, J= 8.1 Hz, 1H), 6.97 (d, J= 8.1 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 5 15.5, 19.9, 20.0, 21.0, 22.2, 22.4, 23.5, 33.8, 34.2, 35.7, 37.9, 40.2, 42.4, 53.4, 60.7, 60.9, 68.3, 124.1, 124.4, 133.0, 137.4, 138.2, 140.8, 151.9, 152.3 H R E S I M S calcd for C 2 5 H 3 8 0 3 N a 409.2719, found 409.2715 Chapter 3: Structure Activity Relationship Study of Pelorol. 177 3.34b 1 H N M R ( C D 3 C N ) 5 0.75 (td, J= 13.0, 3.5 Hz, 1H), 0.86 (s, 3H), 0.91 (s, 3H), 1.07 (td, J= 13.4, 3.8 Hz, 1H), 1.14 (s, 3H), 1.18 (t, J= 7.6 Hz, 3H), 1.34 (brd, J= 13.1 Hz, 1H), 1.64 (s, 3H), 1.73 (dd, J= 13.0, 6.6 Hz, 1H), 2.62 (q, J= 7.6 Hz, 2H), 3.26 (d, J= 3.5 Hz, 1H), 3.80 (s, 3H), 3.91 (s, 3H), 5.56 (d, J= 3.5 Hz, 1H), 6.85 (d, J= 7.9 Hz, 1H), 6.93 (d, J= 7.9 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 8 15.5, 19.6, 19.8, 20.3, 22.1, 22.9, 23.5, 33.7, 34.0, 35.3, 37.0, 39.7, 42.4, 52.7, 60.9, 61.1, 67.8, 124.1, 125.0, 132.8, 136.3, 138.9, 141.0, 152.1,152.8 H R E S I M S calcd for CasHasOaNa 409.2719, found 409.2714 Chapter 3: Structure Activity Relationship Study of Pelorol. 178 Preparation of indene 3.35 and the elimination product 3.35a 3.35 T he procedure for the preparation of indene 3.35 from the benzyl alcohol 3.34a and 3.34b was the same as that used in the preparation of indene 3.4 from the benzyl alcohol 3.3a and 3.3b, and it afforded 17 mg of 3.35 (84%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.83 (s, 3H), 0.92 (s, 3H), 1.20 (t, J= 7.6 Hz, 3H), 1.20 (s, 3H), 1.22 (s, 3H), 2.14 (dd, J= 9.8, 3.0 Hz, 1H), 2.63 (q, J= 7.6 Hz, 2H), 3.82 (s, 3H), 3.92 (s, 3H), 6.31 (s, 1H), 6.75 (s, 1H) 1 3 C N M R (CDCI3) 5 15.5, 18.6, 19.3, 19.4, 21.6, 23.3, 25.0, 33.6, 33.8, 38.3, 39.0, 39.3, 42.3, 51.3, 56.8, 60.8, 60.9, 113.7, 116.4, 132.8, 133.9, 145.7, 148.8, 151.3, 166.9 H R E S I M S calcd for C25H37O2 [M+H] + 369.2794, found 369.2795 Chapter 3: Structure Activity Relationship Study of Pelorol. 179 3.35a (3.35a) 1 H N M R (CDCI 3 ) 5 0.86 (s, 3H), 0.93 (s, 3H), 1.02 (s, 3H), 1.18 (t, J= 7.6 Hz, 3H), 1.47 (s, 3H), 2.24 (brd, J= 9.8 Hz, 1H), 2.61 (m, 2H), 3.78 (s, 3H), 3.83 (s, 3H), 5.56 (brs, 1H), 6.25 (s, 1H), 6.78 (s, 1H) 1 3 C N M R (CDCI3) 6 15.1, 19.3, 19.7, 21.9, 22.9, 23.1, 25.3, 32.6, 33.8, 38.1, 38.7, 42.4, 48.2, 60.1, 60.8, 115.7, 123.1, 126.3, 128.7, 131.5, 132.7, 136.6, 150.4, 150.5, 151.4 H R E S I M S calcd for C25H37O2 [M+H] + 369.2794, found 369.2791 Chapter 3: Structure Activity Relationship Study of Pelorol. 180 Preparation of tetracycle 3.36 3.36 T he procedure for the preparation of tetracycle 3.36 from the indene 3.35 was the same as that used in the preparation of tetracycle 3.6 from the indene 3.4, and it afforded 13 mg of 3.36 (88%) as a colorless oil. 1 H N M R (CDCI 3 ) 5 0.85 (s, 3H), 0.86 (s, 3H), 1.17 (s, 3H), 1.18 (t, J= 7.6 Hz, 3H), 1.43 (s, 3H), 1.68 (brd, J= 14.3 Hz, 1H), 1.80 (dd, J= 11.3, 8.7 Hz, 1H), 2.60 (m, 2H), 2.74 (dd, J= 15.4, 11.6 Hz, 1H), 2.85 (dd, J= 15.5, 8.1 Hz, 1H), 3.80 (s, 3H), 3.86 (s, 3H), 6.61 (s, 1H) 1 3 C N M R (CDCI3) 5 15.4, 18.4, 18.8, 22.0, 23.3, 23.8, 25.7, 30.0, 33.0, 33.5, 36.4, 38.5, 39.2, 42.5, 45.4, 47.8, 60.0, 60.7, 62.8, 116.5, 130.7, 136.3, 148.5, 149.0, 150.8 H R E S I M S calcd for C 2 5 H 3 9 0 2 [M+H] + 371.2950, found 369.2952 Chapter 3: Structure Activity Relationship Study of Pelorol. 181 Preparation of benzyl alcohol 3.51 3.51 A solution of 1.6 M BuLi in pentane (1.95 mL, 3.12 mmol) was added slowly to a stirred solution of 3.50 (742 mg, 2.85 mmol) in dry T H F (22.0 mL) at - 7 8 ° C . After stirring for 30 minutes, a solution of 2.54 (0.62 g, 2.8 mmol) in dry T H F (6.0 mL) was added. T h e mixture was stirred at - 7 8 ° C for another 30 minutes and was allowed to warm to - 2 0 ° C in 2 hours, after which H 2 0 (10.0 mL) was added and the mixture was extracted with E t 2 0 (60mL, twice). T h e combined extracts were washed with brine, dried ( M g S 0 4 ) , and concentrated to yield a residue. Silica gel column chromatography give 460 mg of 3.51 (41 %) as colorless oil. 1 H N M R ( C D 3 C N ) 5 0.87 (s, 3H), 0.93 (s, 3H), 1.02 (s, 3H), 1.40 (s, 3H), 3.28 (s, 3H), 3.55 (d, J= 3.9 Hz, 1H), 3.78 (s, 3H), 3.83 (s, 3H), 4.33 (s, 2H), 5.63 (d, J= 3.9 Hz, 1H), 6.88 (d, J= 1.7 Hz, 1H), 6.90 (d, J= 1.7 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 8 20.0 (Cx2), 21.0, 22.2, 22.2, 33.9, 34.2, 35.8, 38.0, 40.3, 42.5, 53.4, 56.5, 58.1, 60.7, 68.5, 75.3, 112.0, 120.9, 132.8, 134.5, 139.2, 141.3, 147.3, 153.6 Chapter 3: Structure Activity Relationship Study of Pelorol. 182 H R E S I M S calcd for C 2 5 H 3 8 0 4 N a 425.2668, found 425.2671 Preparation of indenes 3.52 and 3.53 and uncyclized product 3.54 T o a stirred solution of 3.51 (260 mg, 0.65 mmol) in C H 2 C I 2 (30 mL) at -2 0 ° C , T F A (2.72 g, 1.78 mL) was added slowly under argon for 5 minutes. The mixuture was further stirred for 13 minutes and queched with Sat. N a H C 0 3 10.0 mL, then diluted with C H 2 C I 2 (100 mL) poured into ice water. Th e aqueous phase was extracted with C H 2 C I 2 twice (100 mL) and the combined extracts were washed with saturated NaHCC>3, H 2 0 , and dried ( M g S 0 4 ) . Concentration of the C H 2 C I 2 gave a brown oil, which was passed through a normal phase Seppak eluted with Hexane : E t O A c (90:10) to give an yellow oil. Th e mixture was subjected to H P L C separation with a chiral column to afford 77 mg of 3.52 (31%), 67 mg of 3.53 (27%) and 46 mg of 3.54 (19%) as colorless oils. (3.52) 1 H N M R (CDCI 3 ) 5 0.90 (s, 3H), 0.92 (s, 3H), 1.14 (s, 3H), 1.49 (s, 3H), 1.71 (dt, J= 13.4, 3.4 Hz, 1H), 2.01 (dd, J= 12.5, 5.6 Hz, 1H), 2.31 (ddd, J= 12.5, 9.3, 2.4 Hz, 1H), 3.42 (s, 3H), 3.84 (s, 3H), 3.84 (s, 3H), 4.49 (d, J= 11.3 Hz, 1H), 4.54 (d, J= 11.3 Hz, 1H), 6.47 (s, 1H), 6.66 (s, 1H) Chapter 3: Structure Activity Relationship Study of Pelorol. 183 1 3 C N M R (CDCU) 5 17.6, 19.5, 21.5, 26.0, 26.1, 28.0, 33.2, 33.7, 39.0, 40.3, 42.3, 45.2, 52.3, 56.0, 58.3, 61.2, 71.2, 108.6, 117.8, 127.5, 136.9, 141.2, 145.7, 151.3, 170.4 H R E S I M S calcd for C 2 5 H360 3 Na 407.2562, found 407.2560 (3.53) 1 H N M R (CDCI 3 ) 5 0.83 (s, 3H), 0.91 (s, 3H), 1.23 (s, 3H), 1.41 (s, 3H), 1.30 (dd, J= 12.8, 5.0 Hz, 1H), 1.86 (brd, 1H), 2.40 (dt, J= 12.6, 3.0 Hz, 1H), 3.41 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 4.48 (d, J= 11.3 Hz, 1H), 4.56 (d, J= 11.3 Hz, 1H), 6.33 (s, 1H), 6.69 (s, 1H) 1 3 C N M R (CDCI 3 ) 8 18.6, 19.2, 19.5, 21.6, 23.7, 33.5, 33.7, 37.8, 38.5, 39.3, 42.2, 52.6, 56.0, 56.1, 58.2, 61.2, 71.1, 108.9, 114.1, 127.7, 136.1, 141.4, 145.2, 151.2, 168.3 H R E S I M S calcd for C25H37O3 [M+H] + 385.2743, found 385.2744 Chapter 3: Structure Activity Relationship Study of Pelorol. 