University of Wollongong Thesis Collections University of Wollongong Thesis Collection
University of Wollongong Year
The structure of Uniflorines A and B and the total synthesis of Casuarine, Australine and their epimers
Thunwadee Ritthiwigrom University of Wollongong
Ritthiwigrom, Thunwadee, The structure of Uniflorines A and B and the total synthesis of Casuarine, Australine and their epimers, Doctor of Philosophy thesis, School of Chemistry, Faculty of Science, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3134
This paper is posted at Research Online.
The Structure of Uniflorines A and B and The Total Synthesis of Casuarine, Australine and their epimers
A thesis submitted in fulfilment of the requirements for the award of the degree of
Doctor of Philosophy from University of Wollongong
Thunwadee Ritthiwigrom MSc
School of Chemistry
February, 2010
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FOR MUM AND DAD
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DECLARATION
I, Thunwadee Ritthiwigrom, declare that this thesis, submitted in fulfilment of the requirements for the award of Doctor of Philosophy, in the Department of Chemistry, University of Wollongong, is wholly my own work unless due reference is provided. This document has not been submitted for qualifications at any other academic institution.
Thunwadee Ritthiwigrom
February, 2010
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TABLE OF CONTENTS
DECLARATION...... II
TABLE OF CONTENTS...... III
LIST OF FIGURES ...... VII
LIST OF SCHEMES...... X
LIST OF TABLES ...... XIII
LIST OF APPENDICES...... XIV
LIST OF ABBREVIATIONS...... XV
ABSTRACT...... XVII
ACKNOWLEGEMENTS...... XIX
PUBLICATIONS ARISING FROM THIS THESIS ...... XX
CHAPTER 1 INTRODUCTION ...... 1
1.1 DEFINITION AND CLASSIFICATION OF POLYHYDROXYLATED ALKALOIDS...... 1 1.2 DISTRIBUTION AND GLYCOSIDASE INHIBITION ...... 2 1.2.1 Pyrrolidine Alkaloids...... 8 1.2.2 Piperidine Alkaloids ...... 8 1.2.3 Pyrrolizidine Alkaloids ...... 9 1.2.4 Indolizidine Alkaloids ...... 11 1.2.5 Nortropane Alkaloids...... 12 1.3 POLYHYDROXYLATED ALKALOIDS AS GLYCOSIDE INHIBITORS...... 13 1.4 THERAPEUTIC APPLICATION...... 14 1.4.1 Anti-diabetic agents...... 14 1.4.2 Anti-cancer agents...... 15 1.4.3 Anti-viral agents ...... 16 1.5 AIMS OF PROJECT ...... 17 1.5.1 Determination of the correct structures of uniflorines A and B...... 18 1.5.1.1 Isolation and biological activities of uniflorines A and B...... 18 1.5.1.2 Previous syntheses of the proposed uniflorine A structure ...... 19 1.5.2 The development of a new synthetic strategy to prepare polyhydroxylated pyrrolizidines in an efficient and flexible manner...... 27 1.5.3 Testing of the synthesized compounds as glycosidase inhibitors...... 28
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CHAPTER 2 DETERMINATION OF THE CORRECT STRUCTURE OF UNIFLORINES A AND B ...... 29
2.1 SYNTHESIS OF 76, THE C-2 EPIMER OF THE PROPOSED STRUCTURE OF UNIFLORINE A 32 29 2.1.1 The Petasis reaction ...... 30 2.1.2 N-Boc and O-Tr protection...... 31 2.1.3 Ring Closing Metathesis (RCM)...... 32 2.1.4 O-Bn protection...... 33 2.1.5 Syn-dihydroxylation (DH) ...... 35 2.1.6 Formation of the cyclic sulfate and ring-opening...... 36 2.1.7 Debenzoylation...... 39 2.1.8 N-Boc and O-Tr deprotection...... 40 2.1.9 Mitsunobu cyclization...... 41 2.1.10 Debenzylation...... 42 2.2 SYNTHESIS OF (+)-UNIFLORINE A (ENT-6-EPI-CASUARINE)...... 52 2.2.1 Petasis reaction and N-Boc protection...... 52 2.2.2 Trans-acetalation ...... 53 2.2.3 Ring Closing Metathesis (RCM)...... 54 2.2.4 Dihydroxylation (DH) ...... 55 2.2.5 O-Benzylation...... 55 2.2.6 Acetonide and N-Boc deprotection and O-TBS protection...... 56 2.2.7 Mitsunobu cyclization...... 57 2.2.8 O-TBS and O-Bn deprotection ...... 58 2.3 SYNTHESIS OF THE CORRECT STRUCTURE OF UNIFLORINE A (78)...... 63
CHAPTER 3 SYNTHESIS OF CASUARINE ...... 70
3.1 ISOLATION AND BIOLOGICAL ACTIVITIES OF CASUARINE...... 70 3.2 PREVIOUS SYNTHESES OF CASUARINE ...... 74 3.3 TOTAL SYNTHESIS OF CASUARINE 15...... 79 3.3.1 The synthesis of the common precursor 103...... 81 3.3.2 Benzylation and Acetonide and N-Boc deprotection ...... 82 3.3.3 O-TBS and N-Fmoc protection...... 83 3.3.4 Epoxidation ...... 84 3.3.5 Cyclization of 162...... 85 3.3.5.1 N-Fmoc deprotection and a Mitsunobu cyclization reaction ...... 85 3.3.5.2 O-Mesylation and N-Fmoc deprotection ...... 86
3.3.6 Epoxide ring-opening by NaHSO4...... 88 3.3.7 Debenzylation...... 89
CHAPTER 4 SYNTHESIS OF AUSTRALINE, 7-EPI-AUSTRALINE AND 1-EPI- CASTANOSPERMINE...... 93
4.1 ISOLATION AND BIOLOGICAL ACTIVITIES OF AUSTRALINE ...... 93
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4.2 PREVIOUS SYNTHESES OF AUSTRALINE...... 95 4.3 THE SYNTHESIS OF AUSTRALINE 13 FROM PRECURSOR 163 ...... 105
4.3.1 Reduction with lithium aluminium hydride (LiAlH4) ...... 105 4.3.2 Inversion of the hydroxyl group at C-7 position...... 106 4.3.3 Hydrogenolysis ...... 107 4.3.3.1 Synthesis of australine 13 from hydrogenolysis of 241 ...... 107 4.3.3.2 Synthesis of 7-epi-australine 86 from hydrogenolysis of the mixture of 237 and 238.... 110 4.3.3.3 Synthesis of 1-epi-castanospermine 242 from hydrogenolysis of 239...... 112
CHAPTER 5 SYNTHESIS OF 3-EPI-CASUARINE, 1,2,6-TRIHYDRO-XYL-7,8- EPOXYINDOLIZIDINE AND 1,2,7-TRIHYDROXYL-6,8-EPOXYINDOLIZIDINE...... 115
5.1 ISOLATION AND BIOLOGICAL ACTIVITIES OF 3-EPI-CASUARINE ...... 115 5.2 PREVIOUS SYNTHESES OF 3-EPI-CASUARINE...... 115 5.3 THE SYNTHESIS OF 3-EPI-CASUARINE FROM PRECURSOR 161 ...... 117 5.3.1 Mitsunobu reaction for inversion of the hydroxyl group and cyclization...... 117
5.3.2 Epoxide ring-opening by NaHSO4 ...... 119 5.3.3 Hydrogenolysis ...... 123 5.3.3.1 Synthesis of 3-epi-casuarine 79 from hydrogenolysis of 261 ...... 123 5.3.3.2 Synthesis of tricyclic derivative 265 from hydrogenolysis of 262...... 126 5.3.3.3 Synthesis of tricyclic derivative 266 from hydrogenolysis of 264...... 127 5.3.3.4 Attempts to improve the yield of 3-epi-casuarine 79 ...... 129
CHAPTER 6 SYNTHESIS OF 3-EPI-AUSTRALINE, 3,7-DIEPI-AUSTRALINE, 7- DEOXY-3,6-DIEPI-CASUARINE AND 1,6-DIEPI-CASTANOSPERMINE ...... 132
6.1 ISOLATION AND BIOLOGICAL ACTIVITIES OF 3-EPI-AUSTRALINE...... 132 6.2 PREVIOUS SYNTHESES OF 3-EPI-AUSTRALINE ...... 132 6.3 THE SYNTHESIS OF 3-EPI-AUSTRALINE FROM PRECURSOR 259 ...... 134
6.3.1 Reductive ring-opening of pyrrolizidine 259 with LiAlH4 ...... 134 6.3.2 Mitsunobu reaction for inversion of the hydroxyl group ...... 134 6.3.3 Hydrogenolysis ...... 135 6.3.3.1 Synthesis of 3-epi-australine 80 from hydrogenolysis of 280...... 135 6.3.3.2 Synthesis of 3,7-diepi-australine 281 from hydrogenolysis of 278...... 137 6.3.3.3 Synthesis of 7-deoxy-3,6-diepi-casuarine 282 from 279 ...... 140 6.3.3.4 Synthesis of 1,6-diepi-castanospermine 284...... 142
6.3.3.4.1 Reductive ring-opening of 260 with LiAlH4 ...... 142 6.3.3.4.2 Hydrogenolysis of 283...... 142
CHAPTER 7 GLYCOSIDASE INHIBITOR TESTING...... 146
CHAPTER 8 CONCLUSIONS...... 149
CHAPTER 9 EXPERIMENTAL...... 154
9.1 GENERAL EXPERIMENTAL ...... 154
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9.1.1 General reaction conditions ...... 154 9.1.2 Chromatography...... 154 9.1.3 Melting points...... 155 9.1.4 Polarimetry...... 155 9.1.5 Mass spectrometry...... 155 9.1.6 Nuclear magnetic resonance spectroscopy...... 155 9.2 CHAPTER 2 EXPERIMENTAL ...... 156 9.2.1 Total synthesis of C-2-epimer of proposed uniflorine A...... 156 9.2.2 Total synthesis of (+) and (-) uniflorine A (6-epi-casuarine) ...... 169 9.2.2.1 Total synthesis of (+) uniflorine A from D-xylose ...... 169 9.2.2.2 Total synthesis of (-) uniflorine A from L-xylose ...... 179 9.3 CHAPTER 3 EXPERIMENTAL FOR THE TOTAL SYNTHESIS OF CASUARINE...... 183 9.4 CHAPTER 4 EXPERIMENTAL FOR THE TOTAL SYNTHESIS OF AUSTRALINE, 7-EPI- AUSTRALINE AND 1-EPI-CASTANOSPERMINE ...... 193 9.5 CHAPTER 5 EXPERIMENTAL ...... 200 9.5.1 Total synthesis of 3-epi-casuarine and its derivatives...... 200 9.5.2 Attempts to improve the yield of 3-epi-casuarine 79 ...... 209 9.6 CHAPTER 6 EXPERIMENTAL FOR THE TOTAL SYNTHESIS OF 3-EPI-AUSTRALINE AND ITS DERIVATIVES ...... 216
REFERENCES...... 225
APPENDIX...... 237
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LIST OF FIGURES
FIGURE 1.1 NORJIRIMYCIN 1, THE FIRST POLYHYDROXYLATED ALKALOID ISOLATED...... 1 87 FIGURE 1.2 DMDP 3 FROM THE LEAVES OF DERRIS ELLIPTICA...... 8 88,89 FIGURE 1.3 FAGOMINE 11 FROM THE SEEDS OF FAGOPYRUM ESCULENTUM...... 9 FIGURE 1.4 THE PYRROLIZIDINE STRUCTURES OF AUSTRALINE 13 AND CASUARINE 15...... 9 FIGURE 1.5 HYACINTHACINE FROM FIVE SPECIES OF THE HYACINTHACEAE FAMILY; ...... 10
FIGURE 1.6 THE STRUCTURES OF HYACINTHACINES B3 17 AND B7 18...... 10 100 FIGURE 1.7 SWAINSONINE 21 FROM THE LEAVES OF SWAINSONA CANESCENS...... 11
FIGURE 1.8 CASTANOSPERMUM AUSTRALE (LEGUMINOSAE) IN AUSTRALIA, A SOURCE OF 101 CASTANOSPERMINE 22 AND FAGOMINE 11...... 11
FIGURE 1.9 SCOPOLIA JAPONICA (ROOTS) AND DUBOISIA LEICHHARDTII (LEAVES) FROM SOLANACEAE 102,103 FAMILY...... 12
FIGURE 1.10 COMPARISON OF DMDP 3 WITH THE OXONIUMION TRANSITION STATE (TAKEN FROM
104 WRODNIGG)...... 13
FIGURE 1.11 THE STRUCTURES OF SOME GLYCOSIDASE ENZYME INHIBITORS HAVE MADE IT TO MARKET AS ANTIDIABETICS...... 15 141 FIGURE 1.12 EUGENIA UNIFLORA...... 18 FIGURE 1.13 THE PROPOSED ALKALOID STRUCTURES 32 AND 33 AND THE ALKALOID 34 ISOLATED
FROM A WATER-SOLUBLE EXTRACT OF EUGENIA UNIFLORA...... 19 FIGURE 1.14 THE PROPOSED STRUCTURE OF UNIFLORINE A 32 AND THE STRUCTURE OF
CASTANOSPERMIME 22...... 21 FIGURE 1.15 STRUCTURE OF THE C-1 EPIMER 45 AND THE C-1 AND C-2 DIEPIMER 46 OF STRUCTURE 32...... 21 FIGURE 1.16 PROPOSED UNIFLORINE A STRUCTURE AND ITS EPIMERS...... 23 FIGURE 1.17 SUMMARY OF SYNTHESIZED EPIMERS OF UNIFLORINE A...... 27 FIGURE 1.18 THE 3-HYDROXYMETHYLPYRROLIZIDINE RING SYSTEM...... 27 FIGURE 2.1 TLC ANALYSIS OF BENZYLATION REACTION OF 39...... 34 FIGURE 2.2 SIMPLIFIED STRUCTURE OF 81 FROM PC SPARTAN (6-31 G*)...... 36 1 FIGURE 2.3 THE H NMR (500 MHZ, CDCL3) SPECTRUM OF 91...... 42 1 FIGURE 2.4 THE H NMR (500 MHZ, D2O) SPECTRUM OF 76...... 43 13 FIGURE 2.5 THE C NMR (125 MHZ, D2O) SPECTRUM OF 76...... 43
FIGURE 2.6 J1,2 AND J1,8A VALUES OF 32, 76, 45 AND 46...... 44 FIGURE 2.7 THE TRANSPOSED ATOM NUMBERING FROM INDOLIZIDINE 33 TO THAT OF PYRROLIZIDINE
CASUARINE 15...... 47 FIGURE 2.8 OUR PROPOSED STRUCTURE FOR UNIFLORINE A...... 50 FIGURE 2.9 REVISED STRUCTURES FOR UNIFLORINES A AND B...... 51 13 FIGURE 2.10 THE C NMR CHEMICAL SHIFTS OF DIOXOLANE AND DIOXANE DERIVATIVES...... 54 FIGURE 2.11 COSY AND HMBC CORRELATIONS OF 94 AND 94A...... 54 1 FIGURE 2.12 THE H NMR (500 MHZ, D2O) OF ENT-78...... 59
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13 FIGURE 2.13 THE C NMR (125 MHZ, D2O) OF ENT-78...... 59 FIGURE 2.14 THE ROESY CORRELATIONS OF (+)-UNIFLORINE A 78...... 60 163 FIGURE 3.1 CASUARINA EQUISETIFOLIA...... 70
FIGURE 3.2 THE STRUCTURES OF CASUARINE 15 AND CASUARINE 6-O-α-D-GLUCOSIDE 15A...... 71 164-166 FIGURE 3.3 EUGENIA JAMBOLANA...... 72 167-169 FIGURE 3.4 MYRTUS COMMUNIS...... 73 FIGURE 3.5 THE STRUCTURE OF CASUARINE 15 AND 3-EPI-CASUARINE 79...... 73 1 FIGURE 3.6 THE H NMR (500 MHZ, CDCL3) SPECTRA OF 157 AND 158...... 83
FIGURE 3.7 STRUCTURE OF C28H39NO4SI 163 WITH LABELLING OF SELECTED ATOMS...... 87 1 FIGURE 3.8 THE H NMR (500 MHZ) IN D2O OF (+)-CASUARINE 15...... 90 13 FIGURE 3.9 THE C NMR (125 MHZ) IN D2O OF (+)-CASUARINE 15...... 90 178,179 FIGURE 4.1 CASTANOSPERMUM AUSTRALE...... 93 FIGURE 4.2 THE STRUCTURE OF AUSTRALINE 13, CASTANOSPERMINE 22, 6-EPICASTANOSPERMINE 23 54 AND FAGOMINE 11...... 94 1 FIGURE 4.3 THE H NMR (500 MHZ, CDCL3) SPECTRA OF 237 AND 241...... 107 1 FIGURE 4.4 THE H NMR (500 MHZ, D2O) OF (+) AUSTRALINE 13...... 108 13 FIGURE 4.5 THE C NMR (125 MHZ, D2O) OF (+) AUSTRALINE 13...... 108 1 FIGURE 4.6 THE H NMR (500 MHZ, D2O) OF (-) 7-EPI-AUSTRALINE 86...... 110 13 FIGURE 4.7 THE C NMR (125 MHZ, D2O) OF (-) 7-EPI-AUSTRALINE 86...... 110 1 FIGURE 4.8 THE H NMR (500 MHZ, D2O) OF (+) 1-EPI-CASTANOSPERMINE 242...... 112 13 FIGURE 4.9 THE C NMR (125 MHZ, D2O) OF (+) 1-EPI-CASTANOSPERMINE 242...... 112 1 FIGURE 5.1 THE H NMR (500 MHZ, CDCL3) SPECTRA OF 258(A) AND 162(B)...... 118 FIGURE 5.2 THE NOE CORRELATIONS OF 261...... 120 FIGURE 5.3 STRUCTURES OF 262 AND 264 FROM PC SPARTAN (AM1)...... 121 1 FIGURE 5.4 THE H NMR (500 MHZ, CDCL3) OF THE TRICYCLIC 262...... 122 1 FIGURE 5.5 THE H NMR (500 MHZ, CDCL3) OF THE TRICYCLIC 264...... 122 1 FIGURE 5.6 THE H NMR (500 MHZ, CDCL3) OF THE STARTING 259 AND 263...... 123 1 FIGURE 5.7 THE H NMR (500 MHZ, D2O) OF 3-EPI-CASUARINE 79...... 124 13 FIGURE 5.8 THE C NMR (125 MHZ, D2O) OF 3-EPI-CASUARINE 79...... 124 1 FIGURE 5.9 THE H NMR (500 MHZ, D2O) OF TRICYCLIC DERIVATIVE 265...... 126 13 FIGURE 5.10 THE C NMR (125 MHZ, D2O) OF TRICYCLIC DERIVATIVE 265...... 126 1 FIGURE 5.11 THE H NMR (500 MHZ, D2O) OF TRICYCLIC DERIVATIVE 266...... 128 13 FIGURE 5.12 THE C NMR (125 MHZ, D2O) OF TRICYCLIC DERIVATIVE 266...... 128 FIGURE 6.1 THE STRUCTURE OF 3-EPI-AUSTRALINE 80...... 132 1 FIGURE 6.2 THE H NMR (500 MHZ, D2O) OF (-)-3-EPI-AUSTRALINE 80...... 135 13 FIGURE 6.3 THE C NMR (125 MHZ, D2O) OF (-)-3-EPI-AUSTRALINE 80...... 136 1 FIGURE 6.4 THE H NMR (500 MHZ, D2O) OF (-)-3,7-DIEPI-AUSTRALINE 281...... 137 13 FIGURE 6.5 THE C NMR (125 MHZ, D2O) OF (-)-3,7-DIEPI-AUSTRALINE 281...... 138 1 FIGURE 6.6 THE H NMR (500 MHZ, D2O) OF (-)-3,7-DIEPI-AUSTRALINE⋅HCL [281⋅HCL]...... 138 13 FIGURE 6.7 THE C NMR (125 MHZ, D2O) OF (-)-3,7-DIEPI-AUSTRALINE⋅HCL [281⋅HCL]...... 138
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1 FIGURE 6.8 THE H NMR (500 MHZ, D2O) OF (+)-7-DEOXY-3,6-DIEPI-CASUARINE 282...... 140 13 FIGURE 6.9 THE C NMR (125 MHZ, D2O) OF (+)-7-DEOXY-3,6-DIEPI-CASUARINE 282...... 141 FIGURE 6.10 THE NOE CORRELATIONS OF 282...... 141 1 FIGURE 6.11 THE H NMR (500 MHZ, D2O) OF (-)-1,6-DIEPI-CASTANOSPERMINE 284...... 143 13 FIGURE 6.12 THE C NMR (125 MHZ, D2O) OF (-)-1,6-DIEPI-CASTANOSPERMINE 284...... 143
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LIST OF SCHEMES
SCHEME 1.1 TOTAL SYNTHESIS OF THE PROPOSED STRUCTURE OF UNIFLORINE A 32...... 20 SCHEME 1.2 TOTAL SYNTHESIS OF THE C-1 EPIMER 45 AND THE C-1 AND C-2 DIEPIMER 46 OF
STRUCTURE 32...... 22 SCHEME 1.3 TOTAL SYNTHESIS OF STRUCTURE 32...... 24 SCHEME 1.4 THE SYNTHESIS OF 56 AND 57...... 25 SCHEME 1.5 TOTAL SYNTHESIS OF THE C-1 AND C-2 DIEPIMER 46 OF STRUCTURE 32...... 26 SCHEME 1.6 THE TARGET NATURAL PRODUCTS DESCRIBED IN THIS THESIS...... 28 SCHEME 2.1 RETRO-SYNTHESIS OF 76, THE C-2 EPIMER OF THE STRUCTURE 32...... 29 SCHEME 2.2 SYNTHESIS OF AMINO TETROL 35...... 30 SCHEME 2.3 PROPOSED REACTION MECHANISM OF THE PETASIS REACTION...... 31 SCHEME 2.4 SYNTHESIS OF DIENE 37...... 32 SCHEME 2.5 THE PROPOSED MECHANISM OF THE RCM REACTION...... 33 SCHEME 2.6 SYNTHESIS OF DIHYDROPYRROLE 39...... 33 SCHEME 2.7 PROPOSED MECHANISM FOR THE FORMATION OF STRUCTURES 69, 70 AND 81...... 34 SCHEME 2.8 SYNTHESIS OF DIOL 82...... 35 SCHEME 2.9 PROPOSED REACTION MECHANISM OF THE SYN-DIHYDROXYLATION REACTION...... 36 SCHEME 2.10 SYNTHESIS OF BENZOATE 85...... 37 SCHEME 2.11 SYNTHESIS OF STRUCTURE 87A...... 37 SCHEME 2.12 SYNTHESIS OF STRUCTURE 87...... 38 SCHEME 2.13 MECHANISM OF FORMATION OF THE CYCLIC SULFATE 87A AND ITS RING-OPENING ...... 39 SCHEME 2.14 SYNTHESIS OF DIOL 89...... 39 SCHEME 2.15 PROPOSED MECHANISM FOR FORMATION OF THE INDOLIZIDINE 43...... 40 SCHEME 2.16 SYNTHESIS OF COMPOUNDS 90 AND 91...... 40 SCHEME 2.17 SYNTHESIS OF INDOLIZIDINE 91 BY MITSUNOBU CYCLIZATION...... 41 SCHEME 2.18 PROPOSED REACTION MECHANISM OF THE MITSUNOBU REACTION OF 90...... 42 SCHEME 2.19 SYNTHESIS OF 76, THE C-2 EPIMER OF STRUCTURE 32...... 43 SCHEME 2.20 THE TOTAL SYNTHESIS OF COMPOUND 76...... 45 158 SCHEME 2.21 PROPOSED SYNTHESIS OF (+)-UNIFLORINE A...... 52 SCHEME 2.22 SYNTHESIS OF STRUCTURES 92 AND 93...... 53 SCHEME 2.23 SYNTHESIS OF STRUCTURES 94 AND 94A...... 53 SCHEME 2.24 SYNTHESIS OF DIHYDROPYRROLE 95...... 55 SCHEME 2.25 SYNTHESIS OF TETROL 96...... 55 SCHEME 2.26 SYNTHESIS OF O-BENZYL DERIVATIVE 97...... 55 SCHEME 2.27 SYNTHESIS OF STRUCTURES 99 AND 99A...... 56 SCHEME 2.28 THE PROPOSED REACTION MECHANISM FOR THE SYNTHESIS OF 99...... 57 SCHEME 2.29 SYNTHESIS OF BICYCLIC COMPOUNDS 100 AND 100A...... 57 SCHEME 2.30 THE PROPOSED REACTION MECHANISM FOR THE SYNTHESIS OF 100 AND 100A...... 58 SCHEME 2.31 SYNTHESIS OF (+)-UNIFLORINE A ENT-78...... 59
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SCHEME 2.32 TOTAL SYNTHESIS OF (+)-UNIFLORINE A ENT-78...... 62 SCHEME 2.33 THE SYNTHETIC ROUTE FOR (-) UNIFLORINE A...... 63
SCHEME 2.34 THE PROPOSED CYCLIZATION REACTION MECHANISM IN THE PRESENCE OF ET3N·HCL.. 66 162 SCHEME 2.35 TOTAL SYNTHESIS (-)-UNIFLORINE A BY GOTI ET AL...... 68 SCHEME 3.1 THE SYNTHESIS OF 120...... 74 170 SCHEME 3.2 THE TOTAL SYNTHESIS OF CASUARINE 15 BY DENMARK ET AL...... 75 171 SCHEME 3.3 THE TOTAL SYNTHESIS OF CASUARINE 15 BY IZQUIERDO ET AL...... 76 58 SCHEME 3.4 THE TOTAL SYNTHESIS OF CASUARINE 15 BY FLEET ET AL...... 78 172 SCHEME 3.5 THE TOTAL SYNTHESIS OF CASUARINE 15 BY GOTI ET AL...... 79 SCHEME 3.6 SIMILARITIES BETWEEN THE B RING OF 76 AND THE A RING OF 78...... 79 SCHEME 3.7 RETRO-SYNTHETIC ANALYSIS OF CASUARINE 15...... 80 SCHEME 3.8 SYNTHETIC ROUTES TO PREPARE NATURAL PYRROLIZIDINES FROM 77...... 81 SCHEME 3.9 THE SYNTHESIS OF THE COMMON CHIRAL 2,5-DIHYDROPYRROLE PRECURSOR 103...... 82 SCHEME 3.10 THE SYNTHESIS OF 157 AND 158...... 82 SCHEME 3.11 THE SYNTHESIS OF 160...... 84 SCHEME 3.12 THE SYNTHESIS OF THE EPOXIDE 161...... 84 SCHEME 3.13 PROPOSED EPOXIDATION MECHANISM OF 161...... 85 SCHEME 3.14 THE SYNTHESIS OF 163...... 85 SCHEME 3.15 THE SYNTHESIS OF THE PYRROLIZIDINE 163...... 86 SCHEME 3.16 PROPOSED REACTION MECHANISM FOR THE SYNTHESIS OF 163...... 87 SCHEME 3.17 THE SYNTHESIS OF 163...... 88 SCHEME 3.18 RING-OPENING REACTIONS OF EPOXIDE 163 VIA CONFORMATIONS A AND B...... 89 SCHEME 3.19 THE SYNTHESIS OF CASUARINE 15...... 90 SCHEME 3.20 TOTAL SYNTHESIS OF CASUARINE 15 FROM THE PRECURSOR 103...... 92 SCHEME 4.1 THE TOTAL SYNTHESIS OF AUSTRALINE 13 AND (-)-7-EPI-ALEXINE 175 BY PEARSON ET 181,182 AL...... 95 SCHEME 4.2 THE SYNTHESIS OF AUSTRALINE HYDROCHLORIDE 13 FROM CASTANOSPERMINE BY 183 FURNEAUX ET AL...... 97 184,185 SCHEME 4.3 THE TOTAL SYNTHESIS OF AUSTRALINE 13 BY WHITE ET AL...... 98 170 SCHEME 4.4 THE TOTAL SYNTHESIS OF AUSTRALINE 13 FROM DENMARK ET AL...... 99
SCHEME 4.5 SYNTHESIS OF THE TRIOL AMINAL 208 (YIELDS ARE GIVEN FOR THE SYNTHESIS OF THE ENANTIOMERIC COMPOUNDS)...... 100 186 SCHEME 4.6 THE TOTAL SYNTHESIS OF AUSTRALINE 13 BY WONG ET AL...... 101 187 SCHEME 4.7 TOTAL SYNTHESIS OF AUSTRALINE HYDROCHLORIDE 13·HCL BY TROST ET AL...... 102 188 SCHEME 4.8 THE TOTAL SYNTHESIS OF AUSTRALINE 13 BY MARCO ET AL...... 104 188 SCHEME 4.9 THE PROPOSED MECHANISM FOR THE FORMATION OF 233 FROM 232...... 104 SCHEME 4.10 SYNTHESIS OF AUSTRALINE 13 BY ROUTE 2 FROM CHAPTER 3 IN SCHEME 3.8...... 105
SCHEME 4.11 THE SYNTHESIS OF RING-OPENING OF 163/163A WITH LIALH4...... 105 SCHEME 4.12 THE SYNTHESIS OF THE C-7 INVERTED PYRROLIZIDINE 237...... 106 SCHEME 4.13 THE SYNTHESIS OF AUSTRALINE 13...... 108
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SCHEME 4.14 THE SYNTHESIS OF 7-EPI-AUSTRALINE 86...... 110 SCHEME 4.15 THE SYNTHESIS OF 1-EPI-CASTANOSPERMINE 242...... 112
SCHEME 4.16 THE SYNTHESES OF (+) AUSTRALINE 13, (-) 7-EPI-AUSTRALINE 86 AND (+) 1-EPI- CASTANOSPERMINE 242...... 114 191 SCHEME 5.1 THE TOTAL SYNTHESIS OF 3-EPI-CASUARINE 79 BY IZQUIERDO ET AL...... 116 58 SCHEME 5.2 THE TOTAL SYNTHESIS OF 3-EPI-CASUARINE 79 BY FLEET ET AL...... 117 SCHEME 5.3 THE SYNTHESIS OF PYRROLIZIDINE 259 AND INDOLIZIDINE 260...... 118 SCHEME 5.4 THE SYNTHESIS EPOXIDE RING-OPENING OF 259...... 119 SCHEME 5.5 THE PROPOSED MECHANISM OF THE UNDESIRED PRODUCT 262 AND 264...... 120 SCHEME 5.6 THE SYNTHESIS OF 3-EPI-CASUARINE 79...... 124 SCHEME 5.7 THE SYNTHESIS OF TRICYCLIC DERIVATIVE 265...... 126 SCHEME 5.8 THE SYNTHESIS OF TRICYCLIC DERIVATIVE 266...... 127 SCHEME 5.9 ATTEMPTS TO SYNTHESIZE 3-EPI-CASUARINE 79...... 129 SCHEME 5.10 THE TOTAL SYNTHESIS OF 3-EPI-CASUARINE 79...... 131 SCHEME 6.1 THE TOTAL SYNTHESIS OF 3-EPI-AUSTRALINE 80 FROM THE PRECURSOR 198...... 133
SCHEME 6.2 THE TOTAL SYNTHESIS OF 3-EPI-AUSTRALINE 80 FROM THE MIXTURE ANOMERIC STEREOISOMERS...... 133
SCHEME 6.3 REDUCTIVE RING-OPENING OF 259 WITH LIALH4...... 134 SCHEME 6.4 THE SYNTHESIS OF THE C-7 INVERTED PYRROLIZIDINE DERIVATIVE 280...... 134 SCHEME 6.5 THE SYNTHESIS OF 3-EPI-AUSTRALINE 80...... 135 SCHEME 6.6 THE SYNTHESIS OF 3,7-DIEPI-AUSTRALINE 281...... 137 SCHEME 6.7 THE SYNTHESIS OF 7-DEOXY-3,6-DIEPI-CASUARINE 282...... 140
SCHEME 6.8 REDUCTIVE RING-OPENING OF 260 WITH LIALH4...... 142 SCHEME 6.9 THE SYNTHESIS OF 1,6-DIEPI-CASTANOSPERMINE 284...... 142 SCHEME 6.10 TOTAL SYNTHESIS OF 3-EPI-AUSTRALINE 80 AND THE COMPOUNDS 281, 282 AND 284
FROM THE PRECURSOR 259...... 145 SCHEME 8.1 OUTLINE OF THE SYNTHESIS OF COMPOUND 76...... 149 SCHEME 8.2 OUTLINE OF THE SYNTHESIS OF UNIFLORINE A 78...... 149 SCHEME 8.3 SYNTHETIC ROUTES TO PREPARE NATURAL PYRROLIZIDINES FROM 103...... 150 SCHEME 8.4 OUTLINE OF THE SYNTHESIS OF CASUARINE 15...... 151 SCHEME 8.5 OUTLINE OF THE SYNTHESIS OF AUSTRALINE 13...... 151 SCHEME 8.6 OUTLINE OF THE SYNTHESIS OF 3-EPI-CASUARINE 79...... 151 SCHEME 8.7 OUTLINE OF THE SYNTHESIS OF 3-EPI-AUSTRALINE 80...... 152 SCHEME 8.8 A SUMMARY OF THE SYNTHESES OF THE NATURAL AND UNNATURAL POLYHYDROXYLATED
INDOLIZIDINES AND PYRROLIZIDINES WHICH ARE REPORTED IN THIS THESIS...... 153
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LIST OF TABLES
TABLE 1.1 STRUCTURE, DISTRIBUTION AND GLYCOSIDASE INHIBITION OF SOME POLYHYDROXY-LATED ALKALOIDS...... 2 TABLE 1.2 TYPES OF HYACINTHACINE ALKALOIDS...... 10 TABLE 1.3 TYPES OF CALYSTEGINE ALKALOIDS ...... 13
TABLE 1.4 INHIBITION OF α-GLUCOSIDASES BY UNIFLORINE A, UNIFLORINE B AND 34 ...... 19 TABLE 2.1 SYNTHESIS OF THE CYCLIC SULFATES 87A/B FROM THE DIOL 82...... 37 TABLE 2.2 SYNTHESIS OF BENZOATE 87 FROM THE DIOL 82...... 38 TABLE 2.3 THE SYNTHESIS OF INDOLIZIDINE 91...... 41
TABLE 2.4 COUPLING CONSTANTS (D2O) FOR COMPOUNDS 32, 45, 46 AND 76...... 44 1 TABLE 2.5 PHYSICAL AND H NMR SPECTRAL DATA FOR UNIFLORINE A AND 76...... 46 13 TABLE 2.6 C NMR CHEMICAL SHIFTS (PPM) FOR 76...... 47
TABLE 2.7 NMR (D2O) AND OPTICAL ROTATION DATA FOR CASUARINE 15 AND UNIFLORINE B 33. .... 48 1 56 142 TABLE 2.8 THE H NMR CHEMICAL SHIFTS (PPM, D2O) FOR CASUARINE 15 AND UNIFLORINE A. .. 49 13 TABLE 2.9 THE C NMR (D2O) CHEMICAL SHIFTS (PPM) FOR CASUARINE AND UNIFLORINE A...... 51 TABLE 2.10 THE O-TBS PROTECTION OF DIOL 98...... 56 142 158 TABLE 2.11 PHYSICAL AND SPECTRAL DATA FOR (-)-UNIFLORINE A AND ENT-78...... 61 TABLE 2.12 COMPARISON OF THE RESULTS FOR THE SYNTHESIS OF ENT-78 AND 78...... 64 TABLE 2.13 OVERALL YIELDS OF UNIFLORINE A AND ITS ENANTIOMER...... 67 TABLE 2.14 A CHRONOLOGICAL HISTORY OF THE SYNTHESIS AND DISCOVERY OF UNIFLORINE A AND RELATED COMPOUNDS...... 69 TABLE 3.1 THE MITSUNOBU CYCLIZATION OF 163...... 85 TABLE 3.2 RESULTS OF THE EPOXIDE RING-OPENING REACTIONS OF 165...... 89
56 TABLE 3.3 PHYSICAL AND SPECTRAL DATA FOR (+)-CASUARINE AND 15...... 91
54,55,60 TABLE 4.1 PHYSICAL AND SPECTRAL DATA FOR (+)-AUSTRALINE AND 13...... 109 170 TABLE 4.2 PHYSICAL AND SPECTRAL DATA FOR (-)-7-EPI-AUSTRALINE AND 86...... 111
190 TABLE 4.3 PHYSICAL AND SPECTRAL DATA FOR (+)-1-EPI-CASTANOSPERMINE AND 242...... 113
58 TABLE 5.1 PHYSICAL AND SPECTRAL DATA FOR (+)-3-EPI-CASUARINE AND 79...... 125 TABLE 5.2 THE HMBC CORRELATIONS OF TRICYCLIC 265...... 127 TABLE 5.3 THE HMBC CORRELATIONS OF TRICYCLIC 266...... 128 52 TABLE 6.1 PHYSICAL AND SPECTRAL DATA FOR (-)-3-EPI-AUSTRALINE AND 80...... 136 193 TABLE 6.2 PHYSICAL AND SPECTRAL DATA FOR (+)-1,2-DIEPI-ALEXINEHCL SALT [ENT-281] AND 281HCL SALT [3,7-DIEPI-AUSTRALINEHCL SALT]...... 139 TABLE 6.3 THE HMBC CORRELATIONS OF 7-DEOXY-3,6-DIEPI-CASUARINE 282...... 141 194 TABLE 6.4 PHYSICAL AND SPECTRAL DATA FOR (-)-1,6-DIEPI-CASTANOSPERMINE AND 284...... 144
TABLE 7.1 THE GLYCOSIDASE INHIBITION OF UNIFLORINE A 78 (MEAN % INHIBITION AT 143 µG/ML)...... 147 TABLE 7.2 THE GLYCOSIDASE INHIBITION OF SYNTHESISED COMPOUNDS (MEAN % INHIBITION AT 800
µG/ML)...... 148
xiv
1 13 TABLE 9.1 THE REFERENCES USED FOR H AND C NMR SPECTROSCOPY...... 156
LIST OF APPENDICES
1 APPENDIX 1 COMPARISON OF PUBLISHED H NMR DATA OF AUSTRALINE 13...... 238
13 APPENDIX 2 COMPARISON OF PUBLISHED C NMR DATA OF AUSTRALINE 13...... 239
xv
LIST OF ABBREVIATIONS
[α]D specific rotation Ac acetyl Ar aromatic Bn benzyl Boc tert-butyloxycarbonyl br broad Bz benzoyl
CDCl3 deutero-chloroform
CHCl3 chloroform Cy cyclohexyl d doublet (NMR) d day (s) δ chemical shift (NMR) DEAD diethylazodicarboxylate DEPT Distortionless Enhancement by Polarisation Transfer DMAP N,N-Dimethyl-4-aminopyridine DMDP 2,5 -Dihydroxymethyl-3,4-dihydroxypyrrolidine DMF dimethylformamide EI electron impact ionisation eq. equatorial ESI-MS electrospray ionisation mass spectrometry FCC flash column chromatography gCOSY gradient Correlated Spectroscopy gHSQC gradient Heteronuclear Single Quantum Correlation gHMBC gradient Heteronuclear Multiple Bond Correlation HR high resolution Hz Hertz LR low resolution MS mass spectrometry m multiplet m.p. melting point
xvi
[M+] molecular ion m/z mass/charge ratio NMR nuclear magnetic resonance NMO N-methylmorpholine-N-oxide petrol petroleum spirit bp 40-60 °C ppm parts per million py pyridine q quartet
Rf relative mobility rt room temperature s singlet t triplet TBAF tetra-n-butylammonium fluoride TBDPS tert-butyldiphenylsilyl TBS tert-butyldimethylsilyl TFA trifluoroacetic acid THF tetrahydrofuran TPAP tetrapropylammonium perruthenate Tr trityl, triphenylmethyl
xvii
ABSTRACT
The polyhydroxylated alkaloids uniflorines A and B were isolated in 2000 from the leaves of the tree Eugenia uniflora L. The common name for this tree is Surinam Cherry and the water soluble extracts of its leaves have been used as an antidiabetic agent in Paraguayan traditional medicine. Uniflorines A and B showed moderate activity in inhibiting the α-glucosidases, rat intestinal maltase (IC50 values of 12 and 4.0 µM, respectively) and sucrase (IC50 values of 3.1 and 1.8 µM, respectively). Uniflorines A and B were deduced from NMR analysis to have pentahydroxyindolizidine structures. In 2004 Davis and Pyne reported the synthesis of the proposed structure of uniflorine A. Unfortunately, the NMR spectroscopic data of this synthetic material did not match with those of natural uniflorine A. Davis and Pyne then assumed that the structure of uniflorine A must be a diastereomer of the initially proposed structure. Several other diastereomers of the proposed structure, that were epimeric in the A-ring, have been synthesised by other researchers. One remaining A-ring diastereomer was the C-2 epimer of the proposed structure. In Chapter 2 of this thesis we report the synthesis of this compound in 11 synthetic steps and in 0.5% overall yield from L-xylose. However the NMR spectroscopic data for this synthesic compound did not match with those of the natural product. We then re-examined the NMR spectroscopic data of the natural product and revised the structures of uniflorines A and B from initially proposed pentahydroxyindolizidines to 1,2,6,7-tetrahydroxy-3-hydroxymethylpyrrolizidines. Uniflorine B was the known alkaloid casuarine, while uniflorine A was tentatively assigned as 6-epi-casuarine. This was confirmed by the synthesis of the enantiomer of 6-epi-casuarine and then 6-epi-casuarine itself. These syntheses are reported in Chapters 2.2 and 2.3. The total synthesis of uniflorine A (6-epi-casuarine) was achieved in 11 steps and in 13% overall yield from L-xylose. The NMR spectroscopic data of this synthetic compound matched with those of the natural product uniflorine A. Thus we had successfully determined the correct structures of uniflorines A and B. Glycosidase inhibitor testing of uniflorine A at 143 µM showed it had 94-97% inhibition against the α-D-glucosidases of Saccharomyces cerevisiae and Bacillus sterothermophilus and the amyloglucosidase of Aspergillus niger. The IC50 values
xviii were only determinated for the two aforementioned α-D-glucosidases and were found to be modest at 34 and 28 µM, respectively. In addition, we describe a flexible method for the diastereoselective total synthesis of several natural and unnatural polyhydroxylated indolizidines and pyrrolizidines from a common precursor. The synthesis of the alkaloid casuarine, which was obtained in total of 13 synthetic steps and in 8% overall yield from L-xylose, is described in Chapter 3. A key step in this synthesis was a regioselective epoxide ring-opening reaction with hydrogensulfate ion. This reaction secured the correct configurations at C-6 and C-7 of the target molecule. In Chapter 4 we describe the successful synthesis of australine in a total of 14 steps and 6% overall yield from from L-xylose. Key steps in this synthesis were a regioselective epoxide ring-opening reaction with LiAlH4 followed by a Mitsunobu reaction that secured the correct configuration C-7 of the target molecule. The synthesis of the natural product 3-epi-casuarine was completed in 13 steps and in 0.4% overall yield. This synthesis required an inversion of configuration at C-3’ of the butyl side chain which was achieved using the Mitsunobu reaction. The low overall yield was due to a low yielding epoxide ring-opening reaction due to a competing intramolecular epoxide ring-opening reactions involving the 3-α- hydroxymethyl substituent. Natural 3-epi-australine was obtained in total of 14 synthetic steps and in 2% overall yield, all from L-xylose. This synthesis required an inversion of configuration at C-3’ of the butyl side chain which was achieved using the Mitsunobu reaction. Key steps in this synthesis were a regioselective epoxide ring-opening reaction with
LiAlH4 followed by a Mitsunobu reaction that secured the correct configuration C-7 of the target molecule. From this work a number of novel unnatural indolizidine and pyrrolizidine compounds were also obtained as side products. Some of these compounds were screened against 10 different glycosidases at 800 µg/mL. Unfortunately, none showed strong inhibition with only four compounds showing approximately 40-50% inhibition at this relative high concentration.
xix
ACKNOWLEGEMENTS
I wish to express my deepest thanks to my supervisor, Prof. Stephen G. Pyne, for his excellent supervision, endless patience, understanding and motivation during the course of my PhD. I am also grateful for the opportunity he provided me to further my study in University of Wollongong.
I would like to thank the staff members of the School of Chemistry: Wilford Lie for assistance with NMR spectroscopy, Thitima Urathamakul, Karin Maxwell, Roger Kanitz, John Korth and the late Larry Hick for the running of high-resolution mass spectra, and to Roza Dimeska for assisting with the running of IR spectra.
I would also like to give special thanks to Assoc. Prof. Duang Buddhasukh,
Prof. Vatcharin Rukachaisirikul and Sarawek family and all CMU staff; Piyarat, P Keaw, P Sunanta and Putinan, and my close friends; Boay, Noklek, Ae and Nong
Pare for their love and support, Andrew Davis, Pitchaya Mungkornasawakul, Morwenna Baird, Thitima Urathamakul, Kittiya Sompol, Ian Morgan, Christopher Au, Arife Yazici, Leena Burgess, P Mam, P Noi, P Aom (2), P Nan, P Tip, Suriya, Nong Jang and members of Pyne group for all their help, support and all the fun and fantastic times I have had with them here in Wollongong.
Many thanks also go to Chiang Mai University, the Thai Government, the University of Wollongong and P Ya and Nar Toi for the financial support during my PhD.
Last but now least, I am deeply thankful to my dearest parents, without whom I would not have come this far, for all their unconditional love and support. To my sisters and brother for all their love, understanding and taking care of Mum and Dad while I was away.
xx
PUBLICATIONS ARISING FROM THIS THESIS
1. Thunwadee Ritthiwigrom; Anthony C. Willis; Stephen G. Pyne. Total synthesis of uniflorine A, casuarine, australine, 3-epi-australine, and 3,7-diepi australine from a common precursor. J. Org. Chem. 2010, 75, 815-824.
2. Thunwadee Ritthiwigrom; Stephen G. Pyne. Synthesis of (+)-Uniflorine A: A Structural Reassignment and a Configurational Assignment. Org. Lett. 2008, 10, 2769-2771.
3. Andrew S. Davis; Thunwadee Ritthiwigrom; Stephen G. Pyne. Synthetic and spectroscopic studies on the structures of uniflorines A and B: structural revision to 1,2,6,7-tetrahydroxy-3-hydroxymethylpyrrolizidine alkaloids. Tetrahedron. 2008, 64, 4868-4879.
4. Stephen G. Pyne; Christopher W. G. Au; Andrew S. Davis; Ian R. Morgan; Thunwadee Ritthiwigrom; Arife Yazici. Exploiting the borono-Mannich reaction in bioactive alkaloid synthesis. Pure Appl. Chem. 2008, 80, 751- 762.
Chapter 1 [Introduction]
CHAPTER 1 INTRODUCTION
1.1 Definition and Classification of Polyhydroxylated Alkaloids
It is difficult to give an exact definition of an alkaloid. The term ‘alkaloid’ was coined by Meissner in 1819.1 His definition was very simple: alkaloids are plant- derived substances that react like alkalis. Winterstein and Trier defined alkaloids as compounds with heterocyclic bound nitrogen atoms, with more or less basic character, with pronounced physiological action, of complex molecular structure, which are found in plants (and animals).2 The total number of alkaloids that have been isolated from plants, animals, microorganisms, fungi or natural traditional medicines is enormous. Over 100 polyhydroxylated alkaloids alone have been isolated from plants and microorganisms.3 These alkaloids can be considered as analogues of monosaccharides or oligosaccharides in which the ring oxygen has been replaced by nitrogen. These alkaloids are therefore often refered to as azasugars. They can inhibit glycosidases by mimicking the pyranosyl and furanosyl moiety of the natural substrates, for example the first known polyhydroxylated alkaloid; norjirimycin 1, which was isolated from a Streptomyces filtrate by Inouye et al. in 1966 (Figure 1.1),4 is an azasugar analogue of glucose in the pyranose configuration. The monocyclic and bicyclic polyhydroxylated alkaloids that inhibit glycosidases are subdivided into five structural groups; pyrrolidine (resembling furanose sugars), piperidine (resembling pyranose sugars), pyrrolizidine (fused pyrrolidines with N at the bridgehead), indolizidine (fused piperidine and pyrrolidine) and nortropane.
OH
6 4 5 NH HO 1 HO 2 OH 3 OH 1
Figure 1.1 Norjirimycin 1, the first polyhydroxylated alkaloid isolated.
1 Chapter 1 [Introduction]
1.2 Distribution and glycosidase inhibition
Table 1.1 shows the structures of some of the polyhydroxylated alkaloids, azasugars, amino sugars and sugar analogues that have been isolated from microorganisms, plants and insects and their glycosidase inhibitory activities.3
Table 1.1 Structure, distribution and glycosidase inhibition of some polyhydroxy- lated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Pyrrolidines HO OH Angylocalyx spp.(Leguminosae) Potent inhibitor of α- seeds/leaves/bark5 glucosidases (including Arachniodes standishii maltase, isomaltase, N CH2OH 5,6 H (Polypodiaceae) leaves sucrase and trehalase, Morus bombycis (Moraceae) glucosidase II), α-D- 1,4-dideoxy-1,4-imino-D- 7 leaves arabinosidase, arabinitol (D-AB1) Eugenia spp. (Myrtaceae) β-glucosidases (including leaves/bark8 2 lactase and cellobiase) and Hyacinthoides non-scripta 9 α-mannosidases (Hyacinthaceae) bulb/leaves (mannosidase II and Scilla campanulata lysosomal).12-15 10 (Hyacinthaceae) bulb Adenophora triphylla var. japonica (Campanulaceae) whole plant11 HO OH Derris elliptica (Leguminosae) β-Glucosidases (including 16 leaves cellobiase) and α- Lonchocarpus spp glucosidases HOH2C N CH2OH 17 H (Leguminosae) seeds/leaves (including invertase, 2R,5R-dihydroxymethyl- Endospermum sp. sucrase, isomaltase, 3R,4R- (Euphorbiaceae) leaves18 trehalase), human dihydroxypyrrolidine Omphalea diandra lysosomal β-mannosidase, 18 (DMDP) (Euphorbiaceae) leaves β-galactosidase (including 3 Streptomyces sp. KSC-5791 lactase) 19 (Actinomycetes) and β-xylosidase15,17,24-26 Nephthytis poissoni (Araceae) fruit/leaves8,20 Aglaonema spp (Araceae) leaves 20,21 Hyacinthoides non-scripta (Hyacinthaceae) bulb/leaves9 Campanula rotundifolia (Campanulaceae) leaves18 Hyacinthus orientalis (Hyacinthaceae) bulb22 Scilla campanulata (Hyacinthaceae) bulb10 Adenophora spp. (Campanulaceae) roots23
2 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Pyrrolidines
HO OH HO OH O
OH OH HOH C HOH2C N 2 N H H broussonetinine A broussonetinine B O 4 5 Broussonetia kazinoki Potent inhibitors of β- 27-32 (Moraceae) branches galactosidase and α- mannosidase28 HO OH HO OH O
OH OH HOH C HOH2C N 2 N H H H H OH H OH H O broussonetine E broussonetine F 6 7 Broussonetia kazinoki Potent inhibitors of β- 27-32 (Moraceae) branches glucosidase, β-galactosidase and β-mannosidase and good inhibitors of α-glucosidase28 Piperidines OH Streptomyces β-Glucosidases and α- 4,33,34 HO OH roseochromogenes R-468 glucosidases (including Streptomyces lavandulae SF- sucrase, maltase, 4,33,34 425 isomaltase and amylase), β- HO N CH2OH H Streptomyces nojiriencis SF-426 galactosidases and N-acetyl- 4,33,34 nojirimycin β- D - 1 glucosaminidases6,35 OH Morus sp. (Moraceae) roots36 Potent inhibitor of α- HO OH Bacillus amyloliquefaciens, B. glucosidases (including polymyxa, B. subtilis37 trehalase, invertase, Streptomyces lavandulae subsp. sucrase, maltase, N CH2OH 38 H trehalostaticus no. 2882 isomaltase), more weakly 1-deoxynojirimycin (DNJ) Omphalea queenslandiae inhibitory to 39 8 (Euphorbiaceae) leaves glucosidase I and II, and β- Endospermum medullosum glucosidases, also α- 39 (Euphorbiaceae) leaves mannosidases, Morus bombycis (Moraceae) -fucosidase, - and - 7 α α β leaves galactosidases (including Hyacinthus orientalis lactase)15,17,24,35 (Hyacinthaceae) bulb22 Adenophora triphylla var. japonica (Campanulaceae) whole plant11
3 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Piperidines OH Omphalea diandra α-Glucosidases (including 40 HO OH (Euphorbiaceae) leaves maltase, sucrase and Endospermum medullosum trehalase), 39 (Euphorbiaceae) leaves β-glucosidases, lactase and HOH2C N CH2OH H Nephthytis poissoni (Araceae) α-galactosidases 40,41 8 α-homonojirimycin (HNJ) leaves and moderate inhibitor of 9 Aglaonema treubii (Araceae) glucosidase I and II42 21 leaves/roots Hyacinthus orientalis (Hyacinthaceae) bulb22 OH Lonchocarpus sericeus α-Mannosidases 43 HO OH (Leguminosae) seeds/leaves (mannosidase I) and α- Streptomyces lavandulae fucosidase17,34,46 44 GC-148 N CH2OH H Omphalea diandra 1-deoxymannojirimycin (Euphorbiaceae) leaves40 (DMJ) Endospermum medullosum 10 (Euphorbiaceae) leaves39 Derris malaccensis (Leguminosae)45 Angylocalyx spp. (Leguminosae) seeds/leaves/bark8 Hyacinthus orientalis (Hyacinthaceae) bulb22 Adenophora triphylla var. japonica (Campanulaceae) whole plant11 OH Fagopyrum esculentum Weak inhibitor of α- 47 OH (Polygonaceae) seeds glucosidases (including Xanthocercis zambesiaca isomaltase and sucrase)21 (Leguminosae) seeds48 N CH2OH 49 H leaves/roots fagomine Morus bombycis (Moraceae) 11 leaves7 Morus alba (Moraceae) roots50 Lycium chinense (Solanaceae) roots51 Pyrrolizidines
HO H OH Alexa spp. (Leguminosae) Disaccharidase-type α- 52 1 seeds/leaves glucosidases (trehalase, 7 7a OH amyloglucosidase) N 3 5 and β-galactosidase52,53 CH2OH alexine 12
4 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Pyrrolizidines
HO H OH Castanospermum australe Disaccharidase-type α- (Leguminosae) seeds/leaves54,55 glucosidases OH (amyloglucosidase, N sucrase, maltase) and CH2OH glucosidase I, β- 7a-epi-alexine glucosidase, β- (australine) galactosidase54,55,42 13
HO H OH Alexa spp. and Castanospermum α-Glucosidases australe (Leguminosae) (amyloglucosidase), 53 OH seeds/leaves weakly inhibitory to β- N glucosidases53 CH2OH 1,7a-diepi-alexine 14 HO H OH Casuarina equisetifolia α-Glucosidases (including (Casuarinaceae) bark56 trehalase, HO OH Eugenia jambolana (Myrtaceae) amyloglucosidase and N leaves57 glucosidase I)55,59,60
CH2OH Myrtus communis L 58 casuarine (Myrtaceae) shrub 15 H OH Muscari armeniacum Potent inhibitor of lactase, (Hyacinthaceae) bulbs61 moderate inhibitor of OH amyloglucosidase N and α-fucosidase and a poor inhibitor of α- and β- CH2OH hyacinthacine A glucosidases and 1 55 16 β-mannosidase HO H OH Muscari armeniacum Good inhibitor of lactase (Hyacinthaceae) bulbs61 and amyloglucosidase and OH poor inhibitor N of α-fucosidase55 CH OH H3C 2 hyacinthacine B3 17 62 HO H OH Scilla socialis Weak amyloglucosidase (Hyacinthaceae) bulbs62 OH N
CH OH H3C 2 hyacinthacine B7 18
5 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Pyrrolizidines
HO H OH Hyacinthoides non-scripta Moderate inhibitor of (Hyacinthaceae) fruits/stalks10 amyloglucosidase10 HO OH Muscari armeniacum N (Hyacinthaceae) bulbs61 CH OH H3C 2 hyacinthacine C1 19 H OH Good inhibitor of β- glucosidase, β-galactosidase OH 32 N and β-mannosidase H HO CH2OH OH O broussonetine N 20 Broussonetia kazinoki (Moraceae) branches32 Indolizidines OH Swainsona canescens H OH Potent inhibitor of α- (Leguminosae) leaves63 mannosidases69,70 7 8a 1 Astragalus spp. (Leguminosae) OH 64 5 N 3 leaves/stems Oxytropis spp. (Leguminosae) swainsonine 64 21 leaves/stems Rhizoctonia leguminicola (Basidiomycetes)65 Metarhizium anisopliae (Deuteromycetes)66 Ipomoea sp. aff. calobra (Convolvulaceae) seeds67 Ipomoea carnea (Convolvulaceae) leaves/stems68 OH Castanospermum australe H OH α-Glucosidases (including HO (Leguminosae) amyloglucosidase, sucrase, seeds/leaves/bark71 maltase, isomaltase, N Alexa spp. (Leguminosae) trehalase, amylase, HO seeds/leaves/bark72 castanospermine glucosidase I and II) and β- 22 glucosidases (including lactase and cellobiase), β- glucocerebrosidase and β- xylosidase25,26,73 OH Castanospermum australe Amyloglucosidase, neutral H OH 74,76 HO (Leguminosae) seeds/leaves/bark α-mannosidase 74,75 N HO 6-epi-castanospermine 23
6 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Nortropanes
HN OH Calystegia sepium Potent inhibitor of β- (Convolvulaceae) leaves/roots77,78 HO OH glucosidases, α- and β- Convolvulus arvensis galactosidases 77,79 calystegine A3 (Convolvulaceae) leaves/roots (including lactase) and 24 Atropa belladonna (Solanaceae) trehalase82 77,78,80 leaves/roots Solanum spp. (Solanaceae) OH HN tubers/leaves81 Potent inhibitor of β- OH glucosidases, HO OH Ipomoea batatus (Convolvulaceae) leaves/roots8 disaccharidase-type α- calystegine B2 Datura wrightii (Solanaceae) glucosidases 25 leaves81 (including maltase, sucrase Physalis alkekengi var. francheti and trehalase) and α- and (Solanaceae) roots82 β-galactosidase Hyoscyamus niger (Solanaceae) (including lactase)83 leaves/roots80 Mandragora offcinarum (Solanaceae) leaves/roots/fruits80 Scopolia spp. (Solanaceae) leaves/roots80,83 Ipomoea sp. aff. calobra (Convolvulaceae) seeds67 Calystegia japonica (Convolvulaceae) roots8 Lycium chinense (Solanaceae) roots50 B2 only in Morus alba (Moraceae) fruits84 Ipomoea carnea (Convolvulaceae) leaves/stems68 OH Scopolia japonica (Solanaceae) Moderate inhibitor of α- 83 HN roots galactosidases and OH Duboisia leichhardtii (Solanaceae) trehalase, but weak 85 HO OH leaves inhibitor of β-glucosidases
(including cellobiase) and calystegine B4 83 26 lactase HN OH Morus alba (Moraceae) roots50 Potent inhibitor of OH Ipomoea batatus disaccharidase-type α- HO OH 8 HO (Convolvulaceae) roots glucosidases and calystegine C1 Scopolia japonica (Solanaceae) β-glucosidases, moderate 83 27 roots inhibitor of α- and β- Duboisia leichhardtii (Solanaceae) galactosidases82,83,85 85 leaves Lycium chinense (Solanaceae) roots51 Ipomoea carnea (Convolvulaceae) leaves/stems68
7 Chapter 1 [Introduction]
Table 1.1 (cont’d.) Structure, distribution and glycosidase inhibition from some poly- hydroxylated alkaloids.
Alkaloids Source and Reference Glycosidase inhibited Nortropanes HN OH Hyoscyamus niger (Solanaceae) Weak inhibitor of OH leaves/roots86 disaccharidase-type α- H N 2 OH Lycium chinense (Solanaceae) glucosidases and potent roots51 calystegine N1 inhibitor of β-glucosidases 28 (including cellobiase), lactase and α-galactosidase86
1.2.1 Pyrrolidine Alkaloids
The pyrrolidine alkaloids are monocyclic 5 membered ring compounds which contains an N atom. An example is 2R,5R-dihydroxymethyl-3R,4R- dihydroxypyrroli-dine (DMDP) 3, which is a relatively widespread secondary metabolite. It was first isolated from the leaves of the Leguminosae, Derris elliptica (Figure 1.2).16 Later it was discovered in many unrelated species of plants and microorganisms.3 It was found to be an inhibitor of β-glucosidases (including cellobiase) and α-glucosidases (including invertase, sucrase, isomaltase, trehalase), human lysosomal β-mannosidase, β-galactosidase (including lactase) and β- xylosidase.15,17,24-26 Please see print copy for image HO OH
HOH2C N CH2OH H DMDP 3
Figure 1.2 DMDP 3 from the leaves of Derris elliptica.87
1.2.2 Piperidine Alkaloids
Piperidine alkaloids have a monocyclic 6 membered ring which contains an N atom such as fagomine 11 which was first isolated from the seeds of Fagopyrum esculentum (Polygonaceae) (Figure 1.3).47 Later it was isolated from many different
8 Chapter 1 [Introduction] species of plants.3 It was shown to be a weak inhibitor of α-glucosidases, including isomaltase and sucrase.49 Please see print copy for image
OH OH
N CH2OH H fagomine 11
Figure 1.3 Fagomine 11 from the seeds of Fagopyrum esculentum.88,89
1.2.3 Pyrrolizidine Alkaloids
Pyrrolizidine alkaloids have two fused pyrrolidine rings with an N atom at the ring junction. More than 350 pyrrolizidine alkaloids have been discovered from 6000 plant species.3,90,91 The distribution, glycosidase inhibition and the synthesis of the polyhydroxylated pyrrolizidine alkaloids australine 13, casuarine 15 and their derivatives is discussed in more detail in Chapters 3-6 (Figure 1.4). A recent group of polyhydroxylated pyrrolizidine alkaloids are the hyacinthacines. Nineteen hyacinthacine alkaloids have been isolated from five species of the Hyacinthaceae family; Hyacinthoides nonscripta, Muscari armeniacum, Scilla campanulata, Scilla sibirica and Scilla socialis (Figure 1.5). These alkaloids have been classified in to three groups as hyacinthacines A1–A7, B1–B7 and C1–C5 based on their total number of hydroxy and hydroxymethyl groups in the ring B (Table 1.2).10,55,62,92,93 Au and 94 Pyne recently reported the synthesis of hyacinthacines B3 17 and B7 18. The synthesis of the latter structure 18 indicated that the structure proposed for the natural product was incorrect (Figure 1.6).62
OH HO H HO H OH
OH HO OH N N
CH2OH CH2OH casuarine australine 13 15 Figure 1.4 The pyrrolizidine structures of australine 13 and casuarine 15.
9 Chapter 1 [Introduction] Please see print copy for image
Figure 1.5 Hyacinthacine from five species of the Hyacinthaceae family; (a) Hyacinthoides nonscripta (b) Muscari armeniacum (c) Scilla campanulata (d) Scilla sibirica (e) Scilla socialis.95-99
Table 1.2 Types of hyacinthacine alkaloids
Alkaloid Type Ring B X = H, m = 0 and n = 0 or H OH Hyacinthacine A n(HO) X = H, m = 1 and n = 0 B A OH X = H, m = 1 and n = 1 or N Hyacinthacine B CH OH X = OH, m = 1 and n = 0 X m 2 X = H, m = 0 or 1 and n = 2 or General hyacinthacine structure Hyacinthacine C X = OH, m = 1 and n = 1
HO H OH HO H OH
OH OH N N
H3C CH2OH H3C CH2OH hyacinthacine B3 hyacinthacine B7 17 18
Figure 1.6 The structures of hyacinthacines B3 17 and B7 18.
10 Chapter 1 [Introduction]
1.2.4 Indolizidine Alkaloids
Indolizidine alkaloids have a fused piperidine-pyrrolidine bicyclic ring structure with N at the ring junction. Over 170 indolizidine alkaloids are known. Swainsonine 21 was first isolated from the leaves of Swainsona canescens (Leguminosae) (Figure 1.7)63 and was also found from two species in the same family of Astragalus spp64 and Oxytropis spp.64 Furthermore it was discovered in four unrelated species of plants and micro-organisms.63-68 Swainsonine 21 was shown to be a potent inhibitor of α-mannosidases.69,70 Please see print copy for OH image H OH
OH N swainsonine 21
Figure 1.7 Swainsonine 21 from the leaves of Swainsona canescens.100
Another example of an indolizidine alkaloid is castanospermine 22 which was first isolated from Leguminosae Castanospermum australe (seeds, leaves and bark)71 and has been isolated from the dried pods of Alexa spp. (Figure 1.8).72 Castaospermine 22 was found to inhibit α-glucosidases (including amyloglucosidase, sucrase, maltase, isomaltase, trehalase, amylase, glucosidase I and II) and β-glucosidases (including lactase and cellobiase), β-glucocerebrosidase and β-xylosidase.73,25,26 Please see print copy for image OH H OH HO
N HO castanopermine 22 PleasePlease seesee printprint copy for image Figure 1.8 Castanospermum australe (Leguminosae)copy for imagein Australia, a source of castanospermine 22 and fagomine 11.101
11 Chapter 1 [Introduction]
1.2.5 Nortropane Alkaloids
The calystegine group of alkaloids include 13 different polyhydroxylated, 1- hydroxy-nortropane molecules (calystegines A-C), one trihydroxylated 1-amino- 86 51 nortropane (calystegine N1) and the N-methyl derivatives of calystegines B2 and 51 7,51,68,83,85 C1 and the glycoside derivatives of calystegines B2 and C1. The individual members of this group have shown inhibition of different glycosidases (Table 1.1).
Calystegine B4 26 for example, showed the strongest inhibitory activity against pig 83 kidney trehalase, with an IC50 of 4.8 µM and was first isolated in 1996 from the root extracts of Scopolia japonica83 and later from the leaves of Duboisia 85 leichhardtii in the same Solanaceae family (Figure 1.9). While calystegines B2 25 strongly inhibited almond β-glucosidase (IC50 = 2.6 µM), Caldocellum saccharolyticum β-glucosidase (IC50 = 2.4 µM), green coffee bean α-galactosidase
(IC50 = 1.9 µM), Aspergillus niger α-galactosidase (IC50 = 3.9 µM), rat intestinal β- galactosidase (IC50 = 7.8 µM) and porcine kidney trehalase (IC50 10.0 µM). The known calystegines have been structurally subdivided based on their number of hydroxyl or amino groups (Table 1.3).90
Please see print copy for image
OH
HN OH
HO OH
calystegine B4 26
Figure 1.9 Scopolia japonica (roots) and Duboisia leichhardtii (leaves) from Solanaceae family.102,103
12 Chapter 1 [Introduction]
Table 1.3 Types of calystegine alkaloids
Alkaloid Type Number of hydroxyl, amino and methyl groups
Calystegine A m + n = 2, R’ = OH, R = H m (HO) R N Calystegine B m + n = 3, R’ = OH, R = CH3 or H H R'
(OH)n Calystegine C m + n = 4, R’ = OH, R = CH3 or H General calystegine alkaloid
Calystegine N m + n = 3, R’ = NH2, R = H
1.3 Polyhydroxylated alkaloids as glycoside inhibitors
The particular distributions of the polyhydroxylated alkaloids in plants may be for phylogenetic reasons. These alkaloids can be released into the soil by producer plants and microorganisms from where these alkaloids can be readily taken up and accumulated in plant tissues of completely different neighbouring species. It could be the case that microorganisms closely associated with specific plants may also produce polyhydroxylated alkaloids which could be taken up by the plant.8 Many of these polyhydroxylated alkaloids are inhibitors of glycosidases.15,35 Glycosidases are enzymes that catalyse the hydrolysis of the glycosidic bonds in complex carbohydrates and glycoconjugates. As seen in Figure 1.10, DMDP 3 could inhibit a glycosidase enzyme by adopting a similar conformation to that of the furanosyl moiety of the natural substrate. Because DMDP would be protonated at physiological pH it can mimic the charge and conformation of the oxonium ion intermediate formed in the active site of the glycosidase enzyme, upon glycoside hydrolysis. Protonated DMDP binds tightly to the enzyme active site and acts as a competitive inhibitor by blocking this site to substrate (glycoprotein or complex carbohydrate).
