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ISOLATION AND STRUCTURAL STUDIES OF NATURAL PRODUCTS OF SOLIDAGO OHIOENSIS RIDDELL, AMPHIACHYRIS DRACUNCULOIDES (DC.) NUTT., ANDAGERATINA COLESTINUML.
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Pamela L. Boner, B.S. (Pharm.)
*****
The Ohio State University 1997
Dissertation Committee: Approved by
Raymond W. Doskotch, Ph.D., Adviser
Robert W. Brueggemeier, Ph.D.
Robert W. Curley, Ph.D.
Larry W. Robertson, Ph.D. Adviser College of Pharmacy UMI Number: 9801647
UMI Microform 9801647 Copyright 1997, by UMI Company. Ail rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
The genus Solidago (Fam. Asteraceae) is known to produce clerodane diterpenes. Solidago ohioensis Riddell, commonly known as Ohio goldenrod, has not been studied chemically. This study of S. ohioensis was initiated to examine the phytochemicals produced by the species and subject them to spectral and chemical studies. Extensive column chromatography of the terpene fractions of the ethanolic extract of the aerial parts and roots on silica gel and reversed-phase adsorbents with a variety of solvent systems, and final crystallization when possible gave ten trans- clerodane diterpenes, and two monoterpene glucosides. Their structures were established by physical and chemical methods. High field ID and 2D NMR techniques were used to make complete and NMR spectral assignments.
Nine diterpenes and both glucosides were isolated from the aerial extract. Three were known compoimds, the furan diterpene hardwickiic acid, and two butenolides, patagonic acid, and 15-methoxypatagonic acid. The new diterpenes were two hydroxybutenolides of the known patagonic, and clerodermic acids; three substituted tetrahydrofurans, methyl ohioenate B and C, and ohioenic acid D; and the cinnamoyl ester, methyl ohioenate A. The glucosides were the known (-)-bomyi P-D-
glucopyranoside and its monoacetate, a new compound. From the roots were isolated
the known hardwickiic acid and its corresponding C-19 alcohol hardwickiol.
It appears that hardwickiic acid, the major diterpene, is the biosynthetic
precursor to the other diterpenes via 1,4-conjugate addition of oxygen to the furan ring.
Opening to the dialdehyde with subsequent oxidations and reductions to give the
appropriate functionalities leads to the other isolated diterpenes. Patagonic acid, the a-
substituted butenolide was prepared from hardwickiic acid via singlet oxygen addition.
Hardwickiic acid, the most abundant of the isolated clerodanes, is known to have
antimicrobial activity.
Amphiachyris dracunculoides (DC.) Nutt., from Oklahoma, (Fam. Asteraceae) was subjected to a phytochemical study avoiding all alcoholic solvents in the extraction and isolation procedure to avoid formation of addition artifacts with alcoholic substituents. This resulted in the isolation of six c/s-clerodane diterpenes. One was a new compound amphiacrolide R with the amphiacrolide skeleton and a C-3 ketone. The other five were identical to known amphiacrolides. Their structues were established by physical and chemical methods. The isolation of amphiacrolide E and I (the ethyl, and methyl ether of the same dihemiacetal) in a fractionation sequence designed to exclude methanol and ethanol shows that these compounds obtained in a previous study are not isolation artifacts.
The 95% ethanolic extract of Ageratina colestinum L. displayed good antimicrobial activity against several microorganisms. Four antifungal polymethoxylted
in flavones were isolated in good yield and identified as the known compounds linderoflavone B, eupalestin, 5'-methoxynobiIetin, and nobiletin. Complete ’^C NMR assignments were made. This is the first report of the antifungal activity for three of these flavones.
IV DEDICATION
To Mom and Dad
with love ACKNOWLEDGMENTS
I wish to thank;
My husband, Ferdi, for his love, support, friendship, and many dinners prepared during the writing of this dissertation.
Dr. Raymond W. Doskotch, for his advise, guidance, and help in maturing as a scientist.
The faculty of the Division of Medicinal Chemistry and Pharmacognosy, for their help and willingness to share their experience and knowledge.
Joan Dandrea and Kathy Brooks for their assistance with making the graduate school experience a little easier.
Mr. John W. Fowble, and Mr. John Miller, for all the help with instrumentation and computers.
Dr. Charles E. Cottrell, and Dr. D. Chang at the Ohio State University Chemical Instrumentation, for the high field NMR and mass spectral data.
Dr. Larry W. Robertson, Panadda (Bee) Yaipakdee, Rosaria Rojas, and Haiyan Pu for all the antimicrobial testing.
My lab buddy. Dr. Michael J. Pcolinski, for his friendship, laughter, and scientific training.
Dr. Aime Quinn, Dr. Jill O’Reilly, and Nancy Gilbert for their lively and entertaining discussions and friendships throughout the years.
My family for their encouragement and understanding, which made this work possible.
XenoBiotics Laboratories, The Ohio State University, the College of Pharmacy, and the Division of Medicinal Chemistry and Pharmacognosy, for their financial support.
vi VITA
September 19, 1960 Bom - Mishawaka, Indiana
June 1983 ...... B.S. Pharmacy, Purdue University, West Lafayette, Indiana
1983-198 4 ...... Pharmacist, Memorial Hospital, South Bend, Indiana
1984-1986 ...... Pharmacist, Center Pharmacy, Miami Shores, Florida
1986-1989 ...... Pharmacist, Pharmacy T emporaries, Miami, Florida
1989-1990...... Pharmacist, Miami Children’s Hospital, Miami, Florida
1991-1997...... Graduate Teaching Associate, College of Pharmacy, Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University
FIELDS OF STUDY
Major Field: Pharmacy Specialization: Natural Products Chemistry
VII TABLE OF CONTENTS
ABSTRACT...... ii
DEDICATION...... v
ACKNOWLEDGMENTS...... vi
VITA...... vii
LIST OF FIGURES...... xii
LIST OF TABLES...... xix
LIST OF ABBREVIATIONS...... xxii
CHAPTER 1...... 1 Introduction ...... I CHAPTER 2...... 16 Diterpenes and Monoterpenes Isolated from Solidago ohioensis...... 16 Extraction of plant material ...... 16 Structure Elucidation of Diterpenes ...... 21 Hardwickiic acid (6 8 ) ...... 21 Hardwickiol (79) ...... 36 Methyl ohioenate A (80) ...... 39 Butenolides from S. ohioensis ...... 51 Patagonic acid (83) ...... 52 15-Methoxypatagonic acid (85) ...... 61 15-Hydroxypatagonic acid (87) ...... 63
16-Hydroxyclerodermic acid (8 8 )...... 70 Methyl ohioenate B (91) ...... 82 Methyl ohioenate C (92) ...... 8 8
VIII Ohioenic acid D (93) ...... 89 Synthesis of patagonic acid (83) ...... 93 Monoterpenes from Solidago ohioensis ...... 108 (-)-Bomyl P-D-glucopyranoside (94) ...... 108
(-)-Bomyl 6 -O-acetyl-p-D-glucopyranoside (97) ...... 118 CHAPTER 3...... 121 Isolation of c/s-clerodane diterpenes from Amphiachyris dracunculoides ...... 121 Extraction o f the plant material ...... 123 Amphiacrolide R (101) ...... 127 CHAPTER 4...... 141 Antifungal Flavones from Ageratina colestinum L...... 141 Antimicrobitil assay ...... 142 Plant materials ...... 143 Partitioning of the 95% ethanol extract ...... 143 Isolation of flavones ...... 146 Linderoflavone B (106) ...... 146 Eupalestin (107) ...... 148 5'-Methoxynobiletin (108) ...... 149 Nobiletin (109) ...... 154 CHAPTERS...... 158 Experimental ...... 158 Equipment and reagents ...... 158 Reagents ...... 158 Thin layer chromatography (TLC) ...... 158 Column chromatography adsorbents ...... 159 p-Anisaldehyde spray reagent ...... 159 Measurement of physical constants and spectra ...... 160 Plant materials ...... 161 Initial processing of plant material ...... 161 Solidago ohioensis leaves and stems ...... 161 Solidago ohioensis roots ...... 162 Partitioning o f the ethanolic extract residue ...... 162 Solidago ohioensis leaves and stems ...... 162 Solidago ohioensis roots ...... 163 Investigation of the 90% methanol- soluble terpenoids ...... 163 Isolation o f the terpenes from the 90% methanol fraction Solidago ohioensis leaves and stems ...... 163 Isolation of the terpenes from the 90% methanol fraction Solidago ohioensis roots ...... 163
IX Sample preparation for column chromatography ...... 164 Silica gel 60 adsorbent ...... 164 Reversed-phase adsorbent ...... 164 Isolation of the Terpenes from Solidago ohioensis Leaves and Stems ...... 165 Adsorption chromatography of the terpenes fraction ...... 165 Hardwickiic acid (6 8 ) ...... 165 Methyl ohioenate A (80) ...... 166 Méthylation of Fraction B-13 ...... 167 Acetate of methyl ohioenate A (81) ...... 168 Hydrolysis of methyl ohioenate A (80) to diol 82 ...... 169 Patagonic acid (83) ...... 170 15-Methoxypatagonic acid (85) ...... 171 15-Methoxypatagonic acid methyl ester ( 8 6 ) ...... 172 Chromatography of Fraction D ...... 173 Méthylation of Fraction D-3 ...... 173 Methyl ohioenate B (91) ...... 174 Methyl ohioenate C (92) ...... 174 15-Hydroxypatagonic acid (87) ...... 175 Reduction of 15-hydroxypatagonic acid (87) to patagonic acid (83) ...... 176 Patagonic acid methyl ester (84) ...... 177 16-Hydroxyclerodermic acid (8 8 ) ...... 178 Reduction of 16-hydroxyclerodermic acid ( 8 8 ) to clerodermic acid (89) ...... 179 Clerodermic acid methyl ester (90)...... 179 Adsorption chromatography of column fractions G and H ...... 180 Ohioenic acid D (93) ...... 180 (-)-Bomyl 6-0-acetyl-P-D-glucopyranoside (97) ...... 181 (-)-Bomyl P-D-glucopyranoside (94) ...... 182 (-)-Bomyl 2,3,4,6-tetra-G-acetyl-P-D-glucopyranoside (95)...... 183 (-)-Bomyl P-D-glucopyranoside tetramethyl ether (96) ...... 184 Isolation of the Terpenes from Solidago ohioensis roots ...... 184 Adsorption chromatography of the terpene fraction ...... 184
Hardwickiic acid (6 8 ), and hardwickiol (79) ...... 185 Reduction of hardwickiic acid ( 6 8 ) to hardwickiol (79) ...... 186 Synthesis of Patagonic Acid (83) ...... 186 Terpenes from Amphiachyris dracunculoides (DC.) Nutt...... 187 Plant material ...... 187 Initial processing of plant material ...... 188 Chromatography of tiie acetone extract residue ...... 188 Amphiacrolide B (98) ...... 189 Amphiacrolide C (99) ...... 189 Amphiacrolide D (100) ...... 190 Amphiacrolide R (101) ...... 191 Amphiacrolide R acetate (102)...... 192
X Amphiacrolide R methyl ether (103) ...... 193 Amphiacrolide E (104) ...... 193 Amphiacrolide I (105) ...... 195 Antimicrobial Compounds from Ageratina colestinum L...... 195 Plant material ...... 195 Initial processing of plant material ...... 196 Biological screening ...... 196 Partitioning of the ethanolic extract residue Ageratina colestinum L...... 197 Separation of the terpenes from the flavonoids in the methanol partition fraction of Ageratina colestinum L...... 198 Adsorption chromatography of the terpenes ...... 198 Isolation of flavones ...... 198 Linderoflavone B (106) ...... 200 Eupalestin (107) ...... 201 5 '-Methoxynobiletin (108) ...... 201 Nobiletin (109) ...... 202 APPENDIX SPECTRA...... 203
LIST OF REFERENCES...... 269
XI LIST OF FIGURES
Figure 1 ; Clerodane (1) and labdane (2) ring systems ...... 2
Figure 2; Structure of clerodin (3)...... 3
Figure 3 : Biosynthesis of isoprene units from acetyl CoA ...... 4
Figure 4: Biosynthesis of cis and bans clerodanes ...... 5
Figure 5; Suggested mechanism for the cyclization of geranylgeranyl pyrophosphate
to cis-clerodane compounds from T. cordifolia ...... 8
Figure 6 : Extraction and fractionation of the ethanolic extract residue of the dried aerial parts of Solidago ohioensis ...... 18
Figure 7: Extraction and fractionation of the ethanolic extract residue of the dried roots of Solidago ohioensis ...... 19
Figure 8 : Structure of hardwickiic acid ( 6 8 ) ...... 26
Figure 9: Proton coupled units of hardwickiic acid ( 6 8 )...... 26
Figure 10: Upfield region of the COLOC spectrum hardwickiic acid ( 6 8 ) ...... 27
Figure 11 : Connection of partial structures A and B to form decalin ring ...... 28
Figure 12: Partial structure F of hardwickiic acid ( 6 8 ) ...... 29
Figure 13: Connection of partial structures C, D, and E to decalin ring ...... 30
Figure 14: NOE difference spectrum for irradiation of Me-20
of hardwickiic acid ( 6 8 )...... 32
XI1 Figure 15: NOE by difference results for Me-20, Me-17, and Me-18 irradiations, of
hardwickiic acid, expressed as percent enhancements ...... 33
Figure 16: NOE by difference results for H-10, and H- 6 P irradiations, of hardwickiic acid, expressed as percent enhancements ...... 34
Figure 17: ‘^C NMR assignments for hardwickiic acid ( 6 8 ) ...... 35
Figure 18: Structure of hardwickiol (79) ...... 40
Figure 19: Structure of methyl ohioenate A (80), its acetate 81, and parent diol 82 ...... 40
Figure 20: /ram-Decalin ring of methyl ohioenate A (80) with selected COLOC interactions ...... 48
Figure 21 : Partial structure G of methyl ohioenate A (80) ...... 49
Figure 22: Structure of patagonic acid (83) and methyl patagonate (84) ...... 56
Figure 23: Selected COLOC interactions for butenolide ring of patagonic acid (83)... 56
Figure 24: Proposed MS fragmentation of patagonic acid (83) ...... 57
Figure 25: Structure of 15-methoxypatagonic acid (85), methyl ester ( 8 6 ), and 15-hydroxypatagonic acid (87) ...... 6 6
Figure 26: Summary of chemical conversions performed with 15-hydroxypatagonic acid (87) ...... 69
Figure 27: Structure of 16-hydroxyclerodermic acid ( 8 8 ) ...... 72
Figure 28: 'H NMR temperature studies upfield region 16-hydroxyclerodermic acid
(8 8 ) in pyr-ds (270 MHz) ...... 75
Figure 29: Upfield region INADEQUATE experiment 16-hydroxyclerodermic acid (8 8 ) ...... 77
Figure 30: NOE results for H-14, H-16, and HO-16 irradiations, of 16- hydroxyclerodermic acid expressed as percent enhancements ...... 78
Figure 31 : Structures of methyl ohioenate B (91), C (92) and ohioenic acid D (93) .....83
XIII Figure 32: Downfield region of the COLOC spectrum
methyl ohioenate B (91) in CDCI 3 ...... 87
Figure 33: Selected COLOC interactions of methyl ohioenate B (91) ...... 8 8
Figure 34: Selected COLOC interactions of ohioenic acid D (93) ...... 92
Figure 35: *H NMR spectrum ohioenic acid D methyl ester in CDCI 3 ...... 95
Figure 36: '^C NMR spectrum ohioenic acid D methyl ester in CDCI 3 ...... 96
Figure 37: Proposed MS fragmentation of ohioenic acid D (93) ...... 97
Figure 38: Retrosynthetic analysis of diterpenes from Solidago ohioensis ...... 99
Figure 39: Routes to dialdehyde from hardwickiic acid ( 6 8 )...... 100
Figure 40: Synthesis of patagonic acid (83) ...... 1 0 2
Figure 41 : Reaction conditions attempted to synthesize clerodane dialdehyde by photooxygenation ...... 105
Figure 42: Proposed hydrolysis products and derivatives from diethoxytetrahydrofriran clerodanes ...... 106
Figure 43 : Extraction of the tops of Solidago ohioensis ...... 107
Figure 44: c/5 -Clerodanes from Linaria japonica ...... 108
Figure 45: Structures of (-)-bomyl P-D-glucopyranoside (94), tetraacetate 95, tetramethyl ether 96,
and (-)-bomyl 6 -O-acetyl-p-D-glucopyranoside (97) ...... 110
Figure 46: Partial structures J, K, and L of tetraacetate 95 ...... 115
Figure 47: NOE results for Me - 8 (0.83 ppm) and Me-9 (0.84 ppm), of tetraacetate 95, expressed as percent enhancements ...... 117
Figure 48: NOE results for H-3a, H-5a, and H- 6 a, of tetraacetate 95, expressed as percent enhancements ...... 117
Figure 49: cw-Clerodanes from Amphiachyris dracunculoides ...... 124
XIV Figure 50: Extraction of the above ground portion of Amphiachyris dracunculoides ...... 125
Figure 51 : Proton coupled units of amphiacrolide R (101) ...... 129
Figure 52: Selected NOE by difference enhancements by percent for methyl amphiacrolide R (103) in CDCI 3 ...... 139
Figure 53: Amphiacrolide R with H-4a and H-4P ...... 140
Figure 54: Conformational drawing for amphiacrolide R and the octant projection showing the lower right positive quadrant predicting a positive Cotton effect curve...... 140
Figure 55: Extraction and fractionation of the ethanolic extract residue of the dried whole plant of Ageratina coelestinum ...... 144
Figure 56: Structure of the flavones isolated from Ageratina coelestinum ...... 153
Figure 57: *H NMR spectrum of hardwickiic acid ( 6 8 )...... 204
Figure 58: IR spectrum of hardwickiic acid ( 6 8 ) ...... 205
Figure 59: ‘H NMR spectrum of hardwickiol (79) ...... 206
Figure 60: IR spectrum of hardwickiol (79) ...... 207
Figure 61: 'H NMR spectrum of methyl ohioenate A (80) ...... 208
Figure 62: IR spectrum of methyl ohioenate A (80) ...... 209
Figure 63: ’H NMR spectrum of methyl ohioenate A acetate (81) ...... 210
Figure 64: IR spectrum of methyl ohioenate A acetate (81) ...... 211
Figure 65 : ’ H NMR spectrum of methyl ohioenate A diol (82) ...... 212
Figure 6 6 : IR spectrum of methyl ohioenate A diol (82) ...... 213
Figure 67: ‘ H NMR spectrum of patagonic acid (83) ...... 214
Figure 6 8 : IR spectrum of patagonic acid (83) ...... 215
XV Figure 69: ‘H NMR spectrum methyl patagonate (84) ...... 216
Figure 70: IR spectrum methyl patagonate (84) ...... 217
Figure 71 : ‘H NMR spectrum of 15-methoxypatagonic acid (85) ...... 218
Figure 72: IR spectrum of 15-methoxypatagonic acid (85) ...... 219
Figure 73: 'H NMR spectrum 15-methoxypatagonic acid methyl ester ( 8 6 )...... 220
Figure 74: IR spectrum 15-methoxypatagonic acid methyl ester ( 8 6 ) ...... 221
Figure 75: ‘H NMR spectrum of 15-hydroxypatagonic acid (87) ...... 222
Figure 76: IR spectrum of 15-hydroxypatagonic acid (87) ...... 223
Figure 77: NMR spectrum 16-hydroxyclerodermic acid ( 8 8 ) ...... 224
Figure 78; IR spectrum 16-hydroxyclerodermic acid ( 8 8 )...... 225
Figure 79: 'H NMR spectrum clerodermic acid (89) ...... 226
Figure 80: IR spectrum clerodermic acid (89) ...... 227
Figure 81: *H NMR spectrum methyl clerodermate (90) ...... 228
Figure 82: IR spectrum methyl clerodermate (90) ...... 229
Figure 83: 'H NMR spectrum methyl ohioenate B (91) ...... 230
Figure 84: IR spectrum methyl ohioenate B (91) ...... 231
Figure 85: ‘H NMR spectrum methyl ohioenate C (92) ...... 232
Figure 8 6 : IR spectrum methyl ohioenate C (92) ...... 233
Figure 87: NMR spectrum ohioenic acid D (93) ...... 234
Figure 8 8 : IR spectrum ohioenic acid D (93) ...... 235
Figure 89; 'H NMR spectrum of (-)-bomyl P-D-glucopyranoside (94) ...... 236
XVI Figure 90: IR spectrum of (-)-bomyl P-D-glucopyranoside (94) ...... 237
Figure 91: *H NMR spectrum of (-)-bomyl 2,3,4,6-tetra-O-acetyI-P-D-glucopyranoside (95) ...... 238
Figure 92: ER spectrum of (-)-bomyl 2,3,4,6-tetra-O-acetyi-P-D-gIucopyranoside (95) ...... 239
Figure 93 : *H NMR spectrum of (-)-bomyl 2,3,4,6-tetra-O-methyI-P-D-gIucopyranoside (96) ...... 240
Figure 94: IR spectrum of (-)-bomyl 2,3,4,6-tetra-O-methyI-P-D-gIucopyranoside (96) ...... 241
Figure 95: 'H NMR spectrum (-)-bomyl 6 -O-acetyI-p-D-gIucopyranoside (97) ...... 242
Figure 96: IR spectrum (-)-bomyl 6-0-acetyl-P-D-glucopyranoside (97) ...... 243
Figure 97: ‘H NMR spectrum amphiacrolide B (98) ...... 244
Figure 98: IR spectrum amphiacrolide B (98) ...... 245
Figure 99: 'H NMR spectrum amphiacrolide C (99) ...... 246
Figure 100: IR spectnun amphiacrolide C (99) ...... 247
Figure 101 : 'H NMR spectrum amphiacrolide D (100) ...... 248
Figure 102: IR spectrum amphiacrolide D (100) ...... 249
Figure 103: NMR spectrum amphiacrolide R (101) ...... 250
Figure 104: IR spectrum amphiacrolide R (101) ...... 251
Figure 105: CD spectnun of amphiacrolide R (101) ...... 252
Figure 106: *H NMR spectrum amphiacrolide R acetate (102) ...... 253
Figure 107: IR spectnun amphiacrolide R acetate (102) ...... 254
Figure 108: ‘H NMR spectrum amphiacrolide R methyl ether (103) ...... 255
XVII Figure 109: IR spectrum amphiacrolide R methyl ether (103) ...... 256
Figure 110: ‘H NMR spectrum amphiacrolide E (104) ...... 257
Figure 111: IR spectrum amphiacrolide E (104) ...... 258
Figure 112: NMR spectrum amphiacrolide I (105) ...... 259
Figure 113: IR spectrum amphiacroide I (105) ...... 260
Figure 114: 'H NMR spectrum linderoflavone B (106) ...... 261
Figure 115: UV spectrum linderflavone B (106) ...... 262
Figure 116: 'H NMR spectrum eupalestin (107) ...... 263
Figure 117: UV spectrum eupalestin (107) ...... 264
Figure 118; ‘H NMR spectrum 5'-methoxynobiIetin (108) ...... 265
Figure 119: UV spectrum 5'-methoxynobiletin (108) ...... 266
Figure 120: 'H NMR spectrum nobiletin (109) ...... 267
Figure 121: UV spectrum nobiletin (109) ...... 268
XVIII LIST OF TABLES
Table I : Clerodane diterpenes isolated from Solidago species ...... 10
Table 2: Compounds isolated from Solidago ohioensis ...... 20
Table 3. 'H and *^C NMR assignments for hardwickiic acid ( 6 8 ) in CDCI 3 ...... 24
Table 4: ‘H and '^C NMR assignments for hardwickiic acid ( 6 8 ) in pyridine-ds 25
Table 5: *H and ‘^C NMR assignments for hardwickiol (79) in CDCI 3 ...... 37
Table 6 : '^C NMR assignments for methyl ohioenate A (80), its acetate (81), and parent diol (82) in CDCI 3 ...... 44
Table 7; '^C NMR assignments for methyl ohioenate A (80), and acetate 81 in pyridine-ds ...... 45
Table 8 : 'H NMR assignments for methyl ohioenate A (80), its acetate 81,
and parent diol 82 in CDCI 3 ...... 46
Table 9: NMR assignments for methyl ohioenate A (80), and its acetate 81 in pyridine-ds ...... 47
Table 10: Comparison of ‘H NMR assignments (H-14, -15, -16) for butenolides to
hardwickiic acid ( 6 8 ) ...... 53
Table 11: Comparison of ‘^C NMR assignments (C -13, -14, -15, -16) for butenolides to hardwickiic acid ( 6 8 )...... 53
Table 12: ’^C NMR assignments for patagonic acid (83)
and methyl patagonate (84) in CDCI 3 ...... 58
XIX Table 13: H NMR assignments for patagonic acid (83),
and methyl patagonate (84) in CDCI 3 ...... 59
Table 14: • *H and NMR assignments for patagonic acid (83) in pyridine-ds ...... 60
Table 15: 'H and NMR assignments for 15-methoxypatagonic acid (85) in CDCI 3 ...... 64
Table 16: ‘H and '^C NMR assignments for
15-methoxypatagonic acid methyl ester ( 8 6 ) in CDCI 3 ...... 65
Table 17: ’H and ’^C NMR assignments 15-hydroxypatagonic acid (87) in pyridine-ds ...... 6 8
Table 18: 'H and NMR assignments 16-hydroxyclerodermic acid ( 8 8 ) in pyridine-ds ...... 76
Table 19: 'H and '^C NMR assignments for clerodermic acid (89) in CDCI3...... 79
Table 20: 'H and NMR assignments for clerodermic acid (89) in pyridine-ds ...... 80
Table 21: ’H and *^C NMR assignments methyl clerodermate (90) in CDCI3...... 81
Table 22: *H and '^C NMR assignments methyl ohioenate B (91) in CDCI 3 ...... 84
Table 23: *H NMR data comparisons for H-14, -15, -16 of methyl ohioenate B (91), C (92), and ohioenic acid D (93) to hardwickiic acid ( 6 8 ),
and 15-methoxypatagonic acid (85) in CDCI 3 ...... 8 6
Table 24: Comparisons o f‘^C NMR assignments for C-13, -14, -15, -16 to methyl ohioenate B (91), C (92), and ohioenic acid D (93),
to hardwickiic acid ( 6 8 ), and 15-methoxypatagonic acid (85) in CDCI 3 ..... 8 6
Table 25: 'H and NMR assignments for methyl ohioenate C (92) in CDCI3 90
Table 26: 'H and ’^C NMR data ohioenic acid D (93) in pyridine-ds ...... 94
Table 27: 'H and ‘^C NMR assignments
(-)-bomyl P-D-glucopyranoside (94) in CDCI 3 ...... 1 11
Table 28: ’H and ’^C NMR assignments for
(-)-bomyl 2,3,4,6-tetra-(9-acetyl-P-D-glucopyranoside (95) in CDCI 3 ...... 112
XX Table 29: 'H and ’^C NMR assignments for
(-)-bomyl 2,3,4,6 -tetra-O-methyI-P-D-glucopyranoside (96) in CDCI 3 113
Table 30: 'H and '^C NMR assignments for
(-)-bomyl 6 -O-acetyl-P-D-glucopyranoside (97) in CDCI 3 ...... 1 20
Table 31 : Compounds isolated from Amphiachyris dracunculoides Oklahoma collection ...... 126
Table 32: *H and NMR assignments for amphiacrolide R (101) in CDCI 3 130
Table 33: ‘H and NMR assignments for amphiacrolide R (101) in pyridine-ds ..131
Table 34: ‘H and '^C NMR assignments for
amphiacrolide R acetate (102) in CDCI 3 ...... 136
Table 35: 'H and ‘^C NMR assignments for amphiacrolide R methyl ether (103) in CDCI 3 ...... 137
Table 3 6 : Antimicrobial activity of Ageratina coelestinum partition fractions 145
Table 3 7 : Antimicrobial activity of Ageratina coelestinum MeOH column fractions ...... 145
Table 38: Flavones isolated from Ageratina coelestinum ...... 147
Table 39: Sources of flavones isolated from Ageratina coelestinum ...... 150
Table 40: 'H NMR assignments for flavones isolated from Ageratina coelestinum in CDCI 3 ...... 151
Table 41 : NMR assignments for flavones isolated from Ageratina coelestinum in CDCI 3 ...... 152
Table 42: Antimicrobial activity of flavones from Ageratina coelestinum ...... 156
XXI LIST OF ABBREVIATIONS
Ac Acetate group
AczO Acetic anhydride
23 [cc]d Specific rotation, at the sodium-D line at specified temperature in °C ang Angelic acid (cw-2-methyl-2-butenoic acid) anhyd Anhydrous
BB-decoupIed Broad Band decoupled *^C NMR experiment c Concentration in mg/10 ml
CD Circular dichroism spectroscopy
CDCI3 Deuterochloroform
CHCI3 Chloroform
CH3 CN Acetonitrile
Cr(acac) 3 Chromium(ni) acetylacetonate
COLOC correlation via LOng range Coupling heteronuclear NMR experiment concd Concentrated
XXII ‘h ,'h -c o s y Two-dimensional 'H,'H Correlation SpectroscopY
5 Nuclear magnetic resonance chemical shift in ppm
DEPT Distortionless Enhancement by Polarization Transfer
EIMS Electron Ionization Mass Spectrometry
Et Ethyl group
EtzO Diethyl ether
EtOH Ethanol
95% EtOH 95% ethanol
OEt Ethoxy group
EtOAc Ethyl acetate
FABMS Fast Atom Bombardment Mass Spectrometry
GGPP Geranylgeranyl pyrophosphate
HCl Hydrochloric acid
INADEQUATE Incredible Natural Abundance DoublE QUAntum Transfer Experiment
IR Infrared spectroscopy
J Absolute value of the coupling constant in Hz
K2 CO3 Potassium carbonate
X Wavelength
L1AIH4 Lithium aluminum hydride loge Logarithm of the molar extinction coefficient
M Molar concentration in moles per liter
VT Molecular ion in mass spectrometry xxiii Me Methyl group
MeOH Methanol
MHz Megahertz
mp Melting point
OMe Methoxy group
m/z The mass of an ion divided by its charge (usually unity in MS)
Frequency maximum of IR absorption bands
NaBH4 Sodium borohydride
NH4CI Ammonium chloride nm nanometer
NMR Nuclear Magnetic Resonance spectrometry
NOE Nuclear Overhauser Effect
GAc Acetate group ppm Parts per million pyr Pyridine pyr-ds Deuteropyridine
RP Reversed Phase
SFORD Single-Frequency Off-Resonance Decoupled ‘^C NMR experiment
Si Silica gel
[Q]x ellipticity (degcm^dmol') at x nm
TEA Trifluoroacetic acid xxiv THF Tetrahydrofliran
TLC Thin layer chromatography tig Tiglic acid (/ra/7 J-2 -methyi-2 -butenoic acid)
u v Ultraviolet spectroscopy
XXV CHAPTER 1
INTRODUCTION
During the last thirty years over 650 diterpenoids with the clerodane (I) ring system (Figure 1) have been isolated [I]. Diterpenes are C 2 0 compounds, built up from
four basic C5 units called isoprenes, and two of the most common bicyclic classes are the labdanes (2) and clerodanes (1) [2]. The numbering system is that proposed by
Rowe [3]. There is confusion in the literature regarding the absolute stereochemistry of isolated clerodane diterpenes. The compounds with the same absolute stereochemistry as clerodin (Figure 2) are referred to as «eo-clerodanes and the enantiomers as ent-neo- clerodanes [4] which correspond to the steroid ring stereochemistry. Clerodanes are also separated into the cis and trans compounds which defines the stereochemistry of the decalin ring junction.
Biosynthetically, the clerodanes appear to be derived from the labdanes via a series of methyl and hydride shifts [1]. The labdane isoprene units are derived from R- mevalonic acid [5]. Mevalonate is biosynthesized from acetyl CoA by the pathway shown in Figure 3. Mevalonate is the first committed step in terpene or steroid
biosynthesis [ 6 ]. Mevalonate undergoes the reactions outlined in Figure 3 to form
isopentenyl pyrophosphate and dimethylallyl pyrophosphate. These are the building blocks referred to as isoprene units which form all terpenes and steroids.
Four of these isoprene units condense to form geranylgeranyl pyrophosphate (4)
(GGPP). There are a number of cyclase enzymes which convert geranylgeranyl pyrophosphate into a variety of diterpenes [7]. For a review of the recent literature on the biosynthetic pathways leading to terpenoids smaller than C 3 0 , the enzymes and enzyme mechanisms, and genes encoding those enzymes, see Dewick [7] and references therein.
17 17 2 2
3 3
19 18 19
Figure 1: Clerodane (1) and labdane (2) ring systems OAc 3
Figure 2: Structure of clerodin (3)
When geranylgeranyl pyrophosphate is folded in the chair-chair conformation and cyclized in a concerted manner the trans-zxAi ring system of the labdanes result as shown in Figure 4. This is the stereochemistry found in the sterols. An enantiomeric folding of geranylgeranyl pyrophosphate would give the enantiomeric (jent-) labdanes.
The trans clerodanes can arise via a concerted migration of the labdane carbonium ion giving intermediate 6 . The cis compounds require a stepwise process, with a pause at intermediate 7. Depending on which of the C-4 methyl groups migrate, intermediate 7 can lead to either the cis or trans series of clerodane diterpenes [ 8 ]. This proposed biosynthetic pathway is supported by the isolation of partially rearranged labdanes in which the C-20 methyl group has migrated from C-10 to C-9 and are members of the class of diterpenes known as chettaphananes [9-12]. o
^ S C o A h MGC oA O ^ S C oA H M G C oA Acetyl CoA synthase reductase | | _ HO. O O Y Ï SCoA 3-hydro>Q^-3-raethylgIutaryICoA i?-mevabnic acid Acetoacetyl CoA (HMG CoA)
mevalonate '% .OH phosphomevalonate kinase kinase
ATP*" pqJ CO2H ATF^ p p q J CO2H
mevalonate 5-phosphate mevalonate 5-dphosphate
Gopenteryl mevalonate pyrophosphate decarboxylase isomerase PPO
isopentenyl diphosphate dimethylallyl diphosphate
Figure 3: Biosynthesis of isoprene units from acetyl CoA OPP
OPP
concerted migration concerted migration from
C 5 to C|o, C 9 , and Cg
OPP OPP
6 7
Me-19 to C5
Me-18 to C5
Figure 4: Biosynthesis ofcis and trans clerodanes The biosynthesis of a series of clerodane furanoditerpene lactones in Tinospora cordifolia has been investigated by feeding (4/?)-[4-^Hi,2-''*C]mevalonate [13].
Retention of all labels in the furanoditerpenes indicated that two hydride and two methyl shifts had occurred during formation of the ring system (Figure 5). Chemical degradation of the labeled furanoditerpene showed that C-19 was not labeled, and that the labeled methyl from the original gem-dimethyl had migrated from C-4 to C-5.
The vast majority of clerodanes have been isolated from Angiosperms
(Magnoliophyta) [1]. Clerodanes are represented in all but one sub-class of dicotyledons. Below the sub-class only a small number of Orders and Families have been shown to produce clerodanes. Based on taxonomic trends one might except to find clerodanes spread throughout all the Orders. There are two reasonable explanations.
The ability to biosynthesize clerodanes may have developed independently within the genera, or possibly a number of families may produce clerodanes that have not been isolated yet [1 ].
Clerodanes have been isolated from two species of monocotyledons, one genus of gymnosperm, three genera of liverworts, one genus of fungi, and one bacterium [ 1 ].
This diversity seems to support the independent development of clerodane biosynthesis.
The Compositae or Asteraceae is one of the largest and most familiar families of dicotoyledons. This family contains over 1,000 genera and 15,000 species and has been the subject of research for centuries [14]. It is generally accepted that the Compositae are a family grouped on the basis of uniformity of flower structure. The Compositae occupy a wide range of habitat types and are found in abundance on every continent
except Antarctica.
The constituents of this family has been widely investigated for a variety of
reasons leading to a great deal of information on the chemical constituents present.
These represent several classes of compounds including terpenes, polyacetylenes and
polysaccharide fructans, but no single class of compounds is unique to this family.
Many of the substances produced by this family are toxic or show other
physiological or pharmacological activity, and may be one reason why plants of this
family are rarely used as food for animals or humans. The most useful economic plant of this family is the sunflower {Helianthns annus). Many members of this family accumulate essential oils or other terpenoids, and these terpenoids with other phenolics are useful as flavoring agents and for their medicinal properties. A study of the early plant herbals reveals a large number of plants of the Compositae were used for their medicinal properties. For example, leaf extracts of the dandelion {Taraxacum oficinale) have long been known to be diuretic [14].
Despite being one of the largest plant families the Compositae are the source of relatively few products of medicinal importance. Only about 30 plant species are employed as crude drugs with about 20 pure substances commercially available [14].
Modem isolation techniques have led to the discovery of many new compoimds. These have served as lead compoimds in the synthesis of other biologically active substances.
