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Chemical and biodegradation studies on usnic acid Sanchez Flores, Ignacio Humberto 1974

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CHEMICAL AND BIODEGRADATION STUDIES ON USNIC ACID BY IGNACIO HUMBERTO SANCHEZ FLORES B.Sc. (cum laude), Universidad Nacional Autonoma de Mexico, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry We accept t h i s t h e s i s as conforming to the required standard: The U n i v e r s i t y of B r i t i s h Columbia December, 1974 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C o lumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f C - H Ir/' 1 I C;T $ Y' The U n i v e r s i t y o f B r i t i s h Columbia Vancouve r 8, Canada - i i -ABSTRACT This thesis describes i n v e s t i g a t i o n s on the biodegradation of the l i c h e n substance (+)-usnic a c i d (35), a n a t u r a l a n t i b i o t i c , by several s o i l fungi and b a c t e r i a . Part A of t h i s work i s concerned with the biotransformation of 35 into (+)-6-desacetylusnic a c i d (107) by a Pseudoroorias species. I t s structure e l u c i d a t i o n , laboratory preparation and subsequent conversion i n t o 35 are described. Thorough i n v e s t i g a t i o n s on the condensation of 35 with a s e r i e s of a l i p h a t i c and aromatic amines allowed the determination of the c h a r a c t e r i s t i c g-diketoenamine structure present i n these compounds, as 2 11 exemplified by ( - ) - A ' -enaminousnic acid (132), and concluded with the preparation of the useful (+)-isoxazolo[4,5-b]usnic acid (158) and (+)-2H-[l,2]oxazocinousnic a c i d (159), a novel eight-membered heterocycle. However, the removal of the aromatic a c e t y l grouping under strong a l k a l i n e conditions res u l t e d i n solvent a d d i t i o n to the C.-C. double bond with concomitant 4 4a i r r e v e r s i b l e rearrangement to the i s o u s n i c a c i d s e r i e s , an important process that had not been recognized by previous workers. A l l the attempts to c o n t r o l or avoid t h i s isomerization were unsuccessful, r e s u l t i n g i n m u l t i p l e synthesis of the isomeric (+)-8-desacetylisousnic a c i d (178), which was e a s i l y converted v i a i t s diacetate 179 into (+)-isousnic acid (113), thus c o n s t i t u t i n g the f i r s t t o t a l synthesis of t h i s n a t u r a l l y occurring m a t e r i a l . The o r i g i n a l synthetic goal was f i n a l l y accomplished by methylation of the isoxazole 158 with methyl i o d i d e - s i l v e r oxide i n chloroform and Beckmann rearrangement of the corresponding r i n g A oxime to produce (+)-N-acetyl-6-amino-7,9-di-0-methylisoxazolo[4,5-b]usnic acid (228), one of the various previously unknown ring-aminated d e r i v a t i v e s of usnic acid that were prepared during the course - i i i -of t h i s study. S u b s t i t u t i o n of the amide f u n c t i o n a l i t y by hydrogen resulted from the use of isoamyl n i t r i t e i n dioxane. Boron tribromide demethylation and subsequent regeneration of the protected B-triketone system furnished i n good y i e l d the metabolite (+)-6-desacetylusnic a c i d (107) , which was converted back to 35 by a one-step a c e t y l a t i o n - F r i e s rearrangement. Simultaneous i n v e s t i g a t i o n s produced the f i r s t high y i e l d i n g preparations of mono-, d i - , and mixed diether d e r i v a t i v e s of usnic a c i d , compounds whose preparation was not achieved by e a r l i e r workers. Part B discusses the i s o l a t i o n and structure determination of (+)-2-desacetylusnic acid (108), a product r e s u l t i n g from the biodegradation of 35 by Mucor globosus, I t s synthesis was accomplished by means of the B a e y e r - V i l l i g e r oxidation of (+)-usnic a c i d (35) with 30% hydrogen peroxide i n pyridine, at room temperature to produce the a-acetoxy d e r i v a t i v e 251 followed by i t s chromium (II) c h l o r i d e reduction i n acetone s o l u t i o n . In Part C i s described the i s o l a t i o n and i d e n t i f i c a t i o n of three metabolites obtained during the biodegradation of 35 by M b r t i e r e l l a i s a b e l l i n a . Thorough a n a l y s i s of t h e i r spectroscopic c h a r a c t e r i s t i c s , complemented by chemical degradations, showed that one of them was i d e n t i c a l with (+)-2-desacetylusnic a c i d (108), a product i s o l a t e d from the Mucor globosus fermentation (Part B). The main component, (+)-2-acetoxyusnic acid (251), has a novel structure r e s u l t i n g from the net i n s e r t i o n of oxygen at the 2-p o s i t i o n , and proved i d e n t i c a l with the product obtained from the Baeyer-V i l l i g e r oxidation of (+)-usnic acid (Part B). The f i n a l component was shown to be (+)-la-hydroxy-2-desacetylusnic acid (252), a compound a r i s i n g from both deacylation and reduction of the carbonyl group at the one-position. 14 Part D provides the synthesis of (+)-6- COCH^-usnic acid (255), - iv -the f i r s t r a d i o a c t i v e precursor s p e c i f i c a l l y l a b e l l e d at the aromatic a c e t y l grouping. This product w i l l f i n d immediate a p p l i c a t i o n i n the determination of the biodegradation pathways involved during the described transformations. - V -TABLE OF CONTENTS Page T i t l e Page Abstract ..... .. i i Table of Contents v Li s t of Figures •. v i Li s t of Tables ." x i i i Acknowledgements xvi Dedication x v i i Summary x v i i i Introduction... 1 Biodegradation.. 2 Usnic Acid.. .. • 28 Discussion.. .70 Part A.. 71 Part B ... ..... 268 Part C . 290 Part D 315 Experimental 319 Part A 322 Part B. • ... 438 Part C .. 449 Part D 462 Bibliography 465 - v i -LIST OF FIGURES Figure Page 1. Oxidative Metabolism of Aromatic Compounds 6 2. The Convergent Sequences of the g-Ketoadipate Pathway .. 7 3. Oxidative Metabolic Pathway of Catechol (1) by Microorganisms 10 4. Oxidative Metabolic Pathways of Protocatechuic Acid (2) by Microorganisms 11 5. The Central Sequences of the g-Ketoadipate Pathway and t h e i r Regulation i n P_. putida 13 6. The Quinate and Shikimate Sequences of the 3-Keto-adipate Pathway 14 7. The Shikimate and Central Sequences of the g-Keto-adipate Pathway and t h e i r Regulation i n Acineto-bacter calcoaceticus. Brackets denote Coordinate Synthesis of Enzymes 16 8. Pathways f o r the Formation of Catechol from Benzene 20 9. Naphthalene Hydroxylation i n B a c i l l u s sp 21 10. Conversion of Benzoic, S a l i c y l i c and A n t h r a n i l i c Acids to Catechol (1) by Pseudomonas sp 21 11. Mechanism for the Hydroxylation of Phenylalanine (23) 12. Ring F i s s i o n of Catechol (1) and Protocatechuic Acid (2)' 23 13. A l t e r n a t e Ring F i s s i o n of Catechol (1) and Proto-catechuic Acid (2) by Pseudomonas sp 24 14'.' Proposed Mechanism for the Action of Catechol-2,3-dioxygenase :(MetapyrQcatechase) 25 15. Ring F i s s i o n Products from Catechol (1) and Proto-catechuic Acid (2) and t h e i r Respective Pyridine Acids 27 - v i i -Figure Page 16. Alkaline.Degradation of Usnic and Usnetic Acids .. 35 17. Schopf and Heuck's S t r u c t u r a l Proposals (1972) . . . 36 18. Robertson and Curd's S t r u c t u r a l Proposal for Usnic Acid (35) 44 19. Synthesis of a-Coumaranone 70 47 20. Summary on the Chemical Degradations of Usnic Acid (35) 55 21. Barton's Synthesis of (±)-Usnic Acid (35) 58 22. Riedl's Attempted Synthesis of Usnic Acid (35) . . . 59 23. P e n t t i l a ' s i n v i t r o Biosynthesis of (±)-Usnic Acid (35) 60 24. Possible C y c l i z a t i o n s of the Hypothetical 3,5,7-Trioxooctanoic Acid (100) 64 25. The Biosynthesis of Usnic A c i d . Feeding E x p e r i -ments with 14c_Sodium Formate-, 2-14c-Sodium Acetate and 2- 1 4C-Diethyl Malonate .-. . 66 26. The Incorporation of 2'--^C-Methylphloroacetophenone (104) into Usnic Acid 68 27. Biosynthesis of Usnic Acid i n Lichens 69 28. ' The Infrared Spectrum of (+)-Usnic Acid (35) 73 29. ' Proton Magnetic Resonance Spectrum of (+)-Usnic Acid (35) 74 30. The Mass Spectrum of (+)-Usnic Acid (35) 77 31. The Postulated Fragmentation of (+)-Usnic Acid (35) i n the Mass Spectrometer 78 32. The C i r c u l a r Dichroism Spectra of (+)-Usnic Acid (35) (A), (-)-Usnic Acid (111) (B) and (+)-Isousnic Acid (112) (C) 82 33. Proton Magnetic Resonance Spectrum of (+)-6-Des-acetylusnic Acid (107) 85 I - v i i i -Figure Page 34. The Mass Spectrum of (+)-6-Desacetylusnic Acid (107) 86 35. The Postulated Fragmentation of (+)-6-Desacetylusnic Acid (107) i n the Mass Spectrometer 87 36. The S t r u c t u r a l E l u c i d a t i o n of (+)-6-Desacetylusnic Acid (107) 90 37. The Infrared Spectra of Pyrousnic Acid Diacetate (114) Obtained from: A) (+)-6-Desacetylusnic Acid (107) and B) (+)-Usnic Acid (35) 92 2 11 38. The Mass Spectrum of (-)-A * -Enaminousnic Acid (132) 109 2 11 39. The Postulated Fragmentation Pattern for (-)-A ' -Enaminousnic Acid (132) 110 40. Computer Generated Drawing of the X-Ray Model of (-)-A2> H-Enaminousnic Acid (132) .112 41. Proton Magnetic Resonance Spectrum of (-)-N-Methyl-AZ >L±-Enaminousnic Acid (134) H4 42. Dihedral Angles Observed f o r the Dihydrousnic Acid Series 130 43. Summary on the Condensation of (+)-Usnic Acid (35) and (-)-Dihydrousnic A c i d (154) with A l i p h a t i c and Aromatic Amines 133 44. Proton Magnetic Resonance Spectrum of (+)-Isoxazolo [4,5-b]usnic Acid (158) 135 45. Proton Magnetic Resonance Spectrum of (+)-2H-[l,2] Oxazocinousnic Acid (159) 138 46. Proton Magnetic Resonance Spectrum of (+)-Isoxazolo [5,4- a]usnic Acid (160) 141 47. The Postulated E q u i l i b r a t i o n of "Usnic Acid Oxime" (161) 143 48. The I n t e r r e l a t i o n of (+)-Isoxazolo[4,5-b]usnic Acid (158), (+)-2H-[l,2]0xazocinousnic Acid (159) and (+)-Isoxazolo[5,4-a]usnic Acid (160) 150 49. The Base Treatment of (+)-Isoxazolo[4,5-b]usnic Acid (158) 151 - i x -Figure Page 50. Proton Magnetic Resonance Spectrum of (+)-a-Ethoxy-isoxazolo[4,5-b]usnic Acid (164) 157 51. Proton Magnetic Resonance Spectrum of (+)-g-Hydroxy-desacetylisoxazolof4,5-b]isousnic Acid (165) 159 52. Proton Magnetic Resonance Spectrum of (+)-a-Hydroxy-desacetylisoxazolo[4,5-b]isousnic Acid (166) 162 53. The Synthetic Route to (+)-8-Desacetylisousnic Acid (178) 165 54. The Infrared Spectrum of: A) (+)-8-Desacetylisousnic Acid (178) and B) (+)-6-Desacetylusnic Acid (107) ••• 170 55. Proton Magnetic Resonance Spectrum of (+)-8-Desacetyl-isousnic Acid (178) 171 56. The Synthesis of (+)-Isousnic Acid (112) 175 57. Proton Magnetic Resonance Spectrum of (+)-Isousnic Acid (112) 177 58. Proposed Mechanism f o r the Base Cleavage of (+)-Isoxazolo[4,5-b]usnic A c i d (158) 181 59. The Base Cleavage of (+)-2H-[l,2]oxazocinousnic Acid (159) 185 60. The Acid-Catalyzed Dehydration of (+)-ct-Hydroxy-desacetyl-2H-[l,2]oxazocinoisousnic Acid (183) 188 61. Proton Magnetic Resonance Spectrum of (+)-N-Acetyl-desace ty1-2H-[1,2]oxazocinoisousnic Acid Acetate (105) 190 62. The Preparation of (+)-8-Desacetylisousnic Acid (178) from (+)-2H-[l,2]oxazocinousnic Acid (159) 194 63. Proton Magnetic Resonance Spectrum of (-)-Usnic Acid Isomethoxide Monoacetate (82) 202 64. Proton Magnetic Resonance Spectrum of (+)-7-0-Benzyl-usnic Acid (202) 216 65. Proton Magnetic Resonance Spectrum of (±)-7,9-Di-0-benzylusnic Acid (203) .... 218 - x -Figure Page 66. Proton Magnetic Resonance Spectrum of (+)-7-0--p_-Bromobenzylusnic Acid (204) 220 67. Proton Magnetic Resonance Spectrum of (±)-7-0-Benzyl-9-0-methylusnic Acid (205) 222 68. Proton Magnetic Resonance Spectrum of (±)-9-0-Methylusnic Acid (206) 223 69. Proton Magnetic Resonance Spectrum of (±)-N-Acetyl-6-amino-9-0-methylusnic Acid (210) 230 70. Proton Magnetic Resonance Spectrum of (±)-N-Acetyl-6-aminousnic Acid (213) 234 71. ' Proton Magnetic Resonance Spectrum of (+)-7,9-Di-O-methylisoxazolo[4,5-b]usnic Acid (220) ...... 240 72. Proton Magnetic Resonance Spectrum of the C-A l k y l a t e d Isoxazole 221 242 73. Ri e d l ' s Synthesis of C-Alkylated Derivatives of Phloroacetophenone (91) 244 74. The Proposed Synthesis of (+)-6-Desacetylusnic Acid (107) 245 75. Proton Magnetic Resonance Spectrum of (+)-N-Acetyl-6-amino-7,9-di-0-methylisoxazolo[4,5-b] usnic Acid (228) 2 4 7 76. Proton Magnetic Resonance Spectrum of (+)-6-Desacetyl-7,9-di-0-methylisoxazolo f 4,5-b]usnic Acid (229) 253 77. Proton Magnetic Resonance Spectrum of (+)-6-Desacetyl-7-0-methylisoxazolo[4,5-b]usnic Acid (232) 2 5 7 78. Proton Magnetic Resonance Spectrum of Synthetic (+)-6-Desacetylusnic Acid (107) 2 6 3 79. Proton Magnetic Resonance Spectrum of Synthetic (+)-Usnic Acid (35) 267 80. Proton Magnetic Resonance Spectra of (+)-2-Desacetylusnic Acid (108) 270 - x i -Figure Page 81. The Mass Spectrum of (+)-2-Desacetylusnic Acid (108) 271 82. The Postulated Fragmentation of (+)-Desacetyl-usnic Acid (108) i n the Mass Spectrometer 273 83. Computer Generated Stereo View of the C r y s t a l Structure of (+)-2-Desacetylusnic Acid (108) 275 84. The C r y s t a l Structure of (+)-2-Desacetylusnic Acid (108) Viewed Down c (Y=b and X=a s i n &) 276 85. The C r y s t a l Structure of (+)-2-Desacetylusnic A c i d (108) Viewed Down b 277 86. Proton Magnetic Resonance Spectrum of the a-Coumaranone Diacetate 70 ,. 279 87. The Synthesis of (+)-2-Desacetylusnic Acid (108) 285 88. Proton Magnetic Resonance Spectrum of (+)-2-Acetoxyusnic Acid (251) 288 89. Proton Magnetic Resonance Spectrum of Synthetic (+)-2-Desacetylusnic Acid (108) 289 90. A Representative" E x t r a c t i o n Procedure f o r the Biodegradation of (+)-Usnic Acid (35) by. M o r t i e r e l l a i s a b e l l i n a 291 91. Proton Magnetic Resonance Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252) 294 92. Proton Magnetic Resonance Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252) (Upon Addit i o n of D 20) 295 93. Proton Magnetic Resonance Spectrum of (+)-lct-Hydroxy-2-desacetylusnic Acid (252) (Recorded at 60 MHz) 296 94^ Mass Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252) 300 95. The Postulated Fragmentation of (+)-la-Hydroxy-2-desacetylusnic Acid (252) i n the Mass Spectro-meter 301 - xi:i. -Figure Page 96. Proton Magnetic Resonance Spectra of (+)-2-Desacetylusnic Acid (108) Isolated from M o r t i e r e l l a i s a b e l l i n a 303 97. Proton Magnetic Resonance Spectrum of Natural (+)-2-Acetoxyusnic Acid (251) 3 0 4 98. Mass Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid T r i a c e t a t e (253) 3 0 6 99. The Postulated Fragmentation of (+)-lct-Hydroxy-2-desacetylusnic A c i d i n the Mass Spectrometer . 3^7 100. Sequential Study on the Biodegradation of (+)-Usnic Acid (35) by M o r t i e r e l l a i s a b e l l i n a 3 ^ 3 101. Product D i s t r i b u t i o n i n the Acid F r a c t i o n of the Biodegradation of (+)-Usnic Acid (35) by M o r t i e r e l l a  i s a b e l l i n a 3 1 4 102. The Synthesis of Radioactively Labelled (+)-Usnic Acid (35) 3 1 6 - x i i i -LIST OF TABLES Table Page 1. Compounds Metabolized v i a the Two Main Central Sequences 5 2. Aromatic Compounds and t h e i r Ring-Fission Substrates . 18 3. O p t i c a l Properties of Usnic Acid 30 4. Occurrence of (+)-Usnic Acid i n Lichens 31 5. Occurrence of (-)-Usnic Acid i n Lichens 32 6. Enzymatic Coupling of Phenols 61 7. C i r c u l a r Dichroism Properties of (+)-Usnic Acid (35), (-)-Usnic Acid (111) and (+)-Isousnic Acid (112) 80 8. Summary of Selected Proton Magnetic Resonance Data f o r the A l i p h a t i c Amine-Usnic Acid Condensation Products ( i n ppm, 6 units) 119 9. Selected Proton Magnetic Resonance Data for the Aromatic Amine-Usnic Acid Condensation Products257 ( i n ppm, 6 units) 121 10. The Thermal C y c l i z a t i o n of "Usnic Acid Oxime" 144 11. The Racemization of (+)-Usnic Acid (35) i n Various Solvents297 146 12. The Racemization of (+)-Usnic Acid (35) and some Derivatives i n Dioxan279,299 146 13. The Observed Chemical S h i f t f o r the A c e t y l Groupings i n the Usnic and Isousnic Acid Series 154 14. The C i r c u l a r Dichroism Properties of the Cleavage Products of (+)-Isoxazolo[4,5-b]usnic Acid (158) .. 156 15. The Chemical S h i f t of the C^-Methylene Protons and the Cg^-Methyl Group for the C^g-a- and (3-Series . . 