B - OH O2C δ+ OH-O C O 2 HO HO NH + HO 2 O HO - R O2C OH OH
HO2C B
Figure 1.10 Comparison of DMDP 3 with the oxoniumion transition state (taken from Wrodnigg).104
13 Chapter 1 [Introduction]
1.4 Therapeutic Application
DNJ 8, DMJ 10, swainsonine 21 and castanospermine 22 are available commercially; they have became standard reagents used to study the potential therapeutic and biochemical applications of this class of glycosidase inhibitors. Glycosidases are involved in a number of important biological processes, including intestinal digestion, post-translational modification of glycoproteins and the lysosomal catabolism of glycoconjugates. For these reasons sugar-mimicking polyhydroxylated alkaloids could have significant therapeutic potential in many diseases such as diabetes, cancer, viral infection and obesity.
1.4.1 Anti-diabetic agents
A large number of the naturally occurring polyhydroxylated alkaloids are potent inhibitors of the various α-glucosidase-specific disaccharidases implicated in mammalian digestion for example, sucrase, maltase, isomaltase, etc. as evident from Table 1.1. These enzymes digest dietary carbohydrate to monosaccharides which are absorbed through the surface of the epithelial cells of the brush border in the small intestine.3,90 After 1970, it was realised that inhibitors of these enzymes could be used therapeutically in the oral treatment of the non-insulin dependent diabetes mellitus (NIDDM or type II diabetes).36,37 A few examples of some glycosidase enzyme inhibitors that have made it to market as anti-diabetics are acarbose 29, a potent inhibitor of pig intestinal sucrase with an IC50 0.5 µM, This compound has been shown to be effective in carbohydrate loading tests in rats and healthy volunteers, by increasing insulin secretion and reducing postprandial blood glucose.90 After intensive clinical development, acarbose 29 was commercially available in Germany in 1990 and then in the United States in 1996 (Figure 1.11).
14 Chapter 1 [Introduction]
HOH2C
HO HO OH H3C HN O HO HO CH2OH O O HO HO CH2OH O acarbose 29 O HO OH HO
HOH2C OH
HO HO OH HO CH2OH OH OH OH N HN CH2CH2OH CH2OH voglibose 30 miglitol 31
Figure 1.11 The structures of some glycosidase enzyme inhibitors have made it to market as antidiabetics.
Voglibose 30 is another drug for treatment of type II diabetes and has been available in Japan since 1994. It is an N-substituted voliolamine derivatives which is produced by reductive amination of valiolamine.90 Valiolamine was isolated from Streptomyces hygroscopicus var. limoneus and was found to inhibit pig intestinal maltase and sucrase, with an of IC50 2.2 and 0.049 µM, respectively. However its derivative 30 showed much better inhibition, with IC50 values towards maltase and sucrase of 0.015 and 0.0046 µM, respectively. Another example is miglitol 31 which is N-substituted DNJ. This drug partially inhibits intestinal disaccharidases to reduce the level of postprandial glucose. It was introduced onto the market in the United States in 1999 and also Japan in 2006.90
1.4.2 Anti-cancer agents
Cancer is a group of diseases characterized by unmanageable growth and spread of aberrant cells. Transformation of normal cells to cancer cells and in tumour cell invasion and migration is associated with both change in glycosidase activity and in glycans on the cell surface.105 Many tumour cells show abnormal N-linked glycosylation due to an altered expression of glycosyltransferases.106 The levels of
15 Chapter 1 [Introduction] glycosidases are elevated in the sera of many patients with different tumours.107 Cancer cells have been shown to upregulate certain glycosidases which may be involved in the degradation of the extracellular matrix during metastasis.108 The worldwide incidence of cancer is estimated as 6 million new cases per year.109 In the USA, the leading cause of death for people younger than 85 years is cancer.110 Compounds that are able to prevent the formation of aberrant asparagine-linked oligosaccharides during glycoprotein processing and specifically inhibit the enzymes that are upregulated during cancer cell transformation are being actively pursued as a therapeutic strategy for cancer. Although, a number of polyhydroxylated alkaloids has been reported to have anti-cancer potential, the alkaloid swainsonine 8 has been the most investigated.111-117 The anti-metastatic effect of swainsonine has been shown to be largely due to augmentation of natural anti-tumour defences. Swainsonine increases the susceptibility of cancer cells to natural killer cells and lymphokine activated killer cell cytotoxicity.118 Direct anti-tumour activity was also evident as human tumour cells reverted to normal when treated with swainsonine.119 A phase I study of swainsonine in patients with advanced malignancies showed that minimal toxicity was caused when administered intravenously to cancer patients at dosages that inhibit both Golgi α-mannosidases.120 In a pharmacokinetic study in mice it appeared that central nervous system toxicity was not problematic at swainsonine levels that prevented metastasis.121 Ester derivatives of swainsonine at positions 2 or 8,122 were found to be poor inhibitors of α-mannosidase in vitro but entered the cell at a rate comparable to swainsonine where they were converted to swainsonine by intracellular esterases. In vivo, the analogues were found to have comparable activities to swainsonine as stimulators of bone marrow cell proliferation. This led to the suggestion that these or similar analogues could be useful pro-drugs which could be preferentially hydrolysed to release swainsonine only once they were inside tumour and/or lymphoid cells.122
1.4.3 Anti-viral agents
Another therapeutic application of polyhydroxylated alkaloids are as anti- viral agents. DNJ 8 and castanospermine 22 have been shown to reduce the infectivity of human immunodeficiency virus (HIV) in vitro at concentrations which are not cyctotoxic to lymphocytes. In contrast, the specific inhibitors of processing
16 Chapter 1 [Introduction]
α-mannosidases, DNJ 8 and swainsonine 21, had no effect on HIV.123-125 The alkaloids 8 and 22 also decrease the infectivity of other retroviruses e.g. the feline equivalent of HIV126 and human cytomegalovirus (CMV) which is an opportunistic pathogen in AIDS and caused by HIV infection.127 The interaction between the heavily glycosylated viral cover glycoproteins gp120 and gp41 with the CD4 receptor, a membrane bound glycoprotein found on the surface of T-lymphocytes, is necessary for HIV infection. In the presence of DNJ 8 and castanospermine 22 the glycosylation patterns of the viral coat glycoproteins are altered. Although this does not prevent formation of viral particles, they no longer have the ability to interact correctly with the CD4 receptor and so they are non-infectious.125,128 In 1988,124,129 N-butyl-DNJ (SC-48334) was shown to be more potent in vivo than DNJ. It had enhanced anti-HIV activity and was developed as a drug candidate and evaluated in phase II clinical trials.128,130-133 Further, the pharmacokinetic behaviour of a more lipophilic derivative of DNJ (N-benzyl-DNJ) has been tested in rats.134 In the same manner, synthetic modification of castanospermine 22 showed that the 6-O-acyl derivatives were more potent inhibitors of HIV than the natural product 135,136 and the 6-O-butanoyl derivative (in past clinical trials for AIDS) was found to be ca. 20 times more active than castanospermine 22 and 50 times more active than N-butyl- DNJ.125 The lipophilic nature of the acyl and butanoyl derivatives improved their uptake by cells with these compounds being intracellularly converted to castanospermine 22.137,138 In vitro combination studies with castanospermine 22 and AZT or other similar dideoxynucleside drugs showed synergistic activity against HIV type 1 and 2 replication,139,140 whereas N-butyl-DNJ and AZT are used in combination therapy in vivo in human patients but caused diarrhoea, abdominal pain and weight loss.132
1.5 Aims of Project
The aims of this project are, (1) To determinate of the correct structures of the polyhydroxylated alkaloids uniflorines A and B; (2) To develop a new synthetic strategy to prepare polyhydroxylated pyrrolizidines in an efficient and flexible manner; and (3) To test the synthesized compounds as glycosidase inhibitors.
17 Chapter 1 [Introduction]
1.5.1 Determination of the correct structures of uniflorines A and B.
1.5.1.1 Isolation and biological activities of uniflorines A and B
Eugenia uniflora; Surinam Cherry, Brazilian Cherry, or Cayenne Cherry is a plant in the family Myrtaceae, native to tropical America. It is a small tree or large shrub with a conical form, growing slowly to 8 m in height. The glossy green leaves thrive up to 4 cm in length and the new leaves are copper-coloured. The plant has fragrant white flowers and fruit that grows to 2 cm. The taste of these fruits ranges from sour to sweet depending on the level of ripeness (the green to orange range is strikingly tart, while the darker red to black range is quite sweet) (Figure 1.12).141
Binomial name Eugenia uniflora L. Scientific classification Kingdom: Plantae Order: Myrtales Family: Myrtaceae Genus: Eugenia Species: E. uniflora
141 Figure 1.12 Eugenia uniflora.
In 2000, Arisawa et al.142 made a phytochemical investigated of Nangapiry, a Paraguayan natural medicine made from the water-soluble extract (WSE) of the chopped dry leaves of E. uniflora. The WSE was found to inhibit an increase of plasma glucose levels in a sucrose tolerance test (STT) on mice, at a single oral dose of 100 mg/kg, whereas it did not inhibit an increase of plasma glucose levels in a glucose tolerance test (GTT). These results suggested that the WSE may contain glucosidase inhibitors. Three active compounds in the WSE were isolated and identified as uniflorines A and B and the known alkaloid, (+)-(3α,4α,5β)-1- methylpiperidine-3,4,5-triol 34. The structural elucidation of these compounds was
18 Chapter 1 [Introduction] based on the interpretation of the NMR spectroscopic and mass spectrometric data. The structure of uniflorine A was proposed as (-)-(1S,2R,6S,7R,8R,8aR)-1,2,6,7,8- pentahydroxyindolizidine 32 and the structure of uniflorine B was proposed as (+)-
(1S,2R,5R,7R,8S,8aS)-1,2,5,7,8-pentahydroxyindolizidine 33 (Figure 1.13).
HO HO OH OH H OH H HO 8 HO 4 OH HO 8 1 8a 1 5 3 7 8a 7 2 2 OH 6 OH 6 2 6 3 1 5 N 3 5 N HO N OH 32 33 34
Proposed structure for Proposed structure for (+)-(3 ,4 ,5 )-1-methylpiperidine- uniflorine A uniflorine B 3,4,5-triol piperidine.
Figure 1.13 The proposed alkaloid structures 32 and 33 and the alkaloid 34 isolated from a water-soluble extract of Eugenia uniflora.
Uniflorines A and B showed moderate activity in inhibiting the α-glucosidases maltase and sucrase while compound 34 revealed a weak activity towards these enzymes (Table 1.4).
Table 1.4 Inhibition of α-glucosidases by uniflorine A, uniflorine B and 34
IC50 uniflorine A uniflorine B compound 34 maltase 12.0 µM 4.0 µM 500 µM sucrase 3.1 µM 1.8 µM 270 µM
1.5.1.2 Previous syntheses of the proposed uniflorine A structure
In 2004, the first total synthesis of the proposed structure of uniflorine A 32 was achieved by Davis and Pyne,143 in eight steps from L-xylose. The synthesis began with the borono-Mannich reaction which gave the desired amino-tetraol 35 in 73% yield as a single diastereomer (Scheme 1.1). Protection of the secondary amino and then the primary hydroxyl group of 35 afforded the N-Boc derivative 36 and subsequently the O-trityl compound 37, respectively. A ring-closing metathesis (RCM) reaction of 37 with Grubbs’ first generation catalyst 38a afforded the 2,5- dihydropyrrole 39. The stereodirecting effect of the C-2 substituent in 39 ensured that the pentaol 40 was obtained as a single diastereomer by using a osmium(VIII)
19 Chapter 1 [Introduction] catalyzed syn-dihydroxylation (DH) reaction. The pentaol 40 was converted to its penta-O-benzyl derivative 41 under standard conditions. Deprotection of 41 using TFA, in the presence of anisole as a cation scavenger, revealed a mixture of the expected amino-alcohol 42 (37%) and the unexpected indolizidine 43 (54%). Compound 42 was then converted to indolizidine 43 by treatment under Appel cyclization reaction conditions using PPh3, CBr4, NEt3. Debenzylation of 43 under hydrogenolysis conditions, using PdCl2/H2 in MeOH, followed by ion-exchange and recrystallization furnished compound 32.
PCy Cl 3 Ph HO Ru HO OOH H Cl H HO Ph PCy HO a 38a 3 e HO OH RN d N HO HO Boc OH OR1 OTr L-xylose 35;R=R1 =H 39 b 36;R=Boc,R1 =H c 37;R=Boc,R1 =Tr
RO BnO BnO H OR H OBn H OBn RO BnO BnO g OR OBn OBn N HN + N RO Boc BnO BnO OTr OH 40;R=H 42 43 f 41;R=Bn h
RO H OR RO i OR N RO
32;R=H j 44;R=Ac
Scheme 1.1 Total synthesis of the proposed structure of uniflorine A 32. Reagents and conditions: (a) (E) PhCH=CHB(OH)2, allylamine, EtOH, rt, 16 h; ion- exchange, 73%; (b) (Boc)2O, Et3N, MeCN, DMF, 0 °C (4 h) then rt (14 h), 51%; (c) TrCl, py, rt, 18 h, 68%; (d) Grubbs’I catalyst, CH2Cl2, reflux, 18 h, 86%; (e) K2OsO42H2O, NMO, acetone/water, rt, 30 h, 88%; (f) NaH, BnBr, n-Bu4NI, THF, 50 °C, 3 d, 76%; (g) TFA, anisole, CH2Cl2, 0 °C, 2 h, 42 (37%) and 43 (54%); (h) PPh3, CBr4, NEt3, CH2Cl2, 0 °C, 2 h,
54%; (i) PdCl2, H2 (1 atm), MeOH, rt, 2 h; ion-exchange then recrystallization, 63% (j)
Ac2O, py, rt, 4 h, 88%.
20 Chapter 1 [Introduction]
The structure of 32 was unequivocally established by a single crystal X-ray structural analysis of its pentaacetate derivative 44. However, the spectral and physical data of the synthetic material 32 and 32-hydrochloride salt were incompatible with the data reported for the natural product.142 Therefore, it was concluded that the proposed structure of uniflorine A was incorrect. Importantly, the 1H NMR spectroscopic data for natural uniflorine A, indicated J1,8a was 4.5 Hz, more consistent with the relative syn-H-8a, H-1 stereochemistry, whereas in the 1H NMR spectrum of synthetic 32
J1,8a was 7.7 Hz and J8,8a was 9.0 Hz, consistent with their anti-H-8a, H-1 and anti-H- 8, H-8a stereochemical relationships. It was concluded that if uniflorine A was an indolizidine alkaloid, it should have the same relative, H-8a and H-1 stereochemistry as castanospermine 22 (Figure 1.14).
HO OH HO OH H H HO HO 8 8 8a 1 7 8a 1 7 2 OH 2 6 6 3 5 N 3 5 N HO HO 32 22 Proposed structure for castanospermine uniflorine A
Figure 1.14 The proposed structure of uniflorine A 32 and the structure of castanospermime 22.
In 2005, Mariano et al.144 published the syntheses of the C-1 epimer 45 and the C-1 and C-2 diepimer 46 of the structure 32, in order to elucidate the correct structure of uniflorine A. This was based on Pyne’s proposal that uniflorine A could be one of the two pentahydroxylated indolizidines 45 or 46 (Figure 1.15) both of which have the same B-ring stereochemistry and the syn relationships between H-1 and H-8a. HO HO OH H OH H HO HO 8 8 1 8a 1 7 8a 7 2 2 OH 6 OH 6 3 5 N 3 5 N HO HO
45 46
Figure 1.15 Structure of the C-1 epimer 45 and the C-1 and C-2 diepimer 46 of structure 32.
The preparation of one of these compounds, indolizidine 46, was reported in 1996 by Fleet et al.,145 before uniflorine A was discovered. Mariano et al.144 reported that the ring rearrangement metathesis (RRM) reaction of the enantiomerically
21 Chapter 1 [Introduction] enriched cis,trans-N-allylacetamidocyclopentendiol derivative 47 (Scheme 1.2), proceeded smoothly to furnish the corresponding 6-allyltetrahydropyridine 48. This highly regioselective process was performed using the second generation Grubbs’ ruthenium alkylidine catalyst 38b in the presence of ethylene.
Ac BnO Ac BnO Ac H H N N N a b c BnO OTBS TBSO HO
47 N 48 49 MesN Mes Cl Ru CHPh Cl 38b PCy3 BnO Ac BnO Ac BnO Ac H H H N d N f N g HO HO HO RO OR OBn O BnO OR OBn
50 51;R=H 53 e 52;R=Bn
HO HO HO H OH HO BnO N i BnO N j OH H H N HO BnO OBn BnO OBn OBn OBn 54 55 45
h
HO H OH HO OH N HO 46 Scheme 1.2 Total synthesis of the C-1 epimer 45 and the C-1 and C-2 diepimer 46 of structure 32.
Reagents and conditions: (a) 38b, ethylene, CH2Cl2, reflux, 16 h, 30%; (b) TBAF, THF, rt, 2 h, 98%; (c) VO(acac)2, t-BuOOH, CH2Cl2, rt, 15 h, 50%; (d) NaOBz (aq.), 130 °C, 12 h,
55%; (e) NaH, BnBr, DMF, 0 °C, 2 h, 94%; (f) OsO4, NMO, acetone/water, 25 °C, 4 h, 78%;
(g) i: 6N HCl, THF, 70°C, 4 h; ii: PPh3, DEAD, anh. py, 0 °C, 4 h, 74%; (h) PdCl2, H2 (1 - atm), MeOH, 25°C, 4 h, ion-exchange (Dowex 1-X8, OH ), 94%; (i) i: benzoic acid, PPh3,
DEAD, THF, 25 °C, 12 h, ii: NaOMe, MeOH, 25 °C, 12 h, 73%; (j) PdCl2, H2 (1 atm), MeOH, 25°C, 4 h, ion-exchange (Dowex 1-X8, OH-), 98%.
22 Chapter 1 [Introduction]
Allylic alcohol 49 was obtained by treatment of 48 with TBAF, followed by N- acetylation of the resulting product. The regio- and diastereoselective epoxidation of 49 produced the corresponding epoxy-alcohol 50. Trans-diaxial ring-opening of 50 under mild basic conditions furnished the trihydroxypiperidine 51. Benzylation of 51 provided the tetrabenzyl ether 52. The diol 53 was prepared from a diastereoselective DH reaction of the alkene 52. Acid hydrolysis of the amide 53 gave an amino diol that was cyclized under Mitsunobu reaction conditions to give the indolizidine 54. The known indolizidine 46145 was obtained from 54 using a hydrogenolytic benzyl deprotection reaction. Indolizidine 45 was also generated from the same starting material 54 by using a Mitsunobu hydroxyl inversion reaction to form 55 followed by hydrogenolytic benzyl deprotection. The indolizidines 45 and 46, and their HCl salt forms, also had NMR spectroscopic data different to those reported for uniflorine A.
Dhavale et al.146 reported the syntheses of the putative structure of uniflorine A 32 and its epimeric analogues 56 and 57 in 2006 (Figure 1.16).
HO HO HO OH H OH H OH H HO HO HO 7 8a 1 OH OH OH 3 N HO 5 N HO N HO
56 57 Proposed started uniflorine A 32 C-1, C-2 and C-8a- C-8a epimer of 32 triepimer of 32
Figure 1.16 Proposed uniflorine A structure and its epimers.
Their syntheses began with a 1,3-addition reaction of vinylmagnesium bromide to the nitrone 58 which provided 59a (79% yield) and 59b (11% yield). Separate treatment of 59a and 59b with zinc in acetic acid-water gave 60a and 60b by N-O bond reductive cleavage. N-alkylation of 60a and 60b with allyl bromide afforded 61a and 61b followed by a RCM reaction using Grubbs’ first generation catalyst 38a gave 62a and 62b, respectively. A syn-DH reaction of 62a using either AD-mix-α or AD-mix-β gave the diol 63a. Debenzylation of 63a using ammonium formate and 10% Pd/C and then selective amino group protection using benzyl chloroformate affored the N-Cbz protected derivative 64a. In the last step, the 1,2- acetonide group of 64a was cleaved with TFA/H2O and then an intramolecular
23 Chapter 1 [Introduction] reductive amination reaction gave the structure 32 (Scheme 1.3). The spectroscopic and physical data of the structure 32 were the same as that of synthetic 32 prepared by Davis and Pyne143 and were different from those of the natural product. While, the syn-DH reaction of 62b using AD-mix-α gave 63b and 63c in a 2:8 ratio, AD-mix-β afforded only 63c as a single diastereomer. Following the same reaction sequence, with 63b and 63c the new compounds 2(S)-hydroxy-8a-epi-castanospermine 56 and 2(R)-hydroxy-1,8a-diepi-castanospermine 57, were obtained, respectively (Scheme 1.4). A comparison of the NMR spectroscopic data of 56 and 57 with those of the natural product, uniflorine A, showed that these three compounds were different.
Bn NH R R O Bn N H Bn N H O a O O OBn OBn + OBn H H H O O O O O O
58 59a;R=OH,79% 59b;R=OH,11% b b 60a;R=H,88% 60b;R =H,88% c c 61a;R=allyl,92% 61b;R =allyl,91%
d 84%d 82%
Bn Bn Bn N N N HO H e H H O O O OBn 60% OBn OBn H H H HO O O O O O O 63a 62a 62b
f 78%
Cbz HO OH N H HO H g HO O OH OH H 91% HO N HO O O 32 64a Scheme 1.3 Total synthesis of structure 32.
Reagents and conditions: (a) vinylmagnesium bromide, TMSOTf, THF, -78 °C, 2 h, 90%;
(b) Zn, Cu(OAc)2, AcOH, 80 °C, 1 h; (c) K2CO3, allylbromide, DMF, rt, 12 h; (d) Grubb’s catalyst (1st generation) 38a, CH2Cl2, rt,12 h. (e) AD-mix-α, MeSO2NH2, t-BuOH/H2O (1:1
(f) (i) HCOONH4, 10% Pd/C, MeOH, 80 °C, 1 h; (ii) CbzCl, NaHCO3, MeOH/H2O (5:1), 2 h; (g) i: TFA/H2O (3:2), 25 °C, 2.5 h; ii: 10% Pd/C, MeOH, 80 psi, 12 h.
24 Chapter 1 [Introduction]
Bn Cbz HO OH N N H HO H HO H HO O b O c OBn OH OH H 73% H 83% HO O HO O HO N 63b O 64b O 56 a 62b + dr = 2:8; 59% Bn Cbz N N HO OH HO H HO H H O b O c HO OBn OH 79% 84% OH HO H H N O HO O HO 63c O O 64c 57
Scheme 1.4 The synthesis of 56 and 57.
Reagents and conditions: (a) AD-mix-α, MeSO2NH2, t-BuOH/H2O (1:1 (b) (i) HCOONH4,
10% Pd/C, MeOH, 80 °C, 1 h; (ii) CbzCl, NaHCO3, MeOH/H2O (5:1), 2 h; (c) i: TFA/H2O (3:2), 25 °C, 2.5 h; ii: 10% Pd/C, MeOH, 80 psi, 12 h.
The synthesis of the C-1 and C-2 diepimer 46 of structure 32 from L-xylose was also reported by Davis and Pyne143 (Scheme 1.5). Amino tetraol 35, obtained from the boronic acid-Mannich reaction of L-xylose, allylamine and (E)-styrene boronic acid, was converted to its N-Troc derivative 65 (75% yield) using trichloroethyl succinimidylcarbonate (Troc-OSu). Regioselectively protection of the primary hydroxyl group of 65 with trityl chloride furnished the O-trityl compound 66. An attempted O-benzylation reaction gave a mixture of the oxazol-2-one 67 and the oxazin-2-one 68 that were not separated but were treated with Grubbs’ second- generation ruthenium catalyst 38b. Two major bicyclic compounds were isolated, the pyrrolo[1,2-c]oxazol-3-one 69 and the pyrrolo[1,2-c]oxazin-1-one 70. Treament of 70 under DH reaction conditions provided the diol 71 as a single diastereomer. The diol 71 was O-benzylated and the trityl group was removed by acid hydrolysis using
TFA/H2O to give the primary alcohol 73. Base hydrolysis of 73 under microwave irradiation conditions afforded the pyrrolidine 74. The indolizidine 75 was achieved in good yield by cyclization of 74 under Appel reaction conditions using
Ph3P/CBr4/Et3N. Debenzylation of 75 under standard conditions (PdCl2/H2) gave 46, after ion-exchange chromatography and then recrystallization from ethanol/water. The spectroscopic data of the structure 46 was the same as those that was synthesized by Fleet et al.145 and the optical rotation of this compound was of the same sign but of a significantly different magnitude to that reported.
25 Chapter 1 [Introduction]
HO H HO Ph TrO OBn ac 35 HH N BnO HO Ph Troc O RO N 65;R=H O b 67 66;R=Tr + OBn OBn H H TrO Ph
TrO OBn O N HH BnO O O 68 N O 69 66 d (39% from )
+ OBn OBn OBn OBn OR1 H H 2 H H TrO e R O OR1 O N O N
O O 70 (31% from 66) 71;R1 =H,R2 =Tr f 72;R1 =Bn,R2 =Tr g 73;R1 =Bn,R2 =H
BnO RO OR H OBn H h HO i HO OBn OR HN N BnO RO HO 75;R=Bn 74 j 46;R=H C-1 and C-2 diepimer 32 Scheme 1.5 Total synthesis of the C-1 and C-2 diepimer 46 of structure 32.
Reagents and conditions: (a) Troc-OSu, NaHCO3, dioxane, rt, 4 h, 75%; (b) TrCl, py, o CH2Cl2, rt, 9 h, 75%; (c) NaH, BnBr, Bu4NI, THF, 50 C, 4 d; (d) Grubbs' II catalyst 38b,
CH2Cl2, reflux 24 h, 69 (39% from 66) and 70 (31% from 66); (e) K2OsO42H2O, NMO, acetone/H2O, rt, 16 h, 67%; (f) NaH, BnBr, Bu4NI, THF, 67%; (g) TFA, anisole, 61%; (h) o o NaOH, MeOH, H2O, 110 C, MW, 1 h, 62%; (i) CBr4, Ph3P, Et3N, CH2Cl2, 0 C, 2 h, 85%;
(j) PdCl2, H2, MeOH, rt, 4 h, 62%.
26 Chapter 1 [Introduction]
If uniflorine A was epimeric at C-1 and/or C-2 with compound 32 then the only remaining possible structure for the natural product was 76, the C-2 epimer of 32 (Figure 1.17). Thus the first part of this thesis (Chapter 2) describes the diastereoselective synthesis of 76 and a comparison of its NMR spectroscopic data with those of uniflorine A.
HO HO HO OH HO H OH H OH H H OH HO HO HO HO 8 8a 1 OH OH OH OH 5 N 3 N N N HO HO HO HO 32 45 46 76 Proposed structure C-1 epimer of 32 C-1 and C-2 diepimer of 32 C-2 epimer of 32 for uniflorine A Synthesis: Synthesis: Synthesis: Synthesis: Mariano et al. 2005144 Fleet et al. 1996145 This thesis147 Pyne et al. 2004143 Mariano et al. 2005144 Dhavale et al. 2006146 Pyne et al. 2008147
Figure 1.17 Summary of synthesized epimers of uniflorine A.
1.5.2 The development of a new synthetic strategy to prepare polyhydroxylated pyrrolizidines in an efficient and flexible manner.
We also planned to develop a new general method for preparing polyhydroxylated 3-hydroxymethylpyrrolizidines (Figure 1.18). An efficient, flexible method would allow for the total synthesis of these alkaloids and their analogues for biological assays and structure-activity relationship studies.
m(HO) (OH)n N
n=2 OH m=1or2 Figure 1.18 The 3-hydroxymethylpyrrolizidine ring system.
Scheme 1.6 summarizes the natural product synthetic targets described in this thesis. These were prepared from a common intermediate, generalized in structure 77.
27 Chapter 1 [Introduction]
HOH OH HOH OH
HO OH HO OH N N
OH OH uniflorine A 78 casuarine 15 Chapter 2 Chapter 3
HOH OH OH OP H OH L-xylose OP N OH NP' OH 77 australine 13 Chapter 4
HOH OH HOH OH
OH HO OH N N
OH OH 3-epi-australine 80 3-epi-casuarine 79 Chapter 6 Chapter 5
(P = Protecting group)
Scheme 1.6 The target natural products described in this thesis.
1.5.3 Testing of the synthesized compounds as glycosidase inhibitors.
The natural products and their analogues prepared in this project were to be screened by Prof. Robert J. Nash at the Institute of Grassland and Environmental Research, Aberystwyth, UK against 10-15 different glycosidases. These results are reported in Chapter 7.
28 Chapter 2 [Uniflorines A and B]
CHAPTER 2 DETERMINATION OF THE CORRECT STRUCTURE OF UNIFLORINES A AND B
2.1 Synthesis of 76, the C-2 epimer of the proposed structure of uniflorine A 32
The planned synthesis of 76, the C-2 epimer of the proposed structure uniflorine A 32 as shown in Scheme 2.1, was similar to that used in the synthesis of structure 32, however the stereochemistry at the C-2 position required inversion. The synthesis of 76 involved the following four key reactions: • N-alkylation to provide the piperidine ring. • A regioselective ring-opening of the cyclic sulfate A with benzoate ion to allow inversion of the configuration at C-2. • A RCM reaction and then a syn-DH reaction to form the dihydroxylated pyrrolidine ring. • The Petasis reaction, which would allow a highly stereoselective synthesis of the anti-1,2 amino alcohol 35. This reaction would establish the absolute configurations at C-6, C-7, C-8 and C-8a of the target molecule.
O HO OP 1. RCM H OH H O HO 1. Ring opening of cyclic sulfate PO S O 2. DH 2. N-alkyla tion 3. SO2X2 OH O N P'N HO PO OP'' 76 A HO OH H HO Ph HO Ph Petasis reaction CHO (HO)2B HN HO HO H2N OH OH 35 P = Protecting group
Scheme 2.1 Retro-synthesis of 76, the C-2 epimer of the structure 32.
29 Chapter 2 [Uniflorines A and B]
2.1.1 The Petasis reaction
The Petasis reaction148 is very useful reaction for the preparation of the chiral anti-1,2-amino alcohols. These have been used as building blocks for the synthesis of polyhydroxylated pyrrolizidines and indolizidines, including the epimers of 149 94 150,151 152 australine, hyacinthacine B3, swainsonine, castanospermine and the proposed structure of uniflorine A 32.143 The boronic acid-Mannich reaction (Petasis reaction) of the three components; L-xylose, allylamine and (E)-styrene boronic acid gave the desired amino alcohol 35 in an excellent yield of 91% after purification by ion-exchange chromatography (Scheme 2.2). The NMR spectroscopic data of this compound matched with those earlier reported.143
HO H B(OH)2 HO Ph 1. Ph L-xylose NH2 HN HO EtOH,rt,48h, HO 35 2. Ion-exchange, 91%
Scheme 2.2 Synthesis of amino tetrol 35.
The exact mechanism of the Petasis reaction is not known. The proposed reaction mechanism (Scheme 2.3) starts with ring-opening of L-xylose to give the aldehyde form, which condenses with allyl amine to afford an iminium ion. The boronic acid then coordinates to the hydroxyl group of the iminium ion at the C-2 position to give the boronate intermediate B. The iminium ion adopts the reactive conformation C to minimize 1,3-allylic strain150 between the allylic (α) substituents and the iminium ion. Intramolecular transfer of the strenyl group to the iminium ion provides the desired anti-amino tetraol 35. The coordination of boron to the other hydroxyl group positions is also possible; however these processes would be reversible and the corresponding intermediates would be less likely to lead to products because of the remoteness of the strenyl group to the iminium ion.
30 Chapter 2 [Uniflorines A and B]
OH HO O OH H HO B(OH)2 HO Ph CHO Ph
HO OH HO HN NH HO OH 2 OH HO 35 B(OH)2 4 32 Ph NH2 OH 1 1 HO R H R1 H H B H R H NN H OH O Ph N 22 H O H 2 HO H RR HO N R HO H HO B Ph OH H HO Ph Ph AB CB
Scheme 2.3 Proposed reaction mechanism of the Petasis reaction.
2.1.2 N-Boc and O-Tr protection
The amino tetraol 35 was converted to the N-Boc derivative 36 by using
(Boc)2O, Et3N in THF with stirring at rt for 2 d (Scheme 2.4). TLC analysis showed two spots at Rf 0.68 and 0.4 in EtOAc. These two compounds were separated successfully by column chromatography. The 1H NMR spectrum of the lower spot demonstrated a singlet at 1.44 ppm with an integration of 9H, corresponding to a single tert-butyl group. Therefore, the lower spot was the mono Boc protected 36. This was confirmed by ESI-MS analysis which showed a protonated molecular ion peak at m/z 394 [M+H]+. However, the 1H NMR spectrum of the higher spot gave rise to a singlet at the chemical shift of 1.48 ppm, with an integration of 18H. Moreover, the chemical shift of C1-H2 was at lower field at 3.81 ppm because of the deshielding effect of the electron withdrawing Boc carbonyl group. After confirmation by mass spectrometry (m/z 494 [M+H]+), it was concluded that the higher spot product had two Boc groups as shown in structure 36a. However, 36a could be converted to 36 using K2CO3 in MeOH giving a 56% yield of 35 over two steps.
31 Chapter 2 [Uniflorines A and B]
OH OH EtOAc Eluent HO Ph HO Ph Solvent front 3 5 a HN N HO 1 HO Boc 36a OH BocO 35 36a + b 36 OH OH HO Ph HO Ph c N N HO HO Boc Boc TrO HO SM Co RM 37 36 SM = Starting mater ial Co = Co-spot of SM and RM RM = Reaction mixture Scheme 2.4 Synthesis of diene 37.
Reagents and conditions: (a) (Boc)2O, Et3N, THF, rt, 48 h, 36a (23%) and 36 (46%); (b)
K2CO3, MeOH, rt, 17 h, 56%; (c) TrCl, py, CH2Cl2, 40 h, 81%.
The primary hydroxyl group of 36 was protected with a trityl group by treatment with triyl chloride (1.05 eq.) and pyridine (1.05 eq.) in CH2Cl2 for 40 h. After purification by column chromatography, the trityl ether 37 was obtained in 81% yield. The 1H NMR spectrum of 37 showed signals at 7.46-7.19 ppm (m, 20 aromatic H; increasing 15H from 36) and signals for the two H-1 protons at 3.37 (dd, J 9.3 and 2.7 Hz) and 3.22-3.15 ppm (m). The chemical shifts of the H-1 protons in the trityl ether 36 moved to higher field due to the trityloxy group. The NMR spectroscopic data of compounds 36 and 37 were the same as those reported in the earlier literature.143
2.1.3 Ring Closing Metathesis (RCM)
The RCM reaction153 is one of the key reaction steps in our proposed approach, for the preparation of the pyrrolidine ring. The proposed reaction mechanism for the general RCM reaction is showed in Scheme 2.5. In the case of our diene 37 the first [2+2] cycloaddition between the ruthenium catalyst and the substrate most likely occurs at the less hindered terminal alkene moiety. Andrew Davis,143 who worked in Pyne’s laboratory, investigated the RCM reaction of 37 using 10% Grubbs’ I catalyst (38a) with heating at reflux for 24 h in CH2Cl2. The
32 Chapter 2 [Uniflorines A and B] best yield for this reaction was 96%. Following the same method, the pyrrolidine compound 39 was obtained in 90% yield after purification by column chromatography (Scheme 2.6). The success of the RCM reaction on the diene 37 was indicated by changes in the 1H NMR spectrum from five alkene protons and 20 aromatic protons in the starting material to two alkene protons and 15 aromatic protons in the product. The NMR spectroscopic data of 39 corresponded to those previously reported.143
[Ru] 2+2 cycloaddition
cycloreversion
[Ru]
[Ru]
2+2 cycloreversion cycloaddition [Ru]
[Ru] = Ru carbene complex
Scheme 2.5 The proposed mechanism of the RCM reaction.
OH Cl PCy3 Ph Ru HO H Cl HO Ph HO H PCy3 38a N N HO CH2Cl2,reflux,1d HO Boc Boc 90% OTr OTr 37 39
Scheme 2.6 Synthesis of dihydropyrrole 39.
2.1.4 O-Bn protection
The resulting pyrrolidine 39 was tri-O-benzylated by treatment with NaH (3.3 eq.), benzyl bromide (6 eq.) and 10% tetrabutylammonium iodide catalyst in dry THF solution under standard conditions.154 The progress of the reaction was monitored by TLC analysis (Figure 2.1). After 25 h, TLC analysis showed only a spot for the starting material. After 48 h, TLC analysis showed incomplete consumption of the starting material and several new product spots. It was assumed
33 Chapter 2 [Uniflorines A and B] that partially O-benzylated products had been formed. After 96 h, there was no significant difference of the reaction mixture by TLC analysis. Therefore, the reaction was worked up and the crude reaction mixture was treated again under the same conditions for 4 d. TLC analysis showed no difference between the starting mixture and the final product mixture.
40%EtOAc/Petrol 40% EtOAc/Petrol 40%EtOAc/Petrol
81 69
70
SM Co RM SM Co RM SM Co RM 25 h 48 h 96 h Figure 2.1 TLC analysis of benzylation reaction of 39. TrO OBn BnO BnO HO BnO H HH H H H BnO TrO HO NaH, BnBr BnO + O + N n-Bu4NI, THF N N O N 0 BnO HO Boc 50 C, 4 d Boc OTr OTr O O 39 81 (56%) 69 (3%) 70 (9% ) Ph I
O O H TrO
O N Ph O I O
Ph I Ph I
O O H TrO
O N O O
Scheme 2.7 Proposed mechanism for the formation of structures 69, 70 and 81.
34 Chapter 2 [Uniflorines A and B]
Separation and purification of the crude reaction mixture by column chromatography gave the tri-O-benzylated product 81 in 56% yield and the two undesired products the di-O-benzylated oxazolidinone 69 in 3% yield and the di-O- benzylated oxazinanone 70 in 9% yield. The possible mechanisms for the formation of there products are displayed in Scheme 2.7. The 1H NMR spectrum of 81 showed an increase of 15 aromatic protons from the starting material and also showed six doublet signals (J ca.11.4-11.0 Hz) for the diastereotopic methylene protons of the benzyl groups at 4.84, 4.73, 4.66, 4.59, 4.33 and 4.21 ppm. The NMR spectroscopic data of the di-O-benzylated oxazolidinone 69 and that of di-O-benzylated oxazinanone 70 matched with those that were previously reported (Scheme 1.5).143,147
2.1.5 Syn-dihydroxylation (DH)
Syn-DH of the double bond of 81 employing K2OsO42H2O (5 mol%) and
NMO as co-oxidant in a mixture of acetone and H2O at rt for 3 d affored the diol 82 in 75% yield and as a single diastereomer.
BnO BnO H H OH BnO BnO . 2 K2OsO4 2H2O, NMO 442 N OH acetone/water, 72 h N BnO Boc BnO Boc OTr 75% OTr 81 82
Scheme 2.8 Synthesis of diol 82.
The stereochemical outcome of this DH reaction was expected due to the stereodirecting effect of the C-2 pyrrolidine substituent of 81 which hindered the β- face of 81 to attack by the osmium(VIII) reagent (Figure 2.2). The 1H NMR spectrum of 82 showed that the two olefinic proton resonances at the chemical shift 5.84-5.80 (m, 1H) and 5.76-5.74 (m, 1H) ppm of the starting material 81 had disappeared and two new methine proton signals at 4.12-4.04 (m, H-3) and 3.62-3.54 (m, H-4) ppm were observed. The 1H NMR spectrum of 82 showed peak broadening and signals for two rotamers because of hindered rotation about the N-Boc group. The proposed reaction mechanism of the DH reaction is shown in Scheme 2.9.
35 Chapter 2 [Uniflorines A and B]
Figure 2.2 Simplified structure of 81 from PC Spartan (6-31 G*).
Scheme 2.9 Proposed reaction mechanism of the syn-dihyroxylation reaction.
2.1.6 Formation of the cyclic sulfate and ring-opening
In 2003,155 Minyan Tang in Pyne’s group reported that the ring-opening of the cyclic sulfate 84 with cesium benzoate proceeded with inversion of configuration at C-6 to give the C-6 benzoate 85 (Scheme 2.10). In the case of diol 82 our attempts to make the cyclic sulfate 87a using the two step method show in Scheme 2.11, involving first formation of the corresponding cyclic sulfite (SOCl2, Et3N) followed 156 by oxidation with catalytic ruthenium tetraoxide (RuCl3·3H2O, NaIO4) gave mixtures of the desired compound 87a and the O-Tr deprotected cyclic sulfate 87b. These results are summarized in Table 2.2. The best yield of 87a was 47% when the ruthenium catalysed oxidation step was buffered by the addition of Na2HPO4, (Table 2.1, entry 3).
36 Chapter 2 [Uniflorines A and B]
PMBO H OH PMBO PMBO HO OH H H H O H H OBz H SO2 1 a b O 2 OH O O O OH OH N 3 N N N O OBn O O OH 83 84 OBn 85 OBn 86
Scheme 2.10 Synthesis of benzoate 85.
Reagents and conditions: (a) i: SOCl2, Et3N, CH2Cl2, 0 °C 30 min, ii: RuCl3·3H2O, NaIO4,
2:2:3 CCl4/CH3CN/H2O, rt, 2 h, 80 % over 2 steps; (b) i: PhCO2H, Cs2CO3, DMF, 40 °C, 23 h, ii: H2SO4 (conc.), THF, H2O, rt, 18 h, 56% over 2 steps.
BnO H OH BnO O O BnO H S a BnO OH N O BnO N Boc BnO Boc OTr OTr 82 O O BnO BnO H O H O S O BnO S O b BnO O + O N N BnO Boc BnO Boc OTr OH 87a 87b Scheme 2.11 Synthesis of structure 87a. o Reagents and conditions: (a) SOCl2, Et3N, CH2Cl2, 0 C, 1 h then rt 2 h; (b)
RuCl3·3H2O, NaIO4, Na2HPO4, CCl4:CH3CN:H2O/2:2:3 rt 17 h, 47%.
Table 2.1 Synthesis of the cyclic sulfates 87a/b from the diol 82.
Entry Reagents Results (yield)
a. SOCl2 (1.5 eq.), Et3N (16 eq.), CH2Cl2 87a (3%) and 1 b. RuCl3·3H2O (0.05 eq.), NaIO4 (2 eq.), CCl4:CH3CN:H2O/2:2:3 87b (26%)
a. SOCl2 (5 eq.), Et3N (15 eq.), CH2Cl2 87a (36%) and 2 b RuCl3·3H2O (0.15 eq.), NaIO4 (4 eq.), CCl4:CH3CN:H2O/2:2:3 87b (15%)
a. SOCl2 (5 eq.), Et3N (15 eq.), CH2Cl2 87a (47%) and 3 b. RuCl3·3H2O (0.1 eq.), NaIO4 (4 eq.), Na2HPO4 (2 eq.), 87b (6%) CCl4:CH3CN:H2O/2:2:3
The cyclic sulfate 87a was found to be sensitive to purification on silica gel and was thus taken through the next step without purification. The cyclic sulfate 87a could be prepared more directly from 82 by treatment with sulfuryl chloride under 156 basic conditions (SO2Cl2, Et3N) (Scheme 2.12). This method was more efficient than the two step method. Treatment of crude 87a with cesium benzoate in DMSO at
37 Chapter 2 [Uniflorines A and B]
40 oC for 19 h followed by acid hydrolysis of the resulting sulfate ester adduct gave the benzoate 88a in yields ranging from 43-54% from the diol 82. The results of this two step process are summarized in Table 2.2. The benzoate 88a resulted from the regioselective nucleophilic ring-opening of the cyclic sulfate 87a at the less hindered C-4 pyrrolidine position giving rise to inversion of stereochemistry at C-4. Attack of benzoate at the C-3 position of the pyrrolidine ring was hindered by the C-2, β- substituent. This reaction also produced a small amount (7-9%) of the corresponding O-trityl deprotected analogue 88b formed under the acidic hydrolysis conditions (Scheme 2.12). In each case a significant amount of the starting diol 82 (16-23%) was also isolated resulting from hydrolysis of unreacted 87a. A mechanism for the above two step process is shown in Scheme 2.13. O BnO BnO H OH H O BnO BnO S O a OH 2 4 O N N BnO Boc BnO Boc OTr OTr 82 87a
BnO BnO OH H OH H BnO BnO b + OBz OBz N N BnO BnO Boc Boc OTr OH 88a 88b Scheme 2.12 Synthesis of structure 87. o Reagents and conditions: (a) SO2Cl2, Et3N, CH2Cl2, 0 C, 1 h then rt 2 h; (b) i: PhCO2Cs, o DMSO, 40 C, 19 h; ii: H2SO4/H2O, THF, rt, 20 h, 88a (54%) and 88b (7%) (2 steps).
Table 2.2 Synthesis of benzoate 87 from the diol 82.
Entry Reagents Results
a. SOCl2 (5 eq.), Et3N (15 eq.), CH2Cl2 82 (21%) and 1 b. I: PhCO2H (5 eq.), CsCO3 (5.5 eq.), THF 87a (43%) II: H2SO4/H2O, THF
a. SOCl2 (5 eq.), Et3N (15 eq.), CH2Cl2 82 (23%),
2 b. I: PhCO2H (5 eq.), CsCO3 (5.5 eq.), THF 88a (53%) and
II: H2SO4/H2O, THF 88b (9%)
a. SOCl2 (5 eq.), Et3N (15 eq.), CH2Cl2 82 (16%),
3 b. I: PhCO2H (5 eq.), CsCO3 (5.5 eq.), THF 88a (54%) and
II: H2SO4/H2O, THF 88b (7%)
38 Chapter 2 [Uniflorines A and B]
O Cl S O Cl O S O Cl O O H OH H O H S O 2 OH OH 4 O N N N Boc Boc Boc O PhC OCs 82 87a OH H OSO3 H H3O 2 2 4 OBz 4 OBz N N Boc Boc 88a Scheme 2.13 Mechanism of formation of the cyclic sulfate 87a and its ring-opening by cesium benzoate.
2.1.7 Debenzoylation
The benzoate group of 88a was removed by methanolysis using K2CO3 (2 eq.) in MeOH to give the diol 89 in 44% yield. The 1H NMR spectrum of the product 89 showed thirty aromatic protons, five aromatic protons less than that of the starting material 88a while the 13C NMR spectrum of 89 showed one carbonyl carbon signal at 154.5 ppm for the N-Boc group when compared with the starting material that had two carbonyl carbon signals at 166.9 ppm (benzoate carbonyl carbon) and 153.5 ppm (carbonyl of Boc group).
BnO BnO H OH H OH BnO BnO OCOPh K2CO 3,MeOH OH N N BnO Boc rt, 24 h, 44% BnO Boc OTr OTr 88a 89
Scheme 2.14 Synthesis of diol 89.
39 Chapter 2 [Uniflorines A and B]
2.1.8 N-Boc and O-Tr deprotection
Davis143 reported that deprotection of compound 41 using TFA, in the presence of anisole, as a cation scavenger,157 gave not only gave the desired product 42 (Scheme 1.1) but also the cyclized indolizidine 43. Their proposed mechanism for the formation of 43 is shown in Scheme 2.15.
BnO H OBn BnO H OBn 41 N 43 BnO But TrO O O H
Scheme 2.15 Proposed mechanism for formation of the indolizidine 43.
Not surprisingly, when the same reaction conditions were applied to compound 89 a separable mixture of the desired amino alcohol 90 (66%) and the indolizidine 91 (8%) was obtained (Scheme 2.16). The desired amino alcohol 90 was highly polar and required the addition of ammonia solution to the eluting solvent during separation of these compounds by column chromatography. The 1H NMR spectra of these products showed sharp and clear signals that indicated that the N- Boc group had been successfully removed. The structures of the amino alcohol 90 and the indolizidine 91 were also confirmed by mass spectrometric analysis.
BnO BnO BnO H OH H OH H OH BnO BnO BnO TFA, anisole OH + OH OH N N N CH2Cl2,rt,20h BnO BnO BnO Boc H OH OTr 89 90 (66%) 91 (8%) Scheme 2.16 Synthesis of compounds 90 and 91.
40 Chapter 2 [Uniflorines A and B]
2.1.9 Mitsunobu cyclization
The next step, as shown in Scheme 2.17, was the cyclizaton of the amino alcohol 90 to the indolizidine 91. The Mitsunobu reaction has been successfully used by others in forming related pyrrolidines149 and indolizidines.152 BnO BnO H OH H OH BnO BnO DIAD, PPh3,py OH OH N 0-5 oC, 48 h, 25% N BnO H BnO OH 90 91
Scheme 2.17 Synthesis of indolizidine 91 by Mitsunobu cyclization.
The Mitsunobu reaction normally uses PPh3 and diethyl azodicaboxylate (DEAD), however, diisopropyl azodicaboxylate (DAID) is less expensive and less toxic than DEAD and also works well. The Mitsunobu reaction of 90 using DIAD,
PPh3 in pyridine gave a complex mixture of products from which 91 could be isolated in only 25% yield (Scheme 2.17). The cyclization of 90 was attempted under Appel reaction conditions,143 however a complex mixture of products resulted (Table 2.3). The amino alcohol 90 was first treated with the Dess-Martin periodinane reagent with the expectation of obtaining the corresponding aldehyde and the crude reaction mixture was then treated with NaCNBH3 to affect cyclization via a reductive alkylation reaction. This reaction gave 91 in only 2% yield. This compound was identical to the indolizidine formed in Scheme 2.16. The proposed mechanism of the Mitsunobu reaction is outlined in Scheme 2.18.
Table 2.3 The synthesis of indolizidine 91.
Reaction Entry Reagents Results
Mitsunobu 1 PPh3 (2 eq.), DAID (2 eq.), py, 24 h 91 (25%)
PPh3 (2.5 eq.), CBr4 (2.5 eq.), NEt3 (4.0 eq.), Complex mixture 1 o CH3CN, 0 C 2 h, then rt 24 h Appel PPh3 (2.5 eq.), CBr4 (2.5 eq.), NEt3 (4.0 eq.), Complex mixture 2 o CH3CN, 0 C 2 h, then rt 48 h. o I: Dess-Matrin (1.1 eq.), CH3CN, 0 C 2 h 91 (2%) Oxidation and 1 II: NaCNBH3, HOAc (pH~5), 18 h then Reductive o I: Dess-Matrin (1.1 eq.), CH3CN, 0 C 2 h 91 (2%) Amination 2 II: NaCNBH3, HOAc (pH~7), 18 h
41 Chapter 2 [Uniflorines A and B]
Scheme 2.18 Proposed reaction mechanism of the Mitsunobu reaction of 90.
BnO H OH BnO 8a 1 7 OH 5 N 3 BnO 91 H-7 H-8 H-5 H-3 H-1 H-2 H-5 H-3 H-8a H-6
4.00 3.50 3.00 2.50 2.00 ppm (t1)
Bn Bn Bn
7.0 6.0 5.0 4.0 3.0 2.0 ppm (t1) 1 Figure 2.3 The H NMR (500 MHz, CDCl3) spectrum of 91.
2.1.10 Debenzylation
The last step was a debenzylation reaction of the indolizidine 91. Under standard reaction conditions using PdCl2/H2 in MeOH, (1S,2S,6S,7R,8R,8aR)octahy- droindolizidine-1,2,6,7,8-pentol 76 was obtained in 72% yield after purification by basic ion-exchange chromatography (Scheme 2.19).
42 Chapter 2 [Uniflorines A and B]
BnO HO H OH H OH BnO HO PdCl2,H2 (1 atm ) OH OH N MeOH, rt,4 h; N BnO HO ion-exchange 72% 91 76
Scheme 2.19 Synthesis of 76, the C-2 epimer of structure 32.
The 1H NMR spectrum of 76 showed six methine protons and two methylene protons while the 13C NMR spectrum showed signals for eight different carbons. These NMR spectra are shown in Figures 2.4 and 2.5, respectively. The NMR assignments were based on COSY, HSQC and HMBC experiments.
HO H OH HO H-8 H-7 8a 1 H-5β 7 OH H-3α 5 N 3 H-8a HO H-2 H-1 H-5α H-3β H-6 76
4.00 3.50 3.00 2.50 ppm (t1)
4.0 3.0 2.0 1.0 0.0 ppm (t1) 1 Figure 2.4 The H NMR (500 MHz, D2O) spectrum of 76.
C-7 HO C-8 C-8a H OH C-1 C-6 C-3 HO C-2 C-5 8a 1 7 OH 5 N 3 HO 76
80.0 75.0 70.0 65.0 60.0 55.0 ppm (f1) 13 Figure 2.5 The C NMR (125 MHz, D2O) spectrum of 76.
1 The H NMR spectrum of 76 showed J1,8a (7.0 Hz) and J1,2 (2.5 Hz) values consistent with its proposed stereochemistry and the 1,2-diaxial like arrangement of H-8a and H-1 and the 1,2-diequatorial like trans-relationship between H-1 and H-2 (Figure 2.6). Moreover, the B ring coupling constants of 76 were essentially the same as those of 32 and its epimers 45 and 46 (Table 2.4).
43 Chapter 2 [Uniflorines A and B]
J1,8a =7.5 J1,8a =7.0 HO HO J =2.5 H HOH H H OH 1,2 HO HO 8 1 H BA OH 7 OH N 5 N 3 HO HO 32 76 C-2 epimer of 32 J =0 J =5.8 HO 1,2 HO 1,2 H H OH H H OH HO HO H H OH N N OH HO HO 45 46 C-1 epimer of 32 C-1 and C-2 diepimer of 32
Figure 2.6 J1,2 and J1,8a values of 32, 76, 45 and 46.
Table 2.4 Coupling constants (D2O) for compounds 32, 45, 46 and 76.
Coupling constants (Hz) C-1 and C-2 C-1 epimer of 32144 RING J 76 32143 diepimer of 32145 45 46 J1,2 2.5 7.5 0 5.8
J1,8a 7.0 7.5 6.3 3.5
A J2,3 6.3 6.5 6.3 8.1
J2,3 <1 7.0 4.4 2.6
J3,3 11.0 10.5 10.3 10.9
J5,5 10.8 10.5 10.9 10.7
J5,6 10.4 10.8 9.1 10.7 J 5.3 5.5 5.2 5.3 B 5,6 J6,7 9.0 9.0 9.1 9.9
J7,8 9.0 9.0 9.1 9.5
J8,8a 9.0 9.0 10.2 9.5
In summary, we have successfully developed a synthesis of compound 76, the C-2 epimer 32 in 11 steps and 0.5% overall synthesis yield from L-xylose. The several syntheses are summarized in Scheme 2.20.
44 Chapter 2 [Uniflorines A and B]
TrO OH OBn OBn OBn RO H H HH H H HO Ph BnO TrO a d RO L-xylose + O RN N + O N HO N RO Boc 1 OR OTr O O 35;R=R1 =H 39;R=H b e 36;R=Boc,R1 =H 81;R=Bn 69 70 c 37,R=Boc,R1 =Tr
BnO BnO O BnO OH H OH H O H BnO BnO S O BnO g(i) g(ii) f O OR OH N N N BnO BnO Boc BnO Boc Boc OTr OTr OTr 87a 82 88a ;R=COPh h 89;R=H
BnO OH BnO OH HO OH H H H BnO BnO HO i k OH + OH OH N N N BnO BnO HO H HO 76 C-2epimerofproposeduniflorineA 32 90 91 j
Scheme 2.20 The total synthesis of compound 76.
Reagents and conditions: (a) (E) PhCH=CHB(OH)2, allyl amine, EtOH, rt, 48 h; ion- exchange, 91%; (b) (Boc)2O, Et3N, THF, RT, 48 h, 59%; (c) TrCl, py, CH2Cl2, 40 h, 81%; o (d) Grubbs' I cat. 38a, CH2Cl2, reflux, 24 h, 90%; (e) NaH, BnBr, n-Bu4NI, THF, 50 C, 4 d,
81 (56%), 69 (3%) and 70 (9%); (f) K2OsO42H2O, NMO, acetone/water, 72 h, 75%; (g) i: o o SO2Cl2, Et3N, CH2Cl2, 0 C, 1 h then rt 2 h; ii: 1. PhCO2Cs, DMSO, 40 C, 19 h; 2.
H2SO4/H2O, THF, rt, 20 h, 54% (2 steps); (h) K2CO3, MeOH, rt, 24 h, 44%; (i) TFA, anisole, o CH2Cl2, rt, 20 h, 90 (66%) and 91 (8%); (j) DIAD, PPh3, py, 0-5 C, 48 h, 25%; (k) PdCl2, H2 (1 atm), MeOH, rt, 4h; ion-exchange, 72%.
45 Chapter 2 [Uniflorines A and B]
Table 2.5 Physical and 1H NMR spectral data for uniflorine A and 76.
Our synthesis 76 Uniflorine A142 C-2-epi-32
HO Structure H OH HO ? 8a 1 7 OH 5 N 3 HO Physical appearance Colourless micro crystals Colourless viscous liquid
25 Optical rotation [α]D - 4.4 (c 1.2, H2O) [α] D - 9.2 (c 0.2, H2O) Mass spectrometry ISMS m/z 206 (M+H+) ESI-MS +VE m/z 206 (M+H+, 100%) 1 H NMR 500 MHz, D2O 500 MHz, D2O
4.35 (1H, m, H-2) 4.14 (ddd, 1H, J2,3β = 6.3, J1,2 = 2.5,
J2,3α < 1 Hz, H-2)
4.18 (1H, t, J1,8a = J1,2 = 4.5 Hz, H-1) 3.96 (dd, 1H, J1,8a = 7.5, J1,2 = 2.5 Hz, H-1)
3.64 (ddd, 1H, J5β,6 = 10.4, J6,7 = 9.0, 3.94 (1H, t, J7,8 = J8,8a = 7.7 Hz, H-8) J5α,6 = 5.3 Hz, H-6)
3.81 (1H, dd, J6,7 = 9.0, J7,8 = 7.7 Hz, 3.47 (t, 1H, J7,8 = J8,8a = 9.0 Hz, H-8) H-7)
3.76 (1H, dd, J5β,6 = 3.8, J5α,5β = 11.8 3.29 (t, 1H, J6,7 = J7,8 = 9.0 Hz, H-7) Hz, H-5β)
3.61 (1H, dd, J5α,6 = 6.4, J5α,5β = 11.8 3.11 (dd, 1H, J5α,5β = 10.8, J5α,6 = 5.3 Hz, H-5α) Hz, H-5α)
3.14 (1H, dd, J8,8a = 7.7, J1,8a = 4.5 2.87 (brd, 1H, J3α,3β = 11.0 Hz, H-3α) Hz, H-8a)
3.04 (1H, dd, J2,3α = 5.1, J3α,3β = 12.1 2.76 (dd, 1H, J3α,3β =10.9, J2,3β = 6.7 Hz, H-3α) Hz, H-3β)
2.98 (1H, m, J2,3β = 5.1, J3α,3β = 12.1 2.16 (t, 1H, J5α,5β = J5β,6 =10.8 Hz, H- Hz, H-3β) 5β)
2.76 (1H, m, J5α,6 = 6.4, J5β,6 = 3.8, 2.11 (dd, 1H, J8,8a = 9.0, J1,8a = 8.0 Hz,
J6, 7 = 9.0 Hz, H6) H-8a)
46 Chapter 2 [Uniflorines A and B]
Table 2.6 13C NMR chemical shifts (ppm) for 76.
Our synthetic 76 Uniflorine A142 C-2-epi-32 ∆δ Nucleus 125 MHz, D2O, 125 MHz, D2O, (ppm)
TMS Ref. (δ =0 ppm) CH3CN Ref. (δ =1.47 ppm) C-1 78.1 82.7 -4.6 C-2 74.2 78.1 -3.9 C-3 60.0 59.7 0.3 C-5 65.3 55.6 9.7 C-6 72.5 70.3 2.2 C-7 79.9 79.2 0.7 C-8 81.2 74.1 7.1 C-8a 73.6 72.1 1.5
The 1H and 13C NMR spectral data for 76, as shown in Tables 2.5 and 2.6, did not match with those reported for uniflorine A. The optical rotation of 76 and uniflorine A were of the same sign but the values were different by a factor of two,
25 142 [α] D - 9.2 (c 0.2, H2O) for 76 and lit. [α]D - 4.4 (c 1.2, H2O). From the previous synthesis of 32 and its epimers we concluded that uniflorine A was not an indolizidine alkaloid. Uniflorine A was originally isolated along with uniflorine B which was assigned as structure 33 (Figure 2.7).142
HO HO OH H OH H HO 8 1 8 7 8a 1 8a 2 HO 2 7 OH 6 OH 3 5 5 N 3 N
OH 6 OH 33 Proposed structure Transposed atom numbering of unflorine B from structure 33 to that of casuarine
HO H OH 7 7a 1 HO OH 5 N 3
8 OH 15 casuarine
Figure 2.7 The transposed atom numbering from indolizidine 33 to that of pyrrolizidine casuarine 15.
47 Chapter 2 [Uniflorines A and B]
Our analysis of the NMR spectral data uniflorine B and its optical rotation indicated that this compound was the known 1,2,6,7-tetrahydroxy-3- hydroxmethylpyrrolizidine alkaloid casurine 15.56 The optical rotation and 1H NMR 3 spectral data of both compounds matched very closely, the JHH values for the two samples were in very close accord as were their 1H NMR chemical shifts (Table 2.7).
Table 2.7 NMR (D2O) and optical rotation data for casuarine 15 and uniflorine B 33.
Chemical shifts (ppm) Coupling constants (Hz) Nucleus casuarine56 uniflorine B142 casuarine56 uniflorine B142 15 33 15 33 b b H-1 4.16 4.17 (H-8) J1, 2 8.0 8.1 (J7, 8)
H-2 3.80 3.79 (H-7) J1, 7a 8.0 8.1 (J8, 8a)
H-3 3.04 3.04 (H-5) J2, 3 8.0 multiplet
H-5α 2.91 2.92 (H-3) J3, 8 3.8 3.7 (J5,6)
H-5β 3.27 3.26 (H-3) J3, 8’ 6.6 6.8 (J6,7)
H-6 4.21 4.22 (H-2) J5α, 6 4.0 3.9 (J2,3)
H-7 4.19 4.19 (H-1) J5β, 6 4.7 4.5 (J2,3’)
H-7a 3.07 3.06 (H-8a) J6, 7 a 4.5 (J1,2)
H-8 3.77 3.78 (H-6) J7, 7a 3.5 3.2 (J1,8a)
H-8’ 3.61 3.61 (H-6) J8, 8’ 11.9 11.3 (J6,6’’)
J5,5’ 12.2 12.2 (J3,3’) cauarine56 uniflorine B142 15 33 ∆ δ Nucleus Optical rotation 125 MHz, D2O 125 MHz, D2O, (ppm) TMS Ref. (δ = 0 ppm) Acetone Ref. (δ = 29.80 ppm) C-1 77.8 80.9 (C-8)b 3.1 cauarine56 C-2 76.6 79.8 (C-7) 3.2 24 [α] D +16.9
C-3 70.0 73.0 (C-5) 3.0 (c 0.8, H2O) C-5 58.0 61.1 (C-3)c 3.1 C-6 77.4 80.6 (C-2) 3.2 uniflorine B142
C-7 78.8 81.9 (C-1) 3.1 [α]D + 16.3
C-7a 72.1 75.2 (C-8a) 3.1 (c 1.1, H2O) C-8 62.2 65.5 (C-3)c 3.1 aCould not be determined due to peak overlap. bOriginal assignment based on an indolizidine structure.142 cBoth signals were assigned as C-3 in the original paper.142
The 13C NMR chemical shifts for uniflorine B however, were consistently 3.0-3.2 ppm downfield of the corresponding 13C NMR resonances for casuarine 15.
48 Chapter 2 [Uniflorines A and B]
Alternative referencing between the two samples could account for this consistent 13 13 discrepancy. Both C NMR spectra were determined at 125 MHz in D2O. The C NMR spectrum of casuarine 15 was referenced to acetone at 29.80 ppm56 while that of uniflorine B was apparently referenced to TMS (a standard not known for its water (D2O) solubility) as an internal standard.
The 13C NMR chemical shifts of the methylene carbons of uniflorine A at 65.3 and 60.0 ppm correspond more closely to C-3 and C-5, respectively, of a 1,2,6,7-tetrahydroxy-3-hydroxymethylpyrrolizidine (c.f. casuarine C-3 (70.0 ppm) and C-5 (58.0 ppm), Table 2.8) than to C-3 and C-5 of a 1,2,6,7,8-pentahydroxy- indolizidine (the methylene resonances in 32 are at C-3 (59.2 ppm) and C-5 (55.4 ppm)) Thus we assumed that uniflorine A, like uniflorine B, was also a 1,2,6,7- tetrahydroxy-3-hydroxmethylpyrrolizidine alkaloid. On this assumption we reassigned the published 1H NMR data for uniflorine A as shown in Table 2.8 by analogy with the known chemical shifts, coupling constants and assignments for casurine 15.56 A comparison of this reassigned NMR data for uniflorine A with casuarine 15 (Table 2.8) indicated that this compound was clearly different. This NMR reassignment required the numbering of uniflorine A to be transposed to that shown on the pyrrolizidine structure D in Figure 2.8. Unlike uniflorine B, this transposition retains the same sequential order of the C-atoms.
1 56 Table 2.8 The H NMR chemical shifts (ppm, D2O) for casuarine 15 and uniflorine A.142 nucleus casuarine 15 uniflorine A H-1 4.16 3.94 (H-8)a H-2 3.80 3.81 (H-7) H-3 3.04 2.76 (H-6) H-5 2.91 2.98 (H-3β) H-5’ 3.27 3.04 (H-3α) H-6 4.21 4.35 (H-2) H-7 4.19 4.18 (H-1) H-7a 3.07 3.14 (H-8a) H-8 3.77 3.61 (H-5α) H-8’ 3.61 3.76 H-5β) aOriginal assignments based on an indolizidine structure.142
49 Chapter 2 [Uniflorines A and B]
Further support for this structure came from the results of the reported NOE studies and 13C NMR data of uniflorine A.142 The original paper reported NOE correlations for uniflorine A between H-1 and H-8, H-3 and H-6 and H-5 and H-7. This would correspond to NOE correlations between H-7 and H-1, H5 and H-3 and H-8 and H-2 in the proposed structure 78 (Figure 2.8). These NOE correlations are consistent with the proposed relative configurations at C-1, C-2, C-3 and C-7 in 78. However they do not provide information on the relative stereochemistry of H-6 and
H-7a, although the latter is inferred from the magnitude of J1,7a (7.7 Hz).