However, the scientific and medicinal potential of the Compositae family has yet to be fully exploited. ,OPP OPP
Figure 5: Suggested mechanism for the cyciization of geranylgeranyl pyrophosphate to cis-clerodane compounds from T. cordifolia. denotes * and T denotes from (4iî)-[4-^Hi,2-*‘*C] mevalonate
Only around 10% of the species in this family have been studied chemically with work largely confined to root extracts. Polyacetylenes, and coumarins seem to be quite characteristic constituents of this family, and the diterpenoids are largely of the labdane and clerodane type.
Thirty-one genera have been shown to produce clerodanes [1] of both the cis and trans series, with the trans ring series dominating. These clerodanes contain a number of modifications with the simple C-11 through C-16 open chained compounds being the most abundant.
The genus Solidago has been studied chemically by several groups of workers, and shown to produce clerodanes of both the cis and trans series (Table 1). The genus also contains other classes of diterpenes including labdanes [15-18], abietanes [18-20], and kauranes [18,21]. Solidago species have been reported as medicinal plants of the
Chippewa Indians and used to treat fever, ulcer, boils, diseases of women, urinary, and lung disorders [ 2 2 ].
Solidago ohioensis Riddell, with the common name Ohio goldenrod has not been reported to have been studied chemically. Solidago ohioensis is a perennial growing to about 3 feet with alternate leaves having ciliate margins being oblong or lancolate in shape and may be toothed or entire. The leaves get progressively smaller upward on the stem, and the well-developed basal leaves have longer petioles. The flowers have yellow corollas, with disk flowers bisporangiate and ray flowers carpellate with seeds forming in both. It is found in wet habitats in the limestone regions of Ohio; for example, in the fens around Springfield, Ohio.
This study of S. ohioensis was initiated to examine the phytochemicals produced by this species and to subject them to spectral and chemical studies. Since, the literature already recorded that Solidago species yielded diterpenes it was not surprising that S. ohioensis afforded tra«j-clerodanes, the nature of which will be discussed. With the advances made in separation and instrumentation techniques, especially two dimensional high-field NMR, the characterization of natural products on limited quantities of material is now more easily accomplished. Thus, minor constituents and those from limited quantities of plant material can be successfully studied. Table 1: Clerodane Diterpenes Isolated from Solidago species
Species Compound Reference
S. altissima L. 8 , kolavenol ( 1 1 ) [ 1 ] kolavenic acid ( 1 2 ) 13,14,17 solidagonic acid (18) solidagolactone IV (23) or elongatolide A elongatolide B (25) solidagolactone VI (29) or elongatolide D 34
9,15,16,35 [23]
solidagolactone II (21) or [23,24] elongatolide C solidagolactone m ( 2 2 ) solidagolactone V (24) solidagolactone VII (25) or elongatolide E
S. elongata Nutt. 19, 21,23,25,29,36-39, [25] 41-42
S. gigantea sub serotina solidagoic acid A (43) [25] (Kuntze) Cronqu solidagoic acid B (44) 46-56 [26]
S. gigantea Ait. 54-56 [27]
S. vigauria L. 21-22,24,26-28,54-60,30 [28] solidagolactone VIE (31) continued on next page
10 Table 1 continued
Species Compound Reference
S. arguta Ait. 61-67 [29]
S', serotina Ait. 40,67,69-75 [30]
S. juncea Ait. hardwickiic acid ( 6 8 ) [20] junceic acid (76), 77
S', shortii Torr & Gray 32-33 [21]
S. canadensis L. 78 [18]
S. rugosa Mill. 11,68 [31]
continued on next page
II <0 00
roroN>roK)ioroio OONO)OI^WM-& illo p !F o 0 (D BMM# HI% X X X ? R w O P II ,, II II II N, T 1 Oi 1 S ^ | 9 *S§§ R O (Û Y 0 0
o 0 ) ! f 00 I0 ) Q. 0 0
ISO -& -k -& O (O OO N O) cn
CO CO CO CO 0 CO K) - » O % % o m t 2 < 2 < 2 < 2< 01 II p 9 I 1 % g %I a illX T3 % Table 1 continued
34 35R=H 38 X=OAc, a-Me-18 36 R=OAc 39 X=OAng, a-Me-18 37 R=OAng 40X=K p-Me-18
OH
X
41X=H 43 X=Me. Xi=C02H 50 Xi=H, X2=0, 42 X=OAng 44 X=CH20Ang, Xi=C02H 3-4 = double 45 X=Me, Xi=CHO 51 Xi=OKX2=H2, 46 X=Xi=CHO 3-4 = double 47 X=Me, Xi=CH20H 52 X-]=OH, X2 —H2 , 48 X=ChtOK Xi=Me 3-4 = epoxde 49 X=Xi=CH20H 53 X=Xi=OK X2=H2. 3-4 = single
continued on next page
13 Table 1 continued
54X=H 57 X=OAng, Xi=X2 = 0 H, Xa=H 61 X=CH2 0 H, Xi=H 55 X=OH 58 X—OTig, X^ —X2 ~OH, X3 —H 62 X=CH2 0 Ac, Xi=H
56 R=OGIu 59 X^OAng, X i—H, X2 '*"X 3 — O 63X=Me,Xi=H
60 X=OTig, Xi=H, X2 +Xs= O 64X=CH20KX i=OH 65 X=CH20Ac, Xi=OH
X
66 67X=Me 69 X=Xi=OH. X2 =H 6 8 X=CQzH 70 X=H, Xi+Xg= O
continued on next page
14 Table I continued
CHO CHO
71 13-14=E 73 13-14 = 2 72 13-14 = Z 74 13-14= E
75X=Me, X 3,X4=H, 3-4 =a-epoxide, p-Me-19 78 76 X=CQ2H, X3=X4=H, 3-4 = double 77 X=CQ 2H, X2=X4=H, 3-4 = p-epoxide, a-Me-19
15 Chapter 2
DITERPENES AND MONOTERPENES ISOLATED FROM SOLIDAGO OHIOENSIS
Extraction of Plant Material
The dried and powdered leaves and stems of Solidago ohioensis Riddell were treated with 95% ethanol to extract all but the polymeric carbohydrates and the lignins.
After solvent removal under reduced pressure at 40°C the residue was partitioned between pairs of solvent systems into four fractions differing in the polarity of the constituents (Figure 6 ). Solute concentrations were kept below 10% to minimize interface formation and cosolubilization. In order to avoid degradation of the natural products, the isolation procedure was conducted using mild conditions by avoiding heat, strong acids or bases. The roots were processed similarly into four fractions (Figure 7).
Thin-layer chromatographic (TLC) analysis of the fractions from the partitioning of Solidago ohioensis tops and roots revealed that the 90% methanol- soluble fraction (henceforth, called the methanol fraction) contained the most terpenes
16 as indicated by the nature of the color developed from the p-anisaidehyde spray reagent
(blue or purple spots after heating). The investigation of the diterpenes and monoterpenes in the methanol fraction of the leaves and roots is the subject of this chapter.
Chromatography of the methanol-solubles on Sephadex LH-20 with methanol separated the flavonoids and other phenolic compounds from the terpenes. The phenolics constituted about 9% of the weight of the methanol fraction from the leaves and 16% of the 90% methanol fraction from the roots.
Extensive column chromatography of the terpene fractions on silica gel and reversed-phase adsorbents with a variety of solvent systems, and final crystallization when possible, yielded a total of ten diterpenes and two monoterpenes. Nine diterpenes and two monoterpenes were isolated from the leaves (Table 2). Two diterpenes were isolated from the roots. Six of the diterpenes are previously unknown (new) natural products. Their structures and stereochemistries were determined and are of the trans- clerodane type. The structural elucidation of these compoimds is discussed individually on the basis of their structural similarity, not fraction of origin.
17 Powdered Plant Material 2.0 kg
95% EtOH Percolation (SOL)
Ethanol Extract Residue 307 g
CHCI3 /H 2O Partitionmg
CHC^ Solubles Fraction 125 g
EtOAc Hexane / 90% MeOH Extraction Partitioning
HzO EtOAc 90% MeOH Hexane Solubles Solubles Solubtes Solubles 25 g 72 g 53 g
Figure6 ; Extraction and fractionation of the ethanolic extract residue of the dried aerial parts of Solidago ohioensis
18 Powdered Root Material 0.9 kg
95% EtOH Percolation (SOL)
Ethanol Extract Residue 123 g
CHCIj/t^O Partitioning
HzO CHCy Solubks Fraction 62 g
EtOAc Hexane / 90% MeOH Extraction Partitioning
EtOAc 90% MeOH Hexane Solubles Solubles Solubles Solubles
6 g 30 g 32 g
Figure 7: Extraction and fractionation of the ethanolic extract residue of the dried roots ofSolidago ohioensis
19 Table 2: Compounds isolated from Solidago ohioensis
Compound % Yield* Rf'’ Color "
hardwickiic acid ( 6 8 ) 0.0300 0.23 blue
hardwickiic acid ( 6 8 ) ^ 0.4700 0.23 blue
methyl ohioenate A (80) 0.0035 0 . 2 2 pink
patagonic acid (83) 0.0078 0.38 dark blue
15-methoxypatagonic acid (85) 0 . 0 0 1 1 0.33 dark blue
methyl ohioenate B (91) 0.0027 0.32 purple
methyl ohioenate C (92) 0.0031 0.35 purple
15-hydroxypatagonic acid (87) 0.0040 0.28" pink
16-hydroxyclerodermic acid (8 8 ) 0.0400 0.37 pink ohioenic acid D (93) 0.0030 0.51 purple
(-)-bomyl 6-0-acetyl-P-D-glucopyranoside (97) 0.0007 0.65 dark blue
(-)-bomyl P-D-glucopyranoside (94) 0.0170 0.74 dark blue
a / . c J . J ^ _____ b r-> r . 0 ___ 1 /-ITT /-i-VT/TT / ~ \ A C sprayed with p-anisaldehyde spray reagent, and heated at 110-120°C for 10 min. w/w of dried roots. " RP - 8 silica gel, CH3 CN/H2 O (1:1) + 0.1% IF A.
2 0 Structure Elucidation of Diterpenes
Hardwickiic Acid (6 8 )
(-)-Hardwickiic acid has been reported from several plant families including
Euphorbiaceae, [32-35] Compositae [20,31,36-39] and Labiatae [40]. It was first isolated from Hardwickia pimata and the absolute stereochemistry determined [41-43].
It was previously isolated as a minor constituent of two Solidago species, S. juncea [20] and S. rugosa [31]. This is the first report of hardwickiic acid from Solidago ohioensis.
Hardwickiic acid (6 8 ) mp 90-91°C, [a]o -128.9° has the molecular formula
C2 0 H2 8 O3 (MW 316.2031, calculated value 316.2038) from High Resolution Electron
Impact Mass Spectrometry (HRMS). The infrared (IR) spectrum showed absorptions for hydroxyl (3400-3000 cm'*), a,P-unsaturated carboxylic acid carbonyl (1680 cm'' ), and vibrations of a furan ring (1500,1455, 1411,1384 cm ') [44]. Of the seven double bond equivalents required by the molecular formula, one is a carbonyl and three are double bonds, necessitating three rings, with one of the rings from the furan. The 'H and '^C NMR spectral data in deuterochloroform (CDCI 3 ) and deuteropyridine (pyr-ds) are found in Tables 3 and 4. The structure of hardwickiic acid ( 6 8 ) is shown in Figure
8 .
Although complete 'H and '^C NMR assignments are reported in the literature
[35,45], extensive NMR studies were repeated to confirm and extend these results and to gain experience with this class of compounds, before studying the new derivatives.
Heymann et al. [45] have published complete 'H NMR assignments at 400 MHz for the
21 (+) enantiomer, and have assigned coupling patterns to the five overlapping protons (H-
la, -7a, -?P, - 8 , and -lib). Our results at 500 MHz show these to be overlapped with no clear coupling patterns. The authors state that they base their assignments on 'H,‘H and C,H-corr data.
The Broad Band (BB)-decoupled NMR spectrum showed that hardwickiic acid contained 20 carbons. The Single-Frequency Off-Resonance decoupled (SFORD)
NMR spectrum revealed that these consisted of three methyls, six methylenes, six methines, and five quaternary carbons. These signals confirmed the existence of three olefins and one carbonyl.
A two-dimensional Carbon-Hydrogen correlation ( C,H-corr) identified the proton(s) on the relevant carbons and also helped to assign the proton spectrum especially the methylene groups [46-50]. Partial structures as units that are *H-coupled were obtained from the two-dimensional Correlation SpectroscopY (*H,‘H-COSY) experiment. This information in combination with that from the C,H-corr results established the partial structures for hardwickiic acid designated A-E and are shown in
Figure 9.
The correlation via LOng range Coupling (COLOC) experiment allowed the assignment of the methyl, carbonyl, and quaternary carbons [49,51]. This is an NMR experiment that shows long-range coupling which could be through two, three, and sometimes four bonds, with three-bond couplings predominating. A plot of the experimental results is shown in Figure 10.
2 2 COLOC results indicated a coupling of the 2.32 and 2.21 ppm methylene protons of partial structure A to the 141.76 ppm quaternary carbon. The 6.87, and 1.40 ppm methine protons of A, as well as, the 2.45 ppm methylene proton of B were coupled to the 37.80 ppm quaternary carbon suggested these two partial structures were linked through this quaternary carbon. The methine proton at 1.40 ppm and the methyl protons at 0.84 ppm showed coupling to the 39.03 ppm quaternary carbon resulting in the completion of the decalin ring system as shown in Figure 11. Further support for this ring system came from COLOC information obtained for the methyl groups. The
1.29 ppm methyl protons showed a two-bond coupling to the 37.80 ppm quaternary carbon, three-bond coupling to the methylene carbon at 36.04 ppm, and quaternary carbon 141.76 ppm. The three-bond coupling of the 6.87 ppm olefinic proton and the four-bond coupling of the 1.18 ppm methylene proton to the carbonyl carbon located it at C-19. This left one methyl to be assigned. The singlet methyl protons at 0.77 ppm showed a two-bond coupling to the 39.03 ppm quaternary carbon, and a three-bond coupling to the 36.47 ppm methine carbon to give, combined with the other results, partial structure F shown in Figure 12.
23 Table 3. ‘H and NMR assignments for hardwickiic acid 6( 8 ) in CDCI3 *
position (C) 5C mult.*’ position (H) ' 5H H pattern'* 7 Hz
1 17.67 t la 1.52 hm IP 1.70 dd 7.2,11.6
2 27.69 t 2 a 2.33 ddd' 6 , 6 , 2 0
2P 2 . 2 1 hm 3 140.47 d 3 6.87 dd 3,4.3 4 141.76 s 5 37.80 s
6 36.04 t 6 a 2.45 ddd 3.2, 3.2, 13
6 P 1.18 ddd 4.1, 13, 13 7 27.49 t 7a 1.42 hm 7P 1.48 hm
8 36.47 d 8 1.58 hm 9 39.08 s
1 0 46.91 d 1 0 1.40 brd 1 2
11 38.86 t 1 1 a 1 . 6 8 ddd' 4.8, 13.5, 13.5
1 1 b 1.55 hm
1 2 18.39 t 1 2 a 2.32 ddd' 4.1, 12.6, 12.6
1 2 b 2.19 ddd' 5.2, 13.5, 13.5 13 125.79 s 14 111.18 d 14 6.26 dd 0.7, 1.6
15 142.91 d 15 7.35 dd 1 .6 , 1 . 6 16 138.60 d 16 7.21 brs 17 16.15 q 17 0.84 d 6 . 6 18 20.74 q 18 1.29 s 19 173.02 s
2 0 18.46 q 2 0 0.72 s Mise (OH) 11.85 brs
* 'H NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution o f 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI3 center solvent peak set at 77.2 ppm) and data resolution o f 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE difference NM R studies.
24 Table 4: H and NMR assignments for hardwickiic acid 6( 8 ) in pyridine-ds®
position (C) 5C mult.*’ position (H) ' ÔH H pattern** y Hz
1 17.90 t la 1.46 ddd' 4.7, 13.3, 13.2 IP 1.58 hm 2 27.35 t 2a 2.19 ddd' 5, 5, 20 2P 2.08 hdddd 3 136.66 d 3 6.99 dd 3.3,3.6 4 143.57 s 5 38.03 s 6 36.52 t 6a 2.87 ddd 3, 3, 13 6P 1.36 ddd' 5, 12, 12 7 27.73 t 7a 1.55*' hm 7P 1.39*’ hm 8 36.52 d 8 1.55 hm 9 39.03 s 10 47.03 d 10 1.40 d 12 11 39.03 t 11a 1.69 ddd 5.2, 13.6, 13.6 11b 1.56 ddd' 4.3, 13.6, 13.7 12 18.48 t 12a 2.32 ddd 4.1, 13.5, 13.8 12b 2.20 ddd 4.1, 13.5, 13.8 13 126.29 s 14 111.25 d 14 6.47 dd 1, 1 15 143.33 d 15 7.60 ddd I, 1, 1.5 16 139.11 d 16 7.52 brs 17 16.12 q 17 0.77 d 6 18 21.02 q 18 1.50 s 19 169.79 s 20 18.48 q 20 0.73 s
® 'h NMR spectrum taken at 500 MHz and reported as ppm (pyrni, peak set at 7.19 ppm) and data resolution o f 0,3 Hz. '^C NMR spectrum taken at 67.9 MHz (upfield center residual solvent peak set at 123.5 ppm) and data resolution of 1.0 Hz. ^ '^C NMR multiplicities (SFORD); q=quartet, t=triplet, d=doublet, s=singlet. ' a and (3 denote geminal protons where a is below the plane o f the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE difference NMR studies. '' Chemical shifts may be interchanged within a column.
25 HOC II G
Figure 8: Structure of hardwickiic acid (68)
6.87 2.21 1.70 1.40 2.18 1.45 1.58 H H H H H H H D a D—?
H H H H 2.32 1.52 2.45 1.45
B
2.19 1.68 H H
D a
H H H H H 2.32 1.55 7.21 7.35 6.26
Figure 9: Proton coupled units of hardwickiic acid (68)
2 6 ro
H
"1“ I ■ 1 I’ I— if •I i | i f SI If M
Figure 10: Upfield region of the COLOC spectrum hardwickiic acid6 ( 8 ) in CDCL3 (6.35 tesla , 270MHz for 'H) 1.52 1.70 2.21 H 1.40
39.03 2.32 H 37.80
6.87 141.76
2.45 1.18
Figure 11: Connection of partial structures A and B to form decalin ring
Partial structures C, D, and E remained to be connected to the decalin system.
COLOC results established the presence of a fliran with an alkyl substituent at the p position, on the basis that the olefinic proton at 7.21 ppm of D showed coupling to the two olefinic carbons at 142.91 and 111.18 ppm in partial structure E. This left the two methylene carbons of partial structure C to be assigned. Since the furan ring and the decalin system still have one quaternary carbon each at 125.79 ppm and 39.03 ppm, respectively, the dimethylene unit must connect the components. COLOC data showed a three-bond coupling of the 7.21 ppm furan proton to the 18.39 ppm methylene carbon and three-bond coupling of the 0.77 ppm methyl protons to the 38.86 ppm methylene
28 carbon as shown in Figure 13. This assigned all the carbons and protons, and supported the clerodane skeleton for hardwickiic acid.
0.77 CH
39.03
36.47
37.80 141.76 36.04
6.87 CH3 173.02 1.29 1.18
Figure 12: Partial structure F of hardwickiic acid (6 8 )
2 9 7.35 142.91 138.60
111.18 125.79
6.26 18.39'
38.86
39.03
CH. COgH
Figure 13: Connection of partial structures C, D, and E to decalin ring
The stereochemical 'H NMR assignments in hardwickiic acid were based on the
Nuclear Overhauser Effect (NOE) difference experiments which identify the protons proximate to an irradiated proton [52,53]. An example of such an irradiation is shown in Figure 14. Irradiation at Me-20 (0.77 ppm) produced enhancement of 2.4% for Me-
17 (0.84 ppm), and 9% for Me-18 (1.29 ppm). This irradiation also revealed the
30 multiplicities and chemical shifts of the H-11 protons; 12% for the H-1 lb (1.55 ppm. ddd), and 4% for H-1 lb (1.67 ppm, ddd). This result and those obtained from irradiation of the other methyl protons are shown in Figure 15, and support their location on the same face (P-side) of the molecule.
Irradiation of H-10 (1.40 ppm) produced enhancements of 6 % for H-ip (1.70
ppm), 6 % for H- 6 P (1.18 ppm), and 9% for H- 8 p (1.596 ppm). Irradiation of H- 6 P
gave enhancements of 4% for H-10 (1.40 ppm), 2% for H - 8 (1.576 ppm), 1% for H-7P
(1.51 ppm), and 23% for H- 6 a (2.45 ppm). These groups must therefore be located on the same face of the molecule, and opposite the face of Me-18 as shown in Figure 16.
No NOE enhancement were seen for H-10 to any of the methyls or vice versa. This requires a trans A/B ring jimction.
The NMR chemical shift data also supports a trans ring assignment. The angular methyls can be used to distinguish between cis and trans A/B ring junctions in clerodane diterpenes [54,55]. In frnw-clerodanes, the C-18 methyl carbon resonates in the region of 11-20 ppm. In all cases of the cis series the C-18 methyl is less shielded by 11-12 ppm and resonates above 20 ppm. In our case the chemical shift for Me-18 is
20.75 ppm (CDCb) and 21.02 ppm (pyr-dg) shown in Figiure 17.
31 expansion
2.a t.a t.a 1.7 l.f t.a
—T- —r- 4.5 4 !o a!o a!s 2.0 1.5 1.0 PPM
Figure 14: NOE difference spectrum at 500 MHz in CDCI3 for irradiation of Me-20 of hardwickiic acid (6 8 ) CH Me-20 Irradiation
CH
H CH3 Me-17 Irradiation
CH
= J
H CH; Me-18 Irradiation CH
HO2 C CH
Figure 15: NOE by difference results for Me-20, Me-17, and Me-18 irradiations, expressed as percent enhancements
33 'Hh ^CH CH:s H-10 Irradiation
CH
HO2C CH;
H-6p Irradiation
Figure 16: NOE by difference results for H-10, and H-6 P irradiations, expressed as percent enhancements
With the relative stereochemistry of hardwickiic acid ( 6 8 ) established, its absolute stereochemistry needed to be resolved. Both enantiomers of hardwickiic acid are known and stereochemistry established [41-43,56]. The specific rotation [a]o
-128.9° (c 4.5, MeOH) [literature [a]o -114.7°(CHCl3)] for 6 8 indicates the levo rotating isomer (as shown in Figure 17) is the enantiomer present in Solidago ohioensis.
34 142.91 138.60 111.18 125.79
1839 \ 12
38.86
17.67 H
27.69 39.03 46.91 36.47
37.80 140.47 27.49
141.76 i 36.04 CH3
19 CO2 H 20.75 173.02
Figure 17: NMR assignments for hardwickiic acid 6( 8 )
Hardwickiic acid has exhibited a number of biological activities. These include antimicrobial activity against Mycobacterium smegmatis, the gram-positive bacteria
Bacillus subtilis, and the filamentous fimgi Trichophyton mentagrophytes [35], and insecticidal activity against Aphis craccivora [33]. Also, weak antitumor activity has been reported for (+)-hardwickiic acid [57]. Since hardwickiic acid is concentrated in the roots of Solidago ohioensis the antimicrobial activity may give this plant an advantage in the wet areas where it grows.
35 Hardwickiol (79)
Hardwickiol (79), a homogeneous solid, has the molecular formula C 2 9 H3 0 O2
(MW 302.2446) from HRMS. Hardwickiol (15,16-Epoxy-3,13(16),14-clerodatrien-19-
ol) has been reported as a reduction product (via LiAlH») of hardwickiic acid ( 6 8 )
[35,41,58]. Bohlmann, however, gave it the common name bacchalineol [58]. This is
the first report of the isolation of hardwickiol as a natural product. The IR spectrum
shows absorptions for hydroxyl (3338 cm’*) and a furan (1502, 1452, 1382 cm’*). No
absorption in the carbonyl region was present.
The *H and *^C NMR spectral data for hardwickiol (79) were very similar to
those for hardwickiic acid ( 6 8 ) except for the following features. First, the BB
decoupled *^C NMR spectrum showed that hardwickiol (79) contained twenty carbons,
while the SFORD *^C NMR spectrum revealed that these consisted of three methyls,
seven methylene, six methines, and four quaternary carbons. This differed from
hardwickiic acid ( 6 8 ) by an additional methylene carbon and one less quaternary carbon.
Thus twenty-nine hydrogens are bound to carbons leaving one hydrogen associated with
a hydroxyl. The absence of peaks above 150 ppm, confirmed the IR data that carbonyl carbons were absent.
36 Table 5: ‘H and u c NMR assignments for hardwickiol (79) in CDCb“
position (C) ÔC mult position (H) ÔH H pattern'* J Hz
1 18.35 t la 1.50 hm 1.67 hm
2 26.73 t 2 a 2.18 ddd 4.6,4.6, 16.2
2 P 2.07 dddd 2 .4, 2.4, 8 .6 , 18.3
3 1 2 2 . 2 2 d 3 5.58 dd 2.7,2.7 4 148.05 s 5 37.96 s
6 36.46 t 6 a 1.76 ddd 3.1, 3.1, 12.5
6 P 1.34 ddd 4.4,11,12.5 7 27.41 t 7a 1.43' hm 7p 1.50' hm
8 36.46 d 8 1.56 hm 9 38.87 s
1 0 46.45 d 1 0 1.45 d 1 1 . 8
1 1 38.72 t 1 1 a 1 . 6 6 ddd 5.1, 13.6, 13.6
1 1 b 1.50 hddd
1 2 18.35 t 1 2 a 2.31 ddd 4.3, 13.5, 13.5
1 2 b 2 . 2 2 ddd 5.4, 13.2, 13.3 13 125.89 s
14 111.18 d 14 6.25 d 0 . 6
15 142.83 d 15 7.34 dd 1 .6 , 1 . 6 16 138.54 d 16 7.20 brs 17 16.16 q 17 0.83 d 6.7 18 21.50 q 18 1.08 s 19 63.07 t 19 4.10 s 2 0 18.40 q 2 0 0.75 s
“ ‘h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution o f 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. '^C NM R multiplicities (SFORD); q=quartet, t=triplet, d=doublet, s=singlet. ' a and (3 denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Chemical shifts may be interchanged within a column.
37 Secondly, a few significant '^C NMR chemical shift differences were noted. An
upfield shift for C-3 to 122 ppm fi'om 140 ppm suggested an olefin no longer in
conjugation with a carbonyl. The upfield shift firom 173 ppm for the C-19 carbonyl to
63 ppm was attributable to an alcohol bearing carbon.
Thirdly, the 'H NMR spectrum showed an upfield shift for H-3 from 6.87 ppm
to 5.58 ppm. Two protons associated with C-19 resonated in the alcoholic region of the
proton spectrum (4.10 ppm). The Me-18 protons (1.08 ppm) were more shielded in
hardwickiol (79) than in the acid at 1.29 ppm. Thus, hardwickiol has a primary alcohol
at C-19.
NOE difference experiments on hardwickiol (79) revealed the same relative
stereochemistry as in hardwickiic acid ( 6 8 ). Reduction of hardwickiic acid ( 6 8 ) with
LiAlPIt gave an alcohol identical by TLC, NMR, IR and specific rotation with
hardwickiol (79) isolated from the plant. The absolute stereochemistry of hardwickiol
(79) is levorotatory the same as hardwickiic acid ( 6 8 ) and its structure is found in
Figure 18.
Complete NMR spectral assignment are given in Table 5 and were made from
detailed 2D (COSY, C,H-corr, and COLOC) studies. The *H NMR data in the literature
is incomplete and the ‘^C NMR assignments differed from that published [35].
McChesney and Clark have assigned C-2 as 27.4 ppm and C-7 as 26.6 ppm. In our study the carbon at 26.7 ppm was coupled in the C,H-corr to a methylene with protons at 2.18 and 2.07 ppm, which were coupled to the olefinic proton (H-3) at 5.58 ppm.
Also, from the COLOC data a three-bond coupling from Me-17 (0.84 ppm) to C-7 (27.4
38 ppm) was observed. Thus, the assignments in the literature for C-2 and C-7 must be interchanged.
Methyl ohioenate A (80)
Methyl ohioenate A (80) with molecular formula C30H40O5 (MW 480) as determined by HRMS was characterized by extensive spectral studies including 1D and
2D NMR, by comparison to previously isolated hardwickiic acid ( 6 8 ), and preparation of derivatives. Initial attempts to isolate ohioenic acid A were unsuccessful due to its tendency to tail from a chromatographic column and not elute in a clean sharp zone.
TLC plates showed severe streaking. This suggested the presence of a carboxylic acid similar to hardwickiic acid ( 6 8 ). Treatment with diazomethane formed the methyl ester in the crude fraction which eliminated the streaking and allowed ohioenic acid A , a new compound, to be isolated as the methyl ester.
The presence of an a,p-unsaturated ester, a cinnamoyl ester, and a hydroxyl was indicated by their characteristic UV absorption A, max (MeOH) 216, 223, and 275 nm; and IR bands at 2471 (hydroxyl), 1712 (a,p-unsaturated ester, intense absorption), and
1635 cm'* (double bond, intense absorption). As will be further shown, the additional 9 carbons in the molecular formula are attributed to a cinnamoyl ester substituent on the diterpene. HRMS supported the presence of a cinnamoyl ester, with cleavage of the cinnamoyl unit at the ester bond between the carbonyl and the oxygen gave the m/z
3 9 HO
Figure 18: Structure of hardwickiol (79)
OR OH
T CH oO C-C=C OH
MeOC MeOCII 80R=H 81 R=Ac
Figure 19: Structure of methyl ohioenate A (80), its acetate (81), and parent diol (82)
40 131 fragment as the base peak, while cleavage on the other side of its carbonyl gave the
CH=CHPh^ fragment at m/z 103. The phenyl fragment was also observed at m/z 11.
The BB-decoupled NMR spectra showed that methyl ohioenate A (80) contained 30 carbons while the Distortionless Enhancement by Polarization Transfer
(DEPT) experiment revealed them to be four methyls, eight methylenes, eleven methines, and seven quaternary carbons. The data indicated 39 protons on carbons thus leaving one associated with a hydroxyl. The DEPT experiment indicated that the hydroxyl was primary and acétylation with acetic anhydride and pyridine gave the monoacetate (81). The '^C and ‘H NMR spectral assignments for methyl ohioenate A
(80) in CDCI 3 and pyr-d; are given in Tables 6 , 7, 8 , and 9 and its structure in Figure 19.
The 'H and NMR spectra had a number of similarities to that of hardwickiic
acid (6 8 ). Comparison of the chemical shifts and their multiplicities to those of
hardwickiic acid ( 6 8 ) showed that the carbons corresponding to C-1 through C-12, and
C-17 through C-20 were nearly identical (Table 6 ). This suggested that methyl ohioenate A (80) differed from hardwickiic acid ( 6 8 ) in the side chain carbons C-13 through C-16.
The *^C chemical shifts also suggested the presence of a phenyl ring, two ester carbonyls, two carbons bearing oxygen, and three double bonds. These would account for nine of the eleven degrees of unsaturation required by the molecular formula, thus necessitating two additional rings.
41 The 'h NMR spectrum of 80 was nearly identical to hardwickiic acid ( 6 8 ) in the upfield region (0.5-2.5 ppm). The spectrum showed signals for a P-proton on an a,P- unsaturated carbonyl (6.56 ppm), four allylic protons (1.90, 2.02, 2.15, and 2.25 ppm) and three methyl groups attached to sp^ carbons (0.75, 0.87, and 1.25 ppm). A carboxymethyl group for the methyl ester was present at 3.48 ppm.
Extensive 2D NMR studies (‘H,‘H-C0SY, C,H-corr, and COLOC) confirmed the presence of a /ra«j-clerodane ring system with a dimethylene side chain as shown in
Figure 20. The '^C, and 'H NMR data and NOE enhancements matched those discussed imder hardwickiic acid ( 6 8 ) where the assignments are discussed in detail. The one difference in the bicyclic ring was the presence of a methyl ester instead of a carboxylic acid. Long-range coupling data (COLOC) supported the assignments of the C-
19 methyl ester (Figure 20) with three-bond couplings firom the carboxymethyl protons at 3.48 ppm, and the H-3 olefinic proton at 6.56 ppm to the C-19 carbonyl at 167.84 ppm. COLOC data also established the attachment of the dimethylene unit to the bicyclic ring at C-9 (Figure 20) by the presence of a three-bond coupling to the methylene carbon at 37.5 ppm fi'om the C-20 methyl protons at 0.75 ppm.
Extensive 2D NMR studies especially COLOC established partial structure G
(Figure 21). The assignments for the *^C and *H NMR chemical shift values for the C-
13, -14, -15,-16, and the trawj-cinnamoyl portion of compoimd 80 were confirmed firom the COLOC experiment and are given in Figure 21. The Z stereochemistry of the
13,14- double bond was determined by NOE difference studies. Irradiation of Hi-15 gave enhancements of 9.7% for H-14, and 6.3% for Hi-16. Irradiation of H-14 gave
42 enhancements for Hi-15, and Hi-12 but none to Hi-16. Irradiation of Hi-16 produced an 11% enhancement for Hi-IS but none to H-14. This required Hi-15, and Hi-16 to be cis, and H-14 and Hi-16 on opposite sides giving a Z-double bond The E- stereochemistry of the 2',3'-double bond was determined by the large (16 Hz) coupling of the olefinic protons in the *H NMR spectrum, and lack of NOE enhancements on each other when individually irradiated.
Partial structure G was connected to the decalin system fi’om long-range '^C-’H coupling data (COLOC). The C-13 quaternary carbon (138.26 ppm) was coupled to the protons of two methylene groups, one at 1.90 and 2.02 ppm, and the other an oxygenated carbon at 4.84 and 4.78 ppm. This connected the bicyclic ring to the cinnamoyl ester at C-16. Couplings were also observed firom C-12 (28.12 ppm) to the
H-16 protons at 4.81 and 4.78.
Preparation of the monoacetate 81 confirmed the presence of a primary alcohol at C-15 with the characteristic downfield shift of the H-15 methylene protons fi-om 4.26 ppm to 4.71 ppm. The ‘^C and *H NMR spectral assignments for methyl ohioenate A acetate (81) in CDCI 3 are given in Tables 6 , and 8 , and in pyr-dg in Tables 7, and 9.
43 Table 6 : NMR assignments for methyl ohioenate A (80), its acetate (81), and parent diol (82) in CDCI 3 ®
compound position mult.*’ 68 80 81 82
1 t 17.67 17.66 17.68 17.73
2 t 27.69 27.44 27.39 27.37 3 d 140.00 136.90 136.93 136.94 4 s 141.76 142.72 142.69 142.78 5 s 37.80 37.79 37.80 37.82
6 t 36.04 36.07 36.07 36.14 7 t 27.49 27.37 27.45 27.49
8 d 36.47 36.47 36.47 36.49 9 s 39.08 38.95 39.00 38.92
1 0 d 46.91 46.76 46.77 46.77 II t 38.86 37.24 37.06 37.38
1 2 t 18.39 28.30 28.59 29.19 13 s 125.79 138.40 140.87 144.76 14 d I I I .18 128.98 123.80 126.30 15 d 142.91 58.63 60.67 61.20 16 d 138.60 62.05 61.90 58.77 17 q 16.15 16.08 16.12 16.08 18 q 20.74 20.87 20.67 20.90 19 s 173.02 168.02 168.00 168.12 2 0 q 18.46 18.52 18.53 18.49 OMe q 51.28 51.29 51.30 I' s 167.10 166.78
2 ' d 117.76 117.78 3' d 145.64 145.49 4' s 134.42 134.49 5', 9' d 128.29 128.29
6 ', 8 ' d 129.11 129.12 T d 130.65 130.62 CO s 171.00 Me q 21.15
resolution of 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet.
44 Table 7: NMR assignments for methyl ohioenate A(80) and acetate (81) in pyridine-ds®
compound position mult.** 80 81
1 t 17.68 17.67 2 t 27.40 27.37 3 d 137.46 137.47 4 s 142.38 142.35 5 s 37.99 37.98 6 t 36.21 36.18 7 t 27.57 27.54 8 d 36.49 36.46 9 s 38.97 38.99 10 d 46.93 46.90 11 t 37.61 37.21 12 t 28.62 28.73 13 s 136.48 141.08 14 d 131.67 122.71 15 d 58.49 60.86 16 d 62.13 61.94 17 q 16.06 16.06 18 q 20.94 20.81 19 s 167.52 167.50 20 q 18.57 18.51 OMe q 51.03 51.04 r s 166.77 166.66 2' d 118.75 118.54 3' d 145.16 145.40 4' s 134.92 134.87 5', 9’ d 128.58 128.63 6', S' d 129.37 129.38 T d 130.75 130.81 CO s 170.66 Me q 20.91
* C NMR spectrum taken at 67.9 MHz (pyr-ds upfield center peak set at 123.5 ppm) and data resolution o f 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet.