184 (3.54) 1 H N M R (CDCI 3 ) 5 0.88 (s, 3H), 0.97 (s, 3H), 1.04 (s, 3H), 1.21 (td, J= 13.1, 3.9 Hz, 1H), 1.41 (dd, J= 3.5, 1.9 Hz, 3H), 1.60 (dt, J= 10.0, 3.5 Hz, 1H), 1.70 (tt, J= 13.5, 3.1 Hz, 1H), 1.84 (brd, J= 12.3 Hz, 1H), 3.29 (s, 3H), 3.69 (s, 3H), 3.81 (s, 3H), 4.31 (s, 2H), 5.61 (brs, 1H), 6.27 (brs, 1H), 6.61 (d, J= 1.5 Hz, 1H), 6.83 (d, J= 1.5 Hz, 1H) 1 3 C N M R ( C D 3 C N ) 5 20.1, 20.2, 22.2, 23.6, 26.1, 33.0, 34.6, 39.1, 39.6, 43.1, 49.2, 56.4, 58.2, 60.4, 75.0, 111.6, 116.8, 123.5, 130.1, 132.2, 134.7, 135.5, 146.8, 152.9, 153.3 H R E S I M S calcd for C 2 5H360 3 Na 407.2562, found 407.2567 Chapter 3: Structure Activity Relationship Study of Pelorol. 185 Preparation of tetracycle 3.55 Catalytic hydrogenation of 3.52 (70 mg, 0.18 mmol) was carried out in C H 2 C I 2 ( 10 mL) under H 2 (45 psi) in presence of 5% R h / C ( 300 mg) overnight. The reaction mixture was filtered and concentrated to afford an oil, which was purified by passing through a normal phase Seppak to give 58 mg of 3.55 (83%) as a colorless oil. 1 H N M R (CDCI3) 5 0.36 (s, 3H), 0.74 (s, 3H), 0.86 (s, 3H), 0.93 (dd, J= 11.6, 3.2 Hz, 1H), 1.17 (s, 3H), 1.72 (brd, J= 14.2 Hz, 1H), 2.49 (m, 1H), 2.75 (d, J= 16.8 Hz, 1H), 2.88 (dd, J= 16.6, 7.0 Hz, 1H), 3.38 (s, 3H), 3.79 (s, 3H), 3.81 (s, 3H), 4.38 (d, J= 11.1 Hz, 1H), 4.50 (d, J= 11.1 Hz, 1H), 6.67 (s, 1H) 1 3 C N M R (CDCI3) 8 15.5, 18.5, 19.8, 22.0, 28.3, 32.4, 33.1, 33.6, 35.3, 37.4, 40.8, 42.1, 48.9, 53.2, 55.9, 57.9, 59.9, 62.7, 72.4, 112.2, 127.6, 137.8, 142.6, 144.2, 150.3 H R E S I M S calcd for C 2 5H 3 80 3 Na 409.2719, found 409.2715 Chapter 3: Structure Activity Relationship Study of Pelorol. 186 Preparation of tetracycle 3.56 The procedure for the preparation of tetracycle 3.56 from the indene 3.53 was the same as that used in the preparation of tetracycle 3.55 from the indene 3.52, and it afforded 49 mg of 3.56 (81%) as a colorless oil. 1 H NMR (CDCI 3 ) 5 0.84 (s, 3H), 0.86 (s, 3H), 1.22 (s, 3H), 1.59 (s, 3H), 1.67 (brt, J= 13.6 Hz, 1H), 1.82 (dd, J= 12.2, 8.4 Hz, 1H), 1.98 (brd, J= 11.4 Hz, 1H), 2.70 (dd, J= 15.5, 12.2 Hz, 1H), 2.91 (dd, J= 15.8, 8.2 Hz, 1H), 3.37 (s, 3H), 3.81 (s, 3H), 3.82 (s, 3H), 4.47 (s, 2H), 6.75 (s, 1H) 1 3 C NMR (CDCI3) 5 18.4, 18.5, 21.9, 24.5, 26.2, 30.2, 33.0, 33.5, 36.6, 37.6, 38.3, 42.4, 47.7, 47.9, 56.0, 58.0, 60.2, 62.4, 72.1, 112.4, 128.6, 134.7, 144.5, 145.2, 150.3 HRESIMS calcd for C 2 5 H380 3 Na 409.2719, found 409.2717 Chapter 3: Structure Activity Relationship Study of Pelorol. 187 3.9 References 1. Koehn, F. E . ; Carter, G . T. Nature Rev. Drug. Discov. 2005, 4, 206-220. 2. Aoki, S.; Kong, D.; Matsui, K.; Rachmat, R.; Kobayashi , M . Chem. Pharm. Bull. 2004, 52, 935-937. 3. Prokofeva, N. G . ; Utkina, N. K.; Chaikina, E . L ; Makarchenko, A . E . Comp. Biochem. Physi. Part B. 2004, 139, 169-173. 4. Brittain, R. T.; Drew, G . M . ; Levy, G . P. Br. J. Pharmacol. 1982, 77, 105-114. 5. Hua, D. H . ; Huang, X. ; Chen , Y. ; Battina, S. K.; Tamura , M . ; Noh, S. K.; Koo, S. I.; Namatame, I.; Tomoda , H. ; Perchellet, E . M . ; Perchellet, J . P. J. Org. Chem. 2004, 69, 6065-6078. 6. Trauner, D.; Bats, J . W. ; Werner, A . ; Mulzer, J . J. Org. Chem. 1998, 63, 5908-5918. 7. Mukku, V . J . R. V . ; Edrada, R. A. ; Schmitz, F. J . ; Shanks, M . K.; Chaudhuri, B.; Fabbro, D. J. Nat. Prod. 2003, 66, 686-689. 8. Sanchez, I. H. ; Mendoza, S.; Calderon, M . ; Larraza, M . I.; Flores, H . J . J. Org. Chem. 1985, 50, 5077-5079. Chapter 4: Absolute Configuration of Contignasterol. 188 Chapter 4: Absolute Configuration of Contignasterol. 4.1 Introduction Contignasterol (4.1 ),1 isolated from Petrosia contignata collected in Papua New Guinea, was the first reported example of an emerging family of marine sponge OH 4.7 clathriol 4.8 IPL576-092 Aversion of this chapter has been published. Yang, Lu; Andersen, Raymond J . (2002) Absolute Configuration of the Antiinflammatory Sponge Natural Product Contignasterol. Journal of Natural Products. 65(12), 1924-1926. Chapter 4: Absolute Configuration of Contignasterol. 189 steroids that are characterized by having in common the T a r e cis C / D ring junction and 15-keto, 6ot-hydroxyl, and 7p-hydroxyl functionalities. Other known members of this family include xestobergsterols A to C (4.2 to 4.4),2,3 14-tamosterone sulfate (4.5),4 haliclostanone sulfate (4.6),5 and clathriol (4.7).6 Contignasterol 7 and other members of this family of s tero ids 2 , 6 , 8 inhibit the anti-lge stimulated release of histamine from sensitized rat mast cells in a dose-dependent manner. O n the basis of this promising biological activity, 9 contignasterol was chosen as the lead structure for an analog synthesis program that identified IPL576-092 as a novel antiasthma/antiinflammatory agent . 1 0 IPL576-092 has been advanced to phase II human clinical trials. T h e initial structure elucidation of contignasterol (4.1) assigned the relative stereochemistry by analysis of the coupling constants and N O E data obtained for contignasterol tetraacetate. 1 Diagnostic coupling constants indicated that H-3 and H-4 were equatorially oriented and H-5, H-6, H-7, H-8 and H-9 were all axially oriented. N O E experiments in conjunction with scalar coupling constants showed that the hemiacetal ring in the side chain occupied a chair conformation. A N O E between C H 3 - 1 8 and 14-H helped to establish the 14-H p configuration. T o complete the structure elucidation of contignasterol (1), we set out to solve the absolute configuration of this compound ten years after it was originally identified. The relative stereochemistry at C-17 was assumed to be the "natural p configuration". Our efforts to establish the relative configuration between C-17, C -Chapter 4: Absolute Configuration of Contignasterol. 190 Figure 4.1 Separate coupling systems A, B, and C of Contignasterol 20 and C-22 with N O E experiments were unsuccessful, thus the contignasterol structure was divided into three parts with the separate coupling systems shown in Figure 4.1. These were: A) the steroid core, B) C-20, and C) the hemiacetal ring in the side chain. Our goal was to determine the absolute configuration of the moieties A and C independently and then try to relate one or the other to moiety B. Chapter 4: Absolute Configuration of Contignasterol. 