HO HO H OH H OH HO 1 8 8a 8 7 1 HO 2 7 OH OH 3 N 6 5 N 3 HO 32 D 5 OH originally pr oposed structure our proposed skeletal of unflorine A structure of uniflorine A showing transposed numbering * H H OH HO H OH HO H 7 7 1 H 7a 1 HO 2 HO 2 OH 5 3 5 N 3 N OH * 8 H H H 8 OH H * OH 78 78 6-epi-casuarine *reported NOE proposed str ucture of correlations uniflorine A showing stereochemistry and systematic numbering Figure 2.8 Our proposed structure for uniflorine A.
Analysis of the 13C NMR chemical shifts for casuarine 1556 and uniflorine A142 (Table 2.9) indicated that casuarine 15 and uniflorine A had the closest matching chemical shifts. Assuming that the 13C NMR spectrum of uniflorine A was also incorrectly referenced by 3 ppm then the chemical shifts of these compounds are even more similar, except for the chemical shift of C-6 (77.4 ppm for 15 and 74.2 - 3 = 71.2 ppm for uniflorine A), consistent with uniflorine A being 6-epi-casuarine 78.
50 Chapter 2 [Uniflorines A and B]
13 Table 2.9 The C NMR (D2O) chemical shifts (ppm) for casuarine and uniflorine A.
Casuarine56 uniflorine uniflorine A Nucleus 15 A142 -3 ppm C-1 77.8 81.2 (C-8)a 78.2 C-2 76.6 79.9 (C-7) 76.9 C-3 70.0 72.5 (C-6) 69.5 C-5 58.0 60.0 (C-3) 57.0 C-6 77.4 74.2 (C-2) 71.2 C-7 78.8 78.1 (C-1) 75.9 C-7a 72.1 73.6 (C-8a) 70.6 C-8 62.2 65.3 (C-5) 62.3 a Original assignments based on an indolizidine structure.142
To summarize the above investigations, we successed in the synthesis of 76, the C-2 epimer of structure 32. However the spectroscopic data of this compound did not match with those of the natural product uniflorine A. We now believe that uniflorines A and B are not indolizidine alkaliods but the pyrrolizidine alkaloids, 6- epi-causuarine 78 and casuarine 15, respectively based upon an analysis of the NMR spectroscopic data given for the natural products (Figure 2.9).
OH HO OH H OH H HO 7 8 8a 1 7a 1 OH HO 5 OH 5 N 3 N 3 HO 8 OH 32 78 Originally proposed Revised structure structure of of (-)-uniflorine A (-)-unflorine A OH H OH HO H OH HO OH HO OH N N
OH OH 15 33 Casuarine Originally proposed [Uniflorine B structure of is casuarine] unflorine B
Figure 2.9 Revised structures for uniflorines A and B.
51 Chapter 2 [Uniflorines A and B]
2.2 Synthesis of (+)-uniflorine A (ent-6-epi-casuarine).
To verify our hypothesis that uniflorine A was actually 6-epi-casuarine, we developed a synthesis of its enantiomer, (+)-uniflorine A, from D-xylose. The proposed synthsis of (+)-uniflorine A is shown in Scheme 2.21. The first investigation to synthesize this compound used D-xylose instead of L-xylose, since D- xylose is twelve times cheaper than L-xylose. Moreover, we also synthesized (-) uniflorine A and this will be will summarized at the end of this Chapter.
OH OH OH O D-xylose Ph OH Ph O + 1. Petasis 4. RCM NH2 NR OH 3. Trans-acetalation NBoc OH + B(OH) Ph 2 92;R=H 2. N-Boc 94 protection 93;R=Boc
OH O OR OBn OH H RO O BnO H O H O OR 5. DH 7. Acetonide and N-Boc deprotection RO BnO NBoc OH NBoc OR NH OBn
96;R=H 98;R=H 95 6. O-Bn 8. O-TBS protection protection 97;R=Bn 99;R=TBS
BnO OBn H HO H OH 9. Mitsunobu 11. O-Bn cyclization deprotection BnO OBn OH N HO N
OR OH ent-78 100;R=TBS 10. O-TBS deprotection (+)-uniflorine A 101;R=H
Scheme 2.21 Proposed synthesis of (+)-uniflorine A.158
2.2.1 Petasis reaction and N-Boc protection
The amino tetraol 92 (Scheme 2.22) was prepared in high yield (91%) similar to it enantiomer 35 (Scheme 2.1) from the Petasis reaction using D-xylose rather than L-xylose. This compound was then converted to its N-Boc derivative in an improved yield of 75% using MeOH rather than THF as the solvent. This variation prevented formation of the O-Boc derivative due to the presence of more reactive alcohol
52 Chapter 2 [Uniflorines A and B] methanol. The NMR spectroscopic data of 92 and 93 were identical to that of their enantiomers, 35 and 36, respectively.143
OH OH OH OH D-xylose (Boc)2O, Et3N + EtOH, rt, 48 h Ph OH Ph OH NH 2 ion-exchange MeOH, rt, 3 d NH OH NBoc OH + 91% 75% B(OH)2 Ph 92 93
Scheme 2.22 Synthesis of structures 92 and 93.
2.2.2 Trans-acetalation
The terminal diol functionality of 93 was selectively protected as its acetonide derivatives using 2,2-dimethoxypropane (1.06 eq.) and pyridinium para- toluenesulfonate (0.1 eq.) as a catalyst in anhydrous acetone with stirring under an atmosphere of N2 for 20 h (Scheme 2.23).
Scheme 2.23 Synthesis of structures 94 and 94a.
TLC analysis showed two product spots at Rf 0.38 and 0.25 in 30:70 EtOAc/petrol. These two compounds were readily separated by column chromatography. The 1H NMR spectrum of both compounds showed two different methyl signals. The less polar and major product showed methyl signals at 1.43 and 1.36 ppm and the more polar and minor product at 1.42 and 1.39 ppm, corresponding to an isopropylidene group. Moreover, the ESI mass spectrum of these compounds confirmed their formulas as C24H35NO6. This reaction could produce dioxolane and/or dioxane derivatives (Figure 2.10). Redlich et al.159 reported that the quarternary carbon of dioxolane and dioxane derivatives had 13C NMR chemical shifts around 109 and 99 ppm, respectively (Figure 2.10).
53 Chapter 2 [Uniflorines A and B]
δC δC
109 ppm 99 ppm O O OO
dioxolane dioxane
Figure 2.10 The 13C NMR chemical shifts of dioxolane and dioxane derivatives.
The major and minor products showed signals at 109.2 and 109.1 ppm, respectively in their 13C NMR spectra indicating both were dioxolane derivatives. From COSY and HMBC experiments the major and minor products were determined to be the acetonides 94 and 94a, respectively (Figure 2.11).
OH O OH O Ph 7 5 3 Ph 7 5 3 O 1 COSY correlations NBoc OH N O HMBC correlations Boc 1 OH 94 94a
Figure 2.11 COSY and HMBC correlations of 94 and 94a.
The HMBC correlations of 94 indicated that the isopropylidene group was connect to the terminal 1,2-diol as the quaternary carbon of the isopropylidene group showed a three bond correlation to the methylene protons (H-1) while the COSY and HMBC correlations of 94a indicated the isopropyldiene group was connected to the oxygens at positions 2 and 3. The regioisomer 94a could be recycled back to 93 by hydrolysis with TFA (0.5 eq.) in MeOH/water (3:1, 10mL/mmoL) at rt for 36 h and the crude product was reprotected to give 94 in 49% overall yield.
2.2.3 Ring Closing Metathesis (RCM)
The diene 94 was converted to the 2,5-dihydropyrrole 95 in 94% yield using
Grubbs’ first generation ruthenium catalyst 38a at reflux in CH2Cl2 for 20 h (Scheme 2.24). The 1H NMR spectrum of the 2,5-dihydropyrrole 95 showed two alkene protons at 5.95 (brd, 1H, J 6.5) and 5.86 (brd, 1H, J 6.5) ppm.
54 Chapter 2 [Uniflorines A and B]
OH O OH O H Ph O O 38a, CH2Cl2 NBoc OH reflux, 20 h NBoc OH 94% 94 95
Scheme 2.24 Synthesis of dihydropyrrole 95.
2.2.4 Dihydroxylation (DH)
The 2,5-dihydropyrrole 95 underwent an osmium(VIII)-catalyzed syn-DH reaction to furnish the tetrol 96 as a single diastereomer in 68% yield (Scheme 2.25). The stereochemical outcome of this DH reaction was expected due to the stereodirecting effect of the C-2 pyrrolidine substituent in 96. The structure of 96 was fully supported by it NMR spectroscopic data and its ESI-MS analysis (m/z 364 [M+H+]). The configuration of this diol was established from ROESY NMR studies on the final product ent-78.
OH O OH O HO H H O O K2OsO4 H2O, NMO HO NBoc OH acetone/H2O, rt NBoc OH 24 h, 68%
95 96
Scheme 2.25 Synthesis of tetrol 96.
2.2.5 O-Benzylation
The O-benzylation reaction of tetrol 96 afforded only the O-benzyl protected derivative 97 in 86% yield using standard reaction conditions. Surprisingly, this reaction did not produce the corresponding oxazolidinone or oxazinanone side products. Their formation may have been hindered by the bulky isopropylidene ring.
OH O OBn O HO H BnO H O O NaH, BnBr, Bu4NI HO BnO NBoc OH THF, 24 h, 86% NBoc OBn
96 97
Scheme 2.26 Synthesis of O-benzyl derivative 97.
55 Chapter 2 [Uniflorines A and B]
2.2.6 Acetonide and N-Boc deprotection and O-TBS protection
Treatment of 97 under acidic conditions (HCl/MeOH) resulted in N-Boc and acetonide hydrolysis and gave the amino diol 98 in 78% yield. Regioselective O- silylation of 98 with TBSCl (1.1 eq.) /Et3N/DMAP gave the primary silyl ether 99 in 60% yield along with the bis-O-TBS derivative 99a (12%) and unreacted diol 98 (22%) (Table 2.10, entry 6). The other methods shown in Table 2.10 (entries 1-5) provided lower yields of the desired compound 99. DMAP was found to be essential for this reaction to occur. The proposed reaction mechanism of O-TBS protection catalyzed by DMAP is shown in Scheme 2.28.
BnO OBn O BnO OBn OH H O H OH a BnO BnO NBoc OBn NH OBn
97 98
OBn OH OBn OTBS BnO H BnO OTBS H OTBS b BnO + BnO NH OBn NH OBn
99 99a Scheme 2.27 Synthesis of structures 99 and 99a.
Reagents and conditions: (a) HCl/MeOH, rt, 24 h, 78%; (b) TBSCl, Et3N, DMAP,
CH2Cl2, rt, 5 h, 60%.
Table 2.10 The O-TBS protection of diol 98.
Entry Reagents and conditions Results 1 TBSCl (1.0 eq.), imdazole (1.5 eq.), DMF, 0 °C, 2 d No reaction
2 TBSCl (1.2 eq.), imdazole (2.5 eq.), DMF, N2, rt, 2 d No reaction
3 TBSCl (1.5 eq.), Et3N (1.5 eq.), THF, rt, 4 d No reaction
4. TBSCl (1.2 eq.), Et3N (1.1 eq.), DMAP (0.05 eq.), 99 (14%) and 98 (63%)
CH2Cl2, N2, rt, 1 d
5 TBSCl (2.4 eq.), Et3N (2.2 eq.), DMAP (0.1 eq.), 99 (33%) and 99a (58%)
CH2Cl2, rt, 15 h
6 TBSCl (1.1 eq.), Et3N (1.1 eq.), DMAP (0.05 eq.), 99 (60%), 99a (12%) and
CH2Cl2, N2, rt, 5 h 98 (22%)
56 Chapter 2 [Uniflorines A and B]
Et3N H N N N O TBS Cl HO HO OTBS BnO BnO H + Et NH BnO OBn H 3 N N N BnO OBn NH TBS NH BnO Si Cl + BnO HO OH 99 N BnO H BnO OBn NH 98 N BnO
Scheme 2.28 The proposed reaction mechanism for the synthesis of 99.
2.2.7 Mitsunobu cyclization
The primary silyl ether 99 underwent cyclization under Mitsunobu reaction conditions using pyridine155,160 as the solvent to give a mixture (ca 4 : 1) of the desired pyrrolizidine 100 and an indolizidine product 100a in a combined yield of 30% after purification of the crude reaction mixture by column chromatography (Scheme 2.29). The undesired indolizidine product arose from first base catalysed O- TBS migration to the secondary hydroxyl group in 100 followed by Mitsunobu cyclization onto the primary carbon of the butyl side chain (Scheme 2.30). These cyclized products could be separated by a second and more careful column chromatographic separation.
OBn OH OBn BnO H BnO H OBn BnO H OTBS OBn BnO DIAD, Ph3P, py BnO OBn + BnO NH OBn N N rt, 3 d, 30% OTBS OTBS 99 100 100a
Scheme 2.29 Synthesis of bicyclic compounds 100 and 100a.
57 Chapter 2 [Uniflorines A and B]
O O i Pri O NN OPri Pr O NN OPri
O O PPh3
PPh3
PPh3 BnO H OBn BnO OBn O H OTBS -Ph3PO BnO OBn BnO N N OBn OTBS H 10068 O H 96 i PriO N N OPr O PHPh3 OO PPh3 O i Pr O i N N OPri i Pr O N N OPr H i i N N OPr O Pr O O PPh3 H O BnO OBn O BnO H OBn OH OBn O BnHO OTBS H OTBSOTBS O TBS O BnO BnO NH OBn NH OBn OBn TBS-migration OBn
OPri O OBn OTBS N PPh3 BnO H OPPh3 HN BnO O NH OBn PriO
-Ph3PO
OBn BnO H OBn BnO N OTBS 68a 96a100a
Scheme 2.30 The proposed reaction mechanism for the synthesis of 100 and 100a.
2.2.8 O-TBS and O-Bn deprotection
Acid hydrolysis of 100 gave the primary alcohol 101 in 66% yield.
144,147 22 Hydrogenolysis of 101 using PdCl2/H2 gave ent-78 ([α] D + 6.6 (c 0.35, H2O)
142 (Lit. for (-)-uniflorine A, [α]D -4.4 (c 1.2, H2O)), in 74% yield after ion-exchange chromatography (Scheme 2.31).
58 Chapter 2 [Uniflorines A and B]
BnO OBn H BnO H OBn BnO H OBn
BnO OBn a BnO OBn b BnO OBn N N N
OTBS OH OH 100 101 ent-78 (+)-uniflorine A Scheme 2.31 Synthesis of (+)-uniflorine A ent-78.
Reagents and conditions: (a) HCl/MeOH, rt, 5 h, 66%; (b) PdCl2, H2 (1 atm), MeOH, rt, 3 h;. ion-exchange, 74%.
The total synthesis of ent-78 was completed in 11 synthetic steps from D- 1 xylose. The H NMR spectal data (D2O) of ent-78 (Figure 2.12) and that of the natural product uniflorine A were essentially identical (∆δH = 0.00-0.02 ppm). The 13 C NMR signals of ent-78 (Figure 2.13) (in D2O with MeCN as an internal reference at 1.47 ppm) however, were all consistently 2.1-2.2 ppm upfield of those reported for the natural product (Table 2.11). HOH OH
7 1 HO7a OH 5 N 3 8 OH ent-78 H-7 H-1 H-8’ H-5β H-5α H-3 H-6 H-2 H-8 H-7a
4.00 3.50 3.00 pm (t1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1)
1 Figure 2.12 The H NMR (500 MHz, D2O) of ent-78.
HOH OH 7 1 C-7 C-7a HO7a OH C-2 C-6 5 N 3 C-1 C-8 C-5 8 C-3 OH ent-78
80 70 60 50 40 30 20 10 0 ppm (f1) 13 Figure 2.13 The C NMR (125 MHz, D2O) of ent-78.
59 Chapter 2 [Uniflorines A and B]
It was noted earlier147 (Table 2.7) that while the 1H NMR spectral data reported for uniflorine B and casuarine were also essentially identical, the 13C NMR shifts reported for casuarine were all consistently 3.0-3.2 ppm upfield of the corresponding 13C NMR resonances reported for uniflorine B.142 We suggested that alternative referencing between the two samples accounted for this consistent discrepancy.147 Thus the consistent differences in the 13C NMR chemical shifts between synthetic ent-78 and that of (-)-uniflorine A can also be ascribed to the differences in referencing between the different samples. Unfortunately we have not been able to obtain a copy of the NMR spectra of uniflorine A for comparison purposes from the original authors.142 The observed cross-peaks in the ROESY spectrum of ent-78 were fully consistent with the configurational assignment of ent- 78 as shown in Figure 2.14. Thus our synthesis of ent-78, the enantiomer of (-)- uniflorine A, provides unequivocal proof that (-)-uniflorine A is 6-epi-casuarine. This synthesis also establishes the absolute configuration of (-)-uniflorine A as that shown in the Figure 2.4. (-)-Uniflorine A and 3-epi-casuarine58 therefore represent the two known natural product stereoisomers of casuarine.58 * HO H H H OH 7 1 H * HO 7a 2 *ROESY 5 N 3 H OH * correlations 8 H H H H * OH ent -7 8
Figure 2.14 The ROESY correlations of (+)-uniflorine A 78.
60 Chapter 2 [Uniflorines A and B]
Table 2.11 Physical and spectral data for (-)-uniflorine A142 and ent-78.158
Uniflorine A 142 Our synthetic ent-78
Structure HO H OH HOH OH 7 7a 1 HO OH 7 7a 1 5 N 3 HO OH 5 N 3 8 OH 8 78 OH ent-78 (Revised structure) Physical Appearance Colourless Microcrystals brown foamy solid.
22 Optical Rotation [α]D -4.4 (c 1.2, H2O) [α] D +6.6 (c 0.35, H2O). Melting Point 174 - 178 °C - Mass Spectrometry ISMS m/z 206 (M+ + H) ESI-MS +ve m/z 206 (M + H+, 100 %) 1 H NMR 500 MHz, D2O 500 MHz, D2O
4.35 (1H, m, H2) 4.34 (1H, app. q, J5α,6 = J5β,6 = J6,7 = 4.8 Hz, H-6)
4.18 (1H, t, J1,8a = J1,2 = 4.5, H1) 4.17 (1H, t, J6,7 = J6,7a = 4.5 Hz, H-7)
3.94 (1H, t, J7,8 = J8,8a = 7.7, H8) 3.92 (1H, t, J1,2 = J1,7a = 7.5 Hz, H-1)
3.81 (1H, dd, J6,7 = 9.0, J7,8 = 7.7, 3.79 (1H, t, J1,2 = J2,3 = 8.5 Hz, H-2) H7)
3.76 (1H, dd, J5β,6 = 3.8, J5α,5β = 3.76 (1H, dd, J3,8’ = 3.8, J8,8’ = 11.8 Hz, H- 11.8, H5β) 8’)
3.61 (1H, dd, J5α,6 = 6.4, J5α,5β = 3.61 (1H, dd, J3,8 = 6.5, J8,8’ = 11.5 Hz, H-8) 11.8, H5α)
3.14 (1H, dd, J8,8a = 7.7, J1,8a = 4.5, 3.12 (1H, dd, J1,7a = 7.5, J7,7a = 5.0 Hz, H- H8a) 7a)
3.04 (1H, dd, J2,3α = 5.1, J3α,3β = 3.02 (1H, dd, J5β,6 = 5.8, J5α,5β = 11.8 Hz, H- 12.1, H3α) 5β)
2.98 (1H, m, J2,3β = 5.1, J3α,3β = 2.96 (1H, dd, J5α,6 = 5.3, J5α,5β = 12.3 Hz, 12.1, H3β) H-5α)
2.76 (1H, m, J5α,6 = 6.4, J5β,6 = 3.8, 2.74 (1H, m, H-3)
J6, 7 = 9.0, H6) 13 C NMR 125 MHz, D2O (Ref. TMS) 125 MHz, D2O (Ref. CH3CN) ∆δ (ppm) 81.2 (d, C-8) 79.1 (C-1) 2.1 79.9 (d, C-7) 77.8 (C-2) 2.1 78.1 (d, C-1) 76.0 (C-7) 2.1 74.2 (d, C-2) 72.1 (C-6) 2.1
73.6 (d, C-8a) 71.5 (C-7a) 2.1 72.5 (d, C-6) 70.3 (C-3) 2.2 65.3 (t, C-5) 63.2 (C-8) 2.1 60.0 (t, C-3) 57.8 (C-5) 2.2
61 Chapter 2 [Uniflorines A and B]
In conclusion we have successfully prepared (+)-uniflorine A (ent-78) in 11 synthetic steps and 1.4% overall yield from D-xylose. The total synthesis is summarized in Scheme 2.32.
OH OH OH O D-xylose Ph OH O + a c Ph d NH2 NR OH NBoc OH + B(OH) Ph 2 92;R =H b 94 93;R =Boc
OH O OBn OH H RO OR O BnO O H H OR e O g RO BnO NBoc OH NBoc OR NH OBn 96;R=H 98;R=H 95 f h 97;R=Bn 99;R=TBS
BnO OBn H HO H OH i k BnO OBn OH N HO N
OR OH ent-78 100;R=TBS j (+)-uniflorine A 101;R=H
Scheme 2.32 Total synthesis of (+)-uniflorine A ent-78.
Reagents and conditions: (a) (E) PhCH=CHB(OH)2, allyl amine, EtOH, rt, 3 d; ion- exchange, 91%; (b) (Boc)2O, Et3N, MeOH, rt, 3 d, 75%; (c) DMP, PPTS, acetone, rt, 20 h, o 55%; (d) Grubbs' I 38a, CH2Cl2, 50 C, 20 h, 94%; (e) K2OsO4H2O, NMO, acetone/H2O, rt,
24 h, 68%; (f) NaH, BnBr, Bu4NI, THF, 24 h, 86%; (g) HCl/MeOH, rt, 24 h, 78%; (h)
TBSCl, Et3N, CH2Cl2, rt, 5 h, 60%; (i) DIAD, Ph3P, py, rt, 3 d, 30%; (j) HCl/MeOH, rt, 5 h,
66%; (k) PdCl2, H2 (1 atm), MeOH, rt, 3 h;. ion-exchange, 74%.
62 Chapter 2 [Uniflorines A and B]
2.3 Synthesis of the correct structure of uniflorine A (78)
Scheme 2.33 shows the total synthesis of the correct structure of natural uniflorine A 78. This synthesis was the same as the previous one except that the starting material was D-xylose rather than L-xylose. The yields and physical properties of each product are summarized in Table 2.12.
OH OH OH O L-xylose Ph OH Ph O + a c d NH2 NR OH NBoc OH + B(OH) Ph 2 35;R =H b 102 36;R =Boc
OH O OR O OBn OH H RO BnO H O H O OR e g RO BnO NBoc OH NBoc OR NH OBn
104;R=H 106;R=H 103 f h 105;R=Bn 107;R=TBS
BnO H OBn HO H OH i k BnO OBn HO OH N N
OR OH 78 108;R=TBS j (-)-uniflorine A 109;R=H Scheme 2.33 The synthetic route for (-) uniflorine A.
Reagents and conditions: (a) (E) PhCH=CHB(OH)2, allyl amine, EtOH, rt, 3 d; ion-
exchange, 92%; (b) (Boc)2O, Et3N, MeOH, rt, 3 d, 80%; (c) DMP, PPTS, acetone, rt, 20 h, o 64%; (d) Grubbs' I 38a, CH2Cl2, 50 C, 18 h, 97%; (e) K2OsO4H2O, NMO, acetone/H2O, rt,
18 h, 72%; (f) NaH, BnBr, Bu4NI, THF, 24 h, 96%; (g) HCl/MeOH, rt, 18 h, 81%; (h)
TBSCl, DMAP, imidazole, CH2Cl2, rt, 48 h, 85%; (i) DIAD, Ph3P, Et3NHCl, py, rt, 3 d,
76%; (j) HCl/MeOH, rt, 18 h, 90%; (k) PdCl2, H2 (1 atm), MeOH, rt, 24 h;. ion-exchange, 87%.
63 Chapter 2 [Uniflorines A and B]
Table 2.12 Comparison of the results for the synthesis of ent-78 and 78.
Synthetic step D-xylose series L-xylose series OH OH OH OH Ph OH Ph OH
1. Petasis reaction NH OH NH OH 92 (yield = 91%) 35 (yield = 92%) 25 25 [α] D +27 (c 0.06, MeOH) [α] D -17 (c 0.3, MeOH)
OH OH OH OH Ph OH Ph OH
2. N-Boc protection NBoc OH NBoc OH 93 (yield = 75%) 36 (yield = 80%) 25 25 [α] D +29 (c 2.3, CHCl3) [α] D -50 (c 3.0, CHCl3)
OH O OH O Ph O Ph O
3. Diol protection NBoc OH NBoc OH
94 (yield = 55%) 102 (yield = 64%) 23 22 [α] D +41 (c 10.1, CHCl3) [α] D -36 (c 6.5, CHCl3)
OH O OH O H O H O
4. RCM NBoc OH NBoc OH
95 (yield = 94%) 103 (yield = 97%) 22 21 [α] D -37 (c 5.75, CHCl3) [α] D +125 (c 4.5, CHCl3)
OH O OH O HO H HO O H O HO HO 5. DH NBoc OH NBoc OH 96 (yield = 68%) 104 (yield = 72%) 22 22 [α] D -32 (c 4.8, CHCl3) [α] D +32 (c 4.9, CHCl3)
OBn O OBn O BoO H BnO O H O 6. Acetonide and N- BnO BnO NBoc OBn Boc deprotection NBoc OBn 97 (yield = 86%) 105 (yield = 96%) 25 [α] 23 -50 (c 5.4, CHCl ) [α] D +45 (c 4.26, CHCl3) D 3
64 Chapter 2 [Uniflorines A and B]
Table 2.12 (cont’d.) Comparison of the results for the synthesis of ent-78 and 78. Synthetic step D-xylose series L-xylose series
BnO OBn OH BnO OBn OH H OH H OH BnO BnO 7. Benzylation NH OBn NH OBn 98 (yield = 78%) 106 (yield = 81%) 21 22 [α] D +35 (c 1.45, CHCl3) [α] D -27 (c 3.7, CHCl3)
BnO OBn OH BnO OBn OH H OTBS H OTBS 8. O-TBS BnO BnO NH OBn NH OBn protection 99 (yield = 60%) 107 (yield = 85%) 22 22 [α] D +21 (c 0.7, CHCl3) [α] D -17 (c 4.6, CHCl3)
BnO OBn BnO H OBn H
BnO OBn BnO OBn 9. Mitsunobu N N cyclization OTBS OTBS 108 (yield = 76%) 100 (yield = 30%) 20 ND [α] D -34 (c 0.4, CHCl3) OBn BnO H OBn BnO H
BnO OBn BnO OBn N 10. O-TBS N deprotection OH OH 101 (yield = 66%) 109 (yield = 90%) 23 20 [α] D +34 (c 1.3, CHCl3) [α] D -35 (c 1.3, CHCl3)
HO H OH HO OH H HO OH N HO OH N 78 OH (yield = 87%) OH 11. Debenzylation ent-78 (yield = 74%) (-)-unif lorine A (+)-unif lorine A 23 [α] D -3.7 (c 1.2, H2O) 22 [α] +6.6 (c 0.35, H O) 142 D 2 Lit ; [α]D -4.4 (c 1.2, H2O) mp. 163.2-167.8 °C Lit142; mp. 174-178 °C * ND = No determined
65 Chapter 2 [Uniflorines A and B]
For each related reaction step in these separate syntheses the yields were very similar, except for steps 8-10 (Table 2.12). In step 8 (O-TBS protection) the base was changed from Et3N to imidazole, resulting in an improved yield for 107 of 85%. In step 9 the yield for the Mitsunobu cyclization was dramatically improved to 76% with little or no formation of the undesired product (indolizidine). Recently, 161 Anderson and Chapman reported that the addition of Et3NHCl salt to the
Mitsunobu reaction can improve the yield. Following this method we added Et3NHCl salt (1.0 eq.) to the Mitsunobu reaction in the cyclization of 99 to 100. This modification gave 100 in a much improved yield of 46%. In the enantiomeric series 107 was converted to 108 in 76% yield under same reaction conditions. A proposed reaction mechanism for the role of Et3NHCl salt in the reaction is showed in Scheme
2.34. We believe that the Et3NHCl salt may act as a buffer and prevent the deprotonation of the secondary hydroxyl group in 108 that results in O-TBS migration.
O O
PriO NN OPri PriO NN OPri
O O PPh3
PPh3
O H PriO N N OPri HNEt PPh 3 O 3 O OBn OH BnO H PriO NN OPri OTBS BnO + Et3N O PPh3 NH OBn
PPh3 BnO H OBn OBn O Et NH BnO H 3 OTBS CO Pri BnO OBn H 2 N BnO + N NH N OBn i Pr O2C 108 OTBS H -Ph3PO NEt 3
Scheme 2.34 The proposed cyclization reaction mechanism in the presence of Et3N·HCl.
66 Chapter 2 [Uniflorines A and B]
The reaction step 10 was improved from 66% to 90% yield. Compounds 101 and 109 were obtained using the same reaction conditions, however the improved yield for 109 was most likely due to the larger scale of this reaction.
The overall yields for the total synthesis ent-78 and 78 are shown in Table 2.13.
Table 2.13 Overall yields of uniflorine A and its enantiomer.
HO H OH HO H OH
HO OH HO OH N N Structure ent-78 OH 78 OH
From D-xylose From L-xylose
Overall yeild 1.4% 13%
During the writing of this thesis, Goti et al.162 reported the total synthesis of (-)-uniflorine A 78 in 9 steps and 11% overall yield. Their key steps were a 1,3- dipolar cycloaddition reaction, a Tamao-Fleming reaction and a Mitsunobu reaction. Their total synthesis (Scheme 2.35) began with a stereoselective cycloaddition reaction of the nitrone 110 with the alkene 111 in CH2Cl2 to give the isoxazolidine 112. The lactam 113 was obtained from cleavage of the N-O bond in 112 with Zn/HOAc followed by attack of the resulting amine onto the ester carbonyl group to form the lactam 113 in 93% yield. The OH group at the C-6 position of the isoxazolidine 112 was protected by acetylation. The Tamao-Fleming reaction
(Hg(CF3CO2)2, TFA, AcOH, AcOOH) was used to convert the silyl group in 113 to the OH group in lactam 114 with retention of configuration. Benzylation of the OH group at C-7 position of lactam 114 with BnOC(=NH)CCl3, and CF3SO3H in Et2O also resulted in deprotection of the acetyl group at C-6 and provided compound 115 in 75% yield. The pyrrolizidine 117 was obtained by inversion of the stereochemistry at the C-6 position of compound 115 by a Mitsunobu reaction with BzOH, PPh3, DIAD in THF that gave 116 in 75% yield, followed by reduction of the lactam carbonyl group and deprotection of the benzoylated group with LiAlH4. Debenzylation of the pyrrolizidine 117 gave (-) uniflorine A 78 in 71% yield using
67 Chapter 2 [Uniflorines A and B] standard hydrogenolysis conditions after purification by ion-exchange chromatography with Dowex 50WX8 resin. The 1H NMR and 13C NMR spectroscopic data was idectical with those from our previous synthesis.158 This
21 synthetic material 78 had mp 117-180 °C and [α] D -6.9 (c 0.42, H2O).
BnO OBn SiMe2Ph PhMe2Si H OBn a b OBn + N CO Et EtO2C OBn 2 O N O OBn 110 111 112
PhMe2Si H OBn HO H OBn c d AcO OBn AcO OBn N N O OBn O OBn
113 114
BnO H OBn BnO H OBn e f HO OBn BzO OBn N N OBn O O OBn
115 116
BnO H OBn HO H OH g HO OBn HO OH N N OBn OH
117 78 Scheme 2.35 Total synthesis (-)-uniflorine A by Goti et al.162
Reagents and conditions: (a) CH2Cl2, rt, 36 h, 79%; (b) i: Zn, AcOH/H2O, 60–65 °C, 5 h,
93%; ii: Ac2O, Py, rt,15 h, 100%; (c) Hg (CF3CO2)2, TFA, AcOH, AcOOH, CHCl3, 82%; (d) i: BnOC(=NH)CCl3, CF3SO3H, Et2O, rt, 3 h; ii: Ambersep 900 OH, MeOH, rt, 15 h, 75% (2 steps); (e) BzOH, PPh3, DIAD, THF, rt (75%); (f) LiAlH4, THF, reflux, (45%); (g) H2, 10%
Pd/C, MeOH, HCl, rt, then Dowex 50WX8, 6% NH4OH (71%).
A chronological history of the synthesis and discovery of uniflorine A and related compounds is shown in Table 2.14.
68 Chapter 2 [Uniflorines A and B]
Table 2.14 A chronological history of the synthesis and discovery of uniflorine A and related compounds.
Year Isolation Synthesis Structure
145 HO Fleet et al. H OH HO 8 8a 1 7 2 6 OH 5 N 3 1996 HO 46
142 HO Arisawa et al. H OH HO 8 1 7 8a 2000 2 6 OH 5 N 3 HO Proposed structure32 143 HO Pyne and Davis H OH HO 8 8a 1 7 2004 2 6 OH 5 N 3 HO 32 144 HO HO Mariano et al. H OH H OH HO 8 HO 8 8a 1 1 7 7 8a 2005 2 2 6 OH 6 OH 5 N 3 5 N 3 HO HO 45 46 146 HO HO HO Dhavale et al. H OH H OH H OH HO 8 HO 8 HO 8 8a 1 8a 1 8a 1 7 7 7 2006 2 2 2 6 OH 6 OH 6 OH 5 N 3 5 N 3 5 N 3 HO HO HO 32 56 57 147 HO HO Pyne et al. H OH H OH HO 8 HO 8 1 8a 1 7 8a 7 2008 2 2 6 OH 6 OH (This thesis) 5 N 3 5 N 3 HO HO 46 76 Pyne and HO H OH 158 HO OH 2009 Ritthiwigrom N
(This thesis) OH ent-78 Pyne and HO H OH HO OH 2009 Ritthiwigrom N
(This thesis) OH 78 162 Goti et al. HO H OH HO OH 2009 N
OH 78
69 Chapter 3 [Casuarine]
CHAPTER 3 SYNTHESIS OF CASUARINE
3.1 Isolation and biological activities of casuarine
Casuarine equisetifolia;163 or commonly called, Australian pine, Filao or beach she oak is a plant in the family Casuarinaceae, native to South East Asia, islands of the western Pacific Ocean (including French Polynesia, New Caledonia, Vanuatu), Australia (northern Territory, north and east Queensland, and northeastern New South Wales) and West Africa. It is an evergreen tree that grows up to 6-35 m in height. The slender leaves contain much-branched green to grey-green twigs 0.5-1 mm in diameter. The flowers are unisexual with female and male flowers that differ in appearance and which are found on same tree. The female flowers are on short peduncles while the male flowers are spikes 0.7-4 cm long. The immature fruits are greenish colour and mature fruits are light brown colour. The fruit has an oval woody structure of 10-24 mm in length and 9-13 mm in diameter (Figure 3.1). Please see print copy for Binomial name image Casuarina equisetifolia L. Scientific classification Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Fagales Family: Casuarinaceae Genus: Casuarina Species: C. equisetifolia
Figure 3.1 Casuarina equisetifolia. 163
70 Chapter 3 [Casuarine]
The first pentahydroxylated pyrrolizidine alkaloid, with 6 contiguous stereogenic centres and functional groups on all of the 8 carbon atoms, was isolated in 1994 from the bark of Casuarina equisetifolia L. This alkaloid was named casuarine 15 (1R,2R,3R,6S,7R,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizi- dine, by Nash et al.56 This investigation started with a GC-MS analysis of the per- trimethylsilylated bark extract which revealed a pentahydroxylated pyrrolizidine alkaloid and its glycoside as the major nitrogen containing compounds. The 75% aqueous ethanol bark extract was purified by ion-exchange column chromatography + with Amberlite CG 120 (NH4 form) which was eluted with 0.1 M NH4OH to afford first the glycoside of casuarine 15a and then casuarine 15 itself. The absolute configuration of casuarine 15 was established by X-ray crystallographic analysis.56
HO HO HO H OH OH HO O OH H HO OH HO O OH N N
OH OH 15 15a
Figure 3.2 The structures of casuarine 15 and casuarine 6-O-α-D-glucoside 15a.
The bark of Casuarina equisetifolia L was prescribed as a remedy for Mrs E. Langford to treat breast cancer in Western Somoa.57 Extracts of the wood, bark and leaves of this plant have also been claimed to be useful for the treatment of diarrhoea, dysentery and colic.56
Eugenia jambolana and E. cumini and Syzygium cumini and S. jambolanum 164-166 are plants in the family Myrtaceae, native to Bangladesh, India, Nepal, Pakistan and Indonesia. An evergreen tree it grows up to 30 m in height and lives more than 100 years. The seed, fruits and leaves have been well known in India for various alternative therapeutic uses including diabetes and bacterial infections and the fruits and leaves also have been used as controlling blood pressure and gingivitis (Figure 3.3). 164-166
71 Chapter 3 [Casuarine] Please see print copy for Binomial name image Eugenia jambolana L. Scientific classification Kingdom: Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Rosids Order: Myrtales Family: Myrtaceae Genus: Syzygium Species: S. cumini
Figure 3.3 Eugenia jambolana.164-166
In 1996, Wormald et al.57 isolated casuarine 15 and the glucoside alkaloid 15a (Figure 3.3) from the leaves and the seeds of Eugenia jambolana.
In 2000,142 Arisawa et al. reported uniflorines A and B from the leaves of the tree (Eugenia uniflora L), these compounds have been shown to be 6-epi-casuarine 78 and casuarine 15, respectively.
Casuarine was shown to be a potent inhibitor of glucosidase I with 72% inhibition at 5 µg/mL60. Kato et al.55 reported that casuarine was a good inhibitor against rat intestinal maltase and amyloglucoside (IC50 = 0.7 µM) and rice α- glucosidase (IC50 = 1.2 µM) and was less active against rat intestinal isomaltase (IC50 = 3.9 µM).
Myrtus communis L., commonly known as Myrtle or True Myrtle, belongs to the family Myrtaceae. It originates from the Mediterranean, North African and Western Asia regions. Myrtle is a shrubby plant which can grow between 0.5-3 m in height. It has thick, interlaced, globe-like, irregular, large and expansive foliage in the upper section of the tree. Its evergreen, aromatic, coriaceous, lanceolate, dark
72 Chapter 3 [Casuarine] green leaves are 2-3 cm in length, which are normally attached to the branches in an opposite direction. The myrtle tree has white with yellow tinted flowers of 2-3 cm in diameter and the fruit has small, purplish or blackish globe-like berries that can grow up to 1 cm in diameter.167-169 Please see print copy for image Binomial name Myrtus communis
L. Scientific classification Kingdom: Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Rosids Order: Myrtales Family: Myrtaceae Myrtus Genus: L. Species: Myrtus communis
Figure 3.4 Myrtus communis.167-169
Casuarine 15 was the major alkaloid that was isolated from Myrtus communis L. in 2006 by Fleet et al.58 Another pyrrolizidine alkaloid isolated from this plant was 3-epi-casuarine 79; (1R,2R,3S,6S,7R,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahy- droxypyrrolizidine (Figure 3.5) the absolute configuration of which was established by X-ray crystallographic analysis.58 3-Epi-casuarine 79 was found to be a weaker inhibitor of α-D glucoside than casuarine 15.
HO H OH HO H OH
HO OH HO OH N N
OH OH 15 79 Figure 3.5 The structure of casuarine 15 and 3-epi-casuarine 79.
73 Chapter 3 [Casuarine]
3.2 Previous syntheses of casuarine
The first synthesis of casuarine 15 was achieved by Denmark et al.170 in four additional steps from a tandem [4+2]/[3+2] nitroalkene cycloaddition reaction as a key step in 20% overall yield. The synthesis started with the synthesis of the chiral vinyl ether 120 (Scheme 3.1). The chiral alkoxy aldehyde 118 was converted to the silyl enol ether 119 in 99% yield as a 10:1 (Z/E) mixture. O-Benzoylation of 119 with benzoyl fluoride and a catalytic amount of TBAF (2 mol%) provided 120 as a mixture of Z and E vinyl ethers, which were separated by silica gel chromatrography in yields of 81% and 6%, respectively (Scheme 3.1).
Ph abPh Ph Ph Ph Ph O O O
CHO OTMS OBz 118 119 120 Scheme 3.1 The synthesis of 120.
Reagents and conditions: (a) TMSCl, Et3N, CH3CN, 81 °C, 99%; (b) BzF, TBAF, THF, 0 °C, 2 h, (81% Z: 6% E).
The chiral nitronate 122, the 1,3-dipolar component of the [3+2] cycloaddition, was prepared from an endo-diastereoselective [4+2] cycloaddition reaction of the nitroalkene 121 and the chiral vinyl ether 120 in the presence of 2.5 equiv of SnCl4 in toluene at -78 °C (Scheme 3.2). The nitronate 122 was not stable and was then immediately treated with the β-silyl enone 123 to give a 45:7:3:2:1:1 mixture of six isomeric cycloadducts in 76% yield. Purification by HPLC afforded the desired nitroso acetal 124 in 55% overall yield. Reduction of the ketone group of 124 with L-selectride at -78 °C led to a 10:1 mixture of epimeric alcohols 125 in 87% yield. Mesylation of the secondary alcohol 125 gave the mesylate 126 in 97% yield, which was converted to the pyrrolizidine 127 in 64% yield via hydrogenolysis over Raney nickel and then hydrolysis of both benzoate groups under basic hydrolysis conditions. The final step was transformation of the C-1 silyl group to the final hydroxyl subsituent (Tamao-Fleming reaction) by first dearylation of the silyl group with mercuric trifluoroacetate in trifluoroacetic acid, followed by oxidation
74 Chapter 3 [Casuarine] with peracetic acid to afford pure casuarine 15 in 84% yield after ion-exchange column chromatography.
O OTDS
O O OG* O O OOG* O N N SiMe2Ph N 2 (a) Z-120 123 TDSO OBz H OBz OBz b PhMe Si H OBz 3 OBz 124 121 122 76% (45:7:3:2:1:1) Ph HPLC OG* 55% (41 :0:0:2 :1:1) Ph O
OTDS RO H O OOG* N HO H OH c e N f TDSO OBz HO OH H HO OH PhMe3Si N OBz H PhMe3Si OH 125; R=H 127 15 OH d 126; R=Ms + Ph Ph
HO
128 Scheme 3.2 The total synthesis of casuarine 15 by Denmark et al.170
Reagents and conditions: (a) Z-120, SnCl4, toluene, -78°C (b) 123, CHCl3; (c) L-Selectride,
THF, -78 °C, 87% (10:1); (d) Ms2O, py, 1 h, 97%; (e) i: Raney Nickel, MeOH, 260 psi H2; ii: K2CO3, MeOH, rt, 64%; (f) Hg(OTFA)2, TFA, HOAc, AcO2H, 84%.
A synthesis of casuarine 15 and its 6,7-diepimer 15, in a stereocontrolled manner, was reported by Izquierdo et al.171 The synthesis of casuarine 15 began with N-Cbz protection of the DMDP derivative 129 that gave the Cbz compound 130 in 93% yield (Scheme 3.3). Primary alcohol oxidation and then a Wittig reaction of 131 gave the pyrrolidinic propenoate 132 (2 steps). Dihydroxylation of 132 using osmium tetraoxide, NMO in the presence of O-(4-chlorobenzoyl)hydroquinine (DHQ-CLB) as a chiral ligand gave a mixture of 133 and 134 in yield of 27% and 58%, respectively. The configuration of both diol products could not be determined at this stage. After two more steps, an NOE experiment confirmed that 134 was the desired intermediate to make casuarine 15. N-deprotection of 134 using catalytic hydrogenolysis provided pyrrolidine 135 which was subsequently transformed to 136 by refluxing in methanol in the presence of a catalytic amount of NaOMe. Reduction of the lactam carbonyl group of 136 gave 137 in 89% yield using BH3·SMe2 complex
75 Chapter 3 [Casuarine] in THF. O-TBDPS deprotection and then debenzylation gave 138 in 95% yield. Hydrogenolysis then gave the final compound, however it was not pure. The product was further purified by acetylation that gave 139 in 41% yield. Base catalysed deacetylation of 139 afforded casuarine 15 in 93% yield. This synthesis was achieved in 8 steps from the DMDP derivative 129 in 12.6 % overall yield.
Cbz OH BnO R 1 OBn OTBDPS H R a R N d CO2Me OBn BnO R2 R3 HN N Cbz OTBDPS BnO OBn TBDPSO 129 130; R=CH2OH b 133; R=R2 =OH;R1=R3 =H 131; R=CHO 2 1 3 c 134; R=R =H;R =R =OH 132; R=(E )-CH=CHCO2Me
OH 2 1 H OBn R O H OR e 134 HO f OBn R2O OR1 HN N MeO O OTBDPS X OR
135 136; R = TBDPS; R1 =Bn;R2 =H;X=O g 137; R=TBDPS;R1 =Bn;R2 =H;X=H h 2 2 1 138; R=R =H;R=Bn;X=H2 i 1 2 139; R=R =R =Ac;X=H2 j 1 2 15; R=R =R =H;X=H2
Scheme 3.3 The total synthesis of casuarine 15 by Izquierdo et al.171
Reagents and conditions: (a) CbzCl, Me2CO, K2CO3, rt, 93%; (b) TPAP, NMO, 4°A Ms,
CH2Cl2; (c) Ph3P=CHCO2Me, CH2Cl2, rt, 85% (from 130); (d) OsO4, NMO, DHQ-CLB, acetone/H2O, rt, 2 d, (133:134 = 27%:58%); (e) H2, 10% Pd-C, MeOH; (f) cat. MeONa, + - MeOH, rt, 85%; (g) H3B:SMe2, THF, then MeOH, ∆, 89%; (h) n-Bu4N F ·3H2O, THF, rt, - 95%; (i) i: H2, 10% Pd-C, MeOH, then Amberlite IRA-400 (OH form), ii: Ac2O, py, DMAP, 41%; (j) cat. NaOMe, MeOH, rt, 93%.
76 Chapter 3 [Casuarine]
In 2006, Fleet et al.58 published the synthesis of casuarine 15 from D- gluconolactone 140 (Scheme 3.4). D-gluconolactone 140 was reacted with 2,2- dimethoxypropane and the open chain diacetonide, was subsequently esterified with trifluoromethanesulfonic anhydride to afford the triflate 141 in 72% yield. The trifate group of 141 was displaced with sodium azide in dimethyl formamide to give the azide 142 in 97% yield. The unsaturated ester 143 was obtained from reduction of the azidoester 142 with DIBAL, followed by treatment of the resulting aldehyde with the Wittig reagent, Ph3P=CHCO2Me, in 75% yield over the two steps. It was observed that the unsaturated ester 143 had an E:Z ratio of 10:1. After isolation of the pure E isomer of compound 143 it was converted to a mixture of the diols 144 and 145 using an OsO4 catalysed DH reaction. A mixture of the diols 144 and 145 was obtained in a ratio of 1:4 in 72% yield. Hydrogenation of this mixture gave a mixture of amines which cyclized to the pyrrolizidine framework upon heating in toluene. Finally, after treatment of the reaction mixture with TBSCl, the lactam 146 was separated in 70% yield over the 3 steps. The terminal acetonide of 146 was removed by acid hydrolysis to afford the diol 147 in 69% yield. Selective protection of the primary hydroxyl group of diol 147 with TBSCl gave the secondary alcohol 148 in 81% yield. The remaining secondary hydroxyl group of 148 required inversion of configuration. This was achieved by treatment with triflic anhydride to afford the unstable triflate 149 which was reacted with caesium trifluoroacetate. Base hydrolysis of the resulting trifluoroacetate gave the inverted alcohol 150 in 20% yield over the 3 steps. The desired lactam mesylate 151 was obtained in 90% yield by treating 150 with methanesulfonyl chloride. Reduction of the lactam carbonyl group of 151 with BH3·THF gave the amine 152 (57% yield). Finally pure casuarine 15 was obtained after 2 more steps, O-silyl group hydrolysis with TFA and then cyclization by treatment with sodium acetate (91% yield over the two steps).
77 Chapter 3 [Casuarine]
OH O O O O O MeO C O HO OH F3CO2SO 2 a b MeO2C c
MeO2C N3 N3 O O CH2OH O O O O O O 140 141 142 143
O OH O OH O TBSO d e O f MeO2C O + MeO2C O TBSO OH N3 OH N3 NH O O O O O O O 144 145 146
O O O O TBSO TBSO TBSO TBSO O g O h O i O TBSO TBSO TBSO TBSO NH NH NH NH OH OH OH OSO2CF3 O OH O OTBS O OTBS O OTBS 147 148 149 150
O O OH TBSO TBSO HO HO OH j O k O lmOH H TBSO TBSO HO HO OH NH NH NH N OMs OMs OMs O OH OTBS OTBS OH 15 151 152 153
Scheme 3.4 The total synthesis of casuarine 15 by Fleet et al.58
Reagents and conditions: (a) Me2C(OMe)2, p-TsOH, MeOH; then (CF3SO2)2O, py, CH2Cl2,
72%; (b) NaN3, DMF, 97%; (c) t-Bu2AlH, -78 °C; then Ph3P=CHCO2Me, toluene (75% over two steps); (d) cat. OsO4, NMO, t-BuOH/H2O, 72%; (e) H2, Pd/C, THF; then toluene, ∆; then t-
BuMe2SiCl, imidazole, THF (70% over three steps); (f) 60% HOAc, H2O/MeOH, 69%; (g) t-
BuMe2SiCl, py, 81%; (h) (CF3SO2)2O, py, CH2Cl2; (i) CF3CO2Cs, 2-butanone; then K2CO3,
MeOH (20% from 148); (j) CH3SO2Cl, Et3N, CH2Cl2, 90%; (k) BH3·THF, THF, 57%; (l) 90%
CF3CO2H, H2O; (m) NaOAc, H2O (91% over two steps).
In early 2009, Goti et al.172 published the synthesis of casuarine 15 and the first total synthesis of its 6-O-α-glucoside 15a. Their syntheses started with the same starting materials 110 and 111 which underwent a 1,3-dipolar cycloaddition reaction to afford 112 (Scheme 2.35). N-O bond cleavage of 112 with Zn/HOAc then attack of the amine on to the ester carbonyl group resulted in the lactam 154. This compound was converted to 155 using the Tamao-Fleming reaction similar to that 170 employed by Denmark (Scheme 3.2). Reduction of lactam 155 with LiAlH4 gave 156 in 76% yield, which was debenzylated under standard hydrogenolysis conditions to give pure casuarine 15 in five steps and 44% overall yield.
78 Chapter 3 [Casuarine]
O BnO BnO O CO Et N 2 a O N BnO N b + BnO CO2Et BnO OH SiMe Ph 2 H H BnO OBn BnO SiMe2Ph BnO SiMe 2Ph 110 111 112 154
BnO O BnO HO H OH c N d N e BnO OH BnO OH HO OH N BnO H OH BnO H OH 155 156 15 OH Scheme 3.5 The total synthesis of casuarine 15 by Goti et al.172
Reagents and conditions: (a) CH2Cl2, rt, 36 h, 79%; (b) Zn, AcOH/H2O, 60-65 °C, 5 h, 93%;
(c) Hg(CF3CO2)2, TFA, AcOH, AcOOH, CHCl3, 76%; (d) LiAlH4, THF, reflux, 78%; (e) H2, Pd/C, MeOH, HCl, 100%.
3.3 Total synthesis of casuarine 15
We desired to synthesize casuarine 15 using the chemistry we have developed during the synthesis of the ring B of 76, the C- 2 epimer of the proposed structure of uniflorine A (Scheme 2.20) and the synthesis of the ring A of uniflorine A 78 (Scheme 2.33). We planned to combine these two ring syntheses to produce casuarine 15 (Scheme 3.6).
OH HO H HO H OH OH HO B HO A OH N N OH 76 78 OH
HO H OH
HO B A OH N
OH casuarine 15
Scheme 3.6 Similarities between the B ring of 76 and the A ring of 78.
79 Chapter 3 [Casuarine]
The proposed synthesis of casuarine 15 was similar to that for the synthesis of uniflorine A 78, however the stereochemistry at the C-6 position required inversion. The stereochemistry of the B ring of 76 was the same as that found in casuarine 15. The retro-synthetic analysis of casuarine 15 involved the following five key reactions is outlined in Scheme 3.7. • A regioselective ring-opening of epoxide A with an oxygen nucleophile • N-alkylation of B to provide the pyrrolizidine • A diastereoselective epoxidation of 77 • A ring-closing metathesis reaction (RCM) of C • The Petasis reaction
OP OH HO H OH H OP H O OP'' Ring opening O N-alkylation HO OH OP NP' OP N of epoxide N 15 OH A OP' B
OH OH OP OP H H Petasis Ph OH OP Ph (HO) B Epoxidation RCM OP Reaction 2 NP' CHO OH NP' OH OP OP'' NH2 OH 77 C
(P = Protecting group)
Scheme 3.7 Retro-synthetic analysis of casuarine 15.
It was noted that the precursor 77 from the retro-synthesis of casuarine 15 can also be used as a common precursor for the synthesis of other pyrrolizidine alkaloids. The synthesis of casuarine 15, australine 13, 3-epi-casuarine 79 and 3-epi-australine 80 using the synthetic strategy shown in Scheme 3.8 will also be described in this thesis.
80 Chapter 3 [Casuarine]
OH OP H OP
NP' OH 77 Deprotection Protection Epoxidation
Required inversion at C-3'
OP OH OP OH H H O OP'' O OP'' 1' 3' 1' 3' NP' OP NH OP
Cyclization Cyclization
H OP H OP O O OP OP N N
OP' OP'
+ + Ring opening with H3O Ring opening with H3O Deprotection Deprotection HO OH - - H Ring opening with H HO OH Ring opening with H Inversion of OH at C-7 H Inversion of OH at C-7 HO OH Deprotection Deprotection N HO OH HO OH H N HO H OH 15 OH 79 OH OH OH casuarine N 3-epi-casuarine N Route 1: Chapter 3 Route 3: Chapter 5 13 OH 80 OH australine 3-epi-australine Route 2: Chapter 4 Route 4: Chapter 6
(P = Protecting group)
Scheme 3.8 Synthetic routes to prepare natural pyrrolizidines from 77.
3.3.1 The synthesis of the common precursor 103
The common intermediate 103 for these syntheses could be readily prepared on a 4 g scale from L-xylose in four steps and in 46% overall yield (Scheme 3.9) using the chemistry described in Chapter 2.
81 Chapter 3 [Casuarine]
OH OH L-xylose + Ph OH NH2 a c NR OH + B(OH)2 Ph 35; R = H b 36; R = Boc
OH O OH O H Ph O d O
NBoc OH NBoc OH
102 103
Scheme 3.9 The synthesis of the common chiral 2,5-dihydropyrrole precursor 103.
Reagents and conditions: (a) EtOH, rt, 3 d, ion-exchange, 92%; (b) (Boc)2O, Et3N, MeOH, rt, 3 d, 80%; (c) DMP, PPTS, acetone, rt, 20 h, 64%; (d) Grubbs' I cat.38a, CH2Cl2, reflux, 18 h, 97%.
3.3.2 Benzylation and Acetonide and N-Boc deprotection
The diol group of 103 was protected under standard conditions to give the dibenzyl ether 157 in 92% yield (Scheme 3.10). Treatment of 157 with HCl in methanol resulted in N-Boc and acetonide hydrolysis and provided the amino diol 158 in 76% yield. The 1H NMR spectroscopic data of the desired dibenzyl ether 157 demonstrated an increase of 10 aromatic protons from the starting material 103 and also displayed four diastereotopic benzylic methylene proton doublet signals (all 1H, d, J ca. 11.5 Hz) at 4.81, 4.74, 4.55 and 4.35-4.25 ppm. The 1H NMR spectrum of 158 showed sharp and clear signals that indicated that the N-Boc group had been successfully removed along with the isopropylidene group (Figure 3.6).
OH O OBn O OBn OH H H O O H OH a b NBoc OH NBoc OBn NH OBn
103 157 158
Scheme 3.10 The synthesis of 157 and 158.
Reagents and conditions: (a) NaH, BnBr, n-Bu4NI, THF, 18 h, 92%; (b) HCl/MeOH, rt, 30 h, 76%.
82 Chapter 3 [Casuarine]
OBn O H O NBoc OBn
157
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1)
OBn OH H OH NH OBn
158
Absence of Boc and isopropylidene
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1)
1 Figure 3.6 The H NMR (500 MHz, CDCl3) spectra of 157 and 158.
3.3.3 O-TBS and N-Fmoc protection
Regioselective O-silylation of 158 at the primary hydroxyl group gave the TBS ether 159 (81% yield), which was efficiently N-protected as its Fmoc derivative 160 using 9-fluorenylmethyl chloroformate (FmocCl) in THF and a saturated solution of Na2CO3 in 94% yield (Scheme 3.11) according to the method of Carpino and Han.173 We decided to protect the secondary amino group of 159 with the Fmoc group since it could be removed under basic conditions without removing the primary O-TBS group.
83 Chapter 3 [Casuarine]
OBn OH OBn OH OBn OH H H OH H OTBS OTBS a b NH OBn NH OBn N OBn Fmoc 160 158 159
Cl O O
=FmocCl
Scheme 3.11 The synthesis of 160. Reagents and conditions: (a) TBSCl, DMAP, imidazole, THF, rt, 1 d, 81%; (b) FmocCl, o THF, sat. Na2CO3, 0 C, 3 h, 94%.
3.3.4 Epoxidation
We had initially planned to synthesize the 3β, 4β diol of 160 and then prepare its cyclic sulfate. However, the yields for doing this transformation in Chapter 2.1 were not consistent. We then turned our attention to ring-opening of the epoxide 161. Epoxidation of the alkene moiety of 160 using 1,1,1-trifluoroacetone and oxone174-176 provided the β-epoxide 161 in 81% yield as a single diastereomer (Scheme 3.12) due to the steric influence exerted by C-2 α-side chain. The stereochemistry of the epoxide 161 was confirmed by X-ray crystallographic analysis of the cyclized product 163. The mechanism of the epoxidation is shown in Scheme 3.13. The 1H NMR spectrum of epoxide 161 showed the epoxide methine signals at 3.86-3.80 (m, 1H) and 3.63 ppm (d, 1H, J 2.0 Hz) and the structure was further supported by ESI- MS analysis (m/z 744 [M+Na+]).
OBn OH OBn OH H H OTBS O OTBS CF3COCH3, oxone, NaHCO 3 N OBn N OBn o Fmoc MeCN/H2O, 0 C, 2 h, 81% Fmoc 160 161
Scheme 3.12 The synthesis of the epoxide 161.
84 Chapter 3 [Casuarine]
O F C 3 KHSO5 F3C O O N Fmoc
Dioxirane 160
F3C O N Fmoc + O
161
Scheme 3.13 Proposed epoxidation mechanism of 161.
3.3.5 Cyclization of 162
3.3.5.1 N-Fmoc deprotection and a Mitsunobu cyclization reaction
Next we attempted the conversion of the epoxide 161 to the epoxy- pyrrolizidine 163 (Scheme 3.14). The epoxide 161 was converted to the secondary amino alcohol 162 using piperidine (2 eq.) in MeCN with stirring at rt for 2 h to give 162 in 92% yield after purification by column chromatography. Attempts to cyclize 162 by treatment under the Mitsunobu reaction conditions (Table 3.1) gave the pyrrolizidine 163 (Table 3.1 entry 1) in the best yield of only 14%. From these results, we thought that the epoxide ring, which would make the pyrrolidine ring more rigid, and the pyrrolizidine product higher in energy due to increased ring strain, was affecting the efficiency of the Mitsunobu cyclization reaction. OBn OH OBn OH H H H OBn O OTBS O OTBS O ab OBn N OBn NH OBn N Fmoc OTBS 161 162 163 Scheme 3.14 The synthesis of 163.
Reagents and conditions: (a) piperidine, MeCN, rt, 2 h, 92%; (b) DIAD, PPh3, Et3NHCl, 1 d, 14%.
Table 3.1 The Mitsunobu cyclization of 163.
Entry Reagents and conditions Results
1 DIAD (2.5 eq.), PPh3 (2.5 eq.), Et3NHCl (1 .0 eq.), rt, 1 d 163 (14%)
2 DIAD (2.5 eq.), PPh3 (2.5 eq.), Et3NHCl (1 .5 eq.), rt, 2 d Complex mixture
3 DIAD (3.5 eq.), PPh3 (3.5 eq.), Et3NHCl (1 .5 eq.), rt, 3 d Complex mixture
85 Chapter 3 [Casuarine]
3.3.5.2 O-Mesylation and N-Fmoc deprotection
We then changed our method to prepare the pyrrolizidine 163 by first O- mesylation of the free secondary hydroxyl of 161 and then N-Fmoc deprotection (Scheme 3.15).
OBn OH OBn OMs H H O OTBS O OTBS a N OBn N OBn Fmoc Fmoc
161 164
OBn H OBn H O O OBn b OBn + N N OTBS 163 OTBS 163a [X-ray] [163:163a =91:9] Scheme 3.15 The synthesis of the pyrrolizidine 163.
Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, N2, 0 °C, 3 h, 94%; (b) piperidine, MeCN, rt, 15 h, 96%.
The O-mesylate derivative 164 was obtained in 94% yield from treatment of 161 with an excess of methanesulfonyl chloride (MsCl) and Et3N in CH2Cl2. TLC analysis of this product (Rf 0.5) showed a spot very close to the spot of the starting material at Rf 0.49 in 30% EtOAc/petrol. The structure of 164 however was fully confirmed by its NMR spectroscopic data and its ESI-MS analysis (m/z 822 [M+Na+]). Treatment of the mesylate 164 with piperidine resulted in smooth N-Fmoc deprotection and then cyclization of the free cyclic secondary amine to give in 96% yield a 91:9 mixture of the desired pyrrolizidine 163 and the undesired indolizidine 163a, respectively. A proposed mechanism for the synthesis of 163 is shown in Scheme 3.16. Product 163 could arise via cyclization of a N-centred incipient anion intermediate or from the corresponding amine (not shown). We assume that 163a arose from O-TBS migration under the basic conditions of the O-mesylation reaction, however, this was difficult to ascertain since NMR analysis of the mesylate 164 was made difficult because of N-Fmoc rotamers. Fortunately, a small amount of pure 163 could be obtained by further separation by column chromatography. The
86 Chapter 3 [Casuarine] structure of the epoxide 163 was confirmed by a single crystal X-ray analysis (Figure 3.7).
OBn OMs H H OBn O OTBS O OBn N OBn N Fmoc
164 163 OTBS
CO2 +
OBn OBn OMs H H O O
N O N O O N H O H
Scheme 3.16 Proposed reaction mechanism for the synthesis of 163.
Figure 3.7 Structure of C28H39NO4Si 163 with labelling of selected atoms. Anisotropic displacement ellipsoids show in 30% probability levels. Hydrogen atoms are drawn as circles with small radii.
87 Chapter 3 [Casuarine]
3.3.6 Epoxide ring-opening by NaHSO4
Several attempts in our laboratory to ring-open the epoxide group of compounds related to 163 using aqueous acid conditions (for example, H2SO4, water)174 led to complex mixtures and low yields of diol products. However, when 163 was treated under the conditions recently reported by Saracoglu,177 using
NaHSO4 as both the acid catalyst and the nucleophilic species in CH2Cl2 at reflux for 2 d, followed by the addition of water to hydrolyze the intermediate sulfate, then the diol 165 was obtained as an 86:14 mixture of regioisomers in which the O-TBS had also been hydrolyzed. We found from ESI-MS analysis that after 5 h of reaction that the TBS group of 163 was deprotected (m/z 368 [M+H+]). (Table 3.2, entry 4). Purification of the above reaction mixture by column chromatography gave a 92:8 mixture of the diastereomeric diols 165 and 6,7-diepi-165, respectively, in 51% yield (Scheme 3.17). The regiochemistry of this ring-opening reaction was consistent with that reported on related epoxy-pyrrolizidines174 and was expected from stereoelectronic considerations as shown in Scheme 3.18. For trans-1,2- diaxial like - ring-opening of epoxide 163 by HSO4 , the two reactive conformations, A and B are possible. Attack on conformation A at C-7 is inhibited by pseudo 1,3-diaxial - interactions between the nucleophile (HSO4 ) and the pseudoaxial protons H-1α and H-5α and thus addition to conformation B at C-6 predominates resulting in 165 as the major regioisomeric product.
H OBn HOH OBn O NaHSO4,CH2Cl2,reflux,2d OBn HO OBn N water, rt, 1 h, 51% N
163 OTBS 165 OH [163:163a =91:9] [165:6 ,7 -d iepi-165 =92:8] Scheme 3.17 The synthesis of 163.
88 Chapter 3 [Casuarine]
Table 3.2 Results of the epoxide ring-opening reactions of 165.
Entry Reagents and conditions Results
I: NaHSO4 (4.0 eq.), CH2Cl2, rt 3 d 1 No reaction II: H2O, rt, 1 h
I: NaHSO4 (5.0 eq.), CH2Cl2, rt 3 d, then reflux 10 h 2 165 (35%) II: H2O, rt, 1 h
I: NaHSO4 (5.0 eq.), CH2Cl2, reflux 2 d 3 165 (47%) II: H2O, rt, 1 h
I: NaHSO4 (5.0 eq.), CH2Cl2, N2, reflux 2 d 4. 165 (51%) II: H2O, rt, 1 h
H HO H O OBn attack OBn OBn at C-7 OBn H N H N OR OR H H5α 1α H OSO3 A HSO4
ring flip HO attack H H O H H OBn OBn at C-6 H N OBn H N OBn OR OR H H B OSO3 HSO4 ring flip
HO H H2O OBn 165 O3SO H N OBn OR H (R = TBS or H)
Scheme 3.18 Ring-opening reactions of epoxide 163 via conformations A and B.
3.3.7 Debenzylation
23 Hydrogenolysis of 165 over PdCl2/H2 gave casuarine 15 ([α] D +18.1 (c 1.0,
56 24 H2O), lit. [α] D +16.9 (c 0.8, H2O)), in 93% yield after purification by ion-exchange chromatography (Scheme 3.19). The 1H and 13C NMR spectra of our synthetic casuarine 15 are shown in Figures 3.8 and 3.9, respectively.
89 Chapter 3 [Casuarine]
HOH OBn HOH OH PdCl2,H2 (1 atm), MeOH, rt, 1.5 h HO OBn HO OH N ion-exchange, 93% N
165 OH 15 OH (165:6,7-diepi-165 = 92:8) Scheme 3.19 The synthesis of casuarine 15.
HOH OH
7 1 HO OH 5 N 3 8 H-7 15 OH H-6 H-1 H-2 H-8 H-8’ H-5β H-7a H-3 H-5α
4.00 3.50 3.00 ppm (t1)
*
5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) * Acetone 1 Figure 3.8 The H NMR (500 MHz) in D2O of (+)-casuarine 15.
HOH OH
7 1 C-1 HO OH C-6 5 N 3 C-7 C-2 C-3 C-8 8 C-5 15 OH C-7a
90 80 70 60 50 40 ppm (t1) 13 Figure 3.9 The C NMR (125 MHz) in D2O of (+)-casuarine 15.