45 Table 8 : H NMR assignments for methyl ohioenate A (80), its acetate (81), and parent diol (82) in CDCls^
80 81 82 jKJsition 5H pattern' J Hz 5H pattern' y Hz 5H pattern' J Hz
la 1.47 tun 1.45 hm 1.45 hm Ip 1.66 dd 6.9. 13 1.64 dd 7, 13.1 1.65 dd 7, 13.2 2o 2.25 hm 2.23 hm 2.23 hm 2P 2.15 dddd 3. 7.9, 11. 19.4 2.15 hm 2.16 dddd 3.1. 7.4, 8.8. 22 3 6.56 dd 3.6. 3.6 6.56 dd 3,3 6.58 dd 3.7, 3.7 6a 2.26 ddd 3.2, 3.2, 13.3 2.25 ddd' 3.1, 3.1, 12.9 2.28 ddd 5, 5,13 6P 1.12 ddd 3.9, 12.7,12.7 1.11 ddd 4, 12.6. 12.6 1.10 ddd 4, 12.6, 12.6 7a 1.38 hm 1.43 hm 1.40 hm 7P 1.47"' hm 1.43 hm 1.40 hm S 1.50 hm 1.49 hm 1.50 hm 10 1.32 d 12 1.31 d 12 1.32 dd 0.9. 12.2 11a 1.53 hddd 1.54 ddd 4.7, 13, 13 1.50 hddd 11b 1.50 hddd 1.43 hddd 1.40 hddd 12a 2.02 ddd 4.1, 13.3,13.3 2.03 ddd 4.9,13, 13 2.00 ddd 4. 13.2. 13.2 12b 1.90 ddd 4.8, 13.3, 13.3 1.91 ddd 4.9, 13, 13 1.87 ddd 4.7. 13.7. 13.7 14 5.70 dd 7,7 5.57 dd 7,7 5.58 dd 6.9. 6.9 15 4.26 d(2H) 4 4.71 d(2H) 7 4.18 d(2H) 7 16 4.81 d 12.4 Abq 4.81 d 12.9 ABq 4.14 s(2H) 4.78 d 12.4 Abq 4.75 d 12.9 ABq 17 0.87 d 6 0.81 d 6 0.80 d 6 18 1.25 s 1.25 s 1.25 s 20 0.75 s 0.75 s 0.73 s 2' 6.42 d 16 6.43 d 16 3' 7.70 d 16 7.70 d 16 5'. 9' 7.52 m 7.52 m 6', 7', 8- 7.40 dd 3,3 7.39 dd 3.2, 3.2 OMe 3.48 s 3.67 s 3.67 s Ac 2.07 s
“ ‘h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution o f 0.3 Hz. a and (3 denote geminal protons where a is below the plane o f the paper and (3 is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br^broadened. ** Chemical shifts may be interchanged within a column. ' Pattern revealed by NOE studies.
46 Table 9: 'H NMR Assignments for methyl ohioenate A (80), and its acetate (81) in pyridine-ds®
80 81 position ÔH pattern‘d y Hz ÔH pattern‘d y Hz
la 1.40 hm 1.35 hm IP 1.63 hm 1.57 hm
2 a 2.14 hm 2 . 1 2 hm
2P 2.14 hm 2 . 1 2 hm 3 6.59 dd 3.6,3.6 6.59 dd 3.6, 3.6
6 a 2.50 ddd 2.8, 5.9, 12.7 2.49 ddd 2.9,3.0, 13
6 P 1.14 ddd 3.5, 12.9, 12.9 1.13 ddd 3.4, 12.7, 12.7 7a 1.32“ hm 1.30 hm 7P 1.44“ hm 1.40 hm
8 1.50 hm 1.45 hm
1 0 1.31 dd 3.5, 13.2 1.29 d 1 2
1 la 1.60 ddd 4.6, 13.7, 13.7 1.57 ddd 5.1, 13.5, 13.5
1 1 b 1.50 hddd 1.45 hddd
1 2 a 2.14 hddd 2 . 1 2 hddd
1 2 b 2 . 0 0 ddd 4.4, 13.1, 13.1 1.98 ddd 4.9, 13.4, 13.4 14 6.04 dd 6.4, 6.4 5.79 dd 6.9, 6.9
15 4.67 d(2H ) 6 4.97 d(2H) 7 16 5.10 d 13.4 ABq 5.08 d 13.1 Abq 5.07 d 13.4 ABq 5.05 d 13.1 Abq 17 0.77 d 6.5 0.76 d 6.3 18 1.34 s 1.33 s
2 0 0.69 s 0.67 s
2 ' 6.76 d 16.1 6.79 d 16.1 3' 7.94 d 16.0 7.96 d 16.0 5', 9' 7.62 m 7.64 dd 1.8, 7.5
6 ', 7', 8 ' 7.36 m 7.36 m OMe 3.66 s 3.65 s
Ac 2 . 0 2 s a I H NMR spectrum taken at 500 MHz in and reported as ppm (pyr-d^ upfield peak set at 7.19 ppm) and data resolution o f 0,3 Hz. a and (3 denote geminal protons where a is below the plane of the paper and p is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. ' Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. Chemical shifts may be interchanged within a column.
47 28.12
37.05
17.48 38.76 27.19 46.57
37.61 136.71 2726 6.56 142.54 35.89 CH 20.69
Figure 20: ^ra/is-Decalin ring of methyl ohioenate A (80) with selected COLOC interactions
48 58.46
^ /H 2 C— OH
,C: 1/8./5128.78 i| 128.94 ^ 6 1 . 130.48
145.47 128.94 H 4.78
Figure 21: Partial structure G of methyl ohioenate A (80)
4 9 Hydrolysis of methyl ohioenate A (81) with sodium methoxide in methanol to the parent diol 82 (structure in Figure 19) confirmed the presence of the cinnamoyl ester at C-16. The and ‘H NMR spectral assignments for the diol 82 in CDCI 3 are given in Tables 6 , and 8 . Comparisons were made to bincatriol and crolechinol, two closely related /raw-clerodanes containing the C-15, C-16 diol, but having an alcohol in place of the carboxylic acid at C-19 [32]. The characteristic upfield shift of the Hz-16 methylene protons from 4.78 and 4.81 ppm to 4.14 ppm confirmed the presence of a primary alcohol at C-16. Shoolery’s rules permit calculation of a proton chemical shift position of a methylene group attached to two functional groups by the additive effect of the shielding constants [59]. Shoolery’s rules predict the Hz-16 methylene protons in the methyl ohioenate A (80) to be 4.68 ppm (observed 4.78 and 4.81 ppm) and those of the diol 82 to be 4.11 ppm (observed 4.14).
50 Butenolides from S. ohioensis
Four compounds were isolated that gave NMR spectra suggesting they were very closely related. They were identified from the data that follows, as patagonic acid (83), and 15-methoxypatagonic acid (85), two known compounds; 15-hydroxypatagonic acid
(87), and 16-hydroxycIerodermic acid ( 8 8 ) two new compounds.
The 'H and NMR data for these compounds contained common features and many similarities to the previously isolated hardwickiic acid ( 6 8 ). There were signals corresponding to one secondary (Me-17), and two tertiary (Me-18, and Me-20). methyl groups, a carboxylic acid (C-19), and a trisubstituted double bond (C-3 and C-4). From the BB-decoupled '^C NMR spectrum and the SFORD multiplicities these compounds contained three quaternary carbons (C-4, -5, -9), six methylene carbons (C-1, -2, - 6 , -7.
-11, and -12) and three methine carbons (C-3, - 8 , and -19). Comparison of the spectral data to those of hardwickiic acid ( 6 8 ) showed that carbons C-1 through C-12, and C-I7 through C-20 were nearly identical. This suggested that these compounds differed only in the side chain C-13, -14, -15, -16. Tables 10 and 11 compares the ‘H and ’^C NMR data for the four side chains of these compounds. Each of these substances is now individually discussed.
Patagonic Acid (83)
(-)-Patagonic acid, [a]o^' -78° was reported independently from two different laboratories in 1988. It was obtained first from Baccharis patagonica [60] and then
51 Grangea maderaspatana [39] both of the family Asteracea. Subsequently, it was
obtained from Eperua leucantha (Leguminosae) as its methyl ester and given a complete *^C NMR spectral assignment [61], and also from Salvia regia
(Labiatae)[40]. The Salvia compound was identified by direct comparison (TLC, IR, and ‘H NMR) to an authentic sample of clerodermic acid (the ^-substituted butenolide).
However, the publication has the structural drawing of the a-substituted butenolide, patagonic acid.
Complete high field *H and NMR spectral assignments have been made for the (+) enantiomer isolated from Sindora sumatrana (Leguminosea) [45]. Heymann et al. have assigned coupling patterns for the five overlapping protons (H-la, -7a, -?p, - 8 , and -I lb). Our data shows these signals to be overlapping with no clear coupling patterns. This is the first report of patagonic acid (83) from any Solidago species.
The structure of patagonic acid (83) (Figure 22) was identified from spectral data, and by comparison to hardwickiic acid ( 6 8 ) and literature values [39,61]. The '^C and ‘H NMR spectral assignments for patagonic acid (83) in CDCI 3 are in Tables 12. and 13, and in pyr-dg in Table 14.
Patagonic acid (83) has the molecular formula C 2 oH2 g0 4 (MW 332.1966, calculated value 332.1988) from HRMS. The presence o f an a,P-unsaturated-y-lactone. and a,p-unsaturated carboxylic acid was indicated by the characteristic IR absorptions at 1753 and 1684 cm'*, respectively. An intense and broad IR absorption from 3400-
52 Table 10: Comparison of H NMR assignments (H-14, -15, -16) for butenolides to hardwickiic acid (68)*
compound position 68 83 85'’ 87 88 89
14 6.26 lH,m 7.18 lH,m 6.75 IH, nm 7.19 IH, brs 6.14 lH,s 6.02 1H, nm 15 7.35 lH,dd (1,1) 4.76 2H, nm 5.72 lH,nm 6.49 IH, brs 16 7.21 IH, nm 6.48 1H,S 4.83 2H.S a Itt -«.m 4T» ____ . ^ 1 - resolution of 0.3 Hz and chemical shifts in ppm with reference to pyr-d 4 at 7.19 ppm (upfield peak). Taken in CDCI3 with reference to CHCI3 peak at 7.26 ppm. Abbreviations are: m=multipiet, s=singiet, nm=narrow multiplet, brs=broadened singlet
Table 11: Comparison of'^C NMR assignments (C -13, -14, -15, -16) for butenolides to hardwickiic acid (68)*
position 6 8 83 85” 87 8 8 89
13 126.3 s 134.3 s 139.5 s 137.8 s 171.6 s 169.8 s 14 111.3 d 145.1 d 141.4d 145.4 d 117.2d 114.9d 15 143.3 d 70.6 1 102.7 d 98.4 d 171.7s 174.3 s 16 139.1 d 174.5 s 171.5s 172.5 s 1 0 0 . 2 d 73.4 t
‘^C NMR taken in pyridine-ds at 67.9 MHz with multiplicities determined by SFORD and chemical shifts relative to pyr-ds upfield center peak set at 123.5 ppm, and data resolution of 1.0 Hz. ^ taken at 125 MHz in CDCI 3 and reported as ppm (CDCI 3 center peak set at 77.2 ppm) and data resolution of 1 .0 Hz.
53 2500 cm ' suggested a carboxylic acid. Of the seven double-bond equivalents required by the molecular formula, three were accounted for by the a , 3 -unsaturated-y-lactone, two by the unsaturated acid, and two by the clerodane ring. The SFORD '^C NMR spectrum supported 27 hydrogens bound to carbons leaving one associated with oxygen.
The ‘H NMR spectrum of patagonic acid (83) exhibited signals at 4.76 ppm
(2H) and 7.18 ppm for the methylene and olefinic protons, respectively, of an a- substituted-y-butenolide moiety. Similarly, the ‘^C NMR spectrum showed signals which could be assigned to a butenolide unit (134.3, 145.1, 70.6, and 174.5 ppm) identical to those reported for patagonic acid [60] and related compounds [62,63].
A COLOC experiment confirmed the presence of an a-substituted-y-butenolide.
For example the H-14 olefinic proton (7.19 ppm) showed coupling to the C-16 lactone carbonyl (174.5 ppm) and the oxygen bearing C-15 (70.6 ppm) carbons. The H-I5 methylene protons (4.76 ppm) showed couplings to the C-13 quaternary (134.5 ppm), and C-14 olefinic (145.1 ppm) carbons as shown in Figure 23. A coupling from the H-
12 methylene protons (2.05 and 2.20 ppm) to the C-13 (134.5 ppm) quaternary carbon connected the butenolide ring to the dimethylene side chain of the clerodane ring system
(Figure 23).
The structure of patagonic acid was supported by the characteristic mass spectral fragmentation pattern. The facile elimination of a molecule of water may involve the loss of an allylic y-hydrogen at C-2 and the hydroxyl of the carbonyl group giving ion
54 fragment m/z 314 as the base peak. A proposed fragmentation scheme for the other observed peaks is given in Figure 24.
Estérification of patagonic acid (83) with diazomethane produced methyl patagonate (84) and confirmed the presence of a carboxylic acid. The position of the carboxylic acid at C-19 was supported by the characteristic upfield shift of the carbonyl carbon from 172.7 ppm to 168.0 ppm in the methyl ester. The NMR showed a methoxy group (3.68 ppm) which gave a long-range coupling (COLOC) to the ester carbonyl at 168 ppm. The structure of methyl patagonate (84) is given in Figure 22.
The ’^C and ‘H NMR assignments in CDCb are given in Tables 12, and 13 and are in agreement with the literature values [39]. Patagonic acid was prepared from hardwickiic acid and will be discussed later.
55 ROC II G
83R=H 84 R=Me Figure 22: Structure of patagonic acid (83) and methyl patagonate (84)
4.76
70.6 174.6
4.76 145.1
134.5
7.09 220
2.05
Figure 23: Selected COLOC interactions for butenolide ring of patagonic acid (83)
56 +•, H -CO
c O
m/z203 (15%) m/z 175 (15%)
+
AV
HO-C C C III 04- m/z 332 (0.3%) m/z 111 (7%)
O
+ •
C III 04- m/z299 (5%) m/z271 (7%)
Figure 24: Proposed MS fragmentation of patagonic acid (83)
57 Table 12: NMR assignments for patagonic acid (83) and methyl patagonate (84) in CDCI3 ®
compound
position mult. 83 84 6 8
1 t 17.57 17.66 17.67 2 t 27.64 27.33 27.69 3 d 140.71 137.16 140.47 4 s 141.43 142.47 141.76 5 s 37.77 37.80 37.80 6 t 35.92 36.03 36.04 7 t 27.39 27.42 27.49 8 d 36.46 36.50 36.47 9 s 38.95 38.95 39.08 1 0 d 46.87 46.74 46.91 1 1 t 36.19 36.18 38.86
1 2 t 19.24 19.26 18.39 13 s 135.11 135.16 125.79 14 d 143.80 143.64 111.18 15 t 70.39 70.31 142.91 16 s 174.65 174.48 138.60 17 q 16.06 16.08 16.15 18 q 2 0 . 6 8 20.87 20.74 19 s 172.70 167.97 173.02 2 0 q 18.36 18.36 18.46 OMe 51.30
“ C NMR spectrum taken at 67.9 MHz (CDCI3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet,s=singlet.
58 Table 13: 'H NMR assignments for patagonic acid (83) and methyl patagonate (84) in CDCb*
83 84
jjosition *’ 6 H H pattern ‘ JH z 5H H pattern ' J Hz
la 1.50 hm 1.50 hm
IP 1.69 dd 6.1, 13.2 1 . 6 8 hm 2 a 2.32 ddd 5.0,5.3, 19.8 2.29 ddd 3.3, 13.1, 13.3
2 P 2.24 dddd 3.2, 7.5, 10,20 2 . 2 2 hm
3 6 . 8 6 dd 3,4.3 6.60 dd 3.7, 3.7
6 a 2.44 ddd 3,3, 13 2.30 hm
6 P 1.15 ddd 4.2, 12.6, 12.7 1.13 ddd 4.2, 12.6, 12.6 7a 1.45 hm 1.50 •* hm 7P 1.43 ■* hm 1.40“* hm
8 1.53 hm 1.53 hm
1 0 1.35 d 1 2 1.36 dd 1 , 1 2
1 1 a 1.65 ddd 4.8, 13.6, 13.6 1.65 ddd 4.8, 13.1, 13.1
1 1 b 1.50 hm 1.50 hm
1 2 a 2 . 2 0 dddd 1 .8 ,3.8, 1 1 .2 , 1 1 . 2 2 . 2 0 hdddd 1.6, 5.4, 14, 14
1 2 b 2.05 dddd 1.9,2.1, 12.8, 12.8 2.05 dddd 2, 4.8, 14.2, 14.2
14 7.09 nm 7.08 nm 15 4.76 (2H) nm 4.76 (2H) nm 16 17 0.82 d 6.4 0.82 d 6.4
18 1.24 s 1.26 s
2 0 0.76 s 0.77 s
Mise 8.55 (OH) brs 3.68 (OCH 3 ) s
' 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. a and (3 denote geminal protons where a is below the plane of the paper and p is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ' Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ** May be interchanged within a column.
59 position (C) 5C mult. position (H) ' ÔH H pattern ** J Hz
1 17.84 t la 1.50 hm IP 1.68 dd 6.8, 14.7 2 27.37 t 2a 2.26 hm 2P 2.18 hm 3 136.67 d 3 6.95 dd 3.6, 3.6 4 143.49 s 5 38.09 s 6 36.43 t 6a 2.85 ddd 2.5, 2.6, 12.5 6P 1.32 d dd' 3.6, 12.9, 12.9 7 27.69 t 7a 1.47'' hm 7P 1.31 *■ hm 8 36.60 d 8 1.47 hm 9 38.99 s 10 47.15 d 10 1.38 d 12.1 11 36.43 t 11a 1.62 ddd' 4.8, 13.1. 13.1 11b 1.50 hddd 4.5, 13.4, 13.4 12 19.41 t 12a 2.20 hddd 3.1, 14.5, 14.5 12b 2.08 ddd 2.8, 13.8, 13.8 13 134.30 s 14 145.15 d 14 7.17 nm 15 70.62 t 15 4.73 (2H) nm 16 174.58 s 16 17 16.07 q 17 0.79 d 5.7 18 21.01 q 18 1.49 s 19 169.78 s 20 18.41 q 20 0.73 s
® 'h NMR spectrum taken at 500 MHz and reported as ppm (pyr-cL, upfield peak set at 7.19 ppm) and data resolution of 0.3Hz. '^C NMR spectrum taken at 67.9 MHz ( pyr-ds center of upfield peak set at 123.5 ppm) and data resolution of I.O Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singiet. a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically unequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE studies. ^ May be interchanged within a column.
6 0 15-Methoxypatagonic Acid (85)
l5-Methoxypatagonic acid (85) was isolated originally from Grangea maderaspatana (Asteraceae) as a C-15 epimeric mixture of methyl esters [39].
Complete ‘H NMR assignments were not made at that time and no ‘^C assignments are reported. This is the first report of 15-methoxypatagonic acid isolated as the free acid and the first report from any Solidago species.
The structure of 15-methoxypatagonic acid (85) was identified from spectral data, comparisons with hardwickiic acid ( 6 8 ), patagonic acid (83), and related compoimds in the literature [39,64], Also, conversion to the methyl ester allowed direct comparison to the literature values. The ’H and '^C NMR assignments for 15- methoxypatagonic acid in CDCI 3 are given in Table 15 and the structure in Figure 25.
15-Methoxypatagonic acid (85) [a]o -55° has the molecular formula
C2 1 H3 0 O5 (MW 362.2090, calculated value 362.2093) from HRMS. However, the highest mass peak was at m/z 376.2247 corresponding to C 2 2 H3 2 O5 (1.4%). This peak is undoubtedly formed by transfer of MeO from the methyl acylal to give the methyl ester when the sample is heated in the probe. There is also a prominent peak at m/z
344 (60%) corresponding to C 2 1 H2 8 O4 probably resulting from the facile loss of H 2 O from the carboxylic acid observed in the fragmentation of this class of compounds
(Figure 24). The base peak m/z 329 (100%) is probably due to the loss of H 2 O with subsequent loss of a methyl.
61 The presence of an a,(3-unsaturated-Y-lactone, and a,p-unsaturated carboxylic acid was indicated by their characteristic IR bands at 1770 and 1710 cm'*, respectively.
An intense and broad IR absorption from 3400-3100 cm ' suggested a carboxylic acid.
The ‘H NMR spectrum of compoimd 85 was similar to that of patagonic acid
(S3) but also showed that it was a mixture of epimers. The two proton signal at 4.76 ppm was replaced by a narrow multiplet at 5.75 ppm which integrated for one proton
(Table 10). Two methoxy signals were present at 3.566 and 3.573 ppm. Also, the H-
17 and H-18 signals were doubled indicating the presence of epimeric 15-methoxy derivatives of patagonic acid (83) were present.
The '^C NMR spectrum of compound 85 was close to that of hardwickiic acid
(6 8 ) and patagonic acid (83) (Table 11). However, compound 85 differed from hardwickiic acid at carbons 13 through 16. The downfield shift of C-16 from 139.1 to
171.5 ppm indicated the presence of a carbonyl carbon. The C-15 methylene carbon
(70.6 ppm) in patagonic acid was changed to a methine carbon at 102.7 ppm in (85).
In addition, the C-4, -11, and -12 signals were doubled supporting the epimeric 15- methoxy derivatives.
In the COLOC experiment where the methoxy protons at 3.56 (3.57) ppm showed a two-bond coupling to C-15 (102.7 ppm) confirmed the presence of a methoxy group at C-15. NOE difference experiments with 15-methoxypatagonic acid
(85), details of which are not given here revealed the same relative stereochemistry as in hardwickiic acid ( 6 8 ).
6 2 Diazomethane treatment formed the methyl ester 8 6 and confirmed the
presence of a carboxylic acid. The position of the carboxylic acid at C-19 was
supported by the characteristic upfield shift of the C-19 carbonyl carbon from 172.5
ppm to 167.9 ppm. The NMR indicated an additional methoxy group (3.68 ppm)
which showed, in the COLOC experiment, three-bond coupling to the ester carbonyl at
167.9 ppm. The structure of methyl 15-methoxypatagonate ( 8 6 ) is Figure 25 and the
’H and ‘^C NMR spectral data in CDCI 3 (Table 16) agree with literature values [39].
15-Hydroxypatagonic Acid (87)
The structure of 15-hydroxypatagonic acid (87), a new clerodane diterpene, was established from spectral data, comparison with the spectral data discussed under hardwickiic acid ( 6 8 ), and the previously discussed butenolides. Comparison was also made to spectral data of compounds in the literature with the same hydroxybutenolide unit [64,65]. The *H and ’^C NMR assignments for 15- hydroxypatagonic acid in pyr-ds are in Table 17 and its structure is Figure 25.
15-Hydroxypatagonic acid (87) [a]o^' -93° has the molecular formula
C2 0 H2 8 O5 (MW 348.1934, calculated value 348.1937) from HRMS. As already seen for the a-substituted butenolides, the base peak m/z 330 (100%) results from the facile
loss of H 2 O, and a prominent peak at m/z 315 (47%) corresponding to the subsequent loss of methyl.
63 Table 15: H and NMR assignments for 15-methoxypatagonic acid (85) in CDCb"
position (Q SC mult. jositfon (H)^' 6H H pattern J Hz
1 17.57 t la 1.48 hm
1 3 1.67 hm 2 27.62 t 2a 2.30 ddd 5.3, 5.3. 20.4
2 3 2.22 hm 3 140.54 d 3 6.85 dd 2.1, 2.4 3 140.62 ' 4 141.43 s 5 37.78 s 6 35.92 t 6a 2.43 ddd 3.1, 3.1, 13.2 63 1.15 ddd 4.1, 12.5, 12.5 7 27.38 t 7a 1.41 hm 73 1.41 hm 8 36.48 d 8 1.50 hm 9 38.99 s 10 46.89 d 10 1.33 d 11.9 11 36.03 t 11a 1.63 hm 11 35.99' lib 1.45 hm 12 19.18 t 12a 2.22 hm 12 19.20' 12b 2.05 ddd 3.6, 16, 16 13 139.50 s 14 141.46 d 14 6.75 dd 1.2, 1.5 15 102.68 d 15 5.72 dd 1.2, 1.4 16 171.45 s 16 17 16.06 q 17 0.81 d 6.3 17 0.82' 18 20.68 q 18 1.24 s 18 1.25' 19 171.53 s 20 18.33 q 20 0.76 s OMe 57.13 q 3.56 s OMe 57.21' q 3.57' s
“ H NMR spectrum taken at 500 MHz and reported as ppm (CHClj peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 125 MHz (CDCI3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. '^C NMR multiplicities (DEPT): q=quartet, t=triplet, d=doublet, s=singlet ' a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Values for epimer.
64 Table 16: and NMR assignments for 15-methoxypatagonic acid methyl ester (86) in CDCI3*
position (C) SC mult. *’ position (H) ' 5H H pattern J Hz
1 17.65 t la 1.50 hm IP 1.67 hm 2 27.31 t 2 a 2.29 hm
2 p 2 . 2 2 hm 3 137.18 d 3 6.60 dd 2.1, 2.4 3 137.09 4 142.47 s 4 142.43 *■ 5 37.60 s
6 35.95 t 6 a 229 hddd 3.2, 3.2. 13.6
6 36.00 ^ 6 p 1 . 1 2 ddd 4.1, 12.5, 12.5 7 27.39 t 7a 1.43' hm 7P 1.48' hm 8 36.50 d 8 1.50 hm 9 38.97 s
1 0 46.75 d 1 0 1.33 d 1 1 . 6
1 1 36.03 t 1 1 a 1.65 hm
1 1 b 1.50 hm
1 2 19.17 t 1 2 a 2 . 2 0 hm
1 2 19.19'' 1 2 b 2.05 dddd 1.3, 4.7, 14.1, 14.1 13 139.53 s 14 141.42 d 14 6.75 dd 1.3, 1.5 15 102.67 d 15 5.72 dd 1.3, 1.5 16 171.51 s 16 17 17.65 q 17 0.81 d 6 . 2 17 0.82^ 18 20.87 q 18 1.26 s 19 167.94 s 2 0 18.34 q 2 0 0.77 s OMe 57.27 q 3.56 s 57.19'' q 3.57^ s OMe ester 51.33 q 3.68 s
“ 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI 3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ^ ’^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically unequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged within a column. ^ Values for epimer.
65 RO,
85 R=CH3, Ri=H 86 R=R i=CH3 87 R=Ri=H
Figure 25: Structure of 15-methoxypatagonic acid (85), methyl ester (8 6 ), and 15-hydroxypatagonic acid (87)
The presence of an a,p-unsaturated-Y-lactone, and a,p-unsaturated carboxylic acid was indicated by their characteristic IR bands at 1765 and 1683 cm'', respectively.
An intense and broad IR absorption from 3400-2500 cm ' suggested an acid was present. The SFORD '^C NMR indicated 26 hydrogens bound to carbons, thus leaving two associated with oxygens.
The spectroscopic data for compoimd 87 was similar to that of 15- methoxypatagonic acid (85) (Table 10 and 11) except for the following features: (a)
6 6 the 'h NMR spectrum did not contain a methoxy group, (b) the proton (H-15) on the
dioxygenated carbon, and H-14 were shifted downfield by 0.4 and 0.7 ppm
respectively, and (c) the NMR data showed the dioxygenated (hemiacylal) carbon
C-15 (98.42 ppm) resonated ftirther upfield by 4.3 ppm.
The spectroscopic data for compound 87 was also similar to that observed for
patagonic acid (83) except for the following features: (a) the proton H-15 on the
dioxygenated (hemiacylal) carbon was shifted downfield by l.Sppm, (b) the ‘^C NMR
shows that C-15 resonated ftirther downfield by 27.8 ppm indicating the carbon was
further oxygenated. These features were attributed to a hydroxyl group at C-15. Thus,
compound 87 contains a hydroxybutenolide. From the data for compoimds 83, 85, and hardwickiic acid (68) along with NOE results, the structiue and relative stereochemistry of 15-hydroxypatagonic (87) was established to be the same as hardwickiic acid (68). Furthermore, their negative specific rotations would strongly suggest they belong to the same enantiomeric series as hardwickiic acid (Figure 17).
Several chemical conversions were tried on compound 87 and are summarized in Figure 26. Attempts to generate the methyl ester or the methyl ester followed by acétylation of the hydroxyl group were unsuccessful. The compoimd appears to be unstable under the reaction conditions. However, reduction with NaBH 4 gave patagonic acid (83) which was identical (TLC, NMR, IR and specific rotation) with patagonic acid (83) isolated from the plant. Estérification of the reduced compoimd with diazomethane gave the methyl ester (84) identical to the one previously prepared.
67 Table 17: H and NMR assignments 15-hydroxypatagonic acid (87) in pyridine-ds*
position (C) SC mult. **position (H) 5H H pattern ^ J Hz
1 17.77 t la 1.50 hm IP 1.68 hm 2 27.34 t 2a 2.20 hm 2p 2.10 hm 3 136.70 d 3 6.94 brs 4 143.41 s 5 38.06 s 6 36.37 t 6a 2.83 brs 6P 1.27* ddd 3.7, 9.6, 9.6 7 27.63 t 7a -1.47'' hm 7p -1.31 *’ hm 8 36.55 d 8 1.50 hm 9 38.95 s 10 47.09 d 10 1.37 d 12.9 11 36.37 t 11a 1.62 hm 11b -1.31 hm 12 19.22 t 12a 2.25 hm 12b 2.15 hm 13 137.80 s 14 145.40 d 14 7.19 s 15 98.42 d 15 6.49 s 16 172.51 s 16 17 16.02 q 17 0.76 d 6 18 20.98 q 18 1.48 s 19 169.76 s 20 18.38 q 20 0.71 s
NMR spectrum taken at 500 MHz in and reported as ppm (pyr-cL, upfield peak set at 7.19 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (pyr-ds center of upfield peak set at 123.5 ppm) and data resolution of 1.0 Hz ‘^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. 'a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE difference studies. ^ May be interchanged within a column.
6 8 HO. HO. AcO.
pyridine HOC
decomposed mixture and upon handling decomposition products NaBH
CHoN.
Figure 26: Summary of chemical conversions performed with 15-hydroxypatagonic acid (87)
69 16-HydroxycIerodermic Acid (8 8 )
16-Hydroxyclerodermic acid (8 8 ) was the second most abundant diterpene isolated. Its identification was made from spectral data, and comparison with data from hardwickiic acid ( 6 8 ), 15-methoxypatagonic acid (85), and 15-hydroxypatagonic acid
(87). Comparisons to compounds in the literature with the same hydroxybutenolide were made [64-66].
16-Hydroxyclerodermic acid (8 8 ) [a]o^' -92° has the molecular formula
C2 0 H2 8 O5 (MW 348.1965, calculated value 348.1937) from HRMS.
16-Hydroxyclerodermic acid (8 8 ) exhibited an intense and broad IR absorption between
3400-2500 cm'' together with a strong absorption at 1677 cm'' for a conjugated carboxylic acid. The presence of an a,p-unsaturated-y-lactone with an a-hydrogen was indicated by its characteristic IR absorption at 1763 and 1739 cm''. This type of carbonyl band splitting is often observed in the IR spectrum of such compounds when an a-hydrogen is present [67]. The SFORD '^C NMR indicated 26 hydrogens bound to carbons leaving two associated with oxygens.
The 'H NMR data for compound 8 8 indicated two conformations of this compound were present in solution. For example the Me-17 showed two doublets
( 0.71 and 0.78 ppm) each split by a proton at 1.50 ppm ('H,'H-C0SY). Normally, the chemical shifts and coupling constants observed in the 'H NMR will be an average of all the possible conformations of a molecule, because the NMR time scale is slower than the rate of change between the different conformers. For a simple methyl group
70 attached to a disubstituted carbon the rotation about the carbon-carbon bond is rapid enough to give a single doublet pattern even if the average contribution of the various rotamers is not equal. The chemical shift and coupling constants are a result of population-weighted averages dependent on the fractional population of the various species. Thus, the average spectrum observed may be one in which a compound spends more or less time in various conformations but what is observed is a weighted average.
In theory, it is possible to change the rate of distribution between conformers by increasing the temperature. As temperature is increased a limiting value of the chemical shifts and coupling constants is reached. Unfortunately, in practice it is often not possible to attain the necessary temperature where the rate is rapid enough to give a spectrum showing the sharp peaks of an apparent “single” conformer. In our case, as shown in Figure 28 as the temperature was increased to 373K (100°C) the two conformations started to coalesce but was not yet a clean spectrum. A series of NMR solvents (CDCI3, DMSO, and pyr-ds) were tried and in each case a mixture of conformers was observed. Acétylation gave the expected epimeric mixture of acetates which unfortunately still showed two conformations in the 'H NMR spectrum. The 'H and NMR assignments in pyr-ds are given in Table 18 and its structure is Figure 27.
The 'H NMR spectrum at room temperature is given in the appendix in Figure 77.
The spectroscopic data of compound 8 8 was similar to 15-hydroxypatagonic acid (87) except: (a) the ’^C NMR spectrum indicated a ^-substituted hydroxybutenolide with the downfield shift of C-13 to 171.6 ppm and the upfield shift
71 OH
O
Figure 27: Structure of 16-hydroxycierodermic acid (88)
of C-14 to 117.2 ppm (Table 11). The downfield shift of C-13 is in agreement with other compounds containing a p-substituted butenolide [62,66,68]. This was supported by the 'H NMR data which showed a proton at 2.36 ppm not associated with a carbon which corresponded to the 16-hydroxyl proton.
Unequivocal evidence for the structure of 16-hydroxyclerodermic acid ( 8 8 ) and the presence of the p-substituted hydroxybutenolide was obtained from the Incredible
Natural Abundance DoublE Quantum Transfer Experiment (INADEQUATE) NMR experiment [69]. This experiment directly provides information on carbon-carbon connectivity but requires that adjacent carbons both be ’^C. The probability of adjacent
’^C is 0.01% (natural abimdance of is ~ 1%). The relative sensitivity is only 0.016% that of *H. Therefore, the INADEQUATE is the least sensitive of the 2D ‘^C NMR experiments. A 0.6M solution was used in this experiment, and the upfield region of the INADEQUATE spectrum for 16-hydroxyclerodermic acid ( 8 8 ) is shown in Figure
72 29. Interactions from C-I4 (117.20 ppm), C-I 6 (100.22 ppm), and C-12 (21.67 ppm) were seen to 171.60 ppm thus assigning it as the C-13 olefmic carbon. However, before this experiment was performed the C-H long range coupling data (COLOC) established the olefrnic carbon as 171.60 ppm, with a 2-bond coupling from the H-12 proton (2.52 ppm) to C-13 (171.60 ppm).
The NOE difference studies revealed the same relative stereochemistry as in all the other diterpenes isolated from Solidago ohioensis. It also supported the presence of a hydroxyl group at C-16. Irradiation of H-14 (6.14 ppm) produced enhancements of
1% to H-16 (6.48 ppm) and 1% to HO-16 (2.36 ppm). Irradiation of H-16 and HO-16 produced similar enhancements to each other and H-14 ( Figiue 30). This requires these groups to be located near each other.
A sample of 16-hydroxyclerodermic acid ( 8 8 ) was reduced with NaBH, to gave the known diterpene clerodermic acid (89). This reduced product was identical
(specific rotation, ER, UV, MS, *H and NMR) to the literature values. Conversion to methyl clerodermate (90) with diazomethane confirmed the presence of a carboxylic acid group and the spectral data agreed with the literature values.
73 Clerodermic acid has been previously isolated from Clerodendron inermi by two groups [68,70]. The first report in 1990 names it clerodermic acid and gives complete
’^C NMR assignments and some ‘H NMR data. They also report the methyl ester prepared with diazomethane. The second group named it cleroinermin and also report preparation of the methyl ester. Complete 'H and '^C NMR assignments for clerodermic acid (89) in CDCI 3 , and pyr-ds are in Tables 19 and 20, and for methyl clerodermate (90) in CDCI 3 in Table 21. Neither of these compounds exhibited evidence of more than one conformation in solution.