191 4.2 Absolute configuration of Moiety A T he absolute stereochemistry of moiety A was solved by applying the exciton chirality circular dichroism method 1 1 because the two a-diol systems of 3,4-OH and 6,7-OH provided easy access to this method. Contignasterol was treated with 4-methoxybenzoyl chloride and the 3,6,7-tri-p-methoxybenzoate derivative was obtained in high yield as shown in Figure 4.2. 4.1 Contignasterol 4.9 R= p-MeOBz Figure 4.2 Synthesis of contignasterol 3,6,7-tri-p-methoxybenzoate Figure 4.3 CD spectra of contignasterol and contignasterol 3,6,7-tri-p-methoxybenzoate (4.9) in EtOH Chapter 4: Absolute Configuration of Contignasterol. 192 Th e p-methoxybenzoylation of the 4|3-hydroxyl group proved to be quite difficult due to the steric hindrance from the 19-CH3, which simplified the interpretation of the C D spectrum. In this case, when the tri-p-methoxybenzoate derivative was employed for C D measurement, the observed Cotton effects were only affected by the benzoate groups at C-3 , C-6 , and C-7 on the steroid nucleus. The C D spectrum of contignasterol tri-p-methoxybenzoate in E t O H showed a pair of typical exciton-split Cotton effects with opposite signs centered upon the U V absorption (257nm) of the p-methoxybenzoate chromophore: AS264.0 -29.4 and AS246.8 +16.3. The negative longer wavelength Cotton effect clearly defines the negative chirality between the three p-methoxybenzoate chromophores, thus unequivocally assigning the absolute configuration at C-3 as R. Th e C D spectrum of contignasterol (4.1) shows a negative Cotton effect at 300 nm attributed to the n to re* transition of the C-15 ketone. Comparison of this C D Figure 4.4 Negat ive chirality between the three p-methoxybenzoate chromophores on moiety A C0 2Me 4.10 Chapter 4: Absolute Configuration of Contignasterol. 193 data with that for the model 15-keto-14 steroid 4.10, which is reported to have a negative Cotton effect at 300 nm in its O R D curve , 1 2 confirmed that the nucleus of contignasterol has the standard steroidal configuration. Chapter 4 : Absolute Configuration of Contignasterol. 194 4.3 Absolute configuration of Moiety C The secondary hemiacetal hydroxyl group suggested that the determination of side chain absolute configuration might be possible by using a modified Mosher's method.13 A potential complicating factor in the analysis of the side chain configuration was the polyhydroxyl nature of contignasterol, which we anticipated would result in the formation of multiple esters with the chiral reagent. To circumvent this complication, the hydroxyl groups on the nucleus were protected as acetate esters prior to selective exposure of the C-29 alcohol and forming the Figure 4.5 Derivatization of contignasterol hemiacetal alcohol to (R)- and (S)-MTPA esters (4.13 and 4.14) Chapter 4: Absolute Configuration of Contignasterol. 195 C-22 chiral auxiliary esters. This sequence of transformations was accomplished as shown in Figure 4.5. Reaction of contignasterol with excess acetic anhydride in pyridine in the presence of a catalytic amount of (dimethylamino)pyridine gave pentaacetylcontignasterol (4.11), as a mixture of C-29 epimers, in good yield. Treatment of the pentaacetates with BF 3 etherate in aqueous acetonitrile selectively hydrolyzed the C-29 acetates to give the tetraacetate 4.12.14 (R) and (S)-MTPACI were coupled to the hemiacetal hydroxyl group in 4.12 and the two major diastereomers were isolated by normal phase HPLC (hexane:ethyl acetate=4:3). O - M T P A Figure 4.6 se lected A 8 R S 1 H N M R va lues in ppm for M T P A esters 4.13 and 4.14 (A8R S= 5 ( R ) . M TPA - 8 (S)-MTPA) Comparison of the 1HNMR spectra of the (R) and (S)-MTPA esters was carried out between the two diastereomers that had the 29-aHs. The 1HNMR spectra indicated that H28, H24, H 2 5 and H 2 6 ,27of (S)-MTPA ester were more shielded than in the (R)-MTPA ester, which suggested the C-22 configuration in contignasterol was (S). Comparing the H 2 2 and H 2 9 chemical shifts of the (S)-MTPA ester with the corresponding (R)-MTPA ester, we found they were also more shielded. This could be explained by conformational deviation of the MTPA moiety from the ideal position which requires the carbinyl proton and ester carbonyl and Chapter 4: Absolute Configuration of Contignasterol. 196 trifluoromethyl groups of MTPA to be synperiplanar.13 Therefore, it was not safe to derive the absolute configuration from the MTPA esters of the hemiacetal alcohol. Also, there is almost no literature on hemiacetal Mosher esters. Further evidence was required to demonstrate the absolute configuration in the side chain. Contignasterol has a cyclic hemiacetal in its side chain, which exists in equilibrium with the open chain 22-hydroxy-29-aldehyde form (Figure 4.7). The secondary alcohol at C- 22 appeared to be ideally suited for chiral auxiliary analysis of its absolute configuration using either MTPA or MPA esters. The C-22 alcohol could be liberated from the hemiacetal in 4.12 by trapping the C-29 aldehyde with 1,2- ethanedithiol to give the cyclic dithiane 4.15 (Figure 4.8).15 It's worth noting that the reaction condition applied to this transformation would not lead to epimerization of C-22 according to Miranda et al.'s recent report.16 Reaction of 4.15 with the (S)- and (R)-MPA and DCC gave the Mosher esters 4.16 and 4.17,17 while treatment with (R)- and (S)-MTPA acid chlorides gave the MTPA esters 4.18 and 4.19, respectively. Figure 4.7 Chapter 4: Absolute Configuration of Contignasterol. 197 Figure 4.8 chemical conversion of tetraacetate 4.12 into the MPA esters 4.16 and 4.17 and the MTPA esters 4.18 and 4.19 Chapter 4: Absolute Configuration of Contignasterol. 198 Table 4.1. Selected NMR data for (R), (S)-MPA esters (4.16)and (4.17), (R), (S)-MTPA esters (4.18)and (4.19) recorded at 400MHz in CD 2 CI 2 at 300K 5 (R)-MPA 5 (S)-MPA *A5RS S (R)-MTPA 8 (S)-MTPA **A8SR 1 2 1.70, 1.83 1.70, 1.83 0.00, 0.00 3 4.80 4.81 -0.01 4.82 4.82 0.00 4 4.96 4.97 -0.01 4.98 4.98 0.00 5 1.83 1.86 -0.04 1.89 1.89 0.00 6 5.00 5.02 -0.02 5.03 5.04 0.01 7 5.88 5.92 -0.03 5.93 5.94 0.01 8 1.83 1.90 -0.08 1.94 1.94 0.00 9 1.23 1.22 -0.01 10 11 12 13 14 2.14 2.24 -0.10 2.27 2.25 -0.02 15 16a 2.18 2.42 -0.24 2.46 2.42 -0.04 16/? 