160 - xiv -Table Page 16. Proton Magnetic Resonance Data of (+)-Desacetyl-isousnic Acid (178) and (+)-6-Desacetylusnic Acid (107) and their Corresponding Diacetates (in ppm, 6 units) '. 172 t 17. Circular Dichroism Data for (+)-6-Desacetylusnic Acid (107) and (+)-8-Desacetylusnic Acid (178) 173 18. Proton Magnetic Resonance Data of (+)-Usnic Acid (35) and (+)-Isousnic Acid (112) (in ppm, 6 units) 176 *• 19. Proton Magnetic Resonance Data of (+)-2H-[l,2] oxazocinousnic Acid (159) and (+)-2H-[1,2]oxazo-cinoisousn.tc Acid (188) 193 20. Proton Magnetic Resonance Data of (+)-8-Methoxy-isoxazolo[4,5-b]usnic Acid (191) and (+)-ct-Methoxyisoxazolo[4,5-b]usnic Acid (192)..... 198 21. Proton Magnetic Resonance Data of (-)-Usnic Acid Isomethoxide Monoacetate (82) 203 22. Proton Magnetic Resonance Data of Several Ether Derivatives of Usnic Acid 224 23. Selected Proton Magnetic Resonance Data for the Various Amide Derivatives of Usnic Acid 248 24. Selected Studies on the Decomposition of N-Nitrosoacetanilides (231) in Various Solvents 254 25. Proton Magnetic Resonance Data for (+)-6-Desacetyl-isoxazolo[4,5-b]usnic Acid (230) and (+)-6-Tesacetyl-isoxazolo [4,5-b] isousnic Acid (176) and Their Corresponding Acetates 259 26. Proton Magnetic Resonance Data for the Natural and Synthetic (+)-6-Desacetylusnic Acid (107) and Their Corresponding Acetates 264 27. Circular Dichroism Data for Natural and Synthetic (+)-6-Desacetylusnic Acid (107) 265 28. Representative Product Distribution for the Bio-degradation of (+)-Usnic Acid (35) with Mortierella isabellina 292 - XV -Table Page 29 Sequential Study on the Biodegradation of (+)-Usnic Acid (35) by M o r t i e r e l l a i s a b e l l i n a 311 30. The Biodegradation of (+)-Usnic Acid by M o r t i e r e l l a  i s a b e l l i n a . Product D i s t r i b u t i o n i n the Acid F r a c t i o n 312 - x v i -ACKNOWLEDGEMENTS I would l i k e to express my sincere appreciation to Professor James P. Kutney for h i s guidance and encouragement throughout the course of t h i s study. I am e s p e c i a l l y g r a t e f u l to my wife, whose never ending optimism, confidence and understanding made t h i s work a r e a l i t y . I would also l i k e to thank Professor S. Shibata f o r h i s gracious g i f t of several usnic acid samples, and Pr o f e s s o r s R . J . Bandoni and G.H.N. Towers of the Botany Department, U n i v e r s i t y of B r i t i s h Columbia, for the g i f t of the culture of M o r t i e r e l l a i s a b e l l i n a used during Part C of t h i s thesis and f o r the many h e l p f u l suggestions concerning the research p r o j e c t . Thanks are due to Dr. P h i l i p J . Salisbury f o r h i s patience and expertise i n propagating the microorganisms, and to Dr. T. Yee, Mr. Vicente Ridaura and the members of our research group, past and present, f o r i n t e r e s t i n g discussions and suggestions. My sincere thanks to Miss Claudia Thorne f o r the generous cont r i b u t i o n of her time and the able typing of the manuscript. Receipt of f i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia, The National Research Council of Canada and the Universidad Nacional Autonoma ' de Mexico i s g r a t e f u l l y acknowledged. ! - x v i i -DEDICATION To Maria Elena - x v i i i -SUMMARY The following schemes present i n a comprehensive manner the various aspects of the work performed throughout Parts A to D of t h i s t h e s i s - x i x -( 107) (108 ) Scheme A. Summary of the Various Biodegradation Products Derived from (+)-Usnic Acid (35). - XX -(35) COCH OH (132) R = H (13/*) R = Me (136). R = Et fjlH-(1U) R = (135) R=Me (137) R - C H . C - H -( 153) COCH (154) COCH (156) R = Scheme B . Summary of the Condensation Products of (+)-Usnic Acid (35) and (-)-Dihydrousnic Acid (154) with Various Amines. Scheme C. Summary of the Condensation of (+)-Usnic Acid (35) with Hydroxylamine Hydrochloride. - x x i i -( 112 ) Scheme D. Summary of the Transformation of (+)-Isoxazolo[4,5-b] usnic Acid (158) i n t o (+)-Isousnic Acid (112). : - x x i i i -(178) Scheme E. Summary of the Transformation of (+)-2H-[l,2]0xazocinousnic Acid (159) i n t o (+)-8-Desacetylisousnic Acid (178). - x x i v -(107) Scheme F. Summary of the Conversion of (+)-Usnic Acid (35) into (+)-6-Desacetylusnic Acid (1C)7) . - X X V -Scheme G. The Conversion of (+)-Usnic Acid (35) i n t o (+)-2-Desacetylusnic Acid (108). H H Scheme H. The Synthesis of (+)-Usnic Acid (35) and Radioactively Labelled (+)-Usnic Acid (255) from (+)-6-Desacetylusnic Acid (107). - 1 -INTRODUCTION Throughout the centuries the continued endeavours of Man into the vast v a r i e t y of nat u r a l sciences have been aimed at understanding,, and subsequently applying Nature's own processes to the s o l u t i o n of problems, consequently o r i g i n a t i n g our t e c h n o l o g i c a l l y advanced so c i e t y . However, the multiple a p p l i c a t i o n s of our present technology often produce as a byproduct materials and wastes that burden our nat u r a l habitat and endanger many forms of l i f e . Such materials include com-pounds present i n the e f f l u e n t water of i n d u s t r i a l wastes, p e s t i c i d e s , hormone herbicides, sulfonated aromatic detergents and many other nat-u r a l and synthetic chemicals. The s i g n i f i c a n c e of research i n t h i s area can be found both from the chemical and e c o l o g i c a l points of view. From a chemical stand-point we must r e a l i z e that i n s p i t e of the large s t a b i l i t y of the aro-matic nucleus, there are micro-organisms which, under the mildest condi-t i o n s , can completely degrade benzenoid and polyaromatic systems. I f such chemicals were to prove r e s i s t a n t to mi c r o b i a l decomposition, they could accumulate i n the s o i l and eventually, v i a the food chain, become l o c a l i z e d i n animal t i s s u e s , leading to serious e c o l o g i c a l changes. Therefore, since the mi c r o b i a l degradation of aromatic com-pounds constitutes an e s s e n t i a l step i n nature's carbon c y c l e , i t i s of v i t a l importance that we understand how microorganisms degrade not only n a t u r a l products, but also those added to the environment through the a c t i v i t y of man. - 2 Biodegradation The breakdown of aromatic compounds, one of Nature's e s s e n t i a l biochemical steps, i s performed by several kinds of micro-organisms. Bacte r i a are the most v e r s a t i l e i n t h i s respect, but several yeast and fungi are able to degrade a more l i m i t e d range of benzenoid structures ,,1,2 as w e l l . A study of the means by which b a c t e r i a cleave the aromatic r i n g 3 appeared as early as 1903 with a report by Emmerling and Abderhalden, of a s t r a i n of Micrococcus chinicus which oxidized q u i n i c acid by aroma-t i z a t i o n to protocatechuic a c i d . Work on aromatic oxidations i n the next two decades was concerned mainly with the degradation of benzene 4-7 d e r i v a t i v e s by several kinds of microorganisms, and much of t h i s 8 9 early /work, -.summarized .in -excellent reviews -by Happold ..and .Evans, dealt mainly with the biochemical aspects of the biodegradation of mononuclear aromatic compounds. Ear l y work with polynuclear aromatics other than l i g n i n , describing b a c t e r i a l attack on naphthalene and phenanthrene, was f i r s t reported by Tausson.^ ^ 2 However, the m i c r o b i a l oxidation of other 13 14 fused systems has not been extensively studied u n t i l quite r e c e n t l y . ' Certain higher fungi have been shown to be capable of attacking the aromatic r i n g , and include s t r a i n s of Aspergillus,^" 5 P e n i c i l l i u m , ^ 17 18 Oospora, and Neurospora. Nevertheless, a t t e n t i o n with the molds, p a r t i c u l a r l y with regard to aromatics, has been d i r e c t e d more towards t h e i r synthetic and transforming a b i l i t i e s rather than t h e i r degradative t a l e n t s . Certain s o i l and wood-rooting fungi have been shown to carry out s i m i l a r - „. 19-20 transformations. - 3 -Interest in the biodegradation of aromatic compounds centers on (i) the study of the metabolic intermediates involved in the breakdown of the different aromatic substrates, ( i i) the elucidation of the enzyma-tic mechanisms of hydroxylation and ring fission, and ( i i i ) the relatively recent investigations into the control of the enzymes responsible for this metabolic activity. Several reviews dealing with the first two , , 21-25 areas are now available. The ability to utilize aromatic compounds as the sole source of carbon is not universal amongst microorganisms. In the light of present knowledge, microbial attack on the aromatic ring appears to be dependent on induced enzyme formation. Since the presence of other ox-idizable carbon compounds may prevent the induction of the enzymes nec-essary for aromatic degradation, the practical criteria imposed for attack is aerobic growth in a mineral-salts medium with an aromatic compound as the only source of carbon and energy. Bacteria capable of growth under these conditions have been isolated from soi l , sewage, feces, and sea 26 water. Amongst these, there are representatives of the families Coccaceae, Mycobacteriaceae, Pseudomonadaceae, Spirillaceae, Bacteriaceae, 27-29 and Bacillaceae. Of these, the soil pseudomonads appear, by far, the most active. In mammals, unnatural aromatic substances are usually detoxified by hydroxylation, cleavage and conjugation, when necessary, followed by 30 their excretion. Early studies by Jaffe, later confirmed by several 31-33 workers, showed that the administration of benzene to rabbits and dogs gives rise to a small amount of trans, trans-muconic acid in the urine, thus demonstrating that mammals have not entirely lost the capacity - 4 -f o r r i n g cleavage. In the case of the n a t u r a l l y occurring aromatic amino-acids, there are i n l i v e r t i s s u e f a i r l y w e l l characterized enzy-mic systems which hydroxylate phenylalanine to t y r o s i n e , and disrupt 34 the benzene r i n g of the l a t t e r a f t e r manipulation of the side chain. A noteworthy f a c t i s the close s i m i l a r i t y i n the biochemical pathways of r i n g cleavage metabolism of the n a t u r a l l y occurring aromatic amino-acids i n a l l forms of l i f e so far studied. General Metabolic Pathways for the M i c r o b i a l Degradation of Aromatic  Compounds There appears to be a number of general intermediates to which a l l aromatic compounds are converted to before r i n g f i s s i o n can occur. The steps i n the biodegradative pathway p r i o r to formation' of these- key 25—35 intermediates are known as the Peripheral Convergent Sequence. The point of covergence i n numerous cases of aromatic r i n g oxidation i s 1,2-dihydroxybenzene, catechol (1). However, depending on the s u b s t i t u t i o n pattern i n the o r i g i n a l molecule undergoing degra-dation, an alternate compound, 3,4-dihydroxybenzoic a c i d , protocatechuic a c i d (2), may be obtained. Most known pathways of m i c r o b i a l attack on aromatics are directed toward, and a r r i v e at, these two key intermediates, which then follow the convergent branches of the 8-ketoadipate pathway, - 5 -a p r o c e s s known as t h e C e n t r a l Sequence, thus p r o v i d i n g means f o r t h e c o n v e r s i o n o f c e r t a i n a r o m a t i c s and h y d r o a r o m a t i c compounds t o i n t e r m e d -i a t e s o f t h e T r i c a r b o x y l i c A c i d C y c l e (T.C.A. c y c l e ) , as se e n i n F i g u r e 1. The C e n t r a l Sequence c o m p r i s e s two s u b s e t s : ( i ) the C a t e c h o l  Sequence, i n c l u d i n g t h e c o n v e r s i o n o f c a t e c h o l t o t h e e n o l - l a c t o n e o f 6-k e t o a d i p i c acid', and ( i i ) the P r o t o c a t e c h u a t e Sequence, r e s u l t i n g i n t h e c o n v e r s i o n of p r o t o c a t e c h u i c a c i d t o 8 - k e t o a d i p y l - c o e n z y m e A; t h e f i n a l s t e p - r e a c t i o n b e i n g a t h i o l y t i c c l e a v a g e o f t h e l a t t e r t o s u c c i n i c a c i d and a c e t y l - C o A ( F i g u r e 2). The p e r i p h e r a l sequences a r e u s u a l l y d e s i g -n a t e d by t h e i r p r i m a r y s u b s t r a t e s : t h u s , t h e M a n d e l a t e Sequence c o m p r i s e s t h e s e t of r e a c t i o n s by w h i c h m a n d e l i c a c i d i s o x i d i z e d t o c a t e c h o l , e t c . A l i s t o f some i m p o r t a n t s u b s t r a t e s m e t a b o l i z e d v i a t h e s e c e n t r a l sequences can be se e n i n T a b l e 1. C a t e c h o l Sequence P r o t o c a t e c h u a t e Sequence B e n z o i c A c i d m - C r e s o l S a l i c y l i c A c i d j D - C r e s o l P h e n o l p - H y d r o x y b e n z o i c A c i d N a p h t a l e n e D - H y d r o x y m a n d e l i c A c i d P h e n a n t h r e n e p - A m i n o b e n z o i c A c i d A n t h r a c e n e P h t h a l i c A c i d o - C r e s o l Benzene M a n d e l i c A c i d T r y p t o p h a n T a b l e 1. Compounds M e t a b o l i z e d v i a t h e Two Main C e n t r a l Sequences - 6 -Aromatic Substrate Oxygenation Hydroxylated Aromatic Intermediates Induced Enzymes Ring F i s s i o n A l i p h a t i c Intermediates ADP + Peri p h e r a l Convergent Sequences Central S Sequence NADH,. > CO, Synthesized C e l l M a t e r i a l C o n s t i t u t i v e Enzymes Figure 1. Oxidative Metabolism of Aromatic Compounds. - 7 -Peripheral Sequences Quinic Acid Shikimic Acid \ 5-Dehydroshikimic Acid p-Hydroxy-benzoic Acid Central Sequences Protocatechuic Acid Carboxymuconic Acid Carboxymuconolactone D-Mandelic L-Tryptophan Acid L-Mandelic Acid i i Benzoic Acid L-Kynurenine A n t h r a n i l i c Acid Catechol Muconic Acid Muconolactone B-Ketoadipic acid enol-lactone B-Ketoadipic Acid 1 B-Ketoadipyl-CoA J Acetyl-CoA + Succinic Acid Figure 2. The Convergent Sequences of the B-Ketoadipate Pathway. - 8 -Metabolism of Catechol and Protocatechuic Acid The s e r i e s of steps involved during the r i n g cleavage and further transformations of these key intermediates to form a l i p h a t i c compounds such as acetate and succinate, which can then enter the t e r -minal r e s p i r a t o r y cycles, required the e f f o r t s of a number of workers. 11 In 1928 Tausson suggested that the benzene nucleus was being cleaved to muconic a c i d , by phenanthrene-oxidizing b a c t e r i a , and then oxidized 36 to carbon dioxide. Evans and Happold excluded, on the basis of t h e i r experiments, the intermediacy of trans, trans-muconic a c i d , and f i n a l l y , Evans and Smith demonstrated that pure c i s , cis-muconic a c i d (3) was metabolized by i n t a c t c e l l s , and was nonoxidatively converted i n t o 6-oxoadipic a c i d by a crude, c a t e c h o l - o x i d i z i n g , c e l l - f r e e preparation, whereas the c i s , trans- and trans, trans-isomers were- inactive.. 37 I t was then found i n 1951 by Evans and coworkers that the 2 lactone y-carboxymethyl-A -butenolide, muconolactone (4), could be con-verted to 3-oxoadipic acid (6) by the same c e l l - f r e e preparation. This transformation requires a double bond isomerization of muconolactone to y-carboxymethyl-A -butenolide, 8-oxoadipic acid enol-lactone (5), before cleavage can occur. The l a c t o n i z i n g enzyme and the l a c t o n e - s p l i t t i n g 38-39 enzyme have been separated by Sistrom and Stanier. The former r e -quires Mg or Mn ions as cofactors and catalyzes the r e v e r s i b l e i n t e r -conversion of c i s , cis-muconic acid and the (+)-lactone; i t can also con-ve r t the c i s , trans-isomer into an eq u i l i b r i u m mixture with the (-)-lactone, but more slowly than with the n a t u r a l substrate. 