74 293 K
313 K
343 K
373 K
M yviV. «'T I ...... I I 2.5 2.0 l.S 1.0 PPM Figure 28: H NMR temperature studies upfîeid region 16-hydroxyclerodermic acid (88) in pyr-ds (270 MHz)
75 Table 18: H and NMR assignments 16-hydroxyclerodermic acid (88) in pyridine-dg"
position (C) 5C mult. ^ position (H) 5H H pattern y Hz
1 17.85 t la 1.45 hm IP 1.66 hm 2 27.33 t 2a 2.13 hm 2(3 2.18 hm 3 136.67 d 3 6.93 brs 4 143.45 s 5 38.09 s 6 36.38 t 6a 2.84 ddd 2.9, 2.9, 12.9 6P 1.32 ddd' 4, 13, 13 7 27.64 t 7a 1.35^ hm 7P 1.49^ hm 8 36.59 d 8 1.49 hm 9 38.97 s 10 47.11 d 10 1.35 brd 14 11 35.38 t 11a 1.78 hddd 3.4. 11.7, 11.7 11b 1.60 hm 12 21.67 t 12a 2.52 hddd 3, 13.7, 13.7 12b 2.25 hm 13 171.60 s 14 117.20 d 14 6.14 brs 15 171.71 d 15 16 100.22 s 16 6.48 brs 17 16.01 q 17 0.71 d 4.5 17 0.78® d 4.3 18 20.99 q 18 1.49 s 19 169.73 s 20 18.40 q 20 0.75 s OH 2.36 dd 7,7
“ 'H NMR spectrum taken at 500 MHz and reported as ppm (pyr-cl, upfield peak set at 7.19 ppm) and data resolution of 0.3 Hz. ’^C NMR spectrum taken at 67.9 MHz (pyr-ds upfield center peak set at 123.5 ppm) and data resolution of 1.0 Hz. Spectrum acquired at room temperature. ** '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ‘‘ Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE difference studies. ^ Assignments may be interchanged within a column. ® Assignment for other conformer.
76 t
•M
-r -T ~r T «i T # T M II T M ff 7 77 H II tf
Figure 29: Upfield Region INADEQUATE experiment 16-hydroxyclerodermic acid (88)( 11.85 Tesla, 125 MHz for '^C) 0.6
0.7 H-14 irradiation OH 152 H 2.36 225 6.48
G
6.14 H
2.36 H-16 irradiatK>n 0.3 OH 2.52 H
0.7 225 6.48 0.8
H O -16 irradiation OH 2.36 2.52 H 6.48 H -12 225 -12
Figure 30: NOE results for H-14, H-16, and HO-16 irradiations, expressed as percent enhancements 78 Table 19: H and NMR assignments for clerodermic acid (89) in CDCI3
position (C) ÔC mult. position (H) ' 5H H pattern J Hz
1 17.69 t la 1.50 hm IP 1.60 hm 2 27.59 t 2 a 2.36 ddd 4.8, 4.8, 20 2 P 2.16 hm 3 140.02 d 3 6.84 dd 3 ,5 4 141.58 s 5 37.79 s 6 35.93 t 6 a 2.45 ddd 2.7, 2.8, 13 6 P 1.14 ddd 3, 13, 13 7 27.30 t 7a 1.48 hm 7P 1.48 hm 8 36.53 d 8 1.46 hm 9 39.00 s 10 46.94 d 10 1.31 dd 2 , 1 1 .6 11 35.66 t 11a 1.68 ddd 5, 13.6, 13.6 lib 1.57 hddd 4.9, 13.8, 13.8 12 22.44 t 12a 2.30 ddd 4, 12.6, 12.6 12b 2.16 hm 13 170.87 s 14 115.37 d 14 5.84 dd 1.5, 1.5 15 174.10 s 15 16 73.21 t 16 4.73 (2H) d 1 .6 17 16.08 q 17 0.82 d 6 18 2 0 .6 8 q 18 1.26 s 19 172.13 s 2 0 18.35 q 2 0 0.80 s
“ 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. ‘^C NMR spectrum taken at 67.9 MHz (CDCI 3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ** ‘^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened.
7 9 Table 20: and NMR assignments for clerodermic acid (89) in pyridine-dg'
position (C) 5C mult. ** position (H) ' 5H H pattern JH z
1 17.84 t la 1.45 hm IP 1.45 hm 2 27.26 t 2a 2.18 hm 2P 2.06 hm 3 136.48 d 3 6.95 dd 3.4, 3.4 4 143.52 s 5 38.06 s 6 36.39 t 6a 2.85 ddd 2.8, 2.8, 12.8 6P 1.32 hddd 3.0, 13.1, 13.1 7 27.62 t 7a -1.40* hm 7P -1.50' hm 8 36.50 d 8 1.40 hm 9 38.93 s 10 46.98 d 10 1.26 brd 11.1 11 35.53 t lia 1.60 ddd 4.9, 13.6, 13.6 lib 1.50 m 12 22.25 t 12a 2.18 hddd 3.7, 14.3, 14.3 12b 2.06 hddd 4.6, 14.6, 14.6 13 172.14 s 14 114.93 d 14 6.02 dd 1.6, 2.8 15 174.29 s 15 16 73.44 t 16 4.83 (2H) d 1 17 16.03 q 17 0.74 d 4.2 18 20.97 q 18 1.50 s 19 139.79 s 20 18.31 q 20 0.74 s alrrxTikxn__ ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (pyr-ds center of upfield peak set at 123.5 ppm) and data resolution of 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. 'a and P denote geminal protons where a is below the plane of the paper and p is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged within a column.
80 Table 21: H and NMR assignments methyl clerodermate (90) in CDCb“
p o s i t i o n (C) 5C m u l t . j ) O s i t i o n (H) ' 5H H p a t t e r n /H z
1 17.74 t la 1.50 hm IP 1.57 hm 2 27.28 t 2 a 2.30 hm 2P 2.15 hm 3 136.61 d 3 6.57 dd 3, 4.5 4 142.62 s 5 37.77 s
6 35.98 t 6 a 2.30 hm
6 P 1.11 ddd 3, 13, 13 7 27.24 t 7a 1.47 hm 7P 1.47 hm 8 36.52 d 8 1.44 hm 9 38.95 s
10 46.76 d 10 1.26 brdd 3.4, 19.7
11 35.60 t I la 1.67 ddd 5, 13.6, 13.6
11b 1.55 hm
12 22.40 t 12a 2.28 hddd 3.5, 12.3, 12.3
12b 2.15 hm 13 170.93 s
14 115.30 d 14 5.82 dd 1.6 , 1.6 15 174.06 s 15
16 73.19 t 16 4.72 (2H) d 1.6 17 16.05 q 17 0.81 d 6 18 20.83 q 18 1.26 s 19 167.84 s 2 0 18.30 q 2 0 0.79 s OMe 51.35 q 3.68 s
NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 peak set at 7,26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI 3 center peak set at 77.2 ppm) and data resolution of l.O Hz. ’’ '^C NMR multiplicities (SFORD); q=quartet, t=triplet, d=doublet, s=singlet. ' a and (3 denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened.
8 1 Methyl ohioenate B (91)
The structure of methyl ohioenate B (91), a new clerodane diterpene, was established from spectral data, and comparison with the spectral data of previously isolated diterpenes from S. ohioensis. Comparisons to similar compounds in the literature were also made [64]. The ‘H and '^C NMR assignments in CDCI3 are in
Table 22 and it structure is Figure 31.
Initial attempts to isolate ohioenic acid B were unsuccessfiil due to its tailing on column chromatography, and severe streaking on TLC. This suggested the presence of a carboxylic acid. Initial 'H NMR studies indicated a mixture of two closely related compounds. Treatment with diazomethane formed the methyl esters in the crude fraction. This allowed both new compounds ohioenic acid B and its diastereomer ohioenic acid C (discussed next), to be isolated as the methyl esters.
Methyl ohioenate B (91) [a]o*^ -65® has the molecular formula C 25H42O7
(MW 454.24, calculated value 454.29) from HRMS. The IR did not show the presence of the carbonyl of an a,p-unsaturated-y-lactone. However, an a,P~unsaturated ester was indicated by its characteristic ER absorption at 1717 cm''. A broad IR absorption at 3455 cm ' suggested the presence of a hydroxyl group. The SFORD '^C NMR indicated 40 hydrogens attached to carbons leaving two associated with oxygens.
The structural features corresponding to the traw-decalin ring and the dimethylene side chain were present as in the previously isolated diterpenes.
However, some new features were present.
8 2 HO HO
(91) Ri= Me, R2 = OEt (92) Ri= Me, R2 = OEt (93) Ri= H, R2 = OMe
Figure 31: Structures of methyl ohioenate B (91), C (92) and ohioenic acid D (93)
The 'H NMR spectrum showed two additional methyl signals as triplets at 1.19 and 1.22 ppm, and four methylene protons at 3.55, 3.79, 3.45, and 3.77 ppm each as a doublet of quartets. No olefinic protons were associated with C-13, -14, -15, or -16
(Table 23). These changes were best accommodated as two ethoxy groups and two hydroxyl groups on a tetrahydrofuran ring.
8 3 Table 22: H and NMR assignments methyl ohioenate B (91) in CDCI 3*
position (C) 5C mult. position ( H ) ' 5H H pattern y Hz
1 17.23 t la 1.40 hm IP 1.70 dd 7. 13 2 27.30 t 2 a 2.13 dddd 3,7, 11,21
2P 2 . 2 0 hm 3 137.22 d 3 6.56 dd 3.6, 3.6 4 142.61 s 5 37.71 s
6 36.10 t 6 a 2.32 ddd 2.3,2.3, 12.6
6 p 1.08 ddd 3.7, 12.6, 12.6 7 27.45 t 7a 1.38* hm 7P 1.45* hm
8 36.21 d 8 1.45 hm 9 38.53 s
1 0 46.93 d 1 0 1.23 hm
1 1 31.00 t 1 1 a 1.54 ddd 3, 11, 11
1 1 b 1.38 hm
1 2 26.55 t 1 2 a 1.45 hm
1 2 b 1.45 hm 13 81.09 s 14 80.80 d 14 3.93 d 3.7 15 109.18 d 15 4.97 d 4 16 106.71 d 16 4.82 s 17 16.05 q 17 0.79 d 5.8 18 20.82 q 18 1.23 s 19 168.20 s 2 0 18.64 q 2 0 0.76 s C O 2 C H 3 51.30 q 3.66 s
O C H 2 C H 3 64.76 t 3.54 dq 7, 7, 7, 10 3.79 dq 7, 7, 7, 10
O C H 2 C H 3 63.17 t 3.45 dq 7, 7, 7, 10 3.77 qd 7, 7, 7, 10
O C H 2 C H 3 15.42 q 1 . 2 2 dd 7.2, 7.2 O C H 2 C H 3 15.09 q 1.19 dd 7.1, 7.1
' ‘h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. "C NMR spectrum taken at 67.9 MHz (CDCI3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ‘^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and p denote geminal protons where a is below the plane of the paper and p is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, q=quartet. hm=hidden multiplet, and br=broadened. Assignments may be interchanged within a column.
84 The presence of the ethoxy groups was apparent from the '^C NMR spectrum
(Table 24) with two methyls (14.89, and 15.22 ppm) and two methylene carbons
(64.57, and 62.98 ppm). The spectrum also showed one quaternary (80.88 ppm), and three tertiary (80.57, 108.95, and 106.49 ppm) carbons attributed to four oxygen bearing carbons (C-13 to C-16).
From this data and its comparison to the data for hardwickiic acid ( 6 8 ), and 15- methoxypatagonic acid (85) it was concluded that C-13 and C-14 each has a hydroxyl group and C-15 and C-16 each has an ethoxy group. The placement of the ethoxy groups on C-15, and C-16 was supported by the COLOC data as shown in Figure 33;
C-16 (62.98 ppm) showed a three bond coupling to H-16 (4.82 ppm), and C-15 (64.57 ppm) showed a three-bond coupling to H-15 (4.97 ppm). In addition, the substituted tetrahydrofuran ring could be connected to the dimethylene side chain, because quaternary carbon (C-13) showed a two-bond coupling to H-12 (1.45 ppm). The downfield portion of the COLOC data is shown in Figure 32.
The relative stereochemistry of (91) was determined to be the same as that of hardwickiic acid ( 6 8 ) based on NOE difference experiments, which is not presented here.
85 Table 23: NMR data comparisons for H-14, -15, -16 of methyl ohioenate B (91), C (92), and ohioenic acid D (93) to hardwickiic acid 6( 8 ), and 15- methoxypatagonic acid (85) in CDCI3 "
______compound ______position 6 8 85 91 92 93^
14 6.26 m,lH 6.75nm, IH 3.93 d (4),IH 3.95 d (4), 1H 4.56d(4),lH 15 7.35dd(l,I),lH 5.72 nm, IH 4.97d(4), 1H 4.97d(4),IH 5.49 d (4),IH 16 7.21 nm, 1H 4.82 s,lH 4.82s,IH 5.21 s,lH
“ 'h NMR spectrum taken at 500 MHz in CDCI3 and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz unless noted. ’’ 'H NMR spectrum taken at 500 MHz in pyridine-ds and reported as ppm (pyr-d, upfield peak set at 7.19 ppm) and data resolution of 0.3 Hz. Pattern symbols: s=singlet, d=doublet, nm=narrow multiplet, and m=multiplet, (J) coupling constants in Hz.
Table 24: Comparisons of NMR assignments for C-13, -14, -15, -16 to methyl ohioenate B (91), C (92), and ohioenic acid D (93), to hardwickiic acid 6( 8 ) and 15-methoxypatagonic acid (85) in CDCI3 "
compound
sition 6 8 83 85’’ 91 92 93"=
13 125.8 s 135.2 s 139.5 s 80.9 s 81.3 s 81.3 s 14 1 1 1 .2 d 143.9 d 141.4 d 80.6 d 80.7 d 80.6 d 15 142.9 d 70.41 102.7 d 108.9 d 109.2 d 111.3 d 16 138.6d 174.7 s 171.5 s 106.5 d 106.8 d 109.4 d
NMR spectrum taken at 67.9 MHz (CHCI3 peak set at 77.2 ppm) and data resolution of 1.0 Hz unless otherwise noted. ’’ '^C NMR spectrum taken at 125 MHz (CHCI3 peak set at 77.2 ppm) and data resolution of 1.0 Hz. ' '^C NMR spectrum taken at 67.9 MHz in pyridine- ds (pyr-ds upfield center peak set at 123.5 ppm) and data resolution of 1.0 Hz. '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet.
86 i.t
i.i
* «
I.»
* % 23
*.i
I.I.
I.I
JK
IH Ilf II* III 111 t Figure 32: Downfield region of the COLOC spectrum methyl ohioenate B (91) inCDCI 3 (6.35 Tesla, 270 MHz for *H) 106.49 62.98 OCH2CH3 Figure 33: Selected COLOC interactions of methyl ohioenate B (91) Methyl ohioenate C (92) Methyl ohioenate C (92) [a]o'^ -18° has the molecular formula C 2 5 H4 2 O7 from analysis of the ‘H and ‘^C NMR data and HRMS. The molecular ion peak was not observed but instead a prominent peak corresponding to the loss of methoxy was present. The ER did not show the presence of an a ,(3-unsaturated-y-lactone carbonyl. However, an a,p-unsaturated ester was indicated by its characteristic IR absorption at 1716 cm '. A broad absorption at 3404 cm'' suggested the presence of a hydroxyl group. The SFORD '^C NMR supported 40 hydrogens bonded to carbons leaving two associated with oxygens. Methyl ohioenate C has the same molecular formula and almost identical spectral data as that of methyl ohioenate B. The relevant carbon chemical shift comparisons are given in Table 24. Methyl ohioenate C has a slightly 88 higher Rf value on reversed phase (RP- 8 ) silica gel (Table 2). Analysis of the spectral data for methyl ohioenate C revealed that it was a diastereomer of methyl ohioenate B. The 'H and NMR assignments in CDCI3 are in Table 25 and it structure in Figure 31. It is possible that both methyl ohioenate B and C are artifacts formed from the dialdehyde or its hydrate the dihemiacetal during extraction of the plant material with 95% ethanol. More on this will be given later. Ohioenic acid D (93) The structure of ohioenic acid D (93) a new clerodane diterpene was established from spectral data, and comparison with the spectral data of the previously isolated diterpenes. Comparisons to similar compounds in the literature were also made [64]. The ’H and NMR assignments in pyr-ds are in Table 26 and it structure in Figure 31. Ohioenic acid D (93) [a]o*^ -56° has the molecular formula C 2 2 H3 6 O7 (MW 412.9831, calculated value 412.2461) from HRMS. The IR did not show the presence of an a,P unsaturated-y-lactone carbonyl. A broad IR absorption at 3400-2500 cm'' suggested the presence of a carboxylic acid group. The SFORD '^C NMR indicated 33 hydrogens bonded to carbons leaving three associated with oxygens. 89 Table 25: H and NMR assignments for methyl ohioenate C (92) in CDCI3® position (C) 3C mult. position (H) " SH H pattern J Hz 1 17.52 t la 1.45 hm IP 1 . 6 6 dd 7, 13 2 27.36 t 2 a 2.14 dddd 3,7, 11,21 2 P 2.26 hm 3 137.11 d 3 6.59 dd 3.6, 3.6 4 142.72 s 5 37.73 s 6 36.10 t 6 a 2.23 ddd 2.3,2.3, 12.6 6 p 1.09 ddd 3.7, 12.6, 12.6 7 27.42 t 7a 1.32 hm 7P 1.40 hm 8 36.46 d 8 1.40 hm 9 38.57 s 1 0 46.42 d 1 0 1.27 d 1 2 II 31.21 t 1 1 a 1.60 hm 1 1 b 1.35 hm 1 2 26.48 t 1 2 a 1.60 hm 1 2 b 1.35 hm 13 81.28 s 14 80.70 d 14 3.95 d 3.7 15 109.25 d 15 4.97 d 4 16 106.84 d 16 4.82 s 17 16.02 q 17 0.79 d 5.8 IS 20.89 q 18 1.24 s 19 168.22 s 2 0 18.58 q 2 0 0.74 s CO2CH3 51.33 q 3.67 s OCH2CH3 64.67 t 3.53 dq 7, 7, 7, 10 3.80 dq 7, 7, 7, 10 OCH2CH3 63.27 t 3.46 dq 7, 7, 7, 10 3.77 qd 7, 7, 7, 10 OCH2CH3 15.42 q 1.23 dd 7.2, 7.2 OCH2CH3 15.09 q 1.19 dd 7.12, 7.1 ® 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. NMR spectrum taken at 67.9 MHz (CDCI3 center solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ‘^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. 'a and p denote geminal protons where a is below the plane of the paper and p is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. 90 Aside from the spectral features common to all of the isolated diterpenes, ohioenic acid D (93) was similar to methyl ohioenate B and C at C-13, -14, -15, and - 16 (Table 24). However, instead of two ethoxy groups ohioenic acid D (93) showed signals corresponding to two methoxy groups attached to C-15 and C-16. The ‘H and ‘^C NMR spectra showed two three-proton singlets at 3.52 and 3.49 ppm, and the SFORD ‘^C spectra had two quartets at 55.99 and 54.89 ppm. The ‘^C BE and SFORD NMR spectra also showed four carbons in the oxygen bearing region corresponding to C-13, -14, -15, and -16 as shown in Table 24. From this data and comparison to methyl ohioenate B and C it was concluded that ohioenic acid D has a tetrahydrofuran ring with hydroxyl groups at C-13 and C-14 and methoxy groups at C- 15 and C-16. The placement of the methoxy groups on C-15 and C-16 was supported by long range 'H-*^C coupling data (COLOC). The methoxy protons at 3.49 ppm showed a three- bond coupling to C-16 (109.37 ppm), and those at 3.52 ppm showed a three- bond coupling to C-15 (111.30 ppm). In addition, the substituted tetrahydrofuran ring could be connected to the dimethylene side chain. The quaternary carbon (C-13) showed a two- bond coupling to H-12 (1.73 ppm). Estérification of ohioenic acid D with diazomethane gave a mixture of diasteromeric methyl esters in approximately 4:1 ratio (see the *H and *^C NMR data in Figures 35, and 36). Due to the limited amount of material available these were not separated or fully characterized. However, it did confirm the presence of a carboxylic acid. 91 5.49 4.56 H 1.73 1.87 5.21 Figure 34: Selected COLOC interactions of ohioenic acid D (93) Two broadened singlets at 2.70 and 2.44 ppm in the *H NMR corresponded to two hydroxyls. Decoupling of H -15 caused H-14 to collapse to a doublet from a double doublet indicating the presence of a hydroxyl group at H-14. Acétylation of the ester mixture with acetic anhydride and pyridine gave a mixture of diacetates. This confirmed the presence of two acylatable hydroxyls. Normally, under these acétylation conditions the tertiary hydroxyl would not be expected to acetylate. This acétylation maybe due to a transestérification reaction occurring between the secondary and tertiary hydroxyl groups. This supports that the hydroxyls are attached to C-13 and C-14 and most likely orientated cis to each other. 92 Recently, a publication appeared in which this same dimethoxy, dihydroxyl substituted tetrahydrofuran ring was present but the remainder of the clerodane diterpene was different [64]. However, their acétylation with pyridine and acetic anhydride gave only the monoacetate suggesting that their hydroxyls might be tram orientated. The HRMS fragmentation pattern supports the presence of this substituted tetrahydrofuran ring as shown in Figure 37. As seen previously in the fragmentation of patagonic acid (83) these diterpene acids undergo facile loss of H 2 O and scission at the C-9 to C-11 bond. Synthesis of Patagonic Acid (83) Methyl ohioenate B (91), methyl ohioenate C (92), and ohioenic acid D (93) might be artifacts formed from the corresponding C-15 and C-16 dialdehyde or its hydrate the dihemiacetal during extraction and isolation of the plant material. During extraction the plant material was exposed to 95% ethanol and during isolation to methanol. A retrosynthetic analysis of the diterpenes isolated from Solidago ohioensis indicated that a common dialdehyde could be the precursor to all of the isolated compounds. With the appropriate oxidations and reductions of this dialdehyde the various diterpenes could be synthesized. As shown in Figure 38 this common dialdehyde could come from hardwickiic acid (6 8 ) since it was by far the most abundant of the diterpenes. For example, 16- hydroxyclerodermic acid (8 8 ) could be obtained from the dialdehyde if C-16 is an aldehyde, and C-15 is oxidized to the acid followed by ring closure. 93 Table 26: H and NMR data ohioenic acid D (93) in pyridine-ds” position (C) ÔC mult. ** position (H) ' 5H H pattern J Hz 1 17.57 t la 1.35 hm IP 1.67 hdd 2 27.67 ' t 2 a 1.70 hm 2P 1.81 hm 3 136.48 d 3 6.79 dd 3.4, 3.4 4 143.64 s 5 37.96 s 6 36.54 t 6 a 1.82 ddd 3, 3, 13 6 P 1.26 ddd 2.6, 12.3, 12.3 7 27.78 t 7a 1.32 hm 7P 1.52 hm 8 36.65 d 8 1.52 hm 9 38.75 s 1 0 46.85 d 1 0 1.35 d 1 2 11 32.03 t 1 1 a 2.03 ddd 4.2, 13.3, 13.3 1 1 b 1.62 ddd 3, 14, 14 1 2 27.17' t 1 2 a 1.87 ddd 2.6, 13.5, 13.5 1 2 b 1.73 ddd 4.4, 13, 13.4 13 81.30 s 14 80.60 d 14 4.56 d 4.4 15 111.30 d 15 5.49 d 4.5 16 109.37 d 16 5.21 s 17 16.03 q 17 0 . 8 6 d 5.8 18 2 1 . 0 1 q 18 1.48 s 19 169.91 s 2 0 18.76 q 2 0 0.78 s OMe 54.89 q 3.49 s OMe 55.99 q 3.52 s OH 4.94 brs ” 'h NMR spectrum taken at 500 MHz and reported as ppm (pyr-d, upfield peak set at 7.19 ppm) and data resolution o f 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (pyr-d; center upfield solvent peak set at 123.5 ppm) and data resolution o f 1.0 Hz. *’ '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane o f the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield o f b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged within a column. 94 o LA me » eme I.I I.i I.i t.e Figure 35; *H NMR (270 MHz) spectrum ohioenic acid D methyl ester in CDCI 3 da l R : s 5 g 2 g : uA : : i 1 R \ H " 1t g r: W Y / 58 SB 5* 52 SI *B <6 *« *2 ' «0 ' J8 ^^36 ' 34 32 ' 31 ' 28 ' 26 ' 24 ' 22 ' 28 ' Figure 36: "C NMR spectrum (67.9 MHz) ohioenic acid D methyl ester in CDCI3 H -CO m/i 203 m^175 OCH OCH- HO" + OCH HO-C m/z394 m/z191 -OCH; OCH HO- 1.-C0 2.-H , m/2 363 m/z334 Figure 37: Proposed MS fragmentation of ohioenic acid D (93). 97 In 1995, a paper appeared in the literature on a series of diterpene glycosides with the same tetrahydrofuran ring found in ohioenic acid D (93) but differing in the rest of the terpene structure [64]. In addition, they isolated the a-, and P-substituted butenolides with a hydroxy group corresponding to 15-hydroxypatagonic acid (87), and 16-hydroxycIerodennic acid ( 8 8 ), and the methoxybutenolide corresponding to 15- methoxypatagonic acid ( 8 6 ). However, they report no butenolides lacking the hydroxy, or methoxy, or those containing ethoxy groups, and propose that the dimethoxy compounds were artifacts and possibly derived from the dialdehyde or its hydrate. This group had used an initial methanol extract of the dried plant material. There are many examples in the literature o f both labdane and clerodane diterpenes exhibiting various oxidation states of the C-I3, -14, -15, and -16 carbons. The C-I5 and C-I 6 dialdehyde could be a precursor in the biosynthesis of these compoimds. Synthesis of the dialdehyde would: (a) provide an opportunity to study its physical and chemical properties, (b) provide a route to synthesize the diterpenes isolated from Solidago ohioensis via hardwickiic acid ( 6 8 ) and to relate the absolute stereochemistry to that of hardwickiic acid, and (c) provide a TLC standard to be used to isolate the dialdehyde from a new collection of the plant material in which exposure to alcoholic solvents would be avoided. Attempts to synthesize the dialdehyde proved unsuccessful. Initially attempts were carried out using singlet oxygen photochemically with methylene blue as the sensitizer using chloroform as the solvent at room temperature and at 0°C (Figure 39). Triphenylphosphine was used as reducing agent for the endoperoxide, either in the 98 H R H COgH OgH H OH (83) R=H (83) R i =CH20H (88 ) (85) R=OMe (85). (87) R= CH (87) R=OH O H R O H H CH b J 0 2 b H X H (93) R=OMe (68) hardwickiic acid (91), (92) R=OEt X= Figure 38: Retrosynthetic analysis of diterpenes from Solidago ohioensis 99 1 . 0 2 methylene blue CHCI3 650W lamp 2. PhsP H0< HOI II II decompostion polymerization of starting material 0 2 methylene blue CHCI3 multiple products 650W lamp Ph3P HOi 0°C Figure 39: Routes to dialdehyde from hardwickiic acid (68) 100 reaction mixture during irradiaiton or added after irradiation. TLC studies indicated the rapid disappearance of starting material but no isolatable products. Initially, methanol was avoided as a solvent since generation of the dialdehyde in the presence of methanol could lead to the methylated product. However, the literature indicated that methanol stabilizes the intermediate endoperoxide [71-73]. Using methanol/water as the solvent at 0°C gave a mixture of methoxy containing compounds as seen from the *H NMR spectrum. No attempt to separate this mixture was made, but instead hydrolysis with aqueous acid was carried out (Figure 40). The only isolatable product was patagonic acid (83) (50% yield). It was identical (TLC, specific rotation. IR, UV, 'H and '^C NMR) with that of patagonic acid isolated from the plant or from NaBHj reduction of 15-hydroxypatagonic acid (87). This result established the absolute stereochemistry of the butenolides as that of hardwickiic acid (68). This prepared patagonic acid probably forms from the disproportionation of the intermediate methoxy containing compounds, in a way similar to a Caimizzaro reaction in which base conditions cause disproportionation of a dialdehyde to give a carboxylic acid and an alcohol, without a change in the overall oxidation state of the compoimd. Other attempts to synthesize the dialdehyde included varying the solvent, temperature, reducing agent, and sensitizer, and were imsuccessfiil giving no isolatable products, as summarized in Figure 41. However, this conversion to the dialdehyde has been carried out with furan itself in a special fi-eezer at -100° with trapping of the product as the phenylhydrazone [74]. 101 Another approach to the dialdehyde could be by reductive cleavage of the intermediate endoperoxide to the diol by NaBRt with subsequent oxidation to the dialdehyde [75]. However, the literature indicates that the fiiran will be formed on oxidation of the cÂs-but-2-ene-1,4-diol unit [63]. Another possible route to the dialdehyde would be via hydrolysis of the dimethoxy or diethoxytetrahydrofurans 91, 92, 93, but since limited amounts of ohioenic acid D (93) was isolated, this method was precluded. A mixture of the diethoxytetrahydrofurans was available (see isolation of methyl ohioenate B and C). Also, hydrolysis products might reveal the specific difference between these diastereomers. MeQ OH (OMe) 5%aq. HCI MeOH/HaO CH3CN methylene blu^ ► thiourea owmight 250W bulb 0°C 50% yield Figure 40: Synthesis of Patagonic Acid (83) 102 Hydrolysis of the mixture of diethoxytetrahydrofuran clerodanes mixture with aqueous HCI in acetonitrile resulted in a mixture of compounds (Figure 42). Attempts to reduce the intermediate to the diol or trap it as the phenylhydrazone were unsuccessful. It appears that under the conditions used in either the singlet oxygen or hydrolysis reactions, the dialdehyde is unstable. Subsequently, another collection of plant material was processed avoiding all alcoholic solvents. The plant material was percolated to exhaustion with solvents of increasing polarity beginning with hexane, followed by acetone, and finally with isopropanol (Figure 43). This percolation procedure extracted 9% (w/w) of the plant material while the 95% EtOH extraction yielded 15%. Initial TLC studies showed that the acetone extract contained the terpenes. The acetone-soluble extract proved to be unstable giving significant decomposition on handling. For example, of the 53.7 g of acetone solubles only 18 g could be re-dissolved in acetone. The remaining material was not soluble to an appreciable extent in hexane, CHCI 3 , acetone, or MeOH, and TLC studies indicated significant decomposition. The remaining 18 g was chromatographed on silica gel and eluted with CHCI 3 . Again, significant decomposition occurred with poor recovery of the material from the column. There was some evidence by TLC of the previously isolated compounds but in very small quantity. No evidence was foimd of the dialdehyde or its hydrate in this plant material. A new diterpene dialdehyde linaridial was isolated from the ether extract of fresh Linaria japonica and its structure established as shown in Figure 44. Linaridial 103 contains a dialdehyde that the authors report as quite labile and is hardly purified by the ordinary silica gel column chromatography due to its decomposition [76]. Linaridial was not detected in the air dried plant material. From the methanolic extract of the fresh plant was isolated an isomeric mixture of cyclic dimethyl hydrates, and 15,15- dimethyl linaridial (Figure 44). A TLC study indicated that these two compounds were only present in the MeOH extract and absent in the ether extract. They were able to synthesize both of these derivatives under mild methanolic acid conditions from linaridial. They suggest that these derivatives of linaridail are formed secondarily from linaridial during the MeOH extraction process catalyzed by organic acids occurring in the plant. Based on this information it seems that isolation of the dialdehyde from Solidago ohioensis might be possible using fresh plant material. 104 HC CH o , hv HOC temperature °C solvent reducing agent sensitizer -78 toluene HMPT rose bengal -100 MeOH HMPT rose bengal -100 toluene HMPT chlorophyll -78 MeOH/isopropanol/acetone 2:2:1 HMPT rose bengal -78 toluene/acetone 4:1 HMPT rose bengal HMPT= hexamethylphosphorous triamide Figure 41: Reaction conditions attempted to synthesize clerodane dialdehyde by photooxygenation 105 EtO. HO. HO ■OEt OH OH HQ HQ HQ HO HO HO mixture of 2 isomers mixture mixture >5 products phenyihydrazine ÎO2H decomposition Figure 42: Proposed hydrolysis products and derivatives from diethoxytetrahydrofuran clerodanes 106 Powdered Fiant Material 1.1 kg tfexane Percolation (30 L) f ▼ Marc Hexane Extract Solubles Acetone 33 g Percoiation (20 L) t Marc Acetone Extract Solubles 52.7 g Isopropanol Percolation (Il L) Marc Isopropanol Extract Solubles 11g Figure 43: Extraction of the tops Solidago ohioensis 107 CHO CHO CHO OCH linaridial dimethyl linaridial hydrate 15,15-dinietltyllinarkliaI Figure 44: cis-Clerodanes fromLinaria japonica Monoterpenes from Solidago ohioensis (-)-Bomyl P-D-glucopyranoside (94) (-)-Bomyl P-D-glucopyranoside (hereafter referred to as bomeol glucoside) [a]D^*-50° has the molecular formula CieHisOe (MW 317.1961, calculated value 317.1964) as supported by HRMS. The IR spectrum showed absorptions for hydroxyl at 3392, 1270, and 1077 cm'*, and bending vibration of C|-H bond of glucose at 897 cm *. The a and p anomer of glucopyranose can be differentiated by the Ci-H bending vibration. A peak at 891 ± 7 cm * supports a P-glucopyranose [77]. Three double-bond equivalents were required by the molecular formula. No chemical shifts above 105 ppm were observed in the *^C NMR spectrum eliminating any double bonds or carbonyls, and thus requiring three rings. The *H and *^C NMR 108 spectral data for bomeol glucoside (94) in CDCI 3 are in Table 27, and its structure is Figure 45. The BB-decoupled NMR spectral data showed that compound 94 contained sixteen carbons with seven resonating in the region for carbons bearing oxygen. The SFORD ’^C NMR revealed that these consisted of three methyls, four methylenes, seven methines, and two quaternary carbons. The 2D C,H-corr experiment identified the protons on the relevant carbons, and the *H,'H-C0SY experiment revealed two distinct *H-coupled units. One of these units was located in the downfield (3.41-5.30 ppm) region of the spectrum and suggested a sugar was present. Acétylation of compound 94 with acetic anhydride and pyridine gave the tetraacetate 95. The characteristic downfield shift of the H-2', -3', 4' and - 6 ' protons confirmed the presence of one primary and three secondary alcohols. The ‘H and '^C NMR assignments for the tetraacetate 95 in CDCI 3 are in Table 28 and its structure is Figure 45. Methyl iodide and silver oxide gave the tetramethyl ether 96. The characteristic upfield shift of the H-2', 3', -4' and - 6 ' protons, and downfield shift of the corresponding carbons confirmed the presence of one primary and three secondary alcohols. The 'H and '^C NMR assignments in CDCI3 are in Table 29 and its structure is Figure 45. 