1.89 2.11 -0.22 2.24 2.21 -0.03 17 1.20 1.62 -0.42 1.69 1.61 -0.08 18 1.05 1.16 -0.11 1.21 1.17 -0.04 19 1.06 1.08 -0.02 1.10 1.11 0.01 20 1.60 1.94 -0.34 2.10 2.01 -0.09 21 0.82 1.01 -0.19 1.10 0.88 -0.22 22 5.12 5.08 0.04 5.26 5.20 -0.06 23 1.44, 1.82 1.32, 1.55 0.12, 0.27 1.35, 1.70 1.35, 1.67 0.00, -0.03 24 25 1.79 1.59 0.20 1.77 1.84 0.07 26, 27 0.88, 0.77 0.65, 0.63 0.23, 0.14 0.85, 0.73 0.90, 0.76 0.05, 0.03 28 1.57, 1.77 1.44, 1.68 0.13, 0.09 1.47, 1.74 1.53, 1.81 0.06, 0.07 29 4.54 4.33 0.21 4.33 4.43 0.10 30, 31 3.20-3.28 3.17-3.23 0.04 3.12-3.22 3.17-3.26 0.05 * A 5 R S = 8 (R)-MPA " 8 (S)-MPA * * A 8 S R = § (S)-MTPA " 8 (R)-MTPA Chapter 4: Absolute Configuration of Contignasterol. 199 Figure 4.9 1 H N M R comparison of (R)- and (S)-MPA esters recorded at 300K in C D 2 C I 2 Chapter 4: Absolute Configuration of Contignasterol. 200 Figure 4.10 1 H N M R comparison of (R)- and (S)-MTPA esters recorded at 300K in C D 2 C I 2 Chapter 4: Absolute Configuration of Contignasterol. 201 Table 4.2 Selected NMR data for dithioacetal (4.15) recorded at 400MHz in CD 2 CI 2 at 300K 8 1 H 5 1 3 C NOE Dithioacetal Dithioacetal Dithioacetal 1 31.99 2 21.92 3 4.81 68.57 H2, H4 4 4.97 67.32 H5, H3 5 1.87 44.41 H4 6 5.02 70.78 18-CH3, H8 7 5.95 74.84 H9 8 1.87 36.98 H6 9 46.50 H7 10 36.37 11 21.41 12 37.81 13 42.02 14 2.73 50.98 H8, 19-CH3, 21-CH3, H7 15 218.80 16a 2.41 39.21 H17, H16/5 160 2.21 H16a, H22, 21-CH3 17 1.92 46.57 21-CH3, H12a,H16a 18 1.20 14.27 H6, H8, H2j3, H^^0 19 1.09 19.99 H8, H14, H22, 21-CH3(very weak) 20 1.84 41.12 21-CH3 21 0.93 17.98 H17, H20, H23, H22, H14 22 3.49 72.62 21-CH3, H23, H160, H25 23 1.20, 1.59 37.26 21-CH3, H22, H29 24 41.43 25 1.76 29.77 H22, 26, 27-CH3 26, 27 0.90, 0.79 19.99, 17.55 H25 28 1.64, 1.82 40.34 H29, H24 29 4.56 52.97 H28, H23 30, 31 3.25-3.15 38.39, 38.66 MTPAO 2 J A5S R> 0 A8 S R< 0 MP AO 2 ; A5 R S< 0 A5 R S< 0 A 8 s r - 8 ( S ) . m t p a - 8 ( R ) . | V I T P A A8 R S -8 (R)_ M p A -8 ( S )_ M p A Figure 4.11 Models proposed by Mosher with expected sign of A 8 S R and AS ;RS 13,16 Chapter 4: Absolute Configuration of Contignasterol. 202 Table 4.1 lists the 1 H chemical shifts for the esters 4.16, 4.17, 4.18, and 4.19 and the A5 S R and A8 R S value for each proton in both the MTPA and MPA (R/S) pairs. From a comparison of the chemical shifts of the (R) and (S)-MPA esters in the table, it was obvious that A5 R S values on the steroid core structure were al negative ranging from -0.01 ppm to -0.42 ppm and the positive A5 R S values are all on the side chain. The chemical shift difference becomes smaler when the proton is more remote from the chiral center C-22. It is worth noting that the A5 R S value of H-22 is only 0.05 ppm, which means the conformational composition and preference are quite similar in the two MPA esters and the assignment of the absolute configuration is reliable. The A8 R S values could be used to deduce the location of Li and L 2 as shown in Figure 4.11 and thus the absolute configuration at C-22. In this case, the steroid core was represented by L2 in the model shown in Figure 4.11 and the side chain was represented by L1. Thus C-22 of 4.12 has the R configuration and the C-22 of 4.1 should have the same configuration, which is opposite to our initial judgement of the chirality by comparing the (R) and (S)-MTPA ester of the hemiacetal alcohol. The absolute configuration of C-24 is R according to the relative stereochemistry of the side chain, which was previously deduced from NOE experiments. Applying the empirical rules13 to the A5 R S values of (R)- and (S)-MTPA esters in Table 1 gives the same conclusion. Recent work by Riguera's group has compared MTPA with MPA as reagents for determination absolute stereochemistry by NMR, which indicated that MTPA Chapter 4: Absolute Configuration of Contignasterol. 203 Figure 4.12 Selected AS 1 H N M R values in ppm for the MPA esters 4.16 and 4.17 and the MTPA esters 4.18 and 4.19 esters were limited by the intrinsic greater complexity of their conformational composition, leading to diminished A 5 R S values, and were consequently less reliable for configurational assignment of chiral alcohols than M P A . 1 6 T h e A 8 R S values of M P A esters and A 8 S R values M T P A esters of contignasterol derivatives as shown in Figure 4.12 were in accordance with Riguera's conclusion. It was obvious that M P A was the preferred chiral auxiliary for determining the C-22 configuration of contignasterol. T h e decision to carry out the apparently redundant configurational analysis with M T P A was based on the report by Izzo et a l . 1 8 that they had synthesized a series of model compounds (i.e., 4.20) 4.20 4.21 Chapter 4: Absolute Configuration of Contignasterol. 204 containing the four possible C-22/C-24 stereoisomers for the contignasterol side chain and they had used (R) and (S) C-22 M T P A esters (4.21) to verify the side chain configurations in the diastereomers of the model compound 4.20. Using the same chiral auxiliary to make C-22 esters of 4.15 provided a more direct comparison of the C-22 configurations in the model compounds and contignasterol (4.1), and it provided independent confirmation of the C-22 configuration determined with the M P A esters (Figure 4.12). It was worth noting that on the basis of comparisons of the proton N M R data obtained for these four stereoisomers (4.20) with the N M R data for contignasterol derivative (4.22) TBDPSi 4.23 Figure 4.13 Comparison of side chain chemical shifts of the contignasterol derivative 4.22 and Izzo's synthetic product 4.23 shown in Figure 4.13, it was proposed by Izzo et a l . 1 8 that the side chain configuration in the natural product was (22S, 24S). A s shown in Figure 4.13, one of the stereoisomers 4.23 with (22S, 24S) configurations on the side chain had very similar 1 H N M R values as the contignasterol derivative, which led to Izzo's assignment of the side chain's absolute configuration of contignasterol antipodal to our proposal that the side chain had (22R, 24R) configurations. This Chapter 4: Absolute Configuration of Contignasterol. 