40 F i n a l l y , i n 1948, K i l b y i s o l a t e d a compound observed trans-36 i e n t l y by Evans and Happold, which gave p o s i t i v e Rothera and Gerhardt - 9 -reactions characteristics of a B-oxo-acid, and identified i t as being 8-oxoadipic acid (6). Thus the steps in the degradation between cate-chol and 8-oxoadipic acid were complete; the last compound in the chain, however, being the first isolated and identified (Figure 3). The final step before the entry of the compounds into the 41 T.C.A. cycle was shown by Kilby to be a C^~C2 split of the 3-oxo-acid to produce succinate (7) and acetate (8). The enzymology of the C^-C^ 42 splitting was worked out by Katagiri and Hayaishi in 1957. They showed, working with a cell-free enzyme preparation of Pseudomonas sp., that in the primary reaction, catalyzed by a specific thiophorase, succinyl-CoA acts as a CoA donor producing B-oxoadipyl-CoA. During the second reaction, this newly formed CoA is cleaved, in the presence of a specific thiolase to yield succinyl-CoA (SucCoA) and acetyl-CoA. The SucCoA can be utilized again as a CoA donor, completing the cycle. Protocatechuic acid (2) is also degraded to 8-oxoadipic acid, but the steps in this degradation are not so well known (Figure 4) as those of the catechol path. A cell-free, protocatechuic acid-oxidizing enzyme preparation obtained from Neurospora crassa was used by Gross and 43 coworkers to demonstrate the following sequence of reactions: Protocatechuic acid (2) cis, cis-B-carboxymuconic acid (9) (-)-B-carboxymuconolactone (10) -* B-oxoadipic acid (6). Their experiments, using (2 ,6-*4C)-protocatechuic acid as sub-strate , demonstrated that the keto-group carbon was derived solely from carbon-6 of protocatechuic acid. Nevertheless, a Pseudomonas sp. ce l l -free enzyme system produced a B-oxoadipic acid in which the keto-group carbon was randomly derived from carbon-1 and carbon-6 of the labelled - 10 -Figure 3. Oxidative Metabolic Pathway of Catechol "(1) by Microorganisms. - 11 -COOH 1 F i g u r e 4. O x i d a t i v e M e t a b o l i c Pathways o f P r o t o c a t e c h u i c A c i d (2) by M i c r o o r g a n i s m s . - 12 -substrate (2). In addition the Pseudomonas system was not a c t i v e on e i t h e r the (+)- or ( - ) -B-carboxymuconolactone, therefore excluding i t as a possible interinediate. 44 Recently Ribbons and Evans reasoned, from tracer r e s u l t s , employing a Pseudomonas system, that an intermediate with a symmetrical structure i s produced p r i o r to the formation of B-oxo-adipic a c i d . The intermediates considered were butanolido-3,Y,Y'»8'-butenolide, dilactone (11) , or i t s open chain counterpart 3,8'-dihydroxyadipic acid (12). In-deed, when the lactone was synthesized and used as a substrate, only the Pseudomonas system produced B-oxo-adipic a c i d ; unfortunately compound (12) was not prepared nor tested for a c t i v i t y . In b a c t e r i a , where a l l sequences of the pathway are i n d u c i b l e , 35 the patterns of induction are complex, and d i f f e r from group to group. The r e g u l a t i o n of the enzymes operative i n the c e n t r a l sequence was f i r s t 45 elucidated by Ornston i n Pseudomonas putida, and i t was l a t e r shown that f o r P. aeruginosa the c o n t r o l patterns were i d e n t i c a l (Figure 5). One of i t s c h a r a c t e r i s t i c features, coordinate product-induction of four en-zymes of the protocatechuate sequence by 8-keto-adipic a c i d or 8~keto-45 46 adipyl-CoA, has also been shown i n several other Pseudomonas species. ' In other groups of aerobic b a c t e r i a , however, the c e n t r a l sequences of the 47 pathway are subject to d i f f e r e n t patterns of c o n t r o l , and i t has been suggested that each of these c o n t r o l patterns c o n s t i t u t e s a complex group-character, shared by r e l a t e d b a c t e r i a l species, which probably r e f l e c t s 48 a common evolutionary o r i g i n of the genetic system governing the pathway. Thus studies on the biochemistry and r e g u l a t i o n of the Quinate Sequence have shown that P. putida d i s s i m i l a t e s the hydroaromatic compounds, quinic and shikimic acids, through a p e r i p h e r a l sequence of the 8-keto--13 INDUCER ENZYME ENZYME INDUCER Protocate-chuic acid g-Ketoadipic Acid or g-ketoadipyl-CoA H O O C v > ^ s ^ O H H O O C ^ C O O H U^COOH COOH HOOC-V°yO 00 H 00H r^cooH gJs^^OSCoA H OH F ' O00H 00H COOH XT v E 2,E 2* E 3,E 3' cis, c i s -muconic acid c i s , c i s -muconic acid oxygenase lactonizing enzyme isomerase enol-lactone hydrolase transferase Figure 5. The Central Sequences of the 3-Ketoadipate Pathway and their Regulation in P_. putida. - 14 -49 adipate pathway, as seen i n Figure 6, C O O H quinic acid 5 - d e h y d r o quinic acid 5 -dehydro shikimic acid : O O H shikimic acid p-keto adipic acid C O O H Figure 6. The Quiriate and Shikimate Sequences of the 8-Ketoadipate Pathway. Manometric studies with i n t a c t c e l l s of a w i l d type of P. putida have shown that the enzymes of the sequence are a l l induced by growth with e i t h e r quinate or shikimate, but not with p-hydroxybenzoate or proto-35 catechuate. Nevertheless, i n Acinetobacter c a l c o a c e t i c u s , a degree of coordinate c o n t r o l f a r greater than that c h a r a c t e r i s t i c of Pseudomonas sp. governs both branches of the pathway. 4 7'"^ In the protocatechuate branch, a l l the enzymes responsible f o r the conversion of shikimic acid to $-keto-adipyl-CoA are e l i c i t e d by one inducer, protocatechuic a c i d , and - 15 -appear to be subject to coordinate induction (Figure 7). In the catechol branch, a l l enzymes mediating the conversion of c i s , cis-muconic a c i d to 8-keto-adipyl-CoA are induced by c i s , cis-muconic a c i d , and t h e i r synthesis i s coordinate under most p h y s i o l o g i c a l conditions. This extension of coordinate regulatory c o n t r o l has necessitated the evolution i n Acineto- bacter of two separate sets of enzymes governing the terminal common steps of the pathway; one set i s l i n k e d by re g u l a t i o n with the enzymes of the protocatechuate branch, and the other with the enzymes of the catechol branch. •? Hydroxylation As previously stated dihydroxylation i s a p r e r e q u i s i t e f o r aromatic r i n g f i s s i o n . The hydroxyl groups may be ortho to each other, as i n catechol (1) and protocatechuic a c i d (2), or para to each other as i n g e n t i s i c (13) and homogentisic (14) acids. (13) <14> Although catechol i s an intermediate during the degradation of polynuclear aromatic hydrocarbons such as naphthalene, anthracene, and phenanthrene, a dihydroxylated polyaromatic compound i s usually the f i r s t substrate for r i n g f i s s i o n . Substituted aromatic n u c l e i present micro-organisms with a choice as to t h e i r mode of attack; r e s u l t s obtained i n studies of the microbial degradation of phenyl-substituted acids by a - 16 -INDUCER ENZYME O O H 0* > " ^ O H O H shikimate dehydrogenase H O O C \ ^ / 0 H Protocate-chuic acid protocatechuate oxygenase O H H 0 0 C Y ^ 0 0 H ^ ^ C O O H carboxymuconate l a c t o n i z i n g enzyme C O O H H O O C - ^ ' ^ O carboxymuconolactone decarboxylase enol-lactone hydrolase I INDUCER ENZYME O H O H catechol oxygenase c i s , c i s -muconic acid O0 0 H O O H muconate l a c t o n i z i n g enzyme C O O H muconolactone isomerase enol-lactone hydrolase II transferase I O ^ k ^ C O O H J ( ^ X O O H Q A ^ C O S C O A transferase II cxs, cxs-r muconic acid Figure 7 . The Shikimate and Central Sequences f o r the 3-Ketoadipate Pathway and t h e i r Regulation i n Acinetobacter calcoaceticus. Brackets denote Coordinate Synthesis of Enzymes. - 17 -Norcadia sp. i n d i c a t e that the a c i d side chain i s metabolized v i a "3-oxidation" processes, that i s , two carbon atoms are removed at a time."** Therefore, side chains with an odd number of carbon atoms are metabolized to benzoic a c i d , which i s converted to catechol p r i o r to r i n g f i s s i o n . Phenyl-substituted acids that contain an even-numbered carbon side chain are metabolized through phenylacetic a c i d . Further metabolism of the 15 52 l a t t e r may proceed through either homogentisic a c i d ' (14) or homo-protocatechuic a c i d , 3,4-dihydroxyphenylacetic a c i d 5 3 ' 5 4 (15), depending on the species of microorganisms. J-L^ COOH Nevertheless, studies on the metabolism of toluene and i s o -propylbenzene by P. putida have shown that these compounds are converted to o-dihydroxy compounds i n which the side chain i s l e f t i n t a c t . These r e s u l t s add to a growing l i s t of fi n d i n g s that many aromatic com-pounds undergo enzymatic hydroxylation of the aromatic nucleus i n pref-erence to degradation of the a l i p h a t i c side chain. Several examples of substituted aromatic compounds and t h e i r respective r i n g - f i s s i o n sub-st r a t e s are shown i n Table 2. - 18 -Aromatic Compound Ring-Fission Substrate Toluene 2,3-Dihydroxytoluene o-Cresol 2,3-Dihydroxytoluene 3-Pheny.propionic acid 2,3-Dihydroxy-g-phenyl-propionic acid Phenylacetic acid 3,4- or 2,5-Dihydroxy-phenylacetic acid Naphthalene 1,2-Dihydroxynaphthalene Anthracene 1,2-Dihydroxyanthracene Phenanthrene 3,4-Dihydroxyphenanthrene 3-Hydroxy-9,10-secoandrosta- 3,4-Dihydroxy-9,10-secoandrostra-l,3,5(10)-triene-9,17-dione 1,3,5(10)-triene-9,17-dione Estrone 4-Hydroxyestrone Table 2. Aromatic Compounds and t h e i r Ring-Fission Substrates B i o l o g i c a l oxidations may occur by three seemingly d i f f e r e n t processes: ( i ) removal of an e l e c t r o n , as i n the conversion of a ferrous to a f e r r i c i on, ( i i ) removal of hydrogen, as i n the oxidation of a l c o h o l to acetaldehyde, and ( i i i ) a ddition of oxygen to a molecule, as i n the transformation of carbon to carbon dioxide. Although the fundamental p r i n c i p l e governing these three types of oxidative reactions i s b a s i c a l l y the same, they are catalyzed by d i f f e r e n t types of enzymes i n the c e l l . The enzymes that p a r t i c i p a t e i n b i o l o g i c a l oxidations may be c l a s s i f i e d into two major groups: Dehydrogenases catalyse the t r a n s f e r of electrons or hydride ions from donors to a v a r i e t y of acceptors. Nevertheless, when molecular oxygen serves as an electron acceptor, the enzyme i s generally r e f e r r e d to as an "Oxidase". Oxygenases are a group of enzymes which catalyze the incorpora-t i o n of molecular oxygen in t o various organic and inorganic substrates. Since the phenomenon of enzymatic oxygen f i x a t i o n was established through the use of heavy oxygen, ^^0, i n 1 9 5 5 , 5 7 these enzymes have been found to be widely d i s t r i b u t e d i n nature and to play p h y s i o l o g i c a l l y important - 19 -r o l e s i n the biosynthesis, transformation, and degradation of various meta-b o l i t e s . Oxygenases may be c l a s s i f i e d i n t o two groups: the Dioxygenases, which incorporate two atoms of oxygen in t o one molecule of substrate, and the Monooxygenases, which incorporate only one atom of oxygen per mole of substrate; the other atom being reduced to water. These enzymes may therefore be regarded as hybrids of an oxygenase and an oxidase, or as 57 58 Mason c a l l s them, "Mixed-function Oxygenases". ' In order to reduce the other atom of oxygen to water, the presence of a s u i t a b l e e l e c t r o n donor i s required. A t y p i c a l r e a c t i o n may be represented by the follow-ing equation: = Electron Donor Pyridine nucleotides, f l a v i n e n u c leotides, cytochromes, metals (Fe, Cu), ascorbate, and p t e r i d i n e d e r i v a t i v e s may serve as exogenous ele c t r o n donor. In some cases, however, the substrate i t s e l f can act as an endogenous e l e c t r o n donor. Phenol has been excluded as an intermediate i n benzene degrada-t i o n since benzene-grown c e l l s have f a i l e d to metabolize t h i s compound. Gibson and coworkers'''' obtained a c e l l preparation from Pseudomonas putida which, when supplied with NADIL^, ferrous ion and a reducing agent, w i l l metabolize a compound r e f e r r e d to i n the l i t e r a t u r e as cis-benzeneglycol, but not the trans-isomer. Radioisotope-trapping experiments implicated - 20 -cis-benzeneglycol (16) as an intermediate i n the formation of catechol from benzene, suggesting that an epoxide i s not an intermediate during hydroxylation by F. putida. The two pathways are shown i n Figure 8. Figure 8. Pathways for the Formation of Catechol from Benzene. The pathway i n v o l v i n g trans-benzeneglycol, trans-1,2-dihydroxy-3,5-cyclohexadiene (18), obtained by s o l v o l y s i s of the corresponding benzene epoxide (17), was postulated by analogy to the ' f i r s t stages of the m i c r o b i a l degradation of naphthalene (Figure 9). Thus Walker and 59 W i l t s h i r e i s o l a t e d trans-1,2-dihydro-l,2-dihydroxynaphthalene (20) from 60 cultures of a B a c i l l u s sp., and G r i f f i t h s and Evans prepared a c e l l ex-t r a c t that oxidized naphthalene to 20 i n the presence of NADPH^ and ferrous ion. The d i o l 20 was assumed to a r i s e from the corresponding arene oxide precursor*'''" 19 . The f i r s t i s o l a t i o n and c h a r a c t e r i z a t i o n of 1,2-naphthalene oxide (19) from a b i o l o g i c a l system ( l i v e r microsomes) 61a was reported by Jer i n a and coworkers i n 1968. itlus sp (19) (20) Figure 9. Naphthalene Hydroxylation i n B a c i l l u s sp. A further example i s presented by the conversion of a n t h r a n i l i c a c i d (21, R = NH^) to catechol (Figure 10). The r e a c t i o n i s catalyzed 62 by an enzyme i s o l a t e d from a Psuedomonas sp., and i s produced during growth on tryptophan. Recently, studies with i s o t o p i c oxygen showed that both atoms of the oxygen molecule are incorporated, and t h i s led to the 63 6( proposal of a c y c l i c peroxide intermediate 22. Hayaishi and coworkers showed that i n the case of s a l i c y l a t e hydroxylase, (21, R = OH), the presence of f l a v i n adenine dinucleotide (FAD) was also required. (1 ) (21 ) ^ (22) R-H.OH.NH-Figure 10. Conversion of Benzoic, S a l i c y c l i c and A n t h r a n i l i c Acids to Catechol (1) by Pseudomonas sp. - 22 -Recent studies have revealed the mechanism for the hydroxy-65 l a t i o n of phenylalanine (23). When a p u r i f i e d preparation of b a c t e r i a l phenylanine hydroxylase was incubated with L-phenylalanine t r i t i a t e d i n the para p o s i t i o n , the tyrosine (24) produced s t i l l contained a considerable amount of t r i t i u m . S i milar r e s u l t s were obtained when 66 4-deuterophenylalanine was used as a substrate. The r e a c t i o n sequence postulated to explain these r e s u l t s assumes that the hydroxylation i s e i e c t r o p h i l i c (Figure 11), and the intermediates formed show a large i s o t o p i c e f f e c t . ( 24) Figure 11. Mechanism for the Hydroxylation of Phenylalanine (23). Ring F i s s i o n Catechol and protocatechuic acid, the metabolic intermediates of many d i f f e r e n t aromatic compounds, have been the object of intensive i n v e s t i g a t i o n . Hayaishi and Hashimoto^ 7 i s o l a t e d an enzyme, catechol-1,2-dioxygenase, that catalyzes the incorporation of molecular oxygen into the catechol molecule (1) (Figure 12) with the r e a c t i o n product being cis,cis-muconic a c i d (3). An analogous transformation has been reported for protocatechuic a c i d (2). The enzyme, protocatechuic a c i d -3,4-dioxygenase, cleaves t h i s a c i d between the hydroxyl groups to form g-carboxy-cis,cis-muconic acid (9). Both enzymes have been p u r i f i e d , and each contains f e r r i c i r o n at i t s a c t i v e c e n t e r . ^ 8 Figure 12. Ring F i s s i o n of Catechol (1) and Protocatechuic Acid (2). An a l t e r n a t i v e enzymatic cleavage of catechol has been reported 69 by Dagley and Stopher. A Pseudomonas sp. which u t i l i z e d o_-cresol as the only carbon source f o r growth, oxidized, catechol (1) to a-hydroxy-muconic semialdehyde (25). The enzyme, catechol-2,3-dioxygenase or 24 metapyrocatechase, has been extracted and c r y s t a l l i z e d from c e l l s of ]?. a r v i l l a , 7 0 and shown to possess ferrous i r o n at i t s a c t i v e center. Growth of a d i f f e r e n t Pseudomonas sp. on _p_-cresol induced the formation of an enzyme, protocatechuic acid-4,5-dioxygenase, which