The diastereomeric purity of (+)-casuarine 15 was 95:5 from 1H NMR spectroscopic 1 analysis. The H NMR spectroscopic data (D2O) of (+)-casuarine 15 (Figure 3.6) and 56 that of the natural product were essentially identical (∆δH = 0.00-0.01 ppm). The 13 C NMR signals of (+)-casuarine 15 (Figure 3.7) in D2O (with acetone as an internal reference at 29.80 ppm) however, were all consistently 1.0-1.3 ppm downfield of those reported for the natural product (Table 3.3).56
90 Chapter 3 [Casuarine]
Table 3.3 Physical and spectral data for (+)-casuarine 56 and 15.
Casuarine 56 Our synthetic 15 Structure HO H OH 7 HO 1 OH 5 N 3 8 OH Physical Crystallize solid Brown foamy solid Appearance Optical 24 23 [α] D +16.9 (c 0.8, H2O) [α] D +18.1 (c 1.0, H2O) Rotation Melting Point 181-182 °C - 1 H NMR 500 MHz, D2O (pH = 8.35) 500 MHz, D2O 4.21 (m, 1H, J = 4.7, 4.0 Hz, H-6) 4.22-4.18 (m, 2H, H-6 and H-7) 4.19 (m, 1H, J =3.5 Hz, H-7)
4.162 (t, 1H, J = 8.0 Hz, H-1) 4.16 (t, 1H, J1,2 = J1,7a = 8.7 Hz, H-1)
3.796 (t, 1H, J = 8.0 Hz, H-2) 3.79 (t, 1H, J1,2 = J2,3 = 8.0 Hz, H-2)
3.77 (dd, 1H, J8,8’ = 10.0, J3,8 = 3.5 Hz, 3.771 (dd, 1H, J = 11.9, 3.8 Hz, H-8) H-8)
3.61 (dd, 1H, J8,8’ = 11.3, J3,8’ = 6.8 3.611 (dd, 1H, J = 11.9, 6.6 Hz, H-8’) Hz, H-8’)
3.27 (dd, 1H, J5α,5β = 12.3, J5β,6 = 4.3 3.270 (dd, 1H, J = 12.2, 4.7 Hz, H-5β) Hz, H-5β)
3.06 (dd, 1H, J1,7a = 8.0, J7,7a = 3.0 3.071 (dd, 1H, J = 8.0, 3.5 Hz, H-7a) Hz, H-7a) 3.04-3.00 (m, 1H, J = 8.0, 6.6, 3.8 Hz, H-3) 3.04-3.00 (m, 1H, H-3)
2.90 (dd, 1H, J5α,5β = 11.8, J5α,6 = 4.3 2.911 (dd, 1H, J = 12.2, 4.0 Hz, H-5α) Hz, H-5α)
13 C NMR 125 MHz, D2O 125 MHz, D2O ∆δ (ppm) 78.8 (C-7) 79.9 (C-7) -1.1 77.8 (C-1) 78.9 (C-1) -1.1 77.4 (C-6) 78.5 (C-6) -1.1 76.6 (C-2) 77.8 (C-2) -1.2
72.1 (C-7a) 73.1 (C-7a) -1.0 70.0 (C-3) 71.0 (C-3) -1.0 62.2 (C-8) 63.5 (C-8) -1.3 58.0 (C-5) 59.0 (C-5) -1.0
91 Chapter 3 [Casuarine]
In conclusion, we have successfully prepared casuarine 15 in a total of 13 synthetic steps and 8% overall yield from L-xylose or 9 synthetic steps and 18% overall yield from the precursor 103. This overall synthesis is summarized in Scheme
3.20.
OBn O OBn OH H H a O b OR e 103 NBoc OBn NR' OBn
157 158;R=R'=H c 159;R=TBS;R'=H d 160;R=TBS,R'=Fmoc
OBn OR OBn H OBn H H OTBS O g O O OBn OBn + N OBn N N Fmoc OTBS 161;R=H 163 OTBS 163a f [X-ray] [163:163a =91:9] 164;R=Ms
HO OBn H HOH OH hi 163 HO OBn HO OH N N
165 OH 15 OH (165:6,7-diepi-165 = 92:8) (dr = 95:5)
Scheme 3.20 Total synthesis of casuarine 15 from the precursor 103.
Reagents and conditions: (a) NaH, BnBr, n-Bu4NI, THF, 18 h, 92%; (b) HCl/MeOH, rt, 30 h, 76%; (c) TBSCl, DMAP, imidazole, THF, rt, 1 d, 81%; (d) FmocCl, THF, sat. Na2CO3, 0 o o C, 3 h, 94%; (e) CF3COCH3, oxone, NaHCO3, MeCN/H2O, 0 C, 2 h, 81%; (f) MsCl, Et3N, o CH2Cl2, N2, 0 C, 3 h, 94%; (g) piperidine, MeCN, rt, 15 h, 96%; (h) NaHSO4, CH2Cl2, reflux, 2 d; water, rt, 1 h, 51%; (i) PdCl2, H2 (1 atm), MeOH, rt, 1.5 h; ion-exchange, 93%.
92 Chapter 4 [Australine]
CHAPTER 4 SYNTHESIS OF AUSTRALINE, 7-EPI-AUSTRALINE AND 1- EPI-CASTANOSPERMINE
4.1 Isolation and biological activities of australine
Castanospermum australe,178,179 commonly known as Moreton Bay Chestnut or Black Bean, is the only species in the Fabaceae family (Figure 4.1). It is native to the east coast of Australia in Queensland and New South Wales, and to the Pacific islands of Vanuatu and New Caledonia. It is a large evergreen tree that can grow to 40 m in height. It has glossy dark green pinnate leaves up to 15 cm in length and 6-7 cm in width. It has red and yellow pea-shaped flowers, 3-4 cm in length. The plant has cylindrical pods 12-20 cm in length, each of which contains 3-5 large bean- or chestnut-liked seeds. Please see print copy for image
Binomial name Castanospermum australe A. Cunn & C. Fraser ex Hook Scientific classification Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Fabales Family: Fabaceae Subfamily: Faboideae Castanospermum Genus: A.Cunn
Figure 4.1 Castanospermum australe.178,179
93 Chapter 4 [Australine]
Australine 13; (1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrro- lizidine, was first isolated in 1988 by Molyneux et al.54 from the mature seeds of Castanospermum australe that were collected from the Huntington Botanical Gardens, San Marina, California in November 1983. The isolation procedure started with the crude MeOH extract of the mature seeds which was purified by ion- + exchange chromatography on Dowex 50W-X8 (NH4 form). Castanospermine 22 was obtained by crystallization of the concentrated eluate from MeOH. The mother liquors were purified by preparative centrifugal TLC and eluted with
CHCl3/MeOH/NH4OH/H2O (70:26:2:2) (120 ml) and EtOH/NH4OH (98:2) (200 mL) to give 6-epi-castanospermine 23, fagomine 11 and a clear and colourless oil that crystallized from acetone to afford australine 13 as colourless prisms, mp 148-
26 149 °C; [α] D + 19.3 (c 2.09 MeOH) (Figure 4.2). The structure of australine 13 was established by NMR spectroscopic techniques, MS analysis and was confirmed by X-ray crystallography. Australine 13 was a good inhibitor of the α-glucosidase amyloglucosidase (IC50 5.8 µM) and a moderate inhibitor of glucosidase I (IC50 ca. 20 µM). In contrast, it did not inhibit a β-glucosidase, an α- or β-mannosidase, or an α- or β-galactosidase.180
OH OH OH HO H OH HO H HO H OH OH HO CH2OH OH N N N N OH OH 13 OH 22 23 11
Figure 4.2 The structure of australine 13, castanospermine 22, 6-epicastanospermine 23 and fagomine 11.54
In 2003, Kato et al.55 also isolated australine 13 from the same plant as Molyneux et al.54 They showed that australine 13 was a good inhibitor of rat intestinal sucrase (IC50 4.6 µM) and a moderate inhibitor of rice α-glucosidase (IC50
21.0 µM), rat intestinal maltase (IC50 24.0 µM) and fungal amyloglucosidase (IC50 28.0 µM).
94 Chapter 4 [Australine]
4.2 Previous syntheses of australine
Pearson et al.181,182 reported a total synthesis of (+)-australine 13 and (-)-7- epi-alexine 175 via a reductive double-cyclization of the azido-epoxy-tosylate 173 (Scheme 4.1).
OBn OBn OBn HO a OBn OBn b OBn c O HO N3
OBn OBn OBn 166 167 168
HO OBn HO OBn OBn OHC d f OBn OBn e O OBn N N3 Ph 3P N3 3 TMSO OBn 170 OBn OBn 169 171 172α:172β=1:1
TsO OBn HO H OR HO H OR g OBn OR + OR O N N N3
OBn OR OR 173α:173β=2:1 174α;R=Bn 174β;R=Bn h h 13; R = H 175; R = H
Scheme 4.1 The total synthesis of australine 13 and (-)-7-epi-alexine 175 by Pearson et al.181,182
Reagents and conditions: (a) Ph3PCH3Br, n-BuLi, THF, -78 °C to rt, 24 h, 66%; (b) i: Tf2O,
py, CH2Cl2, -40 °C to rt, 3h; ii: n-Bu4N3, PhH, 0 °C to rt , 1 h, 75% overall for 2 steps; (c) i:
O3, MeOH, CH2Cl2, -78 °C, 17 min; ii: Me2S, -78 °C to rt, 2.25 h; (d) i: 170, KN(TMS)2,
Me3SiCl, THF, -78 °C to rt, 4 h; ii: aq. HCl, 35% overall for 4 steps; (e) m-CPBA, CH2Cl2, 0
°C to rt , 24 h, 65%; (f) TsCl, py, DMAP, CH2Cl2, -15 °C, 48 h, 67%; (g) i: H2, 10% Pd/C, ether-EtOH (2:1), rt, 15 h; ii: K2CO3, EtOH, reflux, 20 h, 71% overall for 2 steps; (h) H2, 10% Pd/C, EtOH, rt, 48 h, 87%.
In the first paper, Pearson had claimed the total synthesis of (+)-7-epi- australine 86 instead of (+)-australine because of the errors in the NMR spectroscopic data reported for this natural product in the earlier literature. This
95 Chapter 4 [Australine]
NMR data was later corrected by Wormald et al.60 which indicated that Pearson was the first to synthesize (+)-australine. The synthesis started with 2,3,5-tri-O-benzyl-L- xylofuranose 166 which was obtained in 3 steps from L-xylose. A Wittig olefination reaction of 166 provided the secondary alcohol 167 in 66% yield, which was converted to the inverted azide 168 via the corresponding triflate. The azide 168 was unstable and was immediately converted to the homoallylic alcohol 171 by first ozonolysis to give aldehyde 169 which, without purification, was directly converted to the Z-alkene 171 in 35% yield over the 4 steps using a stereoselective Wittig reaction with the silyloxy substituted ylide 170. Epoxidation of 171 afforded a mixture of the α- and β-epoxides, 172α and 172β in a 1:1 ratio. Tosylation of this mixture furnished a 2:1 mixture of tosylate derivatives 173α and 173β. Selective azide reduction of the mixture of 173α and 173β followed by heating a solution of the resulting amines at reflux in EtOH containing K2CO3 gave a 2:1 mixture of the pyrrolizidine 174α and 174β that was easily separated. Debenzylation of the individual alcohols 174α and 174β by hydrogenolysis over Pd/C gave the desired pyrrolizidine, (+)-australine 13 and (-)-7-epi-alexine 175, respectively.
The conversion of castanospermine 22 to (+)-australine 13 was reported by Furneaux et al. (Scheme 4.2).183 The synthesis started with the selective O-Cbz protection of the hydroxyl group at C-6 of castanospermine by treatment with bis(tributyltin) oxide with heating at reflux in toluene followed by treatment with benzyl chloroformate to provide the carbonate 176. Per-acetylation of 176 afforded the triacetate 177 in 76% overall yield for the 3 steps. Deprotection of the C-6 hydroxyl group of 177 was achieved under standard hydrogenolysis conditions using
(H2, Pd/C, EtOAc/EtOH) to give triacetylcastanospermine 178. The reaction of 178 with Tf2O gave the unstable triflate 179 which was treated in situ with benzyl alcohol to afford the benzyl ether protected pyrrolizidine 181 (35%) and the benzyl ether protected indolizidine 182 (20%). These compounds were formed from the aziridinium ion intermediate 180 by nucleophilic attack of benzyl alcohol. Hydrogenolysis of 181 followed by acetate hydrolysis with aqueous ammonia and then acidification with HCl/MeOH gave australine 13 as its crystalline hydrochloride salt. The yields for these steps were not reported.
96 Chapter 4 [Australine]
HO RO AcO OAc H OH H OR H HO RO AcO 7 8a 1 a d 5 N 3 N N HO R1O TfO 176: 1 179 22 b R= H,R =Cbz c 177: R= Ac,R1 =Cbz 178: R= Ac,R1 =H
AcO OAc AcO OAc AcO OAc H H H AcO AcO e AcO + N N N BnO 181 182 BnO (35%) (20%) 180 f
HO H OH
OH HCl N
13.HCl OH Scheme 4.2 The synthesis of australine hydrochloride 13 from castanospermine by Furneaux et al.183
Reagents and conditions: (a) i: (Bu3Sn)2O, toluene, reflux; ii: CbzCl, -20 °C; (b) Ac2O, py, rt, 76% overall for 3 steps; (c) H2, Pd/C, EtOAc-EtOH, 73%; (d) Tf2O, 2,6-di-t-butyl-4- methylpyridine, 181: 35%, 182: 20%; (e) BnOH; (f) i: H2, PdO2/HCl; ii: aqueous NH3; iii: HCl-MeOH.
In 1998, the synthesis of (+)-australine 13 was reported by White et al.184,185 Their synthesis had two key steps that were a RCM reaction and a transannular cyclization reaction (Scheme 4.3). Their synthesis began with the epoxy alcohol 183 which was reacted with 4-butenyl isocyanate to provide the urethane 184 in 93% yield. Cyclization of the urethane 184 upon exposure to potassium tert-butoxide gave the oxazolidinone 185. Migration of the isopropylidene group from the terminal 1,2- position to the internal 2,3-position of the oxazolidinone 185 with Amberlyst-15 resin furnished 186 in 62% yield. Swern oxidation of the primary alcohol 186 provided the aldehyde and then a subsequent Wittig reaction afforded the diene 187. The RCM reaction of diene 187 using Grubbs’ I catalyst 38a produced the azacyclooctene 188. The acetonide group of 188 was hydrolysed using aqueous hydrobromic acid and the resulting hydroxyl groups were O-benzylated under standard reaction conditions to give the bis-O-benzyl ether 190. Epoxidation of 190 with m-CPBA in CH2Cl2 afforded a single epoxide 191 in 75% yield. Treatment of
97 Chapter 4 [Australine]
191 with a hot aqueous solution of LiOH resulted in cleavage of the oxazolidinone and intramolecular cyclization to give 1,2-di-O-benzylaustraline 192. Debenzylation of 192 produced australine 13 in nearly quantitative yield.
O a O b O O H O OH O O N
183 184 O
O OH c d, e f O O O
O N OH N N O O O O O O O 185 186 187 O HO H OR O OR OBn g i j OR N O OR N N N OBn OH O O O O O O k 192: R = Bn 13: R = H 188 h 189: R = H 191 190: R = Bn
Scheme 4.3 The total synthesis of australine 13 by White et al.184,185
Reagents and conditions: (a) CH2=CH(CH2)2NCO, i-Pr2NEt, C6H6, ∆, 93%;(b) tert-BuOK,
THF, 0 °C, 2 h, 96%; (c) Amberlyst 15, Me2CO, rt, 18 h, 62%; (d) (COCl)2, DMSO, Et3N,
CH2Cl2, -78 °C, 90%; (e) Ph3PMeBr, KHMDS, THF, -78 °C to rt, 18 h, 76%; (f) Grubbs’ I catalyst 38a, CH2Cl2, rt, 5h, 97%; (g) HBr, MeCN, rt, 1 h, 99%; (h) NaH, BnBr, Bn4NI,
THF, 60 °C, 2.5 h, 84%; (i) m-CPBA, CH2Cl2, rt, 6 h, 75%; (j) LiOH, EtOH/H2O (1:1), 95
°C, 18 h; 99%; (k) H2, Pd(OH)2/C, MeOH, rt, 24 h, 99%.
In the same year that Denmark reported the total synthesis of casuarine 15 (Scheme 3.2),170 he also published the total synthesis of (+)-australine 13 using a similar synthetic strategy, in 17% overall yield in a nine step sequence (Scheme 4.4).170
98 Chapter 4 [Australine]
a, b t-Bu O 195 t -Bu O Si c Si O t -Bu Cl t-Bu O 193 194 196 NO 2
Ph O OG* 2 O OG* O N R OHO O N 197 f R1O H H d, e O O O O Si Si t-Bu t-Bu t-Bu t-Bu 198 1 2 G*: g 199: R =R =H 200: R1 = TBS, R2 =H Ph h 201: R1 = TBS, R2 =Ms TBSO MsO OTBS HO H OH HN j N k, l i HO HO OH N H H O O O O OH Si Si 13 t-Bu t-Bu t-Bu t-Bu 202 203
Scheme 4.4 The total synthesis of australine 13 from Denmark et al.170
Reagents and conditions: (a) n-BuLi, THF, -23 °C; (b) (t-Bu)2SiClOTf, -78 to 25 °C, 73%;
(c) potassium nitroacetaldehyde 195, CHCl3/CH3CN, rt, 8 h, 85%; (d) MAPh, CH2Cl2, -50
°C, 2 h; (e) PhH, NaHCO3, ∆, 4 h, 45% overall for 2 steps; (f) K2OsO2(OH)4, K2CO3, H2O,
NaHCO3, K3Fe(CN)6, t-BuOH, DHQ-AQN; rt, 86% (99% ee), (g) TBSCl, py, rt, 4.0 h, 93%;
(h) MsCl, py, rt, 0.5 h; (i) H2, Raney Ni, MeOH, 260 psi, rt, 36 h, 82% overall for 3 steps; (j)
CH3CN, ∆, 16 h, 85%; (k) HF, MeOH, rt; (l) AG 50W-X8, 90% overall for 2 steps. [MAPh = Methylaluminium bis(2,6-dipheylphenoxide)].
The synthesis began with ring-opening of 2,5-dihydrofuran 193 by n-BuLi to give a lithium Z-enolate intermediate which was treated with di-tert-butylchlorosilyl triflate to provide the chlorosilane 194 in 73% yield. Displacement of the chloride of 194 with potassium nitroacetaldehyde 195 afforded the nitro olefin 196 in 85% yield. An exo-selective [4+2] intermolecular cycloaddition between 196 and the chiral vinyl ether 197 in the presence of MAPh at -50 °C provided the trans nitronate which underwent an intramolecular [3+2] cycloaddition when heated in benzene to furnish the desired nitroso acetal 198 in a 45% yield over three steps as a 44:1 mixture of diastereomers. The asymmetric dihydroxylation of 198 using dihydroquinidine anthraquinone ligand [(DHQ)2-AQN] was necessary to prepare the diol 199 in 86%
99 Chapter 4 [Australine] yield and as a 12:1 mixture of diastereomers. The three steps leading to intermediate 202 involved, (i) O-TBS protection of the primary alcohol of 199; (ii) mesylation of the secondary alcohol of 200; and (iii) N-O bond hydrogenolysis and reductive amination of the resulting amino aldehyde. Surprisingly, 202 did not spontaneously cyclise and could be isolated in good yield. Nevertheless, the desired cyclised product 203 could be obtained in 85% yield by heating a solution of 202 in CH3CN. Deprotection of 203 and then ion-exchange chromatography gave australine 13 in 90% overall yield from 202.
The chemoenzymatic total synthesis of australine 13 via a sequential enzymatic aldol reaction and then a bis-reductive amination reaction were accomplished by Wong et al. in 2000.186 This approach allowed for the rapid construction of the natural product without the necessary protection of the hydroxyl functionalities (Scheme 4.5 and 4.6).
OH a OH b O OH O 204 205 206
OH OH OH d c HO HO
NHCHO NHCHO 207 208 Scheme 4.5 Synthesis of the triol aminal 208 (yields are given for the synthesis of the enantiomeric compounds).
Reagents and conditions: (a) (+)-DIPT, cumene hydroperoxide, CHCl3, -45 °C, 86%, ee
>99%; (b) 0.5 N NaOH, 98%; (c) 30% NH4OH, rt, then ethyl formate, EtOH, 90 °C, 95%;
(d) O3, MeOH, -78 °C, then In, allyl bromide, H2O, rt. 56% (after separation of diastereomers). [DIPT = Diisopropyltartrate].
The synthesis started with a Sharpless asymmetric epoxidation of the achiral divinylcarbinol 204 to provide epoxide 205 in high enantiomeric purity (>99%). Rearrangement of epoxide 205 under basic conditions and then regioselective nucleophilic ring-opening of the resulting vinyl epoxide with ammonia, followed by protection of the resulting amine with ethyl formate furnished the formamide 207. Ozonolysis of 207 gave a mixture of hemiacetals which were directly treated with allyl bromide and indium to afford the triol 208 as a 3:1 mixture of diastereomers
100 Chapter 4 [Australine] that could be separated. Oxidative cleavage of the terminal alkene of 208 followed by a enzymatically catalyzed aldol reaction of the resulting aldehyde using dihydroacetone phosphate (DHAP) and fructose-1,6-diphosphate aldolase (FDPA) afforded the pyranose 209 as a 1:1 mixture of anomeric stereoisomers in 30% yield. Ozonolysis of the terminal alkene of 209, followed by acid catalysed hydrolysis of the formamide group and then reductive amination of the resulting amino-aldehyde- ketone with NaCNBH3 generated australine 13 in 52% as a 8:1 mixture of diastereomers.
HO OH OH OH OH H a OH b HO O OH OH N HO NHCHO NHCHO 13 OH 208 209a : 209b =1:1
Scheme 4.6 The total synthesis of australine 13 by Wong et al. 186
Reagents and conditions: (a) NaIO4, H2O, then DHAP, FDPA, RT, 72 h, then Pase, 38 °C,
12 h, 30%; (b) O3, MeOH-H2O, -78 °C, then HCl, rt, 72 h, then NaOAc, NaCNBH3, HOAc, rt, 36 h, 52%. [Pase = Acid phosphatase].
Trost et al.187 succeeded in the enantioselective total synthesis of australine hydrochloride (13·HCl) seven years later (Scheme 4.7). The synthesis of australine hydrochloride started with the reductive dimerization of acrolein 210 with zinc followed by cyclization in neat diethyl carbonate to produce the 1:1 separable mixture of dl-and meso-isomers 211 and 212 in 70% yield. Treatment of the dl- carbonate 211 with phthalimide and 5 mol% π-allylpalladium chloride dimer, 15 mol% (R,R)-213, 5 mol% Na2CO3 in CH2Cl2 give only the expected amino alcohol 214 in 81% yield. The oxazolidinone 215 was easily prepared in two steps from 214 by reduction with NH2(CH2)2NH2 in EtOH and then treatment of the resulting amino alcohol with triphosgene/pyridine. The oxazolidinone 215 was converted to a 95:5 mixture of the homoallylic alcohols 216 and 217 by treatment with racemic butadiene mono-epoxide 0.5 mol%, [Pd2dba3]·CHCl3, 1.5 mol% of 213 in the presence of DBU. A RCM reaction of 216 with Grubbs’ 2nd generation catalyst 38b provided 218 in 77% yield.
101 Chapter 4 [Australine]
O O O a H OO + O O
210 rac-211 212
O O NH HN b PPh2Ph 2P (R,R)-213 O
HO NPhth c, d O NH
214 215
OH OH O O e O N O N + O (R,R)-213 216 217
f
O O O OH OBn g, h i OBn O N O N O N HO HO H H H O 218 219 220
OH HO H OH H l j N k HO N OH HO OBn OBn N H H H Cl OH HO OBn HO OBn 221 222 (+) australine hydrochloride 13.HCl Scheme 4.7 Total synthesis of australine hydrochloride 13·HCl by Trost et al.187
Reagents and conditions: (a) i: Zn, THF, aq. NH4Cl; ii: C2H5OCO2C2H5, 70% (dr = 1:1); (b) phthalimine, (C3H5PdCl)2, (R,R)-213, 5% Na2CO3, CH2Cl216, 16 h, 81%;(c) H2N(CH2)2NH2,
EtOH, 96%; (d) triphosgene, py, CH2Cl2, 78%; (e) butadiene mono-epoxide, 0.5 mol%
[Pd2dba3]·CHCl3, 1.5 mol% 213, 10 mol% DBU, CH2Cl2, (R,R)-213: 95:5 dr 216:217, 99%
(f) 1 mol% Grubbs’ II catalyst 38b, CH2Cl2, 77%; (g) benzyl 2,2,2-trichloriacetimidate,
TfOH, 94%; (h) 9-BBN, THF; NaBO3·H2O, H2O, 74%; (i) Oxone, trifluoroacetone,
NaHCO3, MeCN/ aq. EDTA, 0 °C, 67%; (j) Dowex 1x8-50, BnOH, 100 °C, 51%; (k) MsCl,
Et3N, CH2Cl2, 74%; (l) H2, PdCl2, MeOH, 98%.
Protection of the primary alcohol 218 by benzylation and then hydroboration of the terminal alkene gave the primary alcohol 219. Epoxidation of the double bond of 219
102 Chapter 4 [Australine] furnished the epoxide 220 (stereochemistry not determined). Ring-opening of the epoxide and hydrolysis of the oxazolidinone moiety was achieved by heating in BnOH at 100 °C in the presence of Dowex 1X8-50 (HO- form). The final step was cyclization of 221 that was initiated by mesylation of the primary hydroxyl group which ultimately resulted in the dibenzyl pyrrolizidine 222 in 74% yield. Removal of the benzyl groups using the reaction conditions of Tang and Pyne149
(PdCl2/H2/MeOH) produced australine hydrochloride in 98% yield.
Marco et al.188 published the stereoselective synthesis of australine 13 in 2007 through a one pot, double cyclization strategy (Scheme 4.8). This synthesis started with an aldol reaction between the ketone 223 (derived from L-erythrulose) and the aldehyde 224 (derived from L-malic acid) to give the aldol 225 in 72% yield. The secondary hydroxyl group of 225 was protected with a 2-(trimethylsilyl)- ethoxymethyl group (SEM) to produce ketone 226, which was reduced with LiBH4 to furnish alcohol 227 in 80% yield (dr > 95:5). Removal of the triethylsilyl group with DDQ gave the diol 228, which was converted to the per-benzylated derivative 229 under standard reaction conditions. The reaction of 229 with an excess of MeMgBr at reflux gave the primary O-tert-butyl ether 230 in which the SEM group had also been cleaved. Removal of the TPS group of 230 with TBAF was followed by conversion of the corresponding triol 231 to the trimesylate 232. Intermolecular
SN2 substitution of the primary O-mesylate using benzylamine and NaI as a catalyst in DMSO, with heating at 80 °C and then two intramolecular cyclizations generated the pyrrolizidine derivative 233 in 60% yield form 231 (2 steps). The proposed reaction sequence for the formation 233 from 232 is shown in Scheme 4.9.
Hydrogenolysis of 233 using H2, Pd(OH)2 at rt for 2 d afforded 234 in 75% yield. The final step was acid catalyzed cleavage of the tert-butyl ether group of 234 with TFA. Basification with aqueous ammonia then gave australine 13 in 11 steps and 10% overall yield from the ketone 223.
103 Chapter 4 [Australine]
O O OR a O O O OTES b, OH C O OTES OBn OTPS 223 OBn OTPS 224 225;R=H c 226; R = SEM
OR OSEM Ot BuOBn OR g d O O OR' OBn OTPS OR OBn OBn OR'
227;R=H,R'=TES 230;R=H,R'=TPS e h 228;R= R'=H 231;R= R'=H f i 229;R= R'=Bn 232;R= R'=Ms
RO HO H OH jNl R'O OH N H R'O OR' OH 13 233;R=tBu, R' = Bn k 234;R= tBu, R' = H Scheme 4.8 The total synthesis of australine 13 by Marco et al.188
Reagents and conditions: (a) Chx2BCl, Et3N, Et2O, 0 °C; (b) 224, 72%; (c) SEMCl, iPr2NEt,
CH2Cl2, rt, 24 h, 75%; (d) LiBH4, Et2O, -90 °C; 80%; (e) DDQ, aq. THF, rt 24 h, 74%; (f)
BnBr, NaH, THF, 4 h, 40 °C, 80%; (g) MeMgBr, tol/Et2O, 16 h , ∆, 77%; (h) TBAF,
CH2Cl2, rt 1 h, 97%; (i) MsCl, Et3N, CH2Cl2, rt, 2h; (j) BnNH2, NaI, DMSO, 80 °C, 24 h,
60% overall from 231; (k) H2, Pd(OH)2, rt, 2 d, 75%. (l) i: TFA, CH2Cl2, rt, 15 h; ii: aq. NH3, 78% overall from 234. [TES = Triethylsilyl, TPS = triphenylsilyl, DDQ = 2,3-dichloro-5,6- dicyano-1,4-benzoquinone].
233
232
Scheme 4.9 The proposed mechanism for the formation o f 233 from 232.188
104 Chapter 4 [Australine]
4.3 The synthesis of australine 13 from precursor 163
Our synthesis of australine 13 started from the epoxide 163 described in Chapter 3 (Scheme 3.8). The key steps were a regioselective reductive ring-opening of the epoxide of 163 followed by inversion of the configuration at C-7 and then deprotection (Scheme 4.10).
H OBn HO H OH O 1. Reduction OBn OH N 2.InversionofOHatC-7 N 3. Deprotection 163OTBS 13 OH
Scheme 4.10 Synthesis of australine 13 by Route 2 from Chapter 3 in Scheme 3.8.
4.3.1 Reduction with lithium aluminium hydride (LiAlH4)
A 91:9 mixture of the pyrrolizidine 163 and indolizidine 163a, respectively was treated with LiAlH4 in THF at 0 °C to afford a mixture of the pyrrolizidines 235 and 236 (27% yield; dr 88:12), a mixture of the pyrrolizidines 237 and 238 (64% yield; dr 92:8) and the indolizidine 239 (2% yield) (Scheme 4.11). OBn H OBn H O O OBn + OBn N N OTBS 163 OR 163a 163/163a =91:9
o LiAlH4,THF,0 C, 8 h
OBn HO H OBn H OBn HO H OBn OBn + HO OBn + N N N OTBS OR OR 235;R=H 236;R=H(235:236 =88:12),27% 239 (2%) 237;R=TBS 238;R=TBS(237:238 = 92:8), 64%
Scheme 4.11 The synthesis of ring-opening of 163/163a with LiAlH4.
105 Chapter 4 [Australine]
The formation of the pyrrolizidines 235 and 237 as the major regioisomeric products of this reaction can be rationalized using the previous Scheme 3.18 where the nucleophile is now hydride instead of water, with activation of the epoxide by Li+ rather than H+. These mixtures of regioisomers could not be readily separated but could be in the next synthetic step. The regioisomeric mixture of 235 and 236 could be converted to a mixture of 237 and 238 in 61% yield by treatment with TBSCl,
DMAP, Et3N in THF.
4.3.2 Inversion of the hydroxyl group at C-7 position
It was necessary to invert the configuration of the C-7 hydroxyl group of the alcohol 237. This was achieved using the Mitsunobu reaction189 which gave the C-7α 4-nitrobenzoate ester 240 after purification by column chromatography. Treatment of
240 with K2CO3 in MeOH to hydrolyze the benzoate ester afforded the diastereomerically pure alcohol 241 in 57% yield over the 2 steps (Scheme 4.12). ESI-MS analysis of the starting alcohol 237 and the product 241 were nearly the same whereas the 1H and 13C NMR spectrum were clearly different. The most significant difference was the chemical shifts of the two H-6 protons. After inversion these had closer chemical shifts than those in 237 (see circles in Figure 4.3). The chemical shift of H-1 had changed from 3.86 ppm in 237 to 4.27 ppm in the product 241, (Figure 4.3).
HO H OBn H OBn RO H OBn a 1 OBn + HO OBn 6 OBn N N N
OR OR OTBS
237;R=TBS 238;R=TBS 240;R=4-NO2C6H4CO b (237:238 = 92:8) 241;R=H
Scheme 4.12 The synthesis of the C-7 inverted pyrrolizidine 237. 0 Reagents and conditions: (a) DIAD, PPh3, p-NO2ArCO2H, toluene, 80 C, 1.5 h; (b)
K2CO3, MeOH, rt, 2 h, 57%.
106 Chapter 4 [Australine]
HO H OBn
6 1 OBn N
237 OTBS
H-1
H-6 H-6
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) HO H OBn
6 1 OBn N
OTBS 241
H-1
2xH-6
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) 1 Figure 4.3 The H NMR (500 MHz, CDCl3) spectra of 237 and 241.
4.3.3 Hydrogenolysis
4.3.3.1 Synthesis of australine 13 from hydrogenolysis of 241
Hydrogenolysis of 241 over PdCl2/H2, which also resulted in hydrolysis of the TBS ether due to in situ formation of HCl, gave diastereomerically pure
22 181 25 australine 13 ([α] D +9.4 (c 2.4, H2O), lit. [α] D +8 (c 0.35, H2O)), in 86% yield after ion-exchange chromatography (Scheme 4.13). The 1H and 13 C NMR spectra of our synthetic 13 are shown in Figures 4.4 and 4.5, respectively. The 1H NMR 54,55,60 spectroscopic data (D2O) of 13 and that of the natural product were essentially 13 identical (∆δH = 0.06-0.10 ppm, Table 4.1). The C NMR signals of 13 (in D2O with MeCN as an internal reference at δ 1.47) however, were all consistently 2.0-2.3 ppm
107 Chapter 4 [Australine] upfield of those reported for the natural product.54,55,60 Our 1H and 13C NMR spectroscopic data compared with those of the natural product and those of the previously synthesed compounds are shown in Appendices 1 and 2.
HO H OBn HO H OH
PdCl2,H2 (1 atm) OBn OH N MeOH, rt, 3 h; N ion-exchange 241 OTBS 86% 13 OH
Scheme 4.13 The synthesis of australine 13.
HOH OH 7 1 OH 5 N 3 8 13 OH
H-1 H-2 H-8 H-8’ H-7 H-7a H-5 H-5 H-3 2xH-6
4.00 3.50 3.00 2.50 2.00 pm (t1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) 1 Figure 4.4 The H NMR (500 MHz, D2O) of (+)-australine 13.
HOH OH C-7a 7 1 OH C-3 5 N 3 C-2 C-1 C-7 C-8 C-5 C-6 8 13 OH
80 70 60 50 40 30 20 10 0 ppm (t1)
13 Figure 4.5 The C NMR (125 MHz, D2O) of (+)-australine 13.
108 Chapter 4 [Australine]
Table 4.1 Physical and spectral data for (+)-australine 54,55,60 and 13.
Natural Product 54,55,60 Our synthetic 13
Physical Colourless prisms 54 Yellow oil Appearance
Optical Rotation 25 181 22 [α] D +8.0 (c 0.35, H2O) [α] D +9.4 (c 2.4, H2O) Melting Point 148-149 °C 54 # 1 60 H NMR 500 MHz, D2O, pH = 8.6 500 MHz, D2O
4.43 (ddd, 1H, J7,7a = 4.4, J6,7 = 4.2, J6,7 4.37-4.35 (m, 1H, H-7) = 2.4 Hz, H-7)
4.29 (dd, 1H, J1,2 = 8.2, J1,7a = 7.4 Hz, 4.22 (t, 1H, J1,2 = J1,7a = 7.8 Hz, H-1) H-1)
3.96 (dd, 1H, J2,3 = 9.5, J1,2 = 8.2 Hz, 3.89 (dd, 1H, J2,3 = 9.5, J1,2 = 8.0 Hz, H-2) H-2)
3.79 (dd, 1H, J8,8’ = 12.0, J3,8 = 3.5 Hz, 3.85 H-8)
3.61 (dd, 1H, J8,8’ = 11.5, J3,8’ = 7.0 Hz, 3.68 H-8’)
3.27 (dd, 1H, J1,7a = 7.4, J7,7a = 4.4 Hz, 3.17 (dd, 1H, J1,7a = 7.8, J7,7a = 4.8 Hz, H-7a) H-7a)
3.23 (dd, 1H, J5,6 = 11.5, J5.6 = 6.0 Hz, 3.15-3.12 (m, 1H, H-5) H-5a) 2.80 (m, 2H, H-3 and H-5b) 2.74-2.69 (m, 2H, H-3 and H-5)
2.10 (ddd, 1H, J5,6 = 6.0, J6,7 = 2.4, J5,6 2.05-2.00 (m, 1H, H-6) = 2.1 Hz, H-6a)
2.00 (ddd, 1H, J5,6 = 11.5, J5,6 = 7.5, J6,7 1.97-1.89 (m, 1H, H-6) = 4.2 Hz, H-6b) 55 125 MHz, D2O 125 MHz, D2O 13C NMR ∆δ (ppm) (Ref. TPS) (Ref. CH3CN) (Original 81.8 (C-7 / C-2) 79.5 (C-2) 2.3 assignment 75.9 (C-1) 73.7 (C-1) 2.2 73.5 (C-2 / C-7a) 71.3 (C-7a) 2.2 /reassignment) 73.3 (C-3) 71.1 (C-3) 2.2 72.3 (C-7a / C-7) 70.1 (C-7) 2.2 65.5 (C-8) 63.5 (C-8) 2.0 54.6 (C-5) 52.4 (C-5) 2.2 38.0 (C-6) 35.8 (C-6) 2.2 # Although our compound was pure by NMR analysis it did not crystallize at rt.
109 Chapter 4 [Australine]
4.3.3.2 Synthesis of 7-epi-australine 86 from hydrogenolysis of the mixture of 237 and 238
Hydrogenolysis of the mixture of 237 and 238 (dr 92:8) over PdCl2/H2 followed by purification and neutralized by ion-exchange chromatography gave 7- epi-australine 86 in 90% yield. 1H NMR analysis suggested that 86 was of 92%
23 170 25 purity ([α] D -13.2 (c 1.2, H2O), lit. [α] D -13.04 (c 0.55, H2O, pH 8.37) (Scheme 4.14). The 1H and 13 C NMR spectra are shown in Figures 4.6 and 4.7, respectively. 1 The H NMR spectroscopic data (D2O) of 86 and that of the synthetic compound 170 from Denmark et al were essentially identical (∆δH = 0.16-0.18 ppm, Table 4.2). 13 The C NMR signals of 86 (in D2O with MeCN as an internal reference at δ 1.47) however, were all consistently 1.0-1.1 ppm downfield of those reported earlier.170
HO H OBn HO H OH PdCl2,H2 (1 atm) MeOH, rt, 3 h OBn OH N then conc HCl, rt, 17 h N ion-exchange OTBS 90% OH 237 (dr 92:8) 86 (dr 92:8) Scheme 4.14 The synthesis of 7-epi-australine 86. HOH OH
7 1 H-2 OH H-1 5 N 3 H-7 H-8 H-8’ H-7a 8 H-5 H-5 H-3 H-6 H-6 86 OH
4.00 3.50 3.00 2.50 2.00 ppm (f1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 4.6 The H NMR (500 MHz, D2O) of (-)-7-epi-australine 86.
HOH OH 7 1 C-7 OH 5 N 3 C-2 C-8 C-5 C-6 8 86 OH C-1 C-7a C-3
80 70 60 50 40 30 ppm (f1) 13 Figure 4.7 The C NMR (125 MHz, D2O) of (-)-7-epi-australine 86.
110 Chapter 4 [Australine]
Table 4.2 Physical and spectral data for (-)-7-epi-australine170 and 86.
7-Epi-australine170 Our synthetic 86 Physical Cubic crystals Pale yellow solid Appearance
Optical 20 23 [α] -13.04 (c 0.55, H2O, pH 8.37) [α] -13.2 (c 1.2, H2O) Rotation D D Melting Point 193-194 °C - 1 H NMR 500 MHz, D2O (pH 8.37) 500 MHz, D2O
4.18 (dt, 1H, J = 4.6, J = 2.7 Hz, H-7) 4.34 (brs, 1H, H-7)
3.60 (dd, 1H, J = 11.7, J = 3.9 Hz, H- 3.77 (dd, 1H, J8,8’ = 12.0, J3,8 = 4.5 Hz, H-8) 8)
3.58 (t, 1H, J = 8.5 Hz, H-2) 3.76 (t, 1H, J1,2 = J2,3 = 8.5 Hz, H-2)
3.54 (t, 1H, J = 8.1 Hz, H-1) 3.71 (t, 1H, J1,2 = J1,7a = 8.0 Hz, H-1)
3.46 (dd, 1H, J = 11.7, J = 6.3 Hz, H- 3.64 (dd, 1H, J8,8’ = 11.8, J3,8 = 5.8 Hz, H-8) 8)
2.90 (ddd, 1H, J = 11.5, J =10.3, J = 3.08 (dd, 1H, J5,5 = J5,6 =10.8, J5,6 = 6.0 Hz, 5.9 Hz, H-5) H-5)
2.84 (dd, 1H, J = 7.8, J = 2.0 Hz, H- 3.01 (brd, 1H, J1,7a = 8.0 Hz, H-7a) 7a)
2.70 (ddd, 1H, J = 11.0, J = 7.1, J = 2.89-2.84 (m, 1H, H-5) 3.4 Hz, H-5)
2.50 (ddd, 1H, J = 9.3, J = 6.3, J = 2.69-2.64 (m, 1H, H-3) 3.9 Hz, H-3) 1.90 (m, 1H, H-6) 2.11-2.04 (m, 1H, H-6)
1.60 (ddt, 1H, J = 12.7, J = 6.1, J = 1.80-1.74 (m, 1H, H-6) 3.4 Hz, H-6) 13 C NMR 126 MHz, D2O 125 MHz, D2O ∆δ (ppm) (Ref not given) (Ref. CH3CN) 77.5 (C-1) 78.6 (C-1) -1.1 75.9 (C-2) 77.0 (C-2) -1.1 74.8 (C-7) 75.8 (C-7) -1.0 73.5 (C-7a) 74.5 (C-7a) -1.0 67.9 (C-3) 69.0 (C-3) -1.1 62.3 (C-8) 63.4 (C-8) -1.1 51.4 (C-5) 52.4 (C-5) -1.0 31.2 (C-6)* 32.3 (C-6) -1.1 *data from NMR spectrum
111 Chapter 4 [Australine]
4.3.3.3 Synthesis of 1-epi-castanospermine 242 from hydrogenolysis of 239
Hydrogenolysis of 239 over PdCl2/H2 gave diastereomerically pure 1-epi-
20 190 22 castanospermine 242 ([α] D +6.2 (c 0.15, MeOH), lit. [α] D +3.3 (c 0.3, MeOH) in 94% yield after ion-exchange chromatography (Scheme 4.15). The 1H and 13 C NMR spectra are shown in Figures 4.8 and 4.9, respectively. The 1H NMR spectroscopic 190 data (D2O) of 242 and that of the synthetic compound from Cronin and Murphy 13 were essentially identical (∆δH = 0.02-0.03 ppm, Table 4.3). The C NMR signals of
242 (in D2O with MeCN as an internal reference at 1.47 ppm) however, were all consistently 1.7-1.8 ppm downfield of those reported previously. However the internal reference used in the published 13C NMR spectrum was not reported.190
HO OBn OH H PdCl2,H2 (1 atm) HO H OBn MeOH, rt, 3 h OH N then conc HCl, rt, 21 h N OTBS ion-exchange OH 94% 239 242
Scheme 4.15 The synthesis of 1-epi-castanospermine 242. OH HO H OH 1 8a 7 3 N 5 H-8 H-5 OH H-7 H-5 H-3 H-3 H-8a 242 H-1 H-6 H-2 H-2
4.00 3.50 3.00 2.50 2.00 ppm (f1)
4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 4.8 The H NMR (500 MHz, D2O) of (+) 1-epi-castanospermine 242. OH HO H OH 1 8a 7 3 N 5 C-7 C-8 C-8a OH C-6 C-5 C-3 C-2 C-1 242
80 70 60 50 40 30 ppm (f1) 13 Figure 4.9 The C NMR (125 MHz, D2O) of (+) 1-epi-castanospermine 242.
112 Chapter 4 [Australine]
Table 4.3 Physical and spectral data for (+)-1-epi-castanospermine190 and 242.
1-Epi-castanospermine190 Our synthetic 242 Physical Crystallize solid Brown foamy solid Appearance Optical [α] 22 +3.3 (c 0.3, MeOH) [α] 20 +6.2 (c 0.15, MeOH) Rotation D D 1 H NMR 300 MHz, D2O 500 MHz, D2O 4.26-4.22 (m, 1H, H-1) 4.21 (ddd, 1H, J = 9.4, J = 3.5 Hz)
3.61 (ddd, 1H, J5,6 = 11.0, J6,7 = 9.5, J5,6 = 5.5 3.58 (ddd, 1H, J = 10.8, J = 5.3 Hz) Hz, H-6)
3.39 (t, 1H, J7,8 = J8,8a = 9.0 Hz, H-8) 3.34 (2 overlapping t, 2H, J = 8.93 Hz) 3.33 (t, 1H, J6,7 = J7,8 9.0 Hz, H-7)
3.14 (dd, 1H, J = 11.0, J = 5.3 Hz) 3.16 (dd, 1H, J5,5 = 10.8, J5,6 = 5.8 Hz, H-5)
2.95 (ddd, 1H, J3,3 = 9.5, J2,3 = 8.0, J2,3 = 1.5 2.92 (t, 1H, J = 9.5 Hz) Hz, H-3)
2.54 (q, 1H, J = 18.5, J = 9.2 Hz) 2.57 (dt, 1H, J3,3 = 9.3, J2,3 = 8.5 Hz, H-3),
2.26-2.24 (m, 1H, J = 18.0, J = 9.4 Hz) 2.34-2.28 (m, 1H, H-2)
2.21 (t, 1H, J = 11.0 Hz) 2.24 (t, 1H, J5,5 = J5,6 = 10.8 Hz, H-5)
2.10 (dd, 1H, J = 9.1, J = 6.7 Hz) 2.12 (dd, 1H, J8,8a = 9.5, J1,8a = 6.5 Hz, H-8a)
1.67 (m, 1H, H-2) 1.70 (brt, 1H, J2,2 = J2,3 = 11.5 Hz, H-2)
75 MHz, D2O 125 MHz, D2O 13C NMR ∆δ (ppm) (Ref not given) (Ref . CH3CN) 81.1 79.3 (C-7) 1.8 76.1 74.3 (C-1) 1.8 75.7 74.0 (C-8) 1.7 75.2 73.4 (C-8a) 1.8
72.3 70.5 (C-6) 1.8 57.2 55.5 (C-5) 1.7 53.2 51.4 (C-3) 1.8 34.8 33.0 (C-2) 1.8
In conclusion, the epoxide 163 has also provided ready access to australine
13, in a total of 14 synthetic steps and in 6% overall yield from L-xylose, and 7-epi- australine 86 in 92% purity and 10% overall yield in 13 synthetic steps. While the epoxide 163a has also allowed the synthesis of 1-epi-castanospermine 242. These syntheses are summarized in Scheme 4.16.
113 Chapter 4 [Australine]
163/163a [163/163a = 91:9]
a
OBn OBn OBn HO H H HO H OBn OBn + HO OBn + N N N OTBS OR OR 235;R=H 236;R=H(235:236 =88:12),27% 239 (2%)
237;R=TBS 238;R=TBS(237:238 = 92:8), 64% f
e b OH HO OH HO H RO H OBn H OH OH OBn N N N OH OH 242 86 OTBS
240;R=4-NO2C6H4CO c 241;R=H
d
HO H OH
OH N
13 OH
Scheme 4.16 The syntheses of (+) australine 13, (-) 7-epi-australine 86 and (+) 1-epi- castanospermine 242. o Reagents and conditions: (a) LiAlH4, THF, 0 C, 8 h, 235:236 [dr = 88:12] (27%), 237:238 0 [dr = 92:8] (64%) 239 (2%) (b) DIAD, PPh3, p-NO2ArCO2H, toluene, 80 C, 1.5 h (c)
K2CO3, MeOH, rt, 2 h, 57% (d) (PdCl2, H2 (1 atm), MeOH, rt, 3 h, ion-exchange, 86%; e)
PdCl2, H2 (1 atm), MeOH, rt, 3 h, then conc. HCl rt, 17 h, ion-exchange, 90%; (f) PdCl2, H2 (1 atm), MeOH, rt, 3 h, then conc. HCl rt, 21 h, ion-exchange, 94%.
114 Chapter 5 [3-Epi-casuarine]
CHAPTER 5 SYNTHESIS OF 3-EPI-CASUARINE, 1,2,6-TRIHYDRO-XYL- 7,8-EPOXYINDOLIZIDINE AND 1,2,7-TRIHYDROXYL-6,8- EPOXYINDOLIZIDINE
5.1 Isolation and biological activities of 3-epi-casuarine
Fleet et al.58 reported the isolation of 3-epi-casuarine 79 in the same year as the isolation of casuarine 15 from Myrtus communis L which is described in more detail in Chapter 3.1.
5.2 Previous syntheses of 3-epi-casuarine
In 2006, Izquierdo et al.191 published the synthesis of 3-epi-casuarine 79 in the same year that Fleet et al.58 reported its isolation as a natural product and also its synthesis. The synthesis of 3-epi-casuarine 79 by Izquierdo et al.191 involved the same methodology that they used for the synthesis of casuarine 15 (Scheme 5.1) except using the pyrrolidine 243 as the starting material. N-Cbz protection of 243 gave the Cbz carbamate 244 in only 25% yield. The primary alcohol of 244 was oxidized using TPAP and NMO to afford the aldehyde 245 which after a Wittig reaction gave the (E)-pyrrolidinic propenoate 246 (93% yield). Cis-DH reaction of 246 using osmium tetraoxide and NMO in the presence of O-(4- chlorobenzoyl)hydroquinine (DHQ-CLB) as a chiral ligand gave the diols 247 (13% yield) and 248 (84% yield). The configuration of both diol products could not be determined at this stage. After two more synthetic steps an NOE experiment confirmed that 249 was the desired intermediate to prepare 3-epi-casuarine 79. N- deprotection of 248 using catalytic hydrogenolysis provided pyrrolidine 249 which was subsequently transformed to 250 by refluxing in methanol in the presence of a catalytic amount of NaOMe. Acetylation under standard reaction conditions then produced the acetate derivative 251 in 88% yield. Reduction of the lactam carbonyl group of 251 using BH3·SMe2 complex in THF gave 252 in 96% yield. O-TBDPS deprotection and then debenzylation provided 253 in 73% yield. Hydrogenolysis then gave the final compound, however it was not pure. The product was further purified by per-acetylation that gave 254 in 88% yield. Base catalysed deacetylation of 254
115 Chapter 5 [3-Epi-casuarine] afforded 3-epi-casuarine 79 in 66% yield. This synthesis was achieved in 12 steps from the pyrrolidine derivative 243 in an overall yield of 2.2%.
R' BnO R 1 OTBDPS H R CO Me R N d 2 BnO 2 3 N R R BnO OBn Cbz TBDPSO 243; R=CH2OH; R' = H a 247; R=R2 =OH;R1=R3 =H 244 ; R=CH2OH; R' = Cbz 2 1 3 b 248; R=R =H;R =R =OH 245; R = CHO; R' = Cbz c 246; R=(E)-CH=CHCO2Me;R' = Cbz
OH 1 H OBn RO H OR e 248 HO f OBn RO OR1 HN N MeO O OTBDPS X OPG
249 250; R = H; PG = TBDPS; R1 =Bn;X=O g 251; R = Ac; PG = TBDPS; R1 =Bn;X=O h 1 252; R = H; PG = TBDPS; R =Bn;X=H2 i 1 253; R=PG=H;R =Bn;X=H2 j 79; R=R1 =PG=H;X=H g 2 k 1 254; R=R =PG=Ac;X=H2
Scheme 5.1 The total synthesis of 3-epi-casuarine 79 by Izquierdo et al.191
Reagents and conditions: (a) CbzCl, Me2CO, K2CO3, rt, 25%; (b) TPAP, NMO, 4°A Ms,
CH2Cl2, 64%; (c) Ph3P=CHCO2Me, CH2Cl2, rt, 93%; (d) OsO4, NMO, DHQ-CLB, acetone/H2O, rt, 2 d, (247:248 = 13%:84%); (e) H2, 10% Pd-C, MeOH; (f) cat. NaOMe,
MeOH, rt, 63%; (g) Ac2O, py, DMAP, 88%; (h) H3B:SMe2, THF, then MeOH, ∆, 96%; (i) + - - n-Bu4N F ·3H2O, THF, rt, 73%; (j) i: H2, 10% Pd-C, MeOH, then Amberlite IRA-400 (OH form), ii: Ac2O, py, DMAP, 70%; (k) cat. NaOMe, MeOH, rt, 66%.
In 2006, Fleet et al. reported the synthesis of casuarine 15 from D- gluconolactone 140 (Scheme 3.4) together with the synthesis of 3-epi-casuarine 79 from D-gluconolactone 140 in the same publication (Scheme 5.2).58 He also followed the methodology that he used to synthesize casuarine 15 up to the precursor 147. Regioselective protection of the primary hydroxyl group of diol 147 with TBSCl and then reaction at the secondary hydroxyl group by treatment with methanesulfonyl chloride generated 255 in 66% yield. Reduction of the lactam carbonyl group of 255 with BH3·THF gave the protected amine 256 (57% yield). Finally pure 3-epi- casuarine 79 was obtained after 2 more steps; (i) O-silyl group hydrolysis with TFA
116 Chapter 5 [3-Epi-casuarine] to produce 257; and then (ii) cyclization by treatment with sodium acetate (89% yield over the two steps).
O O OH TBSO TBSO O a O b HO OH TBSO TBSO NH NH OH OMs O O CH2OH O OH O OTBS 140 147 255
O OH TBSO HO HO OH O cdOH H TBSO HO HO OH NH NH N OMs OMs OTBS OH OH 79 256 257 Scheme 5.2 The total synthesis of 3-epi-casuarine 79 by Fleet et al.58
Reagents and conditions: (a) t-BuMe2SiCl, py; then CH3SO2Cl, Et3N, CH2Cl2, 66%; (b)
BH3·THF, THF, 57%; (c) 90% CF3CO2H, H2O; (d) NaOAc, H2O (89% over two steps).
5.3 The synthesis of 3-epi-casuarine from precursor 161
5.3.1 Mitsunobu reaction for inversion of the hydroxyl group and cyclization
The synthesis of naturally occurring 3-epi-casuarine 79 from the epoxide 161 required an inversion of the configuration of the butyl side chain secondary hydroxyl in 161 (Scheme 5.3). This was achieved by the Mitsunobu reaction of 161 with 4- nitrobenzoic acid. Base treatment (K2CO3/MeOH) at rt for 24 h of the resulting secondary 4-nitrobenzoate ester resulted in benzoate hydrolysis and N-Fmoc cleavage giving the amino alcohol 258 in 55% overall yield for the two steps. However the 1H NMR spectrum of 258 showed ca. 16% of the secondary O-TBS derivative of 258 had formed from O-TBS migration (Figure 5.1(a)). These compounds could not be separated by TLC. A comparison of the 1H NMR spectra of 258 with 162, indicated that the secondary hydroxyl group of 258 had inverted because of the differences in their chemical shift (Figure 5.1(a) and (b)). This mixture underwent cyclization under Mitsunobu reaction conditions with toluene as a solvent to provide a separable mixture the desired pyrrolizidine 259 in 70% yield and
117 Chapter 5 [3-Epi-casuarine] the indolizidine 260 in 4% yield (90% purity). The NMR spectroscopic and ESI mass spectrometric data confirmed the structures of 259 and 260.
OBn OH OBn OH H OTBS H OTBS 9steps O a O L-xylose N OBn NH OBn Fmoc 161 258
OBn H OBn H b O O OBn OBn + N N OTBS OTBS 259 260 (70%) (4%)
Scheme 5.3 The synthesis of pyrrolizidine 259 and indolizidine 260. o Reagents and conditions: (a) i: p-NO2ArCO2H, PPh3, toluene, 80 C, 5 h; ii: K2CO3, MeOH, o rt, 24 h, 55% (2 steps); (b) DIAD, Ph3P, toluene, 80 C, 3 d, 259 (70%) and 260 (4%).
OBn OH H O OTBS 1' 3' NH OBn (a) 258
1.00 0.19 84% (1° O-TBS) 16% (2° O-TBS) 4.550 4.500 pm (t1) H-2’, H-3’, H-4’
1 2 1 1 4 8 0
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) OBn OH H O OTBS 1' 3' (b) NH OBn H-2’, H-3’, H-4’ 162
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 5.1 The H NMR (500 MHz, CDCl3) spectra of 258(a) and 162(b).
118 Chapter 5 [3-Epi-casuarine]
5.3.2 Epoxide ring-opening by NaHSO4
The epoxy-pyrrolizidine 259 was treated under the same epoxide ring- opening reaction conditions used to prepare casuarine 15 in Chapter 3.3.6 (Scheme 3.17) except that heating at 50 °C was continued for 7 d. We attribute the slower rate of epoxide ring-opening of 259, when compared to that of 163, due to the steric hindrance of the -CH2OTBS substituent in 259. TLC analysis of the crude reaction mixture showed many very polar products. Column chromatography of the reaction mixture gave 5 fractions, however the 1H NMR spectra of these fractions indicated that they were not pure. The fractions were then combined and the mixture was acetylated. Separation by column chromatography gave the desired product 261 in 7% yield, the undesired products 262 (8%) and 264 (17%) and the epoxide compound 263 in 9% yield (Scheme 5.4).
Scheme 5.4 The synthesis epoxide ring-opening of 259.
Unfortunately, several attempts to improve the yield of this step were not successful. The 1H NMR spectroscopic data of the 3-epi-casuarine derivative 261 showed three 3H singlet signals at 2.11, 2.05 and 2.01 ppm for the acetate groups and the NOE correlations shown in Figure 5.2. The definite NOE correlations between H-7 and H-1 indicated that H-7 and H-1 were on the same face of the molecule and also H-7 showed a correlation to H-5α that confirmed that they were on the same face of the ring. Furthermore H-5α showed a correlation to H-8 which indicated that the C-3 group had the desired α-configuration. A NOE cross peak between H-3 and H-2 and a weak cross peak between H-3 and H-7a were also observed. A NOE
119 Chapter 5 [3-Epi-casuarine] correlation between H-5β and H-6 and a weak cross peak between H-5β and H-7a were also found. Compound 261 was therefore determined to be the desired protected 3-epi-casuarine derivative.
AcO H H HOBn H 7 1 H AcO OBn 5 N 3 H Hα 8 H β H OAc H
NOE Correlations
Figure 5.2 The NOE correlations of 261.
A mechanism for formation of the undesired products of 262 and 264 is shown in Scheme 5.5. Acid catalysed hydrolysis of the O-TBS ether of 261 before epoxide ring-opening would give the intermediate alcohol A. Intramolecular epoxide ring-opening could then give a mixture of 262 and 264.
H OBn H OBn O NaHSO4,CH2Cl2 O OBn OBn N 50 oC N 261 OTBS A OH
H OBn H OBn HO HO OBn OBn N N
OH OH
H OBn RO H OBn 7 1 1 RO OBn 6 OBn N 8 N O 8 O 8
R=H R=H Ac O, py Ac O, py 2 R=Ac(262; 8%) 2 R=Ac(264; 17%)
Scheme 5.5 The proposed mechanism of the undesired product 262 and 264.
120 Chapter 5 [3-Epi-casuarine]
The 1:2 ratio of 262 and 264 was consistant with their relative heats of formation as calculated using PC Spartan (AM1). The calculated heat of formation of 262 (-122.29 kcal/mol) was more than that of 264 (-129.78 kcal/mol) suggesting 262 had more ring strain than 264. The identities of 262 and 264 were established by 1D and 2D NMR analysis. The 1H NMR spectrum of 262 showed that the most downfield proton (H-6) at 5.24 (dd, 1H, J = 6.3, 2.8 Hz) ppm was coupled to two of the diastereotopic H-5 protons at 3.65 (dd, 1H, J = 14.5, 6.5 Hz, H-5α) and 2.85 (dd, 1H, J = 14.3, 2.8 Hz, H-5β) ppm (Figure 5.4). The H-5α and H-5β protons were individually assigned based upon their vicinal coupling constants to H-6 and from NOESY NMR spectra. Using PC Spartan (AM1) the dihedral angle (φ) between H-6 and H-5β was -120.3° and between H-6 and H-5α was 0.2°. Based on the Karplus equation J5β,6 should be 3-6 Hz and J5α,6 8.5-12.5 Hz. The larger observed vicinal coupling constant (6.5 Hz) between H-6 and the two H-5 protons was therefore assigned to J5α,6 and the smaller one (2.8 Hz) to J5β,6. While the most downfield 1 proton in the H NMR spectrum of 264 (H-7) resonates at 4.97 (brs, W1/2 = 1 Hz, 1H) ppm (Figure 5.5) and showed a weak COSY cross peak to H-5α (W-coupling). The calculated dihedral angle from PC Spartan (AM1) between H-7 and H-6 was 99.2° and between H-7 and H-7a was -128.8°. From the Karplus equation J6,7 should be 0-
3 Hz and J7,7a 3-7 Hz. The coupling constants observed for H-7 were consistent with the calculated J values and the structure of 264. The structures and relative configurations of 262 and 264 were further supported by NOESY NMR experiments in Figure 5.3.
7 7 7a 7a 5 2 5 2 8 8
NOE correlations 262 264
Figure 5.3 Structures of 262 and 264 from PC Spartan (AM1).
121 Chapter 5 [3-Epi-casuarine]
The definite NOE between H-7a and H-5β in 262 indicated that these protons were on the same face of the ring. The correlation between H-5α and H-8 indicated that H-3 was on the opposite face to H-5α. The NOE correlation between H-3 and H- 2 indicated that they were on the same face of the ring. Furthermore NOE correlations between H-6 and H-8 further supported the structure of 262 as shown in Figure 5.3. NOE experiments on 264 showed correlations between H-7 and H-1, H- 7a and H-5β and H-2 and H-3. H-5α showed a weak correlation to H-8. These correlations further supported the structure assigned to 264 (Figure 5.3).
H OBn 6 1 AcO OBn N O 8 262
5.300 5.250 5.200 5.150 ppm (f1)
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 5.4 The H NMR (500 MHz, CDCl3) of the tricyclic 262.
AcO H OBn
7 1 OBn N O 8 264
050 5.000 4.950 4.900 ppm (f1)
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 5.5 The H NMR (500 MHz, CDCl3) of the tricyclic 264.
122 Chapter 5 [3-Epi-casuarine]
For compound 263, the 1H NMR spectrum was similar to that of 259 except for the chemical shifts of H-8 and H-3. In 263 the signals for these protons were observed at lower field due to the deshielding effect of the acetate group (Figure 5.6).
H OBn O OBn N
259 OTBS
TBS group 2xH-8
H-3
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1) H OBn Ac group O OBn 2xH-8 H-3 N 263 OAc
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1)
1 Figure 5.6 The H NMR (500 MHz, CDCl3) of the starting 259 and 263.
5.3.3 Hydrogenolysis
5.3.3.1 Synthesis of 3-epi-casuarine 79 from hydrogenolysis of 261
Hydrogenolysis of 261 over PdCl2/H2 in MeOH for 4 d gave
25 58 23 diastereomerically pure 3-epi-casuarine 79 ([α] D +2.0 (c 0.4, H2O), lit. [α] D +5.7
(c 0.5, H2O) in 77% yield after purification by ion-exchange chromatography (Scheme 5.6). The 1H and 13C NMR spectra are shown in Figures 5.7 and 5.8, 1 respectively. The H NMR spectroscopic data (D2O) of 79 and that of the natural 58 13 product were essentially identical (∆δH = 0.01-0.02 ppm, Table 5.1) as were the C 58 NMR chemical shifts (∆δC = 0.0-0.1 ppm).
123 Chapter 5 [3-Epi-casuarine]
AcO H OBn HO H OH PdCl2,H2 (1 atm) MeOH, 4 d AcO OBn HO OH N N ion-exchange, 77% 261 OAc 79 OH
Scheme 5.6 The synthesis of 3-epi-casuarine 79. . HO H OH 7 1 HO OH 5 N 3 8 79 OH
2xH-5, H-7a
H-1 H-2 H-7 H-8’ H-6 H-8 * H-3
4.00 3.50 ppm (f1)
* impurity
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1)
1 Figure 5.7 The H NMR (500 MHz, D2O) of 3-epi-casuarine 79.
HO H OH 7 1 HO OH 5 N 3 8 79 OH C-2 C-3 C-7 C-6 C-8 C-1 C-7a C-5
85.0 80.0 75.0 70.0 65.0 60.0 55.0 50.0 45.0 ppm (f1)
13 Figure 5.8 The C NMR (125 MHz, D2O) of 3-epi-casuarine 79.
124 Chapter 5 [3-Epi-casuarine]
Table 5.1 Physical and spectral data for (+)-3-epi-casuarine58 and 79.
Natural Product58 Our synthetic 79
Physical Crystallize solid58 Pale yellow solid Appearance
Optical Rotation 23 58 25 [α] D +5.7 (c 0.5, H2O) [α] D +2.0 (c 0.4, H2O)
1 60 H NMR 500 MHz, D2O, pH = 8.6 500 MHz, D2O 4.26 (dd, 1H, 1.6, 1.4 Hz, H-1) 4.28 (brs, 1H, H-1)
4.17 (dd, 1H, 3.3, 1.4 Hz, H-2) 4.19 (dd, 1H, J2,3 = 3.5, J1,2 = 1.5 Hz, H-2)
4.10 (ddd, 1H, 8.5, 7.7, 7.7 Hz, H- 4.11 (dt, 1H, J5,6 = J6,7 = 7.8, J5,6 = 7.5 Hz, 6) H-6)
4.03 (d, 1H, 7.7 Hz, H-7) 4.05 (t, 1H, J6,7 = J7,7a = 8.0 Hz, H-7)
4.00 (dd, 1H, J8,8 = 11.8, J3,8 = 6.3 Hz, H- 3.98 (dd, 1H, 11.8, 6.4 Hz, H-8) 8)
3.93 (dd, 1H, 11.8, 7.3 Hz, H-8) 3.94 (dd, 1H, J8,8 = 11.5, J3,8 = 7.5 Hz, H-8)
3.26 (ddd, 1H, 7.3, 6.4, 3.3 Hz, H- 3.27 (ddd, 1H, J3,8 = 6.8, J3,8 = 6.5, J2,3 = 3) 3.5 Hz, H-3)
3.09 (overlapping, 2H, 2xH-5) 3.10 (d, 3H, J7,7a = 8.0 Hz, 2xH-5, H-7a) 3.087 (dd, 1H, 7.7, 1.6 Hz, H-7a) 58 125 MHz, D2O 125 MHz, D2O 13C NMR ∆δ (ppm) (Ref. dioxane) (Ref. CH3CN) 80.4 (C-1) 80.4 (C-1) 0.0 79.7 (C-2) 79.7 (C-2) 0.0 79.2 (C-7) 79.2 (C-7) 0.0 75.9 (C-6) 75.9 (C-6) 0.0 75.5 (C-7a) 75.5 (C-7a) 0.0 65.0 (C-3) 64.9 (C-3) 0.1 57.4 (C-8) 57.4 (C-8) 0.0 51.6 (C-5) 51.6 (C-5) 0.0
125 Chapter 5 [3-Epi-casuarine]
5.3.3.2 Synthesis of tricyclic derivative 265 from hydrogenolysis of 262 The acetate group of 262 was first removed using amberlyst (OH- form) in MeOH and then hydrogenolysis over PdCl2/H2 in MeOH for 1 d gave the deprotected
23 tricyclic derivative 265 [α] D +8.6 (c 0.5, MeOH), in 82% yield after ion-exchange chromatography (Scheme 5.7). The 1H and 13 C NMR spectra are shown in Figures 5.9 and 5.10, respectively.