109 0R1 CH RO- RO 94 R= Ri= H 95 R= Ri= Ac 96 R= Ri= Me 97 R=H, R i= Ac Figure 45: Structures of (-)-bomyi P-D-glucopyranoside (94), tetraacetate (95), tetramethyl ether (96), and (-)-bomyl 6-<7-acetyl-P-D-glucopyranoside The 2D C,H-corr, and ‘H,‘H-COSY NMR studies combined with th e 'H NMR spectral data of the tetraacetate 95 established partial structures J and K shown in Figure 46. COLOC studies connected the remaining two quaternary and three methyls to partial structure J with the requirement of a bicyclic ring. This ordering of the partial units provided the bomeol (or camphane) ring system. For example Me-10 (0.85 ppm) showed a three-bond coupling to C-2 (84.24 ppm) and C-5 (26.60 ppm) and to one or both of the quaternary carbons (48.14, and 49.27 ppm). This connected C-2 and C-5 through one of the quaternary carbons. The other methyl protons at 0.83, and 0.84 ppm showed coupling to both quaternary carbons and a three-bond coupling to C-4 (45.04 ppm). This completed the bicyclic 2.2.1 ring partial structure L in Figure 46. A coupling no Table 27: H and ’^C NMR assignments (-)-bornyl P-D-glucopyranoside (94) in CDCb* position (C) SC mult. **position H ' ÔH H pattern J Hz 1 49.25 " s 2 84.80 d 2 3.97 d 8.2 3 36.47 t 3a 1.09 dd 2.4, 13.2 3P 2.14 dd 9.4, 12 4 45.01 d 4 1.63 d 4 5 28.38 t 5a 1.22 dd 7.3, 7.3 5P 1.68 m 6 26.87 t 6a 1.97 m 6P 1.22 dd 7.3, 7.3 7 48.37' s 8-Me 19.07 q 0.82 s 9-Me 19.99 q 0.83 s 10-Me 13.75 q 0.85 s r 102.09 d r 4.25 d 7.6 2' 73.65 d 2' 3.41 dd 8.1,8.1 3' 77.39 d 3’ 3.58 m 4’ 67.97 d 4' 3.58 m 5’ 75.51 d 5' 3.26 d 8.9 6' 61.99 t 6' 3.83 s OH 5.00 brs OH 5.30 brs “ 'h NMR spectrum taken at 270 MHz and reported as ppm (CHCI 3 solvent peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI 3 solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ^ '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Chemical shifts may be interchanged within a column. Ill Table 28: *H and NMR assignments (-)-bornyl 23»4,6-tetra-0-acetyi-P-D- giucopyranoside (95) in CDCI3* position (C) 5C mult. ^ ^ stio n H ÔH H pattern J Hz I 49.27 ' s 2 84.25 d 2 3.96 ddd 2.3, 2.3, 9.2 3 36.31 t 3a 0.95 dd 3.1, 16.2 3P 2.14 dddd 3.4, 4.4, 8.3, 14.2 4 45.04 d 4 1.64 dd 4.5, 4.5 5 28.50 t 5a 1.08 ddd 4.5, 9.6, 11.9 5P 1.71 ddddd 4.2, 4.2, 4.2, 12, 12 6 26.60 t 6a 1.89 ddd 4.1, 11.1, 11.1 6P 1.19 dddd 1.7, 4.3, 12.3, 12.3 7 48.14" s 8-Me 19.02 q 0.83 s 9-Me 19.89 q 0.84 s 10-Me 13.40 q 0.85 s r 99.86 d r 4.46 d 8 2' 71.62 d 2' 4.99 dd 8, 9.6 3' 73.03 d 3' 5.20 dd 9.5, 9.5 4' 69.02 d 4' 5.08 dd 9.7, 9.7 5’ 71.82 d 5' 3.65 ddd 2.7, 4.9, 9.5 6’ 62.35 t 6' 4.12 dd 2.6, 12.7 4.26 dd 5, 12.2 MeCO 20.76 q 2.05 s MeCO 169.29 s MeCO 20.80 q 2.02 s MeCO 169.58 s MeCO 20.80 q 2.00 s MeCO 170.47 s MeCO 20.87 q 2.07 s MeCO 170.80 s * 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI3 center peak set at 77.2 ppm). '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ‘ a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged within a column. 112 Table 29: (-)-bornyl 23»4,6-tetra-0-methyl-P-D-gIucopyranoside (96) in CDCI 3' position (C) 5C mult. *= position (H) ' SH H pattern "* J Hz 1 49.19"= s 2 83.78 d 2 3.99 ddd 1.5, 2.9, 11.1 3 36.30 t 3a 1.09 dd 3.2, 13.1 3P 2.14 dddd 1.4, 4.1, 8 .8 , 17.7 4 45.12 d 4 1.64 dd 4.5, 4.5 5 28.37 t 5a 1.21 m 5P 1.72 ddddd 4.5,4.5, 4.5, 12.5, 12.5 6 27.04 t 6 a 2.0 0 m 6 P 1.25 m 7 48.17"= s 8 -Me 19.14 q 0.83 s 9-Me 19.97 q 0.85 s 10-Me 13.56 q 0.87 s r 1 0 2 .2 0 d V 4.18 d 8 2 ’ 84.14 d T 2.97 m 3' 86.71 d 3' 3.13 hm 4' 79.71 d 4' 3.13 hm 5' 75.05 d 5' 3.21 hm 6 ' 71.72 t 6 ' 3.55 dd 4.6, 11 3.62 dd 2 .2 , 11.1 2'-MeO 60.62*’ q 3.60 s 3'-MeO 60.93 *’ q 3.61 s 4'-MeO 60.50 q 3.52 s 6 '-MeO 59.70 q 3.40 s “ 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz ( CDCI3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. ** ’^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and p denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ^ Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged. ^ Assignments may be interchanged. 113 from H-4 to the quaternary carbon at 49.27 ppm would place it at C-I assuming a three- bond coupling. Because the chemical shifts for the quaternary carbons are so close they could be interchanged. This left only the downfield C-2 methine to link partial structures K and L via a glycosidic bond. The relative stereochemistry was established through NOE difference studies with bomeol glucoside tetraacetae and the *H NMR coupling data. Irradiation of Me - 8 (0.83 ppm) produced enhancements of 3.1% for H-4 (1.64 ppm), 6 .6 % for H-3p (2.14 ppm), and 12% for H-2 (3.96 ppm). Irradiation at Me-9 (0.84 ppm) gave enhancements of 6 .8 % for H- 6 p (1.19 ppm), 3.4% for H-4 (1.64 ppm), 5.8% for H-5p (1.17 ppm), and 2.7% for H-2 (3.96 ppm). The methyl groups must be on the same face of the molecule as H-2, -3p, -Sp, and - 6 p shown in Figure 47. 114 3.96 2.14 1.64 1.70 1.89 8425 36.31 45.04 28.50 26.60 0.95 1.08 1.18 5.08 4.99 69.02 AcO AcO. 71.62 73.03 AcO 99.86 3.65 520 4.46 CH 3 0.83 0.84 H 3 1.64 26.60 0.85 L Figure 46: Partial structures J, K, and L of tetraacetate 95 115 Irradiation of H-3a (0.94 ppm) produced enhancements of 4.2% for H-5a ( 1 .08 ppm), and 4.0% for H-4 (1.64 ppm). Irradiation of H- 6 a (1.89 ppm) gave enhancements of 3.4% for H-5a (1.08 ppm), and irradiation of H-5a (1.08 ppm) produced enhancements of 4.6% for H-3a (0.94 ppm), 5.0% for H- 6 a (1.89 ppm), 4.4% for H-4, and 26.9% for H-SP (1.71 ppm). No NOE enhancements were seen from any of the a protons to either Me - 8 or Me-9 or vice versa. Figine 48 summarizes the a- faced NOE interactions. With the relative stereochemistry established for bomeol glucoside its absolute stereochemistry could be addressed. Both the (-)-, and (+)-bomeol glucosides have been synthesized by several methods with the best yields obtained from a modified Koening- Knorr procedure [78,79]. Lipschitz determined the specific rotation for (-)-bomyl P-D- glucopyranoside as [a]o -55.6°, and (+)-boraeol P-D-glucopyranoside as [a]o-15.9° [80]. The [a]o -50° establishes our (-)-bomeol glucoside as the enantiomer isolated from S. ohioensis. 116 0.83 H,C 3 2.14 1.71 3.96 1.19 CH Figure 47: NOE results for Me-8 (0.83 ppm) and Me-9 (0.84 ppm), expressed as percent enhancements 0.84 CH 0.95 1.19 3.96 H 1.08 CH 22 1.89 Figure 48: NOE results for H-3a, H-5a, and H-6a, expressed as percent enhancements 117 (-)-Bomyl P-D-glucopyranoside as a natural product has been obtained only from one other source, the roots of Ophiopogon Japonicus (Liliaceae) [81]. However, the specific rotation they report as [a]o -7.3°, corresponds to (+)-bomyl p-D- glucopyranoside for which as previously stated by Lipschitz is -15.9°. They hydrolyzed the glycoside but characterized the isolated bomeol only by TLC, and GC, with no specific rotation reported. With this uncertainty in the work of Kaneda et al., it would appear our work is the first report of the isolation of bomyl glucoside. Monoterpene glucosides have enjoyed a renewed interest in their biological activity. Several have been shown to inhibit aldose reductase [82] which is connected with the onset of diabetic retinopathy. (-)-Bomyl 6-0-acetyl-P-D-glucopyranoside (97) (-)-Bomyl 6-0-acetyl-P-D-gIucopyranoside (97) (hereafter referred to as bomeol 6 -acetylglucoside) [a]o^’ -40.0°, has the molecular formula CigHsoO? (MW 358.19). The IR was similar to bomeol glucoside (94) with an additional carbonyl absorption (1743 cm '). The 'H and '^C NMR spectral data (Table 30) for bomeol 6 -acetyl glucoside (97) were similar to those for bomeol glucoside (94) except for the following. First, the BB-decoupled '^C NMR spectrum showed that bomeol 6 -acetylglucoside (97) contained eighteen carbons, while SFORD '^C NMR spectrum revealed that these consisted of four methyl, five methylene, seven methines, and three quatemary carbons. 118 This differed by an additional quatemary and methyl carbon from bomeol glucoside 94. with the new methyl resonating at 2.09 ppm in the 'H NMR spectrum suggestive of an acetyl group. Thus, twenty-seven hydrogens are botmd to carbons leaving three hydrogens associated with hydroxyls. The latter was supported by loss of three protons (3.87, 3.65, and 2.97 ppm) upon D 2O exchange. Secondly, a significant downfield shift of C- 6 ' to 63.74 ppm from 61.99 ppm in the NMR spectrum placed an acetyl group on the 6 ' position of the glucose unit, and was also supported in the 'H NMR spectrum by an upfield shift for H- 6 ' from 3.83 ppm to 4.31, and 4.40 ppm. These changes were nearly identical to those observed at the 6 ' position of the sugar in the tetraacetate of bomeol glucoside (95). NOE difference experiments with bomeol 6 -acetyl-glucoside (97) revealed the same relative stereochemistry as in bomeol glucoside (94) and its tetraacetate (95). The [a]o^' -40.0° established the absolute stereochemistry as (-)-bomeol-6-0-acetyl-(3-D- glucopyranoside as the enantiomer present in S. ohioensis and its stmcture is Figure 45. A search of the literature did not reveal this glucoside to have been previously reported, thus this is the first report of its existence. 119 Table 30: *H and NMR assignments for (-)-bomyi 6-0-acetyI-|3-D- glucopyranoside (97) in CDCI 3® position (C) 5C mult. position (H) ' 5H Hpattem y Hz 1 49.28 ' s 2 84.96 d 2 3.98 ddd 1.4, 3.3, 9.9 3 36.71 t 3a 1.05 dd 3.3,13.2 3(3 2.19 dddd 3.1,4.5.8 , 15.1 4 44.74 d 4 1.65 dd 4.5,4.5 5 28.43 t 5a 1.21 m 5(3 1.72 ddddd 4.4,4.4, 4.4, 13.8, 13.8 6 26.80 t 6 a 1.96 ddd 3.6,7.5, 12.4 6 P 1.21 m 7 48.40 ' s 8 -Me 19.01 q 0.84 s 9-Me 2 0.01 q 0.85 s 10-Me 13.49 q 0.87 s r 101.94 d r 4.23 d 7.8 2 ' 73.86 d 2 ' 3.37 dd 8.5, 8.5 3' 76.32 d 3' 3.55 dd 8.5, 8.5 4' 70.47 d 4' 3.43 m S' 73.86 d 5' 3.43 m 6 ' 63.74 t 6 ' 4.31 dd 1.6, 11.4 4.40 dd 5, 12 Me 21.08 s 2.09 s CO 171.83 s ^ ’H NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. ’^C NMR spectrum taken at 67.9 MHz (CDCI3 solvent peak set at 77.2 ppm) and data resolution of 1.0 Hz. ^ '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. a and (3 denote geminal protons where a is below the plane of the paper and (3 is above, while a and b denote magnetically nonequivalent geminal protons, where a is downfield of b. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Assignments may be interchanged within column. 120 CHAPTERS ISOLATION OF c«-CLERODANE DITERPENES ¥KOU AMPHIACHYRIS DRACUNCULOIDES (JiC.) NUTT. The genus Amphiachyris (Asteracea; Astereae) is a North American genus comprised of two species, Amphiachyris dracunculoides (DC.) Nutt, and Amphiachyris amonea (Shinners) Solbrig. Taxonomists have differed as to where these plants should be grouped. Amphiachyris has previously been placed within other genera including Gutierrezia, and Xanthocephalum, but the latest findings by Lane establishes Amphiachyris as a distinct genus [83]. Amphiachyris dracunculoides collected in Kansas was subjected to an extensive phytochemical study by Harraz [84-86] and eighteen clerodane diterpenes were isolated. Their structures and stereochemistries were determined and are of the cw-clerodane type, seventeen of which were new natural products. The structure of amphiacrolide B (98), the most abundant diterpene from Amphiachyris dracunculoides, is shown in Figure 49. All of the compounds isolated contained a P-substituted-y-lactone in the side chain and nine of the compoimds contained a second lactone ring attached to the decalin system as in amphiacrolide B (98). 121 Two of the isolated compounds amphiacrolide E (104) and amphiacrolide I (105) were the ethyl and methyl ethers of the same parent diterpenoid (Figure 49). These compounds were quite possibly isolation artifacts since exposure to both 95% ethanol and methanol occurred in the isolation procedure. Thus, compounds with the dialdehyde or its hydrate the dihemiacetal could be present in Amphiachyris dracunculoides. Attempts by Harraz to obtain the dialdehyde or the hydration product under a variety of conditions was unsuccessful, however, the dialdehyde is known from Jones oxidation of 18,19-epoxy-19a-hydroxy-cw-cleroda- 3,13(14)-diene-15,16-olide (also known as amphiacrolide C) from Gutierrezia texana [87]. Our research of Solidago ohioensis had failed to yield any of the corresponding dialdehyde in which the side chain unit was involved. However, Amphiachyris dracunculoides might be a source of these compounds, and a collection was available from Oklahoma. Previous work by Harraz was carried out on two collections from Kansas, and differences in the natural products present in the collections were found. Thus, the possibility of finding new clerodanes, as well as the elusive dihemiacetal made a reinvestigation of this plant attractive. During this study all alcoholic solvents were avoided in the extraction and isolation procedure, to avoid the production of any isolation artifacts, and this resulted in the isolation of six cis-clerodane diterpenes. One was a new compound containing the usual side chain lactone, and the lactone attached to the decalin ring. The other five were identical to the previously isolated amphiacrolides B (99), C (100), D (101), E 122 (104), and I (105). This chapter describes the isolation and identification of these compounds. Extraction of the Plant Material The dried and powdered leaves of Amphiachyris dracunculoides (DC.) Nutt were extracted by percolation with solvents of increasing polarity. The sequence was hexane, acetone, isopropanol, and finally 95% ethanol, that gave four residues of increasing polarity (Figure 50). Thin-layer chromatographic analysis of the residues revealed that the acetone-soluble residue was most promising for containing diterpenes. Extensive column chromatography of the acetone-soluble residue on silica gel and reversed-phase adsorbents with a variety of solvent systems and crystallization when possible gave six pure diterpenes (Table 31). The structures of amphiacrolide B (98), C (99), D (100) , E (104) and I (105) were determined by direct comparison (TLC, specific rotation, mp when crystalline, IR. 'H and ‘^C NMR) to authentic samples. Only amphiacrolide R (101) will be discussed in detail. The isolation of amphiacrolide E and I in a fractionation sequence that was purposely designed to exclude methanol and ethanol shows that these ethoxy and methoxy containing compounds are not isolation artifacts, but must be of natural origin. The earlier studies could not resolve this problem. It also explains why the presumed precursor, the dihemiacetal could not be detected. Apparently, it is converted to the alkyl derivatives. 123 98 99 100 Ri=H, R2=0H 101 R=OH 104 R i=" OEt, Ra= ""OH 102 R= “ OAc 105 Ri=""OMe, Ra= "OH 103 R= -OMe Figure 49: cis-CIerodanes fromAmphiachyris dracunculoides 124 Powdered Plant Material 1.5 kg Hexane Percolation (20 L) r Marc Pfexane Extract Solubles Acetone L Percolation (17 L) r f Marc Acetone extract Solubles 236 g Isopropanol Percolation (11 L) T 'f Marc Isopropanol Extract 11 95% EtOH Percolation (IIL) 1 ir Marc 95% ETOH Extract Solubles 93 g Figure 50: Extraction of the above ground portionof Amphiachyris dracunculoides 125 Table 31: Compounds isolated from Amphiachyris dracunculoides Oklahoma collection Compound % yield * Rf" Color‘d amphiacrolide B (98) 0.208 0.41 greenish amphiacrolide C (99) 0 . 2 1 2 0.42 purple amphiacrolide D (100) 0.144 0.52 purple amphiacrolide R (101) 0.024 0.59 purple amphiacrolide E (104) 0.188 0.43 reddish ® w/w of the dried plant material. RP - 8 TLC plate developed in CH 3CN/H2O (3:2). ' TLC plate sprayed with / 7-anisaldehyde reagent, and heated at 110-20°C for 10 min. 126 Amphiacrolide R (101) The structure of amphiacrolide R (101) was established from spectral data and by comparison with spectral data of the known amphiacrolides. Amphiacrolide R. [cc]d'^ +110°, a homogeneous solid has the molecular formula C 20H28 O5 (MW 348.1929, calculated value 348.1937) as supported by HRMS. The mass spectrum showed fragmentation peaks at m/z 99, 98, and 11 1 due to an unsubstituted dimethylene attached to a butenolide ring [ 8 8 ] as seen in all the amphiacrolides isolated previously. The UV spectrum, determined in MeOH, showed end absorption at 214 nm indicating a butenolide [89,90]. A low intensity second absorption at 281 iim (log s 2.02) suggested an n-> 7r* transition for a non-conjugated ketone [90]. The CD spectrum determined in MeOH showed a large positive Cotton effect at 283 nm ([0] 9900). The IR spectrum showed absorption at 3413 cm ' due to a hydroxyl group. The presence of peaks at 1778, 1747, and 1636 cm"' correspond to an a,|3-unsaturated-y- lactone with an a-hydrogen [91,92]. Another absorption in the carbonyl region at 1716 cm'' was consistent with a ketone in a six-membered saturated ring. Of the seven double-bond equivalents required by the molecular formula three were accounted for by the butenolide and one by the ketone carbonyl, necessitating three additional rings. The 'H and '^C NMR spectral assignments for amphiacrolide R in 127 CDCI] and pyr-dg are given in Tables 32, and 33, respectively and the structure in Figure 49. The BB- and SFORD-decoupled NMR revealed twenty carbons consisting of two methyl, eight methylenes, five methines, and five quaternary carbons. The ’H,'H-C0SY revealed four proton-coupled units for Amphiacrolide R (Figure 51): (a) the five- proton spin system of C-I, -2, and -10 (A); (b) H-14 and H 2-I 6 of the butenolide with the two methylenes H 2- 1 1, and H 2-I 2 (B); (c) the five protons of C- 6 . -7, - 8 and Me-17 (C); and (d) Hi-19 with H-4 and long range to H-18, the carbinyl proton with an acylatable hydroxyl (D). The use of two different NMR solvents (CDCI 3 and pyr-ds) was helpful in clarifying overlapping spin patterns. These spin-coupled units are readily accommodated by a clerodane ring skeleton, as seen previously in compounds isolated from Amphiachyris dracunculoides. The '^C and 'H NMR data were similar to amphiacrolides C and D except for the following features: (a) only one olefin was present corresponding to the a,p- unsaturated butenolide; (b) the downfield shift of the C-2 methylene carbon (from 26.6, or 27.1 ppm) to 39.4 ppm suggested a deshielded environment, a neighboring carbonyl; (c) C-3 was shifted downfield to 210.5 ppm indicating a ketone carbonyl; (d) C-4 was now a methine in place of a quaternary carbon. 128 220 1.19 1.96 5.81 39.4Y I Y I Y I 3 8 .8 ^ 114.0 4.72 1.64 H 73.1 23.1 1.52 “n=x^ 37.5 4.72 2.47 1.89 2.39 2.39 B 1.32 1.72 1.59 r ~ ] 4.54 3.05 Y Y Y 54.91I Y I 62.8 I 1 0 0 . L H H C H 3 H H H 1.79 1.62 0.91 2.36 3.81 5.17 C D Figure 51: Proton coupled units of amphiacrolide R (101) 129 Table 32: H and NMR assignments for amphiacrolide R (101) in CDClj^ position (C) ÔC mult. position (H) ' ÔH H pattern ** J Hz 1 22.16 t la 1.85 hm IP 1.85 hm 2 39.42 t 2a 2.20 ddd 6.6, 12.2, 19 2P 2.47 ddd 1.8, 4.6, 19 3 210.47 s 4 54.98 d 4P 2.36 d d ' 3,8 5 52.61 s 6 26.47 t 6a 1.72 hm 6P 1.62 hm 7 25.99 t 7a 1.79 hm 7P 1.32 m 8 32.52 d 8 1.59 hm 9 37.62 s 10 38.82 d 10 1.96 dd 2, 12.3 11 37.59 t I la 1.64 hm 11b 1.52 hddd 6, 14, 14 12 23.11 t 12a 2.39 hm 12b 2.39 hm 13 171.34 s 14 114.84 d 14 5.81 dd 1.4, 1.4 15 174.24 s 15 16 73.19 t 16 4.72 (2H) d 1.4 17 15.92 q 17 0.91 d 6.9 18 100.11 d 18 5.17 d 1 19 62.82 t 19a 3.81 dd 8.3, 8.3 19P 4.54 dd 2.8, 8.8 20 19.33 q 20 0.93 s “ 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 peak set at 7.26 ppm) and data resolution of 0.3 Hz. NMR spectrum taken at 125 MHz (CDCI 3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. ** '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons. ** ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE difference studies. 130 Table 33: 'H and NMR assignments for amphiacrolide R (101) in pyridine-ds' c 5C mult. H" 5H H pattern J Hz I 22.51 t la 1.75 ddd* 3.7, 3.7, 11.8 IP 1.83 hdddd 4.8, 12.6, 12.6, 12.6 2 39.92 t 2 a 2.35 ddd* 6 , 13, 19 2 P 2.51 ddd 2, 5, 18 3 209.97 s 4 55.59 d 4P 2.42 dd 4,4 5 52.69 s 6 26.97 t 6 a 1.80 hm 6 P 1.70 hm 7 26.51 t 7a 1.80 hm 7P 1 .2 2 hm 8 33.05 d 8 1.60 hm 9 37.95 s 10 39.51 d 10 2.26 dd 2 , 12 11 37.90 t 1 1 a 1.60 ddd* 6 , 14, 14 1 1 b 1.53 hddd 5.2, 12.9, 12.9 12 23.43 t 12a 2.42 hm 1 2 b 2.34 hm 13 172.47 s 14 114.81 d 14 5.96 dd 2 , 2 15 174.30 s 15 16 73.39 t 16 4.74 (2H) d 2 17 16.14 q 17 0.84 d 7 18 100.37 d 18 5.56 s 19 62.85 t 19a 3.98 dd 8 , 8 19p 4.98 dd 3,8 2 0 19.43 q 2 0 0 .8 6 s ' ‘h NMR spectrum taken at 500 MHz and reported as ppm (pyr-d^ upfield peak set at 7.19 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (pyr-ds upfield center peak set at 123.5 ppm) and data resolution of 1.0 Hz. ‘^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. a and P denote geminal protons where a is below the plane of the paper and P is above, while a and b denote magnetically nonequivalent geminal protons. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. * Pattern revealed by NOE difference studies. 131 The long-range coupling experiments (COLOC) confirmed the assignment of the methyl, carbonyl and quaternary carbons and placement of the hydroxyl at C-18. H-10 (1.96 ppm), H - 6 (1.79 ppm), HO-18 (3.05 ppm), and the C-18 carbinyl proton (5.17 ppm) all showed two-bond couplings to the quaternary carbon at 52.6 ppm. The C-2 proton (2.47 ppm) showed a two-bond coupling to the carbonyl carbon at 210.5 ppm. The C-18 carbinyl proton (5.17 ppm) showed a three-bond coupling to C-4 (54.98 ppm). Me-20 showed a two-bond coupling to C-9 (37.62 ppm) and a three-bond coupling to C-10 (38.82 ppm). The chemical shifts for the C-13 olefin and C-15 carbonyl were based on comparisons to amphiacrolides B, C, D, E, and I along with long-range coupling of Hz- 12 to C-13 but not to C-15. The methylene protons and relative stereochemical assignments were made from extensive NOE difference studies, and from the NOESY experiment, as well as the coupling constants of the proton multiplets. The H-2 multiplet at 2.20 ppm with J values of 6 , 12, and 19 Hz must be the axial proton (a-orientation) with two large couplings for a geminal and one trans diaxial coupling and a smaller axial-equatorial coupling. The other H-2 proton at 2.47 ppm had J values of 2, 4, and 19 Hz in support of the P position with two smaller equatorial-axial couplings and one large geminal coupling. Irradiation of Me-17 produced enhancement of 4.5% for H-7P (1.32 ppm), and 9.2% for H - 8 (1.59 ppm). Irradiation of Me-20 produced enhancements of 5.1% for H- 6 P, 5.5% for H-II, 8.7% for H-12, and 3.4% for H-10 (1.96 ppm). This places 132 the two methyl groups on the P face. Irradiation of H-10 produced enhancements for H- 2a, and H - 8 placing it on the a face and opposite of the methyl groups. The hemiacetal proton H-18 in CDCI 3 and pyr-ds relaxed to H- 6 a, H-10, and one or both H 2-II. This requires an equilibrium mixture of both C-18 epimers to be present in solution. The relative stereochemistry of H-4 could not be firmly established from the NOE studies, because the H-4 methine proton is partially overlapped with the H 2-I 2 protons in both the CDCI 3 and pyr-ds ût the 'H NMR spectrum. However, irradiation of H-2P in CDCI 3 gave a 1% enhancement to H-4 revealing a clear double doublet. This suggested that H-4 was p. In order to gain further support for the proposed structure and to confirm the H-4 relative stereochemistry, a series of chemical transformations and derivatizations were undertaken. When Amphiacrolide R was treated with acetic anhydride in pyridine, a monoacetate derivative 102 was obtained. Its 'H NMR spectrum showed a 3-proton methyl singlet at 1.97 ppm, and conversion of the H-18 doublet at 5.17 ppm to a sharp singlet at 6.11 ppm. The *H and NMR data is given in Table 34 and its structure in Figure 49. Treatment of amphiacrolide R with hydrochloric acid in methanol gave the methyl ether derivative 103. Its *H NMR spectrum showed an additional 3-proton singlet at 3.20 ppm (OCH 3 ) and conversion of the H-18 doublet at 5.17 ppm to a sharp 133 singlet at 4.65 ppm. The and '^C NMR assignments are given in Table 35 and its structure in Figure 49. NOE difference studies on both the acetate and methyl ether assigned the H-18 proton as a. For example with the methyl ether (103) irradiation at H-18 produced a 1.8% enhancement for H-10. Irradiation of MeO-18 produced a 6 % enhancement for H- 19p (Figure 52). NOE experiments also confirmed the placement of the Me-17 and Me- 2 0 on the p-face as seen in studies on the parent compotmd 1 0 1 . NOE enhancements from both H-20 protons to H-4 were observed in the acetate and the methyl ether. No enhancements from H-2P to H-4 were observed in either the acetate or methyl ether giving no conclusive evidence to assign H-4. Since no conclusive NOE enhancements were seen another way to determine the H-4 stereochemistry was needed. Figure 53 illustrates the two possible H-4 configurations. Two different derivatives could provide the needed support. First, if the other epimer with H-4a was available NOE difference studies could be used. If H-4 is a a 1,3-diaxial relationship with H-2a, and H-10 would result, and they would be expected to produce significant NOE enhancements to each other. Secondly, generating the alcohol at C-3 would give a carbinyl proton that could be used in NOE studies and also would show a coupling pattern from which the dihedral angles could be obtained from the coupling constants. Taking the case where H-4 is P and the reduction produces the a-positioned hydroxyl the carbinyl proton would show a pattern (assuming the expected chair conformation) resulting from one axial-axial and 134 two axial-equatorial interactions. This pattern would be observed as resulting from apparent J values of 8 , 3, and 3 Hz. In a similar analysis of the ^-positioned hydroxyl the pattern would result from approximate J values of 3, 3, and 3 Hz. If the H-4 is a- positioned the expected pattern for the a-hydroxyl would result from approximate J values of 8 , 8 , and 3 Hz. The P-hydroxyl compotmd on the other hand would be expected to give a pattern similar to that in the case where H-4, and HO are p. Differentiation between these two would be make from the NOE studies involving H-2, -4, and -10. Attempts to reduce the C-3 ketone to the alcohol with sodium borohydride yielded a mixture of alcohols. Due to the limited amount of material no attempt was made to separate these isomers. Reduction of amphiacrolide R with K selectride® in order to prevent (or greatly reduce) mixture formation resulted in decomposition of the starting material. More detailed reduction studies were prevented by a limited amount of starting material. Another observation that could provide the C-4 epimer came from the acétylation of amphiacrolide R. A very minor product (1 mg) was formed and initial *H NMR studies suggested it was the C-4 epimer of the major acetate. Attempts to generate more of this epimer by enolization of the major acetate under basic conditions with either pyridine or potassium carbonate were imsuccessful. 135 Table 34: 'H and NMR assignments for amphiacrolide R acetate (102) in CDCI3* c 5C mult. ** H = ÔH H pattern JH z 1 2 2 .1 1 t la 1.82 hm IP 1.90 dddd 4.7, 13.1, 13.1, 13.1 2 39.36 t 2 a 2.15 hm 2P 2.57 ddd 1.9, 4.4. 19.4 3 208.58 s 4 54.36 d 4P 2.41 dd 2.6, 7.8 5 51.64 s 6 27.08 t 6 a 1.82 hm 6 p 1.73 ddd 6 .8 , 12.9, 12.9 7 25.91 t 7a 1.82 hm 7P 1.34 m 8 31.42 d 8 1.60 hm 9 37.99 s 10 39.97 d 10 1.58 hm 11 37.23 t 1 1 a 1.58 ddd* 4.8, 13.2, 13.2 1 1 b 1.46 ddd 4.7, 13.2, 13.2 12 23.15 t 1 2 a 2.18 hddd 6 , 12.9, 12.9 1 2 b 2.26 ddd 4.8, 13.9, 13.9 13 170.09 s 14 115.53 d 14 5.81 dd 2 , 2 15 173.74 s 15 16 73.06 t 16 4.72 ABq 2, 17 4.67 ABq 2, 17 17 15.88 q 17 0 .8 8 d 7 18 100.38 d 18a 6 .1 1 s 19 64.40 t 19a 3.92 dd 8 , 8 19P 4.62 dd 3,9 2 0 18.69 q 2 0 0.94 s COCH3 169.96 s COCH3 21.29 q 1.97 s “ 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI3 solvent peak set at 7.26 ppm) and data resolution of 0.3 Hz. NMR spectrum taken at 67.9 MHz (CDCI3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. ^ '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. ' a and (3 denote geminal protons where a is below the plane of the paper and P is above. ** Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. ' Pattern revealed by NOE studies. 136 Table 35: H and NMR assignments for amphiacrolide R methyl ether (103) in CDCb* c 5C mult. ** H" ÔH H pattern J Hz 1 22.30 t la 1.73 hm ip 1.85 dddd 5, 13, 13, 13 2 39.55 t 2 a 2.15 ddd 7, 11,20 2 P 2.45 ddd 2,5,19 3 209.92 s 4 55.21 d 4P 2.28 dd 3,8 5 52.22 s 6 27.03 t 6 a 1.70 hm 6 P 1.61 hm 7 26.31 t 7a 1.80 hm 7P 1.32 dddd 2.2, 6 .8 , 6 .8 , 15.2 8 32.68 d 8 1.55 hm 9 37.89 s 10 38.86 d 10 1.93 dd 3,12 II 37.75 t lia 1.52 hm lib 1.58 hm 12 23.23 t 12a 2.33 hm 1 2b 2.33 hm 13 171.01 s 14 115.22 d 14 5.82 dd 2 , 2 15 174.03 s 15 16 73.25 t 16 4.73 (2H) d 2 17 16.18 q 17 0.92 d 7 18 106.24 d 18a 4.65 s 19 62.84 t 19a 3.84 dd 8 , 8 I9p 4.41 dd 3,8 2 0 19.53 q 2 0 0.93 s OCH3 54.05 q 3.20 s ® 'h NMR spectrum taken at 500 MHz and reported as ppm (CHCI 3 solvent peak set at 7.26 ppm) and data resolution of 0.3 Hz. '^C NMR spectrum taken at 67.9 MHz (CDCI 3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. ^ '^C NMR multiplicities (SFORD): q=quartet, triplet, d=doublet, s=singlet. ' a and P denote geminal protons where a is below the plane of the paper and P is above. Pattern symbols: s=singlet, d=doublet, t=triplet, hm=hidden multiplet, and br=broadened. 137 Application of the Octant Rule allows determination of the configuration of cyclohexanones when its conformation is known. It was used extensively by Djerassi in the early 1960’s [93,94], and applied extensively in steroid configuration studies. In the Octant Rule, the space surrounding the carbonyl chromophore is divided into eight regions (octants), and the octant occupied by a particular pertuber determines the sign of its contribution to the rotatory strength of the n n:* transition [95]. Two planes divide the space surrounding the carbonyl into quadrants and a third surface (non symmetry derived) divides the space into octants at the center of the carbonyl. Each of the quadrants is assigned a positive or negative contribution for a perturber lying in that quadrant. Atoms lying in a symmetry plane make no contribution, and atoms symmetrically located across the carbonyl symmetry plane will cancel. Contributions are additive, and the magnitudes of the contributions fall off rapidly with increasing distance from the carbonyl chromophore or closeness to the nodal surface. In amphiacrolide R with H-4 being (3 or above the plane the ring containing C- 18 and -19 would be in the lower right or positive quadrant, thus the rule predicts a positive cotton effect. This is illustrated in Figure 54. Carbons-2, -3, -4, - 8 , -9, and -10 lie in symmetry planes and make no contribution. Contributions from C- 6 , and -7 are canceled by C-17 and -20. Thus, the major contributing perturber is the ring with C-18, and -19 which lies in the lower right positive octant. The CD spectrum of amphiacrolide R (Appendix Figure 105) shows a large positive Cotton effect, which can be represented by the stereochemistry shown in Figure 54. Therefore, H-4 must be positioned (3 or on the top surface. 138 Me Me, OCH Figure 52; Selected NOE by difference enhancements by percent for methyl amphiacrolide R (103) in CDCI3 139 ,0 M e- M e- Me, Me. OH OH H-4p H-4a Figure 53: Amphiacrolide R with H-4a and H-4f3 O Me- Me— .0 Me, Me O OH OH Figure 54: Conformational drawing for amphiacrolide R and the octant projection showing the lower right positive quadrant predicting a positive Cotton effect curve. 140 CHAPTER 4 ANTIFUNGAL FLAVONES FROM AGERATINA COLESTINUML. Until the 1980’s, drug companies were actively developing new antimicrobials. If pathogen resistance emerged there was always another antimicrobial available [96]. In the 1960’s and 1970’s there was a feeling that infectious diseases had been conquered and were no longer a major threat. This was fueled by the dwindling support of the NIH, which reached a low in 1975 [97]. Also, major pharmaceutical companies greatly reduced or eliminated their antibiotic screening programs. Only five new antibiotics were approved in 1991, and two in 1990 [96]. There have been many stories in the media about the onslaught of new drug resistant bacteria. With the rise in AIDS many fungi and bacteria not considered pathogenic have emerged and are becoming resistant to existing drugs. In the process of searching for novel antimicrobials from plants several strategies may be employed. Recorded uses of certain plant extracts in the treatment of wounds could be important, or selection of plants based on the known distribution of natural products, or simply based on the availability and ease of collection of the plant material. 