oxidized proto-21 catechuic a c i d (2) to a-hydroxy-y-carboxymuconic semialdehyde (26), as seen i n Figure 13. Figure 13. Alt e r n a t e Ring F i s s i o n of Catechol (1) and Protocatechuic Acid (2) by Pseudomonas sp. - 25 -Electron spin resonance (ESR) studies showed that, i n the pres-ence of catechol and oxygen, the i r o n was converted to the f e r r i c s t a t e . These r e s u l t s , and the f a c t that ferrous ion binds to oxygen to form I | | | | per ferry'! i o n , an e q u i l i b r i u m state between Fe 0^ and Fe 0^ , led H a y a i s h i 2 4 ' 7 ^ to the p o s t u l a t i o n of the reaction mechanism shown i n Figure 14. ( 2 5 ) ( 2 7 ' ) Figure 14. Proposed Mechanism f o r the Action of Catechol-2,3-dioxygenase (Metapyrocatechase). When the catechol molecule approaches p e r f e r r y l ion, i t w i l l p r e f e r e n t i a l l y combine with the f e r r i c oxygen form and•thereby favor the I | j _ | | formation of more Fe 0^ from Fe 0^• Thus, i n t h i s ternary complex of i r o n , oxygen, and catechol, the relevant peroxide intermediate 27 i s formed. Concomitantly, ferrous ion i s released and binds to another mole-cule of oxygen. Subsequent intramolecular rearrangement of the c y c l i c - 26 -peroxide '27 w i l l lead to the formation of the f i n a l product, a-hydroxy-muconic semialdehyde (25). Although the flow of electrons may be schem-a t i c a l l y analyzed as being due to a sequence of re a c t i o n s , the e n t i r e event i s presumed to take place by a concerted mechanism i n a ternary 24 complex of ferrous ion-oxygen-substrate on the enzyme surface. Hayaishi's fi n d i n g s were subsequently confirmed by Senoh and 71 72 coworkers ' with c r y s t a l l i n e 3,4-dihydroxyphenylacetate-2,3-oxygenase, an enzyme c a t a l y z i n g the addition of two oxygen atoms to 3,4-dihydroxy-phenyl a c e t i c a c i d (28), to produce a-hydroxy-5-carboxymethylmuconic semi-aldehyde (29). The s i m i l a r i t y of k i n e t i c , a n a l y t i c a l , and ESR studies to those obtained f o r metapyrocatechase i n d i c a t e that such findings may indeed be generalized to include other dioxygenases of t h i s type. 2 f ^ ^ C O O H Fe , CH-COOH C H 2 C O O H (28) (29) The muconic semialdehydes, (25) and (26), can undergo nonenzy-mic r i n g closure i n the presence of ammonia to form the py r i d i n e d e r i v a -t i v e s p i c o l i n i c (30) and 2 , 4 - l u t i d i n i c (31) a c i d s , r e s p e c t i v e l y (Figure 15). On the other hand, furt h e r metabolism of the former r e s u l t s i n the formation of 2-oxo-4-hydroxyvaleric (32) and formic acids. The v a l e r i c acid thus formed undergoes subsequent a l d o l cleavage to produce pyruvic 73 acid (33) and acetaldehyde (34). - 27 -0 ( 1) (30) HOOC (2) ... (26) (31) R = COOH Figure 15. Ring F i s s i o n Products from Catechol (1) and Protocatechuic Acid (2) and t h e i r Respective Pyridine Acids. CHjCOCOOH ( 33) . , COOH * S0H XHO M) (25) HCOOH (32) CH^HO ( 3D r ^ V 0 H rV H H 0 ^ Y ° L i —* L ^00H—v ' c o c ^ / ^ O H XHO * - 28 -Fate of Substituents The fate of substituents on monocyclic aromatic rings i s quite varied; ( i ) Aromatic methyl ethers are demethylated to the corresponding hydroxy d e r i v a t i v e . The methoxyl-C can be trapped as formaldehyde, and 74 i t i s terminally oxidized to CC^. ( i i ) Methyl substituents may be oxidized step-wise to the carboxylic acid (eg. the c r e s o l s ) , but i n some cases they remain i n t a c t u n t i l a f t e r . 75 r i n g f i s s i o n . ( i i i ) Chlorine atoms usually remain on the r i n g unless removal i s obligatory f o r the i n t r o d u c t i o n of hydroxyls which are necessary to f i s s i o n . ( i v ) N i t r o and s u l f o n i c a c i d groups can be eliminated and replaced by hydroxyl. (v) A carboxyl group may be o x i d i a t i v e l y decarboxylated; i n some cases anaerobic decarboxylation occurs, eg. of 4,5-dihydroxyphthalate to protocatechuate. (vi) A l i p h a t i c side chains are dealt with i n a v a r i e t y of ways. They may be eliminated, by 8 - o x i d a t i o n 7 7 or other mechanisms, or even re-78 main i n t a c t , eg. phenylpropionic a c i d . Since microorganisms do not follow a r i g i d set of r u l e s , i t i s not s u r p r i s i n g to f i n d exceptions to the proposed schemes. Neverthe-l e s s , the g e n e r a l ity shown by such biodegradation sequences provides a p r a c t i c a l working model f o r the p r e d i c t i o n of possible intermediates i n the metabolism of the various aromatic compounds. 79 Preliminary i n v e s t i g a t i o n s performed by Bandoni and Towers, with a number of microorganisms i s o l a t e d from surface and subsurface s o i l samples c o l l e c t e d at Rubble Creek i n the G a r i b a l d i Park area, indicated that - 2 9 -both d- and 1-usnic acid (35) undergo chemical changes when incubated with c e r t a i n fungi and b a c t e r i a . This a b i l i t y to degrade 35 was shown by Phycomycetes, Ascomycetes, and Fungi Imperfecti. The same workers were able to obtain an i s o l a t e of Cladosporium which twice degraded up to 60% of the usnic acid provided (0.4 g/1) i n a 10-day period, but a f t e r subculturing for 6 months, i t l o s t completely the a b i l i t y to degrade the substrate. How-ever, species of Absidia, M o r t i e r e l l a , Mucor, and P e n i c i l l i u m looked more promising i n t h i s respect. Of the b a c t e r i a l i s o l a t e s , only a species of Pseudomonas appeared to degrade usnic a c i d , but i t was not subjected to further t e s t i n g . Since usnic acid (35) shows a marked a n t i b i o t i c a c t i v i t y towards 80 many microorganisms, i t s degradation would be one of s p e c i a l i n t e r e s t . Bearing t h i s i n mind, i t was therefore decided to pursue further the b i o -degradation studies i n i t i a t e d by Bandoni and Towers on t h i s compound. At the same time, r e a l i z i n g the implications that the chemical work developed during the s t r u c t u r a l e l u c i d a t i o n and synthesis of usnic a c i d could have on the c h a r a c t e r i z a t i o n and possible laboratory prepara-t i o n of any metabolites i s o l a t e d during the course of our studies, i t i s i n order to present a b r i e f d e s c r i p t i o n of such extensive and f a s c i n a t i n g endeavours. Usnic A c i d Occurrence and I s o l a t i o n The yellow pigment usnic a c i d (35), the most widely d i s t r i b u t e d l i c h e n substance, i s an o p t i c a l l y a c t i v e dihydrodibenzofuran possessing - 30 -a highly a c i d i c enol f u n c t i o n a l i t y . COCH. (35) Both enantiomeric forms are present i n nature, and rec e n t l y 81 82 the racemic and a "quasi-racemic" modification have been i s o l a t e d as w e l l (Table 3) Form 20 Op t i c a l Rotation, QaJ Melting Point, ° d-usnic . ,83 a c i d +495° (CHCI3) 203-204 1-usnic acid^3 -495° (CHCI3) 203 d l - u s n i c acid84 194 Table 3. O p t i c a l Properties of Usnic Acid. Usnic a c i d can be obtained by extr a c t i n g the pulverized l i c h e n 85 86 87 material with b o i l i n g ether or benzene, ' or acetone. The crude residue, d i s s o l v e d i n benzene or chloroform, i s p u r i f i e d by p r e c i p i t a t i o n with e t h y l a l c o h o l and r e c r y s t a l l i z a t i o n from benzene. 88 89 Usnic acid i s found i n many species of Cladonia, ' C e t r a r i a , 89 90 91 Placodium, A l e c t o r i a , Haematomma, Usnea, RamaUna, Evernia, Parmelia, ' Lecanora, and Nephroma. The racemate has been i s o l a t e d from C e t r a r i a 81 i s l a n d i c a and Cladonia s i l v a t i c a , but i t i s r e l a t i v e l y uncommon; a "quasi-racemate" (9:1 mixture of (+)-and (-)-usnic acids) has also been 82 83b i s o l a t e d from Acroscyphus sphaerophoroides by Japanese workers. ' Although Usnea sp. have often been u t i l i z e d as sources, Haematomma coccineum var. abortivum (Hepp) contains as much as 20 per cent of i t s dry weight f ^ - 81,122 of t h i s substance. - 31 -An exhaustive l i s t i n g of a l l lichens containing these sub-stances i s not wit h i n the scope of t h i s work, nevertheless, the following tables w i l l i l l u s t r a t e the occurrence of (+)-and (-)-usnic acids i n some l i c h e n f a m i l i e s . Table 4. Occurrence of (+)-Usnic Acid i n Lichens Genus Species Reference A l e c t o r i a ochroleuca 92 Cladonia arbuscula 93 m i t i s 94 Usnea dasypoga 94,95 aspera 96 longissima 96 o r i e n t a l i s 96 i m p l i c i t a 97 aureola 98 lac e r a t a 98 rubicunda 98 p u s i l l a 99 eulychniae 100 barbata 101,102 compacta 103 misaminensis 103 rubicunda 103 (var. c e r a t i n e l l a ) f l o r i d a 94 h i r t a 94 Parmelia scabrosa 104 t a r a c t i c a 96 d i l a t a t a 97 Ramalina fraxinea 104 terebrata 105 tumidula 106 c h i l e n s i s 107 inanis 108 t i g r i n a 108 boulhautiana 109 c a l i c a r i s 101 l i n e a r i s 103 subamplicata 103 ca p i t a t e 94 r e t i c u l a t a 87,110 - 32 -Genus Species References Evernia p r u n a s t r i 111 d i v a r i c a t a 94 Lobaria s c r o b i c u l a t a 112 Lecanora h a n d e l i i 113 badia 114 polytropa 114 sulphurea 114 Nephroma g y e l n i k i i 115 Table 5. Occurrence of (-)-Usnic Acid i n Lichens Genus Species References A l e c t o r i a japonica 92 sarmentosa 92 ochroleuca 94 Cladonia aberrans 116 a l p e s t r i s 93,119 r e t i c u l a t a 117 e n d i v i a e f o l i a 118 impexa 120 leucophea 120 tenuis 120 deformis 94 Lecanora melanophthalma 105 Centr a r i a s t r a c h e y i 121 ( f . ectocarpisima) n i v a l i s 94,111 Haematomma coccineum 122 More r e c e n t l y , a non-lichen derived usnic acid-polyene complex has been i s o l a t e d from a s t r a i n of actinomycetes, Actinomycetes s t r a i n 123 C-2167, which morphologically resembles the species A. candidus. The acid can be freed from the complex by p r e c i p i t a t i o n s with water from a c e t i c - 33 -acid , and petroleum ether from ether, followed by repeated c r y s t a l l i z a t i o n s , S t r u c t u r a l E l u c i d a t i o n In 1843, Rochleder and Heldt*°* obtained a yellow pigment from the lichens Ramalina fraxinea Ach. and Usnea barbata F r . , and gave i t 124 the name "Usnein", l a t e r changed by Knop to the present name of "Usnic Acid". 124 E a r l y studies on usnic a c i d were c a r r i e d out by Salkowski, Paterno,* 2"' Hesse,* 2* 1 and Zo p f , * 2 7 but f a i l e d to reach any d e f i n i t i v e conclusions on the chemical structure of th i s substance. 128 In 1900-1902, Widman found that usnic a c i d was an o p t i c a l l y a c t i v e substance, and both d- and 1-forms occurred i n nature. He estab-l i s h e d i t s molecular formula as being C^gH^O^, and forwarded, the s t r u c -t u r a l formula 36 , as well as 37 for decarbousnic a c i d , which had been 125 obtained by Paterno by heating usnic a c i d with e t h y l a lcohol i n a sealed tube. H00C 3 (37, b ° 0 H Widman's usnonic a c i d , ^gH^Og, & P e t m a n S a n a t e oxidation product of usnic acid was given formula 38 . Consequently, u s n o l i c a c i d , C.oH..,0-,, and l o l b / 129 decarbousnol, C^H^O,., which had been obtained by Stenhouse and Groves, 125 and by Paterno from usnic acid and decarbousnic a c i d , r e s p e c t i v e l y , on treatment with cone. H2S0^, were represented by formulae 39 and 40 . - 34 -C8H11 (38) C8H11 C o H 8n11 (40) These formulae, postulated without much experimental evidence, were assumptions based on the idea that usnic a c i d might be an acetoacetic acid r e l a t e d compound, since i t decomposes into acetone and carbon dioxide by the a c t i o n of concentrated a l k a l i . 130 In 1925, Schopf and Kraus found three a c t i v e hydrogen atoms i n usnic a c i d by Zerewitinoff's method, and derived the presence of car-boxyl or a c i d anhydride groupings i n decarbousnic a c i d . At the same time, the p o s s i b i l i t y that an enol moiety was responsible for the a c i d i c p roperties of both usnic and decarbousnic acids was f i r s t suggested. The a l k a l i n e decomposition of usnic acid had been previously investigated by several workers, ' and was known to produce C ^ , 124 acetone and a compound i n i t i a l l y c a l l e d pyrousnetic acid by Paterno, 131 or u s n i d i n i c acid by 0. Hesse. This compound, usnetic a c i d , has the 124 formula C^H^O^, and decomposes on heating to produce usnetol, 132 ^13^14^4' w u ^ - c n e a r l i e r workers c a l l e d u s n i d o l . The former, under a l k a l i n e treatment y i e l d s pyrousnic a c i d , C}_2H1205' w n * c n c a n ^ e f u r t n e r decarboxylated to usneol, ^ 1 1 ^ 1 2 ^ 3 (Figure 16). - 35 -C18 H16°7 + N a ° H usnic acid -» C0 o + CH„COCH.„ + C..H-.0, 2 3 3 14 14 6 usnetic a c i d C12 H12°5 + C H 3 C 0 0 H pyrousnic a c i d C11 H12°3 + C ° 2 usneol NaOH C13 H14°4 + C ° 2 usnetol Figure 16. A l k a l i n e Degradation of Usnic and Usnetic Acids, Following these r e s u l t s , and the fac t that C-methylphloroglucinol (41) and a c e t i c a c i d were obtained on ozonolysis of usnetol and usneol, 102 Schopf and Heuck forwarded the s t r u c t u r a l formulae 42 and 43 for usnetol and usneol, 44 and 45 for usnetic and usnic a c i d s , r e s p e c t i v e l y (Figure 17). 133 In 1933, Robertson and Curd attempted confirmation of S c h o p f s usneol formula 43, and synthesized 4,6-dimethoxy-2,3,7-tri -methyl coumarone (46), the dimethyl ether of 43, but i t did not agree with usneol-dimethyl ether. OMe 2.ZnCl 2 OMe ( 46) - 36 -0 OH 0 ( U ) (US) Figure 17. Schb'pf and Heuck's Structural Proposals (1927). - 37 -154 Robertson and Curd, therefore, proposed the alternate struc-ture 47, and prepared the dimethyl and diethyl ethers of 2,3,5-trimethyl-4,6-dihydroxycoumarone (48a,b), which proved identical with the dimethyl and diethyl ethers of usneol. : O C H , a) R = Me b) R =Et Thereafter, they synthetically confirmed usnetol as being 2,3,5-trimethyl-4,6-dihydroxy-7-acetylcoumarone (49, R=H), and indicated that its yellowish tint was due to the o-hydroxyacetophenone moiety and • • •j . 135 not to a semiqumoid structure as previously considered. MeCN ZnCl, COCH - 38 -Moreover, they suggested formulae 50 and 51 for pyrousnic and usnetic acids, based on the fact that usneol is derived from pyro-usnic acid by decarboxylation and gave a negative ferric chloride reaction, indicating a carboxymethyl side chain at the 2- or 3-position>instead of at the 7-position. H 2C00H HO X H 2 C 0 0 H h C H o ( 50) a) 2-CH 2 COOH, 3 -CH 3 ( 51 ) b) 2 - C H 3 ,3-CH 2 COOH 136-8 Asahina and Yanagita"""" " subsequently determined the position of the carboxymethyl side chain as being at the 2-position, by alkaline peroxide oxidation of pyrousnic and usnetic acids, and obtaining elsholt-ziaic acid (52). - 39 -The same workers also proved, unequivocally, the position of the acetic acid moiety in pyrousnic acid (50a), by converting methyl pyrousnetate dimethyl ether (53) into 3,5-dimethyl-4,6-dimethoxycoumarone (54), identical with a synthetic sample obtained from C-methylphloro-glucinol dimethyl ether (55). N-OH C H 2 C O O C H 3 C H 3 0 \ ^ > ^ 0 » Y ^ C - C O O C H 3 NaOCH3 (53) EtOH N-OH R = CH3 ( 5 5 ) - 40 -Subsequently, Robertson and Curd synthesized pyrousnic acid dimethyl ether (56) from the coumarone 54 by the following route: (56) 136 Asahina and Yanagita proposed, i n 1936-1937, a 1,3-diketone side chain for decarbousnic acid since i t shows a c i d i c properties, gives 140 a v i o l e t - c o l o r e d condensation product with o^-phenylenediamine, and e a s i l y converts into usnetic acid (51a) with l i b e r a t i o n of acetone. According to the new formulation, decarbousnic acid would be represented by (57), and the previously obtained decarbousnic a c i d oxime anhydride and 128 phenylhydrazone anhydride, by the isoxazole 58 and pyrazole 59 d e r i v a t i v e s , r e s p e c t i v e l y . COCH (57 ) R.R* =H COCH (58) HO COCH 0-^-CH. N (59) Further confirmation to the structure of decarbousnic acid proposed by Asahina and Yanagita^ 3^ and by Robertson, B i r c h and Flynn''"4"'" 142 was obtained by Schopf and Ross. Ozonolysis of decarbousnic acid diacetate (60) afforded a diacetate of a C-methylphlorodiacetophenone (61), i d e n t i c a l 143 with a synthetic sample prepared by Dean and Robertson, who showed that the corresponding t r i a c e t a t e 62, the d i r e c t ozonolysis product, hydrolyzes r e a d i l y to the diacetate i s o l a t e d by the German workers. - 42 -COCH AcO OAc OCH, 0 0 1)0-2) H 2 0 1) 0-2) H 2 O.A A c 2 0 Py COCH. COCH 3 OHc Ac 20 AcO COCH NaOAc Ac 2O^NaOAc COCH COCH. Formula 57 also explains the f a c t that o p t i c a l l y i n a c t i v e decarbousnic acid i s obtained from either d- or 1-usnic a c i d , as i t does not contain any asymmetric centers. 