Scheme 5.7 The synthesis of tricyclic derivative 265.
OH 7 H 1 HO OH 5 N 3 O 8 2xH-8 265 H-1 H-7 H-7a H-6 H-2 H-5α H-3 H-5β
4.50 4.00 3.50 3.00 ppm (f1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1)
1 Figure 5.9 The H NMR (500 MHz, D2O) of tricyclic derivative 265.
H OH 7 1 HO OH 5 N 3 C-6 C-7a O 8 C-7 C-2 C-1 C-3 C-8 265 C-5
90 80 70 60 50 ppm (f1)
13 Figure 5.10 The C NMR (125 MHz, D2O) of tricyclic derivative 265.
126 Chapter 5 [3-Epi-casuarine]
The structure of 265 was confirmed by HMBC experiments which are summarized in Table 5.2.
Table 5.2 The HMBC correlations of tricyclic 265.
1 Structure H NMR (500 MHz, D2O) HMBC correlations
OH 4.41 (dd, 1H, J5,6 = 6.0, J5,6 = 2.5 Hz, 56.8 (C-5), 70.6 (C-7a) and 81.4 7 H 1 H-6) (C-7) HO 7a OH 53 N 4.27 (brd, 1H, J1,2 = 4.0 Hz, H-1) 79.9 (C-2) or 81.4 (C-7) O 8 4.27 (brdd, 1H, J2,3 = 5.5, J1,2 = 4.5 59.5 (C-8) and 75.8 (C-1) 265 Hz, H-2) 3.87 (brs, 1H, H-7) 56.8 (C-5) and 59.5 (C-8) 3.62-3.54 (m, 2H, 2 x H-8) 63.7 (C-3), 79.9 (C-2) and 81.4 (C-7)* 3.62-3.54 (m, 1H, H-5α) 63.7 (C-3), 70.6 (C-7a) and 74.4 (C-6) 2.96 (brs, 1H, H-7a) 63.7 (C-3) and 79.9 (C-2)
2.94 (brd, 1H, J2,3 = 5.0 Hz, H-3) 59.5 (C-8), 70.6 (C-7a), 75.8 (C- 1) and 79.9 (C-2)
2.63 (dd, 1H, J5,5 = 14.0, 5.0, J5,6 = 63.7 (C-3), 70.6 (C-7a) and 74.4 2.0 Hz, H-5β) (C-6) * Important correlation that confirmed the structure
5.3.3.3 Synthesis of tricyclic derivative 266 from hydrogenolysis of 264
Following the same two step deprotection method described above,
23 compound 264 was converted to the tricyclic derivative 266 [α] D +7.6 (c 0.34, MeOH), in 53% yield after purification by ion-exchange chromatography (Scheme 5.8). The 1H and 13 C NMR spectra are shown in Figures 5.11 and 5.12, respectively. The structure of 266 was confirmed by HMBC experiments which are summarized in Table 5.3.
AcO OBn HO OH H Amberlyst O H, MeO H, rt, 16 h H PdCl2,H2 (1 atm), MeOH, 12 h OBn OH N N ion-exchange, 53% O O 264 266
Scheme 5.8 The synthesis of tricyclic derivative 266.
127 Chapter 5 [3-Epi-casuarine]
H-6, H-7 HO H OH H-8’ 7 1 OH H-5α H-7a H-5β 5 N 3 H-2 H-8 H-3 H-1 O 8 266
4.00 3.50 3.00 ppm (f1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 5.11 The H NMR (500 MHz, D2O) of tricyclic derivative 266.
HO H OH
7 1 OH 5 3 C-2 N C-1 8 C-6 C-7 C-7a C-3 C-8 C-5 O 266
80 70 60 50 40 ppm (f1) 13 Figure 5.12 The C NMR (125 MHz, D2O) of tricyclic derivative 266.
Table 5.3 The HMBC correlations of tricyclic 266.
1 Structure H NMR (500 MHz, D2O) HMBC correlations
HO H OH 4.25 (t, 1H, J 1,2 = J 2,3 = 7.0 Hz, H- 54.9 (C-8), 64.3 (C-3) and 79.5 (C-1) 7 1 2) 7a OH 5 N 3 4.22 (s, 2H, H-6 and H-7) 47.7 (C-5), 54.9 (C-8) and 77.9 (C-7) O 8 4.16 (dd, 1H, J1,2 = 7.5, J1,7a = 4.5 76.5 (C-7a) 266 Hz, H-1)
3.76 (dd, 1H, J8,8 = 12.5, J3,8 = 5.5 77.7 (C-2) Hz H-8)
3.69 (d, 1H, J8,8 = 12.5 Hz, H-8) 64.3 (C-3) and 77.7 (C-2)
3.63 (d, 1H, J5,5 = 14.0 Hz, H-5α) 64.3 (C-3) and 79.9 (C-7)
3.54 (t, 1H, J2,3 = J3,8 = 6.0 Hz, H-3) 47.7 (C-5), 77.7 (C-2) and 79.5 (C-1)
3.08 (d, 1H, J1,7 = 4.0 Hz, H-7a) 64.3 (C-3) and 79.5 (C-1)
2.80 (d, 1H, J5,5 = 14.0 Hz, H-5β) 64.3 (C-3), 79.9 (C-7) and 80.2 (C-6)
128 Chapter 5 [3-Epi-casuarine]
5.3.3.4 Attempts to improve the yield of 3-epi-casuarine 79
The epoxide ring-opening reaction of 259 with NaHSO4 was low yielding due to formation of the undesired products 262 and 264. We therefore desired to change the TBS group to a TBDPS group which is more stable under acidic conditions.192
OBn OH OBn OH H H OR a OR c
NH OBn N OBn Fmoc 158 267;R=H b 268; R= TBDPS
OBn OH OBn OR1 H H 2 O OTBDPS O OR d e N OBn NH OBn Fmoc 270 ; R1 =H,R2 = TBDPS 269 270b; R1 =TBDPS,R2 =H
OBn OBn OBn HO H H H OBn O O OBn f OBn + HO N N N OTBDPS OTBDPS 271 OTBDPS 272 273
Scheme 5.9 Attempts to synthesize 3-epi-casuarine 79. o Reagents and conditions: (a) FmocCl, THF, sat. Na2CO3, 0 C, 5 h, 81%; (b) TBDPSCl, o DMAP, imidazole, THF, rt, 1 d, 78%; (c) CF3COCH3, oxone, NaHCO3, MeCN/H2O, 0 C,
10 h, 67%; (d) p-NO2ArCO2H, PPh3, toluene, rt, 2 d; K2CO3, MeOH, rt, 1 d, 62%; (e) DIAD, o Ph3P, toluene, 80 C, 3 d, (93%), 271:272 = 3:2 (f) NaHSO4, CH2Cl2, reflux, 14 d; water, rt, 1 h, 76 (7%).
This synthesis began with N-Fmoc protection of 158 which provided 267 in good yield and then the primary alcohol of 267 was protected by treatment with TBDPSCl/DMAP/imidazole to give the TBDPS derivative 268 in 78% yield. Epoxidation of 268 furnished epoxide 269 in 67% yield. Inversion of configuration of the secondary hydroxyl group of 269 using the Mitsunobu reaction followed by base hydrolysis gave the amino alcohol 270 and 270b in 62% yield over the 2 steps. Compounds 270 and 270b were a 90:10 mixture of OTBDPS ethers. In the minor
129 Chapter 5 [3-Epi-casuarine] component the primary OTBDPS group had migrated to the secondary alcohol position under the basic hydrolysis conditions. Cyclization of the mixture of 270 and 270b using Mitsunobu reaction conditions generated a 3:2 mixture of the epoxy- pyrrolizidine 271 and the epoxy-indolizidine 272 in 93% yield. Ring-opening of this epoxide mixture under the same reaction conditions as described in Chapter 5.3.2 gave only diol indolizidine 273 in 7% yield and a complex mixture of other products after column chromatography. The desired diol product that could arise from ring- opening of 271 could not be isolated pure. While the TBDPS group is more stable than the TBS group under acidic conditions; the TBDPS group more easily migrates under basic conditions.192
In conclusion, we have successfully synthesized 3-epi-casuarine 79 in 13 synthetic steps and in 0.4 % overall yield, however the method requires a better protecting group at the C-3 hydroxymethyl position. A benzyl or MOM ether may have been a better choice. However, this synthesis provided the two novel polyhydroxylated alkaloid like structures, 265 and 266, for testing as glycosidase inhibitors. The overall synthesis is summarized in Scheme 5.10.
130 Chapter 5 [3-Epi-casuarine]
L-xylose
9 steps
OBn OH H O OTBS
N OBn Fmoc 161 a
OBn OH H O OTBS
NH OBn 258 b OBn H OBn H O O OBn OBn + N N OTBS 259 OTBS 260 (70%) (4%)
c
AcO OBn H H OBn H OBn AcO H OBn O AcO OBn + AcO OBn + OBn + OBn N N N N O O OAc 261 262 263 OAc 264 (7%) (8%) (9%) (17%)
d e f
HO H OH H OH HO H OH
HO OH HO OH OH N N N O O OH 79 265 266
Scheme 5.10 The total synthesis of 3-epi-casuarine 79. o Reagents and conditions: (a) i: p-NO2ArCO2H, PPh3, toluene, 80 C, 5 h; ii: K2CO3, o MeOH, rt, 24 h, 50%; (b) DIAD, Ph3P, toluene, 80 C, 3 d; (c) i: NaHSO4, CH2Cl2, o 50 C, 7 d; ii: Ac2O, py, DMAP, 24 h; (d) PdCl2, H2 (1 atm), MeOH, 4 d; ion- exchange, 77%; (e) Amberlyst OH, MeOH, rt, 12 h; PdCl2, H2 (1 atm), MeOH, 24 h; ion-exchange, 82%; (f) Amberlyst OH, MeOH, rt, 16 h; PdCl2, H2 (1 atm), MeOH, 12 h; ion-exchange, 53%.
131 Chapter 6 [3-Epi-australine]
CHAPTER 6 SYNTHESIS OF 3-EPI-AUSTRALINE, 3,7-DIEPI- AUSTRALINE, 7-DEOXY-3,6-DIEPI-CASUARINE AND 1,6-DIEPI- CASTANOSPERMINE
6.1 Isolation and biological activities of 3-epi-australine
Nash et al.52 reported the isolation of 3-epi-australine 80 from the finely ground freeze dried seeds of Castanospermum australe (Chapter 4.1). This was the same plant that Molyneux et al.54 had isolated australine 80 from, also in 1988. 3- Epi-australine, (1R,2R,3S,7S,7aR)-3-hydroxymethyl-1,2,7-trihydroxypyrrolizidine, was isolated by ion-exchange chromatography from the 75% aqueous alcohol seed extract. The absolute configuration of 3-epi-australine 80 was established by single crystal X-ray crystallographic analysis.52
HO H OH
OH N
80 OH
Figure 6.1 The structure of 3-epi-australine 80.
In 2003, Kato et al.55 also isolated 3-epi-australine 80 from the same plant as Nash et al.52 They showed that 3-epi-australine 80 was a poor inhibitor of glycosidase activities.
6.2 Previous syntheses of 3-epi-australine
Denmark et al.170 reported the synthesis of 3-epi-australine 80 in a seven-step sequence in 18.5% overall yield, His synthesis started with 193 which was converted to the same precursor 198 which he used to prepare australine 13 (Chapter 4.2). The alkene 198 was converted to the diol 274 in 93% yield as a 208:1 mixture of diastereomers using dihydroquinine phenanthracene (DHQ-PHN) as a chiral ligand (Scheme 6.1). Primary alcohol protection of 274 with TBSCl generated the O-TBS derivative 275 in 95% yield. Mesylation of the remaining secondary alcohol provided the primary O-TBS ether/secondary mesylate derivative 276. Treatment of mesylate 276, under hydrogenolysis conditions over Raney Ni gave the pyrrolizidine 277 in 78%. Deprotection of 277 with HF in MeOH resulted in clean conversion to
132 Chapter 6 [3-Epi-australine] the hydrofluoride salt of 80, which was then converted to its free base after ion- exchange chromatography.
O OG* R2O H O OG* O N O N
1 O a R O H H 193 O O O O Si Si t-Bu t-Bu t-Bu t-Bu 198 1 2 G*: b 274: R =R =H 275: R1 = TBS, R2 =H Ph c 276: R1 = TBS, R2 =Ms
TBSO HO H OH d N e,f HO OH N O H O Si 80 OH t -Bu t-Bu 277
Scheme 6.1 The total synthesis of 3-epi-australine 80 from the precursor 198.
Reagents and conditions: (a) K2OsO2(OH)4, K2CO3, H2O, NaHCO3, K3Fe(CN)6, t- BuOH, DHQ-PHN; rt, 93%; (b) TBSCl, py, rt, 1.0 h, 95%; (c) MsCl, py, rt, 1.5 h; (d)
H2, Raney Ni, MeOH, 160 psi, rt, 48 h, 78% overall for 2 steps; (e) HF, MeOH, rt; 24 h (f) AG 50Wx8, 96% overall for 2 steps.
The same procedure that was used for the chemoenzymatic total synthesis of australine 13 in Chapter 4.2 (Scheme 4.5 and 4.6) was also used to synthesize 3-epi- australine 80 by Wong et al. (Scheme 6.2).186 He converted a 1:1 mixture of 209a and 209b using ozonolysis and an intramolecular twofold reductive amination reaction to furnish 3-epi-australine 80 in 70% yield and as a single isomer.
OH HO OH OH OH H O OH HO OH NHCHO N 204 209a : 209b =1:1 80 OH
Scheme 6.2 The total synthesis of 3-epi-australine 80 from the mixture anomeric stereoisomers.
Reagents and conditions: (a) O3, MeOH/H2O, -78 °C, then HCl and H2 Pd/C, 70%.
133 Chapter 6 [3-Epi-australine]
6.3 The synthesis of 3-epi-australine from precursor 259
Our synthesis of 3-epi-australine 80 began with the epoxide 259 described in from Chapter 5 (Scheme 5.3). The key steps were the same as those for the synthesis of australine 13 (Scheme 4.11).
6.3.1 Reductive ring-opening of pyrrolizidine 259 with LiAlH4
Reductive ring-opening of the epoxide 259 with lithium aluminium hydride at rt gave a separable mixture of the regioisomeric pyrrolizidines 278 and 279, in yields of 41% and 9%, respectively. The formation of the pyrrolizidine 278 as the major regioisomeric product was expected based on our previous synthesis of australine (Chapter 4). In contrast to the reductive ring-opening reaction of 163 (Scheme 4.11), the more hindered TBS group in these products remained intact. OBn H HO OBn H OBn O H 7 1 LiAlH4, THF OBn OBn + HO OBn 5 N 3 N rt, 12 h N 8 259 OTBS 278 OTBS 279 OTBS (41%) (9%)
Scheme 6.3 Reductive ring-opening of 259 with LiAlH4.
6.3.2 Mitsunobu reaction for inversion of the hydroxyl group
In the final steps of the synthesis, the configuration at C-7 in 278 was inverted by the two step sequence described in Chapter 4 for the synthesis of australine. Treatment of 278 under the Mitsunobu reaction conditions with 4- nitrobenzoic acid followed by base treatment of the resulting 4-nitrobenzoate gave the C-7 inverted alcohol 280 in 64% overall yield.
HOH OBn HO OBn DIAD, PPh3, p-NO2ArCO2H H toluene, rt, 8 h OBn OBn N N K2CO 3,MeOH 278 OTBS rt, 4 h, 64% 280 OTBS
Scheme 6.4 The synthesis of the C-7 inverted pyrrolizidine derivative 280.
134 Chapter 6 [3-Epi-australine]
6.3.3 Hydrogenolysis
6.3.3.1 Synthesis of 3-epi-australine 80 from hydrogenolysis of 280
The pyrrolizidine 280 underwent hydrogenolysis under acidic conditions to
23 deliver diastereomerically pure 3-epi-australine 80 ([α] D -10.5 (c 0.7, H2O) in 88%
23 yield after ion-exchange chromatography. Its hydrochloride salt, 80·HCl, had [α] D -
37 (c 0.7, H2O) which was of the same sign as the natural product but was
52 20 significantly larger in magnitude (lit. for 3-epi-australine·HCl, [α] D -3.5 (c 1.35, 1 13 H2O)). The H and C NMR spectra of 3-epi-australine 80 are shown in Figures 6.2 1 and 6.3, respectively. The H NMR spectroscopic data (D2O) of 80 and that of the 52 13 natural product matched closely (∆δH = 0.13-0.19 ppm, see Table 6.1). The C
NMR signals of 80 (in D2O with MeCN as an internal reference at 1.47 ppm) however, were all consistently 0.4-0.9 ppm downfield of those reported for the natural product.
HO OBn H HOH OH PdCl2,H2 (1 atm) MeOH, rt, 1 d OBn OH N ion-exchange N 88% 280 OTBS 80 OH
Scheme 6.5 The synthesis of 3-epi-australine 80.
HOH OH
7 1 OH 5 N 3 H-1 8 H-7 H-2 80 OH H-8 H-8’ H-7a H-3 H-5β 2xH-6 H-5α
4.00 3.50 3.00 2.50 2.00 ppm (t1)
4.0 3.0 2.0 1.0 0.0 ppm (t1) 1 Figure 6.2 The H NMR (500 MHz, D2O) of (-)-3-epi-australine 80.
135 Chapter 6 [3-Epi-australine]
HOH OH
7 1 OH C-7a 5 N 3 C-2 C-1 C-7 C-3 8 C-8 C-5 C-6 80 OH
80 70 60 50 40 30 ppm (t1) 13 Figure 6.3 The C NMR (125 MHz, D2O) of (-)-3-epi-australine 80.
Table 6.1 Physical and spectral data for (-)-3-epi-australine52 and 80.
3-Epi-australine52 Synthetic 80 Physical Appearance oil brown viscous oil Optical Rotation 20 23 [α] D -3.5 (c 1.35, H2O), HCl salt. [α] D -37 (c 0.7, H2O), HCl salt
1 H NMR 500 MHz, D2O 500 MHz, D2O
4.41 (brt, 1H, J6,7 = J7,7a = 4.0 Hz, H- 4.24 (dt, 1H, J = 4.5, 2.0 Hz, H-7) 7)
4.12 (t, 1H, J = 3.5 Hz, H-1) 4.30 (t, 1H, J1,2 = J1,7a = 3.3 Hz, H-1)
3.96 (dd, 1H, J = 4.5, 3.5 Hz, H-2) 4.15 (t, 1H, J1,2 = J2,3 = 4.0 Hz, H-2)
4.01 (dd, 1H, J8,8’ = 11.8 Hz, J3,8 = 3.80-3.70 (2H, AB part of ABX, 5.8 Hz, H-8)
CH2OH) 3.92 (dd, 1H, J8,8’ = 11.8 Hz, J3,8’ = 6.3 Hz, H-8’)
3.38 (t, 1H, J1,7a = J7,7a = 4.3 Hz, H- 3.25 (dd, 1H, J = 4.5, 4.0 Hz, H-7a) 7a)
3.30 (dt, 1H, J3,8’ = 5.3 Hz, J2,3 = J3,8 3.16 (dt, 1H, J = 6.0, 4.5 Hz, H-3) = 4.5 Hz, H-3) 2.96 (ddd, 1H, J = 11.5, 9.0, 6.0 Hz, 3.15-3.10 (m, 1H, H-5) H-5)
2.74 (m, 1H, H-5) 2.88 (t, 1H, J5,5 = J5,6 = 8.0 Hz, H-5) 1.82 (m, 2H, 2xH-6) 2.00-1.87 (m, 2H, 2xH-6)
13 C NMR 125 MHz, D2O 125 MHz, D2O ∆δ (ppm) 78.5 (C-2) 79.3 (C-2) 0.8 74.8 (C-7a) 75.2 (C-7a) 0.4 74.0 (C-1) 74.7 (C-1) 0.7 69.6 (C-7) 70.4 (C-7) 0.8
63.5 (C-3) 63.9 (C-3) 0.4 56.9 (C-8) 57.8 (C-8) 0.9 44.9 (C-5) 45.3 (C-5) 0.4 34.8 (C-6) 35.6 (C-6) 0.8
136 Chapter 6 [3-Epi-australine]
6.3.3.2 Synthesis of 3,7-diepi-australine 281 from hydrogenolysis of 278
The direct hydrogenolysis of 278 under acidic conditions gave
24 diastereomerically pure 3,7-diepi-australine 281 ([α] D -9.3 (c 1.1, H2O) in 90% yield
21 after ion-exchange chromatography. Its hydrochloride salt, 281·HCl, had [α] D -21 (c
0.63, H2O) which was of opposite sign to that of its synthetic enantiomer, 1,2-diepi-
193 20 1 13 alexine·HCl (lit. [α] D +33 (c 0.1, H2O)). The H and C NMR spectra of 3,7-diepi- australine 281 and its salt are shown in Figures 6.4-6.7, respectively. The 1H NMR spectroscopic data (D2O) of 281·HCl and that reported in the literature for 1,2-diepi- 13 alexine·HCl were essentially identical (∆δH = 0.03-0.04 ppm, see Table 6.2). The C
NMR signals of 281·HCl (in D2O with MeCN as an internal reference at 1.47 ppm) however, were all consistently 1.7-2.1 ppm upfield of those reported for its enantiomer.193
HO H OBn HO OH PdCl2,H2 (1 atm) H MeOH, rt, 18 h OBn OH N N ion-exchange 90% 278 OTBS 281 OH
Scheme 6.6 The synthesis of 3,7-diepi-australine 281.
HOH OH
7 1 OH 5 N 3 H-1 8 281 OH H-2 H-8 H-8’ H-7a H-7 H-3 H-5α H-5β H-6 H-6
4.00 3.50 3.00 2.50 2.00 ppm (t1)
4.0 3.0 2.0 1.0 0.0 ppm (t1) 1 Figure 6.4 The H NMR (500 MHz, D2O) of (-)-3,7-diepi-australine 281.
137 Chapter 6 [3-Epi-australine]
HOH OH
7 1 OH 5 N 3 C-2 8 C-1 C-7a C-7 C-3 281 OH C-8 C-5 C-6
80 70 60 50 40 30 ppm (t1) 13 Figure 6.5 The C NMR (125 MHz, D2O) of (-)-3,7-diepi-australine 281.
HOH OH 7 1 OH 5 N 3 2xH-8 H Cl 8 H-1 OH 281.HCl H-2 H-7 H-3 H-7a H-5α H-5β H-6 H-6
4.50 4.00 3.50 3.00 2.50 2.00 ppm (t1)
4.0 3.0 2.0 1.0 0.0 ppm (t1)
1 Figure 6.6 The H NMR (500 MHz, D2O) of (-)-3,7-diepi-australine⋅HCl [281⋅HCl].
HOH OH
7 1 OH 5 N 3 H Cl 8 OH C-2 . C-7a 281 HCl C-1 C-7 C-3 C-8 C-6 C-5
80 70 60 50 40 30 ppm (t1) 13 Figure 6.7 The C NMR (125 MHz, D2O) of (-)-3,7-diepi-australine⋅HCl [281⋅HCl].
138 Chapter 6 [3-Epi-australine]
Table 6.2 Physical and spectral data for (+)-1,2-diepi-alexineHCl salt [ent-281]193 and 281HCl salt [3,7-diepi-australineHCl salt]. 1,2-Diepi-alexine·HCl salt 193 Synthetic 281·HCl salt [ent-281·HCl] Physical Not reported Pale yellow viscous oil Appearance
Optical 20 21 [α] +33 (c 0.1, H2O), HCl salt [α] -21 (c 0.63, H2O), HCl salt Rotation D D 1 H NMR 500 MHz, D2O 500 MHz, D2O
4.62-4.58 (m, 1H) 4.63 (dt, 1H, J6,7 = 8.0 Hz, J6,7 = J7,7a = 6.0 Hz, H-7) 4.37 (brs, 1H) 4.41 (brs, 1H, H-1)
4.32 (brs, 1H) 4.35 (d, 1H, J1,2 = 2.5 Hz, H-2)
4.13 (dd, 1H, J8,8’ = 12.5, J3,8 = 5.0 Hz, H-8)
4.12-3.98 (m, 3H) 4.10 (d, 1H, J8,8’ = 9.0 Hz, H-8) 4.06-4.02 (m, 1H, H-3)
3.80 (d, 1H, J = 5.8 Hz) 3.84 (d, 1H, J7,7a = 6.5 Hz, H-7a)
3.75 (dd, 1H, J5,5 = 11.3, J5,6 = 6.3 Hz, H-5α) 3.75-3.60 (m, 2H) 3.73 (dd, 1H, J5,5 = 10.8, J5,6 = 6.3 Hz, H-5β) 2.51-2.44 (m, 1H) 2.54-2.48 (m, 1H, H-6) 2.04-1.96 (m, 1H) 2.07-1.99 (m, 1H, H-6)
13 C NMR 75 MHz, D2O 125 MHz, D2O ∆δ (ppm) 81.8 80.1 (C-7a) 1.7 79.4 77.6 (C-1) 1.8 78.9 77.1 (C-2) 1.8 74.8 73.1 (C-7) 1.7
69.8 67.7 (C-3) 2.1 57.6 55.8 (C-8) 1.8 50.3 48.6 (C-5) 1.7 34.9 33.1 (C-6) 1.8
139 Chapter 6 [3-Epi-australine]
6.3.3.3 Synthesis of 7-deoxy-3,6-diepi-casuarine 282 from 279
23 Pure 7-deoxy-3,6-diepi-casuarine 282 ([α] D +28.6 (c 0.23, MeOH) was obtained after 3 more steps; (i) hydrogenolysis of 279 under acidic conditions over
PdCl2/H2 which provided a product that was not pure enough to characterize, (ii) acetylation by treatment with Ac2O in py; and then (iii) deacetylation using Amberlyst A-26 (OH-) in MeOH. In this way 7-deoxy-3,6-diepi-casuarine 282 was obtained in 50% yield over the three steps.
H OBn H OH
HO OBn HO OH N N
279 OTBS 282 OH
Scheme 6.7 The synthesis of 7-deoxy-3,6-diepi-casuarine 282.
Reagents and conditions: i: PdCl2, H2 (1 atm), MeOH, rt, 3 h; then conc. HCl 5 drops; ii: - Ac2O, py, DMAP, rt, 24 h; iii: Amberlyst A-26 (OH ), MeOH, 3 h, 50% (3 steps).
The 1H NMR spectrum of 282 showed 5 methine protons and 6 methylene protons while the 13C NMR spectrum showed signals for eight different carbons. The 1H and 13C NMR spectra are shown in Figures 6.8 and 6.9, respectively. The structure of 282 was confirmed by NOE and HMBC experiments which are summarized in Figure 6.10 and Table 6.3, respectively. H OH
7 1 HO OH 5 N 3 8 282 OH H-1 H-6 H-2 H-3 2xH-8 H-5β H-7a H-5α H-7β H-7α
4.50 4.00 3.50 3.00 2.50 2.00 ppm (f1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1) 1 Figure 6.8 The H NMR (500 MHz, D2O) of (+)-7-deoxy-3,6-diepi-casuarine 282.
140 Chapter 6 [3-Epi-australine]
H OH
7 1 HO OH 5 N 3 8 C-6 C-8 C-7 282 OH C-2 C-3 C-5 C-1 C-7a
80 70 60 50 40 30 ppm (f1) 13 Figure 6.9 The C NMR (125 MHz, D2O) of (+)-7-deoxy-3,6-diepi-casuarine 282.
7 7a 1
3
5
8
NOE correlations
Figure 6.10 The NOE correlations of 282.
Table 6.3 The HMBC correlations of 7-deoxy-3,6-diepi-casuarine 282.
1 Structure H NMR (500 MHz, D2O) HMBC correlations
H OH 4.57 (brs, 1H, H-6) -
7 1 4.22 (brs, 1H, H-2) 71.4 (C-7a) HO OH 5 N 3 4.07 (brts, 1H, H-1) 79.7 (C-2), 65.0 (C-3) and 8 282 OH 38.0 (C-7)
4.00 (dd, 1H, J8,8 = 11.5, J3,8 = 6.5 Hz, H-8) 79.7 (C-2) and 65.0 (C-3)
3.92 (dd, 1H, J8,8 = 11.3, J3,8 = 6.8 Hz, H-8)
3.59 (t, 1H, J1,7a =J7,7a = 7.8 Hz, H-7a) 81.7 (C-1) and 56.2 (C-5) 3.41 (brs, 1H, H-3) 79.7 (C-2) and 57.5 (C-8)
3.31 (dd, 1H, J5,5 = 10.5, J5α,6 = 2.5 Hz, H- 65.0 (C-3) 5α)
2.92 (d, 1H, J5,5 = 10.5 Hz, H-5β) 72.9 (C-6), 38.0 (C-7) and 71.4 (C-7a) 2.22-2.17 (m, 1H, H-7α) 56.2 (C-5) and 72.9 (C-6) 2.04-2.00 (m, 1H, H-7β) 81.7 (C-1) and 72.9 (C-6)
141 Chapter 6 [3-Epi-australine]
6.3.3.4 Synthesis of 1,6-diepi-castanospermine 284
6.3.3.4.1 Reductive ring-opening of 260 with LiAlH4
The indolizidine 260 was treated with LiAlH4 in THF at rt for 20 h to provide only the indolizidine 283 in 60% yield (Scheme 6.8).
OBn OBn H HO H OBn O LiAlH4,THF OBn N rt, 20 h, 60% N OTBS OTBS 260 283
Scheme 6.8 Reductive ring-opening of 260 with LiAlH4.
6.3.3.4.2 Hydrogenolysis of 283
Hydrogenolysis of 283 under acidic conditions with PdCl2/H2 afforded 1,6-
25 194 24 diepi-castanospermine 284 [α] D -74.2 (c 1.5, MeOH), lit. [α] D -72.0 (c 0.7, MeOH) in 95% yield and 95% purity (Scheme 6.9).
HO OBn OH H HO H OBn PdCl2,H2 (1 atm) OH MeOH, rt, 4 d N N OTBS ion-exchange OH 283 95% 284
Scheme 6.9 The synthesis of 1,6-diepi-castanospermine 284.
The 1H and 13C NMR spectra of 1,6-diepi-castanospermine 284 are shown in 1 Figures 6.11 and 6.12, respectively. The H NMR spectroscopic data (D2O) of 284 194 and that of the synthetic material matched closely (∆δH = 0.11-0.15 ppm, see 13 Table 6.4) and the C NMR spectroscopic data (in D2O with MeCN as an internal reference at δ 1.47) of 284 and that reported in the literature for 1,6-diepi- castanospermine were all consistently 0-0.2 ppm downfield.
142 Chapter 6 [3-Epi-australine]
OH HO H OH 1 8a 7 3 N 5 OH H-8 284 H-6 H-7 H-5 H-5 H-3 H-3 H-8a H-1 H-2 H-2
4.00 3.50 3.00 2.50 2.00 ppm (f1)
5.0 4.0 3.0 2.0 1.0 0.0 ppm (f1)
1 Figure 6.11 The H NMR (500 MHz, D2O) of (-)-1,6-diepi-castanospermine 284.
OH HO H OH 1 8a 7 3 N 5 OH C-8a C-1 284 C-8 C-6 C-7 C-5 C-3 C-2
80 70 60 50 40 30 ppm (f1) 13 Figure 6.12 The C NMR (125 MHz, D2O) of (-)-1,6-diepi-castanospermine 284.
143 Chapter 6 [3-Epi-australine]
Table 6.4 Physical and spectral data for (-)-1,6-diepi-castanospermine194 and 284.
1,6-diepi- Synthetic 284 castanospermine194 Physical Not reported Pale yellow viscous oil Appearance Optical Rotation 24 25 [α] D -72.0 (c 0.7, MeOH) [α] D -74.2 (c 1.5, MeOH),
1 H NMR 500 MHz, D2O 500 MHz, D2O 4.13 (ddd, 1H, H-1) 4.29-4.25 (m, 1H, H-1) 3.88 (dd, 1H, H-6) 4.03 (brs, 1H, H-6)
3.54 (dd, 1H, H-8) 3.68 (t, 1H, J7,8 = J8,8a = 9.8 Hz, H-8)
3.38 (dd, 1H, H-7) 3.53(dd, 1H, J6,7 = 3.5, J7,8 = 9.5 Hz, H-7)
2.92 (dd, 1H, H-5) 3.05 (dd, 1H, J5,5 = 12.5, J5,6 = 2.5 Hz, H-5)
2.80 (dd, 1H, H-3) 2.92 (t, 1H, J2,3 = J3,3 = 8.5 Hz, H-3)
2.36 (dd, 1H, H-3’) 2.48 (dt, 1H, J2,3 = 9.0, J2,3 = J3,3= 9.3 Hz, H-3)
2.30 (d, 1H, H-5’) 2.42 (d, 1H, J5,5 = 12.8 Hz, H-5) 2.15 (d, 1H, H-2’) 2.34-2.25 (m, 1H, H-2) 1.88 (dd, 1H, H-4) 1.99 (dd, 1H, J1,8s = 6.5, J8,8a = 9.5 Hz, H-8a) 1.51 (dddd, 1H, H-2) 1.67-1.621 (m, 1H, H-2) 13 C NMR 75 MHz, D2O 125 MHz, D2O ∆δ (ppm) 75.7 75.8 (C-7) 0.1 74.5 74.6 (C-1) 0.1 74.0 74.0 (C-8a) 0.0 72.0 72.2 (C-8) 0.2
69.3 69.3 (C-6) 0.0 55.7 55.7 (C-8) 0.0 51.8 51.7 (C-3) 0.1 32.8 32.9 (C-2) 0.1
In conclusion we have successfully prepared 3-epi-australine 80 in a total of 14 synthetic steps and in 2% overall yield, and the novel pyrrolizidine; 3,7- diepiaustraline 281, in a total of 13 steps and in 3% overall yield. In addition we prepared the novel pyrrolizidine, 7-deoxy-3,6-diepi-casuarine 282, and the known indolizidine 1,6-diepi-castanospermine 284 both in a total of 13 synthetic steps and in 0.3% and 0.2% overall yields, respectively from L-xylose. These overall syntheses are summarized in Scheme 6.10.
144 Chapter 6 [3-Epi-australine]
OBn H OBn H O O OBn OBn + N N OTBS 259 OTBS 260 a b
OBn OBn HOH OBn H HO H OBn OBn + HO OBn N N N OTBS 279 278 OTBS OTBS 283
f g c e 1 HO OH HO OR HOH OH H OH H H OH OR1 OH HO OH N N N N OH OR2 281 OH 282 OH 284
280;R1 =Bn;R2 =TBS d 80;R1 =R2 =H
Scheme 6.10 Total synthesis of 3-epi-australine 80 and the compounds 281, 282 and 284 from the precursor 259.
Reagents and conditions: (a) LiAlH4, THF, rt, 12 h, 278 (41%), 279 (9%); (b) LiAlH4, THF,
rt, 20 h, 60%; (c) DIAD, PPh3, p-NO2ArCO2H, toluene, rt, 8 h; K2CO3, MeOH, rt, 4 h, 64% from 278; (d) PdCl2, H2 (1 atm), MeOH, rt, 24 h; ion-exchange, 88%; (e) PdCl2, H2 (1 atm),
MeOH, rt, 18 h, ion-exchange, 90%. (f) i PdCl2, H2 (1 atm), MeOH, rt, 3 h; then conc. HCl 5
drops; ii: Ac2O, py, DMAP, rt, 24 h; iii: Amberlyst A-26 (OH), MeOH, 3 h, 50% (3 steps);
(g) PdCl2, H2 (1 atm), MeOH, rt, 4 d; ion-exchange, 95%.
145 Chapter 7 [Bioactivity]
CHAPTER 7 GLYCOSIDASE INHIBITOR TESTING
Synthetic uniflorine A 78 was tested for glycosidase inhibition activity by Prof. Robert J. Nash at the Institute of Grassland and Environmental Research, Aberystwyth, UK using commercially available enzymes and p-nitrophenyl substrates from Sigma, with the exception of β-mannosidase which came from Megazyme. All enzymes were assayed at 27 °C in 0.1 M citric acid/0.2 M disodium hydrogen phosphate buffers at the optimum pH for each enzyme. The incubation mixture was 10 µL enzyme solution, 10 µL of 1 mg/mL aqueous solution of the test compound and 50 µL p-nitrophenyl substrate (5 mM). The reactions were stopped by addition of 70 µL 0.4M glycine (pH 10.4) and its absorbance at 405 nm was recorded using a Versamax microplate reader (Molecular Devices).9
The quantity of p-nitrophenolate produced in each assay was measured by UV/vis spectrophotometry in order to determine the effect of varying concentrations of the test compound (inhibitor). Upon treatment with glycine buffer (pH 10.4), the liberated p-nitrophenol is converted to its corresponding p-nitrophenolate anion, which has a known extinction coefficient at 405 nm. A Scheme of the glycosidase inhibitory activity assay is shown in Scheme 7.1.
HOH H OH H O α-D-glucosidase H HO HO O + O NO2 HO H buffer HO H H HO H HO H O H OH p-nitrophenolate α NO 2 -D-glucose UV λ 405 nm p-nitrophenyl-α-D-glucopyranoside
Scheme 7.1. Schematic of the glycosidase inhibitory activity assay.
The results of glycosidase inhibitor testing for 78 are shown in Table 7.1. At
143 µg/mL uniflorine A 78 showed 94-97% inhibition against the α-D-glucosidases of Saccharomyces cerevisiae and Bacillus sterothermophilus and against the amyloglucosidase of Aspergillus niger. The IC50 values were only determinated for the two aforementioned α-D-glucosidases and were found to be modest at 34 and 28 µM, respectively.
146 Chapter 7 [Bioactivity]
Table 7.1 The glycosidase inhibition of uniflorine A 78 (Mean % Inhibition at 143 µg/mL).
Enzyme Source pH % Inhibitions
94 α-D-glucosidase Saccharomyces cerevisiae 6.8 IC50 34 µM Bacillus 97 α-D-glucosidase 6.8 sterothermophilus IC50 28 µM
α-D-glucosidase Rice 4 ND
β-D-glucosidase Almond (Prunus sp.) 5 5 Green coffee bean α-D-galactosidase 6.5 5 (Coffea sp.)
β-D-galactosidase Bovine liver 7.3 0
α-L-fucosidase Bovine kidney 5.5 ND Jack bean (Canavalia α-D-mannosidase 4.5 5 ensiformis)
β-D-mannosidase Cellullomonas fimi 6.5 2
naringinase Penicillium decumbens 4 ND
N-acetyl-β-D- Bovine kidney 4.25 13 glucosaminidase
N-acetyl-β-D- Jack bean 5 5 glucosaminidase
N-acetyl-β-D- Aspergillus oryzae 5 ND hexosaminidase amyloglucosidase Aspergillus niger 4.5 97 β-glucuronidase Bovine liver 5 0 ND = No determined
Compounds; 76 (2-epi-32), 1-epi-castanospermine 242, the tricyclic compounds 265 and 266, 3,7-diepi-australine hydrochloric salt 281·HCl, 7-deoxy- 3,6-diepi-casuarine 282 and 1,6-diepi-castanospermine 284, were screened against 10 different glycosidases at 800 µg/mL. None showed strong inhibition with only compounds 76, 242, 266 and 284 showing approximately 40-50% inhibition at this relative high concentration (Table 7.2).
147 Chapter 7 [Bioactivity]
Table 7.2 The glycosidase inhibition of synthesized compounds (Mean % Inhibition at 800 µg/mL).
β-D- α-D- β-D- α-D- β-D- N-acetyl-β-D- β- α-D-glucosidase Compound No. Structures glucosidase galactosidase galactosidase mannosidase mannosidase glucosaminidase glucuronidase
(Yeast) (Bacillus) Bovine kidney Jack bean Bovine liver
HO OH 76 H HO 20 44 13 -7 7 -25 0 0 0 0 (C-2 epimer of 32) OH N HO HO OH 1-epi- H HO 14 43 24 0 25 -10 0 13 17 0 castanospermine 242 N HO H OH
tricyclic 265 HO OH 0 24 0 0 6 0 0 17 24 0 N
O HO H OH
OH tricyclic 266 N 44 50 27 ND 19 -21 12 30 26 8
O HO OH 3,7-diepi- H OH australine·HCl N 0 -9 0 -11 0 0 17 0 7 ND 281·HCl H OH Cl H OH 7-deoxy-3,6- HO OH -13 -16 -34 -38 40 -29 -17 0 0 ND diepi-casuarine 282 N OH HO 1,6-diepi- H OH HO 36 43 55 45 31 23 30 7 -12 ND castanospermine 284 N HO ND = No determined
148 Chapter 8 [Conclusions]
CHAPTER 8 CONCLUSIONS
The first aim of this project was to synthesize compound 76, the C-2 epimer of the proposed structure of the natural product uniflorine A. This synthesis would help us to elucidate the structure of the natural product. The total synthesis of 76 was reported in Chapter 2.1 and started with the boronic acid-Mannich reaction (Petasis reaction)143 of L-xylose, allylamine and (E)-styrene boronic acid to give the amino- tetraol 35. This compound then allowed for the synthesis of the target compound 76
in a further 11 synthetic steps and in 0.5% overall yield (Scheme 8.1). OH HO OH H H HO Ph HO 10 steps L-xylose OH HN N HO HO OH 76 C-2 epimer of proposed uniflorine A 32 35
Scheme 8.1 Outline of the synthesis of compound 76.
Unfortunately, the NMR spectroscopic data of 76 did not match with those of the natural product uniflorine A. After reviewing the spectroscopic data given in the original isolation paper we revised the structures of uniflorines A and B from the initially proposed pentahydroxyindolizidines142 to 1,2,6,7-tetrahydroxy-3- hydroxymethylpyrrolizidines. Uniflorine B was the known alkaloid casuarine 15, while uniflorine A was tentatively assigned as 6-epi-casuarine 78. This was confirmed by the synthesis of ent-78 and 78 which were reported in Chapters 2.2 and 2.3. The total synthesis of uniflorine A 78 was completed in 11 steps and in 13% overall yield from L-xylose (Scheme 8.2). The NMR spectroscopic data of 78 matched with those of the natural product uniflorine A. We have thus successfully
determined the correct structures of uniflorines A and B. OH H HO H OH 2steps HO Ph 9steps L-xylose HO OH N N HO Boc OH OH 78 36 (-)-unif lorine A
Scheme 8.2 Outline of the synthesis of uniflorine A 78.
149 Chapter 8 [Conclusions]
The second aim of this PhD project was to develop a flexible method to prepare other pyrrolizidine alkaloids and their analogues starting from the common precursor 103 (Scheme 8.3).
OH O H O
NBoc OH 103 Deprotection Protection Epoxidation
Required inversion at C-3'
OP OH OP OH H H O OP'' O OP'' 1' 3' 1' 3' NP' OP NH OP
Cyclization Cyclization
H OP H OP O O OP OP N N
OP' OP'
+ + Ring opening with H3O Ring opening with H3O Deprotection Deprotection HO OH - - H Ring opening with H HO OH Ring opening with H Inversion of OH at C-7 H Inversion of OH at C-7 HO OH Deprotection Deprotection N HO OH HO OH H N HO H OH 15 OH 79 OH OH OH casuarine N 3-epi-casuarine N Chapter 3 Chapter 5 13 OH 80 OH australine 3-epi-australine Chapter 4 Chapter 6 (P = Protecting group)
Scheme 8.3 Synthetic routes to prepare natural pyrrolizidines from 103.
In Chapter 3, we reported the total synthesis of casuarine 15 which was obtained in total of 13 synthetic steps and in 8% overall yield from L-xylose (Scheme 8.4). A key step in this synthesis was a regioselective epoxide ring-opening reaction with hydrogensulfate ion. This reaction secured the correct configurations at C-6 and C-7 of the target molecule.
150 Chapter 8 [Conclusions]
OH O HO OH H H O 4-steps 9-steps 7 L-xylose HO 6 OH NBoc OH N 103 15 OH casuarine Scheme 8.4 Outline of the synthesis of casuarine 15.
In Chapter 4, our total synthesis of the alkaloid australine 13 was reported. The natural product was obtained in a total of 14 steps and 6% overall yield from L- xylose (Scheme 8.5). Key steps in this synthesis were a regioselective epoxide ring- opening reaction with LiAlH4 followed by a Mitsunobu reaction that secured the correct configuration C-7 of the target molecule.
H OBn HO H OH 11-steps O 3-steps 7 L-xylose OBn OH N N
163 OTBS 13 OH australine Scheme 8.5 Outline of the synthesis of australine 13.
In Chapter 5 our total synthesis of naturally occurring 3-epi-casuarine 79 was described which is outlined in Scheme 8.6. This total synthesis was achieved in 13 steps and in 0.4% overall yield from L-xylose. This synthesis required an inversion of configuration at C-3’ of the butyl side chain of 161 which was achieved using the Mitsunobu reaction. The low overall yield was due to a low yielding epoxide ring- opening reaction due to a competing intramolecular epoxide ring-opening reactions involving the 3-α-hydroxymethyl substituent.
OBn OH H 9-steps O OTBS 1step L-xylose 1' 3' N OBn Fmoc 161
OBn OH HO OH H H O OTBS 3-steps HO OH NH OBn N 79 258 OH 3-epi-casuarine Scheme 8.6 Outline of the synthesis of 3-epi-casuarine 79.
151 Chapter 8 [Conclusions]
In Chapter 6, we reported the synthesis of 3-epi-australine 80 which was obtained in total of 14 synthetic steps and in 2% overall yield from L-xylose (Scheme 8.7). This synthesis required an inversion of configuration at C-3’ of the butyl side chain of 161 which was achieved using the Mitsunobu reaction. Key steps in this synthesis were a regioselective epoxide ring-opening reaction with LiAlH4 followed by a Mitsunobu reaction that secured the correct configuration C-7 of the target molecule. OBn OH H 9-steps O OTBS 2steps L-xylose 1' 3' N OBn Fmoc 161
H OBn HO H OH O 3-steps OBn 7 OH N N
259 OTBS 80 OH 3-epi-australine
Scheme 8.7 Outline of the synthesis of 3-epi-australine 80.
From this work a number of novel indolizidine and pyrrolizidine compounds were obtained as side products. These were converted to their corresponding polyhydroxylated derivatives. Scheme 8.8 shows a summary of the synthesised natural and unnatural polyhydroxylated indolizidines and pyrrolizidines which are reported in this thesis.
Finally, testing of uniflorine A 78, as described in Chapter 7, for its inhibitory activity against a series of α-glycosidases. This compound displayed moderate inhibition against the α-D-glucosidases from Saccharomyces cerevisiae and Bacillus sterothermophilus (IC50 34 and 28 µM, respectively). Unfortunately, none of the other compounds tested showed any significant inhibitory activities.
152
HOH OH L-xylose Casuarine 2steps 13 steps, 8% overall yield HO OH HO OH H N HO OH 2steps 7steps Uniflorine A H 11 steps, 13% overall yield 15 HO OH OH 7-Epi-aus trali ne
N OH 13 steps, 10% overall yield OH OBn OBn N OH O H HO H 1step 78 OH O 86 H H O OH HO Ph OBn OBn 1step N HO 2steps N H OH N NBoc OH Australine HO 237 14 steps, 6% overall yield Boc 103 OBn OH 163 OTBS OTBS OH OH H 2steps O OTBS + 2steps N 36 5steps OBn 13 OH N OBn H HO OH Fmoc H 9steps O OBn 2steps 161 OH 1-Epi-castanospermine 13 steps, 0.3% overall yield N N HO OH 2steps OTBS OH H 163a HO 242 HO OH OH H 3-Epi-casuarine N 2steps 13 steps, 0.4% overall yield OBn HO H OBn 76 H HO OH OH O OBn O N H 1,2,6-Trihydroxyl-3-methyl-7,8-epoxyindolizidine + OBn 79 2-Epi-32 N N OH HO OH 13steps, 0.5% overall yield 11 steps, 0.5 % overall yield OTBS N 260 259 OTBS 2steps O HO H OH 2steps 265 1step OH 1,2,7-Trihydroxyl--3-methyl-6,8-epoxyindolizidine 2steps N HO OH 13steps, 0.7% overall yield H O OH OBn OH H H 266 7-Deoxy-3,6-diepi-casuarine N 1step OH HO OBn HO OH 13 steps,0.3% overall yield N N 284 279 HO OH 1,6-Diepi-castanospermine OTBS 282 OH H 13 steps, 0.2% overall yield + 3,7-Diepi-aus trali ne OH N 13 steps, 3% overall yield Final product f rom Chapter 2 HOH OBn 1step Final product f rom Chapter 3 281 OH OBn HO H OH N Final product f rom Chapter 4 3-Epi-aus trali ne OH 14 steps, 2% overall yield 278 OTBS Final product f rom Chapter 5 2steps N Final product f rom Chapter 6 80 OH
Minor final product
Scheme 8.8 A summary of the syntheses of the natural and unnatural polyhydroxylated indolizidines and pyrrolizidines which are reported in this thesis.
Chapter 9 [Experimental]
CHAPTER 9 EXPERIMENTAL
9.1 General experimental
9.1.1 General reaction conditions
In general, all reactions unless otherwise stated were performed in oven dried, single-necked round bottom flasks under an atmosphere of dry nitrogen. Progress of reactions was monitored by thin-layer chromatographic (TLC) analysis. Solvents were purchased as Analytical Reagent (AR) grade. Petroleum spirit refers to the hydrocarbon fraction of bp 40-60 °C. THF was stored over KOH pellets until needed, then distilled over sodium wire under nitrogen, using benzophenone as an indicator. Anhydrous
CH2Cl2 and MeOH were purchased from Aldrich.
Where ‘dried’ is specified, this refers to the drying of organic extract over
MgSO4, unless otherwise indicated, followed by filtration. Where ‘evaporation’ is specified, this refers to the evaporation of solvent under reduced pressure using a rotary evaporator. Purified compounds were dried thoroughly under high vacuum. All reaction yields were obtained only after this drying process.
9.1.2 Chromatography
TLC was performed using aluminium backed Merck F254 sorbent silica gel. Compounds were detected under a 254 nm ultraviolet lamp, or by staining with acidified, aqueous solution of ammonium molybdate and cerium(IV) sulfate, followed by development with a 1400 Watt heat gun. One litre of the molybdate dip contained water (950 mL), concentrated H2SO4 (50 mL), (NH4)6MoO24 (50 g) and Ce(SO4)2 (2 g).
Purification of compounds by flash column chromatography (FCC) was achieved using Merck flash silica gel (40-63 µm) and the technique reported by Still et al.195
Acidic ion-exchange chromatography was performed using DOWEX 50WX4-50 acidic exchange resin. In all cases the compounds were applied as their HCl salts
154 Chapter 9 [Experimental] dissolved in distilled water. The column was first eluted with water and then eluted with 14% ammonia solution (w/w). Basic ion-exchange chromatography was performed using Amberlyst A-26(OH) resin. The compounds were applied to the column and eluted with water.
9.1.3 Melting points
Melting points were obtained using a Gallenkamp MF-370 capillary tube melting point apparatus and are uncorrected.
9.1.4 Polarimetry
Optical rotations were measured using a 1 cm cell, in a Jasco DIP-370 digital polarimeter. Eight to ten measurements were taken and the average was used to calculate the specific rotation.
9.1.5 Mass spectrometry
Low resolution mass spectra were obtained either on a Shimadzu GC mass spectrometer (EI and CI) or a Waters LCZ single quadropole (ESI). High-resolution mass spectra were obtained either on a VG Autospec mass spectrometer (EI and CI) or a Waters QTOF (ESI). HRMS (exact masses) were used in lieu of elemental analysis and 1H and 13C NMR spectroscopy were used as criteria for purity.
9.1.6 Nuclear magnetic resonance spectroscopy
1H and 13C NMR spectra were recorded on Varian Unity-300 (300 MHz 1H, 75 MHz 13C) or a Varian INOVA-500 (500 MHz 1H, 125 MHz 13C) spectrometer in deuterochloroform (CDCl3), unless otherwise specified. NMR assignments were based on COSY, DEPT, HSQC and HMBC experiments. NMR solvents used in the experimental and their associated referencing data are displayed in Table 9.1. Unless otherwise stated, the applied NMR frequency was 500 MHz for 1H NMR experiments and 125 MHz for 13C NMR experiments, with samples dissolved in deuterochloroform. In the case of epoxide compounds NMR assignments are given based on the numbering system of the parent pyrrolidine, pyrrolizine or indolizine and not the systematic numbering.
155 Chapter 9 [Experimental for Chapter 2]
Table 9.1 The references used for 1H and 13 C NMR spectroscopy. 1H NMR 13C NMR Solvent Internal standard Other Other
CDCl3 TMS, s, 0.00 ppm residual CHCl3, s, 7.26 ppm CDCl3 77.0 ppm
CD3OD TMS, s, 0.00 ppm residual MeOH, s, 3.31 ppm CD3OD 49.0 ppm
D2O H2O, s, 4.79 ppm - ∼ 5% CH3CN spike, 1.47 ppm
9.2 Chapter 2 Experimental
9.2.1 Total synthesis of C-2-epimer of proposed uniflorine A
(6E)-5-(Allylamino)-5,6,7-trideoxy-7-phenyl-D-gluco-hept-6-enitol (35).
OH OH To a mixture of L-xylose (10.0 g, 66.6 mmol) and trans-2- H HO 3 Ph phenylvinyl boronic acid (9.85 g, 66.6 mmol) was added 1 57 HO HN 1' absolute ethanol (100 mL) and allylamine (5.0 mL, 66.6 3' 35 mmol). The mixture was stirred at rt for 2 d, followed by the evaporation of all volatiles in vacuo. The residue was dissolved in 1 M HCl (ca. 15 + mL), applied to a column of DOWEX resin (H form) and washed with distilled H2O
(1500 mL). The product was eluted with 7 M NH4OH (1500 mL) and 14 M NH4OH (1500 mL). The fractions containing the product were combined and concentrated to a brown foamy solid 35 (17.77 g, 91%). Rf 0.65 (10:2:1 EtOAc/MeOH/NH4OH).
25 [α] D +27 (c 0.06, MeOH). MS (CI +ve) m/z 294 (M+H+, 100%). + HRMS (CI +ve) calculated for C16H24NO4 (M+H ) 294.1705, found 294.1713. -1 IR υmax (cm ): 3324, 3062, 1669, 1649, 1061. 1 H NMR (300 MHz, CD3OD) δ 7.44-7.29 (m, 5H, Ar), 6.56 (d, 1H, J 16.2 Hz, H-7), 6.17 (dd, 1H, J 9.2, 16.1 Hz, H-6), 5.92 (dddd, 1H, J 5.7, 6.6, 9.9, 17.0 Hz, H-2’), 5.20 (dq, 1H, J 1.5, 17.1 Hz, H-3’), 5.12 (dq, 1H, J 1.4, 9.9 Hz, H-3’), 3.85 (app. t, 1H, J 4.8 Hz, H-4), 3.76 (ddd, 1H, J 3.0, 5.1, 6.3 Hz, H-2), 3.69 (dd, 1H, J 3.2, 5.1 Hz, H-3), 3.62 (d, 1H, J 5.1 Hz, H-1), 3.61 (d, 1H, J 6.2 Hz, H-1), 3.50 (dd, 1H, J 4.8, 9.0 Hz, H-5),
156 Chapter 9 [Experimental for Chapter 2]
3.34 (app. ddt, 1H, J 1.5, 5.6, 13.8 Hz, H-1’), 3.17 (app. ddt, 1H, J 1.5, 6.6, 13.5 Hz, H- 1’). 13 C NMR (75 MHz, CD3OD) δ 137.9 (C), 136.3 (C-7), 135.8 (C-6), 129.5 (CH), 128.7 (CH), 127.4 (CH), 117.9 (C-2’), 74.4 (CH), 73.1 (CH), 72.8 (CH), 64.4 (C-1), 63.4 (C- 5), 50.0 (C-1’).
(6E)-5-[Allyl(tert-butylcarbonyl)amino]-5,6,7-trideoxy-7-phenyl-D-gluco-hept-6- enitol (36); (6E)-5-[allyl(tert-butylcarbonyl)amino]-1-O-(tert-butoxycarbonyl)-5,6,7- trideoxy-7-phenyl-D-gluco-hept-6-enitol (36a);
OH OH OH
OH H H HO 3 Ph BocO 3 Ph 1 5 7 1 5 7 HO N 1' HO N 1' Boc 3' Boc 3' 36 36a To a solution of 35 (17.77 g, 60.65 mmol) in anhydrous THF (120 mL) was added anhydrous Et3N (9.3 mL, 66.71 mmol) and di-tert-butyl-dicarbonate (14.57g, 66.71 mmol). The reaction was stirred under an atmosphere of N2 for 2 d, followed by the evaporation of all volatiles in vacuo. The residue was suspended in water (200 mL) and extracted with EtOAc (3x150 mL). The combined organic portions were dried (MgSO4) and filtered then concentrated in vacuo to give a brown oil. The residue was purified by FCC (80:20 to 100:0 EtOAc/petrol and 20:80 MeOH/EtOAc) to give 36 (10.9 g, 46%) as a brown oil, and 36a (6.9 g, 23%) as a brown oil. Compound 36a was hydrolysed to 36 using the following method. To a solution of 36a (6.9 g, 14 mmol) in MeOH (50 mL) was added K2CO3 (2.709 g, 19.6 mmol). After stirring at rt for 17 h, the mixture was evaporated and then dissolved in EtOAc and washed with water. The water layer was extracted with EtOAc (3x60 mL) and the combined EtOAc extracts were washed with brine, dried and evaporated. The residue was purified by FCC (80:20 to 100:0
EtOAc/petrol and 20:80 MeOH/EtOAc) to give 36 (3.06 g, 56%). Rf 0.40 (EtOAc).
25 [α] D -50 (c 3.0, CHCl3). MS (EI +ve) m/z 394 (M+H+, 30%), 102 (Boc + H+, 100%). + HRMS (CI +ve) calculated for C21H32NO6 (MH ) 394.2230, found 394.2229.
157 Chapter 9 [Experimental for Chapter 2]
-1 IR υmax (cm ): 3390, 1663, 1405, 1180, 1150. 1H NMR (300 MHz) δ 7.37-7.17 (m, 5H, Ar), 6.56 (d, 1H, J 15.9 Hz, H-7), 6.40 (dd, 1H, J 6.6, 15.9 Hz, H-6), 5.85-5.69 (m, 1H, H-2’), 5.13-5.05 (m, 2H, H-3’and H-3’), 4.55-4.42 (m, 1H, H-5), 4.38 (brs, 1H, OH), 4.31 (brs, 1H, OH), 4.14 (brs, 1H, OH), 4.03-3.92 (m, 1H, H-4), 3.87 (brs, 1H, H-2), 3.78-3.69 (m, 4H, H-1, H-1 and 2xH-1’), 3.57 (brs, 1H, H-3), 1.44 (s, 9H, t-Bu). 13C NMR (75 MHz) δ 156.4 (CO), 136.6 (C), 134.7 (C-2’), 134.4 (C-7), 128.4 (CH), 127.6 (CH), 126.4 (CH), 125.1 (C-6), 116.9 (C-3’), 80.8 (C (Boc)), 73.0 (C-2), 72.4 (C-
4), 70.2 (C-3), 63.8 (C-1), 60.2 (C-5), 48.9 (C-1’), 28.3 (C(CH3)3).
36a: Rf 0.68 (EtOAc). MS (CI +ve) m/z 494 (M+H+, 30%), 102 (Boc + H+, 100%). + HRMS (CI +ve) calculated for C26H39NO8 (M+H ) 494.2753, found 494.2752. -1 IR υmax (cm ): 3426, 1741, 1685, 1664, 1394, 1278, 1253, 1162. 1H NMR (300 MHz) δ 7.40-7.22 (m, 5H, Ar), 6.60 (d, 1H, J 16.2 Hz, H-7), 6.41 (dd, 1H, J 7.2, 15.9 Hz, H-6), 5.80 (brs, 1H, H-2’), 5.17-5.11 (m, 2H, H-3’and H-3’), 4.42 (brs, 1H, H-5), 4.23 (dd, 1H, J 5.1, 11.4 Hz, H-1), 4.16 (dd, 1H, J 6.0, 11.4 Hz, H-1), 4.05 (brs, 1H, H-2), 3.98 (brs, 1H, H-4), 3.81 (brs, 2H, H-1’and H-1’), 3.62 (brs, 1H, H- 3), 3.44 (brs, 1H, OH), 3.29 (brs, 1H, OH), 1.48 (s, 18H, t-Bu). 13C NMR (75 MHz) δ 156.5 (CO), 153.4 (CO), 136.4 (CH), 134.7 (C-2’, C-7), 128.4 (CH), 127.6 (CH), 126.4 (CH), 124.6 (C-6), 117.0 (C-3’), 82.0 (C (Boc)), 80.8 (C (Boc)), 73.0 (C-4), 70.9 (C-2), 69.0 (C-3), 67.3 (C-1), 60.4 (C-5), 48.8 (C-1’), 28.2
(C(CH3)3), 27.5 (C(CH3)3).
(6E)-5-[Allyl(tert-butylcarbonyl)amino]-1-O-triphenylmethyl-5,6,7-trideoxy-7- phenyl-D-gluco-hept-6-enitol (37).
OH
OH To a solution of 36 (5.31 g, 13.51 mmol) in anhydrous H TrO 3 Ph 5 CH Cl (30 mL) was added TrCl (3.95 g, 14.16 mmol) and 1 7 2 2 HO N 1' Boc 3' anhydrous pyridine (1.14 mL, 14.16 mmol). The mixture 37 was stirred for 40 h at rt, diluted with water (50 mL) and
158 Chapter 9 [Experimental for Chapter 2] extracted with CH2Cl2 (2×50 mL). The combined CH2Cl2 extracts were washed with satd. CuSO4 solution (30 mL) and brine (30 mL), before being dried and evaporated to give a brown oil. The residue was purified by FCC (30:70 to 40:60 EtOAc/petrol) to give 37 as a pale yellow foamy solid (6.91 g, 81%). Rf 0.45 (40:60 EtOAc/petrol).
26 [α] D -24 (c 1.5, CHCl3). MS (ESI +ve) m/z 658 (M + Na+, 43 %), 243 (Tr, 100%). + HRMS (ESI +ve) calculated for C40H45NO6Na (M+Na ) 658.3145, found 658.3144. -1 IR υmax (cm ): 3431, 1690, 1654, 1448, 1250, 1147, 1075. 1H NMR (300 MHz) δ 7.46-7.19 (m, 20H, Ar), 6.57 (d, 1H, J 15.9 Hz, H-7), 6.36 (dd, 1H, J 6.9, 15.9 Hz, H-6), 5.85-5.65 (m, 1H, H-2’), 5.10-5.01 (m, 2H, 2xH-3’), 4.62-4.52 (m, 1H, H-5), 3.93 (brs, 1H, H-2), 3.91-3.79 (m, 2H, H-4 and H-3), 3.80-3.70 (m, 2H, 2xH-1’), 3.37 (dd, 1H, J 2.7, 9.3 Hz, H-1), 3.22-3.15 (m, 1H, H-1), 1.43 (s, 9H, t-Bu). 13C NMR (75 MHz) δ 156.4 (CO), 143.7 (C), 136.5 (C), 134.8 (C-2’), 134.5 (C-7), 128.6 (CH), 128.5 (CH), 127.8 (CH), 127.7 (CH), 127.0 (CH), 126.5 (CH), 124.9 (C-6), 116.9 (C-3’), 86.9 (C (Tr)), 80.9 (C (Boc)), 73.2 (C-4), 72.2 (C-2), 69.3 (C-3), 64.0 (C-
1), 60.4 (C-5), 48.8 (C-1’), 28.3 (C(CH3)3). tert-Butyl (2R)-2-[(1R,2R,3S)- 1,2,3-trihydrox y-4-triphenylmethyloxybutyl]-2,5- dihydro-1H-pyrrole-1-carboxylate (39).
OH OH To a solution of 38 (16.34 g, 25.73 mmol) in anhydrous CH2Cl2 H TrO 3' 1' (800 mL) was added Grubbs’ I catalyst (1.735 g, 2.108 mmol). 3 HO N1 5 The reaction was stirred and heated at reflux for 24 h under an Boc atmosphere of N2 followed by the removal of all volatiles in 39 vacuo. The residue was purified by FCC (35:65 to 50:50
EtOAc/petrol) to give 39 as a brown foamy solid (12.41 g, 91%). Rf 0.25 (40:60 EtOAc/petrol).
25 [α] D +74 (c 0.72, CHCl3). MS (ESI +ve) m/z 554 (M + Na+, 60%), 243 (Tr+, 100%). + HRMS (ESI +ve) calculated for C32H37NO6Na (M+Na ) 554.2519, found 554.2524. -1 IR υmax (cm ): 3395, 1675, 1398, 1169, 1123.
159 Chapter 9 [Experimental for Chapter 2]
1H NMR (300 MHz) δ 7.44-7.20 (m, 15H, Ar), 5.87 (dd, 1H, J 1.8, 6.4 Hz, H-4), 5.81 (d, 1H, J 6.6 Hz, H-3), 4.67 (brs, 1H, H-2), 4.22 (dd, 1H, J 1.5, 15.9 Hz, H-5), 3.98 (dd, 1H, J 3.0, 15.6 Hz, H-5), 3.85-3.75 (m, 2H, H-2’ and H-3’), 3.53 (m, 1H, H-1’), 3.33 (dd, 1H, J 4.8, 9.6 Hz, H-4’), 3.15 (dd, 1H, J 4.8, 9.6 Hz, H-4’), 1.47 (s, 9H, t-Bu). 13C NMR (75 MHz) δ 157.0 (CO), 143.8 (C), 128.6 (CH), 128.5 (CH), 127.8 (CH), 127.0 (CH), 126.4 (C-3), 86.6 (C (Tr)), 80.8 (C (Boc)), 75.3 (C-1’), 72.5 (C-3’), 69.5 (C-
2’), 67.2 (C-2), 64.3 (C-4’), 54.3 (C-5), 28.4 (C(CH3)3). tert-Butyl (2R)-2-[(1R,2R,3R)-1,2,3-tris(benzyloxy)-4-triphenylmethyloxybutyl]-2,5- dihydro-1H-pyrrole-1-carboxylate (81). OBn OBn OBn TrO 3' H OBn OBn 1' H 7 H H TrO TrO 3' 1' 1' 3 1 3 4a 5 BnO 7q 2' 1' 1 O 3 5 OBn N 5 N ON1 7 Boc O O 81 69 70 To a solution of 39 (12.41 g, 23.37 mmol) in dry THF (240 mL) at 0 oC was added NaH
(3.702 g, 77.12 mmol, 50 % in mineral oil). After H2 evolution had ceased (10 min),
BnBr (16.68 mL, 140.2 mmol) and n-Bu4NI (863 mg, 2.337 mmol) were added. The mixture was stirred at rt for 4 d, then treated with MeOH (20 mL) followed by evaporation of all volatiles in vacuo. The residue was dissolved in Et2O and filtered through celite, followed by further washings of the solids with Et2O. The solvent was evaporated and the residue was purified by FCC (5:95 to 30:70 EtOAc/petrol) to give 81 as a brown oil (10.48 g, 56%), 69 (0.45 g, 3%) as a brown oil and 70 (1.34 g, 9%) as a brown oil.