205 discrepancy presumably reflects the sensitivity of the side chain proton chemical shifts to through-space shielding and deshielding interactions with functional groups in the steroid nucleus of contignasterol (4.1). It is likely that the unfunctionalized nucleus of the model compound 4.23 cannot accurately mimic these interactions, making a simple proton chemical shift comparison between the model compounds and contignasterol an unreliable predictor of the side chain stereochemistry in 4.1. O n e of the recent advances in the assignment of absolute configuration by N M R with chiral auxiliary reagents was that the absolute configuration of a secondary alcohol could be deduced from the 1 H N M R of a single methoxyphenylacetic ester derivative [MPA, either (R) or the (S)] recorded at two different temperatures. 1 9 H Figure 4.14 (R)-MPA ester of chiral secondary alcohol exists as two main conformers (sp and ap) A s shown in Figure 4.14, the (R)-MPA ester of chiral secondary alcohol exists as two main conformers (sp and ap) that are in equilibrium. T h e orientation of the phenyl ring produces different shieldings in the two forms, and consequently produces different chemical shifts for L-i and l_2 in each of the conformers. The Chapter 4: Absolute Configuration of Contignasterol. 206 direct consequence of this phenomenon is that at low temperature, the resonance of one of the substituents of the alcohol, is shifted upfield , and at the same time, another substituent l_2, is shifted downfield. In this way, the spatial arrangement of Li and L 2 around the chiral center can be interpreted by comparing the 1 H N M R spectra both at room and low temperatures. A s a valuable complement to determination of the side chain absolute configuration by N M R , this method was applied in our current research. Variable temperature 1 H N M R spectra of the (R) -MPA ester of the contignasterol tetraacetate in CD2CI2 were recorded at room (300K) and four low temperatures (273K, 253K, 223K, 203K). T M S was used as the internal standard in these experiments. Table 4.3 shows selected chemical shifts of the (R) -MPA ester of contignasterol tetraacetate taken at different temperatures. Two groups of data were distinguishable in the table. O n e group was formed by chemical shifts that shifted upfield in the low-temperature spectrum (A5 T 1 T 2>0). They were H 3 , H 6 , H - H , H- | 6 , H I 8 , H 2 0 , H 2 i that all reside on one side of the C-22 alcohol. This group was represented by l_i in the model shown in Figure 4.14. The other group was formed by resonances that moved downfield in the low-temperature spectrum (A8 T 1 , T 2<0). They were H26.27, H29, H 3 0 , 3 i that reside on the other side of the C-22 alcohol. This group was represented by l_ 2 in the model shown in Figure 4.14. Therefore, the C-22 configuration in contignasterol determined by this method was R, which confirmed the results we obtained from both the M T P A and M P A (R/S) pairs. Chapter 4: Absolute Configuration of Contignasterol. 207 Table 4.3. Selected Chemical Shifts of (R)-MPA ester recorded in C D 2 C I 2 at different temperatures* 8 (R)-MPA 8 (R)-MPA 8 (R)-MPA 8 (R)-MPA 8 (R)-MPA 300K 273K 253K 223K 203K 3 4.81 4.80 4.80 4.80 4.79 4 4.96 4.95 4.96 4.96 4.96 6 5.00 4.98 4.97 4.96 4.96 7 5.88 5.90 5.91 5.92 5.93 14 2.14 2.12 2.11 ** _** 16a 2.18 2.16 2.14 2.13 ** 18 1.06 1.06 1.05 1.05 1.05 19 1.05 1.06 1.05 1.05 1.05 20 1.64 1.60 1.57 1.54 1.52 21 0.82 0.81 0.82 0.82 0.82 22 5.12 5.14 5.15 5.17 5.18 26, 27 0.88, 0.75 0.88, 0.77 0.88, 0.76 0.89, 0.76 0.89, 0.75 29 4.54 4.55 4.56 4.57 4.58 30, 31 -3.25 -3.26 -3.26 -3.27 -3.28 * T M S was used as internal standard ** overlapped with upfield signals Chapter 4: Absolute Configuration of Contignasterol. 208 Figure 4.15 1 H N M R comparison of (R)-MPA ester recorded at 300K, 273K, 253K, 223K, and 203K in C D 2 C I 2 . Chapter 4: Absolute Configuration of Contignasterol. 209 4.4 Absolute configuration of Moiety C H 2 2 H 22 Figure 4.16 Newman projections for the C-20/C-22 bond in the dithiane 5 in which C-22 has the R configuration and H-20 and H-22 are anti. The H-22 resonance (8 3.33; benzene-afe) of 4.1 showed a positive N O E when H -14 (8 2.34) was irradiated in a difference N O E experiment, 1 confirming that the side chain was p at C-17. A coupling constant of 8.4 H z was observed between H-20 (8 1.84) and H-22 (8 3.49) in the dithiane 4.15, and there was no appreciable N O E observed between these resonances in a 1D N O E S Y experiment (Figure 4.17), suggesting that the protons were anti to each other. Strong N O E s observed between H-22 (8 3.49) and both the H-16 (8 2.21) and C H 3 - 2 1 (8 0.94) resonances in 1D N O E S Y experiments carried out on 4.15 were consistent with the proposed anti orientation of H-20 and H-22 and the 22-f? configuration shown in the Newman projections in Figure 4.16a and 4.16b. Intense N O E s observed between the C H 3 - 2 1 (8 0.94) and H-23 (8 1.21)/H-23' (8 1.60) resonances in 4.15 demonstrated that C-20 has the S configuration shown in Figure 3a, which is the normal C-20 configuration for a steroid. Chapter 4: Absolute Configuration of Contignasterol. 210 Therefore, the complete absolute configuration of contignasterol is 3R, 4R, 5R, 6R, IR, 8R, 9S, 10R, 13R, 14R, MR, 20S, 22R, 24R. Chapter 4: Absolute Configuration of Contignasterol. 211 1.84 H 20 3.49 Figure 4.17 The fragment structure of 4.15 with selected N O E S Y correlation Figure 4.18 Expansion of the N O E S Y spectrum for 4.15 Chapter 4: Absolute Configuration of Contignasterol. 212 4.5 Experimental Section General: Circular dichroism spectra were recorded on a J A S C O J-76 spectropolarimeter. 1 H and 1 3 C N M R spectra were recorded at 400 and 100 M H z , respectively, on Bruker AV400 , AV500 , and A M 4 0 0 spectrometers. 1 H chemical shifts are referenced to the residual CH 2 CI 2 -c/2 signal ( 5.32 ppm) and CeHe-de signal ( 7 . 1 5 ppm), and 1 3 C chemical shifts are referenced to the CH2CI2-C/2 solvent peak ( 53.8 ppm). In V T 1 H N M R , 1 H chemical shifts are referenced to the residual T M S signal ( 0.0 ppm). F A B M S were recorded on a Kratos Concept II H Q mass spectrometer. Commercially obtained reagents were used without further purification. All reactions were monitored by T L C (silica gel, 60F-54, Merck). H P L C was performed on a Alltech column (250 * 4.6 mm, n-hexane/ethyl acetate, 1 mL/min) monitored by a RI detector. Isolation of Contignasterol (4.1) Contignasterol (4.1) was isolated as a white solid from the extracts of the sponge Petrosia contignata Thiele using the same procedure as reported in the original paper. 1 Preparation of Contignasterol tri-p-methoxybenzoate (4.9) OMe 4.9 R=p-MeOBz Chapter 4: Absolute Configuration of Contignasterol. 