144 145 Asahina and coworkers, and Robertson and Curd i d e n t i f i e d yet another byproduct obtained during the formation of decarbousnic acid 128 (57) from usnic a c i d . The product, o r i g i n a l l y recorded by Widman as isodecarbousnic a c i d , was shown to be deacetyldecarbousnic a c i d , given the formula C, CH,,0 C, and" named acetusnetol (63) by the Japanese group. 15 l b 5 COCH3 H O ^ Y O v - C ^ C O C h ^ OH (63) To a r r i v e at a structure for usnic a c i d from decarbousnic a c i d re-quires only the i n s e r t i o n of a carbonyl group, but the way i n which t h i s - 43 -should be done was for long a source of controversy. Asahina and 136 Yanagita proposed, i n 1937, the s t r u c t u r a l formula 64 for usnic a c i d , on the basis of t h e i r structure of decarbousnic acid (57). H 0 \ J ^ / 0 -OH (64) 146 Nevertheless, Robertson and Curd c r i t i c i z e d t h i s proposal since i t f a i l e d to explain the disappearance of the asymmetric carbon by 144 147 e n o l i z a t i o n . This led Asahina and Yanagita, and l a t e r Asahina to propose structures 65 and 66 for usnic a c i d , based on the assumption that the phl o r o g l u c i n o l nucleus found i n the degradation products was formed by a secondary r e a c t i o n . However, neither formula can f u l l y explain a l l the reactions of usnic a c i d . COCH. ( 65) ( 66) 0 0 146 F i n a l l y , Curd and Robertson proposed the following scheme, which i s now the accepted one, in c l u d i n g structure 35 for usnic a c i d (Figure 18). - 44 -( 35) (67) Figure 18. Robertson and Curd's S t r u c t u r a l Proposal for Usnic Acid (35). i - 45 -The scheme accounts well for the presence of an o p t i c a l l y a c t i v e centre not racemized by d i l u t e a l k a l i , for the ease with which decarbo-usnic a c i d i s formed-bond (a) i n 67 would break e a s i l y because of the tendency for the system to aromatize to a true benzofuran -and for the formation of h e t e r o c y c l i c compounds when the 8-diketonic system reacts with carbonyl reagents, eg. phenylhydrazine, hydroxylamine. Moreover, structure 35 has a strongly a c i d i c centre i n the triacylmethane grouping, which accounts for the fact that usnic a c i d i s strongly a c i d i c yet f a i l s to y i e l d esters or to suffe r simple decarboxylation. obtained a c r y s t a l l i n e ozonide, C22 H20°12' w n i c n o n h y d r o l y s i s produced acetylpyruvic acid (69) which was i d e n t i f i e d as a d e r i v a t i v e , and a pale yellow compound, Ci2 Hi2°5' w n i c h e x h i b i t s a blue-black c o l o r a t i o n with f e r r i c c h l o r i d e . This r e a c t i o n , s i m i l a r to the one given by usnetic a c i d and usnetol, suggested the presence of a methylphloroglucinol nucleus with a C - a c e t y l group, and led the German workers to the proposal of thea-coumaranone system 70 for such a compound. On ozonization of d i a c e t y l u s n i c acid (68 b), Schopf arid Ross 142 RO ( 68) a) R = H; R'= Ac t C ) R = Ac; R'-H b) R, R'= Ac AcO OAc (70) + + COOH (69) 148 Furthermore, Asahina and Okazaki confirmed these experimental r e s u l t s , adding strong support for the l o c a t i o n of double bond (c) i n 67 , The desired a-coumaranone 70 was f i n a l l y synthesized by Dean and Robert-149 son, as seen i n Figure 19. The l a s t step of the i r synthesis involves opening of the lactone r i n g and spontaneous r e c y c l i z a t i o n to the required lactone, previously shown'0' to be formed i n preference to i t s isomer. Attempts to obtain a r i g i d proof for the p o s i t i o n where carbon dioxide l i b e r a t i o n occurs during the formation of decarbousnic a c i d from 144 usnic a c i d were performed by Asahina and coworkers. On heating usnic acid with absolute e t h y l a l c o h o l , they obtained an eth y l ether,C^gH^QO^, 2)NH 2NH 2 3)HN02,EtOH Figure 19. Synthesis of a-Coumaranone - 48 -which could be decomposed on b o i l i n g d i l u t e a l k a l i to produce, i n one case, acetusnetol (63) and carbon dioxide, whilst i n another, usnetic a c i d (51 a) and a c e t i c a c i d . The ester, ethyl acetusnetate, must have one of the two possi b l e formulae 71 or 72 . (71) or — (72) • (51a) Asahina and Yanagita subsequently found that on b o i l i n g with ethanol for a long time mono- or d i a c e t y l u s n i c a c i d (68 a or 68 b) y i e l d s mono-or d i a c e t y l u s n i c a c i d ethoxide, C o„H„.0-. or C„ ,H„ .CL . (73 or 74, a, b) J 11 24 9 24 26 10 which on subsequent treatment with 60% aqueous a c e t i c acid converts i n t o mono- or di a c e t y l a c e t u s n e t i c a c i d (75 or 76, a, b) . abs. EtOH (68a.b) > A a) R=H; R'= Ac b) R.R'= Ac RO COCH. - COOC,H c I 2 5 CH2C0CHC0CH3 COCH-or C00C 2 H 5 CHCOCH, I 2 COCH. (73a,b) (7(.a .b) COCH, OR' COOH I CH^COCH. 60% CH3COOH A COCH, COOH R 0 \ ^ \ / 0 ^ C HCOC H. or (75a.b) (76 a.b) - 50 -The Japanese workers on attempting to determine the p o s i t i o n of the carboethoxy grouping i n e t h y l acetusnetate followed the findings of 153 Jacobson and Ghosh. According to these authors a-substituted-8-ketonic esters condense with phenol, by the action- of cone, s u l f u r i c a c i d , to produce chromone d e r i v a t i v e s , which on h y d r o l y s i s y i e l d phenol-carboxylic ac i d s , whereas Y -substituted - 3-ketonic esters a f f o r d coumarin d e r i v a t i v e s , not decomposed into phenol-carboxylic acids on h y d r o l y s i s . Asahina and Yanagita c a r r i e d out the condensation of ethylacet-usnetate with r e s o r c i n o l and a-naphthol. However, the condensation pro-duct did not give any phenol-carboxylic a c i d on a l k a l i n e h y d r o l y s i s , suggesting that i t was indeed a coumarin d e r i v a t i v e , and ethylacetusnetate would, therefore, be a y-substituted-B-ketonic ester, represented by formula 71 . Hence, the acetylated d e r i v a t i v e s of usnic a c i d ethoxide would be indicated by 73 £ and b). Nevertheless, i t should be noted that the t h e o r e t i c a l basis of 154-5 t h i s conclusion became doubtful when Robinson and Baker revised Jacobson's work, and stated that even a-phenyl and benzyl-substituted aceto-a c e t i c esters generally give coumarin d e r i v a t i v e s upon a c i d catalyzed con-densation with phenolic compounds. Investigations concerning t h i s point have more recently been c a r r i e d out by Yanagita *"^ and by Shibata and coworkers.*"' 7 158 Furthermore, Asahina and Okazaki found that, on heating with water i n a sealed tube, d i a c e t y l u s n i c a c i d ethoxide (73 b) converts into diacetyldecarbousnic acid (57, R, R' = Ac). - 51 -COCH COOEt I H-CO-CHCOCH. H 2 0 COCH (57 ) R.R* =H Summarizing these f a c t s together with Schopf and Ross'sozonolysis r e s u l t s , i t becomes clear that usnic acid would be formed from usnic acid ethoxide by r i n g closure, with l i b e r a t i o n of ethyl a l c o h o l , strongly supporting 35 as the correct formulation for usnic a c i d . D i r e c t proof for the existence of bond (b) i n 67 was obtained 159 by oxidative r i n g cleavage-of usnic a c i d . Barton and Bruun obtained, on ozonlysis of d i a c e t y l u s n i c acid anhydrophenylhydrazone (77) , 1-phenyl-3-methylpyrazole-4,5-dicarboxylic acid (78), along with 3, 3 ' ,5,5'-tetra-methyl-7,7'-diacetyl-4,4',6,6'- tetraacetoxydicoumaran-2-one (79). This 160 compound, previously obtained by Takahashi and Shibata by permanganate oxidation of d i a c e t y l u s n i c a c i d , i s also formed from 7-acetyl-3,5-dimethyl-4,6-diacetoxycoumaran-2-one (70), the ozonolytic product of usnic a c i d j 159 dxacetate, by treatment with permanganate and ozone. Ph H 0 2 C N ^ N . COCH AcO OAc H 0 2 C (78) (77) COCH. COCH AcO 0\^0 Ov^O OAc OAc ( 7 9 ) - 52 -159 Moreover, Barton and Bruun found, that on treatment with methanolic potassium hydroxide, usnic acid anhydrophenylhydrazone (77) i s r e a d i l y converted to 80 v i a cleavage of the aromatic a c e t y l grouping and methanol a d d i t i o n across the double bond. A more stringent treatment ethanolic or aqueous potassium hydroxide - converts 77 into the pyrazole carboxylic a c i d 81, therefore f u r n i s h i n g , beyond any doubt, the p o s i t i o n of bond (b) i n 67. KOH MeOH (80) (77) K O H E t O H or w a t e r C O OH (81) Worthwhile mentioning at t h i s point i s the f a c t that Michael ad d i t i o n to the double bond i n usnic acid i s f e a s i b l e under both basic (vide supra) and a c i d i c conditions. Takahashi"'"^''" found that on treatment with 10% methanolic-HCl, d- or 1-diacetylusnic acid was converted into an o p t i c a l l y a c t i v e compound, ^21^22^9' w n :'- c n W O U 1 ^ represent an a d d i t i o n of one mole of CH^OH to monoacetylusnic a c i d . This product, monoacetylusnic acid isomethoxide (82), d i f f e r s from a r e a c t i o n product of monoacetylusnic acid with methanol, monoacetyl-usnic acid methoxide (83), in i t s o p t i c a l a c t i v i t y . It was found that d-d i a c e t y l - u s n i c acid produces a levorotatory compound, [ c ] ^ = -83.9°, while - 53 -the 1-isomer produced a dextrorotatory d e r i v a t i v e [ c ] ^ = +86.8°C. Based on several degradations, Takahashi t e n t a t i v e l y forwarded the s t r u c t u r a l formula 84 , which could be derived from Robertson-Schdpf's formula of usnic acid diacetate (68 b) by the methanolytic cleavage of the oxygen bridge and p a r t i a l d e a c e tylation. O C H 3 OCH* 0 - U - O O H O C H -OAc ( 82 ) C O C H H O C O C H C00CH 7 C H ^ C O C H C O C H . ( 83 ) OAc (84) 0 Later on, motivated by Shibata's suggestions, 'Takahashi and co-162 workers revised t h e i r proposed structure 84 and found that mono-acetylusnic acid isomethoxide biphenylhydrazone monoanhydride (85), ^33^32^6^4' w ^ e n t r e a t e d with methanolic hydrochloric acid gave the phenyl-hydrazone monoanhydride 86 . On b o i l i n g t h i s compound with methanolic potassium hydroxide, a product, 02-^220,^2, was obtained and shown to be i d e n t i c a l with Barton's degradation product of usnic a c i d phenylhydrazone monoanhydride (80). These r e s u l t s and the nmr data on usnic a c i d isometh-oxide monoacetate, led the Japanese workers to the pos t u l a t i o n that com-pound 82 e x i s t s i n s o l u t i o n , as two of i t s possible tautomeric forms (82, a, b). : O C H 3 O C H 3 C O C H ^ O C H k<>0H C O C H , HA 0 - 54 -OAc (82 ) OCH 3OMe OAc (86) COCH-NH-NH2,HCl f \ - N - N y \=/ T O M e EtOH KOH CH 3OH HCl 3CH5OH (80 ) In order to f a c i l i t a t e the understanding of the numerous degradative reactions performed on usnic a c i d a summary of the same i s presented on Figure 20. Synthesis * n t. - !63 _ . •• - 143 . . . . 152b Sxnce the researches of Robertson, Schopf, and Asahina produced such a considerable body of evidence i n favour of structure 35 for usnic a c i d , the next l o g i c a l step was to develop a synthesis of such a compound. 164 In 1955, Barton and coworkers. r e a l i z i n g the symmetric nature of usnic a c i d , and recognizing the biogenetic importance of oxida-t i v e p a i r i n g of phenolic r a d i c a l s , postulated i t s biogenesis as a r i s i n g by the coupling of two methylphloroacetophenone (87) u n i t s . An oxidation of monohydric phenols by a l k a l i n e f e r r i c y a n i d e Pummerer and h i s c o l l e a g u e s ^ 5 obtained complex mixtures from which they OH ( 71 ) Figure 20. Summary on the Chemical Degradations of Usnic Acid (35). - 56 -i s o l a t e d c r y s t a l l i n e dimers and trimers of the corresponding aryloxy-166 r a d i c a l s . In 1956, Barton and coworkers determined the structure 88 for the neutral c r y s t a l l i n e dimer obtained by oxidation of p_-cresol, showing that i t a r i s e s from a C-C coupling followed by B-addition of the phenolic hydroxyl to the enone system, and not v i a 0-C coupling as o r i g -i n a l l y proposed by Pummerer's structure 89 . (88) ( 8 9 ) 167 Haynes and coworkers have shown that the i n i t i a l stage of such oxidations appears to be a r e v e r s i b l e r e a c t i o n between the phenol anion and the f e r r i c y a n i d e ion, g i v i n g ferrocyanide and the corresponding a r y l o x y - r a d i c a l , followed by i r r e v e r s i b l e coupling to produce dimeric and polymeric products. The r a t e of oxidation was found to be dependent upon the a l k a l i n i t y of the s o l u t i o n and the Ferricyanide / Ferrocyanide r a t i o . The oxidative coupling of phenolic compounds has r e c e n t l y been reviewed i n , ,. 168 the l i t e r a t u r e . Thus, Barton and c o w o r k e r s * ^ ' i l l u s t r a t e d the p a r t i c u l a r s i g n i f i c a n c e of dimer 86 i n biogenetic concepts by a simple and elegant, two-step synthesis of (±)-usnic a c i d (35). Methylphloroacetophenone (87), obtained v i a a Hoesch r e a c t i o n * ^ ' o n methylphloroglucinol (41), was, therefore, oxidized with sodium carbonate-potassium f e r r i c y a n i d e to give as main c r y s t a l l i n e product (15%) the expected dimer 90 , "hydrated" usnic a c i d . A c e t y l a t i o n of 90 with a c e t i c anhydride containing a trace of s u l f u r i c a c i d gave a reasonable y i e l d of (±)-usnic acid diacetate (68 b), further characterized by h y d r o l y s i s to 35 . A l t e r n a t i v e l y , the dimer 90 was dehydrated i n concentrated s u l f u r i c a c i d to give (±)-usnic a c i d d i r e c t l y - 5 7 -(Figure 21). Conversely, racemic usnic acid has been resolved by Dean and coworkers 171 through i t s (-)-brucine s a l t . At the same, time, and as a continuation of t h e i r studies on the nuclear methylation of phloroacetophenone 172 (91), and on phloroglucides, 173 Ri e d l and coworkers 174 attempted another synthesis for usnic a c i d . Their proposal was di r e c t e d towards the synthesis of a 9b-desmethylusnic a c i d * (92) followed by nuclear methylation. Using p h l o r o g l u c i n o l (93) as s t a r t i n g m aterial, these workers were able to prepare, through c l a s s i c a l methods, the hexamethyldiphloro-g l u c i n (94) and the tetrahydroxydibenzofuran (95), d i r e c t precursors of 92. Nevertheless, further attempts to introduce the 2- and 6-acetyl groupings or the 9b-methyl group f a i l e d completely (Figure 22). The continued i n t e r e s t on the oxidative coupling of phenols led P e n t t i l a and Fales"*" 7^ to achieve the " i n v i t r o " biosynthesis of usnic a c i d . Through the use of the enzyme horseradish peroxidase, they were able to obtain, i n 30% y i e l d , "hydrated" usnic a c i d (90), i d e n t i c a l with Barton's m a t e r i a l . The l a t t e r , upon dehydration provided (+)-usnic acid (35) (Figure 22). The use of a dibenzofuran numbering system i s recommended for usnic a c i d . - 58 -Figure 21. Barton's Synthesis of (+)-Usnic Acid (35). - 59 -( 94 ) (95 ) Figure.22. Reidl's Attempted Synthesis of Usnic Acid (35). - 60 -(35) Figure 23. P e n t t i l a ' s in v i t r o Biosynthesis of (±)-Usnic Acid (35). - 61 -Nevertheless, Japanese workers, studying the enzymatic oxida-tion of methylphloroacetophenone (87), found that whilst p-cresol would react with Rhus laccase or with peroxidase to produce Pummerer's ketone (88), dl-"hydrated" usnic acid (90) was formed only with horseradish per-oxidase (Table 6). However, the oxidative coupling of 87 in lichen should be stereospecific, since usnic acid occurs mainly in an optically active form. Furthermore, i t seems probable to assume that not only the position of oxidative coupling, but also the formation of the ether link-age are enzymatically controlled, since isousnic acid (112) occurs in many 178 lichens along with usnic acid. Table 6. Enzymatic Coupling of Phenols. Enzyme pH (90),% (88),% Laccase (Rhus succedanea) 6.5 — dl-,10 Laccase (Rhus vernicifera) 7.8 — dl-,10 Peroxidase (Horseradish) 6.5 dl-,5 dl-,20 Other synthetic analogues of usnic acid have been prepared by F.lix and 179 180—182 coworkers as part of their studies on annelated furans. These workers found that cycloaddition of dimethyl acetylenedicarboxylate or methyl propiolate to 2-isopropenyl-3-methylbenzofuran afforded a conven-ient and versatile route to 9b-methyl-3,9b-dihydrodibenzofuran (100) and 9b-methyl-l,9b-dihydrodibenzofuran (101, a,b). Thus, the alkylation of o-hydroxyacetophenones, such as 97 , with chloroacetone in the presence of potassium carbonate i s accompanied by an aldol condensation and ring - 62 -closure to afford 2-acetyl-3~methylbenzofuran (98). Wittig reaction on the ketone 98 with methylenetriphenylphosphorane gives the required 2-isopropenyl-3-methylbenzofuran (99) , which in its reaction as a "diene" gave the desired 3,9b-dihydrodibenzofuran (100) upon treatment with acetylene. On treatment with base, the 3,9b-dihydrodibenzofuran (100) rearranges to the thermodynamically more stable 1,9b-dihydro-system (101), R1 XX R2 ( 97 ) OH COCH. C l C H 2 C O C H 3 ' < 2 c o 3 D M F , r t R 1 =R2 = H R 1 = O C H 3 ; R 2 = H R1 = R2 = 0CH-4> 3 P = C H 2 DMF, r t C - C O . C H , III 2 3 C - C 0 2 C H 3 C 0 ? C H 3 xylene, A C 0 2 C H 3 (100 ) NaOCHj 3 ( A C H 3 0 H (99 ) 1 ) K 0 H / H 2 0 - E t O H 2)HCl C O o C H , H C 0 2 C H 3 H C ° 2 H (101 a ) ;101 b) - 63 -such as that exhibited by usnic a c i d i t s e l f . The conversion of s i m i l a r compounds into usnic a c i d i s cu r r e n t l y under a c t i v e i n v e s t i g a t i o n . Biosynthesis The b i o g e n e t i c a l o r i g i n of usnic acid (35), one of the most 142 common l i c h e n substances, was f i r s t suggested by Schopf and Ross as a r i s i n g by the coupling of two molecules of methylphloroacetophenone (87). This pathway was l a t e r demonstrated chemically by Barton and coworkers'*"^4 i n t h e i r synthesis of ( t ) - u s n i c a c i d (vide supra). 