81: Rf 0.46 (20:80 EtOAc/petrol).
29 [α] D +73 (c 4.6, CHCl3). MS (ESI +ve) m/z 824 (M+Na+, 100%). + HRMS (ESI +ve) calculated for C53H55NO6Na (M+Na ) 824.3926, found 824.3925. IR -1 υmax (cm ): 1700, 1449, 1110, 1060. 1H NMR (300 MHz) δ 7.46-7.05 (m, 30H, Ar), 5.84-5.80 (m, 1H, H-4), 5.76-5.74 (m, 1H, H-3), 4.84 (d, 1H, J 11.4 Hz, CHHPh), 4.73 (d, 1H, J 11.4 Hz, CHHPh), 4.66 (d,
160 Chapter 9 [Experimental for Chapter 2]
1H, J 11.4 Hz, CHHPh), 4.59 (d, 1H, J 11.4 Hz, CHHPh), 4.49-4.46 (m, 1H, H-2), 4.33 (d, 1H, J 11.0 Hz, CHHPh), 4.21 (d, 1H, J 11.0 Hz, CHHPh), 4.09 (dq, 1H, J 1.8, 15.3 Hz, H-5), 4.01-3.97 (m, 1H, H-1’), 3.96-3.91 (m, 2H, H-3’and H-5), 3.66 (app. t, 1H, J 5.4 Hz, H-2’), 3.47 (dd, 1H, J 3.9, 10.2 Hz, H-4’), 3.39 (dd, 1H, J 5.3, 10.4 Hz, H-4’), 1.45 (s, 9H, t-Bu). 13C NMR (75 MHz) δ 153.9 (CO), 144.0 (C), 138.7 (C), 138.5 (C), 138.2 (C), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.3 (CH), 127.2 (CH), 126.9 (C-4), 126.5 (C-3) 86.9 (C (Tr)), 80.9
(C-2’), 80.3 (C-1’), 79.7 (C (Boc)), 79.3 (C-3’), 74.8 (CH2), 74.6 (CH2), 73.4 (CH2),
67.0 (C-2). 63.8 (C-4’), 53.5 (C-5), 28.4 (C(CH3)3).
69: Rf 0.26 (30:70 EtOAc/petrol).
25 [α] D +13 (c 4.5, CHCl3). MS (ESI +ve) m/z 660 (M + Na+, 43 %). + HRMS (ESI +ve) calculated for C42H39NO5(M+Na ) 660.2726, found 660.2712. -1 IR υmax (cm ): 1695, 1449, 1125, 1070, 1029. 1H NMR (300 MHz) δ 7.47-7.15 (m, 25H, Ar), 5.86 (dq, 1H, J 2.0, 5.9 Hz, H-7), 5.69 (dq, 1H, J 2.0, 5.9 Hz, H-6), 4.81 (dd, 1H, J 5.7, 8.1 Hz, H-1), 4.74 (d, 1H, J 12.0 Hz, CHHPh), 4.68 (d, 1H, J 11.1 Hz, CHHPh), 4.61 (d, 1H, J 11.1 Hz, CHHPh), 4.39 (d, 1H, J 11.1 Hz, CHHPh), 4.27 (app. ddt, 1H, J 2.1, 3.6, 15.5 Hz, H-5), 4.09-4.03 (m, 1H, H- 7a), 3.73-3.67 (m, 2H, H-1’and H-2’), 3.63 (dddd, 1H, J 1.5, 2.7, 4.8, 15.6 Hz, H-5), 3.56 (dd, 1H, J 4.8, 10.2 Hz, H-3’), 3.47 (dd, 1H, J 4.8, 10.2 Hz, H-3’). 13C NMR (75 MHz) δ 162.2 (CO), 144.0 (C), 138.2 (C), 138.1 (C), 131.7 (C-7), 128.53 (CH), 128.49 (CH), 128.5 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.6 (CH), 127.3 (CH), 127.2 (CH), 125.8 (C-6), 87.4 (C (Tr)), 79.0 (C-1), 78.3, 77.1 (C-1’ or C-2’), 74.6
(CH2), 72.6 (CH2), 67.0 (C-7a), 62.5 (C-3’), 54.7 (C-5).
70: Rf 0.07 (30:70 EtOAc/petrol).
25 [α] D +80 (c 7.8, CHCl3). + + MS (ESI +ve) m/z 660 (M + Na , 45 %), 243 (Ph3C , 100%). + HRMS (ESI +ve) calculated for C42H39NO5(M+H ) 637.2828, found 637.2811.
161 Chapter 9 [Experimental for Chapter 2]
-1 IR υmax (cm ): 1685, 1444, 1105, 1096. 1H NMR (300 MHz) δ 7.44-7.16 (m, 25H, Ar), 5.82 (s, 2H, H-5 and H-6), 4.603 (dd, 1H, J 1.3, 6.3 Hz, H-3), 4.601 (d, 1H, J 11.5 Hz, CHHPh), 4.59 (d, 1H, J 11.8 Hz, CHHPh), 4.56 (d, 1H, J 11.8 Hz, CHHPh), 4.52 (dd, 1H, J 1.2, 9.5 Hz, H-4a), 4.45 (d, 1H, J 11.8 Hz, CHHPh), 4.39 (dd, 1H, J 4.8, 15.3 Hz, H-7), 4.01 (dd, 1H, J 5.3, 15.5 Hz, H-7), 3.86 (ddd, 1H, J 1.5, 6.3, 7.3 Hz, H-1’), 3.60 (dd, 1H, J 6.0, 10.0 Hz, H-4), 3.54 (dd, 1H, J 6.0, 10.0 Hz, H-2’), 3.51 (dd, 1H, J 7.0, 10.0 Hz, H-2’). 13C NMR (75 MHz) δ 151.7 (CO), 143.8 (C), 138.0 (C),136.9 (C), 128.59 (C-5 or C-6), 128.55 (CH), 128.2 (CH), 128.1 (CH), 127.8 (C-5 or C-6), 127.7 (CH), 127.6 (CH), 127.4 (CH), 127.3 (CH), 127.1 (CH), 127.0 (CH), 87.4 (C (Tr)), 75.2 (C-1’), 75.0 (C-3),
73.9 (C-4), 72.7 (CH2), 72.2 (CH2), 62.8 (C-2’), 62.7 (C-4a), 55.1 (C-7). tert-Butyl (2R,3S,4R)-3,4-dihydroxy-2-[(1R,2S,3S)-1,2,3-tris(benzyloxy)-4- triphenylmethyloxybutyl]pyrrolidine-1-carboxylate (82).
To a solution of 81 (3.43 g, 4.28 mmol) in acetone (20 mL) OBn OBn OH H TrO 3' 1' and water (20 mL) was added potassium osmate.dihydrate 3 OH OBn N1 5 (78.7 mg, 0.214 mmol) and 4-morpholine-N-oxide (1.051 g, Boc 82 8.985 mmol). The reaction was stirred for 3 d at rt and evaporated to give a black oil which was purified by FCC (30:70 to 50:50 EtOAc/petrol) to give 82 as a brown foamy solid (2.69 g, 75%).
Rf 0.50 (40:60 EtOAc/petrol).
25 [α] D +34 (c 0.50, CHCl3). MS (ESI +ve) m/z 858 (M+Na+, 25%), 243 (Tr+, 100%). + HRMS (ESI +ve) calculated for C53H57NO8Na (M+Na ) 858.3981, found 858.3991. IR -1 υmax (cm ): 3400, 1690, 1395, 1090, 1075. 1H NMR δ 7.46-7.05 (m, 30H, Ar), 4.86 (d, 1H, J 11.4 Hz, CHHPh), 4.70 (d, 1H, J 11.4 Hz, CHHPh), 4.62 (d, 1H, J 11.4 Hz, CHHPh), 4.53 (d, 1H, J 11.4 Hz, CHHPh), 4.38 (d, 1H, J 10.8 Hz, CHHPh), 4.26-4.21 (m, 3H, CHHPh, H-1’ and H-4), 4.12-4.04 (m, 2H, H-3 and H-3’), 3.83 (app. t, 1H, H-2’), 3.72 (brs, 1H, H-2), 3.62-3.54 (m, 2H, H-4 and H-4’), 3.43-3.40 (m, 1H, H-5), 3.22-3.19 (m, 1H, H-5), 1.43 (s, 9H, t-Bu).
162 Chapter 9 [Experimental for Chapter 2]
13C NMR δ 154.5 (CO), 143.9 (C), 138.5 (C), 137.9 (C), 137.8 (C), 128.8 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.4 (CH), 126.9 (CH), 87.0 (C (Tr)), 81.2 (C-2’), 79.3 (C (Boc)),
79.0 (C-3’), 78.0 (C-1’), 75.2 (CH2), 74.0 (CH2), 73.7 (CH2), 71.2 (C-4), 70.4 (C-3),
65.1 (C-2), 64.5 (C-4’), 51.7 (C-5), 28.4 (C(CH3)3). tert-Butyl (3aS,4S,6aR)-4-[(1R,2S,3S)-1,2,3-tris(benzyloxy)-4- triphenylmethyloxybutyl] tetrahydro-5H- [1,3,2]dioxathiolo[4,5-c]pyrrole-5- carboxylate 2,2-dioxide (87a)
OBn OBn O H SO2 To a solution of 82 (301 mg, 0.361 mmol) in CH2Cl2 (3 TrO 3' 1' 3 O mL) was added Et3N (0.755 mL, 5.42 mmol) followed by OBn N1 5 Boc sulfuryl chloride (0.145 mL, 1.81 mmol) at 0 oC. The 87a mixture was stirred for 1 h at 0 oC under an atmosphere of
N2 and then warmed up to rt for 1 h. The residue was suspended in water (5 mL). The aqueous layer was extracted with CH2Cl2 (3x10 mL) and washed with brine. The combined organic extracts were dried, filtered and evaporated under reduced pressure to give a brown oil that was used in the next step without further purification. (87): Rf 0.36 (20:80 EtOAc/petrol). MS (ESI +ve) m/z 920 (M+Na+, 50%), 243 (Tr+, 100%). + HRMS (ESI +ve) calculated for C53H55NO10SNa (M+Na ) 920.3444, found 920.3439. -1 IR υmax (cm ): 1700, 1395, 1210, 1160, 1070. 1H NMR δ 7.42-6.93 (m, 30H, Ar), 5.44 (d, 1H, J 5.0 Hz, H-3), 5.08-5.03 (m, 1H, H-4), 4.76 (d, 1H, J 11.5 Hz, CHHPh), 4.69 (d, 1H, J 11.0 Hz, CHHPh), 4.57 (d, 1H, J 12.0 Hz, CHHPh), 4.50 (d, 1H, J 11.5 Hz, CHHPh), 4.45 (d, 1H, J 11.5 Hz, CHHPh), 4.41 (brs, 1H, H-2), 4.18 (d, 1H, J 11.0 Hz, CHHPh), 4.15-4.11 (m, 1H, H-2’), 4.04 (brd, 1H, J 6.5 Hz H-1’), 3.89-3.86 (m, 1H, H-3’), 3.73-3.68 (m, 1H, H-5), 3.59-3.55 (m, 1H, H- 4’), 3.46 (dd, 1H, J 4.0, 10.5 Hz, H-4’), 3.39 (dd, 1H, J 6.3, 13.3 Hz, H-5), 1.44 (s, 9H, t-Bu). 13C NMR δ 152.8 (CO), 143.9 (C), 138.2 (C), 137.6 (C), 137.4 (C), 128.7 (CH), 128.5 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.5 (CH), 127.2 (CH), 127.0 (CH), 87.1 (C (Tr)), 84.4 (C-3), 83.5 (C-4), 80.8 (C (Boc)), 79.6 (C-1’), 79.3 (C-2’),
163 Chapter 9 [Experimental for Chapter 2]
77.7 (C-3’), 75.4, 74.2 (CH2), 73.7 (CH2), 64.4 (C-2), 63.3 (C-4’), 51.3 (C-5), 28.4
(C(CH3)3). tert-Butyl (2R,3S,4S)-3-hydroxy-4-phenylcarbonyloxy-2-[(2S,3S)-1,2,3- tris(benzyloxy)-4-triphenylmethyloxybutyl]pyrrolidine-1-carboxylate (88a) and tert-butyl (2R,3S,4S)-3-hydroxy-4-phenylcarbonyloxy-2-[(2S,3S)-4-hydroxy-1,2,3- tris(benzyloxy)-butyl]pyrrolidine-1-carboxylate (88b).
BnO BnO H OH H OH BnO BnO 1' 3 1' 3 3' OBz 3' OBz 1N 5 1N 5 BnO BnO Boc Boc OTr HO 88a 88b The crude cyclic sulfate 87a obtained from the above reaction was dissolved in DMSO (0.5 mL) and benzoic acid (0.221 g, 1.81 mmol) and cesium carbonate (0.647 g, 1.97 mmol) were added and the solution was stirred under an atmosphere of N2 for 20 h at 40 oC. The reaction was suspended in THF (4 mL). Water (1.5 mL) and concentrated sulfuric acid (0.8 mL) were added and the mixture was stirred at rt for 20 h, then poured in to satd. NaHCO3 and extracted with CH2Cl2 (3x10 mL). The combined CH2Cl2 extracts were washed with brine, dried, filtered and evaporated. The crude product was purified by FCC (15:85 to 50:50 EtOAc/petrol) to give 88a as a yellow oil (183.3 mg, 54% from 82), 88b as a brown oil (18.4 mg, 7% from 82) and 82 (37.2 mg, 16%).
88a: Rf 0.48 (30:70 EtOAc/petrol).
24 [α] D +36 (c 1.0, CHCl3). MS (ESI +ve) m/z 961.9 (M+Na+, 62%). + HRMS (ESI +ve) calculated for C60H61NO9Na (M+Na ) 962.4244, found 962.4247. IR -1 υmax (cm ): 3385, 1730, 1696, 1271, 1110, 1070, 1027. 1H NMR δ 7.99 (d, 2H, J 7.5 Hz, 2xH-o-Bz), 7.55 (t, 1H, J 7.5 Hz, H-p-Bz), 7.42-7.07 (m, 32H, Ar), 5.10 (dd, 1H, J 14.5, 8.0 Hz, H-4), 4.79 (d, 1H, J 12.0 Hz, CHHPh), 4.75 (d, 1H, J 11.5 Hz, CHHPh), 4.72 (app. brt, 2H, J 5.5 Hz, H-3 and CHHPh), 4.55-4.52 (m, 2H, 2xCHHPh), 4.44 (brs, 2H, H-1’and CHHPh), 4.28 (brs, 1H, OH), 4.20 (brs, 1H,
164 Chapter 9 [Experimental for Chapter 2]
H-5), 4.11 (dd, 1H, J 14.0, 7.0 Hz, H-3’), 3.89 (brs, 1H, H-2’), 3.85 (brs, 1H, H-2), 3.52 (brs, 1H, OH), 3.46 (brs, 2H, 2xH-4’), 3.15 (brs, 1H, H-5), 1.44 (s, 9H, t-Bu). 13C NMR δ 166.9 (CO), 153.5 (CO), 143.9 (C), 138.1 (C), 137.7 (C), 133.3 (CH), 129.8 (CH), 129.4 (CH), 128.7 (CH), 128.3 (CH), 128.29 (CH), 128.22 (CH), 128.16 (CH), 128.13 (CH), 127.7 (CH), 127.69 (CH), 127.61 (CH), 127.2 (CH), 126.8 (CH), 86.9 (C
(Tr)), 80.5 (C-2’), 79.7 (C-4), 79.3 (C (Boc)), 78.7 (C-3’), 78.2 (C-1’), 75.0, 74.2 (CH2),
73.5 (CH2), 73.2 (C-3), 64.8 (C-2), 63.6 (C-4’), 49.2 (C-5), 28.3 (C(CH3)3).
88b: Rf 0.29 (40:60 EtOAc/petrol). MS (ESI +ve) m/z 697.8 (M+H+, 30%). + HRMS (I ES+ve) calculated for C41H47NO9 (M+H ) 698.3329, found 698.3340. -1 IR υmax (cm ): 3436, 1700, 1403, 1265, 1110, 1069. 1H NMR δ 8.00 (d, 2H, J 7.5 Hz, 2xH-o-Bz), 7.53 (t, 1H, J 7.5 Hz, H-p-Bz), 7.40 (t, 1H, J 7.5 Hz, H-m-Bz), 7.37-7.18 (m, 15H, Ar), 5.14 (dd, 1H, J 14.0, 8.0 Hz, H-4), 4.83-4.44 (m, 6H, 4xCHHPh, H-3 and H-1’), 4.30-4.10 (m, 3H, H-5, CHHPh and H-2), 4.00-3.86 (m, 2H, 2xH-4’), 3.84-3.76 (m, 3H, H-2’, H-3 and CHHPh), 3.24-3.12 (m, 1H, H-5), 1.46 (s, 9H, t-Bu). 13C NMR δ 166.7 (CO), 154.0 (CO), 143.8 (C), 138.6 (C), 137.5 (C), 134.4 (C), 133.3 (CH), 129.8 (CH), 129.4 (CH), 128.7 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 128.29 (CH), 128.21 (CH), 128.16 (CH), 128.0 (CH), 127.8 (CH), 127.6(CH), 127.3 (CH), 126.9 (CH), 81.0 (C-2’), 80.3 (C (Boc)), 78.8 (C-4), 78.2 (C-3’), 78.1 (C-1’), 75.6
(CH2), 74.3 (C-3), 74.2 (CH2), 72.7 (CH2), 64.5 (C-2), 60.7 (C-4’), 49.9 (C-5), 28.7
(C(CH3)3). tert-Butyl (2R 3S,4S)-3,4-dihydroxy-2-[(1R,2S,3S)-1,2,3-tris(benzyloxy)-4- triphenylmethyloxybutyl]pyrrolidine-1-carboxylate (89). BnO H OH BnO To a solution of 88a (34.9 mg, 0.037 mmol) in MeOH (1 mL) 1' 3 3' OH 1N 5 was added K2CO3 (0.010 g, 0.075 mmol). After stirring at rt for BnO Boc 24 h, the mixture was evaporated and dissolved in CHCl then TrO 3 89 washed with water. The aqueous layer was extracted with CHCl3
(3x5mL). and the combined CHCl3 extracts were washed with brine, dried and
165 Chapter 9 [Experimental for Chapter 2] evaporated. The residue was purified by PTLC (30:70 EtOAc/petrol) to give 89 as a colourless oil (13.5 mg, 44%). Rf 0.16 (30:70 EtOAc/petrol).
22 [α] D +10.5 (c 0.7, CHCl3). MS (ESI +ve) m/z 858 (M+Na+, 62%), 243 (Tr+, 100%). + HRMS (ESI +ve) calculated for C53H57NO8Na (M+Na ) 858.3982, found 858.3970. IR -1 υmax (cm ): 3400, 1691, 1392, 1163, 1073. 1H NMR δ 7.48-7.11 (m, 30H, Ar), 4.85 (d, 1H, J 11.5 Hz, CHHPh), 4.68 (d, 1H, J 11.5 Hz, CHHPh), 4.61-4.47 (m, 3H, 3xCHHPh), 4.40 (d, 1H, J 11.5 Hz, CHHPh), 4.24 (d, 1H, J 8.5 Hz, H-1’), 4.01 (brs, 1H, H-3’), 3.78-3.76 (m, 2H, H-3, H-4), 3.72-3.62 (m, 4H, H-4’, H-2’, H-4’, H-2), 3.48 (dd, 1H, J 12.0, 4.5 Hz, H-5), 3.24 (d, 1H, J 11.5 Hz, H-5), 1.26 (s, 9H, tBu). 13C NMR δ 154.5 (CO), 143.9 (C), 137.4 (C), 137.1 (C), 137.08 (C), 128.8 (CH), 128.7 (CH), 128.5 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.1 (CH), 127.0
(CH), 87.0 (C (Tr)), 81.6 (C-2’), 80.0 (C (Boc)), 79.2 (C-1’), 77.4 (C-3’), 77.4 (CH2),
77.1 (CH2), 77.2 (C-4), 73.7 (CH2), 73.3 (C-3), 67.9 (C-2). 63.8 (C-4’), 54.5 (C-5), 29.7
(C(CH3)3).
(2S,3S,4R)-4-[(2R,3S,4S)-3,4-Dihydroxypyrrolidin-2-yl]-2,3,4-tribenzyloxybutan-1- ol (90) and (1S,2S,6S,7R,8R,8aR)-1,2-dihydroxy-6,7,8- tribenyloxyoctahydroindolizine (91).
BnO BnO H OH H OH BnO BnO 1' 1' 2 7 8a 1 3' OH OH HN 5 5 N 3 BnO BnO HO 91 90
To a solution of 89 (237 mg, 0.284 mmol) in anhydrous CH2Cl2 (2.8 mL) was added anisole (0.289 mL, 2.841 mmol) and TFA (2.19 mL, 28.41 mmol). The mixture was stirred under an atmosphere of N2 at rt for 20 h, followed by the evaporation of all volatiles in vacuo. The residue was dissolved in CH2Cl2 (5 mL) and washed with satd.
Na2CO3 solution (10 mL). The aqueous layer was extracted with CH2Cl2 (3x10 mL).
The combined CH2Cl2 extracts were dried (Na2CO3) and evaporated to give a brown oil which was purified by FCC (50:50 to 100:0 EtOAc/petrol and 15:85 MeOH/EtOAc) to
166 Chapter 9 [Experimental for Chapter 2] give 91 as a pale yellow solid (11 mg, 8%) and the amino alcohol 90 (92.3 mg, 66%) as a brown foamy solid.
Rf 0.45 (80:19:1 CHCl3/MeOH/NH4OH).
21 [α] D -14.0 (c 0.35, MeOH). MS (ESI +ve) m/z 494 (M+H+, 100%). + HRMS (ESI +ve) calculated for C29H35NO6 (M+H ) 494.2543, found 494.2531. -1 IR υmax (cm ): 3411, 3283, 1454, 1126, 1058, 1044. 1H NMR δ 7.37-7.25 (m, 15H, Ar), 4.86 (d, 1H, J 11.0 Hz, CHHPh), 4.71 (d, 1H, J 11.0 Hz, CHHPh), 4.68 (d, 1H, J 11.5 Hz, CHHPh), 4.65 (d, 1H, J 11.0 Hz, CHHPh), 4.63 (d, 1H, J 11.5 Hz, CHHPh), 4.57 (d, 1H, J 11.5 Hz, CHHPh), 4.00 (brs, 1H, H-3), 3.90-3.88 (m, 1H, H-4), 3.87-3.83 (m, 2H, H-1’and H-4’), 3.80-3.76 (m, 2H, H-2’ and H-4’), 3.75- 3.73 (m, 1H, H-3’), 3.14 (brs, 1H, H-2), 3.00 (dd, 1H, J 11.5, 4.5 Hz, H-5), 2.81 (brd, 1H, J 11.5 Hz, H-5). 13C NMR δ 137.6 (C), 137.4 (C), 137.3 (C), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 80.9 (C-2’), 80.3 (C-1’), 79.5 (C-3), 78.6 (C-3’), 75.5
(CH2), 74.2 (CH2), 72.9 (CH2), 66.8 (C-2), 60.8 (C-4’), 52.3 (C-5).
Synthesis of 91 from 90: To a solution of 90 (37.0 mg, 0.075 mmol) in pyridine(1 mL) was added triphenylphosphine (39.4 mg, 0.150 mmol) and diisopropyl azodicarboxylate o (0.030 mL, 0.150 mmol) at 0 C. The mixture was stirred under an atmosphere of N2 at 0 oC for 8 h, and at 0-5 oC for 40 h, then warm up to rt and stirred for a further 48 h. The volatiles were removed in vacuo then 1M HCl (5 mL) was added and the mixture was extracted with CH2Cl2 (2x10 mL). The combined CH2Cl2 extracts were washed with water dried, filtered and then evaporated to give a brown oil. Purification by FCC (2:98
MeOH/CHCl3 to 95:4:1 CHCl3/MeOH/ NH4OH) gave 91 (9.0 mg, 25%) as a pale yellow solid.
Rf 0.31 (100% EtOAc).
23 [α] D +34 (c 0.26, CHCl3). MS (ESI +ve) m/z 476 (M+H+, 100%). + HRMS (ESI +ve) calculated for C29H33NO5 (M+H ) 476.2437, found 476.2327. -1 IR υmax (cm ): 3293, 1460, 1103, 1071.
167 Chapter 9 [Experimental for Chapter 2]
1H NMR δ 7.38-7.27 (m, 15H, Ar), 5.00 (d, 1H, J 10.5 Hz, CHHPh), 4.94 (d, 1H, J 11.0 Hz, CHHPh), 4.82 (d, 1H, J 11.0 Hz, CHHPh), 4.70 (d, 1H, J 11.5 Hz, CHHPh), 4.67 (d, 1H, J 12.5 Hz, CHHPh), 4.64 (d, 1H, J 11.5 Hz, CHHPh), 4.04 (dd, 1H, J 5.0, 2.0 Hz, H-2), 3.73 (dd, 1H, J 6.5, 2.0 Hz, H-1), 3.71-3.68 (m, 1H, H-6), 3.54-3.51 (m, 2H, H-7 and H-8), 3.18 (dd, 1H, J 10.5, 5.5 Hz, H-5), 2.85 (d, 1H, J 10.5 Hz, H-3), 2.61 (dd, 1H, J 10.5, 6.5 Hz, H-3), 2.04 (app. t, 1H, J 10.5 Hz, H-5), 1.96 (dd, 1H, J 9.0, 7.0 Hz, H- 8a). 13C NMR δ 138.6 (C), 138.2 (C), 138.1 (C), 128.7 (CH), 128.43 (CH), 128.41 (CH), 128.19 (CH), 128.14 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 87.3 (C-
7), 84.1 (C-1), 81.6 (C-8), 79.4 (C-6), 78.0 (C-2), 75.5 (CH2), 74.8 (CH2), 72.9 (CH2), 72.6 (C-8a), 60.4 (C-3), 54.1 (C-5).
(1S,2S,6S,7R,8R,8aR)-Octahydroindolizine-1,2,6,7,8-pentol (76).
HO To a solution of 91 (11.0 mg, 0.023 mmol) in EtOAc (0.5 mL) H OH HO 8a 1 and MeOH (0.5 mL) was added PdCl2 (6.16 mg, 0.035 mmol). 7 OH 5 N 3 HO The mixture was stirred at rt under an atmosphere of H2 76 (balloon) for 4 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 76 C-2-epimer of proposed uniflorine A (3.4 mg, 72%) as a pale yellow, foamy solid.
25 [α] D -9.2 (c 0.17, H2O). MS (ESI +ve) m/z 206 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H15NO5 (M+H ) 206.1028, found 206.0990. -1 IR υmax (cm ): 3308, 1073, 1043. 1 H NMR (D2O) δ 4.14 (ddd, 1H, J2,3β = 6.3, J1,2 = 2.5, J2,3α < 1 Hz, H-2), 3.96 (dd, 1H,
J1,8a = 7.5, J1,2 = 2.5 Hz, H-1), 3.64 (ddd, 1H, J5β,6 = 10.4, J6,7 = 9.0, J5α,6 = 5.3 Hz, H-6),
3.47 (t, 1H, J7,8 = J8,8a = 9.0 Hz, H-8), 3.29 (t, 1H, J6,7 = J7,8 = 9.0 Hz, H-7), 3.11 (dd,
1H, J5α,5β = 10.8, J5α,6 = 5.3 Hz, H-5α), 2.87 (brd, 1H, J3α,3β = 11.0 Hz, H-3α), 2.76 (dd,
168 Chapter 9 [Experimental for Chapter 2]
1H, J3α,3β = 10.9, J2,3β = 6.7 Hz, H-3β), 2.16 (t, 1H, J5α,5β = J5β,6 = 10.8 Hz, H-5β), 2.11
(dd, 1H, J8,8a = 9.0, J1,8a = 8.0 Hz, H-8a). 13 C NMR (D2O) δ 82.7 (C-1), 79.2 (C-7), 78.1(C-2), 74.1 (C-8), 72.1 (C-8a), 70.3 (C-6), 59.7 (C-3), 55.6 (C-5).
9.2.2 Total synthesis of (+) and (-) uniflorine A (6-epi-casuarine)
9.2.2.1 Total synthesis of (+) uniflorine A from D-xylose (6E)-5-(Allylamino)-5,6,7-trideoxy-7-phenyl-L-gluco-hept-6-enitol (92).
OH OH To a mixture of D-xylose (13.00 g, 86.6 mmol) and trans-2-
Ph 5 OH 1 phenylvinyl boronic acid (12.81 g, 86.6 mmol) was added NH OH 3' 1' absolute ethanol (130 mL) and allylamine (6.5 mL, 86.6 92 mmol). The reaction mixture was stirred at rt for 3 d, followed by the evaporation of all volatiles in vacuo. The residue was dissolved in 1 M HCl (ca 20 mL), applied to a column of DOWEX resin (H+ form, 150 mL) and washed with distilled H2O (2 L). The product was eluted with 7 M NH4OH (2 L) and 14 M
NH4OH (2 L). The fractions containing the product were combined and concentrated to a brown foamy solid (23.02 g, 91%). Rf 0.65 (10:2:1 EtOAc/MeOH/NH4OH).
25 [α] D -17 (c 0.3, MeOH). This compound had the same MS, IR and NMR spectroscopic data as (+)-35. tert-Butyl allyl((2R,3S,4S,5R,E)-1,2,3,4-tetrahydroxy-7-phenylpent-6-en-
5-yl)carbamate (93).
OH OH To a solution of 92 (1.703 g, 5.812 mmol) in anhydrous
Ph 5 3 OH 7 1 MeOH (20 mL) was added anhydrous Et3N (1.62 mL, 11.61
1' N OH 3' Boc mmol) and di-tert-butyl-dicarbonate (5.074 g, 23.25 mmol). 93 The reaction mixture was stirred under an atmosphere of N2
169 Chapter 9 [Experimental for Chapter 2] for 3 d, followed by the evaporation of all volatiles in vacuo. The residue was purified by FCC (80:20 to 100:0 EtOAc/petrol and then 20:80 MeOH/EtOAc) to give 93 as a brown oil (1.713 g, 75%). Rf 0.40 (EtOAc).
25 [α] D +29 (c 2.3, CHCl3). This compound had the same MS, IR and NMR spectroscopic data as (-)-36. tert-Butyl allyl((2R,3S,4S,5R,E)-1,2-O-(1-methylethylidene)-3,4-dihydroxy-
7-phenylpent-6-en-5-yl)carbamate (94) and tert-butyl allyl((1S,2S,E)-1-hydroxy-1- (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-4-phenylbut-3-en-2- yl)carbamate (94a).
OH O OH 5 3 Ph 5 3 1 O Ph O 7 7 1' N 1' N HO 3' O 3' Boc Boc OH 94 94a To a solution of 93 (0.80 g, 2.036 mmol) in anhydrous acetone (10 mL) was added 2,2- dimethoxypropane (0.3 mL, 2.443 mmol) and pyridinium p-toluenesulfonate (0.051 g,
0.203 mmol). The reaction mixture was stirred under an atmosphere of N2 for 20 h, followed by the evaporation of all volatiles in vacuo to give a brown oil. The residue was purified by FCC (30:70 to 50:50 EtOAc/petrol) to give 94 (0.480 g, 55%) as a pale yellow foamy solid, Rf 0.38 (30:70 EtOAc/petrol). A small amount of another regioisomer of 94 (94a), as a brown oil, was also isolated (0.137 g, 16%), Rf 0.25 (30:70 EtOAc/petrol).
23 94: [α] D +41 (c 10.1, CHCl3). MS (ESI +ve) m/z 434 (M+H+, 100%). + HRMS (ESI +ve) calculated for C24H36NO6 (M+H ) 434.2543, found 434.2556. -1 IR υmax (cm ): 3482, 2980, 2896, 1669, 1154, 1073. 1H NMR δ 7.38-7.19 (m, 5H, Ar), 6.55 (br d, 1H, J 15.0 Hz, H-7), 6.46 (dd, 1H, J 16.0, 6.5 Hz, H-6), 5.81 (brs, 1H, H-2’), 5.16-5.09 (m, 2H, H-3’, H-3’), 4.36 (brs, 1H, H-5), 4.30-4.28 (br d, 1H, J 5.5 Hz, H-2), 4.05 (dd, 1H, J 8.3, 6.3 Hz, H-1), 3.90 (brs, 1H, H-
170 Chapter 9 [Experimental for Chapter 2]
4), 3.82-3.78 (m, 1H, H-1), 3.79-3.75 (m, 2H, 2xH-1’), 3.57 (brs, 1H, H-3), 1.46 (s, 9H, t-Bu), 1.43 (s, 3H, CH3), 1.36 (s, 3H, CH3). 13C NMR δ 155.8 (CO), 136.4 (C), 134.6 (C-2’), 133.9 (C-7), 128.2 (CH), 127.4 (CH), 126.2 (CH), 125.2 (C-6), 116.8 (C-3’), 109.2 (C), 80.4 (C), 77.2 (C-2), 71.7 (C-4), 70.2
(C-3), 65.6 (C-1), 60.8 (C-5), 49.4 (C-1’), 28.1 (C(CH3)3), 26.3 (CH3), 25.2 (CH3).
94a: 1H NMR δ 7.36-7.14 (m, 5H, Ar), 6.51 (brs, 2H, H-6 and H-7), 5.82 (brs, 1H, H- 2’), 5.14 (br d, 1H, J 16.5 HZ, H-3’), 5.08 (d, 1H, J 10.8 Hz, H-3’), 4.20-4.13 (m, 4H, H-2, H-4, H-5 and H-1’), 3.83-3.83 (brs, 1H, H-3), 3.84 (brs, 1H, H-1’), 3.72 (dd, 1H, J 12.0, 4.0 Hz, H-1), 3.65 (dd, 1H, J 12.0, 5.0 Hz, H-1), 1.43 (s, 9H, t-Bu), 1.42 (s, 3H,
CH3), 1.39 (s, 3H, CH3). 13C NMR δ 155.2 (CO), 136.5 (C), 134.7 (C-2’), 133.5 (C-7), 128.3 (CH), 127.4 (CH), 126.2 (C-6), 116.5 (C-3’), 109.1 (C), 80.2 (C), 77.3 (C-2), 75.9 (C-3), 70.1 (C-4), 62.4
(C-5), 62.1 (C-1), 50.5 (C-1’), 28.2 (C(CH3)3), 26.9 (CH3), 26.8 (CH3).
(S)-tert-Butyl 2-((1S,2R,3R)-1,2,3,4-tetrahydroxybutyl-3,4-O-(1-methylethylidene))- 2,5-dihydro-1H-pyrrole-1-carboxylate (95).
OH O To a solution of 94 (5.481 g, 12.66 mmol) in anhydrous CH2Cl2 H 4' O 1' (250 mL) was added Grubbs’ I catalyst (0.833 g, 1.012 mmol). 2 5 N HO The reaction mixture was stirred and heated at reflux for 20 h Boc 95 under an atmosphere of N2 followed by the removal of all volatiles in vacuo. The residue was purified by FCC (50:50 to 70:30 EtOAc/petrol) to give 95 as a dark brown viscous oil (3.894 g, 94%). Rf 0.18 (50:50 EtOAc/petrol).
22 [α] D -37 (c 5.75, CHCl3). MS (ESI +ve) m/z 352 (M + Na+, 50%). + HRMS (ESI +ve) calculated for C16H27NO6Na (M+Na ) 352.1736, found 352.1738. -1 IR υmax (cm ): 3436, 2980, 2929, 1671, 1369, 1159, 1068. 1H NMR δ 5.95 (br d, 1H, J 6.5 Hz, H-3), 5.86 (br d, 1H, J 6.5 Hz, H-4), 4.68 (brs, 1H, H-2), 4.25 (dd, 1H, J 12.5, 5.5 Hz, H-3’), 4.21 (brs, 1H, H-5), 4.04 (br d, 1H, J 6.0 Hz,
171 Chapter 9 [Experimental for Chapter 2]
H-4’), 4.01 (br d, 1H, J 5.5 Hz, H-5), 3.82 (app. t, 1H, J 7.5 Hz, H-4’), 3.56-3.53 (m, 2H,
H-2’, H-1’), 1.47 (s, 9H, t-Bu), 1.44 (s, 3H, CH3), 1.37 (s, 3H, CH3). 13C NMR δ 155.9 (CO), 127.9 (C-3), 126.4 (C-4), 109.1 (C), 80.2 (C), 77.3 (C-3’), 73.8
(C-1’), 70.1 (C-2’), 67.0 (C-2), 65.6 (C-4’), 54.0 (C-5), 28.0 (C(CH3)3), 26.2 (CH3), 25.2
(CH3).
(2R,3R,4S)-tert-Butyl 2-((1S,2R,3R)-1,2,3,4-tetrahydroxybutyl-3,4-O-(1- methylethylidene))-3,4-dihydroxypyrrolidine-1-carboxylate (96).
To a solution of 95 (1.680 g, 5.112 mmol) in acetone (25 mL) HO OH O H O 1' 3 ' and water (25 mL) was added potassium osmate•dihydrate HO 2 5 N OH Boc (93.95 mg, 0.255 mmol) and 4-morpholine-N-oxide (1.255 g, 96 10.70 mmol). The reaction mixture was stirred for 24 h at rt and evaporated to give a dark brown oil which was purified by FCC (100% EtOAc to
4:96 MeOH/EtOAc) to give 96 as a brown foamy solid (1.250 g, 68%). Rf 0.33 (5:95 MeOH/EtOAc).
22 [α] D -32 (c 4.8, CHCl3). MS (ESI +ve) m/z 364 (M+H+, 100%). + HRMS (ESI +ve) calculated for C16H30NO8 (M+H ) 364.1971, found 364.1978. -1 IR υmax (cm ): 3405, 2975, 2929, 1667, 1403, 1368, 1144, 1067. 1H NMR δ 4.69 (brs, 1H, OH), 4.56 (brs, 1H, OH), 4.31 (brs, 1H, H-1’), 4.26-4.22 (m, 2H, H-3’, H-4), 4.00 (app. t, 1H, J 7.5 Hz, H-4’), 3.86-3.82 (m, 1H, H-4’), 3.74 (brd, 1H, J 6.0 Hz, H-2), 3.50 (m, 1H, H-2’), 3.45 (m, 1H, H-3), 3.38 (brd, 2H, J 5.5 Hz, 2xH-5),
1.39 (s, 9H, t-Bu), 1.38 (s, 3H, CH3), 1.31 (s, 3H, CH3). 13C NMR δ 156.8 (CO), 109.6 (C), 80.8 (C), 77.3 (C-3’), 72.4 (C-1’), 71.9 (C-3), 70.4
(C-2’), 69.9 (C-4), 65.7 (C-4’), 51.2 (C-5), 28.2 (C(CH3)3), 26.3 (CH3), 25.3 (CH3).
(2S,3R,4S)-tert-Butyl 3,4-di(benzyloxy)-2-((1S,2R,3R)- 1,2-di(benzyloxy)-3,4-O-(1- methylethylidene))- 3,4-dihydroxybutylpyrrolidine-1-carboxylate (97).
OBn BnO H O To a solution of 96 (1.308 g, 3.60 mmol) in dry THF (40 O 1' 3 ' mL) was added n-Bu4NI (133.1 mg, 0.36 mmol) and BnBr BnO 2 5 N OBn Boc 97 172 Chapter 9 [Experimental for Chapter 2]
(3.43 mL, 28.80 mmol) follow by NaH (1.038 g, 21.60 mmol, 50 % in mineral oil) at 0 o C. After H2 evolution had ceased (15 min) the reaction mixture was stirred at rt for 48 h. MeOH (20 mL) was then added followed by evaporation of all volatiles in vacuo. The residue was dissolved in EtOAc and filtered through celite, followed by further washings of the solids with EtOAc. The solvent was evaporated and the residue was purified by
FCC (10:90 to 15:85 EtOAc/petrol) to give 97 as a pale yellow syrup (2.232 g, 86%). Rf 0.14 (10:90 EtOAc/petrol).
25 [α] D +45 (c 4.26, CHCl3). MS (ESI +ve) m/z 746 (M + Na+, 100%). + HRMS (ESI +ve) calculated for C44H54NO8 (M+H ) 724.3849, found 724.3838. -1 IR υmax (cm ): 2970, 2924, 1693, 1398, 1362, 1158, 1098. 1H NMR δ 7.38-7.18 (m, 20H, Ar), 4.77-4.69 (m, 3H, CHO and 2xCHHPh), 4.56-4.36 (m, 5H, CHO and 4xCHHPh), 4.29-4.19 (m, 3H, CHO and 2xCHHPh), 4.07-4.02 (m, 3H, CHO, H-2 and H-5), 3.70-3.64 (m, 2H and 2 x H-4’), 3.21-3.16 (m, 2H, CHO and
H-5), 1.49 (s, 9H, t-Bu), 1.41 (s, 3H, CH3), 1.27 (s, 3H, CH3). 13C NMR δ (major rotamer) 156.8 (CO), 138.4 (C), 137.5(C), 137.4 (C), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 125.7 (CH), 109.1 (C), 80.8 (C), 78.8 (CH), 78.6 (CH), 76.0
(CH2), 75.1 (CH and CH2), 74.1 (CH), 71.8 (CH2), 70.9 (CH2), 65.2 (C-4), 61.6 (C-2),
48.8 (C-5), 28.4 (C(CH3)3), 26.3 (CH3), 25.7 (CH3).
1-[(2R,3S,4S)-3,4-Dibenzyloxy-2-pyrrolidinyl]-(1S,2R,3S)-1,2-dibenzyloxy-butane- 3,4-diol (98).
To a solution of 97 (1.500 g, 2.075 mmol) in MeOH (60 BnO OBn OH H OH 1' 3 ' mL) was added dropwise conc. HCl solution (15 mL) BnO 2 5 NH OBn and the mixture was stirred at rt for 24 h. The reaction 0 98 mixture was basified at 0 C with aqueous NH3 solution
(28%). The mixture was extracted with EtOAc, dried (Na2CO3), evaporated and purified by FCC (100% EtOAc to 93:5:2 EtOAc/ MeOH/NH3) to give 98 (0.943 g, 78%) as a yellow viscous oil. Rf 0.31 (95:4:1 EtOAc/MeOH/NH3).
173 Chapter 9 [Experimental for Chapter 2]
21 [α] D +35 (c 1.45, CHCl3). MS (ESI +ve) m/z 584 (M+H+, 100%). + HRMS (ESI +ve) calculated for C36H42NO6 (M+H ) 584.3012, found 584.3008. -1 IR υmax (cm ): 3411, 3293, 3062, 3027, 2919, 2888, 1449, 1063. 1H NMR δ 7.25-7.11 (m, 20H, Ar), 4.58 (d, 1H, J 12.0 Hz, CHHPh), 4.55 (d, 1H, J 11.0 Hz, CHHPh), 4.49 (d, 1H, J 11.0 Hz, CHHPh), 4.46 (d, 1H, J 10.5 Hz, CHHPh), 4.44 (d, 1H, J 12.5 Hz, CHHPh), 4.37 (d, 1H, J 11.5 Hz, CHHPh), 4.34 (d, 1H, J 10.5 Hz, CHHPh), 4.33 (d, 1H, J 10.5 Hz, CHHPh), 4.09 (dd, 1H, J 6.8, 5.3 Hz, H-3), 3.85-3.82 (m, 2H, H-4 and H-3’), 3.79 (app. t, 1H, J 5.3 Hz, H-2’), 3.62-3.57 (m, 2H, H-2 and H- 4’), 3.38 (dd, 1H, J 11.0, 5.0 Hz, H-4’), 3.18 (brs, 1H, OH), 3.00 (dd, 1H, J 12.0, 3.0 Hz, H-5), 2.95 (dd, 1H, J 12.0, 4.5 Hz, H-5). 13C NMR δ 138.1 (C), 138.0 (C), 137.9 (C), 137.6 (C), 128.4 (CH), 128.37 (CH), 128.3 (CH), 128.2 (CH), 127.9 (CH), 127.86 (CH), 127.8 (CH), 127.77 (CH), 127.7 (CH),
127.6 (CH), 79.6 (C-1’), 79.5 (C-3), 77.8 (C-2’), 76.6 (C-4), 73.7 (CH2), 73.3 (CH2),
71.9 (CH2), 71.5 (CH2), 69.1 (C-3’), 64.1 (C-4’), 61.6 (C-2), 48.5 (C-5).
1-[(2R,3S,4S)-3,4-Dibenzyloxy-2-pyrrolidinyl]-(1S,2R,3S)-1,2-dibenzyloxy-4-(tert- butyldimethylsilyloxy)-butan-3-ol (99).
OBn OH OBn OTBS BnO H BnO H 1' 3' OTBS 1' 3 ' OTBS 2 2 BnO BnO 5 NH OBn 5 NH OBn 99 99a To a solution of the diol 98 (0.458 g, 0.786 mmol) and a crystal of 4-dimethylamino pyridinein CH2Cl2 (2 mL) under N2 at rt was added dropwise a solution of TBSCl (0.13 mg, 0.863 mmol) and triethylamine (0.12 mL, 0.863 mmol) in CH2Cl2 (6 mL). The reaction mixture was stirred for 5 h and the reaction was quenched by the addition of water. The solvent was removed under reduced pressure and the residue was extracted with CH2Cl2 (3x10mL). The organic layer was washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographyed on siliga gel by FCC (30:70 EtOAc/petrol to 2:98 MeOH/EtOAc). This gave the di-TBS derivative of 99a as
174 Chapter 9 [Experimental for Chapter 2] a yellow syrup (0.074 g, 12%), 99 (0.330 g, 60%) as a yellow viscous oil and unreacted starting material (0.10 g, 22 %).
99: Rf 0.30 (100% EtOAc).
22 [α] D +21 (c 0.7, CHCl3). MS (ESI +ve) m/z 698 (M+H+, 100%). + HRMS (ESI +ve) calculated for C42H56NO6Si (M+H ) 698.3877, found 698.3862. -1 IR υmax (cm ): 3324, 3088, 3057, 3027, 2924, 2852, 1460, 1065. 1H NMR δ 7.35-7.24 (m, 20H, Ar), 4.64 (d, 1H, J 11.5 Hz, CHHPh), 4.60 (d, 1H, J 11.0 Hz, CHHPh), 4.57 (d, 1H, J 12.0 Hz, CHHPh), 4.49 (d, 1H, J 12.0 Hz, CHHPh), 4.46 (d, 2H, J 12.0 Hz, 2xCHHPh), 4.39 (d, 2H, J 11.5 Hz, 2xCHHPh), 4.14 (dd, 1H, J 6.5, 5.5 Hz, H-3), 3.93-3.90 (m, 2H, H-2’ and H-3’), 3.92-3.86 (m, 2H, H-1’ and H-4), 3.71-3.67 (m, 1H, H-4’), 3.66-3.60 (m, 2H, H-4’and H-2), 3.06-3.04 (m, 2H, 2xH-5), 0.90 (s, 9H, t-Bu), 0.06 (s, 3H, CH3), 0.05 (s, 3H, CH3). 13C NMR δ 138.3 (C), 138.2 (C), 138.1 (C), 128.35 (CH), 128.34 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.8 (CH), 127.7 (CH), 127.69 (CH), 127.65 (CH), 127.5
(CH), 79.5 (C-3), 78.5 (C-1’), 78.2 (C-2’), 76.7 (C-4), 74.1 (CH2), 72.9 (CH2), 71.9
(CH2), 71.5 (CH2), 69.0 (C-3’), 63.2 (C-4’), 61.9 (C-2), 48.5 (C-5), 25.9 (C(CH3)3), 18.1
(C), -5.3 (CH3), -5.4 (CH3).
99a: 1H NMR δ 7.29-7.18 (m, 20H, Ar), 4.75 (d, 1H, J 11.5 Hz, CHHPh), 4.66 (d, 1H, J 12.0 Hz, CHHPh), 4.61 (d, 1H, J 11.5 Hz, CHHPh), 4.59 (d, 1H, J 11.0 Hz, CHHPh), 4.56 (d, 1H, J 12.0 Hz, CHHPh), 4.50(d, 1H, J 12.5 Hz, CHHPh), 4.47 (d, 1H, J 11.5 Hz, CHHPh), 4.44 (d, 1H, J 12.0 Hz, CHHPh), 3.99 (app. t, 1H, J 4.5 Hz, H-4), 3.98- 3.94 (m, 2H, H-2’ and H-3’), 3.85 (dd, 1H, J 5.0, 5.0 Hz, H-3’ or H-2), 3.74 (brs, 2H, H- 1 and H-3), 3.72 (dd, 1H, J 10.3, 4.8 Hz, H-4’), 3.56 (dd, 1H, J 10.0, 6.5 Hz, H-4’), 3.40 (m, 1H, H-2), 2.97 (dd, 1H, J 11.3, 4.8 Hz, H-5), 2.90 (dd, 1H, J 11.0, 5.0 Hz, H-5), 0.87
(s, 9H, t-Bu), 0.81 (s, 9H, t-Bu), 0.04 (s, 3H, CH3), 0.02 (s, 3H, CH3), -0.06 (s, 3H,
CH3), -0.07 (s, 3H, CH3). 13C NMR δ 138.9 (C), 138.38 (C), 138.3 (C), 128.28 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH), 127.83 (CH), 127.81 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.34
(CH), 127.3 (CH), 80.3 (CH), 79.8 (CH), 79.3 (CH), 78.6 (CH2), 74.1 (CH2 and CH),
175 Chapter 9 [Experimental for Chapter 2]
73.8 (CH2), 71.8 (CH2), 71.5 (C-3’), 65.0 (C-4’), 62.6 (C-2), 49.1 (C-5), 25.9 (C(CH3)3),
18.3 (C), 18.1 (C), -3.9 (CH3), -4.7 (CH3), -5.4 (CH3), -5.5(CH3).
(1S,2S,3S,6S,7R,7aS)-1,2,6,7-Tetrabenzyloxy-3-((tert- butyldimethylsilyloxy)methyl)-hexahydro-1H-pyrrolizine (100) and (1R,2S,6R,7R,8S,8aS)-1,2,7,8-tetrakis(benzyloxy)-6-(tert-butyldimethylsilyloxy)- octahydroindolizine (100a).
OBn BnO H OBn BnO H OBn 1 7 7a 1 8a BnO OBn BnO 7 5 N 3 3 N 5 OTBS 8 100 OTBS 100a
To a solution of 99 (0.320 g, 0.459 mmol) in pyridine(5 mL) was added triphenylphosphine (0.301 g, 1.148 mmol) and diisopropyl azodicarboxylate (0.23 mL, 1.148 mmol). The mixture was stirred at rt for 3 d. The volatiles were removed in vacuo then satd. CuSO4 solution (10 mL) was added. The reaction mixture was extracted with
CH2Cl2 (3x15 mL). The combined CH2Cl2 extracts were washed with water, dried
(Na2CO3), filtered and then evaporated. FCC (100% petrol to 20:80 EtOAc/petrol) gave a mixture (ca 4 : 1) of the desired pyrrolizidine 100 and the indolizidine product 100a in a combined yield of 92 mg (30%). These cyclized products could be separated by a second, more careful, column chromatographic separation that gave 100 as a yellow 1 viscous oil (47.6 mg), Rf 0.39 (20:80 EtOAc/petrol) in about 90% purity from H NMR analysis. MS (ESI +ve) m/z 680 (M+H+, 100%). + HRMS (ESI +ve) calculated for C42H56NO6Si (M+H ) 680.3771, found 680.3791. 1H NMR δ 7.37-7.17 (m, 20H, Ar), 4.66 (d, 1H, J 12.5 Hz, CHHPh), 4.62 (d, 2H, J 12.0 Hz, 2xCHHPh), 4.54 (d, 1H, J 12.5 Hz, CHHPh), 4.48 (d, 1H, J 12.0 Hz, CHHPh), 4.45 (d, 2H, J 12.0 Hz, 2xCHHPh), 4.41 (d, 1H, J 12.0 Hz, CHHPh), 4.04-4.01 (m, 1H, H-6), 3.94 (app. t, 1H, J 4.3 Hz, H-2), 3.85 (app. t, 1H, J 3.3 Hz, H-1), 3.74 (dd, 1H, J 7.8, 4.3 Hz, H-7), 3.69 (dd, 1H, J 7.5, 3.0 Hz, H-7a), 363 (dd, 1H, J 10.0, 7.0 Hz, H-8), 3.56 (dd,
176 Chapter 9 [Experimental for Chapter 2]
1H, J 10.0, 6.5 Hz, H-8), 3.41 (br d, 1H, J 12.0 Hz, H-5α), 2.88-2.83 (m, 1H, H-3, H-
5β), 0.86 (s, 9H, t-Bu), 0.01 (s, 3H, CH3), 0.00 (s, 3H, CH3). 13C NMR δ 138.3 (2xC), 138.2 (C), 137.9 (C), 128.3 (CH), 128.28 (CH), 128.26 (CH), 128.2 (CH), 128.0 (CH), 127.8 (CH), 127.6 (CH), 127.58 (CH), 127.5 (CH), 127.49
(CH), 127.4 (CH), 85.8 (C-1 and C-2), 81.5 (C-7), 76.7 (C-6), 72.1 (CH2), 71.8 (CH2),
71.6 (CH2), 71.3 (CH2), 71.2 (C-3), 71.0 (C-7a), 65.4 (C-8), 57.8 (C-5), 25.8 (C(CH3)3),
18.2 (C), -5.3 (CH3), -5.4 (CH3).
100a: 1H NMR δ 7.32-7.20 (m, 20H, Ar), 4.89 (d, 1H, J 11.0 Hz, CHHPh), 4.83 (d, 1H, J 11.0 Hz, CHHPh), 4.78 (d, 1H, J 11.0 Hz, CHHPh), 4.68 (d, 1H, J 11.0 Hz, CHHPh), 4.63 (d, 1H, J 11.0 Hz, CHHPh), 4.54 (s, 2H, 2xCHHPh), 4.52 (d, 1H, J 11.5 Hz, CHHPh), 4.01 (dd, 1H, J 7.0, 7.0 Hz, H-2), 3.80 (app. t, 1H, J 6.5 Hz, H-1), 3.76 (m, 1H, H-6), 3.40 (app. t, 1H, J 8.8 Hz, H-7), 3.32 (app. t, 1H, J 9.3 Hz, H-8), 3.22 (dd, 1H, J 8.5, 6.5 Hz, H-3), 2.97 (dd, 1H, J 10.8, 5.3 Hz, H-5), 2.56-2.51 (m, 2H, H-3 and H-8a),
2.20 (app. t, 1H, J 10.5 Hz, H-5), 0.89 (s, 9H, t-Bu), 0.07 (s, 3H, CH3), 0.05 (s, 3H,
CH3). 13C NMR δ 138.8 (C), 138.7 (C), 138.4 (C), 138.3 (C), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.3 (CH), 127.26 (CH), 127.2
(CH), 88.2 (C-7), 82.0 (C-8), 80.4 (C-1), 76.1 (C-2), 75.6 (CH2), 74.2 (CH2), 72.3 (C-6),
72.2 (CH2), 71.9 (CH2), 70.8 (C-8a), 57.4 (C-3), 57.0 (C-5), 25.8 (C(CH3)3), 17.9 (C), -
4.5 (2xCH3).
((1S,2S,3S,6S,7R,7aS)-1,2,6,7-Tetrabenzyloxy-hexahydro-1H-pyrrolizin-3-yl)- methanol (101).
BnO H OBn To a solution of 100 (47.6 mg, 0.07 mmol) in MeOH (2 mL)
7 1 BnO 7a OBn was added dropwise conc. HCl solution (0.5 mL) and the 5 N 3 mixture was stirred at rt for 5 h. The mixture was basified at 8 OH 0 101 0 C with aqueous NH3 solution (28%). The mixture was extracted with EtOAc, dried (Na2CO3), evaporated and purified by FCC (50:50 EtOAc/petrol) to give 101 (26.0 mg, 66%) as a pale yellow viscous oil.
177 Chapter 9 [Experimental for Chapter 2]
Rf 0.11 (50:50 EtOAc/petrol).
23 [α] D +34 (c 1.3, CHCl3). MS (ESI +ve) m/z 566 (M+H+, 60%). + HRMS (ESI +ve) calculated for C36H40NO5 (M+H ) 566.2906, found 566.2915. 1H NMR δ 7.37-7.20 (m, 20H, Ar), 4.66 (d, 2H, J 12.5 Hz, 2xCHHPh), 4.64 (d, 1H, J 12.0 Hz, CHHPh), 4.59 (d, 1H, J 10.9 Hz, CHHPh), 4.57 (d, 1H, J 12.0 Hz, CHHPh), 4.51 (d, 1H, J 11.5 Hz, CHHPh), 4.46 (d, 2H, J 12.5 Hz, 2xCHHPh), 4.06-4.04 (m, 2H, H-6 and H-2), 3.85 (app. t, 1H, J 4.5 Hz H-1), 3.76 (dd, 1H, J 7.0, 4.5 Hz, H-7), 3.68 (dd, 1H, J 7.0, 4.5 Hz, H-7a), 3.54 (dd, 1H, J 11.0, 4.5 Hz, H-8), 3.49 (dd, 1H, J 11.0, 4.0 Hz, H-8), 3.33 (dd, 1H, J 12.0, 2.5 Hz, H-5α), 2.82 (dd, 1H, J 10.0, 4.5 Hz, H-3), 2.74 (dd, 1H, J 12.0, 4.5 Hz, H-5β). 13C NMR δ 138.0 (2xC), 137.9 (C), 128.4 (CH), 128.38 (CH), 128.34 (CH), 127.9 (CH), 127.87 (CH), 127.8 (CH), 127.78 (CH), 127.7 (CH), 127.68 (CH), 127.6 (CH), 127.5
(CH), 86.0 (C-1), 84.7 (C-2), 81.9 (C-7), 76.8 (C-6), 72.5 (CH2), 71.8 (CH2), 71.6 (CH2),
71.4 (CH2), 71.3 (C-3), 70.4 (C-7a), 60.5 (C-8), 56.3 (C-5).
(1S,2S,3S,6S,7R,7aS)-Hexahydro-3-(hydroxymethyl)-1H-pyrrolizine-1,2,6,7-tetraol (ent-78). To a solution of 101 (26.0 mg, 0.046 mmol) in EtOAc (0.5 HO H OH mL) and MeOH (0.5 mL) was added PdCl2 (12.2 mg, 0.069 7 1 HO 7a OH 5 N 3 mmol). The mixture was stirred at rt under an atmosphere of
8 OH H2 (balloon) for 3 h. The mixture was filtered through a celite ent-78 pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1.5 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave (+)-uniflorine A ent-78 (7.0 mg, 74%) as a brown foamy solid.
22 142 [α] D +6.6 (c 0.35, H2O), lit. [α]D -4.4 (c 1.2, H2O). MS (ESI +ve) m/z 206 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO5 (M+H ) 206.1028, found 206.1023. -1 IR υmax (cm ): 3288, 2924, 1339, 1212, 1104, 1040.
178 Chapter 9 [Experimental for Chapter 2]
1 H NMR (D2O) δ 4.34 (dt, 1H, J5α,6 = J5β,6 = J6,7 = 4.8 Hz, H-6), 4.17 (t, 1H, J6,7 = J6,7a =
4.5 Hz, H-7), 3.92 (t, 1H, J1,2 = J1,7a = 7.5 Hz, H-1), 3.79 (t, 1H, J1,2 = J2,3 = 8.5 Hz, H-2),
3.76 (dd, 1H, J8,8’ = 11.8, J3,8’ = 3.8 Hz, H-8’), 3.61 (dd, 1H, J8,8’ = 11.5, J3,8 = 6.5 Hz, H-
8), 3.12 (dd, 1H, J1,7a = 7.5, J7,7a = 5.0 Hz, H-7a), 3.02 (dd, 1H, J5α,5β = 11.8, J5β,6 = 5.8
Hz, H-5β), 2.96 (dd, 1H, J5α,5β = 12.3, J5α,6 = 5.3 Hz, H-5α), 2.74 (m, 1H, H-3). 13 C NMR (D2O) δ 79.1 (C-1), 77.8 (C-2), 76.0 (C-7), 72.1 (C-6), 71.5 (C-7a), 70.3 (C- 3), 63.2 (C-8), 57.8 (C-5).
9.2.2.2 Total synthesis of (-) uniflorine A from L-xylose tert-Butyl allyl((2S,3S,4R,5R,E)-1,2-O-(1-methylethylidene)-3,4-dihydroxy-
7-phenylpent-6-en-5-yl)carbamate (102).
To a solution of 36 (4.957 g, 12.61 mmol) in anhydrous OH O
Ph 5 3 1 O acetone (70 mL) was added 2,2-dimethoxypropane (1.86 mL, 7 1' N HO 15.13 mmol) and pyridinium p-toluenesulfonate (0.317 g, 3' Boc 102 1.261 mmol). The reaction mixture was stirred under an atmosphere of N2 for 22 h, followed by the evaporation of all volatiles in vacuo to give a brown oil. The residue was purified by FCC (30:70 to 50:50 EtOAc/petrol) to give 102 (3.485 g, 64%) as a white solid, foamy solid. A small amount of another regioisomer of 102 (102a) was also isolated (1.759 g, 19%).
22 [α] D -36 (c 6.5, CHCl3).
Compound 102 and 102a had the same Rf, MS, IR and NMR spectroscopic data as reported for 94 and 94a.
(2R)-tert-Butyl 2-((1R,2S,3S)-1,2,3,4-tetrahydroxybutyl-3,4-O-(1- methylethylidene))-2,5-dihydro-1H-pyrrole-1-carboxylate (103).
OH O H To a solution of 102 (5.553 g, 12.82 mmol) in anhydrous CH2Cl2 O 1' 3' 2 (260 mL) was added Grubbs’ I catalyst (1.055 g, 1.282 mmol). 5 N HO Boc The reaction mixture was stirred and heated at reflux for 18 h
103 under an atmosphere of N2 followed by the removal of all
179 Chapter 9 [Experimental for Chapter 2] volatiles in vacuo. The residue was purified by FCC (50:50 to 70:30 EtOAc/petrol) to give 103 as a dark brown viscous oil (4.09 g, 97%).
21 [α] D +125 (c 4.5, CHCl3).
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (-)-95.
(2R,3S,4R)-tert-Butyl 2-((1R,2S,3S)-1,2,3,4-tetrahydroxybutyl-3,4-O-(1- methylethylidene))-3,4-dihydroxypyrrolidine-1-carboxylate (104).
OH O To a solution of 103 (4.00 g, 12.16 mmol) in acetone (60 mL) HO H O 1' 3 ' and water (60 mL) was added potassium osmate•dihydrate 2 HO 5 N OH (223.7 mg, 0.608 mmol) and 4-morpholine-N-oxide (2.987 g, Boc 104 25.53 mmol). The reaction mixture was stirred for 18 h at rt and evaporated to give a dark brown oil which was purified by FCC (100% EtOAc to 4:96 MeOH/EtOAc) to give 104 as a brown foamy solid (3.174 g, 72%).
22 [α] D +32 (c 4.9, CHCl3).
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (-)-96.
(2R,3S,4R)-tert-Butyl 3,4-bis(benzyloxy)-2-((1R,2S,3S)- 1,2-bis(benzyloxy)-3,4-O-(1- methylethylidene))- 3,4-dihydroxybutylpyrrolidine-1-carboxylate (105).
OBn O To a solution of 104 (3.077 g, 8.477 mmol) in dry THF (85 BnO H O 1' 3 ' mL) was added n-Bu4NI (313.1 mg, 0.848 mmol) and BnBr BnO 2 5 N OBn Boc (8.07 mL, 67.81 mmol) follow by NaH (2.441 g, 50.86 o 105 mmol, 50 % in mineral oil) at 0 C. After H2 evolution had ceased (15 min) the reaction mixture was stirred at rt for 24 h. MeOH (50 mL) was then added followed by evaporation of all volatiles in vacuo. The residue was dissolved in EtOAc and filtered through celite, followed by further washings of the solids with EtOAc. The solvent was evaporated and the residue was purified by FCC (10:90 to 15:85 EtOAc/petrol) to give 105 as a pale yellow syrup (5.89 g, 96%).
23 [α] D -50 (c 5.4, CHCl3).
180 Chapter 9 [Experimental for Chapter 2]
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (+)-97.
1-[(2S,3R,4R)-3,4-Bisbenzyloxy-2-pyrrolidinyl]-(1R,2S,3R)-1,2-dibenzyloxy-butane- 3,4-diol (106).
OBn OH To a solution of 105 (5.78 g, 7.994 mmol) in MeOH (200 BnO H OH 1' 3 ' mL) was added dropwise conc. HCl solution (40 mL) and BnO 2 5 NH OBn the mixture was stirred at rt for 18 h. The reaction mixture 106 0 was basified at 0 C with aqueous NH3 solution (28%). The mixture was extracted with EtOAc (3x60 mL), dried (Na2CO3), evaporated and purified by FCC (100% EtOAc to 93:5:2 EtOAc/MeOH/NH3) to give 106 (3.788 g, 81%) as a yellow viscous oil.
22 [α] D -27 (c 3.7, CHCl3).
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (+)-98.
1-[(2S,3R,4R)-3,4-Bisibenzyloxy-2-pyrrolidinyl]-(1R,2S,3R)-1,2-dibenzyloxy-4-(tert- butyldimethylsilyloxy)-butan-3-ol (107).
OBn OH BnO H To a solution of the diol 106 (0.312 g, 0.535 mmol), OTBS 1' 3 ' BnO 2 imidazole (0.77 mg, 1.123 mmol) and 4- 5 NH OBn dimethylaminopyridine (6.5 mg, 0.053 mmol) in THF (6 107 mL) under N2 at rt was added TBSCl (0.97 g, 0.642 mmol). The reaction mixture was stirred for 2 d and the reaction was quenched by the addition of water. The solvent was removed under reduced pressure and the residue was extracted with CH2Cl2 (3x20 mL). The combined CH2Cl2 extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (30:70 EtOAc/petrol to 2:98 MeOH /EtOAc). This gave 107 (0.316 g, 85%) as a yellow viscous oil.
22 [α] D -17 (c 4.6, CHCl3).
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (+)-99.
181 Chapter 9 [Experimental for Chapter 2]
(1R,2R,3R,6R,7S,7aR)-1,2,6,7-Tetrabenzyloxy-3-((tert- butyldimethylsilyloxy)methyl)-hexahydro-1H-pyrrolizine (108).
BnO OBn H To a solution of 107 (0.792 g, 1.136 mmol) in pyridine(11 mL) 7 1 BnO 7a OBn was added triphenylphosphine (0.301 g, 1.148 mmol), 5 N 3
8 OTBS triethylamine⋅hydrochloride (0.156 g, 1.136 mmol) and 108 diisopropyl azodicarboxylate (0.56 mL, 2.841 mmol). The mixture was stirred at rt for 3 d. The volatiles were removed in vacuo then satd. CuSO4 solution (20 mL) was added. The reaction mixture was extracted with CH2Cl2 (3x25 mL). The combined CH2Cl2 extracts were washed with satd. CuSO4 solution (20 mL) and water (20 mL), dried (Na2CO3), filtered and then evaporated. FCC (100% petrol to 20:80 EtOAc/petrol) gave 108 as a yellow viscous oil (0.587 g, 76%).
20 [α] D -34 (c 0.4, CHCl3). -1 IR υmax (cm ): 3070, 3040, 2924, 2852, 1454, 1120, 1097.
This compound had the same Rf, MS and NMR spectroscopic data as reported for (+)- 100.