213 Contignasterol (4.1) (15.0 mg) was dissolved in 2 mL of pyridine and 4 mL of 4-methoxybenzoyl chloride. The mixture was stirred at R T for 4 h. 3.0 mL of M e O H was added to the mixture and stirring was continued for 0.5 h. Pyridine and M e O H were removed in vacuo, and the residue was purified by normal-phase H P L C (eluent: hexane/EtOAc (4:6)). 1 H N M R ( C 6 D 6 ) 8 0.69 (d, J= 7.5 Hz, 3H), 0.71 (d, J= 7.0 Hz, 3H), 0.73 (d, J= 6.9 Hz, 3H), 1.16 (s, 3H), 1.42 (s, 3H), 2.64 (s, 1H), 2.96 (s, 3H), 3.00 (s, 3H), 3,12 (s, 3H), 3.44 (s, 3H), 3.94 (dd, J= 9.3, 1.8 Hz, 1H), 4.10 (d, J= 3.8 Hz, 1H), 4.18 (brs, 1H), 5.63 (brd, J= 2.4 Hz, 1H), 5.99 (dd, J= 12.0, 9.1 Hz, 1H), 6.40 (d, J= 8.7 Hz, 2H), 6.49 (d, J= 8.8 Hz, 2H), 6.52 (d, J= 8.5 Hz, 2H), 7.34 (dd, J= 10.5, 9.3 Hz, 1H), 8.08 (d, J= 7.8 Hz, 2H), 8.10 (d, J= 7.8 Hz, 2H), 8.15 (d, J= 9.0 Hz, 2H) Preparation of Contignasterol Pentaacetates (4.11) Chapter 4: Absolute Configuration of Contignasterol. 214 Contignasterol (4.1) (32.8 mg) was dissolved in 2 mL of pyridine and 2 mL of A C 2 O . A few crystals of D M A P were added, and the resulting mixture was stirred at R T for 18 h. The reaction solvents were removed by evaporation under high vacuum, and purification of the products was carried out by normal-phase H P L C , eluting with n-hexane/EtOAc (4:3) to yield the C - 2 9 a (6 mg) and C-29f3 (16 mg) contignasterol pentaacetates (4.11) (total isolated yield was 47.5%). The spectroscopic data for the pentatacetates (4.11) were identical to the literature values. 1 Pentaacetate (C-29|3) 1 H N M R ( C 6 D 6 ) 5 0.73 (d, J= 6.7 Hz, 3H), 0.75 (d, J= 7.0 Hz, 3H), 0.76 (d, J= 6.7 Hz, 3H), 0.93 (s, 3H), 1.24 (s, 3H), 1.52, 1.79, 1.85, 1.88, 1.95 (5XCH 3 CO), 2.39 (brs, 1H), 3.31 (t, J= 9.9 Hz, 1H), 5.11 (brd, J= 2.7 Hz, 1H), 5.46 (dd, J= 12.2, 9.1 Hz, 1H), 5.49 (brs, 1H), 5.60 (dd, J= 9.6, 2.1 Hz, 1H), 6.56 (dd, J= 10.7, 9.1 Hz, 1H), Pentaacetate (C-29a) 1 H N M R ( C 6 D 6 ) 8 0.72 (d, J= 7.0 Hz, 3H), 0.74 (d, J= 7.8 Hz, 3H), 0.77 (d, J= 6.9 Hz, 3H), 0.95 (s, 3H), 1.13 (s, 3H), 1.51, 1.78, 1.81, 1.91, 1.95 (5XCH3CO), 2.63 (brs, 1H), 3.77 (t, J= 8.7 Hz, 1H), 5.12 (brd, J= 2.7 Hz, 1H), 5.48 (m, 2H), 6.24 (brd, J= 2.3 Hz, 1H), 6.54 (dd, J= 10.4, 9.0 Hz, 1H) Chapter 4: Absolute Configuration of Contignasterol. 215 Preparation of Contignasterol Tetraacetate (4.12) OH 4.12 T he pentaacetates (4.11) of contignasterol (either C-29 or C-29) (21.2 mg, 0.0295 mmol) were dissolved in 0.8 mL of M e C N containing 7.3 u,L of H 2 0 and treated with B F 3 - O E t 2 (6 ul_, 0.048 mmol) at 0 C for 1.5 h. T h e reaction was quenched with saturated N a H C 0 3 (1 mL) and extracted with E t O A c (3 * 10 mL), and the E t O A c was dried with M g S 0 4 . After evaporation of the solvent, 18.4 mg (92%) of contignasterol tetraacetate (4.12) was obtained as a white solid, which was used without further purification. The 1 H N M R spectrum of 4.12 was not assigned. It is very complex because 4.12 exists as a slowly equilibrating mixture of C-29 epimers. 1 H N M R ( C 6 D 6 ) 5 0.66 (d, J= 7.9 Hz, 3H), 0.68 (d, J= 7.5 Hz, 3H), 0.72 (d, J= 6.2 Hz, 3H), 0.74 (d, J= 5.9 Hz, 3H), 0.80 (d, J= 6.7 Hz, 3H), 0.82 (d, J= 6.9 Hz, 3H), 0.93 (s, 3H), 0.95 (s, 3H), 1.11 (s, 3H), 1.14 (s, 3H), 1.51, 1.64, 1.78, 1.80, 1.81, 1.84, 1.91, 2.05 (8XCH3CO), 2.63 (t, J= 8.8 Hz, 1H), 2.92 (brs, 1H), 3.16 (brs, 1H), 3.68 (t, J= 9.5 Hz, 1H), 5.13 (m, 3H), 5.47 (m, 4H), 6.56 (t, J= 9.8 Hz, 1H), 6.69 (t, J = 9 . 4 H z , 1H) Chapter 4: Absolute Configuration of Contignasterol. 216 F A B H R M S [M + Na] + mlz 699.3715 (calcd for CarHseNaOn, 699.3720). Preparation of MTPA ester of Contignasterol Tetraacetate (4.13) and (4.14) ACO* j fi Y 0 A c OAc OAc 4.13 (R)-MTPA 4.14 (S)-MTPA (R)- or ( S ) - M T P A chloride (0.5 mL) and 1 mL of pyridine were added to 4.12, and the mixture was stirred at R T overnight. T h e pyridine was removed in vacuo, and the (R)- and ( S ) - M T P A esters were purified by normal-phase H P L C (eluent: hexane/EtOAc (7:3)). (R)-MTPA ester (4.13) 1 H N M R ( C D 2 C I 2 ) 5 0.87 (d, J= 6.7 Hz, 3H), 0.89 (d, J= 6.4 Hz, 3H), 0.99 (d, J= 7.2 Hz, 3H), 1.09 (s, 3H), 1.29 (s, 3H), 1.92, 1.92, 1.97, 2.06 ( 4 X C H 3 C O ) , 2.24 (brs, 1H), 3.57 (s, 3H), 4.81 (brs, 1H), 4.97 (brs, 1H), 5.03 (dd, J= 11.9, 9.1 Hz, 1H), 5.82 (dd, J= 9.9, 2.1 Hz, 1H), 5.96 (dd, J= 10.8, 9.3 Hz, 1H), 7.49 (m,5H) (S)-MTPA ester (4.14) 1 H N M R ( C D 2 C I 2 ) 5 0.87 (d, J= 6.7 Hz, 3H), 0.88 (d, J= 6.7 Hz, 3H), 0.97 (d, J= 7.2 Hz, 3H), 1.10 (s, 3H), 1.24 (s, 3H), 1.92, 1.93, 1.98, 2.06 ( 4 X C H 3 C O ) , 2.30 (brs, 1H), 2.53 (dd, J= 19.8, 10.1 Hz, 1H), 3.41 (s, 3H), Chapter 4: Absolute Configuration of Contignasterol. 217 4.22 (dd, J= 9.6, 2.0 Hz, 1H), 4.82 (brd, J= 2.9 Hz, 1H), 4.98 (brs, 1H), 5.03 (dd, J= 12.0, 9.2 Hz, 1H), 5.98 (dd, J= 10.8, 9.2 Hz, 1H), 7.51 (m, 5H) Preparation of Dithiane 4.15 Contignasterol tetraacetate (4.12) (10 mg in 28.7 u.L of A c O H ) was treated with a solution of 1,2-ethanedithiol (9.6 uL) and B F 3 - O E t 2 (1.2 uL), and the resulting mixture was stirred at room temperature overnight. Aqueous N a H C 0 3 (1 mL of a 1 M solution) and 7 mL of C H 2 C I 2 were added, and the organic layer was separated, evaporated in vacuo, and subjected to normal-phase H P L C (eluent: EtOAc/hexane (1:2)) to give pure dithiane 4.15 (yield 7.8 mg, 70%): 1 H N M R ( C D 2 C I 2 ) 5 0.80 (d, J= 6.9 Hz, 3H), 0.90 (d, J= 6.9 Hz, 3H), 0.94 (d, J= 6.9 Hz, 3H), 1.10 (s, 3H), 1.20 (s, 3H), 1.92 (s, 3 H , C H 3 C O ) , 1.92 (s, 3H, C H 3 C O ) , 1.98 (s, 3 H , C H 3 C O ) , 2.06 (s, 3H, C H 3 C O ) , 2.21 (dd, J= 19.6, 1.9 Hz, 1H), 2.41 (dd, J= 19.6, 10.2 Hz, 1H), 2.74 (brs, 1H), 3.22 (m, 4H), 3.49 (t, J= 8.7 AcO* OAc OAc 4.15 Chapter 4: Absolute Configuration of Contignasterol. 218 Hz, 1H), 4.57 (dd, J= 8.9, 5.7 Hz, 1H), 4.82 (brd, J= 2.8 Hz, 1H), 4.97 (brs, 1H), 5.03 (dd, J= 12.0, 9.1 Hz, 1H), 5.96 (dd, J= 10.6, 9.1 Hz, 1H) 1 3 C N M R 5 14.3, 17.6, 18.0, 20.0, 20.0, 21.0, 21.0, 21.1, 21.1, 21.4, 21.9, 29.8, 32.0, 36.4, 37.0, 37.3, 37.8, 38.4, 38.7, 39.2, 40.3, 41.1, 41.4, 42.0, 44.4, 46.5, 46.6, 51.0, 53.0, 67.3, 68.6, 70.8, 72.6, 74.8, 169.9, 170.0, 170.5, 170.9, 218.8 F A B H R M S [M + H ] + mlz 753.3701 (calcd for C39H61O10S2, 753.3706). Preparation of (/?)- and (S)-MPA Esters 4.16 and 4.17 4.16 (R)-MPA 4.17 (S)-MPA (R)- or (S) -MPA, D C C , and the dithiane 4.15 in the molar ratio of 5:7:1 were added into 1 mL of anhydrous CH2CI2 along with a few crystals of D M A P . The mixture was stirred at R T for 10 h, then filtered to remove the dicyclohexylurea, and the (R)- and (S) -MPA esters were purified by normal-phase H P L C (eluent: hexane /EtOAc (2:1)). Chapter 4: Absolute Configuration of Contignasterol. 