183—185 I n i t i a l studies by Mosbach and coworkers demonstrated that the condensation of an a c e t y l coenzyme A unit with three malonyl-CoA units would form a hypothetical 3,5,7-trioxooctanoic acid (102), which would then c y c l i z e i n several d i f f e r e n t modes. Thus an i n t e r n a l a l d o l condensation through carbons two and seven produces the o r s e l l i n i c a c i d (103) type system, whilst condensation through C-3 and C-8 y i e l d s 3,5-dihydroxyphenylacetic acid (104), and a Cl a i s e n r e a c t i o n through C- l and C-6 gives the a c e t y l p h l o r o g l u c i n o l (91) system, as seen i n Figure 24. Furthermore, incorporation of a C - l u n i t s on (91) would produce methylphloroacetophenone (87). •OH ( 87 ) - 64 -< 1 °3 ) (91) n o * , Figure 24. Possible Cyclizations of the Hypothetical 3,5,7-Trioxooctanoic Acid (100). - 6 5 -Biosynthetic studies on the lichens Usnea longissima Ach., Usnea d i f f r a c t a Wain., Evernia mesomorpha Mull. Arg., Parmelia caperata (L.) Ach., Cladonia m i t i s Sandst, and Cladonia a l p e s t i e s (L.) Rabh., were c a r r i e d out by Shibata and coworkers. ''''^  Administration of s u i t a b l e precursors was done by either of the following methods: ( i ) Incubation, by shaking the fresh l i c h e n i n s t e r i l i s e d Czapek-Dox medium, f o r 3-4 days, under i l l u m i n a t i o n with a white e l e c t r i c lamp (200W/100V); ( i i ) D i r e c t absorption, by dropping a s o l u t i o n of the radioactive precursor i n 10% aqueous ethanol on the surface of the f r e s h t h a l l u s . A f t e r a c e r t a i n metabolic period the l i c h e n was extracted with benzene to i s o l a t e the a c i d , which was r e c r y s t a l l i z e d s e v e r a l times from the same solvent, and then subjected to degradations with sodium hypoiodite., Kuhn-Roth oxidation followed by the Schmith reaction, or ozonization of the corresponding acetate. The r a d i o a c t i v i t y present i n these degradation products was then measured on a s c i n t i l l a t i o n counter and compared with that o r i g i n a l l y fed. 14 I n i t i a l experiments, c a r r i e d out with 2- C-sodium acetate, 14 14 2- C-diethyl malonate, and C-sodium formate (Figure 25), i n d i c a t e d c l e a r l y that two C„-polyketide units were involved i n the biosynthesis o of usnic a c i d , and that the two nuclear methyl groups were derived from C^-units. The next step was to determine the time at which methyl i n t r o d u c t i o n takes place. For t h i s , 2'-*^C-phloroacetophenone (105), prepared v i a a Hoesch r e a c t i o n * 8 7 with 2 - * ^ C - a c e t o n i t r i l e on p h l o r o g l u c i n o l , was fed to - 66 -CH 3COONa : O C H (35) Cr0 3 /"H 2 SO A k CH 3COONa BaC0 3 CH3NH KOH- I 2 CHI-CH2(COOEt ) 2 H-COONa 1) C r 0 3 / H 2 5 0 Z l 2) H N 3 CH3NH-? 0 2 N BaC0 3 14. Figure 25. The Biosynthesis of Usnic Acid. Feeding Experiments with C-Sodium Formate, 2-14C-Sodium Acetate and 2- 1 4C-Diethyl Malonate. - 67 -fresh i s o l a t e d l i c h e n t h a l l i . Since no incorporation of r a d i o a c t i v i t y was observed i t was therefore concluded that the methyl group of methyl-phloroacetophenone (MPA) i s not introduced into phloroacetophenone, but i n t o the Cg-polyketide p r i o r to c y c l i z a t i o n . On the other hand, experiments with l a b e l l e d 2'-"^C-methylphloroacetophenone (106) i n d i c a t e d i t s a ctual r o l e as an intermediate i n the biosynthesis of usnic a c i d (Figure 26). Although methylphloroacetophenone has not been as yet i s o l a t e d from l i c h e n by the usual methods, i t s presence has now been demonstrated by the i s o t o p i c d i l u t i o n method i n extracts of Cladonia m i t i s Sandst, 14 incubated i n the presence of CO^ f o r 10 days. Although Shibata and coworkers'*"''7 and Penttila.and Fales"**7^ have found that MPA i s converted to "hydrated"-usnic a c i d by the enzyme horseradish peroxidase, thus f a r no success has been achieved i n the i s o l a t i o n of the o x i d i z i n g enzyme responsible f o r usnic a c i d biosynthesis i n l i c h e n . Nevertheless, "hydrated" usnic a c i d has been shown to be an a c t u a l intermediate of the b i o s y n t h e t i c pathway involved. In conclusion, much of the biosynthesis of usnic a c i d i s s i m i l a r to i t s laboratory synthesis, and can be summarized as shown i n Figure 27. 188 Furthermore, Shibata and coworkers found that i n l i c h e n t h a l l u s the outer layer i s the most active i n secondary metabolism. A constant seasonal v a r i a t i o n i n biosynthetic a c t i v i t y with i t s peak period appearing i n February and i t s minimum i n July was also observed. - 68 -2 t 1 06 ) (105 ) (a) 1.5x105dpm/mM a) U.UxW1* d p m / m M 2 3V. ( c a l c d . 2 5Vo) Figure 26. The Incorporation of 2'- C-Methylphloroacetophenone (104) i into Usnic Acid. - 69 -Figure 2 7 . Biosynthesis o f Usnic Acid i n Lichens. -70-DISCUSSION The biodegradation of organic molecules i s a f a s t growing f i e l d having large chemical and e c o l o g i c a l implications. Therefore, the present work deals with the i s o l a t i o n , s t r u c t u r a l e l u c i d a t i o n and synthesis of those compounds obtained during the biodegradation of the l i c h e n substance (-f-)-usnic acid (35), a natural a n t i b i o t i c , by several s o i l fungi and b a c t e r i a . The work described i n Part A of t h i s thesis concerns i t s e l f with the biotransformation of (+)-usnic acid (35) into (+)-6-desacetylusnic acid (107) by a Pseudomonas species. The c h a r a c t e r i z a t i o n , laboratory preparation and subsequent conversion of t h i s metabolite into (+)-usnic acid (35) are described. The synthetic routes i n i t i a l l y i nvestigated furnished only the isomeric (+)-8-desacetylisousnic acid (178), which was chemically converted to (+)-isousnic acid (112), the natural isomer of (+)-usnic a c i d . Part B describes the i s o l a t i o n , structure e l u c i d a t i o n and synthesis of (+)-2-desacetylusnic acid (108), the metabolite obtained during the b i o -degradation of (+)-usnic a c i d (35) by Mucor globosus. Part C deals with the products obtained during the biodegradation of (+)-usnic acid (35).by M o r t i e r e l l a i s a b e l l i n a . The importance of s u i t a b l y l a b e l l e d precursors f o r d e t a i l e d biodegradation studies i s obvious, therefore, Part D of t h i s work describes the f i r s t synthesis of a s p e c i f i c a l l y l a b e l l e d usnic a c i d , (-f)-usnic acid (6-C0 1 ACH 3) (255). - 71 -Part A The bacterial isolate showing the a b i l i t y to degrade (+)-usnic 189 acid (35) was obtained by Dr. J.D. Leman from surface s o i l samples collected in the Whistler Mountain area. The culture has since been purified and was i n i t i a l l y identified as Pseudomonas species by Dr. R.J. Bandoni of the Botany Department, University of British Columbia. At the present time, the culture is in the process of being f u l l y characterized. The fermentation was carried out in Erlenmeyer flasks containing an appropriate amount of nutrient medium with 3-4 day old cultures of the bacterial isolate. After the substrate (+)-usnic acid (35) was added, the flasks were agitated on a rotary shaker at room temperature for 7-10 days. Usual work-up and purification of the crude extract by preparative layer chromatography afforded two main components: a) recovered (+)-usnic acid (35) (32.1%) and b) (+)-6-desacetylusnic acid (107) (57.0%). Before discussing the structure elucidation of the metabolite 107, i t is pertinent to consider the spectroscopic properties characteristic of the parent molecule, (H-)-usnic acid (35). 20 83 Thus, the dextrorotatory isomer, [ c l ^ + 495°, shows at 328 190 191 (R-Band), 285 (B-Band) and 231 nm (K-Band) ' the ultraviolet absorption 192 maxima characteristic of a phloroacetophenone derived molecule possessing 193 an unsaturated enolized 2-acyl-cyclohexane-l,3-dione system. Previous investigations related to hydrogen bonding in enolized tricarbonyl systems 193-195 using infrared spectroscopy have recently been summarized and provided the experimental background necessary for the interpretation of the - 72 -196 197 complicated i r spectrum of (-t-)-usnic a c i d (35). ' The hydroxyl s t r e t c h i n g absorptions of enolized f3-tricarbonyl systems are not very d i s t i n c t , having broad bands of low e x t i n c t i o n usually displaced towards low frequencies. (+)-Usnic a c i d shows three d i f f e r e n t hydroxyl absorptions i n the 4000-2000 cm * region, as seen i n Figure 28. The absorption i n the 3200-3000 cm * region as shown by (-f-)-usnic a c i d (35) and i t s 7-0-acetyl d e r i v a t i v e can be a t t r i b u t e d to the C -hydroxyl group. The p o s i t i o n of 9 t h i s band also i n d i c a t e s that t h i s hydroxyl group i s engaged i n an i n t r a -molecular hydrogen bond of considerable strength. The absorption at 2800 cm * agrees w e l l with that observed f o r 2,4-dimethoxy-6-hydroxyacetophenone and therefore can be assigned to the chelated C^-hydroxyl group, whereas the one at 2400 cm * corresponds to the very strongly chelated e n o l i c hydroxyl group at C^. The dienone carbonyl absorbs near 1680 cm * wh i l s t the bands corresponding to the aromatic C-acetyl group and the enol ether coalesce producing a very strong absorption at 1625 cm *. The conjugated chelated carbonyl of the enolized 3-tricarbonyl system gives a broad band at 1540 cm *. Its p o s i t i o n i s comparable with that of enolized 2-acyl-cyclohexane-l,3-diones -1 193 198 which absorb i n the 1565-1520 cm region. ' The pmr spectrum of (+)-usnic acid (Figure 29) i s very character-i s t i c and allows f a c i l e assignment of a l l the observed s i g n a l s . Thus, the two three-proton s i n g l e t s at 61.76 and 2.10 can be a t t r i b u t e d to the Cg^ -a-methyl and aromatic C-methyl group r e s p e c t i v e l y . I t i s pertinent at o t h i s point to mention that the absolute configuration about the C ^ - p o s i t i o n 199 i s the one obtained from the X-ray a n a l y s i s of (+)-2-desacetylusnic a c i d (108) (see Part B). i FREQUENCY (CM') 4000 3600 3200 2800 2400 2000 1300 1600 1400 1200 1000 800 650 Figure 28. The Infrared Spectrum of (+)-Usnic Acid (35). I I I I I I Figure 29. Proton Magnetic Resonance Spectrum of (+)-Usnic Acid. - 75 -The six-proton s i n g l e t at 62.70 corresponds to the C2~acetyl and Cg-aromatic a c e t y l groupings, while the one-proton s i n g l e t at 65.96 i s assigned to the C ^ - o l e f i n i c proton. The signals observed at lower f i e l d correspond to the hydroxyl protons and f u r n i s h straightforward information about the hydrogen bonding i n t h i s molecule. The hydroxyl proton of the enolized g-triketone appears at very low f i e l d (618.83). Its p o s i t i o n can be compared to that of ceroptene (109) (2-cinnamoyl-6,6-dimethyl-5-methoxy-cyclohex-4-ene-l,3-dione) and i t s transformation 198 products and in d i c a t e s the presence of a very strong chelate. The hydroxyl proton of the aromatic chelate gives a s i g n a l at 613.31. This low f i e l d s i g n a l agrees i n magnitude with those recorded for r e l a t e d compounds such as 2-hydroxyacetophenone (612.32) and 2,4-dihydroxy-196 acetophenone (512.70). The p o s i t i o n of these signals i n d i c a t e a rather strong aromatic chelate, although i t should be remembered - that the d i a -magnetic anistropy of the aromatic r i n g contributes to t h e i r l o w - f i e l d p o s i t i o n . F i n a l l y , the s i g n a l at 611.01 corresponds to the C^-hydroxyl proton. I t s f a i r l y low f i e l d p o s i t i o n i n d i c a t e s the presence of an intramolecular hydrogen bond of comparable strength to the one found i n sa l i c y l a l d e h y d e . The a p p l i c a t i o n s of mass spectrometry as a t o o l f o r the s t r u c t u r a l e l u c i d a t i o n of natural products have been known for a long t i m e ^ ^ and the c a r e f u l i n v e s t i g a t i o n of the fragmentation processes involved often leads to the determination of s u b s t i t u t i o n patterns. Therefore, i t was decided to undertake the study of the fragmentation patterns of these highly oxygenated molecules i n the mass spectrometer. - 76 -( 1 0 7 ) ( 1 0 8 ) 0 H H ( 1 0 9 ) The mass spectrum of usnic acid"^" 1" and a few chemically derived 202 203 compounds ' have appeared i n the l i t e r a t u r e , but i n general l i t t l e was known about the behavior of t h i s large family of compounds. A l l the fragmentation processes discussed are supported by high r e s o l u t i o n mass measurements of the various fragments as w e l l as the correspond-ing metastable peaks. (+)-Usnic acid (35), the parent member of the s e r i e s , fragments, 203 2 OA as expected, from two of the possible keto-enol tautomers ' (Figures 30, and 31). The retr o D i e l s - A l d e r process, an important fragmentation pathway, produces the [M-84] fragment _a (m/e 260), which further loses carbon monoxide and methyl r a d i c a l to _b (m/e 232) and _c (m/e 217) r e s p e c t i v e l y . The [ M - l l l ] ion d (m/e 233) a r i s i n g from the alternate tautomer cons t i t u t e s the base peak, and subsequently fragments i n two d i f f e r e n t modes. On the one - 78 -COCH3 HCv^J\ ^  C^H = C=0 S^r t«ywT ^ r - fef m* 207.0 m" 196.5 COCH, H OH H*° OH m"314.6 -CH; h oH,3<vr 3 2 9 ( 1 0 ) |-co 1 [ C ,6 H ,3°6] + 301(7) 213 (40) OH H 3 C O COCH3 [ c , ^ K o 7 ] m/a 344 ( 9 0 % ) m* 158.2 -H20 m 198.3 CHS + C H 3 OH fj [C12H lAT 2 0 3 ( 5 ) J + -CO OH CH, F i g u r e 31. The P o s t u l a t e d F r a g m e n t a t i o n o f ( + ) - U s n i c A c i d (35) i n t