((1R,2R,3R,6R,7S,7aR)-1,2,6,7-Tetrabenzyloxy-hexahydro-1H-pyrrolizin-3-yl) -methanol (109).
To a solution of 108 (1.417 g, 2.087 mmol) in MeOH (50 mL) BnOH OBn
7 7a 1 was added dropwise conc. HCl solution (12.5 mL) and the BnO OBn 5 N 3 mixture was stirred at rt for 18 h. The mixture was basified at 0 8 OH 0 C with aqueous NH3 solution (28%). The mixture was 109 extracted with EtOAc (3x40 mL), dried (Na2CO3), evaporated and purified by FCC (50:50 EtOAc/petrol) to give 109 (1.058 g, 90%) as a pale yellow viscous oil. Rf 0.11 (50:50 EtOAc/petrol).
20 [α] D -35 (c 1.3, CHCl3). -1 IR υmax (cm ): 3446, 3050, 2893, 2858, 1449, 1107, 1097.
This compound had the same Rf, MS and NMR spectroscopic data as reported for (+)- 101.
182 Chapter 9 [Experimental for Chapter 3]
(1R,2R,3R,6R,7S,7aR)-Hexahydro-3-(hydroxymethyl)-1H-pyrrolizine-1,2,6,7- tetraol (uniflorine A (78)).
HO OH H To a solution of 109 (0.636 mg, 1.126 mmol) in MeOH (12 7 7a 1 HO OH 5 N 3 mL) was added PdCl2 (0.300 g, 1.690 mmol). The mixture was stirred at rt under an atmosphere of H (balloon) for 24 h. The 8 OH 2 78 mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (3 mL) and applied to a column of Amberlyst A-26 (OH-) resin (7 cm). Elution with water followed by evaporation in vacuo gave uniflorine A 78
(0.201 g, 87%) as a white solid. mp. 163.2-164.8 °C, lit.142 mp. 174-178 °C.
23 142 [α] D -3.7 (c 1.2, H2O), lit. [α]D -4.4 (c 1.2, H2O).
This compound had the same Rf, MS, IR and NMR spectroscopic data as reported for (+) ent-78.
9.3 Chapter 3 Experimental for the total synthesis of casuarine
(R)-tert-Butyl 2-((1R,2S)-1,2-bis(benzyloxy)-2-((S)-2,2-dimethyl-1,3-dioxolan-4- yl)ethyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (157).
OBn O To a solution of the diol 103 (8.55 g, 25.84 mmol) in dry THF H O ' 1' 3 2 (260 mL) was added n-Bu4NI (954.3 mg, 2.58 mmol) and BnBr 5 NBoc OBn (9.27 mL, 78.0 mmol) follow by NaH (3.74 g, 78.0 mmol, 50 % in 157 o mineral oil) at 0 C. After H2 evolution had ceased (15 min) the reaction mixture was stirred at rt for 18 h. MeOH (50 mL) was then added followed by evaporation of all volatiles in vacuo. The residue was dissolved in EtOAc and filtered through celite, followed by further washings of the solids with EtOAc. The solvent was evaporated and the residue was purified by FCC (10:90 to 40:60 EtOAc/petrol) to give
157 as a yellow syrup (12.21 g, 92%). Rf 0.21 (15:85 EtOAc/petrol).
22 [α] D +56 (c 1.4, CHCl3). MS (ESI +ve) m/z 510 (M+H+, 30%).
183 Chapter 9 [Experimental for Chapter 3]
+ HRMS (ESI +ve) calculated for C30H40NO6 (M+H ) 510.2856, found 510.2854. -1 IR υmax (cm ): 2980, 2929, 1696, 1393, 1107, 1060. 1H NMR δ (major rotamer) 7.36-7.22 (m, 10H, Ar), 5.88 (app. t, 2H, J 8.5 Hz, H-3 and H-4), 4.81 (d, 1H, J 11.5 Hz, CHHPh), 4.74 (d, 1H, J 11.5 Hz, CHHPh), 4.55 (d, 1H, J 11.5 Hz, CHHPh), 4.50 (d, 1H, J 5.0 Hz, H-2), 4.35-4.25 (m, 2H, H-1’ or H-2’ and CHHPh), 4.16-3.98 (m, 2H, 2xH-5), 3.91 (d, 1H, J 4.5 Hz, H-1’ or H-2’), 3.60-3.42 (m,
3H, 2xH-4’ and H-3’), 1.45 (s, 9H, t-Bu), 1.42 (s, 3H, CH3), 1.35 (s, 3H, CH3). 13C NMR δ (major rotamer) 153.9 (CO), 138.1 (C), 137.6(C), 128.48 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 127.9 (CH), 127.7 (CH), 127.6 (C-3 or C-4), 126.3 (C-3 or C-
4), 109.1 (C), 80.8 (C-3’), 79.9 (C), 79.3 (C-1’ or C-2’), 77.1 (C-1’ or C-2’), 74.1 (CH2),
73.7 (CH2), 67.5 (C-2), 65.5 (C-4’), 53.2 (C-5), 28.5 (C(CH3)3), 26.6 (CH3), 25.5 (CH3). 13C NMR δ (minor rotamer) 154.2, 138.4, 137.9, 128.42, 128.39 128.3, 128.1, 127.8, 127.5, 126.9, 125.7, 109.0, 81.4, 79.7, 79.5, 77.5, 73.9, 73.5, 67.9, 65.8, 53.3, 28.4, 26.6, 25.6.
(2S,3R,4R)-3,4-Bis(benzyloxy)-4-((R)-2,5-dihydro-1H-pyrrol-2-yl)butane-1,2-diol (158).
OBn OH To a solution of 157 (10.93 g, 21.47 mmol) in MeOH (500 mL) H OH 1' 3' was added dropwise conc. HCl solution (95 mL) and the mixture 2 5 NH OBn was stirred at rt for 30 h. The reaction mixture was basified at 0 0 158 C with aqueous NH3 solution (28%). The mixture was extracted with EtOAc (3x150 mL), the combined EtOAc extracts were dried (Na2CO3), evaporated and purified by FCC (100% EtOAc to 8:2:1 EtOAc/MeOH/NH3) to give 158
(6.0 g, 76%) as a brown foamy solid. Rf 0.34 (9:0.8:0.2 EtOAc/MeOH/NH3).
21 [α] D +122 (c 3.2, CHCl3). MS (ESI +ve) m/z 370 (M+H+, 100%). + HRMS (ESI +ve) calculated for C22H28NO4 (M+H ) 370.2018, found 370.2009. -1 IR υmax (cm ): 3390, 3288, 3062, 3027, 2955, 2909, 1078, 1059. 1H NMR δ 7.3.4-7.27 (m, 10H, Ar), 5.97 (dd, 1H, J 6.0, 1.5 Hz, H-4), 5.89 (dd, 1H, J 6.0, 1.5 Hz, H-3), 4.73 (d, 1H, J 11.5 Hz, CHHPh), 4.67 (d, 1H, J 11.5 Hz, CHHPh), 4.60 (d, 1H, J 11.0 Hz, CHHPh), 4.53 (d, 1H, J 11.0 Hz, CHHPh), 4.27 (brs, 1H, H-2),
184 Chapter 9 [Experimental for Chapter 3]
3.90-3.88 (m, 1H, H-3’), 3.76-3.70 (m, 3H, 2xH-5 and H-4’), 3.65-3.63 (m, 1H, H-2’), 3.61-3.56 (m, 2H, H-1’ and H-4’). 13C NMR δ 138.0 (C), 137.9 (C), 129.9 (C-4), 128.8 (C-3), 128.42 (CH), 128.4 (CH),
128.2 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 81.6 (C-1’), 80.9 (C-2’), 74.4 (CH2),
74.2 (CH2), 70.6 (C-3’), 66.4 (C-2), 63.7 (C-4’), 52.7 (C-5).
(2S,3R,4R)-3,4-Bis(benzyloxy)-1-(tert-butyldimethylsilyloxy)-4-((R)-2,5-dihydro-1H- pyrrol-2-yl)butan-2-ol (159).
OBn OH To a solution of the diol 158 (0.302 g, 0.816 mmol) and a H OTBS 1' 3' 2 crystal of 4-dimethylaminopyridine in THF (8 mL) under N2 at 5 NH OBn rt was added imidazole (0.121 g, 1.776 mmol) and TBSCl 159 (0.153 g, 1.02 mmol). The reaction mixture was stirred for 24 h and the reaction was quenched by the addition of water. The solvent was removed under reduced pressure and the residue was extracted with EtOAc (3x10 mL). The combined
EtOAc extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (100% EtOAc to 10:2:1
EtOAc/MeOH/NH3) to give 159 (0.320 g, 81%) as a brown viscous oil. Rf 0.65
(9:0.8:0.2 EtOAc/MeOH/NH3).
21 [α] D +93 (c 2.3, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2906. -1 IR υmax (cm ): 3390, 3288, 3062, 3021, 2955, 2929, 1077, 1062. 1H NMR δ 7.36-7.27 (m, 10H, Ar), 5.98 (dd, 1H, J 5.5, 1.5 Hz, H-4), 5.92 (dd, 1H, J 6.0, 2.0 Hz, H-3), 4.75 (d, 1H, J 12.0 Hz, CHHPh), 4.73 (d, 1H, J 10.0 Hz, CHHPh), 4.69 (d, 1H, J 11.0 Hz, CHHPh), 4.52 (d, 1H, J 11.0 Hz, CHHPh), 4.21-4.18 (m, 1H, H- 2), 3.88-3.85 (m, 2H, H-2’ and H-3’), 3.74-3.70 (m, 4H, 2xH-5 and 2xH-4’), 3.57 (app. t, 1H, J 7.0 Hz, H-1’), 0.91 (s, 9H, t-Bu), 0.08 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR δ 138.6 (C), 138.2 (C), 130.2 (C-4), 129.4 (C-3), 128.3 (CH), 128.2 (CH),
128.0 (CH), 127.8 (CH), 127.6 (CH), 127.5 (CH), 82.6 (C-1’), 79.8 (C-2’), 74.6 (CH2),
185 Chapter 9 [Experimental for Chapter 3]
74.0 (CH2), 70.3 (C-3’), 66.2 (C-2), 63.2 (C-4’), 53.1 (C-5), 25.9 (C(CH3)3), 18.1 (C), -
5.4 (CH3), -5.5 (CH3).
(R)-(9H-Fluoren-9-yl)methyl 2-((1R,2R,3S)-1,2-bis(benzyloxy)-4-(tert-butyl- dimethylsilyloxy)-3-hydroxybutyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (160).
OBn OH To a solution of 159 (6.05 g, 0.013 mol) in THF (125 mL) and H OTBS 1' 3' 2 satd. Na2CO3 solution (60 mL) was added 9-fluorenylmethyl 5 N OBn o Fmoc chloroformate (3.89 g, 15.03 mmol) at 0 C. The reaction o 160 mixture was stirred at 0 C for 3 h. Water (20 mL) was added and the solvent was removed under reduced pressure and the residue was extracted with
CH2Cl2 (3x70 mL). The combined CH2Cl2 extracts were washed with brine, dried
(Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (10:90 to 30:70 EtOAc/petrol) to give 160 (8.31 g, 94%) as a colourless viscous oil. Rf 0.47 (20:80 EtOAc/petrol).
24 [α] D +125 (c 2.0, CHCl3). MS (ESI +ve) m/z 706 (M+H+, 20%). + HRMS (ESI +ve) calculated for C43H52NO6Si (M+H ) 706.3564, found 706.3537. -1 IR υmax (cm ): 3061, 3028, 2945, 2924, 1700, 1413, 1107. 1H NMR δ (major rotamer) 7.78-7.59 (m, 4H, Ar), 7.42-7.16 (m, 14H, Ar), 5.97-5.95 (m, 1H, H-3), 5.92-5.89 (m, 1H, H-4), 4.90 (d, 2H, J 11.0 Hz, 2xCHHPh), 4.91-4.89 (m, 1H, H-2), 4.67-4.63 (m, 1H, CHHPh), 4.47 (d, 1H, J 12.0 Hz, CHHPh), 4.45 (d, 1H, J
8.0 Hz, H-1’ or H-2’), 4.39 (dd, 2H, J 7.0, 2.3 Hz, CH2 (Fmoc)), 4.26-4.20 (m, 1H, CH (Fmoc)), 4.26-4.07 (m, 2H, 2xH-5 ), 3.88 (dd, 1H, J 13.5, 7.5 Hz, H-3’), 3.77 (d, 1H, J
7.5 Hz, H-1’ or H-2’), 0.90 (s, 9H, t-Bu), 0.07 (s, 3H, CH3), 0.06 (s, 3H, CH3). 13C NMR δ (major rotamer) 154.3 (CO), 144.0 (C), 143.9 (C), 141.3 (C), 141.2 (C), 138.4 (C), 138.2 (C), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH), 127.6 (CH),
127.5 (CH), 126.9 (CH), 125.0 (CH), 119.9 (CH), 78.3 (C-1’), 77.8 (C-2’), 74.7 (CH2),
74.3 (CH2), 70.8 (C-3’), 66.9 (CH2 (Fmoc)), 66.2 (C-2), 63.6 (C-4’), 53.4 (C-5), 47.2
(CH (Fmoc)), 25.8 (C(CH3)3), 18.1 (C), -5.4 (CH3), -5.5 (CH3).
186 Chapter 9 [Experimental for Chapter 3]
13C NMR δ (minor rotamer) 154.3, 144.0, 143.8, 141.3, 141.2, 138.3, 138.2, 128.2, 127.8, 127.7, 127.6, 126.7, 126.5, 125.5, 124.7, 119.9, 80.2, 78.6, 74.9, 74.8, 71.0, 65.9, 65.5, 63.5, 54.2, 47.7, 25.7, 18.0, -5.4.
(1S,2S,5R)-(9H-Fluoren-9-yl)methyl 2-((1R,2R,3S)-1,2-bis(benzyloxy)-4-(tert- butyldimethylsilyloxy)-3-hydroxybutyl)-6-oxa-3-azabicyclo[3.1.0]hexane-3- carboxylate (161).
OBn OH To a solution of the olefin 160 (2.37 g, 3.37 mmol) in MeCN H OTBS -4 O 1' 3' (35 mL) was added Na2EDTA (13.5 mL, 4 x 10 M) and 2 5 N OBn CF3C(O)CH3 (6.8 mL, 7.60 mmol). The reaction was chilled Fmoc to 0 oC before the portionwise addition of a mixture of 161
NaHCO3 (4.24 g, 50.47 mmol) and oxone (4.14 g, 6.73 mmol) over 15 min. After stirring for 2 h at 0 oC, the mixture was poured into water followed by removed of the volatiles under reduced pressure. The residue was extracted with CH2Cl2 (3x40 mL) and the combined organic extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (10:90 to 20:80 EtOAc/petrol) to give 161 (1.95 g, 81%) as a pale yellow oil. Rf 0.42 (20:80 EtOAc/petrol).
25 [α] D +99 (c 1.1, CHCl3). MS (ESI +ve) m/z 744 (M + Na+, 100%). + HRMS (ESI +ve) calculated for C43H51NO7SiNa (M+Na ) 744.3333, found 744.3360. -1 IR υmax (cm ): 3062, 2945, 2924, 2858, 1700, 1454, 1110. 1H NMR δ (major rotamer) 7.76-7.54 (m, 4H, Ar), 7.41-7.16 (m, 14H, Ar), 4.86 (d, 1H, J 10.5 Hz, CHHPh), 4.67 (d, 1H, J 12.0 Hz, CHHPh), 4.64 (d, 1H, J 11.5 Hz, CHHPh),
4.36 (d, 1H, J 11.0 Hz, CHHPh), 4.36-4.30 (m, 3H, CH2 (Fmoc) and H-2), 4.26 (brs, 1H, H-1’), 4.19-4.15 (m, 1H, CH (Fmoc)), 3.91-3.86 (m, 1H, H-3’), 3.86-3.80 (m, 2H, H-2’ and H-3), 3.74-3.67 (m, 2H, H-4’ and H-5), 3.63 (d, 1H, J 2.0 Hz, H-4), 3.59-3.53 (m,
1H, H-4’), 3.24-3.20 (m, 1H, H-5), 0.88 (s, 9H, t-Bu), 0.04 (s, 3H, CH3), 0.03 (s, 3H,
CH3). 13C NMR δ (major rotamer) 154.9 (CO), 143.7 (C), 141.3 (C), 138.0 (C), 137.8 (C), 128.7 (CH), 128.2 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), 127.1 (CH), 127.0 (CH),
187 Chapter 9 [Experimental for Chapter 3]
125.0 (CH), 124.9 (CH), 119.9 (CH), 79.1 (C-1’), 77.2 (C-2’), 74.9 (CH2), 74.4 (CH2),
70.6 (C-3’), 67.1 (CH2 (Fmoc)), 63.5 (C-4’), 60.1 (C-2), 56.3 (C-3), 55.6 (C-4), 47.8 (C-
5), 47.1 (CH (Fmoc)), 25.8 (C(CH3)3), 18.1 (C), -5.4 (CH3), -5.5 (CH3). 13C NMR δ (minor rotamer) 155.0, 144.0, 141.2, 137.9, 137.7, 127.8, 127.7, 127.69, 127.64, 127.63, 127.5, 127.4, 125.0, 124.7, 120.0, 80.6, 78.0, 75.0, 74.8, 70.8, 66.2, 63.4, 59.8, 56.4, 54.9, 48.2, 47.6, 25.7, 18.07, -5.45, -5.48.
(2S,3R,4R)-4-((1S,2S,5R)-6-Oxa-3-aza-bicyclo[3.1.0]hexan-2-yl)-3,4-bis(benzyloxy)- 1-(tert-butyldimethylsilyloxy)butan-2-ol (164).
OBn OH H To a solution of 161 (0.300 g, 0.416 mmol) in MeCN (5mL) O OTBS 1' 3' 2 was added piperidine (0.08 mL, 0.832 mmol). The reaction 5 NH OBn was stirring for 2 h at rt, the volatiles were removed under 164 reduced pressure and the residue was purified by FCC (40:60 EtOAc/petrol to EtOAc) to give a mixture of 164 as a yellow oil (179.2 mg, 83%), Rf 0.40 (EtOAc).
24 [α] D +65 (c 1.2, CHCl3). MS (ESI +ve) m/z 500 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO5Si (M+H ) 500.2832, found 500.2844. -1 IR υmax (cm ): 3344, 3062, 3032, 2950, 2924, 2852, 1454, 1250, 1093. 1H NMR δ 7.35-7.26 (m, 10H, Ar), 4.81 (d, 1H, J 11.0 Hz, CHHPh), 4.80 (d, 1H, J 11.0 Hz, CHHPh), 4.77 (d, 1H, J 11.0 Hz, CHHPh), 4.55 (d, 1H, J 11.5 Hz, CHHPh), 3.82 (app. t, 1H, J 7.5 Hz, H-2’ or H-1’), 3.79 (app. t, 1H, J 9.0 Hz, H-3’), 3.76 (app. t, 1H, J 6.0 Hz, H-4’), 3.74 (d, 1H, J 5.0 Hz, H-4’), 3.73 (d, 1H, J 5.0 Hz, H-3), 3.53 (dd, 1H, J 8.5, 7.5 Hz, H-2’or H-1’), 3.37 (brs, 1H, H-4), 3.36 (d, 1H, J 12.5 Hz, H-3), 3.04 (d, 1H,
J 13.5 Hz, H-5), 2.75 (d, 1H, J 13.0 Hz, H-5), 0.90 (s, 9H, t-Bu), 0.07 (s, 3H, CH3), 0.06
(s, 3H, CH3). 13C NMR δ 138.5 (C), 137.8 (C), 128.4 (CH), 128.3 (CH), 128.1 (CH), 127.9 (CH),
127.8 (CH), 127.5 (CH), 82.0 (C-1’or C-2’), 80.4 (C-1’ or C-2’), 75.1 (CH2), 74.8
(CH2), 70.6 (C-3’), 63.0 (C-4’), 59.3 (C-2), 57.3 (C-3), 55.5 (C-4), 46.4 (C-5), 25.9
(C(CH3)3), 18.2 (C), -5.3 (CH3), -5.4 (CH3).
188 Chapter 9 [Experimental for Chapter 3]
(1S,2S,5R)-(9H-Fluoren-9-yl)methyl 2-((1R,2S,3S)-1,2-bis(benzyloxy)-4-(tert- butyldimethylsilyloxy)-3-(methylsulfonyloxy)butyl)-6-oxa-3- azabicyclo[3.1.0]hexane-3-carboxylate (162).
OBn OMs To a solution of 161 (0.414 g, 0.574 mmol) in anhydrous H O OTBS 1' 3' 2 CH2Cl2 (6 mL) was added anhydrous Et3N (0.24 mL, 1.723 5 N OBn Fmoc mmol) and methanesulfonyl chloride (0.089 mL, 1.148 o 162 mmol). The reaction mixture was stirred at 0 C under an atmosphere of N2 for 3 h, followed by the evaporation of all volatiles in vacuo. Water
(20 mL) was added and the residue was extracted with CH2Cl2 (3x20 mL). The combined organic extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (10:90 to 30:70
EtOAc/petrol) to give 162 (0.433 g, 94%) as a pale yellow oil. Rf 0.5 (30:70 EtOAc/petrol).
25 [α] D +64 (c 1.1, CHCl3) MS (ESI +ve) m/z 822 (M + Na+, 100%). + HRMS (ESI +ve) calculated for C44H54NO9SSi (M+H ) 800.3289, found 800.3273. -1 IR υmax (cm ): 2950, 2924, 2888, 2852, 1695, 1360, 1328, 1175, 1110. 1H NMR δ (major rotamer) 7.70-7.66 (m, 2H, Ar), 7.45 (app. t, 2H, J 6.8 Hz, Ar), 7.35- 7.11 (m, 14H, Ar), 4.76-4.73 (m, 1H, H-3’), 4.64 (d, 1H, J 10.5 Hz, CHHPh), 4.64-4.61
(m, 2H, 2xCHHPh), 4.34 (d, 1H, J 11.5 Hz, CHHPh), 4.31-4.29 (m, 2H, CH2 (Fmoc)), 4.16 (brs, 1H, H-2), 4.13 (app. t, 1H, J 7.0 Hz, CH (Fmoc)), 4.02-4.00 (m, 2H, H-1’ and H-2’), 3.97-3.94 (m, 2H, 2x H-4’), 3.74-3.72 (m, 1H, H-3), 3.68 (d, 1H, J 12.0 Hz, H-5),
3.58-3.56 (m, 1H, H-4), 3.20 (d, 1H, J 13.0 Hz, H-5), 3.04 (s, 3H, CH3 (Ms)), 0.82 (s,
9H, t-Bu), 0.04 (s, 6H, CH3). 13C NMR δ (major rotamer) 154.9 (CO), 144.0 (C), 143.7 (C), 141.3 (C), 141.2 (C), 137.9 (C), 137.5 (C), 128.5 (CH), 128.4 (CH), 128.1 (CH), 128.0 (CH), 127.8 (CH), 127.7 (CH), 127.0 (CH), 125.0 (CH), 124.9 (CH), 120.0 (CH), 81.5 (C-3’), 79.1 (C-1’),
78.6 (C-2’), 75.7 (CH2), 75.0 (CH2), 67.1 (CH2 (Fmoc)), 61.1 (C-4’), 60.6 (C-2), 56.0
(C-3), 55.6 (C-4), 47.8 (C-5), 47.2 (CH (Fmoc)), 38.4 (CH3 (Ms)), 25.8 (C(CH3)3), 18.1
(C), -5.4 (CH3), -5.5 (CH3).
189 Chapter 9 [Experimental for Chapter 3]
(1aR,4R,5R,6R,6aS,6bS)-5,6-Bis(benzyloxy)-4-((tert- butyldimethylsilyloxy)methyl)hexahydro-1aH-oxireno[2,3-a]pyrrolizine (163) and (1aR,5S,6S,7R,7aS,7bS)-6,7-bis(benzyloxy)-5-(tert- butyldimethylsilyloxy)octahydrooxireno[2,3-a]indolizine (163a). OBn H OBn H O O OBn 7 7a11 8a 7 OBn 5 N 33N 5 OTBS
163 OTBS 163a To a solution of 162 (470.3 mg, 0.589 mmol) in MeCN (6 mL) was added piperidine (0.12 mL, 1.12 mmol). The reaction was stirring for 15 h at rt, the volatiles were removed under reduced pressure and the residue was purified by FCC (10:90 to 30:70 EtOAc/petrol) to give a mixture of 163 and 163a (91:9) as a pale yellow oil (271.0 mg, 96%). A pure sample of 163 was obtained by further purification of this mixture by FCC to give 163 as yellow needles.
163: Rf 0.27 (30:70 EtOAc/petrol). mp. 40.9-43.1 oC (yellow needles)
24 [α] D +12 (c 1.0, CHCl3). MS (ESI +ve) m/z 482 (M+H+, 100%). + HRMS (CI +ve) calculated for C28H40NO4Si (M+H ) 482.2727, found 482.2729. -1 IR υmax (cm ): 3032, 2945, 2924, 2858, 1255, 1109. 1H NMR δ 7.36-7.24 (m, 10H, Ar), 4.61 (d, 1H, J 12.0 Hz, CHHPh), 4.60 (d, 1H, J 11.5 Hz, CHHPh), 4.54 (d, 1H, J 12.0 Hz, CHHPh), 4.51 (d, 1H, J 12.0 Hz, CHHPh), 4.15 (app. t, 1H, J 3.8 Hz, H-2), 3.91 (dd, 1H, J 7.3, 3.8 Hz, H-1), 3.69-3.68 (m, 1H, H-6), 3.66 (dd, 1H, J 10.0, 6.0 Hz, H-8), 3.64-3.62 (m, 2H, H-7 and H-7a), 3.50 (app. t, 1H, J 10.0 Hz, H-8), 3.45 (d, 1H, J 11.5 Hz, H-5), 3.08-3.04 (m, 1H, H-3), 2.98 (d, 1H, J 12.0
Hz, H-5), 0.88 (s, 9H, t-Bu), 0.04 (s, 3H, CH3), 0.03 (s, 3H, CH3). 13C NMR δ 138.1 (C), 137.7 (C), 128.4 (CH), 128.3 (CH), 127.8 (CH), 127.7 (CH),
127.64 (CH), 127.6 (CH), 88.7 (C-2), 85.9 (C-1), 72.1 (CH2), 71.8 (CH2), 70.8 (C-3),
69.0 (C-7a), 64.4 (C-8), 58.5 (C-7), 57.0 (C-6), 55.6 (C-5), 25.9 (C(CH3)3), 18.2 (C), -
5.4 (CH3), -5.43 (CH3).
190 Chapter 9 [Experimental for Chapter 3]
(1S,2S,5R,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-(hydroxymethyl)hexahydro-1H- pyrrolizine-1,2-diol (165).
HO H OBn To a solution of the epoxide 163 (37.4 mg, 0.078 mmol) in 1 7 HO 7a OBn anhydrous CH2Cl2 (4 mL) was added NaHSO4 (46.7 mg, 0.389 3 N 5 mmol). The reaction mixture was stirred and heated at reflux for OH 165 2 d under an atmosphere of N2. The reaction was quenched by the addition of water (5 mL) and stirred for 1 h. The solvent was removed under reduced pressure and the residue was extracted with EtOAc (3x10 mL). The combined extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue. NMR analysis of this crude reaction mixture showed an 86:14 mixture of regioisomers. The crude mixture was purified by FCC (100% EtOAc to 8.5:1:0.5 EtOAc/MeOH/NH3) to give 165 (92:8 mixture of diastereomer) as a pale yellow oil (15.3 mg, 51%).
165: Rf 0.34 (8.6:1.0:0.4 / EtOAc:MeOH:NH3).
23 [α] D +19 (c 1.1, CHCl3). MS (ESI +ve) m/z 386 (M+H+, 100%). + HRMS (ESI +ve) calculated for C22H28NO5 (M+H ) 386.1967, found 386.1967. -1 IR υmax (cm ): 3390, 3027, 2929, 2873, 1449, 1103, 1063. 1 H NMR (CD3OD) δ 7.36-7.24 (m, 10H, Ar), 4.68 (d, 2H, J 12.0 Hz, 2xCHHPh), 4.60 (d, 1H, J 11.5 Hz, CHHPh), 4.54 (d, 1H, J 12.0 Hz, CHHPh), 4.19 (app. t, 1H, J 5.3 Hz, H-1), 4.08 (dd, 1H, J 10.5, 5.5 Hz, H-2), 4.04 (app. t, 1H, J 5.3 Hz, H-7), 3.98 (dd, 1H, J 6.5, 5.5 Hz, H-6), 3.62 (dd, 1H, J 11.0, 4.8 Hz, H-8), 3.51 (dd, 1H, J 11.3, 5.8 Hz, H-8), 3.30 (m, 1H, H-5), 3.27 (app. t, 1H, J 5.0 Hz, H-7a), 3.18 (app. dt, 1H, J 5.8, 5.0 Hz, H- 3), 2.87 (dd, 1H, J 11.3, 5.8 Hz, H-5). 13 C NMR (CD3OD) δ 139.6 (C), 139.5 (C), 129.4 (CH), 129.3 (CH), 128.95 (CH), 129.5 (CH), 128.7 (CH), 128.5 (CH), 87.2 (C-1), 85.6 (C-6), 81.4 (C-7), 79.2 (C-2), 75.2 (C-
7a), 73.3 (CH2), 72.9 (CH2), 72.6 (C-3), 63.5 (C-8), 60.1 (C-5).
191 Chapter 9 [Experimental 3]
(1R,2R,3R,6S,7S)-3-(Hydroxymethyl)hexahydro-1H-pyrrolizine-1,2,6,7-tetraol (casuarine (15)).
HO H OH To a solution of 92% diastereomerically pure 165 (21.0 mg, 0.055 7 1 HO OH mmol) in MeOH (2 mL) was added PdCl2 (10.0 mg, 0.055 mmol). 5 N 3 The mixture was stirred at rt under an atmosphere of H2 (balloon) OH 15 for 1.5 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave casuarine 15 (dr = 95:5) as a brown foamy solid (10.4 mg, 93%).
23 56 24 [α] D +18.1 (c 1.0, H2O), lit. [α] D +16.9 (c 0.8, H2O). MS (ESI +ve) m/z 206 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO5 (M+H ) 206.1028, found 206.0953. -1 IR υmax (cm ): 3284, 2919, 1378, 1128, 1102, 1029. 1 H NMR (D2O) δ 4.22-4.18 (m, 2H, H-6 and H-7), 4.16 (t, 1H, J1,2 = J1,7a = 8.7 Hz, H-
1), 3.79 (t, 1H, J1,2 = J2,3 = 8.0 Hz, H-2), 3.77 (dd, 1H, J8,8’ = 10.0, J3,8 = 3.5 Hz, H-8),
3.61 (dd, 1H, J8,8’ = 11.3, J3,8’ = 6.8 Hz, H-8’), 3.27 (dd, 1H, J5α,5β = 12.3, J5β,6 = 4.3 Hz,
H-5β), 3.06 (dd, 1H, J1,7a = 8.0, J7,7a = 3.0 Hz, H-7a), 3.04-3.00 (m, 1H, H-3), 2.90 (dd,
1H, J5α,5β = 11.8, J5α,6 = 4.3 Hz, H-5α). 13 C NMR (D2O) δ 79.9 (C-7), 78.9 (C-1), 78.5 (C-6), 77.8 (C-2), 73.1 (C-7a), 71.0 (C- 3), 63.5 (C-8), 59.0 (C-5).
192 Chapter 9 [Experimental for Chapter 4]
9.4 Chapter 4 Experimental for the total synthesis of australine, 7-epi-australine and 1-epi-castanospermine
(1R,5R,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-(hydroxymethyl)hexahydro-1H-pyrrolizin- 1-ol (235); (1R,5R,6R,7R,7aR)-6,7-bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-ol (237) and (1R,6S,7S,8R,8aR)-7,8-bis(benzyloxy)-6-(tert- butyldimethylsilyloxy)octahydroindolizin-1-ol (239) OBn HO H OBn HO H OBn HO H 7 OBn 1 7a 1 7a 7 1 8a 7 OBn OBn 3 N 5 3 N 5 3 N 5 OTBS
235 OH 237 OTBS 239 To solution of a 91:9 mixture of 163 and 163a (611.5 mg, 1.271 mmol) in anhydrous THF (13 mL) was added dropwise a solution of lithium aluminium hydride (1M in THF, 1.53 mL, 1.526 mmol). The reaction was stirring for 8 h at 0 oC. The solvent was evaporated and the mixture was chromatographed on silica gel by FCC (50:50
EtOAc/petrol to 8.5:1.0:0.5 EtOAc/MeOH/NH3) to give a mixture of 235 and 236 (235:236 = 88:12) as a yellow viscous oil (106 mg, 27%), a mixture of 237 and 238 (237:238 = 92:8) as a yellow viscous oil (330 mg, 64%), 239 (10.3 mg, 2%) as a pale yellow oil and unreacted starting material (97.5 mg, 16%).
235 (on 88:12 mixture): Rf 0.40 (9:1:0.2 EtOAc/MeOH/NH3).
22 [α] D +6 (c 1.1, CHCl3). MS (ESI +ve) m/z 370 (M+H+, 100%). + HRMS (CI +ve) calculated for C22H28NO4 (M+H ) 370.2018, found 370.2000. -1 IR υmax (cm ): 3385, 2924, 2873, 1449, 1362, 1105. 1H NMR δ 7.36-7.28 (m, 10H, Ar), 4.73 (d, 1H, J 11.5 Hz, CHHPh), 4.65 (d, 1H, J 12.0 Hz, CHHPh), 4.62 (d, 2H, J 10.0 Hz, 2xCHHPh), 4.19-4.17 (m, 1H, H-7), 4.07 (app. t, 1H, J 6.8 Hz, H-2), 3.80 (app. t, 1H, J 6.0 Hz, H-1), 3.56 (app. t, 2H, J 3.3 Hz, 2xH-8), 3.29 (dd, 1H, J 6.0, 3.5 Hz, H-7a), 3.17-3.12 (m, 1H, H-5), 2.82-2.79 (m, 1H, H-3), 2.78-2.73 (m, 1H, H-5), 2.04-1.97 (m, 1H, H-6), 1.78-1.73 (m, 1H, H-6).
193 Chapter 9 [Experimental for Chapter 4]
13C NMR δ 138.1 (C), 138.0 (C), 128.5 (CH), 128.4 (CH), 127.9 (CH), 127.8 (CH),
127.78 (CH), 127.7 (CH), 86.6 (C-1), 83.7 (C-2), 76.7 (C-7), 75.7 (C-7a), 72.9 (CH2),
72.2 (CH2), 69.3 (C-3), 60.5 (C-8), 51.6 (C-5), 33.7 (C-6). To a solution of the diol 235 (0.145 g, 0.039 mmol) and a crystal of 4- dimethylaminopyridine in THF (4 mL) under N2 at rt was added imidazole (0.056 g, 0.083 mmol) and TBSCl (0.071 g, 0.047 mmol). The reaction mixture was stirred for 2 d and the reaction was quenched by the addition of water (10 mL). The solvent was removed under reduced pressure and the residue was extracted with EtOAc (3x20 mL).
The combined organic extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (100%
EtOAc to 10:2:1 EtOAc/MeOH/NH3) to give a mixture of 237 and 238 (237:238 = 82:18) (0.056 g, 61%) as a yellow viscous oil.
237 (on 92:8 mixture): Rf 0.44 (70:30 EtOAc/petrol).
22 [α] D -16 (c 2.3, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (CI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2891. -1 IR υmax (cm ): 3380, 2929, 2852, 1454, 1244, 1098. 1H NMR δ 7.35-7.25 (m, 10H, Ar), 4.64 (d, 1H, J 11.5 Hz, CHHPh), 4.63 (d, 1H, J 12.0 Hz, CHHPh), 4.59 (d, 2H, J 12.0 Hz, 2xCHHPh), 4.17 (app. dt, 1H, J 5.0, 5.0 Hz, H-7), 3.95 (app. t, 1H, J 5.3 Hz, H-2), 3.86 (app. t, 1H, J 4.8 Hz, H-1), 3.65 (dd, 1H, J 10.0, 6.0 Hz, H-8), 3.59 (dd, 1H, J 9.8, 6.3 Hz, H-8), 3.26 (app. t, 1H, J 4.8 Hz, H-7a), 3.24- 3.19 (m, 1H, H-5), 2.91-2.83 (m, 2H, H-3 and H-5), 2.09-2.03 (m, 1H, H-6), 1.74-1.67
(m, 1H, H-6), 0.88 (s, 9H, t-Bu), 0.04 (s, 3H, CH3), 0.03 (s, 3H, CH3). 13C NMR δ 138.4 (C), 138.2 (C), 128.4 (CH), 128.3 (CH), 127.74 (CH), 127.7 (CH),
127.61 (CH), 127.6 (CH), 86.9 (C-1), 85.5 (C-2), 76.6 (C-7), 76.4 (C-7a), 72.2 (CH2),
71.9 (CH2), 70.8 (C-3), 65.6 (C-8), 53.2 (C-5), 34.3 (C-6), 26.0 (C(CH3)3), 18.3 (C), -5.4
(2xCH3).
239: Rf 0.38 (40:60 EtOAc/petrol).
25 [α] D +13.5 (c 0.7, CHCl3).
194 Chapter 9 [Experimental for Chapter 4]
MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (CI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2892. -1 IR υmax (cm ): 3349, 2922, 2850, 1248, 1069. 1H NMR δ 7.39-7.26 (m, 10H, Ar), 4.98 (d, 1H, J 11.0 Hz, CHHPh), 4.88 (d, 1H, J 11.5 Hz, CHHPh), 4.81 (d, 1H, J 11.5 Hz, CHHPh), 4.66 (d, 1H, J 11.0 Hz, CHHPh), 3.93- 3.89 (m, 1H, H-1), 3.84-3.79 (m, 1H, H-6), 3.44-3.38 (m, 2H, H-7 and H-8), 2.99 (dd, 1H, J 10.3, 5.3 Hz, H-5), 2.91 (app. t, 1H, J 8.3 Hz, H-3), 2.42 (app. dt, 1H, J 9.0, 8.5 Hz, H-3), 2.25-2.17 (m, 1H, H-2), 2.10 (app. t, 1H, J 10.5 Hz, H-5), 1.96 (app. t, 1H, J
7.8 Hz, H-8a), 1.65-1.58 (m, 1H, H-2), 0.90 (s, 9H, t-Bu), 0.09 (s, 3H, CH3), 0.07 (s, 3H,
CH3). 13C NMR δ 138.9 (C), 138.3 (C), 128.7 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH),
127.5 (CH), 127.4 (CH), 88.7 (C-7 or C-8), 81.9 (C-7 or C-8), 75.5 (CH2), 75.1 (C-1),
74.8 (CH2), 73.3 (C-8a), 72.8 (C-6), 57.5 (C-5), 51.7 (C-3), 32.0 (C-2), 25.8 (C(CH3)3),
17.9 (C), -4.6 (2xCH3).
(1R,2R,3R,7R,7aR)-3-Hydroxymethyl-hexahydro-pyrrolizine-1,2,7-triol (7-epi- australine (86)). To a solution of a 92:8 mixture of 237 and 238 (37.1 mg, 0.077 HO H OH
7 7a 1 mmol) MeOH (2 mL) was added PdCl2 (20.4 mg, 0.115 mmol). The OH 5 N 3 mixture was stirred at rt under an atmosphere of H2 (balloon) for 3 h, 86 OH follow by dropwise conc. HCl (8 drops) at rt for 17 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1.5 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 7-epi-australine 82 (dr 92:8) as a pale yellow solid (13.0 mg, 90%).
23 170 25 [α] D -13.2 (c 1.2, H2O), lit. [α] D -13.04 (c 0.55, H2O, pH 8.37) MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (EI +ve) calculated for C8H15NO4 (M ) 189.1001, found 189.0999. -1 IR υmax (cm ): 3358, 2980, 1340, 1126, 1027.
195 Chapter 9 [Experimental for Chapter 4]
1 H NMR (D2O) δ 4.34 (brs, 1H, H-7), 3.77 (dd, 1H, J8,8’ = 12.0, J3,8= 4.5 Hz, H-8), 3.76
(t, 1H, J1,2 = J2,3 = 8.5 Hz, H-2), 3.71 (t, 1H, J1,2 = J1,7a = 8.0 Hz, H-1), 3.64 (dd, 1H, J8,8’
= 11.8, J3,8= 5.8 Hz, H-8), 3.08 (dd, 1H, J5,5 = J5,6 10.8, J5,6 = 6.0 Hz, H-5), 3.01 (brd,
1H, J1,7a= 8.0 Hz, H-7a), 2.89-2.84 (m, 1H, H-5), 2.69-2.64 (m, 1H, H-3), 2.11-2.04 (m, 1H, H-6), 1.80-1.74 (m, 1H, H-6). 13 C NMR (D2O) δ 78.6 (C-1), 77.0 (C-2), 75.8 (C-7), 74.5 (C-7a), 69.0 (C-3), 63.4 (C- 8), 52.4 (C-5), 32.3 (C-6).
(1S,5R,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-ol (241).
HO H OBn To a solution of a 92:8 mixture of 237 and 238 (0.164 g, 0.34 1 7a 7 OBn 3 N 5 mmol) in toluene (7 mL) was added triphenylphosphine (0.223 g,
OTBS 0.85 mmol) and para-nitrobenzoic acid (0.142 g, 0.85 mmol). The 241 mixture was stirred and cooled to 0 oC and diisopropyl azodicarboxylate (0.17 mL, 0.85 mmol) was added. The mixture was heated and stirred at 80 oC for 1.5 h. The volatiles were removed in vacuo. The reaction mixture was extracted with CH2Cl2 (3x20 mL). The combined CH2Cl2 extracts were washed with water (20 mL), dried (Na2CO3), filtered and then evaporated to give 240 as a brown oil that was used in the next step without further purification.
(1S,5R,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-yl 4-nitrobenzoate (240). O O H OBn 240: Rf 0.42 (30:70 EtOAc/petrol). 1 7a 7 OBn 22 3 5 N [α] D +39 (c 0.9, CHCl3). O2N OTBS MS (ESI +ve) m/z 633 (M+H+, 100%). 240 + HRMS (ESI +ve) calculated for C35H45N2O7Si (M+H ) 633.2996, found 633.3007. -1 IR υmax (cm ): 2924, 2858, 1724, 1528, 1270, 1102.
196 Chapter 9 [Experimental for Chapter 4]
1H NMR δ 8.11 (d, 2H, J 8.5 Hz, Ar), 8.07 (d, 2H, J 8.5 Hz, Ar), 7.38-7.16 (m, 10H, Ar), 5.46-5.43 (m, 1H, H-7), 4.65 (d, 1H, J 11.5 Hz, CHHPh), 4.62 (d, 1H, J 11.5 Hz, CHHPh), 4.55 (d, 1H, J 12.0 Hz, CHHPh), 4.46 (d, 1H, J 12.5 Hz, CHHPh), 4.20 (app. t, 1H, J 5.0 Hz, H-1), 4.07 (dd, 1H, J 7.8, 5.3 Hz, H-2), 3.78 (dd, 1H, J 10.3, 3.8 Hz, H-8), 3.66 (app. t, 1H, J 4.5 Hz, H-7a), 3.63 (dd, 1H, J 10.5, 7.0 Hz, H-8), 3.30-3.26 (m, 1H, H-5), 3.00 (app. dt, 1H, J 7.5, 4.0 Hz, H-3), 2.90-2.84 (m, 1H, H-5), 2.20-2.16 (m, 2H,
2xH-6), 0.90 (s, 9H, t-Bu), 0.08 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR δ163.9 (CO), 150.7 (C), 138.2 (C), 137.7 (C), 135.2 (C), 130.7 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 123.5 (CH), 87.4
(C-2), 82.0 (C-1), 76.5 (C-7), 72.7 (CH2), 72.1 (CH2), 71.8 (C-3), 71.5 (C-7a), 66.1 (C-
8), 52.7 (C-5), 34.5 (C-6), 26.0 (C(CH3)3), 18.3 (C), -5.3 (2xCH3).
To a solution of crude 240 (0.34 mmol) in MeOH (7 mL) was added K2CO3 (0.075 g, 0.510 mmol). After stirring at rt for 2 h, the mixture was evaporated and dissolved in
CH2Cl2 (15 mL) and the solution was washed with water (15 mL). The aqueous layer was extracted further with CH2Cl2 (3x10 mL) and the combined CH2Cl2 extracts were washed with brine, dried (Na2CO3) and evaporated. The residue was purified by FCC (50:50 EtOAc/petrol to 100% EtOAc) to give diastereomerically pure 241 as a yellow oil (93 mg, 57%). Rf 0.30 (80:20 EtOAc/petrol).
22 [α] D -2.3 (c 1.8, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2873. -1 IR υmax (cm ): 3402, 2923, 2850, 1256, 1100, 1047. 1H NMR δ 7.36-7.28 (m, 10H, Ar), 4.63 (d, 1H, J 12.0 Hz, CHHPh), 4.59 (d, 1H, J 12.0 Hz, CHHPh), 4.56 (d, 1H, J 12.0 Hz, CHHPh), 4.52 (d, 1H, J 11.5 Hz, CHHPh), 4.27 (app. t, 1H, J 3.0 Hz, H-1), 4.16-4.14 (m, 1H, H-7), 4.11 (app. t, 1H, J 3.0 Hz, H-2), 3.67-3.57 (m, 3H, H-7a and 2xH-8), 3.22 (app. t, 1H, J 8.5 Hz, H-5), 3.04 (dd, 1H, J 11.3, 7.3 Hz, H-3), 2.86-2.80 (m, 1H, H-5), 196-1.87 (m, 2H, 2xH-6), 0.87 (s, 9H, t-Bu),
0.02 (s, 3H, CH3), 0.01 (s, 3H, CH3). 13C NMR δ 138.2 (C), 137.6 (C), 128.5 (CH), 128.4 (CH), 127.84 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 85.4 (C-2), 81.9 (C-1), 74.0 (C-7a), 72.3 (C-3), 71.94 (C-7),
197 Chapter 9 [Experimental for Chapter 4]
71.9 (CH2), 71.8 (CH2), 65.1 (C-8), 53.2 (C-5), 36.9 (C-6), 26.0 (C(CH3)3), 18.2 (C), -
5.3 (CH3), -5.4 (CH3).
(1R,2R,3R,7S,7aR)-3-Hydroxymethyl-hexahydro-1H-pyrrolizine-1,2,7-triol (australine (13)).
HO OH H To a solution of 241 (74.6 mg, 0.155 mmol) in MeOH (3 mL) was 7 7a 1 OH 5 N 3 added PdCl2 (41.1 mg, 0.232 mmol). The mixture was stirred at rt
8 OH under an atmosphere of H2 (balloon) for 3 h, follow by the dropwise 13 addition of conc. HCl (10 drops) and stirring was continued at rt for 21 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (2 mL) and applied to a column of Amberlyst A-26 (OH-) resin (4 cm). Elution with water followed by evaporation in vacuo gave australine 13 as a yellow oil (25.1 mg,
86%).
22 181 25 [α] D +9.4 (c 2.4, H2O), lit. [α] D +8 (c 0.35, H2O) MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (EI +ve) calculated for C8H15NO4 (M ) 189.1001, found 189.0994. -1 IR υmax (cm ): 3318, 2944, 2873, 2484, 1388, 1332, 1123, 1041. 1 H NMR (D2O) δ 4.37-4.35 (m, 1H, H-7), 4.22 (t, 1H, J1,2 = J1,7a = 7.8 Hz, H-1), 3.89
(dd, 1H, J2,3 = 9.5, J1,2 = 8.0 Hz, H-2), 3.79 (dd, 1H, J8,8’ = 12.0, J3,8 = 3.5 Hz, H-8), 3.61
(dd, 1H, J8,8’ = 11.5, J3,8’ = 7.0 Hz, H-8’), 3.17 (dd, 1H, J1,7a = 7.8 Hz, J7,7a = 4.8 Hz, H- 7a), 3.15-3.12 (m, 1H, H-5), 2.74-2.69 (m, 2H, H-3 and H-5), 2.05-2.00 (m, 1H, H-6), 1.97-1.89 (m, 1H, H-6). 13 C NMR (D2O) δ 79.5 (C-2), 73.7 (C-1), 71.3 (C-7a), 71.1 (C-3), 70.1 (C-7), 63.5 (C- 8), 52.4 (C-5), 35.8 (C-6). 1 13HCl salt: H NMR (D2O) δ 4.72-4.69 (m, 1H, H-7), 4.51 (app. t, 1H, J = 7.5 Hz, H- 1), 4.18 (dd, 1H, J = 10.0, 8.0 Hz, H-2), 4.02 (dd, 1H, J = 13.0, 2.5 Hz, H-8), 3.95-3.92 (m, 1H, H-7a), 3.92 (dd, 1H, J = 13.8, 4.3 Hz, H-8), 3.84 (app. brt, 1H, J = 9.8 Hz, H-5), 3.45-3.38 (m, 2H, H-5 and H-3), 2.36-2.31 (m, 1H, H-6), 2.05-2.00 (m, 1H, H-6), 2.29- 2.27 (m, 1H, H-6).
198 Chapter 9 [Experimental for Chapter 4]
13 C NMR (D2O) δ 76.2 (C-2), 73.3 (C-7a), 72.1 (C-1), 71.4 (C-3), 68.7 (C-7), 56.5 (C- 8), 52.9 (C-5), 35.0 (C-6).
(1R,6S,7R,8R,8aR)-Octahydroindolizine-1,6,7,8-tetraol (1-epi-castanospermine (242)).
OH To a solution of 239 (9.0 mg, 0.019 mmol) MeOH (1 mL) was added HO H OH 1 8a 7 PdCl2 (6.6 mg, 0.037 mmol). The mixture was stirred at rt under an 3 N 5 OH atmosphere of H2 (balloon) for 3 h, follow by dropwise conc. HCl (4 242 drops) at rt for 21 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The filtrate was evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 1-epi- castanospermine 242 as a colourless solid (3.3 mg, 94%).
22 20 [α] D +3.3 (c 0.3, MeOH), lit. [α] D +6.2 (c 0.15, MeOH) MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO4 (M+H ) 190.1079, found 190.1088. -1 IR υmax (cm ): 3330, 2924, 2822, 1444, 1312, 1086. 1 H NMR (D2O) δ 4.26-4.22 (m, 1H, H-1), 3.61 (ddd, 1H, J5,6 = 11.0, J6,7 = 9.5, J5,6 = 5.5
Hz, H-6), 3.39 (t, 1H, J7,8 = J8,8a = 9.0 Hz, H-8), 3.33 (t, 1H, J6,7 = J7,8 = 9.0 Hz, H-7),
3.16 (dd, 1H, J5,5 = 10.8, J5,6 = 5.8 Hz, H-5), 2.95 (ddd, 1H, J3,3 = 9.5, J2,3 = 8.0, J2,3 =
1.5 Hz, H-3), 2.57 (dt, 1H, J3,3 = 9.3, J2,3 = 8.5 Hz, H-3), 2.34-2.28 (m, 1H, H-2), 2.24 (t,
1H, J5,5 = J5,6 = 10.8 Hz, H-5), 2.12 (dd, 1H, J8,8a = 9.5, J1,8a = 6.5 Hz, H-8a), 1.70 (brt,
1H, J2,2 = J2,3 = 11.5 Hz, H-2). 13 C NMR (D2O) δ 79.3 (C-7), 74.3 (C-1), 74.0 (C-8), 73.4 (C-8a), 70.5 (C-6), 55.5 (C- 5), 51.4 (C-3), 33.0 (C-2).
199 Chapter 9 [Experimental for Chapter 5]
9.5 Chapter 5 Experimental
9.5.1 Total synthesis of 3-epi-casuarine and its derivatives
(2R,3R,4R)-3,4-Bis(benzyloxy)-4-((1S,2S,5R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-yl)- 1-(tert-butyldimethylsilyloxy)butan-2-ol (258).
OBn OH H To a solution of 161 (0.095 g, 0.131 mmol) in toluene (2 mL) O 3' OTBS 2 1' was added triphenylphosphine (0.086 g, 0.328 mmol) and 5 NH OBn para-nitrobenzoic acid (0.055 g, 0.328 mmol). The mixture 258 was cooled to 0 oC and diisopropyl azodicarboxylate (64.5 µL, 0.28 mmol) was added. The mixture was stirred at rt for 5 h. The volatiles were removed in vacuo. The reaction mixture was extracted with CH2Cl2 (3x10 mL). The combined
CH2Cl2 extracts were washed with water (5 mL), dried (Na2CO3), filtered and then evaporated to give 258a as a pale yellow oil that was used in the next step without further purification.
(1S,2S,5R)-(9H-Fluoren-9-yl)methyl 2-((1R,2S,3R)-1,2-bis(benzyloxy)-4-(tert- butyldimethylsilyloxy)-3-(4-nitrobenzoyloxy)butyl)-6-oxa-3- azabicyclo[3.1.0]hexane-3-carboxylate (258a).
R 0.41 (30:70 EtOAc/petrol). NO2 f 22 [α] D +35 (c 2.6, CHCl3). O MS (ESI +ve) m/z 870 (M+H+, 100%). OBn O H + OTBS HRMS (ESI +ve) calculated for C50H55N2O10Si (M+H ) O 1' 3' 5 871.3626, found 871.3611. 2 N OBn Fmoc -1 IR υmax (cm ): 2950, 2940, 2857, 1720, 1701, 1529, 1271, 258a 1101. 1H NMR δ (major rotamer) 8.29-8.23 (m, 2H, Ar), 7.79-7.57 (m, 2H, Ar), 7.42-7.20 (m, 18H, Ar), 5.45 (dd, 1H, J 9.0, 5.5 Hz, H-3’), 4.91 (d, 1H, J 11.0 Hz, CHHPh), 4.84 (d,
1H, J 11.5 Hz, CHHPh), 4.60 (d, 1H, J 11.0 Hz, CHHPh), 4.45 (d, 2H, J 6.5 Hz, CH2 (Fmoc)), 4.37 (d, 1H, J 11.5 Hz, CHHPh), 4.28-4.14 (m, 5H, H-1’ or H-2’, H-3 or H-4, 2xH-4’ and CH (Fmoc)), 4.07 (d, 1H, J 3.0 Hz, H-3 or H-4), 3.78 (brs, 1H, H-1’ or H-
200 Chapter 9 [Experimental for Chapter 5]
2’), 3.76 (d, 1H, J 12.0, Hz, H-5), 3.68 (brd, 1H, J 2.0 Hz, H-2), 3.25 (d, 1H, J 11.5 Hz,
H-5), 0.91 (s, 9H, t-Bu), 0.08 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR δ (major rotamer) 163.9 (CO), 154.8 (CO), 150.5 (C), 143.8 (C), 143.5 (C), 141.2 (C), 141.1 (C), 137.6 (C), 137.4 (C), 135.2 (C), 130.7 (CH), 128.4 (CH), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.5 (CH), 127.0 (CH), 126.9 (CH), 124.7 (CH), 123.4 (CH), 119.9 (CH), 80.7 (C-1’), 79.0 (C-2’), 75.8
(C-3’), 74.5 (CH2), 74.8 (CH2), 67.0 (CH2 (Fmoc)), 61.8 (C-2), 60.5 (C-4’), 56.4 (C-3 or
C-4), 55.7 (C-3 or C-4), 47.6 (C-5), 47.1 (CH (Fmoc)), 25.7 (C(CH3)3), 18.0 (C), -5.4
(CH3), -5.5 (CH3).
To a solution of crude 258a (0.131 mmol) in MeOH (2 mL) was added K2CO3 (0.015 g, 0.109 mmol). After stirring at rt for 24 h, the mixture was evaporated and dissolved in
CH2Cl2. The solution was washed with water (5 mL) and the aqueous layer was extracted with CH2Cl2 (3x10 mL). The combined CH2Cl2 extracts were washed with brine, dried (Na2CO3) and evaporated. The residue was purified by FCC (50:50
EtOAc/petrol to 100% EtOAc) to give 258 (dr = 92:8) as a yellow oil (36 mg, 55%). Rf 0.08 (30:70 EtOAc/petrol).
23 [α] D +53 (c 2.8, CHCl3). MS (ESI +ve) m/z 500 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO5Si (M+H ) 500.2832, found 500.2836. -1 IR υmax (cm ): 3362, 2930, 1449, 1250, 1100. 1H NMR δ 7.33-7.25 (m, 10H, Ar), 4.86 (d, 1H, J 11.0 Hz, CHHPh), 4.71 (d, 1H, J 11.5 Hz, CHHPh), 4.62 (d, 1H, J 11.0 Hz, CHHPh), 4.57 (d, 1H, J 11.0 Hz, CHHPh), 3.81 (brs, 3H, H-2’, H-3’ and H-4’), 3.73 (dd, J 9.5, 3.5 Hz, 1H, H-4’), 3.67 (d, 1H, J 2.5 Hz, H-3 or H-4), 3.55 (dd, 1H, J 9.5, 2.0 Hz, H-1’) 3.42 (d, 1H, J 9.5 Hz, H-2), 3.39 (d, 1H, J 2.5 Hz, H-3 or H-4), 3.02 (d, 1H, J 13.5 Hz, H-5), 2.70 (d, 1H, J 13.0 Hz, H-5), 0.91
(s, 9H, t-Bu), 0.08 (s, 6H, 2xCH3). 13C NMR δ 138.3 (C), 138.2 (C), 128.4 (CH), 128.3 (CH), 128.22 (CH), 128.2 (CH),
128.1 (CH), 128.0 (CH), 127.8 (CH), 127.7 (CH), 79.4 (C-2’), 78.5 (C-1’), 74.52 (CH2),
74.5 (CH2), 71.7 (C-3’), 64.4 (C-4’), 59.9 (C-2), 58.0 (C-3 or C-4), 55.8 (C-3 or C-4),
46.9 (C-5), 25.9 (C(CH3)3), 18.3 (C), -5.28 (CH3), -5.3 (CH3).
201 Chapter 9 [Experimental for Chapter 5]
(1aR,4S,5R,6R,6bS)-5,6-Bis(benzyloxy)-4-((tert- butyldimethylsilyloxy)methyl)hexahydro-1aH-oxireno[2,3-a]pyrrolizine (259) and (1aR,5R,6S,7R,7aS,7bS)-6,7-bis(benzyloxy)-5-(tert- butyldimethylsilyloxy)octahydrooxireno[2,3-a]indolizine (260). OBn H OBn H O O OBn 77a 117 OBn 8a 5 3 N 3 N 5 OTBS OTBS 259 260 To a solution of 258 (dr = 92:8) (0.500 g, 1.002 mmol) in toluene (10 mL) was added triphenylphosphine (0.657 g, 2.505 mmol). The mixture was cooled to 0 oC and diisopropyl azodicarboxylate (0.49 mL, 2.505 mmol) was added. The mixture was heated and stirred at 80 oC for 12 h. The volatiles were removed in vacuo. The reaction mixture was extracted with CH2Cl2 (3x25 mL). The combined CH2Cl2 extracts were washed with water (20 mL), dried (Na2CO3), filtered and then evaporated. The residue was purified by FCC (50:50 EtOAc/petrol to 100% EtOAc) to give to give 259 as a yellow oil (0.337 g, 70%) and 260 as a yellow oil (0.02 g, 4%).
259: Rf 0.26 (70:30 EtOAc/petrol).
25 [α] D +43 (c 1.6, CHCl3). MS (ESI +ve) m/z 482 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H40NO4Si (M+H ) 482.2727, found 482.2717. -1 IR υmax (cm ): 2952, 2930, 2850, 1447, 1250, 1095. 1H NMR δ 7.38-7.25 (m, 10H, Ar), 4.55 (d, 1H, J 12.0 Hz, CHHPh), 4.53 (d, 1H, J 12.0 Hz, CHHPh), 4.51 (d, 1H, J 11.5 Hz, CHHPh), 4.48 (d, 1H, J 12.0 Hz, CHHPh), 4.09 (d, 1H, J 4.0 Hz, H-2), 3.99 (app. t, 1H, J 9.3 Hz, H-8), 3.91 (dd, 1H, J 10.0, 5.0 Hz, H-8), 3.80 (d, 1H, J 4.5 Hz, H-1), 3.68-3.66 (m, 2H, H-6 or H-7 and H-7a), 3.60 (d, 1H, J 2.0 Hz, H-6 or H-7), 3.39 (app. dt, 1H, J 8.5, 4.3 Hz, H-3), 3.19 (d, 1H, J 10.5 Hz, H-5),
3.03 (d, 1H, J 11.5 Hz, H-5), 0.09 (s, 9H, t-Bu), 0.06 (s, 6H, 2xCH3). 13C NMR δ 138.3 (C), 137.6 (C), 128.5 (CH), 128.3 (CH), 127.9 (CH), 127.6 (CH),
127.5 (CH), 127.3 (CH), 86.9 (C-2), 85.3 (C-1), 72.2 (CH2), 71.9 (C-7a), 71.7 (CH2),
65.7 (C-3), 58.5 (C-8), 57.6 (C-6 or C-7), 57.3 (C-6 or C-7), 48.1 (C-5), 25.9 (C(CH3)3),
18.2 (C), -5.4 (CH3), -5.5 (CH3).
202 Chapter 9 [Experimental for Chapter 5]
260: Rf 0.25 (70:30 EtOAc/petrol). 1H NMR δ 7.78-7.26 (m, 10H, Ar), 4.97 (d, 1H, J 11.5 Hz, CHHPh), 4.73 (d, 1H, J 11.5 Hz, CHHPh), 4.65 (d, 2H, J 11.5 Hz, 2xCHHPh), 4.15 (m, 1H, H-6), 3.63 (app. t, 1H, J 7.8 Hz, H-8), 3.55 (d, 1H, J 3.0 Hz, H-1 or H-2), 3.52 (d, 1H, J 10.5 Hz, H-3), 3.45 (d, 1H, J 3.0 Hz, H-1 or H-2), 3.38 (dd, 1H, J 10.0, 3.0 Hz, H-7), 3.20 (d, 1H, J 10.5 Hz, H- 3), 3.15 (d, 1H, J 9.5 Hz, H-8a), 2.96 (dd, 1H, J 15.0, 1.5 Hz, H-5), 2.86 (brd, 1H, J 15.0
Hz, H-5), 0.91 (s, 9H, t-Bu), 0.10 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR δ 138.5 (C), 138.3 (C), 133.2 (CH), 133.0 (CH), 128.6 (CH), 128.4 (CH),
128.2 (CH), 127.8 (CH), 127.6 (CH), 127.5 (CH), 84.1 (C-7), 75.0 (CH2), 72.7 (C-8),
72.2 (CH2), 71.3 (C-6), 61.8 (C-8a), 57.8 (C-1 or C-2), 54.7 (C-1 or C-2), 52.0 (C-3),
50.3 (C-5), 28.6 (C(CH3)3), 18.2 (C), -4.6 (CH3), -4.7 (CH3).
(1S,2S,5S,6R,7R,7aR)-5-(Acetylmethyl)-6,7-bis(benzyloxy)-1,2-diacetyl-hexahy-dro- 1H-pyrrolizine (261), 1,2-bis(benzyloxy)-6-acetyl-3-methyl-7,8-epoxyindolizidine (262), (1aR,4S,5R,6R,6bS)-5,6-bis(benzyloxy)-4-(acetylmethyl)hexahydro-1aH- oxireno[2,3-a]pyrrolizine (263) and 1,2-bis(benzyloxy)-7-acetyl-3-methyl-6,8-epoxy- indolizidine (264).
AcO OBn OBn H H H OBn AcO H OBn O AcO OBn AcO OBn OBn OBn N N N N O O OAc OAc 261 262 263 264
To a solution of the epoxide 259 (100.0 mg, 0.208 mmol) in anhydrous CH2Cl2 (5 mL) was added NaHSO4 (125 mg, 1.04 mmol). The reaction mixture was stirred and heated at reflux for 7 d under an atmosphere of N2. The reaction was quenched by the addition of water (5 mL) and stirred for 1 h. The solvent was removed under reduced pressure and the residue was extracted with EtOAc (3x10 mL). TLC analysis showed 4 majors products. The crude mixture was purified by FCC (100% EtOAc to 8.0:1.5:0.5
EtOAc/MeOH/NH3). The fractions were not pure and they were combined, evaporated and then acetylated. To a solution of the crude product in pyridine(2.0 mL) was added acetic anhydride (0.184 mL, 1.948 mmol) and a crystal of 4-dimethylaminopyridine. The
203 Chapter 9 [Experimental for Chapter 5] mixture was stirred at rt for 24 h. The reaction was quenched by the addition of sat.
NaHCO3 solution, followed by removal of the solvent under reduced pressure. The residue was extracted with CH2Cl2 (3x10 mL). The combined CH2Cl2 extracts were washed with water (10 mL), dried (Na2CO3), filtered and then evaporated. The residue was purified by FCC (90:10 EtOAc/petrol to 8.5:1:0.5 EtOAc/MeOH/NH3) to give 261 as a pale yellow oil (7.0 mg, 7%), 262 as a colorless oil (6.7 mg, 8%), 263 as a pale yellow oil (8.0 mg, 9%) and 264 as a pale yellow oil (14.0 mg, 17%).
261: Rf 0.47 (50:50 EtOAc/petrol).
22 [α] D +22.3 (c 1.4, CHCl3). MS (ESI +ve) m/z 512 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H34NO8 (M +H ) 512.2284, found 512.2277. -1 IR υmax (cm ): 2919, 1738, 1372, 1228, 1042. 1H NMR δ 7.36-7.26 (m, 10H, Ar), 5.32 (app. dt, 1H, J 8.0, 6.8 Hz, H-6), 5.22 (app. t, 1H, J 6.5 Hz, H-7), 4.61 (d, 1H, J 12.5 Hz, CHHPh), 4.55 (d, 1H, J 11.5 Hz, CHHPh), 4.48 (d, 1H, J 11.5 Hz, CHHPh), 4.42 (dd, 1H, J 11.8, 5.8 Hz, H-8), 4.37 (d, 1H, J 12.0 Hz, CHHPh), 4.34 (dd, 1H, J 10.8, 7.8 Hz, H-8), 4.30 (brs, 1H, H-1), 3.95 (d, 1H, J 4.0 Hz, H-2), 3.49-3.45 (m, 1H, H-3), 3.35 (dd, 1H, J 5.5, 5.0 Hz, H-7a), 3.31 (app. t, 1H, J
7.8 Hz, H-5β), 3.13 (app. t, 1H, J 8.0 Hz, H-5α), 2.11 (s, 3H, CH3 (Ac)), 2.05 (s, 3H,
CH3 (Ac)) 2.01 (s, 3H, CH3 (Ac)). 13C NMR δ 170.9 (CO), 170.8 (CO), 170.3 (CO), 137.7 (C), 137.6 (C), 128.5 (CH), 128.4 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 85.7 (C-1), 85.6 (C-2),
79.0 (C-7), 76.5 (C-6), 73.8 (C-7a), 71.8 (CH2), 71.7 (CH2), 62.3 (C-3), 60.6 (C-8), 50.4
(C-5), 20.94 (CH3 (Ac)), 20.9 (CH3 (Ac)), 20.8 (CH3 (Ac)).
262: Rf 0.50 (100% EtOAc).