219 (R)-MPA ester 4.16: 1 H N M R ( C D 2 C I 2 ) 5 0.77 (d, J= 6.9 Hz, 3H), 0.82 (d, J= 6.9 Hz, 3H), 0.88 (d, J= 6.9 Hz, 3H), 1.05 (s, 3H), 1.06 (s, 3H), 1.91 (s, 3 H , C H 3 C O ) , 1.92 (s, 3H, C H 3 C O ) , 1.97 (s, 3H, C H 3 C O ) , 2.06 (s, 3 H , C H 3 C O ) , 2.14 (brs, 1H), 3.25 (m, 4H), 3.45 (s, 3H), 4.54 (dd, J= 10.0, 4.9 Hz, 1H), 4.80 (brd, J= 2.7 Hz, 1H), 4.96 (brs, 1H), 5.00 (dd, J= 12.0, 9.2 Hz, 1H), 5.12 (brt, J= 8.0 Hz, 1H), 5.88 (dd, J= 10.7, 9.1 Hz, 1H), 7.39 (m, 5H) F A B H R M S [M + H] + mlz 901.4229 (calcd for C 4 8 H 6 9 0 1 2 S 2 , 901.4230). (S)-MPA ester 4.17: 1 H N M R ( C D 2 C I 2 ) 5 0.63 (d, J= 6.9 Hz, 3H), 0.65 (d, J= 7.0 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H), 1.08 (s, 3H), 1.16 (s, 3H), 1.92 (s, 3H, C H 3 C O ) , 1.94 (s, 3H, C H 3 C O ) , 1.97 (s, 3 H , C H 3 C O ) , 2.06 (s, 3 H , C H 3 C O ) , 2.24 (brs, 1H), 2.42 (dd, J= 19.7, 10.0 Hz, 1H), 3.21 (m, 4H), 3.40 (s, 3H), 4.33 (d, J= 8.7, 5.7 Hz, 1H), 4.81 (brd, J= 2.6 Hz, 1H), 4.97 (brs, 1H), 5.02 (dd, J= 11.9, 9.1 Hz, 1H), 5.08 (m, 1H), 5.92 (dd, J= 10.9, 9.3 Hz, 1H), 7.41 (m, 5H) F A B H R M S [M + H ] + mlz 901.4229 (calcd for C 4 8H 6 90 1 2 S 2 l 901.4230). Chapter 4: Absolute Configuration of Contignasterol. 220 Preparation of the (R)- and (S)-MTPA Esters 4.18 and 4.19 4 . 1 8 (R)-MTPA 4 . 1 9 (S)-MTPA (R)- or ( S ) - M T P A chloride (0.5 mL) and 1 mL of pyridine were added to the dithiane 4.15, and the mixture was stirred at R T overnight. T h e pyridine was removed in vacuo, and the (R)- and ( S ) - M T P A esters were purified by normal-phase H P L C (eluent: hexane/EtOAc (7:3)). (R)-MTPA ester 4.18: 1 H N M R ( C D 2 C I 2 ) 5 0.74 (d, J= 6.8 Hz, 3H), 0.85 (d, J= 6.8 Hz, 3H), 1.09 (d, J= 6.8 Hz, 3H), 1.10 (s, 3H), 1.21 (s, 3H), 1.92 (s, 3 H , C H 3 C O ) , 1.92 (s, 3 H , C H 3 C O ) , 1.98 (s, 3 H , C H 3 C O ) , 2.06 (s, 3 H , C H 3 C O ) , 2.24 (dd, J= 19.3, 2.3 Hz, 1H), 2.27 (brs, 1H), 2.46 (dd, J= 19.3, 9.7 Hz, 1H), 3.17 (m, 4H), 3.45 (dd, J= 3.1, 1.6 Hz, 1H), 3.55 (s, 3H), 4.33 (dd, J= 9.2, 5.4 Hz, 1H), 4.82 (brd, J= 2.8 Hz, 1H), 4.98 (brs, 1H), 5.03 (dd, J= 12.0, 9.2 Hz,1H), 5.26 (dd, J= 10.6, 2.7 Hz, 1H), 5.93 (dd, J= 10.9, 9.3 Hz, 1H), 7.50 (m, 5H) F A B H R M S [M + H] + m/z 969.4103 (calcd for C 4 9 H 6 8 F 3 0 1 2 S 2 , 969.4104). Chapter 4: Absolute Configuration of Contignasterol. 221 (S)-MTPA ester 4.19: 1 H N M R ( C D 2 C I 2 ) 5 0.76 (d, J= 6.9 Hz, 3H), 0.88 (d, J= 6.9 Hz, 3H), 0.90 (d, J= 6.9 Hz, 3H), 1.11 (s, 3H), 1.17 (s, 3H), 1.92 (s, 3 H , C H 3 C O ) , 1.95 (s, 3 H , C H 3 C O ) , 1.98 (s, 3H, C H 3 C O ) , 2.06 (s, 3 H , C H 3 C O ) , 2.21 (dd, J= 19.4, 2.3 Hz, 1H), 2.25 (brs, 1H), 2.42 (dd, J= 19.5, 9.5 Hz, 1H), 3.22 (m, 4H), 3.45 (dd, J= 3.1, 1.4 Hz, 1H), 3.57 (s, 3H), 4.43 (dd, J= 9.1, 5.3 Hz, 1H), 4.82 (brd, J= 2.8 Hz, 1H), 4.98 (brs, 1H), 5.04 (dd, J= 12.1, 9.2 Hz, 1H), 5.20 (brd, J= 10.5 Hz, 1H), 5.94 (dd, J= 10.8, 9.2 Hz, 1H), 7.48 (m, 5H) F A B H R M S [ M + + H] + m/z 969.4105 (calcd for C 4 9 H 6 8 F 3 0 1 2 S 2 , 969.4104). Chapter 4: Absolute Configuration of Contignasterol. 222 4.6 References 1. Burgoyne, D. L ; Andersen, R. J . ; Allen, I . M.J. Org. C h e m . 1992, 57, 525-528. 2. Shoji, N.; Umeyama, A.; Shin, K.; Takeda , K.; Arihara, S.; Kobayashi , J . J. Org. Chem. 1992, 57, 2996-7. 3. Kobayashi , J . ; Shinonaga, H. ; Shigemori, H . ; Umeyama, A . ; Shoji, N.; Arihara, S. J. Nat. Prod. 1995, 58, 312-318. 4. Fu , X. ; Ferreira, M . L. G . ; Schmitz, F . J . ; Kelly, M . J. Org. Chem. 1999, 64, 6706-6709. 5. Sperry, S.; Crews, P. J. Nat. Prod. 1997, 60, 29-32. 6. Keyzers, R. A . ; Northcote, P. T. ; Webb , V . J. Nat. Prod. 2002, 65, 598-600. 7. Takei , M . ; Burgoyne, D.; Andersen, R. J . J. Pharm. Sci. 1994, 83, 1234-1235. 8. Takei , M . ; Umeyama, A. ; Shoji, N.; Arihara, S.; Endo, K. Experientia 1993, 49, 145-149. 9. Bramley, A . M . ; Langlands, J . M . ; Jones, A . K.; Burgoyne, D. L ; Li, Y . ; Andersen, R. J . ; Salari, H . Br. J. Pharmacol. 1995, 7 75, 1433-1438. 10. Shen, Y . ; Burgoyne, D. L. J. Org. Chem. 2002, 67, 3908-3910. 11. Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton Coupling in Organic Stereochemistry, University Science Books, Mill Valley, C A , 1983. 12. Allinger, N. L. ; Hermann, R. B.; Djerassi, C . J. Org. Chem. 1960, 25, 922. 13. Ohtani, I.; Kusumi, T . ; Kashman, Y . ; Kakisawa, H . J. Am. Chem. Soc. 1991, 773,4092-4096. 14. Askin, D.; Angst, C ; Danishefsky, S. J. Org. Chem. 1987, 52, 622.. 15. Hatch, R .P . ; Shringapure, J . ; Weinreb, S . M . J. Org. Chem. 1978, 43, 4172. 16. Pereda-Miranda, R.; Fragoso-Serrano, M . ; Cerda-Garcia-Rojas , C . Tetrahedron. 2001, 57, 47-53. 17. Latypov, Sh . K.; Seco, J . M . ; Quinoa, E . ; Riguera, R. J. Org. Chem. 1996, 67, 8569-8577. 18. Izzo, I.; Pironti, V . ; Delia Monica, C ; Sodano, G . ; De Riccardis, F . Chapter 4: Absolute Configuration of Contignasterol. 223 Tetrahedron Lett. 2001, 42, 8977-8980. 19. Latypov, S .K.; Seco, J . M . ; Quinoa, E . ; Riguera, R. J. Am. Chem. Soc. 1998, 720, 877-882. Summary. " 4 Summary. Small molecules derived from natural sources or syntheses are invaluable tools for biochemists to probe the behavior of biological systems. Discovering new chemical tools can improve our knowledge of biology and also lead to the development of new drugs. The work presented in chapter two represents a successful example of this type of research. Our biology collaborators hypothesized that a SHIP activator could inhibit inflammatory cell activation and leukemic cell proliferation, based on the fact that SHIP deficient mice overproduce neutrophils, macrophages and osteoclasts and are hyper-responsive to inflammatory stimuli, while the loss of SHIP in humans has been implicated in allergies and various leukemias. Identification of the first known SHIP activator, pelorol (2.1), which inhibits macrophage activation in intact cells supported the intial hypothesis that an activator of SHIP might indeed be a potent anti-inflammatory compound. Our synthesis of pelorol starting from the plant natural product sclareolide confirmed its proposed structure, absolute configuration, and biological activity. In the process, several pelorol analogs were prepared. O n e of them, AQX-16A (2.63), showed significantly enhanced bioactivity. AQX-16A has been shown to have excellent in vivo activity in two mouse models of inflammation, which provides proof of principle validation of the original hypothesis. This work appears to be the first demonstration that small molecule activators of phosphatases can be found and they represent viable drug leads. Summary. Z Z O A rational drug design process always requires modification of the lead compound to identify the pharmacophore and to enhance potency and selectivity. Chapter three described an investigation of the structure activity relationship (SAR) for the pelorol pharmacophore. Since the molecular target of pelorol is chiral, it is not surprising that the individual stereoisomers of pelorol and pelalogs may have differential bioactivity. A series of C-ring stereoisomers of pelorol and its analogs have been synthesized in high yield with a Nazarov cyclization as the key step. The biological activity of these stereoisomers help us understand the relationship between the stereochemistry of pelorol and its bioactivity. It is widely recognized that the stereochemistry of an organic compound often determines its chemical, physical, biological, and pharmcological properties. The final portion of the thesis presents the determination of the absolute stereochemistry of the anti-inflammatory sponge metabolite contignasterol (4.1) by a combination of chemical degradation and spectroscopic methods. Some recent developments in the assignment of absolute configuration by N M R have found their application in this. work. Our assignment of the 22R, 24R contignasterol side chain configurations is antipodal to Izzo's result. T h e nucleus of contignasterol has the standard steroidal configuration. Solution of the absolute configuration of contignasterol cleared the way to the asymmetric total synthesis of this polyoxygenated compound. Appendix. 226 Appendix. Appendix for Chapter 2 and 3 Spectra of Compounds submitted for SHIP assay LZZ xipusddv Appendix. 228 ZHIAI00t> 16*1000 in papjooej ZPZ P yiAJN H t ev ejnBu In -o -at -en b -7.2400 OS 6.4861 In -b -T3 3 b -w bi -w b -t V 5 bi -ro b -3.8094 3.7961 2.7175 2.7072 2.7019 2.6886 h 2.6806 2.6711 2.6650 h 2.6524 2.6338 2.5874 2.5687 2.5508 2.5326 2.5181 2.4857 2.3826 2.3742 2.3666 2.3522 2.3442 2.3366 L o cn -^ - 1.2359 4-1.2169 4-1.1978 4-1.0821 \ 1.0216 L 0.8530 622 •xjpusddv Appendix. 230 Ezi-om-, I.9H/91.-, Z969 61. 4\ 9011'1-3-Jr ues'i.34-£899 t>2^ 988292 ^ = 6 0 9 0 £ £ ^ i.08e"ee-\ 6680"Z£^ 8 9 8 2 " 6 £ ^ -W9l'0fr — 6289217 — v692 Lv — I 3 4 I 1 -o : co - o -o in Z2fr6'gg 2860'Z9-81-9E09-8K)£>9 8 0 8 9 ' 9 Z - = oooo'zz-T eeiezz-/ -o oo -o : cn h-8 96Z0I4I. eeezeei 291-898I. 0 9 9 9 £ H | . 2£8>H-9£fr£09l. SO t o C M . O C O . O t o in _o CD Figure A.4 1 3 C NMR of 2.42 recorded in CDCI 3 at 100MHz ZHIAIOOtMeeioao in pepjooej ZQ"3 jo yiAIN H t 9V ejn6id co b" o r b b" cn 7.2400 -6.4731 T3 •a 3 Ol b" 4^  4^  b" — 4.8507 — 4.7339 CO CO b ro ho b 2.6262 2.6079 2.5900 1-2.5744 h 2.5550 fr 2.5390 -2.5188 2.5002 -2.4816 1^2.4606 -2.4271 2.3929 2.3632 -2.3411 2.3327 1.5575 o cn-1.1838 1.1651 1.1461 1.0624 1.0159 0.8485 •xjpusddv Appendix. 232 Q9H"9 l . -8CZ961.-9014'1-S-Z9W \Z -nswz-WSVv-Z -8090X8-QSZCCS-0Z90Z8 -9££ZQZ-8SH"0fr-QWVZb 3280'8t'-3 ZZ90ZQ-fr8ZQ>9-§1 Z0899Z OOOO'ZZ £6l-eZZ I 881-S-83I. ZZ6808I. Z898Z81. 08QCI.H 8 9 1 8 m Figure A.6 1 3 C NMR of 2.57 recorded in CDCI 3 at 100MHz ZHIAIOOt/ jee|OCO w papjooaj 1.9-3 jo yiAIN H t Z V ejnB|j 00 cn -CO b cn" b " P cn -b " cn cn" 7.2400 r 6.6227 r 6.6200 -^6.5215 -^6.5173 cn b • "o cn" •a b " co cn co b " ro cn -ro b • o cn -i l 2 2. 2. 2 2 2, h2, Pr2. i Fj 0 0 0 8029 7984 8096 7955 7731 7594 5592 5478 5230 5116 2547 8448 8368 8289 8136 8056 7980 7763 7748 7740 7687 7440 7432 7417 5407 2660 8835 8336 7822 •xipuaddy Appendix. 234 ZHIAIOOt/ \<ee\QQO in pepjooej Z9Z jo yiAIN H,. 6V ejnBy cn b b c n b 4* c n •a •a 3 *~ o CO c n CO b in b o c n 7.2400 -6.4134 3.7873 2.6958 2.6749 2.6593 2.5253 -2.4922 -2.4561 2.3373 -2.3297 -2.3149 2.3080 -2.2444 -1.0589 -1.0197 0.8481 •Xjpueddv Appendix. 236 •xjpuaddv Appendix. 238 ZHIAJOOfr ie£ |oao in ps-pjcoaj 61/9 P cdlAJN H t Ct'V ejnBij 00 b cn b P 1 cn cn 1 o •a T3 3 Ol Ol b Ol b co cn co b Ol b 7.2400 6.4248 o Ol -4.7441 -4.6794 -2.8172 2.7963 2.7788 2.7582 2.6722 •2.6418 2.6037 2.2403 1.5906 1.2237 0.8538 0.8386 6£Z •xipuaddv Appendix. 240 899881.-ZSLVQl-01^ 26 12-IZQVbZ-9886'92-66W63-2686 28-06LY2Z -9819"9£-898Z98-88l.e'8e-09682*-3 9999 Zfr-9886 Zfr-801-9 29-8089'9Z-=; 0000'ZZ-f J.U2LI-1 3 2 K 8 9 U 9866>2L 2991.Z2I-8809Z81. 1.6Z80H ZQZZ'm-Figure A.14 l d C NMR of 3.19 recorded in CDCI 3 at 100MHz ZHlAI00t> \v£\OQO u| papjooaj QZ'ZP yiAIN H L g ^ V ^ n B u oo b b b cn b T3 b CO b b o b 7.2400 6.3955 4.9066 4.7841 2.8427 2.8229 2.8016 2.7822 2.6406 2.5999 2.5071 2.4926 l r 2.4793 2.4717 2.4580 2.4450 2.2330 1.5232 1.5153 1.5076 1.4898 1.4814 1.4734 V- 1.4563 J-1.4483 l L 1.4403 1.1529 0.9885 0.9771 0.9604 0.9497 0.8591 0.7758 0.4386 •xjpusddv Appendix. 242 ZHIAI00t7 ;B£|oao in pap jooe j gze jo y|AJN H L Ll\f ejnBn 00 b " b cn cn b cn b " cn b " cn -7.2400 6.6497 6.6303 6.4659 6.4461 4.9321 -4.8187 CO cn -co b -ro cn -CO b " o cn _ r2.6197 -2.6037 -2.5839 2.5683 2.5173 |-2.4861 ' - 2.4511 2.0869 2.0800 2.0656 2.0583 2.0511 (-1.2039 |-1.1921 1.1781 11.1689 r 1.1579 |r-1.1350 1.1255 1.0232 1.0045 0.9657 0.9596 0.9394 0.9303 0.8530 •xipueddv Appendix. 244 eZ02'9l.-, 69££'8l-4 Z9LV614-09ze-ez - v -ULS'tZ — e903-££^ ei.zree4_ z8ze\ze-^= 0661017 — Limzt— 6 W 9 ? — 9809'Z9-l-068>9 Z089"9Z-= OOOO'ZZ -f S619'ZZ-J snvzu-8893CU 0980'82l-Luzon-I909"6frl • Figure A.18 1 3 C NMR of 3.25 recorded in CDCI 3 at 100MHz ZHIAJ0017 ie e iOQQ I N pepjcoai |,e-e]o y|/\|N H, 61. V 0 J n 6 | j T 3 3 oo b o i l b 0 5 cn b cn cn-cn b cn b co cn co b cn b o cn 7.2400 6.6764 6.6562 6.5135 6.4937 -4.9854 -4.8929 2.8256 2.8051 2.7872 2.7666 2.7491 2.7206 2.7114 2.6825 1.8661 1.8452 1.8403 1.8186 1.5750 1.5415 1.4228 1.1754 0.8549 0.8424 gt/j -xipuaddv Appendix. 246 •xjpuaddv Appendix. 248 986881. ^32^61. 9899Z2 Lf96"62 809088 66^988 082688 1.200 Z8-Z992l-t'-ZVZVZv-frZ8829 -5 - o CNI - O CO - o o m 0869'29-1 -o C O - o 1 ^ 9L89 9 Z - p 0000'ZZ-f 981-8'ZZ -I - o CO I I -a - o 0 > . o o 890I-2U 0Z6SCH-086L-62I. m9 6zi 9299'I-K 890L9H f "1 t .o CN . O CO .o . o C D Figure A.22 1 3 C NMR of 3.32 recorded in CDCI 3 at 100MHz 

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