h e Mass S p e c t r o m e t e r . - 79 -hand, dehydration leads to the cyclobutanone ion _e>^UJ (m/e 2.15), while 204 isomerization to the 4-methychromenyl ion _f and subsequent loss of carbon monoxide affords ion j | (m/e 205). Minor pathways are the i n i t i a l l o s s of methyl r a d i c a l g i v i n g r i s e to the [M-15] peaks, ion _h (m/e 329), with subsequent loss of carbon monoxide to _ i (m/e 301). Further proof regarding the importance and c o n t r i b u t i o n of hydrogen transfer from the C^-enolic function to the major fragmentation pathway was secured by preparing (+)-usnic acid difluoroborate (110). As expected 110 fragments only v i a the retro D i e l s - A l d e r pathway to a f f o r d OH 0 (110) fragment a. (m/e 260) as the base peak (see Figure 31) with complete 204 suppression of the a l t e r n a t e process i n v o l v i n g hydrogen t r a n s f e r . Conversely, the acetylated d e r i v a t i v e s of 35 e x h i b i t dual fragmentation pathways i n the mass spectrometer. Since the deacylation r u -. • • i * , • * •-, 205,206 of phenolic acetates v i a loss of ketene i s a f a c i l e process, fragments a r i s i n g from both the acetylated and free hydroxyl moieties are observed. i C i r c u l a r dichroism (CD) i s an important and very s e n s i t i v e - 80 -a n a l y t i c a l t o o l , u seful not only for monochromophoric but for polychromo-^ • n n 207,208 „ . . c phone substances as w e l l . Furthermore, since the information obtained can be q u a n t i t a t i v e l y associated with the asymmetric surroundings 209-212 of a given chromophore, i t was of i n t e r e s t to study the CD curves and Cotton e f f e c t s associated with t h i s s e r i e s of compounds. The r e s u l t s for (+)-usnic acid (35), (-)-usnic acid (111) and (+)-isousnic acid (112) are summarized i n Table 7. ,MeOH ,. x Compound A (Ae) v max 35 , 320 (+11.41) 290 (-10.32) 260 (+ 5.43) 245 (+9.23) 111 325 (-11.77) 293 (+12.26) 260 (-10.65) 245 (+8.39) 112 355 (+ 5.60) 293 (14.30) 266 (-15.18) 244 (+9.58) Table 7. CD Properties of (+)-Usnic Acid (35), (-)-Usnic Acid (111) and (+)-Isousnic Acid (112). - 81 -Interesting to notice i s the c l e a r d i f f e r e n c e s between the CD spectra of 35 and 112, a fac t that can be used f or a f a c i l e q u a l i t a t i v e and quantitative c h a r a c t e r i z a t i o n of the normal- and i s o - s e r i e s (Figure 32). From the previous discussion i t i s evident that the spectroscopic properties shown by (+)-usnic a c i d are very c h a r a c t e r i s t i c thereby allowing the f a c i l e determination of s u b s t i t u t i o n patterns and/or possible m o d i f i -cations to the basic dihydrodibenzofuran skeleton on any of the new b i o -derived products to be studied. It w i l l be seen i n the following d i s c u s s i o n that spectroscopic methods indeed played a s i g n i f i c a n t r o l e i n determining the structures of the various biodegradation products. I t must be mentioned, however, that i n order to present i n a consistent manner the c h a r a c t e r i s t i c data f o r a l l the synthetic and bioderived products formally derived from (+)-usnic acid and r e t a i n i n g i t s basic s k e l e t a l features, the numbering system recommended for the parent molecule ( i . e . the approved dibenzofuran numbering ^ ^) w i l l be used throughout the course of t h i s work. In a l l other instances, the numbering a r i s i n g from the corresponding systematic name w i l l be used. Thus, the numbering recommended for the basic usnic a c i d skeleton i s as follows: 14 - 82 -Figure 32. The Circular Dichroism Spectra of (+)-Usnic Acid (35) (A), (-)-Usnic Acid (111) ( B ) and (+)-Isousnic Acid (112) ( C ) . - 83 -We can now proceed with the discussion of metabolite 107, (+)-6-desacetylusnic a c i d , i s o l a t e d during the biodegradation of 35 by a Pseudomonas species. This product was obtained as bright yellow needles, mp 134.5-2 7 o 136°; [ d p + 689°. The molecular formula of 107 as determined by elemental analysis arid high r e s o l u t i o n mass spectrometry was '-'i g^j_6^6" Me OH The uv spectrum of 107 O 336, 266 and 232 nm) showed a hypsochromic r max s h i f t of the aromatic B band as compared to usnic acid from 285 to 266 nm, 191 suggesting strongly that a change had occurred i n the aromatic chromophore. In the i r spectrum of 107 the 3400-2800 cm * region showed the presence of chelated hydroxyl groupings such as the ones i n the parent molecule (+)-usnic acid (35). Furthermore, absorptions at 1670 and 1540 cm * ind i c a t e d the r e t e n t i o n of the r i n g C chelated B-triketone system. Nevertheless, the reduction i n i n t e n s i t y of the 1630 cm * absorption coupled with a d i s t i n c t band at 895 cm * suggested the existence of a pentasubstituted aromatic 213 r i n g . These r e s u l t s combined with the molecular formula and the u l t r a -v i o l e t spectrum were strongly i n d i c a t i v e of a r i n g A deacylated m a t e r i a l . The. pmr spectrum of 107 confirmed the above conclusions when only three three-proton s i n g l e t s were observed, one at 61.70 corresponding to the Cg -methyl., the other at 62.11 assigned to the aromatic methyl group and f i n a l l y one at 62.61 corresponding to the C^-acetyl grouping. To f u r t h e r confirm the l a t t e r assignment the complementary strongly chelated e n o l i c proton was observed, as expected, at 618.64. To confirm the complete retention of the r i n g C unsaturated 2-acyl-cyclohexane-l,3-dione system, the o l e f i n i c proton was observed at 65.79 as a one-proton s i n g l e t . Whereas the Cg-phenol.ic hydrogen remained at 610.23, two new absorptions were - 84 -observed, an aromatic proton at 66.26 and the C^-phenolic proton at 65.67. The s h i f t to higher f i e l d shown by t h i s absorption c l e a r l y indicated the loss of c h e l a t i o n as a r e s u l t of deacylation i n the aromatic r i n g (Figure 33). In the mass spectrum, besides the molecular ion at m/e 302, the [M-15] and [M-84] fragments were observed at m/e 287 and m/e 218. Further-more, as i n the mass spectrum for (+)-usnic a c i d , the [ M - l l l ] fragment at m/e 191, a r i s i n g from the pathway r e q u i r i n g hydrogen transfer from the C^-enolic function c o n s t i t u t e s the base peak. Moreover, the l a t t e r two fragments, showing complete r e t e n t i o n of the s t r u c t u r a l features character-i s t i c of r i n g A appear c o n s i s t e n t l y 42 mass units lower than the correspond-ing fragments observed for (+)-usnic acid, therefore, supporting the postulated C,-deacylated structure 107 (Figures 34 and 35). o The CD spectrum of 107 showed the following absorptions and associated Cotton e f f e c t s : A M E 0 H (Ae) 328 (+9.99), 284 (-6.66), 262 (-3.53), max 241 nm (+13.11). Upon addi t i o n of base (5% methanolic potassium hydroxide) the following bands were observed: A M A X ( A E ) 345 (+8.11), 310 (+6.86), 285 (+1.66), 264 (-12.90), 236 (-10.19). Unfortunately no d e f i n i t e conclusions could be obtained from these spectra at t h i s time since 107 showing re t e n t i o n of the c h i r a l center and modification of the aromatic chromophore represents indeed the parent member of a new s e r i e s . That i s , compounds lacking the r i n g A a c e t y l group might be expected to reveal CD c h a r a c t e r i s t i c s which are not d i r e c t l y correlated to the normal usnic acid s e r i e s . Further spectroscopic information was obtained through preparation of the acetylated d e r i v a t i v e , (+)-6-desacetylusnic acid diacetate (113). Thus, 113 was prepared i n 44% y i e l d upon a c e t y l a t i o n of the parent molecule - 87 -m i o 3 . 3 190(38) 175(3) b c O H H 3 C O H C O C H , m 2 7 2 . 7 • C H ; a ] C H O 1 t 15 1 t 6 I 2 8 7 ( 5 ) - C O 259 (5) [ C , 6 H , 4 ° e ] m/e 3 0 2 (67%) m 124.1 H O . O H [ C „ H „ ° 3 ] 191 (100) Figure 35. The Postulated Fragmentation of (+)-6-Desacetylusnic Acid (107) in the Mass Spectrometer. - 88 -with a p y r i d i n e - a c e t i c anhydride mixture, and was obtained as yellow 26 c r y s t a l s with molecular formula ^C-H^gOg, m p 208-209 , [ c ] ^ + 1 2 1 ° . In the uv, absorptions at 325, 280, 261 and 220 nm were observed, whereas the i r spectrum had bands at 1750 cm c h a r a c t e r i s t i c f or phenolic 213 -1 acetates, 1685 and 1540 cm for the r i n g C dienone and chelated 8-t r i c a r b o n y l system, r e s p e c t i v e l y , and 1620 and 1605 cm ^ f o r the enol ether and aromatic r i n g absorptions. The pmr spectrum of 113 provided a d d i t i o n a l information showing two three-proton s i n g l e t s at 62.31 and 52.43 assigned to the C - and C Q-acetates, r e s p e c t i v e l y . Worthwhile noting i s the s h i f t to higher f i e l d observed f o r the C2~acetyl grouping (62.61 i n the parent molecule to 62.52 i n the d i a c e t a t e ) . This i s believed to be due to the e l i m i n a t i o n of the hydrogen bonding between the C^-hydroxy group and the C^-ketone. As w i l l be seen i n other spectra for acetylated compounds t h i s i s a general and c h a r a c t e r i s t i c feature of the s e r i e s . Furthermore, deshielding by the C -acetate grouping caused a downfield s h i f t of the aromatic C,-proton from 66.26 to 66.81. The o l e f i n i c proton was observed at 65.81 and the e n o l i c hydroxyl proton at 618.41. In the mass spectrum of 113 we see once more the f a c i l e loss of ketene from the phenolic acetate groupings producing the intense [M-42] and [M-84] peaks at m/e 344 and 302, r e s p e c t i v e l y . The base peak was the molecular ion at m/e 386. Other fragmentations are as previously discussed for the parent molecule (+)-6-desacetylusnic acid (107). Having obtained a l l the spectroscopic evidence i n favor of structure 107 for this biodegradation product, i t was decided to provide a firmer basis for the proposed structure by i t s transformation into a - 89 -known usnic acid derivative. One of the few known derivatives lacking ,125,135 the aromatic acetyl grouping is pyrousnic acid (50) a product Ov/CHjCOOH (50) COCH-H 2 C 0 0 H obtained by heating (+)-usnic acid (35) to 210° in 50% sodium or potassium hydroxide under a stream of. hydrogen for 10 minutes. Under somewhat 102 o milder conditions (75% potassium hydroxide at 88-90 for one hour) usnetic acid (51) a derivative retaining the aromatic acetyl is obtained. Therefore, treatment of 107, the ring A deacylated material, under the latter conditions should produce exclusively pyrousnic acid as seen in Figure 36. Thus, treatment of 107 with 75% aqueous potassium hydroxide at 90° produced, in 35% yield, tan needles of pyrousnic acid (50) having the 135 molecular formula C 1 2 H i 2 0 5 ' mp 197-193° ( l i t mp 199-200°), mixed mp with authentic pyrousnic acid obtained from (+)-usnic acid (35), 196-198° and 26 [a]^ 0°. This sample presented the following spectroscopic properties identical in a l l respects with those of the authentic material. In the uv spectrum, pyrousnic acid showed absorptions at 315 and 253 nm, whereas in the i r broad bands in the 3500-2400 cm ^ region indicated the presence of phenolic hydroxyl groupings showing intra- and intermolecular hydrogen - 90 -bonding as well as the vO-H c h a r a c t e r i s t i c of carboxylic acids. Absorp-tions at 1770, 1725 and 1440 cm * further confirmed the existence of the carboxylic acid moiety. OAc ( I K ) Figure 36. The S t r u c t u r a l E l u c i d a t i o n of (+)-6-Desacetylusnic Acid (107). The pmr spectrum of the carboxymethylbenzofuran 50 i n deutero-methano.l showed two three-proton s i n g l e t s at 62.10 and 62.30 assigned to 214 the aromatic methyl and to the furan r i n g methyl groups, r e s p e c t i v e l y . - 91 -Moreover, a two-proton s i n g l e t corresponding to the C2~methylene at 63.63 and a one-proton s i n g l e t for the C^-aromatic proton at 66.46, were observed. In the mass spectrum, fragments corresponding to the molecular ion (m/e 236), to the los s of hydrogen and carbon dioxide .(m/e 191) and to carbon dioxide (m/e 44, base peak) were observed. Further comparison was obtained through the a c i d catalyzed a c e t y l -a t i o n of 50 to produce pyrousnic acid diacetate (114). Elemental a n a l y s i s and high r e s o l u t i o n mass spectrometry indicated C, .H 0 as the molecular formula. l b l b / The i r spectrum of the diacetate 114 (Figure 37) c l e a r l y indicated the re-tention of the carboxylic acid function (3000-2400, 1705, 1420, 1220 cm - 1) and the expected phenolic acetates (1755 cm * ) , while the pmr spectrum showed two three-proton s i n g l e t s at 62.14 and 62.28 corresponding to the C^- and C^-acetates r e s p e c t i v e l y . In the mass spectrum of 114 the expected molecular ion (m/e 320) and the [M-42] (m/e 278) and [M-84] (m/e 236, base peak) f r a g -ments were observed. In order to complete the s t r u c t u r a l e l u c i d a t i o n of 107, (+)-usnic acid (35) was treated with 75% potassium hydroxide to produce i n 38% y i e l d 102 135 the expected product, usnetic acid ' (51), the C-acetyl analog of 50. The uv of 51 (A 358, 308, 255, 249 nm) showed the bathochromic s h i f t max 191 expected for the newly introduced chromophore, whereas i n the i r spectrum the c h a r a c t e r i s t i c absorptions for the carboxylic acid (1715 cm *) and the chelated aromatic a c e t y l grouping (1625 cm *) were observed. Moreover, the pmr spectrum of 51 showed a sharp three-proton s i n g l e t at 62.67 corresponding to the C^-acetyl grouping. The l o g i c a l step was now to attempt the laboratory preparation of 107, since i t could be used not only as a model for the synthesis of other I FREQUENCY (CM1) 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650 Figure 37. The Infrared Spectra of Pyrousnic Acid Diacetate (114) obtained from; A) (+)-6-Desacetylusnic Acid (107) and B) (+)-Usnic Acid (35). - 93 -metabolites but for Che study of the properties c h a r a c t e r i s t i c of t h i s new s e r i e s of compounds. 159 In 1953, Barton and co-workers studied the behavior of (+)-152 159 usnic acid anhydrophenylhydrazone ' (77b), a product obtained by reaction of phenyHydrazine hydrochloride with (+)-usnic acid (35) i n a l c o h o l i c s o l u t i o n , and found that upon treatment with 20% methanolic potassium hydroxide a product (mp 206-207°) having the molecular formula ^23^22^5^2 W a S ° k t a i n e d • A c e t y l a t i o n ( p y r i d i n e - a c e t i c anhydride) of t h i s product furnished a diacetate (mp 209-210°) with molecular formula C„,H„ rO_N.. II lb I I Moreover, based on Robertson-Curd's s t r u c t u r a l formula for usnic acld,"*"^ and the f a c t that the phenolic material showed the presence of a free aro-matic p o s i t i o n ortho to a hydroxyl group as seen by the ready coupling with d i a z o t i z e d a n t h r a n i l i c acid, these workers proposed structure 80 for the cleavage product, (+)-desacetylmethoxyusnic acid anhydrophenylhydrazone. At the same time, they found that under more vigorous conditions (aqueous or ethanolic potassium hydroxide) the r e a c t i o n product was instead the high melting, o p t i c a l l y i n a c t i v e a c i d 81, which they further characterized as the diacetate methyl ester. H OCH-( 8 0 ) O H ° (77b) KOH EtOH or water (81) - 94 -Furthermore, compound 80 was obtained more recently by 162 Takahashi and co-workers, who found that (-)-usnic acid isomethoxide monoacetate"'(82) reacts with excess phenylhydrazine hydrocholride in ethyl alcohol to produce (+)-monoacetylusnic acid isomethoxide bisphenyl-hydrazone monoanhydride (85), which was then hydrolyzed to (+)-monoacetyl-usnic acid phenylhydrazone monoanhydride (86) by the action of methanolic hydrochloric acid. Base cleavage of the monoanhydride 86 with 20% methanolic potassium hydroxide produced a compound possessing the formula £23^22^5^2' mp 205-206°, identical in a l l respects with Barton's material (vide supra). - 95 -Therefore, in order to complete the laboratory preparation of metabolite 107 i t remained necessary only to regenerate the masked 3-triketone system from 80 by reductive cleavage of the N-phenylpyrazole moiety followed by hydrolysis of the amino functions and then methanol elimination to form the C.