25 [α] D -14.9 (c 0.7, CHCl3). MS (ESI +ve) m/z 410 (M+H+, 100%). + HRMS (ESI +ve) calculated for C24H28NO5 (M+H ) 410.1967, found 410.1984. -1 IR υmax (cm ): 2929, 2868, 1737, 1237, 1102, 1046. 1H NMR δ 7.38-7.26 (m, 10H, Ar), 5.24 (dd, 1H, J 6.3, 2.8 Hz, H-6), 4.62 (d, 1H, J 11.5 Hz, CHHPh), 4.59 (d, 2H, J 12.5 Hz, 2xCHHPh), 4.55 (d, 1H, J 12.0 Hz, CHHPh), 4.37
204 Chapter 9 [Experimental for Chapter 5]
(d, 1H, J 4.0 Hz, H-1), 4.30 (dd, 1H, J 5.5, 4.0 Hz, H-2), 3.95 (d, 1H, J 2.0 Hz, H-7), 3.73 (d, 1H, J 12.0 Hz, H-8), 3.65 (dd, 1H, J 14.5, 6.5 Hz, H-5), 3.59 (dd, 1H, J 12.3, 1.8 Hz, H-8), 3.10 (d, 1H, J 2.0 Hz, H-7a), 3.07 (dd, 1H, J 4.8, 1.8 Hz, H-3), 2.85 (dd, 1H, J
14.3, 2.8 Hz, H-5), 2.03 (s, 3H, CH3 (Ac)). 13C NMR δ 170.0 (CO), 138.0 (C), 137.8 (C), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.87 (CH), 127.8 (CH), 127.7 (CH), 85.5 (C-2), 81.9 (C-1), 78.7 (C-7), 77.1 (C-6),
72.2 (CH2), 71.8 (CH2), 68.6 (C-7a), 62.3 (C-3), 59.8 (C-8), 56.1 (C-5), 20.9 (CH3 (Ac)).
263: Rf 0.34 (100% EtOAc). MS (ESI +ve) m/z 410 (M+H+, 100%). 1H NMR δ 7.41-7.23 (m, 10H, Ar), 4.58 (d, 1H, J 12.0 Hz, CHHPh), 4.54 (d, 1H, J 11.5 Hz, CHHPh), 4.53 (d, 1H, J 11.5 Hz, CHHPh), 4.43 (dd, 1H, J 11.5, 6.5 Hz, H-8), 4.37 (dd, 1H, J 11.0, 4.5 Hz, H-8), 4.36 (d, 1H, J 11.5 Hz, CHHPh), 4.03 (d, 1H, J 3.5 Hz, H- 2), 3.81 (d, 1H, J 4.5 Hz, H-1), 3.71 (d, 2H, J 5.0 Hz, H-7a and H-6 or H-7), 3.63 (d, 1H, J 3.0 Hz, H-6 or H-7), 3.57 (dd, 1H, J 11.0, 7.0 Hz, H-3), 3.25 (d, 1H, J 11.5 Hz, H-5),
3.15 (d, 1H, J 11.0 Hz, H-5), 2.03 (s, 3H, CH3 (Ac)). 13C NMR δ 170.9 (CO), 137.6 (C), 137.4 (C), 128.6 (CH), 128.5 (CH), 128.1 (CH),
127.9 (CH), 127.6 (CH), 127.5 (CH), 87.4 (C-2), 84.8 (C-1), 72.2 (C-7a), 71.8 (CH2),
71.7 (CH2), 62.4 (C-3), 60.3 (C-8), 57.4 (C-6 or C-7), 57.2 (C-6 or C-7), 48.1 (C-5), 20.9
(CH3 (Ac)).
264:Rf 0.32 (100% EtOAc). MS (ESI +ve) m/z 410 (M+H+, 100%).
25 [α] D -16.7 (c 1.6, CHCl3). + HRMS (ESI +ve) calculated for C24H28NO5 (M+H ) 410.1967, found 410.1974. -1 IR υmax (cm ): 2929, 2873, 1735, 1378, 1237, 1120. 1H NMR δ 7.41-7.26 (m, 10H, Ar), 4.97 (brs, 1H, H-7), 4.75 (d, 1H, J 12.0 Hz, CHHPh), 4.66 (d, 1H, J 12.5 Hz, CHHPh), 4.61 (d, 1H, J 12.0 Hz, CHHPh), 4.48 (d, 1H, J 11.5 Hz, CHHPh), 4.24-4.23 (m, 2H, H-1 and H-2), 4.17 (s, 1H, H-6), 3.71 (d, 1H, J 10.5 Hz, H-8), 3.56-3.53 (m, 2H, H-3 and H-8), 3.47 (d, 1H, J 13.0 Hz, H-5α), 3.25 (brs,
1H, H-7a), 2.72 (dd, 1H, J 13.0, 1.0 Hz, H-5β), 2.05 (s, 3H, CH3 (Ac)).
205 Chapter 9 [Experimental for Chapter 5]
13C NMR δ 170.0 (CO), 138.2 (C), 138.0 (C), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 86.0 (C-1), 84.7 (C-2), 81.8 (C-7), 75.3 (C-6), 74.2
(C-7a), 72.5 (CH2), 72.1 (CH2), 62.9 (C-3), 55.7 (C-8), 49.4 (C-5), 21.0 (CH3 (Ac)).
(1R,2R,3S,6S,7S,7aR)-3-(Hydroxymethyl)-hexahydro-1H-pyrrolizine-1,2,6,7-tetraol (3-epi-casuarine (79)).
HO H OH To a solution of 261 (14 mg, 0.027 mmol) in MeOH (1 mL) was
7 1 HO OH added PdCl2 (9.7 mg, 0.055 mmol). The mixture was stirred at rt 5 N 3 under an atmosphere of H2 (balloon) for 4 d. The mixture was 8 OH 79 filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 3-epi-casuarine 79 as a white solid (4.3 mg, 77%).
25 58 23 [α] D +2.0 (c 0.04, H2O), lit. [α] D +5.7 (c 0.5, H2O). MS (ESI +ve) m/z 206 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO5 (M+H ) 206.1028, found 206.1024. -1 IR υmax (cm ): 3300, 2924, 2901, 1361, 1027. 1 H NMR (D2O) δ 4.28 (brs, 1H, H-1), 4.19 (dd, 1H, J2,3 = 3.5, J1,2 = 1.5 Hz, H-2), 4.11
(dt, 1H, J5,6 = 7.8, J5,6 = J6,7 = 7.5 Hz, H-6), 4.05 (t, 1H, J6,7 = J7,7a = 8.0 Hz, H-7), 4.00
(dd, 1H, J8,8 = 11.8, J3,8 = 6.3 Hz, H-8), 3.94 (dd, 1H, J8,8’ = 11.5, J3,8’ = 7.5 Hz, H-8’),
3.27 (ddd, 1H, J3,8 = 6.8, J3,8 = 6.5, J2,3 = 3.5 Hz, H-3), 3.10 (d, 3H, J7,7a = 8.0 Hz, 2xH-5 and H-7a). 13 C NMR (D2O) δ 80.4 (C-1), 79.7 (C-2), 79.2 (C-7), 75.9 (C-6), 75.5 (C-7a), 64.9 (C- 3), 57.4 (C-8), 51.6 (C-5).
1,2-Bis(benzyloxy)-6-hydroxyl-3-methyl-7,8-epoxyindolizidine (265a) and 3-methyl 1,2,6-trihydroxyl-7,8-epoxyindolizidine (265).
H OBn To a solution of 262 (13.4 mg, 0.033 mmol) in MeOH (1 mL) 7 1 HO OBn - 5 N 3 was added Amberlyst A-26 (OH ) resin (40 mg). The reaction O 8 265a
206 Chapter 9 [Experimental for Chapter 5] was stirring for 12 h at rt, the mixture was filtered through a celite pad and the solids were washed with MeOH (10 mL). The filtrate was evaporated in vacuo to give as a pale yellow oil that was used to next step without purification. MS (ESI +ve) m/z 368 (M+H+, 100%). + HRMS (ESI +ve) calculated for C22H26NO4 (M +H ) 368.1844, found 368.1862. -1 IR υmax (cm ): 3385, 2925, 2863, 1454, 1362, 1099, 1070. 1H NMR δ7.37-7.26 (m, 10H, Ar), 4.63 (d, 1H, J 12.0 Hz, CHHPh), 4.59 (d, 1H, J 11.5 Hz, CHHPh), 4.57 (d, 1H, J 11.0 Hz, CHHPh), 4.54 (d, 1H, J 12.0 Hz, CHHPh), 4.49 (dd, 1H, J 6.5, 2.5 Hz, H-6), 4.36 (d, 1H, J 3.5 Hz, H-1), 4.33 (dd, 1H, J 5.5, 4.0 Hz, H- 2), 3.80 (d, 1H, J 2.0 Hz, H-7), 3.68 (d, 1H, J 12.0 Hz, H-8), 3.53 (dd, 1H, J 12.3, 5.8 Hz, H-5), 3.50 (dd, 1H, J 12.3, 2.3 Hz, H-8), 3.24 (brs, 1H, H-7a), 3.02 (brd, 1H, J 3.0 Hz, H-3), 2.86 (dd, 1H, J 14.0, 2.5 Hz, H-5). 13C NMR δ 138.0 (C), 137.9 (C), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH),
127.6 (CH), 85.5 (C-2), 82.0 (C-1), 81.5 (C-7), 75.1 (C-6), 72.2 (CH2), 71.7 (CH2), 67.8 (C-7a), 62.3 (C-3), 59.8 (C-8), 57.9 (C-5).
H OH To a solution of crude 265a in MeOH (1 mL) was added PdCl2
7 1 HO OH (8.71 mg, 0.05 mmol). The mixture was stirred at rt under an 5 N 3 O 8 atmosphere of H2 (balloon) for 24 h The mixture was filtered 265 through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 265 as a pale yellow soild (5.0 mg, 82%).
23 [α] D +8.6 (c 0.5, H2O). MS (ESI +ve) m/z 188 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H14NO4 (M+H ) 188.0923, found 188.0928. -1 IR υmax (cm ): 3395, 2965, 2934, 1316, 1033. 1 H NMR (D2O) δ 4.41 (dd, 1H, J5,6 = 6.0, J5,6 = 2.5 Hz, H-6), 4.27 (brd, 1H, J1,2 = 4.0
Hz, H-1), 4.20 (brdd, 1H, J2,3 = 5.5, J1,2 = 4.5 Hz, H-2), 3.87 (brs, 1H, H-7), 3.62-3.54
(m, 3H, 2xH-8 and H-5α), 2.96 (brs, 1H, H-7a), 2.94 (brd, 1H, J2,3 = 5.0 Hz, H-3), 2.63
(dd, 1H, J5,5 = 14.5, J5,6 = 2.0 Hz, H-5β).
207 Chapter 9 [Experimental for Chapter 5]
13 C NMR (D2O) δ 81.4 (C-7), 79.9 (C-2), 75.8 (C-1), 74.4 (C-6), 70.6 (C-7a), 63.7 (C- 3), 59.5 (C-8), 56.8 (C-5).
1,2-Bis(benzyloxy)-7-hydroxyl-3-methyl-6,8-epoxyindolizidine (266a) and 3-methyl- 1,2,7-trihydroxyl-6,8-epoxyindolizidine (266).
To a solution of 264 (14.0 mg, 0.034 mmol) in MeOH (1 mL) was HO H OBn - 7 1 added Amberlyst A-26 (OH ) resin (42 mg). The reaction was stirred OBn 5 3 N for 16 h at rt, the mixture was filtered through a celite pad and the O 8 266a solids were washed with MeOH (10 mL). The filtrate was evaporated in vacuo to give as a pale yellow oil that was used to next step without purification. MS (ESI +ve) m/z 368 (M+H+, 100%). + HRMS (ESI +ve) calculated for C22H26NO4 (M +H ) 368.1844, found 368.1862. -1 IR υmax (cm ): 3365, 2919, 2863, 1440, 1115, 1014. 1H NMR δ7.42-7.26 (m, 10H, Ar), 4.73 (d, 1H, J 12.0 Hz, CHHPh), 4.61 (d, 1H, J 12.0 Hz, CHHPh), 4.56 (d, 1H, J 11.5 Hz, CHHPh), 4.49 (d, 1H, J 12.5 Hz, CHHPh), 4.24 (app. t, 1H, J 6.3 Hz, H-2), 4.07 (dd, 1H, J 7.0, 4.0 Hz, H-1), 3.98 (s, 1H, H-6), 3.91 (s, 1H, H-7), 3.66 (md, 1H, J 10.5 Hz, H-8), 3.54-3.51 (m, 2H, H-3 and H-8), 3.40 (d, 1H, J 13.0 Hz, H-5), 3.07 (d, 1H, J 3.5 Hz, H-7a), 2.77 (d, 1H, J 12.5 Hz, H-5). 13C NMR δ 138.3 (C), 138.0 (C), 128.5 (CH), 128.4 (CH), 128.2 (CH), 127.9 (CH), 127.7 (CH), 127.6 (CH), 85.6 (C-1), 84.7 (C-2), 81.0 (C-7), 77.3 (C-6), 76.0 (C-7a), 72.6
(CH2), 72.5 (CH2), 62.7 (C-3), 55.4 (C-8), 48.6 (C-5).
HO H OH To a solution of crude 266a in MeOH (1 mL) was added PdCl2 (9.1
7 1 OH mg, 0.051 mmol). The mixture was stirred at rt under an atmosphere 5 N 3 O 8 of H2 (balloon) for 24 h. The mixture was filtered through a celite pad 266 and the solids were washed with MeOH. The filtrate was evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 266 as a yellow soild (3.4 mg, 53%).
23 [α] D +7.6 (c 0.34, H2O).
208 Chapter 9 [Experimental for Chapter 5]
MS (ESI +ve) m/z 188 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H14NO4 (M ) 188.0923, found 188.0928. -1 IR υmax (cm ): 3380, 2919, 1114, 1054, 1033. 1 H NMR (D2O) δ 4.25 (t, 1H, J1,2 = J2,3 = 7.0 Hz, H-2), 4.22 (s, 2H, H-6 and H-7), 4.16
(dd, 1H, J1,2 = 7.5, J1,7a = 4.5 Hz, H-1), 3.76 (dd, 1H, J8,8 = 12.5, J3,8 = 5.5 Hz, H-8), 3.69
(d, 1H, J8,8 = 12.5 Hz, H-8), 3.63 (d, 1H, J5,5 = 14.0 Hz, H-5), 3.54 (t, 1H, J2,3 = J3,8 =
6.0 Hz, H-3), 3.08 (d, 1H, J1,7a = 4.0 Hz, H-7a), 2.80 (d, 1H, J5,5 = 14.0 Hz, H-5). 13 C NMR (D2O) δ 80.2 (C-6), 79.5 (C-1), 77.9 (C-7), 77.7 (C-2), 76.5 (C-7a), 64.3 (C- 3), 54.9 (C-8), 47.7 (C-5).
9.5.2 Attempts to improve the yield of 3-epi-casuarine 79
(R)-(9H-Fluoren-9-yl)methyl 2-((1R,2R,3S)-1,2-bis(benzyloxy)-3,4-dihydroxybutyl)- 2H-pyrrole-1(5H)-carboxylate (267).
OBn OH To a solution of 158 (0.467 g, 1.267 mmol) in THF (20 mL) and H OH 1' 3' 2 satd. Na2CO3 solution (10 mL) was added 9-fluorenylmethyl 5 N OBn o Fmoc chloroformate (393 mg, 1.52 mmol) at 0 C. The reaction mixture 267 was stirred at 0 oC for 5 h. Water (20 mL) was added and the solvent was removed under reduced pressure and the residue was extracted with CH2Cl2
(3x20 mL). The combined organic extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC
(50:50 to 80:20 EtOAc/petrol) to give 267 (0.609 g, 81%) as a colourless viscous oil. Rf 0.22 (60:40 EtOAc/petrol).
22 [α] D +141 (c 3.7, CHCl3). MS (ESI +ve) m/z 592 (M+H+, 70%). + HRMS (ESI +ve) calculated for C37H38NO6 (M+H ) 592.2699, found 592.2690. -1 IR υmax (cm ): 3436, 2939, 2868, 1685, 1454, 1424, 1111. 1H NMR δ (major rotamer) 7.78-7.21 (m, 18H, Ar), 5.93 (dd, 1H, J 6.8, 1.8 Hz, H-3 or H-4), 5.89 (dd, 1H, J 6.8, 1.3 Hz, H-3 or H-4), 4.88 (d, 1H, J 11.0 Hz, CHHPh), 4.87- 4.85 (m, 1H, H-2), 4.59 (d, 1H, J 10.5 Hz, CHHPh), 4.57 (d, 1H, J 11.0 Hz, CHHPh),
4.48 (d, 1H, J 12.0 Hz, CHHPh), 4.39-4.36 (m, 3H, CH2 (Fmoc), and H-1’ or H-2’),
209 Chapter 9 [Experimental for Chapter 5]
4.24-4.16 (m, 2H, H-5 and CH (Fmoc)), 4.06-4.02 (m, 1H, H-5 ), 3.90-3.88 (m, 1H, H- 3’), 3.71-3.66 (m, 1H, H-4’), 3.58-3.53 (m, 2H, H-4’ and H-1’ or H-2’). 13C NMR δ (major rotamer) 154.6 (CO), 144.0 (C), 143.8 (C), 141.3 (C), 138.2 (C), 137.8 (C), 128.5 (CH), 128.4 (CH), 128.1 (CH), 127.7 (CH), 127.0 (C-3 or C-4), 126.7 (C-3 or C-4), 125.0 (CH), 120.O (CH), 79.1 (C-1’ or C-2’), 78.1 (C-1’ or C-2’), 74.7
(CH2), 74.1 (CH2), 70.9 (C-3’), 67.1 (CH2 (Fmoc)), 66.3 (C-2), 64.0 (C-4’), 53.4 (C-5), 47.2 (CH (Fmoc)).
(R)-(9H-Fluoren-9-yl)methyl 2-((1R,2R,3S)-1,2-bis(benzyloxy)-4-(tert- butyldiphenylsilyloxy)-3-hydroxybutyl)-2H-pyrrole-1(5H)-carboxylate (268).
OBn OH To a solution of the diol 267 (0.506 g, 0.857 mmol) and 4- H OTBDPS 1' 3' 2 dimethylaminopyridine (10.5 mg, 0.086 mmol) in THF (15 5 N OBn Fmoc mL) under N2 at rt was added imidazole (0.122 g, 1.799 268 mmol) and TBDPSCl (284.5 µL, 1.113 mmol). The reaction mixture was stirred for 24 h and the reaction was quenched by the addition of satd.
NaHCO3 solution (10 mL). The solvent was removed under reduced pressure and the residue was extracted with CH2Cl2 (3x20 mL). The combined extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (20:80 to 50:50 EtOAc/petrol) to give 268 (0.552 g, 78%) as a colourless viscous oil.
Rf 0.50 (30:70 EtOAc/petrol).
23 [α] D +62 (c 2.6, CHCl3). MS (ESI +ve) m/z 830 (M+H+, 100%). + HRMS (ESI +ve) calculated for C53H56NO6Si (M+H ) 830.3877, found 830.3894. -1 IR υmax (cm ): 3421, 2934, 2852, 1757, 1680, 1429, 1112. 1H NMR δ (major rotamer) 7.72-7.16 (m, 28H, Ar), 5.94 (d, 1H, J 6.5 Hz, H-3 or H-4), 5.90 (d, 1H, J 6.0 Hz,H-3 or H-4), 4.87 (brd, 2H, J 10.0 Hz, CHHPh and H-2), 4.62 (d, 1H, J 11.5 Hz, CHHPh), 4.48-4.43 (m, 3H, H-1’ or H-2’ and 2xCHHPh), 4.38-4.32 (m,
2H, CH2 (Fmoc)), 4.29-4.21 (m, 2H, CH (Fmoc) and H-5), 4.10-4.04 (m, 1H, H-5 ), 3.92
210 Chapter 9 [Experimental for Chapter 5]
(app. dt, 1H, J 7.3, 7.0 Hz, H-3’), 3.83 (d, 1H, J 7.5 Hz, H-1’ or H-2’), 3.75 (dd, 1H, J 9.8, 6.3, H-4’), 3.67-3.61 (m, 1H, H-4’), 1.07 (s, 9H, t-Bu). 13C NMR δ (major rotamer) 154.3 (CO), 144.1 (C), 143.9 (C), 141.3 (C), 141.2 (C), 138.4 (C), 138.1 (C), 135.5 (C), 135.4 (C), 135.2 (CH), 134.8 (CH), 129.6 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.0 (CH), 126.7 (C-3 or C-4), 126.6 (C-3 or C-4), 125.0 (CH), 120.0 (CH), 78.2 (C-1’ or C-2’), 77.7 (C-
1’ or C-2’), 74.8 (CH2), 74.3 (CH2), 70.5 (C-3’), 67.0 (CH2 (Fmoc)), 66.1 (C-2), 64.4
(C-4’), 53.5 (C-5), 47.3 (CH (Fmoc)), 26.8 (C(CH3)3), 19.0 (C).
(1S,2S,5R)-(9H-Fluoren-9-yl)methyl 2-((1R,2R,3S)-1,2-bis(benzyloxy)-4-(tert- butyldiphenylsilyloxy)-3-hydroxybutyl)-6-oxa-3-aza-bicyclo[3.1.0]hexane-3- carboxylate (269).
OBn OH H To a solution of the olefin 268 (0.540 g, 0.652 mmol) in OTBDPS O 1' 3' -4 2 MeCN (10 mL) was added Na2EDTA (4 mL, 4 x 10 M) 5 N OBn Fmoc and CF3C(O)CH3 (2.0 mL, 2.24 mmol). The reaction was 269 chilled to 0 oC before the portionwise addition of a mixture of NaHCO3 (0.820 g, 9.771 mmol) and oxone (0.801 g, 1.30 mmol) over 15 min. After stirring for 10 h at 0 oC, the mixture was poured into water followed by removed of the volatiles under reduced pressure. The residue was extracted with CH2Cl2 (3x20 mL) and the combined organic extracts were washed with brine, dried (Na2CO3) and then evaporated to leave a residue which was chromatographed on silica gel by FCC (10:90 to 40:60 EtOAc/petrol) to give 269 (0.366 g, 67%) as a pale yellow viscous oil. Rf 0.45 (30:70 EtOAc/petrol).
24 [α] D +63 (c 2.8, CHCl3). MS (ESI +ve) m/z 846 (M+H+, 100%). + HRMS (ESI +ve) calculated for C53H56NO7Si (M+H ) 846.3826, found 846.3796. -1 IR υmax (cm ): 3503, 2929, 2863, 1700, 1454, 1429, 1111. 1H NMR δ (major rotamer) 7.77-7.13 (m, 28H, Ar), 4.85 (d, 1H, J 10.5 Hz, CHHPh), 4.67 (d, 1H, J 12.0 Hz, CHHPh), 4.56 (d, 1H, J 11.0 Hz, CHHPh), 4.39-4.33 (m, 3H,
CHHPh and CH2 (Fmoc)), 4.27 (d, 2H, J 8.0 Hz, H-2’and H-2), 4.20 (app. t, 1H, J 7.0
211 Chapter 9 [Experimental for Chapter 5]
Hz, CH (Fmoc)), 3.97 (d, 1H, J 6.5 Hz, H-3’), 3.93 (d, 1H, J 7.5 Hz, H-1’), 3.87 (d, 1H, J 2.5 Hz, H-3 or H-4), 3.79-3.73 (m, 2H, H-4’ and H-5), 3.69-3.61 (m, 2H, H-4’ and H-3 or H-4), 3.26 (app. t, 1H, J 10.5 Hz, H-5), 1.06 (s, 9H, t-Bu). 13C NMR δ (major rotamer) 154.9 (CO), 144.0 (C), 143.8 (C), 141.31 (C), 141.3 (C), 138.0 (C), 137.7(C), 135.5 (CH), 133.2 (C), 133.0 (C), 129.8 (CH), 129.7 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.1 (CH), 127.0 (CH), 125.1 (CH), 125.0 (CH), 120.0 (CH), 79.0 (C-2’), 77.0
(C-1’), 74.8 (CH2), 74.3 (CH2), 70.4 (C-3’), 67.1 (CH2 (Fmoc)), 64.3 (C-4’), 60.1 (C-2),
56.5 (C-3 or C-4), 55.7 (C-3 or C-4), 47.8 (C-5), 47.2 (CH (Fmoc)), 26.9 (C(CH3)3), 19.2 (C).
(2R,3R,4R)-4-((1S,2S,5R)-6-Oxa-3-aza-bicyclo[3.1.0]hexan-2-yl)-3,4-bis(benzyloxy)- 1-(tert-butyldiphenylsilyloxy)butan-2-ol (270).
OBn To a solution of 269 (0.230 g, 0.272 mmol) in toluene (3 H OH O OTBDPS 1' 3' mL) was added triphenylphosphine (0.285 g, 1.089 mmol) 2 5 NH OBn and para-nitrobenzoic acid (0.182 g, 1.089 mmol). The 270 mixture was cooled to 0 oC and diisopropyl azodicarboxylate (214 µL, 1.089 mmol) was added. The mixture was stirred at rt for 2 d.
The volatiles were removed in vacuo. The reaction mixture was extracted with CH2Cl2
(3x10 mL). The combined CH2Cl2 extracts were washed with water (5 mL), dried
(Na2CO3), filtered and then evaporated to give 270a as a colourless foamy solid that was used in the next step without further purification.
(1S,2S,5R)-(9H-Fluoren-9-yl)methyl 2-((1R,2S,3R)-1,2-bis(benzyloxy)-4-(tert- butyldiphenylsilyloxy)-3-(4-nitrobenzoyloxy)butyl)-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylate (270a).
NO2 Rf 0.40 (25:75 EtOAc/petrol).
24 [α] D +35 (c 3.1, CHCl3). O + OBn MS (ESI +ve) m/z 995 (M+H , 100%). H O O OTBDPS + 1' 3' 2 HRMS (ESI +ve) calculated for C60H59N2O10Si (M+H ) 5 N OBn Fmoc 995.3939, found 995.3933. 270a
212 Chapter 9 [Experimental for Chapter 5]
-1 IR υmax (cm ): 2934, 2863, 1705, 1526, 1454, 1112. 1H NMR δ (major rotamer) 8.23.710 (m, 32H, Ar), 5.47 (app. dt, 1H, J 4.5, 4.5 Hz, H- 3’), 4.82 (d, 1H, J 11.0 Hz, CHHPh), 4.76 (d, 1H, J 11.5 Hz, CHHPh), 4.49 (d, 1H, J
11.5 Hz, CHHPh), 4.41-4.37 (m, 2H, CH2 (Fmoc)), 4.26 (d, 1H, J 11.0 Hz, CHHPh), 4.23-4.17 (m, 5H, H-1’, H-2’, 2xH-4’ and CH (Fmoc)), 3.99 (d, 1H, J 3.0 Hz, H-2), 3.90-3.88 (m, 1H, H-3 or H-4), 3.71 (d, 1H, J 12.0, Hz, H-5), 3.61 (d, 1H, J 2.5 Hz, H-3 or H-4), 3.19 (d, 1H, J 12.0 Hz, H-5), 1.01 (s, 9H, t-Bu). 13C NMR δ (major rotamer) 163.8 (CO), 154.9 (CO), 150.5 (C), 143.9 (C), 143.6 (C), 141.3 (C), 137.7 (C), 137.4 (C), 136.0 (CH), 135.6 (CH), 135.5 (CH), 135.3 (CH), 132.9 (C), 132.8 (C), 130.9 (CH), 130.6 (CH), 129.8 (CH), 129.4 (CH), 128.5 (CH), 128.4 (CH), 128.0 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.0 (CH), 124.9 (CH), 124.8 (CH), 123.4 (CH), 119.9 (CH), 79.2 (C-1’ or C-2’), 79.1 (C-1’ or C-2’),
75.8 (C-3’), 75.5 (CH2), 74.9 (CH2), 67.1 (CH2 (Fmoc)), 61.8 (C-2), 61.7 (C-4’), 56.5
(C-3 or C-4), 55.8 (C-3 or C-4), 47.7 (C-5), 47.2 (CH (Fmoc)), 27.8 (C(CH3)3), 19.1 (C).
To a solution of crude 270a (0.272 mmol) in MeOH (3 mL) was added K2CO3 (0.056 g, 0.041 mmol). After stirring at rt for 20 h, the mixture was evaporated and dissolved in
CH2Cl2 (5 mL) then washed with water (5 mL). The aqueous layer was extracted with
CH2Cl2 (3x10 mL) and the combined CH2Cl2 extracts were washed with brine, dried
(Na2CO3) and evaporated. The residue was purified by FCC (20:80 to 50:50 EtOAc/petrol) to give 270 as a yellow viscous oil (106 mg, 62%).
Rf 0.22 (40:60 EtOAc/petrol).
25 [α] D +37 (c 1.4, CHCl3). MS (ESI +ve) m/z 624 (M+H+, 100%). + HRMS (ESI +ve) calculated for C38H46NO5Si (M+H ) 624.3145, found 624.3148. -1 IR υmax (cm ): 3370, 3067, 2924, 2858, 1429, 1111. 1H NMR δ 7.69-7.66 (m, 4H, Ar), 7.43-7.15 (m, 16H, Ar), 4.84 (d, 1H, J 11.5 Hz, CHHPh), 4.65 (d, 1H, J 11.5 Hz, CHHPh), 4.55 (d, 1H, J 12.0 Hz, CHHPh), 4.52 (d, 1H, J 12.0 Hz, CHHPh), 3.91-3.82 (m, 4H, H-2’, H-3’ and 2xH-4’), 3.67 (d, 1H, J 2.5 Hz, H-3 or H-4), 3.55 (dd, 1H, J 9.3, 3.8 Hz, H-1’) 3.41-3.38 (m, 2H, H-2 and H-3 or H-4), 3.02 (d, 1H, J 13.5 Hz, H-5), 2.71 (d, 1H, J 13.0 Hz, H-5), 1.08 (s, 9H, t-Bu).
213 Chapter 9 [Experimental for Chapter 5]
13C NMR δ 138.2 (C), 138.1 (C), 135.7 (CH), 135.6 (CH), 133.3 (C), 133.1 (C), 129.7 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.7 (CH), 127.6 (CH), 79.5
(C-2’), 78.4 (C-1’), 74.5 (CH2), 74.49 (CH2), 72.2 (C-3’), 65.3 (C-4’), 60.3 (C-2), 57.9
(C-3 or C-4), 55.8 (C-3 or C-4), 46.8 (C-5), 26.9 (C(CH3)3), 19.3 (C).
(1aR,4S,5R,6R,6aS,6bS)-5,6-Bis(benzyloxy)-4-((tert-butyldiphenylsilyloxy)methyl)- hexahydro-1aH-oxireno[2,3-a]pyrrolizine (271) and (1aR,5R,6S,7R,7aS,7bS)-6,7- bis(benzyloxy)-5-(tert-butyldiphenylsilyloxy)-octahydrooxireno[2,3-a]indolizine
(272). OBn H OBn H O O OBn 771 1 8a 7a OBn 5 N 3 3 N 5 OTBDPS 8 271 OTBDPS 272
To a solution of 270 (92 mg, 0.148 mmol) in toluene (2 mL) was added triphenylphosphine (0.155 g, 0.591 mmol). The mixture was cooled to 0 oC and diisopropyl azodicarboxylate (116 µL, 0.591 mmol) was added. The mixture was heated at 80 oC and stirred for 3 d. The volatiles were removed in vacuo. The reaction mixture was extracted with CH2Cl2 (3 x 10 mL). The combined CH2Cl2 extracts were washed with water (10 mL), dried (Na2CO3), filtered and then evaporated. The residue was purified by FCC (20:80 to 50:50 EtOAc/petrol) to give a 3:2 mixture ratio of 271 and 272 and as a pale yellow viscous oil (87 mg, 93%). The mixture was used in the next step without further purification.
(1S,2S,6R,7S,8R,8aR)-7,8-Bis(benzyloxy)-6-(tert-butyldiphenylsilyloxy)- octahydroindolizine-1,2-diol (273).
OBn HO H To a solution of the above mixture of epoxides 271 and 272 OBn 1 8a 7 (87.2 mg, 0.144 mmol) in anhydrous CH2Cl2 (2 mL) was HO 3 N 5 OTBDPS added NaHSO4 (172.6 mg, 1.44 mmol). The reaction 273 mixture was stirred and heated at reflux for 14 d under an atmosphere of N2. The reaction was quenched by the addition of water (5 mL) and stirred for 1 h. The solvent was removed under reduced pressure and the residue was
214 Chapter 9 [Experimental for Chapter 5] extracted with EtOAc (3x10 mL). The solvent was evaporated and the mixture was chromatographed on silica gel by FCC (40:60 to 80:20 EtOAc/petrol and 7:1.5:0.5
EtOAc/MeOH/NH4OH) to give only pure 273 as a yellow oil (6.0 mg, 7%) and a complex mixture of other products (55 mg).
273: Rf 0.43 (80:20 EtOAc/petrol).
22 [α] D -10 (c 0.5, CHCl3). MS (ESI +ve) m/z 624 (M+H+, 100%). + HRMS (ESI +ve) calculated for C38H46NO5Si (M+H ) 624.3145, found 624.3119. -1 IR υmax (cm ): 3411, 2924, 2858, 1454, 1429, 1111. 1H NMR δ 7.75-7.25 (m, 20H, Ar), 5.03 (d, 1H, J 12.0 Hz, CHHPh), 4.81 (d, 1H, J 11.5 Hz, CHHPh), 4.56 (d, 1H, J 11.5 Hz, CHHPh), 4.40 (d, 1H, J 11.5 Hz, CHHPh), 4.12- 4.08 (m, 2H, H-6 and H-8), 4.00 (brd, 1H, J 4.5 Hz, H-2), 3.85 (d, 1H, J 6.5 Hz, H-1) 3.28 (dd, 1H, J 9.0, 2.0 Hz, H-7), 2.77 (d, 2H, J 9.5 Hz, H-3 and H-5), 2.49 (dd, 1H, J 9.8, 6.3 Hz, H-3), 1.91-1.87 (m, 2H, H-5 and H-8a), 1.09 (s, 9H, t-Bu). 13C NMR δ 138.5 (C), 138.4 (C), 136.1 (CH), 135.9 (CH), 134.7 (C), 133.3 (C), 129.8 (CH), 129.6 (CH), 128.7 (CH), 128.5 (CH), 128.2 (CH), 128.0 (CH), 127.6 (CH), 127.5
(CH), 127.3 (CH), 85.0 (C-7), 84.3 (C-1), 78.9 (C-8), 77.4 (C-2), 74.8 (CH2), 72.9 (C-
8a), 71.1 (CH2), 68.2 (C-6), 60.0 (C-3), 55.3 (C-5), 26.9 (C(CH3)3), 19.5 (C).
215
9.6 Chapter 6 Experimental for the total synthesis of 3-epi-australine and its derivatives
(1R,5S,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-ol (278) and (2S,5S,6R,7R,7aR)-6,7-bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-2-ol (279).
HOH OBn H OBn
777a 1 7a 1 OBn HO OBn 55N 3 N 3 OTBS OTBS 278 279
To a solution of crude 259 (0.037 g, 0.098 mmol) in anhydrous THF (2 mL) was added dropwise a solution of lithiumaluminium hydride (1M in THF, 0.1 mL, 0.1 mmol). The mixture was stirred at rt for 12 h. The solvent was evaporated and the mixture was chromatographed on silica gel by FCC (80:20 EtOAc/petrol to 10:90 MeOH/EtOAc) to give 278 as a pale yellow oil (15.3 mg, 41%) and 279 (3.3 mg, 9%) as a pale yellow oil.
278: Rf 0.31 (5:95 MeOH/EtOAc).
22 [α] D -4 (c 1.4, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2868. -1 IR υmax (cm ): 3390, 2923, 2858, 1260, 1095. 1H NMR δ 7.34-7.25 (m, 10H, Ar), 4.59 (d, 1H, J 11.5 Hz, CHHPh), 4.57 (d, 1H, J 10.5 Hz, CHHPh), 4.52 (d, 1H, J 12.0 Hz, CHHPh), 4.48 (d, 1H, J 11.5 Hz, CHHPh), 4.16 (app. dt, 1H, J 6.5, 5.5 Hz, H-7), 4.04 (dd, 1H, J 4.5, 2.0 Hz, H-2), 3.95 (dd, 1H, J 10.0, 7.3 Hz, H-8), 3.89-3.86 (m, 2H, H-1 and H-8), 3.35 (app. dt, 1H, J 6.5, 4.8 Hz, H-3), 3.30 (app. t, 1H, J 4.5 Hz, H-7a), 3.09 (ddd, 1H, J 9.3, 7.0, 6.5 Hz, H-5), 2.91-2.87 (m, 1H, H-5), 2.19-2.13 (m, 1H, H-6), 1.84-1.78 (m, 1H, H-6), 0.88 (s, 9H, t-Bu), 0.40 (s,
6H, 2xCH3). 13C NMR δ 138.4 (C), 138.1 (C), 128.4 (CH), 128.3 (CH), 127.7 (CH), 127.6 (CH),
127.5 (CH), 127.3 (CH), 85.9 (C-1), 85.6 (C-2), 77.7 (C-7a), 75.6 (C-7), 72.1 (CH2),
216
71.4 (CH2), 65.3 (C-3), 58.8 (C-8), 46.1 (C-5), 35.6 (C-6), 25.9 (C(CH3)3), 18.3 (C), -5.4
(CH3), -5.5 (CH3).
279: Rf 0.11 (5:95 MeOH/EtOAc).
25 [α] D +10.3 (c 1.1, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2863. -1 IR υmax (cm ): 3236, 2952, 2923, 1250, 1096. 1H NMR δ 7.36-7.24 (m, 10H, Ar), 4.56 (d, 1H, J 12.0 Hz, CHHPh), 4.53 (d, 1H, J 12.0 Hz, CHHPh), 4.48 (d, 1H, J 12.0 Hz, CHHPh), 4.45 (d, 1H, J 12.0 Hz, CHHPh), 4.43 (app. brt, 1H, J 4.0 Hz, H-6), 4.10 (dd, 1H, J 4.5, 2.0 Hz, H-2), 3.91 (d, 2H, J 6.0 Hz, 2xH-8), 3.88-3.83 (m, 2H, H-1 and H-7a), 3.54 (app. dt, 1H, J 6.0, 5.0 Hz, H-3), 3.23 (dd, 1H, J 10.0, 3.5 Hz, H-5), 2.96 (d, 1H, J 10.0 Hz, H-5), 2.18 (dd, 1H, J 13.0, 7.3 Hz,
H-7), 1.86-1.81 (m, 1H, H-7), 0.89 (s, 9H, t-Bu), 0.05 (s, 6H, 2xCH3). 13C NMR δ 138.0 (C), 137.9 (C), 128.5 (CH), 128.4 (CH), 127.8 (CH), 127.7 (CH),
127.6 (CH), 127.4 (CH), 86.8 (C-1), 85.7 (C-2), 73.7 (C-6), 72.4 (CH2), 71.5 (CH2), 68.7
(C-7a), 64.5 (C-3), 58.5 (C-8), 56.2 (C-5), 39.5 (C-7), 25.9 (C(CH3)3), 18.3 (C), -5.4
(CH3), -5.5 (CH3).
(1S,5S,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-ol (280).
HO H OBn To a solution of 278 (0.040 g, 0.083 mmol) in toluene (2 mL) was
7 7a 1 OBn added triphenylphosphine (0.055 g, 0.021 mmol) and para- 5 N 3 nitrobenzoic acid (0.035 g, 0.021 mmol). The mixture was stirred at OTBS 280 0 oC and diisopropyl azodicarboxylate (41.1 µL, 0.021 mmol) was added. The mixture was stirred at rt for 8 h. The volatiles were removed in vacuo then satd. CuSO4 solution (5 mL) was added. The reaction mixture was extracted with
CH2Cl2 (3x10 mL). The combined CH2Cl2 extracts were washed with satd. CuSO4 solution (5 mL) and water (5 mL), dried (Na2CO3), filtered and then evaporated to give 280a as a brown oil that was used in the next step without further purification.
217
(1S,5S,6R,7R,7aR)-6,7-Bis(benzyloxy)-5-((tert- butyldimethylsilyloxy)methyl)hexahydro-1H-pyrrolizin-1-yl 4-nitrobenzoate (280a).
O Rf 0.39 (50:50 EtOAc/petrol). O OBn H 26 [α] D +31 (c 3.0, CHCl3). 7 7a 1 OBn + 5 N 3 MS (ESI +ve) m/z 633 (M+H , 70%). O2N + OTBS HRMS (ESI +ve) calculated for C35H45N2O7Si (M+H ) 280a 633.2996, found 633.2986. -1 IR υmax (cm ): 2926, 2853, 1726, 1528, 1272, 1096. 1H NMR δ 7.95 (s, 4H, Ar), 7.37-7.12 (m, 10H, Ar), 5.63 (app. t, 1H, J 5.8 Hz, H-7), 4.56 (d, 1H, J 12.0 Hz, CHHPh), 4.51 (d, 1H, J 12.0 Hz, CHHPh), 4.49 (d, 1H, J 13.0 Hz, CHHPh), 4.47 (d, 1H, J 13.5 Hz, CHHPh), 4.13 (dd, 1H, J 4.5, 1.5 Hz, H-2), 4.07 (dd, 1H, J 10.3, 7.3 Hz, H-8), 4.05 (dd, 1H, J 4.3, 2.3 Hz, H-1), 4.00 (dd, 1H, J 10.3, 6.8 Hz, H-8), 3.68 (app. t, 1H, J 4.8 Hz, H-7a), 3.40 (app. dt, 1H, J 6.0, 5.0 Hz, H-3), 3.30- 3.25 (m, 1H, H-5), 2.81 (app. brt, 1H, J 6.5 Hz, H-5), 2.30-2.23 (m, 1H, H-6), 2.05 (brd,
1H, J 12.0 Hz, H-6), 0.90 (s, 9H, t-Bu), 0.08 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR δ 163.8 (CO), 150.4 (C), 138.3 (C), 137.7 (C), 135.2 (C),130.6 (CH), 128.4 (CH), 128.3 (CH), 127.6 (CH), 127.5 (CH), 126.9 (CH), 123.3 (CH), 86.9 (C-2), 81.7
(C-1), 74.3 (C-7), 73.7 (C-7a), 72.3 (CH2), 71.7 (CH2), 65.4 (C-3), 59.9 (C-8), 46.2 (C-
5), 34.7 (C-6), 25.9 (C(CH3)3), 18.3 (C), -5.3 (CH3), -5.4 (CH3).
To a solution of crude 280a (0.083 mmol) in MeOH (2 mL) was added K2CO3 (0.023 g, 0.1669 mmol). After stirring at rt for 4 h, the mixture was evaporated and dissolved in
CH2Cl2 then washed with water. The aqueous layer was extracted with CH2Cl2 (3x5 mL) and the combined CH2Cl2 extracts were washed with brine, dried (Na2CO3) and evaporated. The residue was purified by FCC (80:20 EtOAc/petrol to 10:90
MeOH/EtOAc) to give 280 as a pale yellow oil (26 mg, 64%). Rf 0.19 (10:90 MeOH/EtOAc).
24 [α] D -5.3 (c 1.2, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2882.
218
-1 IR υmax (cm ): 3418, 2930, 2850, 1673, 1250, 1089. 1H NMR δ 7.36-7.26 (m, 10H, Ar), 4.68 (d, 1H, J 11.5 Hz, CHHPh), 4.62 (d, 1H, J 12.0 Hz, CHHPh), 4.57 (d, 1H, J 11.5 Hz, CHHPh), 4.56 (d, 1H, J 12.0 Hz, CHHPh), 4.25 (app. t, 1H, J 5.0 Hz, H-1), 4.23 (app. t, 1H, J 5.0 Hz, H-2), 4.12 (app. brt, 1H, J 2.5 Hz, H-7), 3.97 (dd, 1H, J 10.8, 5.3 Hz, H-8), 3.82 (dd, 1H, J 10.8, 5.3 Hz, H-8), 3.49 (app. t, 1H, J 4.3 Hz, H-7a), 3.31 (app. dt, 1H, J 4.8, 4.0 Hz, H-3), 3.04-2.99 (m, 1H, H-5), 2.81
(brt, 1H, J 7.8 Hz, H-5), 1.96-1.94 (m, 2H, 2xH-6), 0.88 (s, 9H, t-Bu), 0.05 (s, 3H, CH3),
0.04 (s, 3H, CH3). 13C NMR δ 138.4 (C), 137.9 (C), 128.4 (CH), 128.3 (CH), 128.0 (CH), 127.8 (CH),
127.6 (CH), 127.5 (CH), 85.5 (C-2), 79.5 (C-1), 73.2 (C-7a), 73.0 CH2), 71.9 (CH2),
71.2 (C-7), 62.5 (C-3), 59.3 (C-8), 43.9 (C-5), 36.9 (C-6), 26.0 (C(CH3)3), 18.6 (C), -5.3
(CH3), -5.8 (CH3).
(1R,2R,3S,7S,7aR)-3-(Hydroxymethyl)hexahydro-1H-pyrrolizine-1,2,7-triol (3-epi- australine (80)).
To a solution of 280 (21 mg, 0.045 mmol) in MeOH (1 mL) was HOH OH
7 7a 1 added PdCl2 (12 mg, 0.065 mmol). The mixture was stirred at rt under OH 5 N 3 an atmosphere of H2 (balloon) for 3 h, follow by the dropwise 80 8 OH addition of conc. HCl (5 drops). Stirring at rt was continued for 21 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 3-epi-australine 80 as a brown viscous oil (7.2 mg, 88%).
23 [α] D -10.5 (c 0.7, H2O), MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO4 (M+H ) 190.1079, found 190.1086. -1 IR υmax (cm ): 3279, 2924, 2888, 1429, 1357, 1058. 1 H NMR (D2O) δ 4.41 (brt, 1H, J6,7 = J7,7a = 4.0 Hz, H-7), 4.30 (t, 1H, J1,2 = J1,7a = 3.3
Hz, H-1), 4.15 (t, 1H, J1,2 = J2,3 = 4.0 Hz, H-2), 4.01 (dd, 1H, J8,8’ = 11.8, J3,8 = 5.8 Hz,
219
H-8), 3.92 (dd, 1H, J8,8’ = 11.8, J3,8’ = 6.3 Hz, H-8’), 3.38 (t, 1H, J1,7a = J7,7a = 4.3 Hz, H-
7a), 3.30 (dt, 1H, J3,8’ = 5.3, J2,3 = J3,8 = 4.5 Hz, H-3), 3.15-3.10 (m, 1H, H-5α), 2.88 (t,
1H, J5,5 = J5,6 = 8.0 Hz, H-5β), 2.00-1.87 (m, 2H, 2xH-6). 13 C NMR (D2O) δ 79.3 (C-2), 75.2 (C-7a), 74.7 (C-1), 70.4 (C-7), 63.9 (C-3), 57.8 (C- 8), 45.3 (C-5), 35.6 (C-6).
23 52 20 80HCl salt: [α] D -37 (c 0.7, H2O), lit. [α] D -3.5 (c 1.35, H2O)
1 H NMR (D2O) δ 4.77-4.73 (m, 1H, H-7), 4.65 (s, 1H, H-1), 4.34 (d, 1H, J 3.5 Hz, H-2), 4.29 (d, 1H, J 5.5 Hz, H-7a), 4.16 (dd, 1H, J 12.0, 4.5 Hz, H-8), 4.13-4.04 (m, 2H, H-8 and H-3), 3.74 (dd, 1H, J 11.3, 5.3 Hz, H-5), 3.71-3.65 (m, 1H, H-5), 2.28 (dd, 1H, J 14.0, 5.0 Hz, H-6), 2.21-2.13 (m, 1H, H-6). 13 C NMR (D2O) δ 79.3 (C-7a), 77.4 (C-2), 74.2 (C-1), 69.3 (C-7), 67.1 (C-3), 56.1 (C- 8), 48.4 (C-5), 35.0 (C-6).
(1R,2R,3S,7R,7aR)-3-(Hydroxymethyl)hexahydro-1H-pyrrolizine-1,2,7-triol (3,7- diepi-australine (281)).
HOH OH To a solution of 278 (20 mg, 0.041 mmol) in MeOH (1 mL) was
7 7a 1 added PdCl (11 mg, 0.062 mmol). The mixture was stirred at rt under OH 2 5 N 3 an atmosphere of H2 (balloon) for 3 h, follow by the dropwise
8 OH 281 addition of conc. HCl (5 drops) at rt for 15 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 3,7-diepi-australine 281 as a white solid (7.0 mg, 90%).
24 [α] D -9.3 (c 1.1, H2O). MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO4 (M+H ) 190.1079, found 190.1074. -1 IR υmax (cm ): 3370, 3309, 2509, 1454, 1202, 1060. 1 H NMR (D2O) δ 4.30 (dt, 1H, J6,7 = J7,7a = 6.5, J6,7 = 6.0 Hz, H-7), 4.16 (brd, 1H, J2,3 =
3.5 Hz, H-2), 4.13 (s, 1H, H-1), 3.97 (dd, 1H, J8,8’ = 11.8, J3,8 = 7.0 Hz, H-8), 3.92 (dd,
1H, J8,8’ = 12.0, J3,8’ = 7.0 Hz, H-8’), 3.28 (ddd, 1H, J2,3 = 9.0, J3,8 = 7.0, J2,3 = 4.0 Hz,
220
H-3), 3.11 (ddd, 1H, J5,5 = 10.0, J5,6 = 10.0, J5,6 = 6.0 Hz, H-5α), 3.06 (dd, 1H, J7,7a =
2.0, J1,7a = 5.5 Hz, H-7a), 2.98 (t, 1H, J5,5 = J5,6 = 8.5 Hz, H-5β), 2.23-2.18 (m, 1H, H- 6α), 1.80-1.72 (m, 1H, H-6β). 13 C NMR (D2O) δ 80.5 (C-1), 79.8 (C-2), 78.2 (C-7a), 75.1 (C-7), 64.9 (C-3), 57.6 (C- 8), 46.4 (C-5), 34.5 (C-6).
21 281HCl salt: [α] D -21 (c 0.63, H2O), its synthetic enantiomer, 1,2-diepi-alexine·HCl
193 20 (lit. [α] D +33 (c 0.1, H2O)).
1 H NMR (D2O) δ 4.63 (dt, 1H, J6,7 = 8.0, J6,7 = J7,7a = 6.0 Hz, H-7), 4.41 (brs, 1H, H-1),
4.35 (d, 1H, J1,2 = 2.5 Hz H-2), 4.13 (dd, 1H, J8,8’ = 12.5 Hz, J3,8 = 5.0 Hz, H-8), 4.10 (d,
1H, J8,8’ = 9.0 Hz, H-8), 4.06-4.02 (m, 1H, H-3), 3.84 (d, 1H, J7,7a = 6.5 Hz, H-7a), 3.75
(dd, 1H, J5,5 = 11.3, J5,6 = 6.3 Hz, H-5), 3.73 (dd, 1H, J5,5 = 10.8, J5,6 = 6.3 Hz, H-5), 2.54-2.48 (m, 1H, H-6), 2.07-1.99 (m, 1H, H-6). 13 C NMR (D2O) δ 80.1 (C-7a), 77.6 (C-1), 77.1 (C-2), 73.1 (C-7), 67.7 (C-3), 55.8 (C- 8), 48.6 (C-5), 33.1 (C-6).
(1R,2R,3S,6S,7aR)-3-(Acetoxymethyl)hexahydro-1H-pyrrolizine-1,2,6-triyl triacetate (282a); and (1R,2R,3S,6S,7aR)-3-(hydroxymethyl)hexahydro-1H- pyrrolizine-1,2,6-triol (282). OAc H H OH 7 1 7 1 AcO OAc HO OH 5 N 3 5 3 N 8 OAc 8 OH
282a 282
To a solution of 279 (12 mg, 0.025 mmol) in MeOH (1 mL) was added PdCl2 (6.6 mg,
0.037 mmol). The mixture was stirred at rt under an atmosphere of H2 (balloon) for 3 h, follow by dropwise addition of conc. HCl (5 drops). The mixture was stirred at rt for 21 h. The mixture was filtered through a celite pad and the solids were washed with MeOH. The combined filtrates were evaporated in vacuo and the residue was dissolved in water (1 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave product as a white solid (4.0 mg). However, the 1H NMR spectrum of this compound showed an impure product and
221
therefore this compound was acetylated. To a solution of the above product in pyridine(0.5 mL) was added acetic anhydride (20 µL, 0.254 mmol) and a crystal of 4- dimethylaminopyridine. The mixture was stirred at rt for 24 h. The reaction was quenched by the addition of sat. NaHCO3 solution, followed by removal of the solvent under reduced pressure and the residue was extracted with CH2Cl2 (3x5 mL) and the combined CH2Cl2 extracts were washed with brine, dried (Na2CO3) and evaporated, then purified by FCC (70:30 EtOAc/petrol to 8.5:1:0.5 EtOAc/MeOH/NH3) to give 282a as a pale yellow oil (3.6 mg). Rf 0.49 (90:10 EtOAc/MeOH). MS (ESI +ve) m/z 358 (M+H+, 100%).
23 [α] D -10.6 (c 0.3, CHCl3). -1 IR υmax (cm ): 2924, 2825, 1736, 1372, 1221, 1039. 1H NMR δ 5.40 (app. t, 1H, J 4.5 Hz, H-6), 5.35 (dd, 1H, J 4.5, 1.5 Hz, H-2), 4.83 (brd, 1H, J 1.5 Hz, H-1), 4.38 (dd, 1H, J 11.5, 6.5 Hz, H-8), 4.23 (dd, 1H, J 12.0, 7.0 Hz, H- 8), 3.75 (app. dt, 1H, J 6.3, 5.5 Hz, H-3), 3.59-3.55 (ddd, 1H, J 7.5, 3.0, 3.0 Hz, H-7a), 3.27 (dd, 1H, J 10.5, 4.0 Hz, H-5), 2.99 (d, 1H, J 10.5 Hz, H-5), 2.35 (dd, 1H, J 14.3, 7.8
Hz, H-7), 2.13-2.05 (m, 13H, 4xCH3 and H-7). 13C NMR δ 170.8 (CO), 170.6 (CO), 170.1 (CO), 169.3 (CO), 82.7 (C-1), 79.8 (C-2),
75.6 (C-6), 69.3 (C-7a), 61.0 (C-3), 59.6 (C-8), 53.6 (C-5), 36.3 (C-7), 21.3 (CH3 (Ac)),
20.9 (2xCH3 (Ac)), 20.8 (CH3 (Ac)).
To a solution of 282a (3.6 mg, 0.010 mmol) in MeOH (1 mL) was added Amberlyst A- 26 (OH-) resin (15 mg). The reaction was stirred for 3 h at rt, the mixture was then filtered through a celite pad and the solids were washed with MeOH (10 mL). The combined filtrates were evaporated in vacuo to give 7-deoxy-3,6-diepi-casuarine 282 as a pale yellow solid (2.3 mg, 50%).
23 [α] D +28.6 (c 0.23, MeOH). MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO4 (M+H ) 190.1079, found 190.1074. -1 IR υmax (cm ): 3400, 2923, 2852, 1460, 1053, 1038. 1 H NMR (D2O) δ 4.57 (brs, 1H, H-6), 4.22 (brs, 1H, H-2), 4.09 (brs, 1H, H-1), 4.00 (dd,
1H, J8,8 = 11.5 Hz, J3,8 = 6.5 Hz, H-8), 3.92 (dd, 1H, J8,8 = 11.3, J3,8 = 6.8 Hz, H-8), 3.59
222
(t, 1H, J1,7a = J7,7a = 7.8 Hz, H-7a), 3.41 (brs, 1H, H-3), 3.31 (dd, 1H, J5,5 = 10.5, J5,6 =
2.5, H-5), 2.92 (d, 1H, J5,5 = 10.5 Hz, H-5), 2.22-2.17 (m, 1H, H-7), 2.04-2.00 (m, 1H, H-7). 13 C NMR (D2O) δ 81.1 (C-1), 79.7 (C-2), 72.9 (C-6), 71.4 (C-7a), 65.0 (C-3), 57.5 (C- 8), 56.2 (C-5), 38.0 (C-7).
(1R,6R,7S,8R,8aR)-7,8-Bis(benzyloxy)-6-(tert- butyldimethylsilyloxy)octahydroindolizin-1-ol (283).
OBn HO H To a solution epoixde of 260 (20.0 mg, 0.042 mmol) in anhydrous OBn 1 8a 7 THF (1 mL) was added dropwise a solution of lithium aluminium 3 N 5 OTBS hydride (62.4 µL, 0.062 mmol). The reaction was stirring for 20 h 283 at rt and then evaporated to leave a residue which was chromatographed on silica gel by FCC (20:80 EtOAc/petrol to 100% EtOAc) to give 283 as a yellow viscous oil (12 mg, 60%).
Rf 0.59 (5:95 MeOH/EtOAc).
25 [α] D -31.1 (c 1.8, CHCl3). MS (ESI +ve) m/z 484 (M+H+, 100%). + HRMS (ESI +ve) calculated for C28H42NO4Si (M+H ) 484.2883, found 484.2871. -1 IR υmax (cm ): 3390, 3030, 2922, 2850, 1248, 1092. 1H NMR δ 7.40-7.25 (m, 10H, Ar), 4.96 (d, 1H, J 11.5 Hz, CHHPh), 4.73 (d, 1H, J 11.5 Hz, CHHPh), 4.70 (d, 1H, J 11.0 Hz, CHHPh), 4.60 (d, 1H, J 12.0 Hz, CHHPh), 4.15 (brs, 1H, H-6), 4.01 (app. dt, 1H, J 9.5, 4.8 Hz, H-1), 3.80 (app. t, 1H, J 9.3 Hz, H-8), 3.35 (dd, 1H, J 9.0, 3.0 Hz, H-7), 2.94-2.90 (m, 2H, H-5 and H-3), 2.40 (app. dt, 1H, J 8.8, 8.0 Hz, H-3), 2.22-2.14 (m, 2H, H-5 and H-2), 1.95 (dd, 1H, J 9.3, 5.8 Hz, H-8a),
1.59-1.53 (m, 1H, H-2), 0.90 (s, 9H, t-Bu), 0.08 (s, 3H, CH3), 0.04 (s, 3H, CH3). 13C NMR δ 138.6 (C), 138.5 (C), 128.5 (CH), 128.4 (CH), 128.3 (CH), 127.8 (CH),
127.5 (CH), 127.4 (CH), 85.2 (C-7), 78.5 (C-8), 75.2 (C-1), 74.7 (CH2), 73.4 (C-8a),
71.7 (CH2), 68.2 (C-6), 56.3 (C-5), 51.5 (C-3), 31.8 (C-2), 25.8 (C(CH3)3), 18.2 (C), -4.5
(CH3), -4.53 (CH3).
223
(1R,6R,7R,8R,8aR)-Octahydroindolizine-1,6,7,8-tetraol (1,6-diepi-castanospermine (284)).
OH To a solution of 283 (37.9 mg, 0.079 mmol) in MeOH (2 mL) was HO H OH 8a added PdCl2 (20.9 mg, 0.112 mmol). The mixture was stirred at rt 1 7 3 N 5 under an atmosphere of H (balloon) for 12 h, follow by the dropwise OH 2 284 addition of conc. HCl (10 drops), The mixture was stirred at rt for 3 d. The mixture was filtered through a celite pad and the solids were washed with MeOH. The filtrate was evaporated in vacuo and the residue was dissolved in water (1.5 mL) and applied to a column of Amberlyst A-26 (OH-) resin (3 cm). Elution with water followed by evaporation in vacuo gave 1,6 diepi-castanospermine 284 in 95% purity, as a yellow viscous oil (14 mg, 95%).
25 52 24 [α] D -74.2 (c 1.5, MeOH), lit. [α] D -72.0 (c 0.7, MeOH). MS (ESI +ve) m/z 190 (M+H+, 100%). + HRMS (ESI +ve) calculated for C8H16NO4 (M+H ) 190.1079, found 190.1072. -1 IR υmax (cm ): 3365, 3308, 2914, 2888, 1444, 1202, 1060. 1 H NMR (D2O) δ 4.29-4.25 (m, 1H, H-1), 4.03 (brs, 1H, H-6), 3.68 (t, 1H, J7,8 = J8,8a =
9.8 Hz, H-8), 3.53 (dd, 1H, J7,8 = 9.0, J6,7 = 3.5 Hz, H-7), 3.05 (dd, 1H, J5,5 = 12.5, J5,6 =
2.5 Hz, H-5), 2.92 (t, 1H, J2,3 = J3,3 = 8.5 Hz, H-3), 2.48 (dt, 1H, J2,3 = J3,3 = 9.3, J2,3 =
9.0 Hz, H-3), 2.42 (d, 1H, J5,5 = 12.8 Hz, H-5), 2.34-2.25 (m, 1H, H-2), 1.99 (dd, 1H,
J8,8a = 9.5, J1,8a = 6.5 Hz, H-8a), 1.67-1.62 (m, 1H, H-2). 13 C NMR (D2O) δ 75.8 (C-7), 74.6 (C-1), 74.0 (C-8a), 72.2 (C-8), 69.3 (C-6), 55.7 (C- 5), 51.7 (C-3), 32.9 (C-2).
224 References
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236 Appendices
APPENDIX
237 Appendices
Appendix 1 Comparison of published 1H NMR data of australine 13.
60 Published Wormald et al. 188 181,182 183 60,184 186 Marco et al. from (in D O, pH Pearson et al. Furneaux et al. White et al. 170 Wong et al. 2 Denmark et al. (in D O + This thesis (in D O)* (in D O) (in D O)* (in D O)* 2 adjusted to (in D O) (D2O) 2 2 2 2 2 NaOD) 8.6) NMR (500 360 MHz 300 MHz 300 MHz 500 MHz 600 MHz 500 HMz 500 HMz Nucleus MHz) 4.29 (dd, J = 4.34 (t, J = 7.3 Hz, 4.14 (t, J = 7.9 4.09 (t, J = 6.8 4.22 (t, 1H, J = 7. H-1 4.08 (t, J = 7.5 Hz) 4.25 (t, J = 8 Hz) 4.04 (t, J = 7.8 Hz) 8.2, 7.4 Hz) H-1 or H-2) Hz) Hz) Hz) 4.01 (dd, J = 7.8, 3.96 (dd, J = 3.71 (dd, J = 9.5, 3.80 (dd, J = 9.4, 8. 3.79 (t, J = 7.5 3.89 (dd, 1H, J = H-2 3.73 (t, J = 8.5 Hz) 10.5 Hz, H-1 or 3.91 (t, J = 9 Hz) 9.5, 8.2 Hz) 8.3 Hz) Hz) Hz) 9.5, 8.0 Hz) H-2) 2.75-2.65 (m, 2.74-2.69 (m, H-3 2.80 (m) 2.70-2.50 (m, 2H) 3.79-3.63 (m, 3H) 2.78-2.72 (m, 2H) 2.58-2.52 (m, 2H) 2.64-2.61 (m, 2H) 2H) 2H) 2.80 (ddd, J = 2.75-2.65 (m, 2.74-2.69 (m, H-5 2.70-2.50 (m, 2H) 3.30-3.20 (m, 2H) 2.78-2.72 (m, 2H) 2.58-2.52 (m, 2H) 2.64-2.61 (m, 2H) 11.5, 6 Hz) 2H) 2H) 3.23 (ddd, J = 2.98 (ddd, J = 3.05 (ddd, J = 9.9, 3.15-3.12 (m, H-5´ 3.10-2.90 (m, 2H) 3.79-3.63 (m, 3H) 3.22-3.15 (m, 2H) 3.12 (m, 1H) 7.5, 2.1 Hz) 9.8, 7.6, 2.2 Hz) 7.5, 2.2 Hz) 1H) 2.10 (dddd, J = 1.95-1.90 (m, 2.05-2.00 (m, H-6 1.90-1.70 (m, 2H) 2.22-2.02 (m, 2H) 2.06-2.02 (m, 1H) 1.87-1.82 (m, 1H) 1.95-1.92 (m, 1H) 6, 2.4, 2.1 Hz) 1H) 1H) 2.00 (dddd, J = 1.90-1.80 (m, 1.97-1.89 (m, H-6´ 11.5, 7.5, 4.2 1.90-1.70 (m, 2H) 2.22-2.02 (m, 2H) 2.01-1.92 (m, 1H) 1.79-1.71 (m, 1H) 1.87-1.82 (m, 1H) 1H) 1H) Hz) 4.43 (ddd, J = 4.19 (dt, J = 2.2, 4.27 (dt, J = 2.2, 4.37-4.35 (m, H-7 4.21 (m) 4.54 (m, H-7) 4.39 (s) 4.29 (m) 4.4, 4.2, 2.4 Hz) 4.2 Hz) 4 Hz) 1H) 3.27 (dd, J = 3.02 (dd, J = 7.6, 3.09 (dd, J = 7.5, 4. 3.15 (dd, J = 7.5, 3.17 (dd, 1H, J = H-7a 3.10-2.90 (m, 2H) 3.30-3.20 (m, 2H) 3.22-3.15 (m, 2H) 7.4, 4.4 Hz) 4.4 Hz) Hz) 4.5 Hz, 1H) 7.8, 4.8 Hz) 3.63 (dd, J = 11, 3 3.86 (dd, J = 13.2, 3.81 (dd, J = 12, 3 3.60 (dd, J = 12, 3.70 (dd, J = 11.8, 3.75 (dd, J = 11.3 3.79 (dd, 1H, J = H-8 3.85 (m) Hz) 3.1 Hz) Hz) 3.7 Hz) 3.5 Hz) 3.8 Hz) 12.0, 3.5 Hz) 3.45 (t, J = 11, 6.5 3.63 (dd, J = 12, 6 3.43 (dd, J = 12, 3.51 (dd, J = 11.8, 3.57 (dd, J = 11.5 3.61 (dd, 1H, J = H-8 3.68 (m) 3.79-3.63 (m, 3H) Hz) Hz) 6.6 Hz) 6.6 Hz) 6.8 Hz) 11.5, 7.0 Hz) * Signals were not assigned
238 Appendices
Appendix 2 Comparison of published 13C NMR data of australine 13 Pearson Furneaux White Denmark Wong Marco Published Kato et al.55 et al.181,182 et al.183 et al.60,184 et al.170. et al.186 et al.188 This thesis from (in D O) (D O) 2 (in D O)* (in D O)* (in D O)* (in D O) (in D O)* (in D O + NaOD) 2 2 2 2 2 2 2 Ref residual Ref TPS Ref dioxane ** ** Ref TPS Ref CH CN Nucleus methanol ** 3 NMR (125 MHz) 90 MHz 75 MHz 100 MHz 100 MHz 150 MHz 125 MHz 125 MHz C-1 75.9 73.9 75.3 73.4 72.7 72.2 77.2 73.7 # C-2 81.8 (C-7 / C-2) 79.5 78.1 79.1 78.4 78.0 82.8 (C-7 / C-2)# 79.5 C-3 73.3 71.4 73.3 70.8 70.2 69.7 75.3 71.1 C-5 54.6 52.7 54.9 52.1 51.6 51.0 55.2 52.4 C-6 38.0 35.9 37.1 35.4 34.9 34.4 38.2 35.8 # C-7 72.3 (C-7a / C-7) 70.3 70.8 69.8 69.1 68.6 72.5 (C-7a / C-7)# 70.1 # C-7a 73.5 (C-2 / C-7a) 71.7 74.2 71.0 70.5 69.8 75.3 (C-2 / C-7a)# 71.3 C-8 65.5 63.1 58.5 62.9 62.2 61.9 66.5 63.5 * Signals were not assigned. ** Not given # (Original assignment / reassignment)
239