141 Antimicrobially active plant components can be isolated by bioassay-directed separation techniques utilizing solvent partitioning and chromatography [35]. The structure of these active compounds are generally different from those of microbial fermentation (for example penicillins). Antimicrobial Assay Using an antimicrobial assay, plant extracts and compounds of interest are tested against various standard assay microorganisms. The agar diffusion assay requires little plant material and short incubation times. The compounds or extracts of interest are placed in wells in the agar plated which have been seeded with the test organisms. Activity is recorded in mm of the clear zone surrounding the sample wells [98]. All antimicrobial screening was carried out by Professor Larry W. Robertson and his associates. The initial screening organisms included the Gram-positive bacteria Bacillus subtilis, the Gram-negative bacteria Escherichia coli, the acid-fast bacterium Mycobacterium smegmatis, the yeast-like fungus Candida albicans, and three filamentous fungi Aspergillus niger. Trichophyton mentagrophytes, and Microsporum gypseum. Among several dozen plant extracts examined in our screening study, the 95% EtOH crude extract of Ageratina colestinum L. displayed antimicrobial activity against T. mentagrophytes, M. gypseum, M. smegmatis, and B. subtilis. 142 Plant Materials Ageratina colestinum L. (Asteraceae) belongs to the tribe Eupatorieae. The tribe contains approximately 2000 species and over 160 genera [14]. The nomenclature of this tribe began with Lirmeaus who recognized the genera Eupatorium and Ageratum. Linneaus also started the taxonomic and nomenclature confusion in the tribe by naming both a Eupatorium altissimum and an Ageratum altissimum from North America [14]. The Ageratina group with over 230 species is almost totally restricted to the western parts of the Americas. Three species of Ageratina extend into eastern North America. Only about 10% of the species of this tribe have been examined chemically. Plants of this genus are known to produce coumarins, sesquiterpene lactones, and flavones [99]. Partitioning of the 95% Ethanol Extract The Ageratina coelestinum extract was partitioned as described under Solidago ohioensis into four fractions shown in Figure 55. The MeOH-soluble fraction showed good activity against M. gypseum, T. mentagrophytes and M. smegmatis (Table 36). Silica gel column chromatography of the MeOH solubles gave 19 fractions, A through S, which were grouped into 13 testing pools and tested against B. subtilis, M. smegmatis, and M. gypseum and with results shown in Table 37. Fraction ABC showed the best activity, but lack of plant material limited further investigation. Fraction DE gave a 12mm zone of inhibition against M. gypseum. Initial TLC studies indicated that Fractions F, and G had some compoimds in common with Fraction DE. 143 Powdered Plant Material 640g 95% EtOH Percolation (50L) Ethanol Extract Residue 78 g CHCI3 / H2 O Partitioning HiO CHQ3 Solubles Fraction 40g EtOAc Hexane / 90% MeOH Extraction Partitioning HoO EtOAc 90% MeOH Herane Solubles Solubles Solubles Solubles 5g 29 g 25 g 15 g Figure 55: Extraction and fractionation of the ethanoUc extract residue of the dried whole plant ofAgeratina coelestinum 144 Table 36: Antimicrobial activity ‘ of Ageratina coelestinum partition fractions Organisms Candida Aspergillus Microsporum Trichophyton Mycobacterium albicans niger gypseum mentagrophytes smegmatis Plant extract 48h 48h 48h 72h 48h Hexane ++ +P -H- ++ +-t- EtOAc ++ +P ++ ++ + 90% MeOH ++ ++P +++ + + + -H-+ Water + + + + + DMSO (blank) +P +P +P +p none * Plant extracts 20 mg/ml in DMSO with 100 pi per well. Code: average radius in mm of the zone of inhibition; +, 1 -2 ; ++, 3-6; +++, 7-12; -h -h -, >12; p following a reading means partial inhibition. Table 37: Antimicrobial activity ' of Ageratina coelestinum MeOH column fractions Organisms Bacillus Mycobacterium Microsporum column subtilis smegmatis gypseum fraction 24h 24h 72h ABC +++(1 0 ) ++(6 ) ++++(16) DE ++(5) ++(3) ++++(1 2 ) F ++(3) ++(6 ) +++(7) G ++(3) +++(7) +++( 8 ) H ++(5) ++(4) +++(8 ) I +++(7) ++(5) +++(8 ) JK ++(6 ) ++(4) +++(7) LM ++(6 ) +++(8 ) ++(5) N ++(6 ) +++(9) ++(4) OP ++(5) +++(7) ++(4) Q ++(4) -H-(3) ++(5) R ++(3) ++(4) +++(7) S ++(3) ++(3) ++(6 ) DMSO (blank) none none none “ Plant extracts 10 mg/ml in DMSO with 100 pl per well. Code: average radius in mm of the zone of inhibition; +, 1 -2 ; ++, 3-6; +++, 7-12; i t-i i-, >12; ]p following a reading means partial inhibition. 145 Isolation of Flavones Fractions D, E, F, and G afforded in good yield a mixture of four polyoxygenated flavonones which could be separated only after repeated recrystallizations and column chromatography (Table 38). The known compounds linderoflavone B (106), eupalestin (107), 5'-methoxynobiletin (108), and nobiletin (109) were identified by their physical and spectral data and comparison to literature values. These flavones were previously isolated from Ageratum, Eupatorium, Ageratina, Citrus, and Lindera species (Table 39). Although the ‘H and some NMR assignments are reported in the literature, extensive NMR studies were repeated to confirm and extend these results and to gain experience with this class of compounds. Linderoflavone B (106) Linderoflavone B (5,6,7,8-tetramethoxy-3',4'-methylenedioxyflavone, also called luicidin dimethyl ether), mp 169-170°C, has the molecular formula CaoHigOg (MW 389.0990, calculated value 386.1002) from HRMS. The UV spectrum in MeOH showed peaks at 249, 269, and 334 nm, and IR absorptions at 1647, 1585, and 1503 cm*' indicating a flavone rather than a flavonol [100]. No acetate was formed with addition of pyridine and acetic anhydride confirming no acylatable hydroxyls were present. 146 Table 38: Flavones isolated from Ageratina coelestinum Compound %Yield" Rf*» Color‘d linderoflavone B (106) 0.08 0.49 yellow eupalestin (107) 0.03 0.33 yellow 5'-methoxynobiletin (108) 0.06 0 . 2 1 yellow nobiletin (109) 0.05 0.18 yellow “ w/w of dried plant material. Silica gel TLC, EtiOrpet ether (4:1) with 4 developments. ' TLC plate sprayed with p-anisaldehyde spray reagent, and heated at 110-120°C for 10 min. The 'h NMR spectrum indicated the presence of four methoxy groups, and two singlets at 6.55 (IH) and 6.03 (2H) ppm corresponding to C-3 and the 3',4' methylenedioxy protons, respectively. Also present were two doublets at 6.87 (J=1.5 Hz), and 7.30 ppm ( J= 8 Hz), and a double doublet at 7.44 ppm ( J= 1.5, 8 Hz) attributed to the H- 2', -5', and - 6 ' of the 3', 4' di-substituted B ring. The '^C NMR spectrum showed all four methoxyl carbons at low field (above 60 ppm), suggesting the presence of four methoxyls two of which are in the A ring occupying both ortho positions [101-104]. The complete carbon skeleton was assigned from the '^C NMR INADEQUATE experiment. A 0.45M solution containing O.IM tris(acetylacetonate)chromium (IH) [Cr(acac)]] was utilized in this experiment. The addition of of Cr(acac )3 decreases the long spin-latttice relaxation times for carbons that 147 are remote from protons [105-108]. The only signals left to assign were the methoxy groups and the methylene of the methylenedioxy. The long-range coupling experiment (COLOC) data assigned the methoxy groups, as well as, confirmed the placement of the methylenedioxy at the 3 ', 4 '- position of the B-ring. The methylene protons at 6.03 ppm showed a three-bond coupling to the 3', and 4' quaternary carbons at 148.25 and 150.57 ppm. Based on these results and values reported in the literature, compoimd (106) was identified as linderoflavone B. The complete ‘H and ‘^C NMR assignments are given in Tables 40, and 41, and the structure illustrated with standard numbering in Figure 56. Eupalestin (107) The next eluted compound was eupalestin (5,6,7,8,5'-pentamethoxy-3',4'- methylenedioxyflavone, also called conyzorigim) has the molecular formula C 2 1 H2 0 O9 (MW 416.1111, calculated value 416.1107) from HRMS. The UV spectrum (MeOH) showed peaks at 245(sh), 271, and 334 nm indicating a flavone rather than a flavonol [100]. The and '^C NMR spectral data were similar to linderoflavone B except; (a) the proton spectra showed only two aromatic doublets at 7.10, and 7.05 ppm along with the H-3 (6.52 ppm) and methylenedioxy (6.52 ppm) protons, and (b) five methoxy groups were present instead of four, with only one of the methoxy groups resonating at high field in the carbon spectrum (56.97 ppm). Thus compound 107 must have the additional methoxy group on the 5'-position giving a tri-substituted B ring. 1 4 8 The *^C NMR spectral data was assigned based on that of linderoflavone B. Chemical shifts of the carbons in ring A are not affected by a methoxy group in ring B, and the carbons in ring B are not affected by the position of methoxy groups in ring A [102]. The COLOC experiment was again instrumental in assigning the methoxy groups, as well as, the methylenedioxy position. Based on these results and values reported in the literature, compound 107 was identified as eupalestin. The complete ‘H and ‘^C NMR assignments are given in Tables 40, and 41, and the structure in Figure 56. 5’-Methoxynobiletin (108) S’-Methoxynobiletin (5,6,7,8,3’,4’,5’-heptamethoxyflavone) has the molecular formula C 2 2 H2 4 O9 (MW 432.1418, calculated value 432.1420) from HRMS. The UV spectrum in MeOH showed peaks at 273, and 323 nm indicating a flavone rather than a flavonol [ 1 0 0 ]. The ‘H NMR spectrum indicated seven methoxy groups, and one proton singlet at 6.61 ppm corresponding to H-3. Also, in the aromatic region the signals of the remaining two protons appeared as a singlet at 7.14 ppm and these were assigned to the 2', and 6 ' protons of a tri-substituted B ring. In the '^C NMR spectrum four methoxyl carbons were observed at low field (61.61, 61.74, 61.85, and 62.20 ppm) indicating all positions in the A ring were substituted by methoxy groups. 1 4 9 Table 39: Sources of flavones isolated irom Ageratina coelestinum Compound Plant source Reference linderoflavone B Lindera lucida [109] Agératum conyzoides [110,111] Eupatorium coelestinum [112] Eupatorium leucolepis [113] Agératum tomentosum var. [114] bracteatum eupalestin Agératum conyzoides [110,111,115] Eupatorium coelestinum [112] Eupatorium leucolepis [113] Agératum tomentosum var. [114] bracteatum 5'-methoxynobiletin Agératum conyzoides [110,111,115] Eupatorium coelestinum [112] nobiletin Agératum conyzoides [110,111] Eupatorium coelestinum [112] Eupatorium leucolepis [113] Citrus nobilis [116,117] Citrus reticulata [103] Citrus subgroup microcarpa [101] Citrus hassaku [104] 150 Table 40: H NMR assignments for flavones isolated from Ageratina coelestinum in CDCI3* compound position 106 107 108 109 3 6.51 s 6.52 s 6.61 s 6.52 s 2' 7.30 d (1.7) 7.05 d (1.6) 7.14 s 7.32 d (2) 5' 6.87 d (8.3) 6.90 d (8.6) 6' 7.44 dd (1.8, 8.2) 7.10 d (1.6) 7.14 s 7.47 dd (2.1, 8.5) OCH2 O 6.03 s 6.04 s, 2H OMe 3.90 3.90 3.93 3.87 3.90 3.90 3.93 3.87 4.06 4.06 4.08 4.02 3.98 3.97 4.00 3.95 [5, 6, 7, 8] [5, 6, 7, 8] [5, 6, 7, 8] [5, 6, 7, 8] 3.94 3.93 3.88 [5’] 3.90 3.87 3.93 [3-, 4'] [3', 4', 5’] “ H NMR spectrum taken at 270 MHz and reported as ppm (CHCI3 solvent peak set at 7.26 ppm) with data resolution of 0.3 Hz. Pattern symbols: s=singiet, d=doubIet, and coupling constants (J) in Hz. 151 Table 41: NMR assignments for Flavones isolated from Ageratina coelestinum in CDCb“ compound position mult.^ 106 107 108 109 2 s 160.89 160.84 160.76 160.93 3 d 107.16 107.54 107.16 106.79 4 s 177.25 177.35 177.17 177.16 5 s 148.44 148.60 148.43 148.35 6 s 144.22 144.40 144.19 144.05 7 s 151.50 151.67 151.53 151.36 8 s 138.18 138.26 138.02 138.01 9 s 147.72 147.83 147.70 147.66 10 s 114.95 115.08 114.85 114.83 r s 125.63 126.82 126.70 124.00 2' d 106.13 100.62 103.55 108.66 3' s 148.57 149.81 153.63 149.31 4' s 150.57 138.54 141.23 151.65 5' d 108.85 144.13 s 153.63 s 111.32 6' d 121.15 107.02 103.55 119.58 OCH2 O t 101.98 102.42 OMe q 62.29 61.93 62.20 62.17 61.85 61.93 61.74 61.72 61.67 61.77 61.61 61.57 62.07 62.11 61.85 61.88 [5, 6, 7, 8] [5,6, 7, 8] [5, 6, 7, 8] [5, 6, 7, 8] 56.97 56.23 55.93 [5'] 60.95 56.03 56.23 [3-, 4'] [3-, 4', 5'] “ ‘^C NMR spectrum taken at 67.9 MHz (CDCI 3 center peak set at 77.2 ppm) and data resolution of 1.0 Hz. ** '^C NMR multiplicities (SFORD): q=quartet, t=triplet, d=doublet, s=singlet. 152 9 ^ OCH: OCH3 CHfeO CH3 O 0 linderoflavone B (106) eupalestin (107) OCH. OCH. OCH3 OCH3 CH30^ M CH30"^Y'^Y^ CH3 O CH3 O o CH3 O 0 5'-methoxynoblletin (108) nobiletin (109) Figure 56: Structure of the flavones isolated fromAgeratina coelestinum The ‘^C NMR data for the A ring matched that of the previously isolated flavones. Full NMR assignments were made based on the linderoflavone B INADEQUATE experimental data. Long-range 'H-'^C-coupling (COLOC) data placed the methoxy groups on the relevant carbons and confirmed the 3', 4', 5'-substitution pattern of the B ring. Based on these results and values reported in the literature 153 compound 108 was identified as 5'-methoxynobiletin. The complete ‘H and *^C NMR assignments are given in Tables 40 and 41, and the structure in Figure 56. Nobiletin (109) The most polar flavone, nobiletin (109) (5,6,7,8,3',4'-hexamethoxyfIavone) has the molecular formula C 2 1 H2 2 O8 (MW 402.1319, calculated value 402.1315) from HRMS. The UV spectrum in MeOH showed peaks at 246, 269, and 331 nm indicating a flavone rather than a flavonol [100]. The ‘H NMR spectral data was similar to that of 5'-methoxynobiletin except: (a) only six methoxy groups were present, and (b) the B ring showed a proton coupling pattern similar to linderoflavone B with two doublets at 6.90, 7.32 ppm and a double doublet at 7.47 ppm. These corresponded to the H-2', -5', and -6' of a di-substituted B ring. The ’^C NMR data for the A ring matched that of the previously isolated flavones. Complete '^C NMR assignments were made based the linderoflavone B INADEQUATE data, and the COLOC experiment which assigned the methoxy groups to the relevant carbons and confirmed the 3', 4', substitution pattern of the B ring. Based on these results and values reported in the literature compound 109 was identified as nobiletin. The complete ‘H and *^C NMR assignments are given in Tables 40, and 41, and the structure in Figure 56. However, our '^C NMR data differed from that reported for nobiletin. There were significant differences from the reported chemical shifts of C-5, and C-6. Machida 154 [104] and linuma [102] both report 144.0 and 138.1 ppm, but our findings of 148.35 and 144.05 ppm apply to all four isolated flavones. Since all other reported NMR chemical shifts for nobiletin were nearly identical to ours, we concluded an error was made in the transcribing of these chemical shifts. The crystalline flavones from Ageratina coelestinum were tested in the antimicrobial assay against C. albicans, M. gypseum, and M. smegmatis with the results shown in Table 44. Attempts to determine minimum inhibitory concentrations (MICs) by the method of Hufford et al. [98] were made. Unfortunately, due to the limited solubility of these compoimds no MICs could be determined. The flavones tended to crystallize in the agar medium thus limiting the availability of the compound. The flavones from A. coelestinum showed the best activity against M. gypseum with 5'- methoxynobiletin being the most potent. Comparisons to compounds currently used in the clinics were made. Griseofulvin and miconazole were approximately 10 fold more potent requiring only 1 0 pg of material to give the same zone of inhibition as 1 0 0 pg of S'-methoxynobiletin. 155 Table 42: Antimicrobial Activity ' of Flavones from Ageratina coelestinum Organisms Candida Microsporum Mycobacterium Compound albicans gypseum smegmatis 48h 72h 48h linderoflavone B (106) ++(4) -H- (6 ) ++(3) eupalestin (107) ++(3) +++(1 0 ) ++(3) 5'-methoxynobiletin (108) ++(4) +++(13) ++(3) nobiletin (109) ++(5) +++(9) ++(4) griseofulvin ++(3p) ++++ (15) nd miconazole nitrate ” ++(5) ++++(13) nd amphotericin B ^ +++(8 ) + + (6 ) nd DMSO (blank) none none + (lp) “ Crystalline compounds 1 mg/ml in DMSO with 100 pg per well. Code: average radius in mm of the zone of inhibition; +, 1-2; ++, 3-6; +++, 7-12; -H-++, >12; p following a reading means partial inhibition; nd=not determined. ’’ Commercial antifungals 100 pg/ml in DMSG with 1 0 pg per well. Of these known flavonoids only nobiletin has been reported to exhibit ftmgistatic activity as tested against Deuterophoma tracheiphita which causes a flmgal disease of citrus varieties [118]. Tangeritin (lacking the 3 -methoxy) was only weakly active. Ageratum conyzoides which is reported to contain linderoflavone B, euplastin, nobiletin, and 5'-methoxynobiletin (Table 41) is a herb used in parts of Nigeria to promote wound healing [115]. Our antimicrobial studies indicate that these compounds could be responsible for the wound healing properties of this plant. 156 Even though these flavones were previously isolated, no biological activity was reported for them. In general, the literature of flavonoids lacks information on the biological activity of this important class of plant compounds. Flavonoids have been in nature for approximately one billion years and are important in plant-insect relationships. Over several decades of research, it has been found that a variety of flavonoids have a range of activities on a number of mammalian enzyme systems [119- 121]. This is of interest since flavonoids account for approximately one gram per day of the Western diet [119]. In light of the renewed interest in the biological activity of flavonoids this study of A. coelestinum will add to that knowledge. Several of the more polar fractions (Table 37) showed good activity against M. smegmatis. However, as in the case of Fraction ABC further investigation was limited due to small quantities of material. Another collection of this plant has been made which should give the additional plant material required for further investigation of these fractions. 157 CHAPTER 5 EXPERIMENTAL Equipment and Reagents Reagents Commercial reagents used were Reagent grade. Solvents for chromatography were redistilled. Acetonitrile and MeOH for polarimetric and UV absorption measurements were Spectroscopic grade (Burdick & Jackson laboratories). Diazomethane was generated using Diazald® (Aldrich). Deuterated chloroform for NMR experiments was 99.8 atom % D (Aldrich). Deuterated pyridine was 99 atom % D (MSD) or 99.0-99.5 atom % D (Aldrich). Thin Layer chromatography (TLC) Silica gel TLC plates were made by coating 5 cm x 20 cm, and 20 cm x 20 cm glass plates with Kieselgel 60 GF 254 (EM Reagents) using a Shandon Southern Unoplan Spreader. Poured layers (0.25mm) were air-dried for 1 hour, activated at 158 I20°C for 30 minutes minutes, and stored at room temperature. For reversed phase TLC 0.25mm layer RP-8 F2 5 4 S plates 5 cm x 20 cm (EM Science). Column Chromatography Adsorbents The adsorbents and their sources are as follows: Silica gel 60, particle size 0.06- 0.200 mm (70-230mesh), and Silica Gel 60, particle size 0.040-0.063 mm (230-400 mesh), EM Reagents; Sephadex LFf-20 particle size 25-100 pm, Pharmacia Fine Chemicals; LiChroprep® RP-8, particle size 40-63 pm; Lobar® LiChroprep® RP-8 pre-packed glass column size A (240 mm x 10 mm), and size B (310 mm x 25 mm); Lobar® pre-packed columns Si 60 40-63 pm size A (240 mm x 10 ram), and size B (310 mm X 25mm), EM Science. /7-Anisaldehyde Spray Reagent To a solution of 5 ml p-anisaidehyde and 90 ml absolute ethanol, 5 ml concentrated H 2 SO4 was added [122]. The solution was stored in the refrigerator at 4°C. Sprayed plates were heated at 110-120"C until the desired color intensity (blue and purple spots) was attained. This visualization procedure was used for all plates unless otherwise noted. 159 Measurement of Physical Constants and Spectra Melting points (uncorrected) were determined using a Thomas-Hoover Melt- Temp apparatus. Fourier transform infrared spectra were obtained using a Laser Precision RPX-40 or Nicolet Protégé 460 spectrophotometer. Optical rotations were determined at the sodium-D line using a Perkin-Elmer Model 241 Polarimeter. Ultraviolet spectra were obtained using a Kontron Instruments Uvikon 860 or a Beckman DU-40 Spectrophotometer. EIMS were obtained using a Kratos MS-30 mass spectrometer by direct inlet system at 70eV. Fast atom bombardment (FAB) mass spectra were obtained using a VG Ltd., VG 70-250S mass spectrometer with Xe and m~ nitrobenzyl alcohol matrix, or GT-13 (glycero 1 zdithiothreito 1, 1:3) [123]. All mass spectra were obtained at the Ohio State University Chemical Instrumentation Center. Nuclear Magnetic resonance spectra were obtained at 11.35 Tesla (500 MHz for 'H and 125 MHz for ‘^C) using a Brucker AM-500 spectrometer; and at 6.35 Tesla (270 MHz for 'H and 67.9 MHz for ‘^C) on an IBM AF-270 spectrometer; and at 5.88 Tesla (250 MHz for ’H and 62.9 MHz for '^C) on an IBM AF-250 Spectrometer. Each NMR spectrometer was equipped with an ASPECT 3000 Data system. The 2D NMR experiments were performed using the automation sequences in the Brucker collection, specifically COLOC.AU, COSY.AU, INAD2D.AU, XHCORRD.AU. Nuclear magnetic resonance spectra were referenced to CHCI 3 7.26 ppm for 'H NMR and 77.2 ppm for '^C NMR for CDCI 3 ; 7.19ppm for ‘H NMR pyr-d 4 , and 123.5 ppm for '^C NMR for pyridine-ds. in both the center of the upfield multiplets. 1 6 0 Plant Materials The entire plant of Solidago ohioensis Riddell was collected on August 16, 1990 and was separated into aerial parts and roots. The collection was made in Clarke Coimty, Ohio along Baldwin Road and Route 4 near Springfield, Ohio by professor Raymond W. Doskotch and Mike Pcolinski and was authenticated by the staff of the Department of Botany of the Ohio State University. Voucher specimens of whole plant are deposited in the Ohio State University Herbarium Columbus, Ohio. Another collection of this plant was made at the same site by professor Raymond W. Doskotch and Pam Boner on August 25, 1994. This second collection was dried and processed as described below to give 1.9 kg of ground tops, and 251 g of ground roots. Initial Processing of Plant Material Solidago ohioensis Leaves and Stems Solidago ohioensis leaves and stems were separated from the roots, and the roots were washed, and all were dried in a forced draft oven at 40°C for 72 hours. The leaves and stems were ground in a Wiley mill, passing through a 2 mm mesh screen to give 2 kg of powdered plant material. This material was packed into glass percolators and continually extracted with 95% ethanol imtil the percolates were devoid of residue. The ethanolic extracts were evaporated imder reduced pressure at 40°C to give 307 g of resinous residue. 161 Solidago ohioensis Roots Solidago ohioensis roots were dried and processed as for the leaves and stems to yield 0.9 kg groimd plant material. The dried root material was exhaustively extracted with 95% ethanol and evaporated to give 116 g of residue. The roots were then heated to boiling in 1 L of 95% ethanol three times and solvent removed. This extract was added to the previous residue to give 123 g. Partitioning of the Ethanolic Extract Residue Solidago ohioensis leaves and stems The dried ethanolic extract residue was partitioned between pairs of solvents of different polarities to give four fractions Figure 6 . The solvents were hexane, CHCI 3 , EtOAc, MeOH-HzO (9:1), and H 2 O. The first solvent pair was CHCI 3/H2 O. The CHCI3 soluble residue was then partitioned between hexane/Me 0 H-H2 0 (9:1) to give the hexane solubles as the least polar fraction and the Me 0 H-H2 0 solubles (90% MeOH solubles) as the next polar fraction. The aqueous phase from the first partitioning was extracted with EtOAc to give the least polar of the water solubles. The most polar fraction was the remaining H 2 O solubles. The extracts were dissolved in equal volumes of the solvent pair to give a total concentration of no greater than 10% (w/v). The yield of isolated compounds is based on the weight of compound relative to the weight of dried plant. 162 Solidago ohioensis Roots Partitioning of the dried ethanolic extract of the roots was carried out under the conditions described under Solidago ohioensis leaves and stems. The scheme is found in Figure 7. Investigation of the 90% Methanol- soluble Terpenoids Isolation of the Terpenes from the 90% Methanol Fraction Solidago ohioensis Leaves and Stems The terpenes in the 90% MeOH fraction (hereafter called the MeOH solubles) were first separated from the phenolics by chromatography on Sephadex LH-20 packed in and eluted with MeOH. The flavonoids and other phenolics were retained longer on the column than the terpenes. In 23-27 g aliquots, the MeOH solubles were chromatographed on a 250 g column (59 cm x 4.5 cm). The aliquots were applied as 30% solutions. The effluent fractions were combined after TLC [CHCbrMeOH (20:1)] were pooled to give three fractions; a tarry forerun, the terpenoids, and the flavonoids. This gave 65 g of terpenes and represented a 9% reduction in the amount of material due to phenolics and tarry forerun. Isolation of the Terpenes from the 90% Methanol Fraction Solidago ohioensis Roots The terpenes were separate from the phenolics as given for the leaves and stems. The MeOH solubles 32.1 g were chromatographed in two 16 g aliquots on a 200 1 6 3 g column (58 cm x 4 cm). This gave 27 g of terpenes and represents a 16% reduction in the amount of material due to phenolics and tarry forerun. Sample Preparation for Column Chromatography Silica Gel 60 Adsorbent When the sample was soluble in the initial column solvent, it was dissolved in that solvent as a 33% solution and applied directly to the column. When the sample was not totally soluble in the column solvent, it was adsorbed onto silica gel 60 by dissolving in a suitable solvent (e.g. MeOH), mixed with silica gel 60 usually in a ratio of 1:2 (dried sample weight to silica gel). The mixture was then stirred for 20 minutes and the solvent evaporated under reduced pressure at 40°C in a rotary evaporator to give a dried residue. The residue was broken up in a mortar and the powder applied to the column. Reversed-Phase Adsorbent For reversed-phase low pressure column chromatography, the column was first equilibrated with column solvent. When the sample was soluble in the less polar solvent it was dissolved in a small amount of the less polar solvent (for size A not > 1ml and for size B not > 5ml) and pumped onto the colunm via a three-way valve. When the sample was not soluble in the less polar solvent, it was adsorbed onto reversed-phase silica gel using the procedure described for silica gel sample. The powder was applied 164 to a pre-column of reversed-phase silica gel. TLC analysis on reversed-phase plates was employed to select the column solvent systems. Isolation of the Terpenes from Solidago ohioensis Leaves and Stems Adsorption Chromatography of the Terpenes Fraction The terpenoid fraction from the Sephadex LH-20 columns was fractionated into major fractions as follows: The terpenoids fraction (65 g) was adsorbed to 80 g of Si gel 60 (70-230 mesh) and chromatographed over 750 g of Si gel 60 (64 cm x 6 cm column). The column was eluted with toluene:MeOH (100:0, 49:1, 19:1, 9:1, 4:1, 7:3, 3:2, 1:1) and 2200 15-ml fractions were collected. The effluent fractions were combined after TLC (column solvent system) to give 24 fractions A through X. Nine diterpenes and two monterpene glycosides were isolated from these fractions as shown in Table 2 . Hardwickiic acid (6 8 ) Column fraction A (1.4 g) was divided into two samples I and II. Sample I (0.63 g) was dissolved in 2ml MeOH and applied to a RP - 8 column (size B) equilibrated in Me0 H:H2 0 :CH3 CN ( 1 :1 :0 .2 ) and eluted with mixtures of MeQH:H 2 Ü:CH3 CN of decreasing polarity (5.5:4.5:1; 3:2:0.5; 6.5:3.5:1; 7:3:1). A total of 650 7-ml fractions were collected and pooled based on TLC analysis [Me 0 H:H2 0 :CH3 CN (4:1:0.5)]. Fractions 470 to 490 gave 122 mg of homogeneous solid (-)-hardwickiic acid ( 6 8 ). Sample II (0.77 g) was dissolved in 2.5 ml of MeOH and chromatographed as above. 165 From fractions 150 to 200 was obtained 436 mg (-)- hardwickiic acid ( 6 8 ). Repeated attempts to crystallize these fractions failed. Subsequently, hardwickiic acid was isolated from the roots and crystallization (CH 3 CN) was successful. The crystals (560 mg) of hardwickiic acid ( 6 8 ) (representing 0.03% from dried plant) had the following properties: mp 90-91 °C (literature value 106-107 °C) [41]; [ajo^' -129° (c 4.5. MeOH) (literature value -114°)[41]; IR (film) v^ax 3500-3100 (OH), 2958- 2870 (C-H, aliphatic), 1680 (a,P-imsaturated acid C=0), 1630 (C=C extended conjugated), [1500, 1455, 1411,1384 (furan ring)], 1262(C-0), 1024 (alcoholic C-0), [937, 873, 781, 756 (furan)]cm'\ UV (MeOH) X end abs (log e) 220 nm (3.87); EIMS m/z 316.2031 (1.5%, M^, C2 0 H2 8 O3 requires 316.2038), 298 (0.1, M^-H 2 0 ), 283 (5, M^-H 2 0 -CH3 ), 221 (23, M^-CôHvO), 203 (20, M^-HzO^-CgHTO), 151 (13), 137 (28), 125 (100), 96 (54), 95 (48, CeHyO^ ), 91 (23), 81 (73.7). The 'H and ‘^C NMR spectral assignments are found in Tables 3, and 4 and the *H NMR and IR spectra in the Appendix in Figures 57, and 58. Methyl ohioenate A (80) Column fraction B (13.9 g) was dissolved in 25 ml of CHCI 3 containing 0.1% TEA, and applied to a 200 g column packed with Si gel 60 (70-23Omesh, activated for Ihr at 120°C) coltunn (65 cm x 1.25 cm). Elution with CHClziacetone with 0.1% TFA (100:0, 9:1, 4:1, 7:3, 1:1, 0:100) gave 2340 10-ml fractions. Comparison of the collected fractions by TLC (RP- 8 , CHsCNiHzO 7:3 with 0.1% TFA) gave 22 fractions 166 B-1 through B-22. Fraction B-13 (3.4 g) showed one major (Rf= 0.46) and two minor spots on TLC (RP- 8 , solvent system as above). Initial ‘H NMR studies indicated that the major spot was still a mixture and was not resolved on TLC due to its tendency to streak. Méthylation of Fraction B-13 To a solution of Fraction B-13 (0.78 g) in dioxane (5 ml) 0°C diazomethane prepared from Diazald©, Aldrich, using the non-alcoholic method was added dropwise until no further Nz evolution was observed. After 30 minutes the solvent was evaporated at reduced pressure to yield an oily residue which was chromatographed on a Si gel 60 column (70-230 mesh, 34 cm x 1.5 cm). Elution with toluene:MeOH (99:1) gave 374 mg of an oily residue which was adsorbed to 0.5 g of Si gel 60 and chromatographed on a Si gel column (27.5 cm x I cm). Elution with hexaneracetone (100:0, 9:1, 4:1) gave 90 5-ml fractions that were pooled to give 5 Fractions A to E. Fraction D (46-60) yielded 18 mg of methyl ohioenate A (80) as a homogenous solid. Fractions B and C were re-chromatographed as for Fraction D using Si gel 60 activated for I hour at 120°C to give 18 mg and 34 mg of methyl ohioenate A (80), respectively, as a solid. Methyl ohioenate A (80) (70mg) had the following properties: [oc]d^° -36.4° (c l.l, CH3 CN); IR(film) Vmax 3471 (OH), 3090(HC=C), 3030 (HC=C), 2921, 2873, 2825 (C-H, aliphatic), 1712 (a,P-unsaturated ester C=0), 1635 (C=C), 1450, 1434, 1251, 1018 (C-0), 768, 756 cm''; UV (CH 3 CN) Xmax (log s) 216 nm (sh) (4.82), 223 nm (sh) (4.64), 275 nm (4.66); EIMS m/z 480.2870(1.3%, M^, C 3 0 H4 0 O5 requires 167 480.2876), 449 (2, NT-OCHs), 448 (10, NT-CHjOH), 415 (7, NT-CHjOH-CHî-HiO), 300 (11), 285 (19, Nr-CH 3 0 H-CH3 -H2 0 -C0 CH=CH-Ph), 203 (12, NT -CH 3 OH- C,5H,703), 175 (9), 139 (18), 131 (100, CgHvO^, 119 (10), 107 ( 1 0 ), 103 (27, CH=CH- Ph^, 77 (19, CeHs^. The *H and NMR spectral assignments are found in Tables 6 , 7, 8 , and 9 and the *H NMR and IR spectra in the Appendix in Figures 61, and 62. Acetate of methyl ohioenate A (81) A mixture of methyl ohioenate A (80) (32 mg, 0.07 mmol), pyridine (0.5 ml), and AczO (0.5 ml) after 20 hours was evaporated to dryness at reduced pressure. The residue was dissolved in CHCI 3 (1 ml) and extracted with H 2 O (3 x 1 mi). The CHCI 3 extract was evaporated at reduced pressure and the residue passed through a short column of Si gel 60 (10 cm x 1 cm) eluted with hexane-.CHCb (1:1, 2:3) to give the acetate (81) (12 mg, 0.02 mmol). The oil had the following properties: [a]o^° -36.4° (c 1.1, CH3 CN); IR(film) Vma.x 2954, 2923(C-H, aliphatic), 1724 (acetate C=0), 1683 (a,p-unsaturated ester C=0), 1635 (C=C extended conjugated), 1450, 1433, 1238 (C- 0), 1162, 1035, 769 cm '; UV (MeOH) (log e) 216 nm (sh) (3.76), 222 nm (sh) (3.75), 275 nm (3.58); EIMS m/z 522.2947 (6.25, M", C 3 2 H4 2 O6 requires 522.2981), 490 (100, NT-CH30H), 475(18, VT-CH30H-CH3), 462 (12), 430 (22, IVTCOzCHg- CH3 -H2 O), 415 (79, NT-C 0 2 CH3 -2 CH3 -H2 0 ), 327 (33, NT-HC=CHPh-C 0 2 CH3 -CH3 - H2 O), 300 (33), 285 (23, M+-C 0 CH=CHPh-CH 3 0 -CH3 -H2 0 ), 203 (43), 169 (36), 139 (40), 103 (25), 91(9), 69 (36). The 'H and '^C NMR spectral assignments are found in 168 Tables 6 , 7, 8 , and 9 and the H NMR and IR spectra in the Appendix in Figures 63, and 64. Hydrolysis of methyl ohioenate A (80) to diol 82 To a solution of methyl ohioenate A (80) (23mg, O.OSmmol) in MeOH (1 ml, dry) at 0°C was added NaOMe (1 mg). After 16 hours the reaction was quenched with NH4 CI to neutral pH. Two drops of 5% HCl was added and mixture was evaporated to dryness under reduced pressure. Water (10 ml) was added and extracted with CHCI 3 (3 X 10 ml). The organic phases were pooled and dried under reduced pressure. TLC analysis of the residue showed one major blue spot Rf= 0.35 (CHChiMeOH 19:1) more polar than compound 80. The residue was passed through a short column of silica gel (5g, 70-23Omesh) eluted with CHCbiMeOH (100:0, 99:1, 49:1, 97:3, 24:1) to give the diol 82 (15 mg, 0.04 mmol). The amorphous solid had the following properties: [ajo"' -56° (c 1 .6 , MeOH); IR(film) Vm« 3288 (OH), 3031 (HC=C), 2999, 2979, 2954, 2931,2871 (C-H, aliphatic), 1714 (a,p-unsaturated ester C=0), 1704, 1455, 1434, 1383, 1250, 1232,1194, 1037, 1022, 1014(C-O), 999, 752 cm"'; UV (MeOH) X end abs (log e ) 210 nm (4.15); EIMS m/z 350.2462 (0.06%, M^, C2 1 H3 4 O4 requires 350.2457), 319 (13,1VT-0CH3), 318 (46, M+-CH 3 OH), 303 (39, M^-CH3 0 H-CH3 ), 300 (44), 285 (100, M+-CH3 OH-CH3 -H2 O), 235 (20, M^-CgHnOz), 233 (20), 219 (24), 203 ( 39, M^- C6 H,,0 2 -CH3 0 H), 175 (17), 150 (20), 139 (49), 105 (12), 91 (9), 79 (7). The 'H and ’^C NMR spectral assignments are found in Tables 6 and 8 and the ‘H NMR and IR spectra in the appendix in Figures 65, and 6 6 . 1 6 9 Patagonie acid (83) Column fraction C (2.2 g) was dissolved in 12 ml CHCI 3 containing 0.1% TFA. The solution was applied to a 40 g Si gel 60 (70-230 mesh) column (45 cm x 1.5 cm) in CHCI3 with 0.1% TFA. Elution with CHCI 3 containing 0.1% TFA gave 250 10-ml fractions. The effluent fractions combined after TLC using column solvent system were pooled to give nine pooled fractions C-1 through C-9. Fraction C - 1 (0.7 g) showed 2 major spots on TLC dark blue Rf = 0.38, and a purple Rf = 0.33 (RP- 8 , CH3 CN:H2 0 (3:2) with 0.1% TFA). Fraction C-1 was dissolved in 3 ml CH 3 CN with 0.1% TFA. filtered through a 22p Millipore® filter and applied via 3-way valve to a RP - 8 Lobar® column (size B) equilibrated in CH 3 CN:H2 0 (1:1) containing 0.1%TFA. The column was eluted with mixtures of CH 3 CN:H2 0 with 0.1%TFA of decreasing polarity (3:2, 7:3) and a total of 300 5-ml fractions were collected and combined after TLC analysis (RP-8 , CH3 CN:H2 0 3:2 with 0.1% TFA) to give 7 fractions C-1 A through C-IG. Fraction C-IB (81-95) gave 107 mg of homogeneous solid patagonic acid (83). Fraction C-2 (117 mg) was chromatographed (same conditions as C-1) to give 48 mg of compound 83. The solid (155 mg) patagonic acid (83) has the following properties: -78° (c 10.4, CH 3 CN) [lit. [a]o -65.9° (c 2.65, CHCI3 )] [60] ; IR(film) v^ax 3400-2500 (OH), 2962, 2933, 2875, I784(sh), 1753 (a,p-unsaturated-y-lactone C=0), 1737(sh), 1722(sh), 1684, 1640(sh), 1615 (sh), 1450, 1420, 1385, 1352, 1261, 1213, 1170, 1076, 1054, 1035, 1003, 758 cm"'; UV (CH 3 CN) X end abs (log s) 213 nm (4.21); EIMS m/z 332.1966 (0.3%, M^, C2 0 H2 8 O4 requires 332.1988), 314 (100, M^- 170 HzO), 299 (5, NT-HzO-CHs), 271 (7, M^-HiO-CHs-CO), 221 (5), 203 (155, NT-HiO- C6 H7 O2 ), 175 (16), 137 (12), 125 (28), 112 (78), 111 (4, CeH^Ozl, 105 (13), 93 (13), 91 (15), 81 (11), 79 (113), 69 (123), 55 (15). The ‘H and "C NMR spectral assignments are in Tables 12, 13, and 14 and the ‘H NMR and IR spectra in the Appendix in Figures 67, and 6 8 . 15-Methoxypatagonic acid (85) Column fractions C-IC and C-ID from above were combined (81 mg) and adsorbed to 0.2 g of Si gel 60 (70-230 mesh) and applied to a 10 g Si gel column (20 cm X 1 cm) in hexane. The column was eluted with mixtures o f hexanerEtOAc (9:1, 4:1, 7:3). The hexane:EtOAc (7:3) eluted 21 mg of the lower Rf = 0.33 (RP- 8 CH3 CN:H2 0 (3:2) with 0.1% TFA) spot as a homogenous solid 15-methoxypatagonic acid (85). The homogeneous solid had the following properties: [a]o'* -55° (c 1.6, MeOH); IR (film) Vm« 3400-3100 (OH), 3000 (sh), 2958, 2923, 2859, 1770 (o,(3- unsaturated-y-lactone C=0), 1710 (a,P-unsaturated acid C=0), 1680 (C=C), 1629, 1456, 1421, 1365, 1340, 1263, 1207, 1120, 1078, 1024, 985, 937, 762 cm"'; UV (MeOH) X end abs (log e) 214 nm (4.1); EIMS m/z 362.2090 (0.30%, MT, C2 1 H3 0 O5 requires 362.2093), 344 (60, M+-H2 O), 329 (100, M^-H2 0 -CH3 ), 297 (1 1 ), 203 (13, M"- H2 O-C7 H9 O3 ), 175 (9) 173 (13), 145 (7), 142 ( 1 1 ), 137 (15), 128 (17), 125 (37), 119 (11), 107 (12), 105 (21), 97 (15), 91 (24), 79 (15), 77 (15), 69 (14), 55 (28). The 'H and 171 NMR spectral assignments are found in Table 15 and the *H NMR and IR spectra in the Appendix in Figures 71, and 72. 15-Methoxypatagonic acid methyl ester ( 8 6 ) At 0°C a 13mg (0.036mmol) sample of 15-methoxypatagonic acid (85) in dioxane (1 ml) was esterified as described for méthylation of Fraction B-13. After 1 hour the solvent was removed under reduced pressure to yield a residue which on TLC showed one purple spot Rf =0.23 (CHCI3 ). The residue was chromatographed on a silica gel 60 column (5g) eluted with CHCI 3 . This gave 5 mg (0.013 mmol) of the ester 8 6 as a colorless gum with the following properties: [a]‘^ d -14° ( c 5.0, CH3 CN) [lit. [a]o -30.8° (c 0.37, CHCI 3 )] [39]; IR (film) Vmax 2924, 2879, 2854, 1768 (a,p- unsaturated y-lactone C=0), 1724 (a,P-unsaturated ester C=0), 1718 (sh), 1684 (C=C), 1456, 1367, 1238, 1207, 1172, 1122, 1078, 1024, 985, 935 cm"'; UV (CH 3 CN) X end abs (log e) 215 nm (3.6); EIMS m/z 376.2246 (1.5%, M*”, C 2 2 H3 2 O5 requires 376.2250), 360 (19), 358 (31), 344 (77, VT-CH30H), 329 (71, NT-CH30H-CH3), 297 (6 , M^-OCH3-2CH3Ji20), 271 (11), 249 (20, M+-C 6 H7 O3 ), 235 (7, M"-C7H903), 217 (20), 203 (13, M^-C7H903-CH30H), 179 (24), 175 (13), 173 (25), 128 (46), 91 (48), 55 (70), 41 (100). The 'H and '^C NMR spectral assignments are found in Table 16 and the 'H NMR and ER spectra in the Appendix in Figures 73, and 74. 172 Chromatography of Fraction D Column fraction D (1.6 g) was divided into three samples designated I, II, and in. Sample I (0.51 g) was dissolved in 3 ml CH 3 CN with 0.1% TEA and filtered through a 0.22 pm Millipore® filter. The solution was applied to a RP - 8 Lobar® column (size B) and eluted with HzOzCHsCN containing 0.1% TFA of decreasing polarity (3:2, 5.5:4.5, 1:1,4.5:5.5,2:3). A total of 330 5-mI fractions were collected and pooled based on TLC. The HzO:CH 3 CN (5.5:4.5) eluted fraction 3 (204 mg) of what appeared as a single spot on TLC (RP - 8 H2 0 :CH3 CN 4.5:5.5 with 0.1% TFA). Initial ’H NMR studies of the sample indicated a 1:1 mixture of two closely related compounds not resolved on TLC due to severe streaking. Sample U (0.55 g) and sample in (0.53 g) were similarly chromatographed and gave 210 mg and 349 mg, respectively, of the same mixture. Méthylation of column Fraction D-3 A sample of D-3 (48 mg) was esterified as described for Fraction B-13 to give an oil which by TLC showed one major spot Rf = 0.49 (hexane:acetone 7:3). Another sample of D-3 (50 mg) was treated in the same manner. The residues were passed through a short column of Si gel 60 (5g) eluted with hexane:acetone (9:1, 4:1) to give 39 mg of a white solid. Subsequent ‘^C NMR revealed it was still a 1:1 mixture of closely related diethoxy containing compounds. The remaining 143 mg of D-3 was esterified and chromatographed as above to yield 84 mg of the mixture. 173 The methyl esters were combined (123 mg) and applied to 25g of Si gel 60 (70- 230 mesh, activated for 1 hr at I20°C) column (60 cm x 1 cm) in CHCI 3 . A total of 910 2-ml fractions were collected and pooled to give 7 fractions A to G. Fractions B (341- 440) and C (441-530) yielded 53 mg of a homogeneous white solid methyl ohioenate B (91) with Rf= 0.4 (hexane:EtOAc, 7:3). Fractions E (581-660) and F (661-860) gave 62 mg of methyl ohioenate C (92) as a homogeneous solid, Rf= 0.36. Methyl ohioenate B (91) Methyl ohioenate B (91) had the following properties: [a]o'^ -65° (c 8.0, CH3 CN); IR (film) Vmax 3455 (OH), 2967, 2922, 2877, 1717 (a,p-unsaturated ester C=0), 1700, 1256, 1233, 111, 1083, 1067, 989 cm '; UV (CH 3 CN) X end abs (log e) 215 nm (3.9); EIMS m/z 423.2750 (1.7%, M^- 0 CH3 , C2 4 H3 9 O6 requires 423.2747), 392 (4, M^-CH30H-CH3-CH3), 344 (14), 329 (20), 301 (5), 259 (5), 235 (7, M^-CioHigO;), 203 (10, M^-CioHigOs-CHsOH), 173 (7), 139 (18), 127 (100), 99 (53); FABMS (GT- 13) m/z 477 (4.9%, M^+Na), 454.24 (0.8, M^, C 2 SH4 2 O7 requires 454.29). The 'H and '^C NMR spectral assignments are in Table 22 and the ‘H NMR and IR spectra in the Appendix in Figures 83, and 84. Methyl ohioenate C (92) Methyl ohioenate C (92) had the following properties: [a]o'^ -18° (c 8.0, CH3 CN); IR (film) Vmax 3404 (OH), 2972, 2952, 2918, 2875, 1716 (a,P-unsaturated 1 7 4 ester C=0), 1434, 1383, 1275, 1238, 1197, 1034, 1012, 978, 966, 756 cm''; UV (CH3 CN) X end abs (log e) 215 tim (3.5); EIMS m/z 422.2665 (1.0%, M^-CHjOH, C2 4 H3 8 O6 requires 422.2668), 376 (13, \r - H 2 0 -CH3 -0 CH2 CH3 ), 330 (26), 258 (5), 235 (10, nT-C.oHiçOs), 203 (14, M^-CioH,90s-CH30H), 175 ( 8 ), 139 (26), 127 (100). 99 (30), 55 (27). FABMS (/w-nitrobenzyl alcohol) 423.31 (10.1%, M^-OCH 3 ). The 'H and '^C NMR spectral assignments are found in Table 25 and the ‘H NMR and IR spectra in the Appendix in Figures 85, and 8 6 . 15-Hydroxypatagonic acid (87) Column fraction E (1.4 g) was divided into three samples designated I, II, and m. Sample I (0.6 g) was dissolved in 4ml of CH 3 CN with 0.1% TFA and filtered through a 22 pm Millipore© filter and applied to a RP - 8 Lobar® column (size B). The column was eluted with mixtures of H 2 0 :CH3 CN containing 0.1% TFA of decreasing polarity (3:2, 5.5:4.5, 1:1) to give 330 5-mI fractions. Comparison of the collected fractions by TLC (RP- 8 , H2 0 :CH3 CN, 1:1 with 0.1% TFA) gave 11 pooled fractions E- 1 through E-11. Fraction E-5 eluted with H 2 0 :CH3 CN (5.5:4.5 with 0.1% TFA) gave 202 mg of a solid . Sample II (0.5 g) and in (0.3 g) were chromatographed under the same conditions to give 191 mg, and 138 mg respectively of solid. These solids (531 mg) upon repeated column chromatography could not be purified to a homogeneous solid. Column fractions (see isolation of 16-hydroxyclerodermic acid below) F-2 through 5 were combined (0.34 g) and chromatographed on a Lobar® size B RP - 8 175 column. The column effluent fractions combined after TLC [MeOHiHaOrCHsCN (7:3:1)] were pooled to give 0.2 g of a material with one major spot Rf =0.38 (pink) along with one minor impurity Rf = 0.45. This material was chromatographed on a Lobar® RP-8 size A column eluted with CHsCN-.HiO (1:1) with 0.1% TFA. A total of 130 1.5-ml fractions were collected and pooled. Fractions 43-61 gave 28 mg of a homogeneous solid. The amorphous solid of 15-hydroxypatagonic acid (87) had the following properties: [ajo’’ -93° (c 10.0, CH 3 CN); IR (KBr) v^ax 3400-2600 (OH), 2959, 2921, 2875, 1750 (lactone C=0), 1734 (sh), 1717(sh) (a,P-unsaturated ester C=0), 1680, 1456, 1261, 1206, 1009 cm '; UV (CH 3 CN) X end abs (log s) 210 nm (4.66), W 250 nm (sh) (3.91), 293 nm (3.81); EIMS 348.1934 (0.8%, M^,C 2 oH2 gOs requires 348.1437) 330 (100, M^-H 2 Û), 315 (47, M^-H 2 0 -CH3 ), 297 ( 8 ), 221 (14, VT- C6 H7 O3 ), 203 (21, VT-C6H703-H20), 175 (8.7, M"-C6H7Û3-H20-C0), 173(9.4), 151 (7.6), 125 (37.2), 113 (9), 105 (13), 91 (15), 81 (9), 79 (11), 55 (11). The 'H and ‘^C NMR spectral assignments are found in Table 17 and the 'H NMR and IR spectra in the Appendix in Figures 75, and 76. Reduction of 15-hydroxypatagonic acid (87) to patagonic acid (83) To a stirred solution of 15-hydroxypatagonic acid (87) (12 mg, 0.03 mmol) in MeOH (1 ml) at 0°C was added NaBH» (19 mg, 0.5 mmol) over 30 minutes. After 20 h 5% HCl was added to pH = 3 and then H 2 O (2 ml). The MeOH was removed under 176 reduced pressure and the reaction mixture was extracted with CHCI3 (3x3 ml). The combined organic extracts were dried imder reduced pressure to give 1 1 mg of solid product. The reduction was carried out under the same conditions 2 more times using 23 mg (0.06 mmol) and 40 mg (0.1 mmol) of 87. The combined residues (60 mg), were passed through a short column of silica gel 60 (5g) eluted with CHCI3 containing 0.1% TFA to give patagonic acid (83) (26 mg, 0.08 mmol) identical by TLC, UV, IR, [a]o, 'H and '^C NMR to patagonic acid isolated from the plant. Patagonic acid methyl ester (84) To a solution of patagonic acid (from NABH4 reduction of compound 87) (22 mg, 0.06 mmol) EtzO (1 ml) at 0°C was added freshly prepared diazomethane (Aldrich, standard prep) was added dropwise until no further N 2 release was observed and the yellow color remained for >5min. After 1 h the solvent was removed under reduced pressure to give a gummy residue. The residue (19 mg) was passed through a short column of silica gel 60 (5g) eluted with CHCI3 to give methyl patagonate (84) (7 mg, 0.02 mmol). The gum had the following properties: [a]o’^ -25° (c 4.0, MeOH), literature value [a]o -62° (c 1.38, CHCI 3 ) [39] ; IR(film) v^ax 2852, 2920, 2871, 1754 (a,P-unsataturated-y-lactone C=0), 1712 (a,P-unsaturated ester C=0), 1251, 1237, 1197, 1065 cm"'; UV (MeOH) X end abs (log s) 214 nm (4.22); EIMS 346.2155 (5.9%, IVT, C21H30O4 requires 346.2144), 314 (100, M^-CHsOH), 271 (1 1 , M^-CH3 0 H- CH3-CO), 235 (7, M"-C 6 H7 0 2 ), 203 (19, M^-C6 H7 0 2 -CH3 0 H), 175 (30, M+-C 6 H7 O2 - CH3OH-CO), 151 (13), 139(35), 119(17), 111 (2, C 6 H7 0 2 ^ , 105 (28), 97 (11),93 (25), 177 91 (39), 81 (18), 79 (27), 77 (19), 69 (29), 55 (44). The 'H and "C NMR spectral assignments are found in Tables 12, and 13 and the ‘H NMR and IR spectra in the Appendix in Figures 69, and 70. 16-Hydroxyclerodermic acid (8 8 ) Column fraction F (4.2 g) was chromatographed on a 100 g column (62 cm x 1 cm) of Si gel 60 (70-230 mesh) in CHCI 3 . The column was eluted with CHC^zMeOH (99:1, 49:1, 97:3, 24:1, 19:1, 93:7, 9:1, 4:1, 3:1) and gave 1200 9-ml fractions. Comparison of the collected fractions by TLC allowed combination into 12 Fractions F- 1 through F-12. Fraction F - 6 eluted with 4% MeOH crystallized upon pooling to give 54 mg of 16-hydroxyclerodermic acid (8 8 ) as needle-like crystals. Fractions F-4 and F- 5 were combined (1.4 g) and crystallized twice (CHCI 3 ) to give 544 mg of 8 8 . Fraction F-9 (89 mg) was crystallized from CHCI3 to give 31 mg crystalline 8 8 . The mother liquors from F-4 through F - 6 (960 mg) were adsorbed to Ig of Si gel 60 (70-230 mesh) and chromatographed over 50g of Si gel 60 (51 cm x 0.75 cm) in CHCI 3 . The column was eluted with CHCl 3 :MeOH (99.5:0.5, 99:1, 98.5:1.5, 49:1, 19:1, 9:1,4:1) to give 730 5-ml fractions. Comparison of the collected fractions by TLC allowed combination into 9 Fractions F 4-6A through F 4-61. Crystallization of subfractions D through H (CHCI3) gave an additional 254 mg of crystalline 8 8 . The crystals (880 mg) had the following properties: mp 176-77°C; [a]o‘’ -92° (c 6.0, CH3 CN); IR (KBr) Vm«3383 (OH), 2958. 2920, 2866, 1763 and 1739 (a,P-imsaturated-y-lactone C=0 with a-H), 1678 (a,P- unsaturated acid C=0), 1647, 1454, 1335, 1261,1192, 1126, 991, 891 cm''; UV (CH3 CN) k end abs (log s) 210 nm (4.18), X^ax 250 nm (2.72); EIMS m/z 348.1965 (0.75%, M", C2 0 H2 8 O5 requires 348.1937), 330 (53, Nr-H 2 Ü), 315 (16, M"-H2 0 -CH3 ,. 312 (8 , M^-H2 0 -H2 0 ), 271 ( 8 , M+-H2 O-CH3 -CO2 ), 2 2 1 (43, M^-C6 H?0 3 ), 203 (58, M"- H2 O- C6 H7 O3 ), 137 (31), 125 ( 1 0 0 ), 105 (2 0 ), 91 (28), 79 ( 2 1 ), 77 (14), 69 (13), 55 ( 2 1 ); FABMS (/w-nitrobenzyl alcohol) m/z 349.18 (3.5%, MH^, 348.16 (2, M^ ), 331 (92, 1 7 8 MUT-HzO), 154 (100). The and '^C NMR spectral assignments are found in Table 18 and the ‘H NMR and IR spectra in the Appendix in Figures 77, and 78. Reduction of 16-hydroxyclerodennic acid ( 8 8 ) to clerodermic acid (89) A 10 mg (0.029 mmol) sample of 16-hydroxyclerodermic acid ( 8 8 ) was reduced as given for 15-hydroxypatagonic acid (87). The reduction was carried out twice more under the same conditions using 16 rag (0.046 mmol) and 53 mg (0.160 mmol) of 8 8 . The combined residues (78 mg) were passed through a short Si gel 60 (5 g) column eluted with CHCb.-MeOH (100:0, 99:1,49:1) to give clerodermic acid (89) (76 rag, 0.23 mmol) as a homogeneous solid. Clerodermic acid ( 8 8 ) had the following properties: [a]D*^ -76° (c 10.0, CH 3 CN), literature values [a]o -70° (c 0.52, CHCI 3 )] [70], [a]o -94°, no concentration given (CHCI 3 ) [6 8 ]; IR (film) v^ax 3400-2600 (OH), 2958, 2921, 2863, 1780 and 1747 (a,p unsaturated-y-lactone C=0 with a-H), 1680 (a,P-unsaturated acid C=0), 1635, 1448, 1421, 1387, 1282, 1263, 1170, 1018 cm'‘; UV (CH 3 CN) X end abs (log e) 215 nm (3.27); EIMS m/z 332.1978 (0.78%, NT, C 2 0 H2 8 O4 requires 332.1988), 314 ( 1 0 0 , M^-H2 Û), 299 (18, M+-H2 O-CH3 ), 271 (4, Nf-H 2 0 -CH3 -C0 ), 236 (13), 235 (17), 221 (15, M^-C6H?02), 203 (29, M^-H20-C6H7O2), 189 (12), 175 (12, M"- H2 O-C6 H7 O2 -CO), 165 (13), 125 (6 6 ), 121 (20), 111 (34, C6H702^, 105 (29), 98 (25), 95 (24), 93 (27), 91 (42), 79 (35), 55 (53). The *H and '^C NMR spectral assignments are in Tables 19 and 20 and the *H NMR and ER spectra in the Appendix in Figures 79, and 80. Clerodermic acid methyl ester (90) The methyl ester of clerodermic acid (89) (25 rag, 0.075 mmol) was prepared with diazomethane under the conditions described under “methyl ester of patagonic acid”. Methyl clerodermate (90) (24 rag, 0.069 mmol) had the following properties: [a]o'^ -59° (c 4.1, CH3 CN), literature values [a]o -28° (c 0.1, CHCI 3 )] [70], [a]o -62° 179 (c 1 .6 8 , CHCI3 )] [39]; IR (film) v^ax 2954, 2921, 2864, 1780 and 1750 (a,p- unsaturated y-Iactone C=0 with a-H), 1712 (a,P-unsaturated ester C=0), 1637, 1456, 1441, 1435, 1252, 1171, 1063, 1024 cm UV (CH 3 CN) X end abs (log e) 215 nm (4.14); EIMS m/z 347.2241 (2.2%, MH^, C 2 1 H3 1 O4 requires 347.2222), 314 (100, VT- CH3 OH), 299 (34, VT-CH3 0 H-CH3 ), 271 (10, M^-CH 3 0 H-CH3 -CO), 235 (9, M"- C6 H7 O2 ), 203 (29, M^-CH30H-C6H702), 175 (25, lvr-CH30H-C6H702-C0), 165 (10), 151 (17), 139 (35), 119 (19), 111 (20, C6 H7 O2 I , 107 (23), 105 (34), 98 (14), 95 (16), 93 (25), 91 (42), 79 (32), 69 (16), 55 (36). The 'H and '^C NMR spectral assignments are Table 21 and the ‘H NMR and ER spectra in the Appendix in Figures 81, and 82. Adsorption Chromatography of Column Fractions G and H Column fractions G and H (1.5 g) were applied to the top of a 35 g column of Si Gel 60 (70-230 mesh) of size 39 cm x 0.75 cm packed in CHCI 3 . Elution with CHCbrMeOH (99.5:0.5, 99:1, 49:1, 97:3, 24:1, 19:1, 9:1, 4:1, 7:3) gave 820-3ml fractions. These fractions were further combined based on TLC analysis (CHCb.'MeOH 19:1) to give 11 Fractions G-1 to G-11. Ohioenic acid D (93) Column Fractions G-5 and G - 6 were combined (0.3 g) and adsorbed onto 0.5 g of Si gel 60 (70-23Omesh). The powder was applied to the top of a 4 g column of Si gel (24.5 cm x 0.25 cm) in hexane:acetone (9:1) and eluted with mixtures of hexane:acetone (9:1, 4:1, 3:1, 7:3, 3:2, 1:1). A total of 410 3-ml fractions were collected and further combined into 8 pools (G-5A through G-5H) based on TLC analysis (hexane:acetone 3:2 with 2 developments). Hexane:acetone (4:1) (G-5B) eluted a column fraction from which 60 mg of ohioenic acid D was crystallized (hexane:acetone). The needle-like crystals of 93 had the following properties: mp 165- 180 178 °C decomposition; [a]o'^ -56° (c 10.0, MeOH); IR (film) Vmax 3400-2500(OH), 2956, 2924, 2871, 2856, 1733 (sh), I707(sh), 1681(a,P-unsaturated acid C=0), 1450, 1383, 1241, 1195, 1110, 1074, 989, 966 cm '; UV (MeOH) X end abs (log s) 215 nm (3.74); EIMS m/z 412.9831 (0.09%, VT, C 2 2 H3 6 O7 requires 412.2461), 394 (1, M*- H2 O), 363 (7, NT-HzO-OCHs), 316 (21), 221 (18, M^-CgHjsOs), 203( 21, M^-CgHnO;- H2 O), 191 (1, CgHisOsl, 175 ( 6 , CgHisOs-HzO-CO), 137 (14), 125 (48, CgHisOs-HzO- OH-OCH3 ), 113 (100). The 'H and NMR spectral assignments are found in Table 26, and the 'H NMR and IR spectra in the Appendix in Figures 87, and 8 8 . (-)-Bomyl 6-0-acetyI-p-D-glucopyranoside (97) Column fractions G-5D and G-5E (50 mg) were combined, chromatographed on Si gel 60 (70-230mesh), and eluted with mixtures of hexaneracetone (9:1, 4:1, 3:1, 7:3) to give 30 mg of an impure solid. This material was chromatographed on a Lobar RP - 8 column (size B) eluted with mixtures of Me 0 H:H2 0 :CH3 CN. The Me0 H:H2 0 :CH3 CN (6.5:3.5:l) solvent eluted 17 mg of a homogeneous solid which by 'H NMR contained a small amount of aliphatic impurity. Column fraction G-5 F (60 mg) was chromatographed on a Si gel 60 column (70-23Omesh, 3 g) and eluted with CHCl3 :MeOH (99:1, 49:1) to give 55 mg of an impure compound and subsequently chromatographed on a Lobar RP - 8 (size B) column under the same conditions as above to give another 16 mg of the homogeneous solid. These two samples were combined and rechromatographed on a Si gel 60 (70-23Omesh, 5g) column eluted and with 18 1 CHClg:MeOH( 100, 99:1). The column fractions eluted with CHCbiMeOH (49:1) crystallized from the solvent to give 14 mg of 97. The rosette-like crystals had the following properties: mp 176-177 °C; [a]D*^-40° (c 1.0, MeOH); IR(film) v^ax 3431 (OH), 2951, 2927, 2877, 1743 (C=0), 1454, 1365, 1241, 1148, 1079, 1037 cm*'; UV(MeOH) Àmax (log s) 226 nm (sh) (2.66), 277 nm (2.16) ; EIMS m/z 359.211 (.001%, MH^,C,gH3 i0 7 requires 359.2070), 247 (4), 221 (1, CgHuO?"), 205 (11, CgHnOe^, 187 (15, CgHuOe^-HzO), 137(100, C,oH, 7 ^ , 127 (15), 109 (24), 81 ( 6 6 ). The 'H and '^C NMR spectral assignments are in Table 30, and the 'H NMR and IR spectra in the Appendix in Figures 95, and 96. (-)-Bomyl P-D-glucopyranoside (94) Column fractions K and L which showed one major spot on TLC, Rf = 0.2 (CHCls'.MeOH ,99:1), were combined (4.4 g) and chromatographed on a 100 g column (61 cm X 1 cm) of Si gel 60 (70-230 mesh) in CHCI 3 . The column was eluted with mixtures of CHCl3 :MeOH of increasing polarity (99:1, 49:1, 7:3, 19:1, 93:7, 9:1, 17:3) and a total of 970 5-ml fractions were collected and combined based on TLC analysis (CHCl3 :MeOH, 99:1). CHCl3 :MeOH 9:1 eluted 344 mg of solid 94 which was homogeneous by TLC and NMR. Numerous attempts at crystallization were unsuccessful. The homogeneous solid had the following properties: [ajo’’ -50° (c 10.0, MeOH), literature [a]o -55.6°, no concentration given) [80]; IR(film) v^ax 3392 (OH), 2983, 2950, 2877, 1471, 1454, 1388, 1270, 1108, 1077, 1024 cm*'; UV (MeOH) W (log s) 219 nm (sh) (2.96), 277 nm (1.99); EIMS /n/z 317.1961 (0.2%, MH^, CiaHzgOe 182 requires 317.1964), 195 (3), 163 (2, CeHgOsl, 153 ( 6 , C,oHnOl, 138 (21), 109 (45), 85 (29), 81 (100), 73 (38); The and '^C NMR spectral assignments are found in Table 27, and the NMR and IR spectra in the appendix in Figures 89, and 90. (-)-Bomyl 2,3,4,6-tetra-O-acetyl-P-D-glucopyranoside (95) A 25 mg (0.08 mmol) sample of compound 94 was acetylated under the conditions described for methyl ohioenate A acetate (80) and yielded the crude peracetate. The residue was passed through a short column of Si gel 60 (5g) eluted with CHCI3 to give the tetraacetate 95 (33 mg, 0.07 mmol). Subsequent crystallization (MeOH) gave 7 mg of needle-like crystals. The crystals of 95 had the following properties; mp 121-123 °C, literature value 118-119.5°C [80]; [ajo'^ -41° (c 15.0, MeOH), literature [a]o -52.7°, no concentration given [80]; IR (film) v^ax 2951, 2873, 1752 (C=0), 1365, 1219,1166, 1038 (C-0) cm*'; UV (MeOH) ^max (log e) 226 nm (sh) (2.95), 277 nm (2.18); EIMS m/z 331 (21.3%, C 14H 19OO, 271 (4, C^HigOg^-OAc), 211 (43, C 1 4 H 19O/-OAC-OAC), 169 (74, C, 4 Hi9 0 9 ^-0 Ac-0 Ac-CH2 C0 ), 137 (85, CioHivl, 109 (58), 81 (75); FABMS (/n-nitrobenzyl alcohol) m/z 507.29 (100% M^+Na), 484.44 (0.3, M^, C 2 4 H3 6 O 1 0 requires 484.2308 ), 331 (41), 169 (461), 137 (67). The ‘H and NMR spectral assignments are found in Table 28, and the *H and IR spectra in the Appendix in Figures 91, and 92. 183 (-)-Borayl P-D-glucopyranoside tetramethyl ether (96) To a solution of (-)-bomyl-P-D-glucopyranoside (94) (50 mg, 0.16 mmol) in dry DMF (1.5 ml) was added CH3 I (0.03 ml) and Ag 2 Û (51 mg). After stirring under argon for 48 hours, CHCI 3 (4 ml) was added. After 30 minutes the suspension was filtered and the yellow ppt was washed with CHCI 3 (3x3 ml). The filtrate was extracted with an equal volume of H 2 O and the organic layer was dried under reduced pressure to give 62 mg of a yellowish oil. The oil was passed through a 5 g column eluted with toluene:CHCl 3 (100, 19:1, 9:1, 7:3, 3:2) to give the tetramethyl ether 96 (15 mg, 0.04 mmol). The oil had the following properties: [a]o'^ -173° ( c 1.5, MeOH): IR (film) Vmax 2981, 2951, 2879, 2834, 1456, 1388, 1375, 1365, 1306, 1095, 1055 cm '; UV (MeOH) Xmax (log e) 221 nm (sh) (3.12), 278 nm (2.69), EIMS m/z 309 (5%), 187(12, C,oHi905^-CH30H), 137 (100, C,oH, 7 ^ , 111 (11), 105 (28), 101 (57); FABMS (m- nitrobenzyl alcohol) m/z 373.22 (1.0%, MH^, 373.26 (0.2, M^, C 2 0 H3 6 O6 requires 372.2512), 309 (1), 219 ( 6 , CioHigOsl, 187 (58, CioHigOg^-^OH), 155 ( 8 ), 137 (100, CioHitO- The 'H and *^C NMR spectral assignments are found in Table 29, and the 'H NMR and IR spectra in the Appendix in Figures 93, and 94. Isolation of the terpenes from Solidago ohioensis roots Adsorption chromatography of the terpene fraction The terpenoid fraction from the Sephadex LH-20 column (27 g) was adsorbed onto 30 g of Si gel 60 (70-230 mesh) and chromatographed on a 300 g column of Si gel 1 8 4 60 (47 cm X 4 cm). The column was eluted with toluenerMeOH (100:0, 99:1, 49:1. 19:1, 9:1, 4:1) and 1120 10-ml fractions were collected. Comparison of the collected fractions by TLC (with coliunn solvent) allowed combination into 15 Fractions A through 0. Two diterpenes were isolated from these fractions. Hardwickiic acid (6 8 ), and hardwickiol (79) Column fractions B-F (7.2 g) all contained the same purple spot Rf = 0.47 by TLC (toluene:MeOH 9:1) which was identical to hardwiickic acid. Crystallization from CH3 CN gave 3.7 g of needle-like crystals identical to the previously isolated hardwiickic acid in mp, specific rotation, IR, *H NMR and TLC mobility. The mother liquors were divided into two samples designated I and II. Sample I (0.63 g) was applied to a Lobar® RP - 8 column (size B) and eluted with mixtures of CH 3 CN:H2 0 of decreasing polarity (1:1, 5.5:4.5, 6:4). A total of 330 5-ml fractions were collected and pooled based on TLC analysis (CH 3 CN:H2 0 , 6:4). Fractions 201-280 gave 344 mg of hardwiickic acid. Fractions 289-330 gave 101 mg of hardwickiol as a homogeneous solid. Sample II (0.6 g) was treated in the same manner and gave 195 mg of hardwickiic acid but no hardwickiol. The homogeneous solid of hardwickiol (79) has the following properties: [a]o'^ -39° (c 1.8, EtOH), literature [a]o^^ -47.2 (c 5.6%, no solvent reported) [41], [a]o^^ -41.8 (c 7.0, CHCI 3 ) [35]; IR(film) Vm« 3348 (OH), 2927, 2869, 2831, 1672, 1502, 1452, 1382, 1242, 1217, 1187, 1161, 118, 1097, 1161, 1128, 1397, 1065, 1024, 1000, 873, 779, 760 cm"'; UV (EtOH) (log s) 223 nm (3.34), 240 nm 185 (3.3); EIMS m/z 302.2246 (12.6%, M", C2 0 H3 0 O2 requires 302.2246), 271 (15, M"- CH2 OH), 253 (3, M+.CH2 OH-H2 O), 207 ( 6 , M^-CôHtO), 189 6 ( 6 , M^-C6 H7 Û-H2 0 ), 147 (16), 133 (27), 121 (19), 119 (34), 111 (29), 109 (13), 107 (36), 95 (42, CgHvOl, 91 (41), 81 (100, CsHsO^. The *H and NMR assignments are in Table 5, and the ’H NMR and IR spectra are in the appendix in figures 59, and 60. Reduction of Hardwickiic acid ( 6 8 ) to hardwickiol (79) A 50 ml 3-necked flask that had been flamed-dried and flushed with argon was fitted with an argon balloon, dropping funnel and drying tube. LiAlHj (80 mg, 2 . 1 mmol) was weighed, placed into round bottom flask and covered with EtiO. Hardwiickic acid (195 mg, 0.6 mmol) was dissolved in 10 ml Et 2 0 and added dropwise. After 16 hours the reaction mixture was quenched with H2Û:15% NaOH:H 2 Û (0.1:0.1:0.3) and stirred for 10 minutes. The gray gelatinous material was filtered off and rinsed with Et 2 0 . The filtrate was collected and dried under reduced pressure to give 119 mg of crude residue. The residue was passed through a short column of Si gel 60 (5g) eluted with hexane:CHCl 3 (1:1, 2:3, 3:7) to give 40 mg (0.13 mmol) of hardwickiol (79) identical to the isolated hardwickiol in specific rotation, IR, *H NMR. UV, MS, and TLC mobility. Synthesis of patagonic acid (83) The light source (GE 120V 250W quartz-halogen lamp ) was placed in a glass jacketed water cooled container which was inside a large silver-lined Dewar cooled to 186 0°C. The reaction vessel was a large test tube fitted with a rubber stopper with vent and bubbler for O 2 . A solution of hardwickiic acid ( 6 8 ) 50 mg (0.16 mmol), thiourea (20 mg), and methylene blue (1 mg) in MeOH:HzO (8:1) was irradiated for 2 minutes while O 2 bubbled in. The reaction showed no starting material by TLC (CHCL.MeOH 19:1). The reaction mixture was dried under reduced pressure at 40°C and passed through a short Si gel 60 column (5g) deactivated with 5% H 2 O. The column was eluted with CHCl 3 :MeOH (99:1) to give 43 mg of a mixture of methoxy containing compounds which were hydrolyzed in CH 3 CN (2 ml) to which was added 5% aq. HCl (2 ml) dropwise. After stirring for 16 hoiu-s a white precipitate formed. The solvent was removed to give 27 mg (50% yield) of patagonic acid (83) as a homogeneous solid which had the same specific rotation, IR, NMR, MS, and TLC mobility as the isolated natural product. Terpenes from Amphiachyris dracunculoides (DC.) Nutt. Plant Material The above-ground portion of Amphiachyris dracunculoides (DC.) Nutt, was collected on October 18, 1985 on the farm of Dr. Loyd Harris in Norman, Oklahoma. The air-dried plant material upon inspection showed some mold had formed during transportation. The moldy plant material was discarded and the rest was dried in a forced air oven at 40°C. The dried plant material was ground in a Wiley mill as previously described for Solidago ohioensis. 187 Initial processing of plant material The powdered air-dried above-ground parts of Amphiachyris dracunculoides (1.5 kg) was packed into a glass percolator and continually extracted in sequence with hexane, acetone, isopropyl alcohol, and finally with 95% ethanol until the percolates were devoid of residue. The extracts were evaporated under reduced pressure at 40°C to give the resinous residues at shown in Figure 50. Chromatography of the acetone extract residue The acetone extract, by TLC (CHCbrEtOAc, 3:1) analysis showed several p- anisaldehyde reacting spots. A column of silica gel 60 (70-230 mesh) was slurry packed in CHCI 3 (60 cm x 3.5 cm). Chloroform was passed through the column for seven days at 25ml/hr to settle the packing for improved resolution. A sample of the acetone extract residue (24.3 g) was dissolved in CHCI 3 (125 ml) and applied to the top of the column. The column was eluted with CHCI 3 containing increasing EtOAc, followed by EtOAc, and EtOAc with increasing concentrations of acetone. The effluent fractions combined after TLC (CHCbrEtOAc, 3:1) were pooled to give 26 Fractions A through X. The column was given a final wash with acetone:MeOH (1:1) to ensure all material was eluted and gave Fraction Y. Direct crystallization and/or additional chromatographic separation resulted in the isolation of six cw-clerodane diterpenes. Five were isolated previously in our laboratory fi"om a Kansas collection of Amphyiacyris dracunculoides and one was a new c/j-clerodane diterpene [84-86]. 188 Amphiacrolide-B (98) Fractions F (0.4 g) and G (0.4 g) showed the same blue zone R f=0.28 by TLC (CHClsiEtOAc, 3:1). Crystallization of the two fractions (acetone-hexane) gave colorless needle-like crystals (321 mg) of compound 98, mp 189-190 °C [literature 189- 191 °C] [84,85]. Comparison of the specific rotation, IR, ‘H and '^C NM R with an authentic sample of compound 98 were made. The ‘H NM R, and IR spectra are found in the Appendix in Figures 97, and 98. Amphiacrolide C (99) TLC analysis (CHCL3 : EtOAc, 3:1) showed column Fractions L and M each had two major spots one purple Rf = 0.26, and one blue Rf =0.16. The fractions were combined (1.0 g) and applied to an Ace Michel-Miller pre-column packed with Si gel 60 (5 g). The pre-column was attached to a Lobar® Si gel 60 size B column and eluted with CHCI 3 with increasing EtOAc. A total of 400 3-ml fractions were collected and pooled based on TLC analysis (CHCl 3 :EtOAc, 1:1) to give 9 Fractions LM-1 through LM-9. The column was washed with EtOAc;MeOH (1:1) to give fraction LM-10. Fractions LM-5 through LM-7 were pooled and crystallized from acetone-hexane to give 328 mg as colorless prisms of the lower Rf spot compound 99, mp 150-151 °C (literature 145-147 °C) [84,85]. Comparison of the specific rotation, IR, ‘H and ‘^C NMR with an authentic sample of compound 99 showed them to be the same. The 'H NMR, and IR spectra are found in the Appendix in Figures 99, and 100. 1 8 9 Amphiacrolide D (100) Column Fraction N (0.7 g), O (0.9 g), and P (1.0 g) showed several spots by TLC analysis (CHCLiEtOAc, 3:2) but each contained the same reddish zone Rf = 0.21. Each sample was chromatographed as described for amphiacrolide C (99). From Fraction N a total of 430 3-ml fractions were collected and pooled based on TLC analysis (as above). Fraction 9 gave 52 mg of the reddish reacting material. Fraction O gave 560 4-ml fractions which were combined into 8 fractions. Fractions 0-3 and 0-4 gave 442 mg of the same reddish reacting material. Fraction P gave 310 4-ml fractions which were combined into 7 pools. Fraction P-4 gave 151 mg of the same material. These fractions were combined (designated NOP) and adsorbed onto Si gel 60 (1 g) and applied to a pre-column and chromatographed on a Lobar® Si 60 size B column. Elution with hexanerEtOAc (7:3) gave 730 3-ml fractions. Comparison of the collected fractions by TLC (RP- 8 , CH3 CN:HzO, 3:2) gave 6 pooled Fractions NOP-1 through NOP-6 . Fraction NOP-2 (330 mg) showed one major and two minor spots by TLC (RP- 8 , same as before), and was chromatographed on a Lobar® RP - 8 (size B) column. The sample was adsorbed onto RP - 8 Si gel and applied to a pre-column and eluted with CH3 CN:H2 0 (2:3) to give 100 2-ml fractions. Fractions 67-84 gave 223 mg of amphiacrolide D (100) amorphous solid. Comparison of the specific rotation, IR,'H and ‘^C NMR with an authentic sample of (100) was made. The ‘H NMR, and IR spectra are found in the Appendix in Figures 101, and 102. 190 Amphiacrolide R (101) Column fraction NOP-4 (55 mg) appeared as approximately 95% pure, but crystallization was unsuccessful. The fraction was chromatographed on a 5 g silica gel 60 (70-230 mesh) column (10 cm x 1.0 cm), packed in CHCI 3 and eluted with CHCbracetone (99:1), (49:1), (19:1). CHCl 3 :acetone (99:1) eluted 37 mg of compound 101 which was homogenous by TLC Rf = 0.46 (EtOAC.-CHCh, 3:1) and 'H NMR. Amphiacrolide R (101) has the following properties: [a]o'^ +110° (c 6.0, MeOH); IR(film) V m a x 3413 (OH), 2932, 2875, 1778 and 1747 (a,P-unsaturated y-lactone C=0), 1734(sh), 1716 (ketone C=0), 1635 (C=C), 1456, 1330, 1175, 1116, 1076, 1025, 889 cm''; UV (MeOH) k end abs (log s) 214 nm (4.02), Imax 281 nm (2.02); CD (C 1.72 x lO'^M, MeOH) [0]33o +15, [0]283 +9907 (max), [0]23S +61; EIMS m/z 348.1929(0.9%, M^, C2 0 H2 8 O5 requires 348.1937), 330 (2, M^-HzO), 289 (20, VC-CH 3 -C0 ), 220 (3, M"- OH-C6 H7 O2 ), 219 (5, M+-H2 O-C6 H7 O2 ), 192 (29, M^-0 H-C6 H7 Û2 -C0 ), 189 (40), 177 (18, M^-0H-C6H702-C0-CH3), 147 (12), 135 (13), 123 (25), 199 (21), 111 (58, C6 H7 O2 I , 107 (31), 98 (46), 95 (33), 93 (45), 91 (44), 55 (75), 41 (100). The CD spectrum is found in appendix Figure 105. The 'H and '^C NMR assignments are in Tables 32, and 33, and the ‘H NMR, and IR spectra are in Appendix in Figures 103, and 104. 191 Amphiacrolide R acetate (102) To solution of Amphiacrolide R (101) 98 mg in pyridine (1.5 ml) under argon was added acetic anhydride (1.5 ml). After stirring for 24 hours the solvent was evaporated under reduced pressure. The residue was dissolved in CHCI 3 (5 ml) and extracted with H 2 O (2x5 ml). The organic layer was dried under reduced pressure. The residue was crystallized (MeOH) to give 48 mg of needle-like crystals of compound 102. The mother liquors were dissolved in MeOH (1 ml) and chromatographed on a Lobar® RP - 8 size A column. The column was equilibrated in MeOHiHzOiCHsCN (3.5:6.5:1.0) and eluted with MeOHzHzOiCHsCN (2:3:0.5). A total of 190 2-ml fractions were collected and pooled based on TLC analysis (RP- 8 , column solvent). Fractions 71-79 gave 6 mg of homogenous solid 102, and fraction 101-140 gave 10 mg of the starting material 101. The crystals of compound 102 had the following properties: mp 166-168 °C; [ajo'^+150° (c 0.4, MeOH); IR(film) v^ax 2933, 1778 and 1743 (a,|3-unsaturated y-lactone C=0), 1719 (acetate C=0), 1637 (C=C), 1446, 1373, 1330, 1174, 1097, 1054, 1029, 962, 887 cm*'; UV (MeOH) X end abs (log e) 215 nm (4.04), Xmax 272 nm (1.81); EIMS m/z 390.2030(0.1%, M^, C2 2 H3 0 O6 requires 390.2042), 331 (32, M^-OAc), 330 (152, VT-HOAc), 302 (23.6, M^-HOAc-CO), 238 (20), 219 (152, VT-HOAc-CgHvOz), 192 (602), 189 (582), 161 (122), 111 (73, C6 H?0 2 ^ , 98 (49, C 5 H6 O2 I , 97 (7, CsHs 0 2 ^ , 95 (21), 91(35), 43.0191 (100, Ac", requires 43.0184). The ‘H and ’^C NMR spectral assignments are found in Table 34, and the 'H NMR, and IR spectra in the Appendix in Figures 106, and 107. 192 Amphiacrolide R methyl ether (103) Amphiacrolide R (101) (35 mg) was dissolved in 1 ml MeOH and 5 drops of IN HCl was added. The reaction was monitored by TLC (CHClgzEtOAc, 1:1) and after 3 days, only a small amount of unreacted starting material remained. The solvent was evaporated under reduced pressure. The residue was passed through a short column of silica gel 60 (70-230 mesh, 5g) and eluted with CHCbiEtOAc (7:3) to give compound 103 (15 mg) and starting ketone (3 mg). The homogenous solid 103 has the following properties: Rf =0.33 (CHCl3 :EtOAc, 1:1); [a]o'^+53.3° (c 3.0, MeOH); IR (ftlm) 2927, 1778 and 1747 (a,P-unsaturated-y-lactone C=0), 1716 (ketone C=0), 1637 (C=C), 1450, 1387, 1128, 1101, 1054, 1031, 889 cm '; UV (MeOH) X end abs (log e) 215 nm (3.96); EIMS m/z 362.2109 (2.8%, M+, C 2 1 H3 0 OS requires 362.2093), 331 (23, M^-0CH3), 330 (123, NC-CHsOH), 302 (24, M^-CH30H-C0), 276 (40), 233 (16, M"- H2 O-C6 H7 O2 ), 219 (14, M^-H0 CH3 -C6 H? 0 2 ), 205 (42), 189 (19), 163 ( 1 0 ), 147 (23), 123 (78) 111 (52, C 6 H7 O2 I , 98 (100, C 5 H6 O2 I , 97 (9, CgHiCh^, 95 (41), 91 (56), 79 (84). The ‘H and '^C NMR spectral assignments are found in Table 35 and the ‘H NMR, and IR spectra in the Appendix in Figures 108, and 109 Amphiacrolide E (104) Column Fractions H, I, J, and K each showed the same major TLC spot Rf = 0.26 as did column Fractions L and M as discussed under amphiacrolide C . These four fractions were combined (1.2 g) and chromatographed as were Fractions L and M (as discussed under Amphiacrolide C). A total of 350 3-ml fractions were collected and 193 pooled to give 9 fractions HIJK-1 through 9. Fractions HIJK - 6 and -7 showed by initial 'H NMR analysis that amphiacrolide E (104) was present along with some impurities. Fractions HIJK - 6 and -7 were combined with LM-1 through -4 (0.9 g) and applied to a Si gel 60 (70-240mesh, activated for 1 hr at 120°C) column (34 cm x 1 cm) which had been equilibrated for 24 hours in hexanerCHCh (1:1). The column was eluted with the same solvent and a total of 320 4-ml fractions were collected and pooled to give 685 mg of Amphiacrolide E (104) plus an impurity. Crystallization was unsuccessful. Fractions N-2, 0-2, P-1 and -2 (as discussed under Amphiacrolide D) showed by 'H NMR the presence of amphiacrolide E plus another compound. These combined fractions (0.7 g), designated as NP were chromatographed as above with hexane: CHCI3 (1:1) to give 690 4-ml fractions. The fractions were pooled based on TLC analysis (hexane:EtOAc, 7:3) to give 4 fractions NP-1 through -4. Pool NP-1 gave 120 mg of compound 104 plus impurity. The two fractions from above containing amphiacrolide E (104) (0.8 g) were combined, adsorbed onto RP - 8 Si gel and applied to a precolumn connected to a Lobar® RP - 8 size B column. The column was eluted with CHsCN:H 2 0 (3:2) and a total of 140 3-ml fractions were collected and pooled based on TLC (RP- 8 , column solvent). Fractions 106-140 were combined and evaporated to give 290 mg of homogeneous oil 104. Comparison of the specific rotation, IR, ‘H and ’^C NMR with an 194 authentic sample of 104 showed them to be identical. The ‘H NMR, and IR spectra are found in the Appendix in Figures 110, and 111. Amphiacrolide I (105) Fraction N P-2 (350 mg) (as discussed under isolation of amphiacrolide E) showed one major purple spot by TLC (hexanerEtOAc, 3:2) Rf = 0.24 and one minor spot Rf = 0.20. Fraction N P -2 was adsorbed onto RP-8 silica gel and chromatographed on a Lobar® RP-8 silica gel column size B. The column was eluted with CHsCNrHaO (2:3) and a total of 180 3-ml fractions were collected. Fractions 91-120 were combined and dried under reduced pressure to give 169 mg of homogenous oil 105. Comparison of the specific rotation, IR, ‘H and ‘^C N M R with an authentic sample of compound 105 showed them to be the same. The 'H NM R, and IR spectra are found in the Appendix in Figures 112, and 113. Antimicrobial compounds from Ageratina colestinum L. (syn Eupatorium colestinum L.) Plant material The entire plant of Ageratina colestinum L. was collected on September 7, 1994 by Professor Raymond Doskotch and Pam Boner in Pike County, Ohio along Route 23 approximately 1.3 miles South of Park Road in a large field opposite Gullions Furniture Store. The collection was authenticated by the staff of the Department of 195 Botany of the Ohio State University and voucher specimens were deposited in the Ohio State University Herbarium in Columbus, Ohio. Initial processing of plant material Ageratina colestinum L. was dried in a forced draft oven at 40°C for 72 hours, and ground in a Wiley mill, passing through a 2mm mesh screen to give 640 g of powdered plant material. This material was packed into glass percolators and continually extracted with 95% ethanol until the percolates were devoid of residue. The ethanolic extracts were evaporated under reduced pressure at 40°C to give 87 g of resinous residue. Biological ccreening Antimicrobial screening was performed in the Medicinal Chemistry and Pharmacognosy Department, Ohio State University, Columbus Ohio by Professor Larry W. Robertson. The crude extract, its fiactions, and pure compoimds were tested in the qualitative antimicrobial screening procedure against the following microorganisms, fi-om the American Type Culture Collection (ATCC): Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 10536), Mycobacterium smegmatis (ATCC 607). Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16888), Trichophyton mentagrophytes (ATCC 9972), Microsporum gypseum (ATCC 11395). The assay was carried out using the conditions of Hufford et al. [98]. Plates for the assay were prepared by addition of 25 ml sterile agar in 100 x 15 mm sterile Petri 196 dishes. The sterile agar plates were streaked with a dilution of the test organism and cultured in nutrient agar and BMM (minimal agar) for bacteria, trypticase soy agar for M. smegmatis, and mycophil or czapek agar for fungi. Wells were bored in the agar (I I mm) using a sterile cork borer. To the wells was added a solution of an extract, fraction, or pure compound. The extracts and fractions were tested at 20 mg/ml in DMSO and pure compounds at 1 mg/ml in DMSO with 100 pi per well. The plates were incubated for 24 to 48 hours at 37°C for bacteria, and 48 to 72 hours at 30°C for fungi. The antimicrobial activity was recorded at the end of the incubation period as the width (in mm) of the zone of inhibition. A DMSO blank was included in each assay. Several standard antifungal agents, amphotericin B, miconazole nitrate, and griseofulvin were tested as a positive control for antifungal activity. The following codes were used to describe the activity; (none) no activity, (+) l-2mm, (++) 3- 6 mm, (+++) 7-12mm, (+ + -H -) > 12mm, (p) following a reading means partial inhibition indicating a reduction in number of colonies but not a clear zone. Partitioning of the ethanolic extract residue Ageratina colestinum L. The dried ethanolic extract residue was partitioned between solvent pairs of different polarities to give four fractions. The solvents and techniques used were the same as discussed imder Solidago ohioensis. The scheme is found in Figure 55. 197 Separation of the terpenes from the flavonoids in the methanol partition fraction of Ageratina colestinum L. The terpenes in the MeOH fraction (24.6 g) were first separated from the phenolics by chromatography on Sephadex LH-20 as described under Solidago ohioensis. The appropriate fractions were pooled and evaporated to give 5.8 g of a nonpolar fraction, 13.7 g terpenes, and 3.4 g of phenolics. This represents a 37% reduction in material due to nonpolar and phenolic material. Adsorption chromatography of the terpenes The terpenoid fraction from the Sephadex LH-20 column was first chromatographed as follow: 13.7 g were dissolved in CHCI 3 as a 20% solution and chromatographed over 200 g of Si gel 60 (51 cm x 3 cm) column. The column was eluted with CHCI 3 and MeOH (100:0, 99.5:0.5, 99:1, 19:1, 9:1, 5:1, 7:3, 3:2, 1:1) and 1020 4-ml fractions were collected. Comparison of the collected fractions by TLC (column solvent) allowed combination into 19 pooled fractions A through X. Four flavones were isolated from these fractions as shown in Table 38. Isolation of flavones Fractions D, E, F, and G showed good anitfungal activity against M. gypseum (Table 37). These fractions contained a mixture of flavones which could be separated with difficulty after repeated recrystallizations and column chromatography. 1 9 8 When Fraction F (1.7 g) was concentrated under reduced pressure crystals were observed. Fraction F with two crystallizations from CHCb-hexane, and one from MeOH gave 77 mg of eupalestin (107). The mother liquors were divided into two samples I and H. Sample I (0.3 g) was chromatographed over Si gel 60 (70-230 mesh, 31cm X 2.5cm) column. The column was eluted with mixtures of EtzOzpet ether (100:0. 9:1, 4:1, 7:3) and a total of 350 5-ml fractions were collected and pooled into 5 Fractions F-1 through F-5 based on TLC analysis (EtzO/ pet ether 4:1 with 4 developments). Fraction F/I - 1 eluted with Et 2 0 :pet ether (4:1) gave a homogeneous solid which was recrystallized from MeOH to give 123 mg of linderoflavone B (106). Fractions F/I-2 through F/I-5 contained a mixture of the other three flavones. Sample II (0.9 g) was chromatographed as above. A total of 580 5-ml fractions were collected and pooled into 4 fractions F/H-l through F/II-4. Fraction F/II-1 gave a homogeneous solid which was recrystallized from MeOH to give 122 mg of linderoflavone B (106). Fraction F/II-4 gave a homogeneous solid which was crystallized from MeOH to give 100 mg of nobiletin (109). Fractions F/II-2 and -3 were a mixture of all four flavones. Column Fractions D and E showed the same four flavones by TLC as Fraction F. Column Fractions D and E were combined with the mother liquors and mixtures of flavones from above (1.9 g) and dissolved in hexane and chromatographed over 100 g of Si gel 60 (45 cm x 2.5 cm). The column was eluted with hexane:EtOAc (7:3, 3:2), and 590 5-ml fractions were collected. The fractions were combined after TLC to give 12 similar pooled fractions designated DE-1 through DE-12. Fraction DE-5 on 199 crystallization from MeOH gave 278 mg of linderoflavone B (106). Fraction DE-9 was a mixture of two flavones which upon crystallization from MeOH gave 112 mg of eupalestin (107) The mother liquors from Fractions DE-9 and DE-10 (0.5 g) showed by TLC a mixture of the same two flavones and were combined and chromatographed over 1 0 g Si gel 60. Elution with hexanerEtOAc (19:1, 9:1, 4:1, 7:3) gave three pooled fractions. The first fraction gave after crystallization from acetone-hexane gave 200 mg of 5'- methoxynobiletin (108). Column fraction G (3.1 g) was adsorbed onto 3 g of Si gel 60 and placed on top of a Si gel 60 (50 g) column (23 cm x 2.0 cm). Elution with hexane:EtOAc (100:0, 19:1, 4:1, 7:3) gave 480 5-ml flections. The fractions were combined after TLC to give 9 similar pooled fractions designated G-1 through G-9. From G-2 was crystallized (MeOH) to yield 20 mg of linderoflavone B (106). Fraction G - 6 on crystallization (hexane-acetone) gave 195 mg of 5'-methoxynobiletin (108), while Fraction G-9 was crystallized (MeOH) to give 200 mg of nobiletin (109). Linderoflavone B (106) The colorless needle-like crystals of linderoflavone B (106) (543 mg, 0.08% from dried plant) had the following properties: mp 169-170 °C (MeOH) (literature value 163-168 °C (MeOH) [109], 160 “C [112], 169-170 °C (CHCh/MeOH) [113]); IR (film) 1647 (ketone C=0), 1585, and 1503 cm ' (aromatics); UV (MeOH) Xmax (log s) 249 2 0 0 (4.70), 269 (4.56), 334 nm (4.75) (literature values A.max 247, 270, 332 run [112]); EIMS m/z 386.0990 (27.8%, Nf, CzoHigOg requires 386.1002), 371 (100, M^-Me), 343 (4, M^-Me-CO), 328 (13, M^-Me-CO-Me). The 'H and '^C NMR assignments are found in Tables 40, and 41, and the *H NMR and UV spectra in the Appendix in Figures 114, and 115. Eupalestin (107) The colorless needle-like crystals of eupalestin (189 mg, 0.03% from dried plant) had the following properties: mp 190 °C (MeOH), literature value 187-188 °C (CHCb-MeOH) [113]; IR (film) v^ax 1638 (ketone C=0), 1612, and 1513 cm ' (aromatics); UV (MeOH) Xmax (log e) 245 (sh) (4.76), 271 (4.35), 334 nm (4.60) (literature values Xmax 246, 272 (sh), 332 nm) [112]; EIMS m/z 416.111 (30.8%, M"", C21H20O9 requires 416.1107), 401 (100, VT-Me), 387 (10, M^-CO-H), 371 (35, M^-Me- CH2O) 358 (11, VT-CHs-CO-CHs), 340 (5, VT-CH3-CH20-0Me). The ‘H and '^C NMR assignments are found in Tables 40, and 41, and the 'H NMR and UV spectra are in Appendix Figures 116, and 117. 5'-Methoxynobiletin (108) The colorless needle-like crystals of 5'-methoxynobiletin (108) (395 mg, 0.06% from dried plant) had the following properties: mp 105 ®C (acetone-hexane), literature value 102 °C (Et20) [112]; IR (film) v^ax 1650 (ketone C=0), 1600, and 1510 cm ' (aromatics); UV (MeOH) Xmax (log s) 273 (4.41), 323 nm (4.49), literature values X.max 201 275, 325 nm [112]; EIMS m/z 432.1418 (21.4%, M^, C 2 2 H2 4 O9 requires 432.1420), 417 (100, M^-Me), 401 (4, M^-OMe), 389 (3, M^-Me-CO), 387 (3, M^-Me-CHzO), 374 ( 6 , Nf-Me-CO-CHs), 359 (8 , M^-Me-CHzO-CO), 356 (7, Nf-Me-CHzO-OMe). The 'H and NMR assignments are found in Tables 40, and 41, and the ‘H NMR and UV spectra in the Appendix in Figures 118, and 119. Nobiletin (109) The colorless cube-like crystals of nobiletin (300 mg, 0.05% from dried plant) had the following properties: mp 138-139 °C (MeOH), literature values 134-135 °C (MeOH) [101], 137-138 °C (MeOH) [104], 134 °C (MeOH) [116]; IR (film) v^ax 1644 (ketone C=0), 1600, and 1519 cm'^ (aromatics); UV (MeOH) X^ax ( log e) 246 (4.49), 269 (4.42), 331 nm (4.53), literature values Xmax 246 (4.02), 268 (3.95), 330 nm (4.13) [104]; EIMS m/z 402.1319 (27.0%, M+, CziHzzOg requires 402.1319), 387 (100, M"- Me), 371 (4, M^-OMe), 359 (4, M^-Me-CO), 357 (3, M^-Me-CHzO), 344 (11, M^-Me- CO-CH3 ). The ‘H and ‘^C NMR assignments are found in Tables 40, and 41, and the ’H NMR and UV spectra in the Appendix in Figures 120, and 121. 202 APPENDIX SPECTRA H NMR spectra were recorded at 500 MHz, and acquired in CDCI 3 , unless otherwise stated. IR spectra were recorded as films, unless otherwise stated. 203 lo g Figure 57; ‘h NMR spectrum of hardwickiie acid ( 6 8 ) 100.000 95. 000 85. 000 75. 000 65. 000 55. 000 45. 000 .. I 35. 00^% 03800 3 4 0 0 3000 2 6 £ 0 2 2 0 0 r ...... Kimirc 58: IK spectrum of hardwickiie acid ( 6 8 ) wo O n r-r T --T- ? t *. C A c.a ? 3 i.i : d Figure 59: 'll NMR spectrum of hardwickio) (79) 110. 000 100. 000 60. 0 0 0 40. 000 20. 000 3400 3000 2600 2200 1800 1400 1000 600. 0 Figure 60: IR spectrum of hardwickiol (79) olO 00 Figure 61: NMK spectrum of methyl ohioenatc A (8(1) 110.000 100. 000 80.000 60. 0 0 0 40. 000 3400 3000 2600 2200 1800 1400 1000 600. 0 l _ r — T f— sa 7 3 7 a a e 0 2. A \ i Figure 63: 'il NMR spectrum of methyl ohloenute A acetate (81) 110. ODD 100. 000 90.000 W 80. 000 70. 000 60. 3400 3000 2600 2200 1800 1400 1000 600. 0 Figure 64: IR spcclruiii of iiictliyl oliiociiatc A acvtatc (81) w r o s.t ...... - rr«...... - Figure 65: 'H NMR spectrum of methyl ohioenatc A diol (82) 110. 000 100. 000 80 . 000 40. 000 20. 00. 0 3400 3000 2600 2200 1800 1400 1000 600. 0 Figure 6 6 : IR spectrum of methyl ohioenatc A diol (82) 0.t 7.59.f ?.t B.S B.f S.S ^S.^a 4.5 4.B 5.5 5.B Figure 67: *H NMR spectrum of patagonic acid (83) 110. 000 100. 000 80. 000 60. 000 40.000 20.000 0. 000, 3400 3000 2600 2200 1800 1400 1000 £00. 0 Figure 6 8 : IR spectrum of patagonic acid (83) w On J_ 6~T—’ n ■1" Figure 69: 'll NMK spectrum methyl pataguiiute (84) 10 4- 102 - 100 - 9 8 - 9 6 - 9 4 - lO 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 BOO (cm-1) Figure 70: IR spectrum methyl patagonate (84) w 00 il -X, / r — f - f i.a d ‘hi 3 .1 Figure 71: 11 NMK spectrum of lS-me(hoxypa(agonic acid (85) 1 2 0 . 0 0 0 r— 100. ODD 80. 000 w ^ 60.000 40.000 20. ..L_, ----- 1 3400 3000 2600 2200 i'800 1400 1000 600. 0 Figure 72: IR spectrum of IS-metiioxypatagonic acid (85) g Figure 73; ‘H NMR spectrum 15-methoxypatagoiiic acid methyl ester ( 8 6 ) 110. 000 100. 000 90. 000 Id 80.000 3400 3000 2600 2200 1800 1400 1000 600 Figure 74: lU spectrum IS-metlioxypatagonic acid methyl ester ( 8 6 ) i J t w 7.S 5 # rrn Figure 75: ‘H NMR spectrum of 15-hydroxypatagonlc acid (87) in pyr-dj 1 1 0 . 0 100. o 80 . 0 Idw 60 . 0 40. 0 20. 0 ___ 4000. 0 3400 3000 2600 2200 1800 1400 1000 600. 0 Figure 76: IR spectrum of IS-liydroxypatagonic acid (87) (KBr) g - r T— ^ T— ^ ^—'—'—'—I—' ' ' j— r-,--T — ,— r ; .1 r Figure 77: 'il NMR spectrum 16-liydroxycleruderinic acid ( 8 8 ) in pyr-dg 40. 000 38. 000 34. 000 30.000 26. 000 U\Id 22. 000 18. 000 14. 000 . ■! ■ ■> ■ I m ^ — « I . • « L 1 .. . 1 1 3400 3000 2600 2 2 0 0 1800 1400 1000 600. 0 Figure 78: IK spectrum 16-hydroxyclcroderniic acid ( 8 8 ) (KBr) lOw On Figure 79: *H NMR spectrum clerodermic acid (89) 110.000 100. 000 80.000 — 60. 000 — 40. 000 20.000 3400 3000 2600 2200 1800 1400 1000 600. 0 Figure 80; IK spectrum clerodermic acid (89) lo oo r^H Figure 81: H NMR spectrum methyl clerodermate (90) 110.000 100.000 90. 000 — 80.000 — 70.000 — VOId 60. 000 — 50.000 — 40.000 — 30. 3400 3000 2600 2200 1800 1400 1000 6 0 0 .0 Figure 82: IK spectrum methyl clerodermate (90) bvJ 6 S 6 2 4.0 ppy S 2 5 2.2 Figure 83: H NMR spectrum methyl ohioenatc B (91) 1 20. 000 1 00, 000 80. 000 60. 000 40. 000 20. 000 0 3600 3200 2800 ’^ 2&"0' 2000 1600 1200 800 600. Figure 84: IR spectrum methyl ohioenate B (91) to w 5 5 TT Ta tT t # Figure 85: NMR spectrum methyl ohioenate C (92) 120. 000 1 00. 000 80, 000 60. 000 40. 000 20. 000 0. 000, .fPP.Pî 0. . 3^.PP. 3200 2000 2400 2000 1600 1200 800 600. 0 Figure 8 6 : IR spectrum methyl ohioenatc C (92) tou> T 5 a 0 Figure 87: H NMR spectrum ohioenic acid D (93) in pyr-ds 110. 000 100. 000 90. 000 80 . 000 70. 000 3400 3000 2600 2200 1800 1400 1000 600 Figure 8 8 : IR spectrum ohioenic acid 1) (93) r TT r T TTT - T 1 Figure 89; *H NMR spectrum of (-)-bornyl p-D-glucopyranosidc (94) at 270 MHz 105. 000 too. 000 90.000 00.000 lO 70.000 60.000 3400 3000 2600 2200 1800 1400 1000 500. Figure 90: IR spectrum of (-)-bornyl P-D-glucopyranoside (94) s JIl 1)1 h ft J Ji aII ua 1/ 4 , 1 1 ' " I ' I * I * " I ' I . • I ’ I • I ’ 1 ' I ' I---’ I ' ' I I 1 ' —I I I f T ' I • I » I S.2 s.a 4.s 4.6 4.4 4.2 4 # 3 6 5 6 3.4 3.2 5.6 2.6 2.6 2.4 2.2 2.8 l.B 1.6 1.4 i ,2 1.6 .6 Figure 91; 'H NMR spectrum of (-)-bornyl 2,3,4,6-tetra-O-acetyl-p-D-glucopyranoside (95) 100.000 90.000 lo 80. 0 0 0 so 70. 000 60. 3400 3000 2600 2200 1800 1400 1000 500. 0 Figure 92; IR spectrum of (-)-bornyl 2,3,4,6-tetra-<7-acetyl-P-D-glucopyranoside (95) O lH_ J —r- ~ T ~ T“ ~~T' T ~ T ~ TT 4.2 4. a j 2 5 e 2.9 2.6 2.4 2.'2 2.0 I a \ 6 : 2 1 Q 3 Figure 93: *H NMR spectrum of(-)-bornyl 2,3»4,6-tetra-0-methyI-P-D-glucopyranoside (96) 103. 000 95. 000 85. 000 75.000 3400 3000 2600 2200 1800 1400 1000 500. 0 Figure 94: IR spectrum (-)-bornyi 2 ,3 ,4 ,6 -tetra-t?-melhyl-p-D-glucopyranoside (96) g nil ■f— |- - I r- —1' , r n— • T~ r "f- r \ . S3 pêïi 2 l t A L i ; A Figure 95: ‘H NMR spectrum (-)-bornyl 6-0-acetyl -P-l)-glucopyraiioside (97) 67. 000 64. 000 60. 0 0 0 56. 000 lO w 52. 000 48. 000 44. 000 40. °% o o : 3400 3000 2600 2 2 0 0 1800 r----- 1 400 1000 600.0 Figure 96; IR spectrum (-)-bornyi 6-<7-acetyI -p-D-glucopyranoside (97) 6.ft 6.0 ft.ft ft.O 4.ft Figure 97: *H NMR spectrum amphiacrolide B (98) 270 MHz 110. 0 ï 100. 0 90. 0 80. 0 L/l 70. O 60. 0 50. O J L. r-r 3800. 0 3400 3000 2600 18001400 880. 0 Figure 98: IR spectrum amphiacrolide B (98) O n WaJ!. r - 5.5 5 0 4 5 3 5 3 0 2 5 2 0 0 PPM 15 ) Figure 99; 'H NMR spectrum amphiacrolide C (99) 270 MHz 110. 0 100. 0 f 80. 0 60. 0 'J 40. 0 — 20. 0 0. 0 3800. 0 3400 3000 2600 2200 1800 1400 880. 0 Figure 100: IR spectrum amphiacrolide C (99) oo T T T Figure 101: *H NMK spectrum aniphiacrolidc I) (100) 250 MHz 1 1 0 . 0 90. 0 70. 0 lo < 3 50. O 30. 0 25. O 3800. 0 3400 3000 1800 1400 880. 0 Figure 102: lit spectrum amphiacrolide D (100) w LAO Figure 103: H NMR spectrum amphiacrolide R (101) 115- 110 - 105- 100 - 95 9 0 - 55- 50 4000 35003000 2500 2000 1500 1000 500 Wavenumbers (cm- 1) Figure 104: IR spectrum amphiacrolide R (101) 12000 10000 - 8000 - ^ 6000 - 4000 - 2000 - 220 240 260 280 300 320 340 wavelength (nm) Figure 105: CD spectrum (MeOH) amphiacrolide R (101) 252 LAw UL_ — I— ' — I— — I— 6.0 — r — — r — 9.9 9.0 4.9 4.0 3.9 3.0 2 5 2.0 1.0 PPM 1.9 Figure 106: ‘H NMR spectrum amphiacrolide R acetate (102) 100. o 90. 0 80. O 70. O 50. O 40. O 30. 0 ____ 4000. 0 3400 3000 26002200 1800 1400 1000 600. 0 Figure 107: IR spectrum amphiacrolide R acetate (102) lO U\Ui 5.9 Figure 108; *H NMR spectrum amphiacrolide R methyl ether (103) 105. O 100. 0 90. O 80 . 0 70. 0 GO. 0 50. 0 40. 0 30. 0 ____ 4000. 0 3400 3000 2600 2200 1800 1000 600. 01400 Figure 109: IR spectrum amphiacrolide R methyl ether (103) LA!0 •-J T T Figure 110: ‘H NMR spectrum amphiacrolide E (104) 250 MHz 110. 0 100. 0 e o . 0 60. o 40. 0 2 0 . 0 I 1 1 1 1 1 1 1 1 I I I I 1 I I I 1 1 *1 1 1___ L_J ___ L 3600. 0 3400 3200 3000 2600 2600 2400 2200 2000 1800 1600 * 4 0 0 i pon 1 nnnann o Figure 111; IR spectrum amphiacrolide E (104) s.« Figure 112: *11 NMR spectrum amphiacrolide 1 (105) 250 MHz 110. 0 100. 0 iT- 90. 0 80. O 70. 0 %o 60. 0 SO. 0 40. 0 30. 0 ± J I i.. 3800. 0 3400 3000 2600 2200 1800 1400 880. 0 Figure 113; IR spectrum amphiacroide I (105) G\I J ^ JL_ ^ ^ ^2 70 60 66 64 62 60 50 5^6 5.4 52 50 40 46 4,4 4.2 4.0 38 36 Figure 114: ‘H NMR spectrum iinderoflavonc B (106) 270 MHz 0 . 9 0.8 0.7 0.6 S 0.5 cm •s to I 0.4 lOo\ 0.3 0.2 0.1 lO o motnotnomoinomomoLooino momouiouioioomoinomomo o o ° - tNcocO'V^inin«ocDt^h-ooooo)o>ooT-T- r^(Mroro^«inin(0(or^h>cocoo>a)o (M (N rv (N R) foncococococococorocorororocofo^ Wavelength (nm) Figure 115; UV spectrum linderflavone B (106) 0.0034 mg/ml MeOH Io\ J uJL -JUU “V- —T -----1----- 5------r— '-----1-----' \ '■ ■ I ' I 1 I------r— . 1----- 1 1----- r~ " ■ : - - , ------,— 7.4 7.2 7.0 6.6 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.6 4.6 4.4 4.2 4.0 3.6 3.6 3.4 Figure 116; 'H NMR spectrum eupalestin (107) 270 MHz 0.8 0.7 0.6 0.5 c I0 0.4 1 0.3 0.2 0.1 o m o m o m o lO o O O %— V (X O) o o T - CM CN CM CM CM CM CO CO CO Wavelength (nm) Figure 117: UV spectrum eupalestin (107) 0.004 mg/ml MeOH IJ V,o Jt ______i -JL. JL.. ,IÜL J------r------1---- —I------1------,------1— — I ------1------.------1------r— 7 4 7 2 70 68 6 6 64 62 60 SB 5 6 54 52 5 0 4 8 4 6 4 2 4 0 3 6 3 6 PPM Figure 118: H NMR spectrum S'-methoxynobiietin (108) 270 MHz 0.9 0.8 0.7 0.6 0) cU 0.5 ra •2 IV OS 0.3 0.2 0.1 g o CO COro 5rO Wavelength (nm) Figure 119: UV spectrum S'-methoxynobiletin (108) 0.004 mg/ml MeOH K) OS'J jtU i, J L “I ' I 7 6 7 4 7 2 7 0 6 66 64 6 2 60 68 56 54 52 50 46 46 4 42 40 36 36 PPM Figure 120: ‘H NIMR spectrum nobiletin (109) 270 MHz 0.6 0.5 0.4 ■fi 0.3 t o On 00 o m o m o I/) o tn'o inomouiomomou) o in o in o If) o o T- T- rsj pjcoro'4-vu>u>(0(Oh>r^cQcoo>o> O O T- CM f\J Wavelength (nm) Figure 121; UV spectrum nobiletin (109) 0.0024 mg/ml MeOH LIST OF REFERENCES 1. Merritt, A. T.; Ley, S. V. Nat. Prod. Rep. 1992,243-287. 2. CRC Handbook o f Terpenoids', Dev, S., Ed.; CRC Press, Inc.: Boca Raton, 1985; Vol. n, pp 25-30. 3. 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