-C. double bond. 4 4a (+)-Usnic acid anhydrophenylhydrazone (77b) was thus prepared in 88% yield by reaction of (+)-usnic acid (35) with phenylhydrazine hydrochloride 152 159 in an ethyl alcohol-water mixture. ' In the ultraviolet spectrum i t 215 showed the bathochromic shift expected for a conjugated 3-keto-l-phenyl-pyrazole-system (A 370, 285, 253, 220 nm), whereas the i r spectrum had max absorptions at 1667, 1620 and 1592 cm ^ characteristic of the dienone system, the chelated aromatic acetyl and the N-phenylpyrazole moiety, respectively. The pmr spectrum of compound 77b showed three-proton singlets at 62.60 for the C^-methyl group - the pyrazole ring methyl group - "and at 62.63 for the chelated aromatic methyl ketone, while the deshielding effect of the N-phenyl substituent was experienced by the olefinic proton (66.25). Furthermore, a five-proton singlet corresponding to the phenyl group was observed at 67.58. The phenolic hydroxyl protons were observed, as expected, at low f i e l d (611.08 for the C^-hydroxyl group and 613.28 for the C^-hydroxyl group). Indeed base cleavage of the anhydrophenylhydrazone 77b under 159 Barton's conditions produced, in 64% yield, the desired (+)-desacetyl-methoxyusnic acid anhydrophenylhydrazone (80), mp 200-202°. Its uv spectrum (A 326, 261 nm) showed an hypsochromic shift due both to the loss of the max acetyl chromophore and to the loss of conjugation between the aromatic ring and the N-phenylpyrazole moiety in view of methanol addition to the C^-C^ double bond. The i r spectrum showed as well the expected changes. The chelated aromatic a c e t y l band was not observed while the enone absorption s h i f t e d to 1647 cm \ an absorption within the range of 216217 _ 1 4-keto-N-phenylpyrazoles. ' The absorption ranges (1640-1610 cm ) observed for 4-keto- and for 4-carboxy-N-phenylpyrazoles and t h e i r corresponding esters (1710-1690 cm "*") suggest considerable conjugation of the carbonyl group with the pyrazole r i n g . The pmr spectrum of compound 80 was very i n d i c a t i v e and helped confirm previous observations. The pyrazole r i n g methyl group (C^-methyl group) was observed at 62.50, as seen for other a l k y l substituted 4-keto-218 pyrazoles, the newly introduced C. -methoxy group appeared as a three-proton s i n g l e t at 63.42, the C^-methylene (63.49) as an AB quartet with coupling constant J = 18 Hz, w e l l w i t h i n range for a methylene group attached both to an aromatic substituent and to a keto group (63.22-191 219 3.50). ' Although no conclusive stereochemical assignment can be made for the C, -methoxy group i t i s expected that the thermodynamically 4a more stable a-configuration w i l l be attained, thus producing a cjLs-B/C junction which should also be favored over the trans-fusion. (80 ) - 97 -Moreover, the observed chemical shift for the C^-phenolic hydroxyl proton (65.07) as compared to i t s usual low f i e l d position (610-11) coupled with the appearance of an aromatic proton at 66.08 confirms the loss of the chelated acetyl grouping. In the mass spectrum a fragment was observed at m/e 374 [M-CH^OHJ while subsequent loss of methyl radical produced the base peak, m/e 359. The molecular ion was seen at m/e 406. In order to obtain further data on compound 80 the corresponding 159 diacetate (115) was prepared in 84% yield by acetylation overnight with a 1:1 mixture of acetic anhydride-pyridine. Its i r spectrum clearly indicated the phenolic acetate absorption at 1761 cm and the non-chelated enone system at 1667 cm \ while i t s pmr spectrum indicated the presence of two such groupings characterized by two three-proton singlets at 62.27 and 62.40 for the C^- and Cg-acetates, respectively. Moreover, the „pyrazole ring methyl group was observed at 62.44, the C. -a-methoxy group at 63.37 and 4a the C^-methylene as an AB quartet with coupling constant J = 18 Hz at 63.49. The deshielding influence of the C^-acetate was clearly experienced by the aromatic proton now seen at 66.57, while the phenyl ring protons appeared as a five-proton singlet at 6 7.55. In the mass spectrometer the expected [M-42] and [M-84] fragments were observed at m/e 448 and m/e 406 in- addition to the molecular ion (m/e 490) and the [M-CH^ OH] fragments arising from d i - , mono- and desacetylated species at m/e 458, 416 and 374 respectively. Loss of methyl radical from the last fragment produced the base peak, m/e 359. As seen for the former compounds of this series no significant fragments 220 221 arising from the decomposition of the pyrazole ring ' (i.e. loss of HCN) were observed although (+)-usnic acid anhydrophenylhydrazone (77b) showed ! - 98 -the [M-H-N ] fragmentation at m/e 387. 159 One desirable modification to Barton's synthesis'*""'^ of deacylated anhydrophenylhydrazone derivatives was to avoid the base catalyzed Michael addition of methanol across the C.-C. double bond since i t would reduce 4 4a the synthetic sequence by at least one step. Nevertheless, when isopropyl alcohol was used as a solvent only complex reaction mixtures were obtained from which the carboxylic acid (116) was obtained in low yield (21%). H OMe OAc (115) (116) -1 Thus the acid 116 showed in the i r an absorption at 1680 cm corresponding to the carboxylic acid moiety. This absorption is well 217 within range for 3-alkyl and 3,4-dialkyl-l-phenylpyrazole-4-carboxylic 216 222 223 •— X acids ' ' which absorb around 1690 cm . The chelated aromatic acetyl was observed at 1614 cm *. Its pmr spectrum showed the pyrazole ring methyl group at 62.43, as expected, and the chelated aromatic methyl ketone at 62.48. Moreover, a characteristic two-proton singlet corresponding to the methylene bridge was observed at 64.49. This value can be compared with the chemical shift (63.93-3.97) calculated through the shielding 224 constants for a methylene group attached to two aromatic substituents. The mass spectrum of 116 shows fragments corresponding to the molecular ion (m/e 434) and to the loss of carbon dioxide [M-441 at m/e 390 besides - 99 -an [M-18] fragment (m/e 416) which subsequently loses methyl radical to give the base peak at m/e 401. Although this was a somewhat discouraging result, preliminary investigations carried out on (-)-usnic acid isomethoxide monoacetate (32) indicated that a clean demethoxylation to (+)-usnic acid monoacetate (117) could be carried out by the use of boron tribromide in methylene chloride, 159 thus allowing us to retain the original cleavage conditions. A crucial step was, nevertheless, the f e a s i b i l i t y of the reductive cleavage of the pyrazole ring. Although i t is known that 223 226227 pyrazoles (as their salts) as well as N-alkyl-indazoles ' can be cleaved under harsh conditions by the use of strong bases (i.e. NaNH^-NaOH), the pyrazole ring seems to be particularly resistant to reduction, especially 228 when i t is unsubstituted on the nitrogen atom. Nevertheless, when the pyrazole ring forms part of a condensed structure and has a 3-hydroxyl substituent the N-N bond may be hydrogenolyzed comparatively smoothly by 229 230 refluxing with Raney-nickel in alcoholic solution. ' Reduction of N-phenylpyrazoles is somewhat easier since sodium 222 in alcohol reduces them to N-phenylpyrazolines. However, this reaction - 100 -N a N H 2 |^|^ C 0 N H2 NaOH EtOH NHR Raney-Ni JC^Ci CONH-HR1 is sometimes complicated by concomitant hydrogenolysis of the N-phenyl 231 substituent. By the use of a palladium catalyst in acetic acid at 20* N-phenylpyrazoles are reduced to N-phenylpyrazolines and then at 80° to « ' v i ^^A^ 228,232 N-phenylpyrazolxdmes. ^ M EtOH Unfortunately, the anhydrophenylhydrazone 77b proved resistant to a l l the cleavage conditions attempted. Nevertheless, when reduced over platinum oxide in tetrahydrofuran solution the formation of (-)-dihydrousnic -101 -acid anhydrophenylhydrazone (118) was observed. ( 7 7 b ) • ( 1 1 8 ) In the i r spectrum of 118 the very strong absorption at 1623 cm was assigned both to the chelated aromatic a c e t y l and to the chelated p y r a z o l y l ketone. The pmr spectrum of 118 was very informative with the pyrazole r i n g methyl group appearing at 62.50 and the chelated aromatic a c e t y l at 62.53, while a two-proton doublet at 63.39 with coupling constant J = 5 Hz was assigned to the C^-methylene group. The C^-methine proton was seen as a t r i p l e t , J = 5 Hz, at 65.03, while one-proton s i n g l e t s at 69.60 and 613.37 corresponded to the C,- and C,-phenolic hydroxyl protons, r e s p e c t i v e l y . The sample showed the following CD c h a r a c t e r i s t i c s : A (Ae) 336 (+1.87), max 303 (-1.64), 275 (+7.50), 255 (+4.22) and 237 nm (+6.56). Since the N-phenylpyrazole system proved to be extremely r e s i s t a n t to allow f a c i l e regeneration of the 8-triketone system c h a r a c t e r i s t i c of metabolite 107 new approaches to i t s laboratory preparation had to be devised. Perhaps the most desirable route would be to protect the highly r e a c t i v e 8-triketone through the formation of some type of stable c y c l i c intermediate, which should display the following properties: i ) s t a b i l i t y to hot (concentrated a l k a l i ; i i ) ease of formation and i i i ) mild, s e l e c t i v e - 102 -removal allowing facile regeneration of the B-triketone. Nitrogen-oxygen bonds in general undergo catalytic cleavage with ease, as exemplified by the many fa c i l e reductions of nitro, nitroso, 2 3 2 hydroxylamine and oxime functions, and i t appears that most nitrogen-oxygen bonds, unless severely hindered, are susceptible to hydrogenolysis. On the other hand, almost a l l known examples of catalytic hydrogenation of isoxazoles are accompanied by hydrogenolysis of the ring, with reduction of d i - and tetrahydroisoxazole derivatives proceeding more readily than 2 3 3 the aromatic system of isoxazole i t s e l f . Platinum catalysts can also be used for the hydrogenolysis of styrylisoxazoles, which tend to show an 2 3 4 increased s t a b i l i t y towards reduction. Imines are the products resulting from the hydrogenolysis of isoxazoles -and can sometimes be isolated, but usually undergo further 2 3 3 transformation to the parent ketones in very good yields, as seen by 235 the following example. Moreover, i f the hydrogenolysis of isoxazole derivatives is carried out in acetic aci.d-aeot.ic anhydride mixtures, diacetyl derivatives may be , . , , . . 236 obtained direct 1 v. - 103 -Furthermore, the well known f a c i l i t y of isoxazole formation from 233 237 238 3- triketones ' ' coupled with the s t a b i l i t y of 3,5-disubstituted and 3,4,5 - t r i s u b s t i t u t e d isoxazoles toward a l k a l i and alkoxides, even with • 237 prolonged treatment under d r a s t i c conditions, made t h i s h e t e r o c y c l i c system i d e a l l y suited f or our synthetic requirements. The formation of such a d e r i v a t i v e , usnic acid oximeanhydride, 85 128 was f i r s t reported by Widman ' and l a t e r on studied by Schopf and co-X 0 2 1A 2 workers. ' However, since no s a t i s f a c t o r y s t r u c t u r a l formula could be proposed at that time, i t was decided to undertake the structure deter-mination of t h i s important d e r i v a t i v e beginning with a systematic study of the products obtained through condensation of (+)-usnic acid (35) with a serie s of a l i p h a t i c and aromatic amines. The main purpose of such a study was to determine the p o s i t i o n at which condensation with a v a r i e t y of amino compounds takes place. This was not, however, a straightforward problem since there are four carbonyls at which condensation could occur, three of them located i n the r i n g C 8-triketone system and the other being the chelated aromatic a c e t y l grouping. Further complications a r i s e i f we take i n t o consideration the p o s s i b i l i t y of n u c l e o p h i l i c r i n g opening with formation of amino d e r i v a t i v e s of usnic a c i d ethoxide (73a) or ethyl acetusnetate (71). The complexity of t h i s study was somewhat lessened by Barton's 159 study of (+)-usnic acid anhydrophenylhydrazone (77b), where he found that on ozonolysis of the corresponding diacetate 77a the N-phenylpyrazole-4,5-d i c a r b o x y l i c acid 78 was formed. The f i n a l c h a r a c t e r i z a t i o n of t h i s product was achieved by thermal decarboxylation to the known 3-methyl-l-phenylpyrazole-239 4- carboxylic acid (119) . - 104 -COCH. AcOv, 0 II OAc 0 ( 77a) H O O C - ^ ^ ( 78) HOOC (119) This result clearly indicates that the C - l l carbonyl group, the exocyclic ketone of the enolized 3-tricarbonyl system, is the position at which in i t ia l condensation of phenylhydrazine took place. Furthermore, a l l attempts to prepare C -^amino compounds from other derivatives such as the bisphenylhydrazone monoanhydride (120) proved unsuccessful yielding only the oxime anhydrophenylhydrazone (121), thus indicating a low reactivity of the'C^-carbonyl roup towards condensation with amino compounds. (120 ) N H 2 0 H (121 ) 105 -Moreover, the formation of derivatives such as 77b and 120 indicates that at least two of the four carbonyls present in usnic acid (35) are active towards condensation with the added advantage that, under certain conditions, the reaction can be carried out preferentially at the C^-acetyl grouping as seen by the high yielding preparation of (+)-usnic acid anhydrophenylhydrazone ( 7 7 b ) (see experimental section). 240 241 Recent studies ' have shown that reaction of a l i c y c l i c vinylogous esters such as 122 with secondary amines produces enamino-diketones of type 123 (i.e. ring amination), isomeric with the vinylogous amides such as .124 formed by direct amination of a l i c y c l i c 3-triketones 242 125. Similar results, namely the formation of exocyclic enamines from unprotected a l i c y c l i c S-triketones, have been observed for dehydroacetic acid (126), while concomitant ring amination was obtained for the corresponding vinylogous ester, methyl dehydroacetate (127). R2N 0 OMe 0 > — < C H 2 ) n ( 124) <CHJ 2 ) n 0 > — < C H 2 ) n 0 < ^ H C H 2 , n ( 125 ) ( 122 ) n=1.2 (123) Nevertheless, the ester 127 reacts with excess secondary amine to 243 244 produce, v_ia the labile acid 128, the stable bisenaminoketone ' 129 and a deoxygenated material, the tolylenediamine 130. Such a reaction is - 106 -assumed to proceed through opening of the ct-pyrone ring by nucleophilic 245 246 cleavage of the C^-0 bond. The latter cyclization is of biosynthetic importance because of its similarity to the polyketide processes leading to naturally occurring phenolic products of the orcinol group. 247 Similarly, the novel lactonic enaminoketone gentiocrucine (131) 248 isolated from Gentiana cruciata by Popov and Marekov, shows an exocyclic enamine function and comparable spectroscopic and chemical characteristics with the alicyclic compounds previously discussed. (131 ) - 107 -The simplest amino d e r i v a t i v e of usnic a c i d , the so - c a l l e d "usnic 128 acid amide" was f i r s t prepared by Widman by r e f l u x i n g (±)-usnic a c i d a n i l i d e (the product obtained by the condensation of (±)-usnic acid with 102 1A 2 a n i l i n e ) with a l c o h o l i c ammonia, and l a t e r on by Schopf and coworkers ' by r e f l u x i n g (±)-usnic acid with concentrated ammonium hydroxide i n eth y l alcohol-benzene. Nevertheless, no conclusive s t r u c t u r a l formula was proposed by those workers, who assumed the product was ei t h e r a s a l t of the " a c i d " 35 or an open chain d e r i v a t i v e such as usnic a c i d ethoxide (73a). 2 6 Thus a sample of th i s material ( f a J D - 230°) was prepared 142 u t i l i z i n g Schopf's conditions and was obtained as large pale yellow ! i n o plates from chloroform mp 252-254° ( l i t . mp 248°). Its uv spectrum - 108 -( A C H 3 C N 333, 291 nm) proved similar to that of (+)-usnic acid (35) max therefore suggesting that no ring cleavage had occurred. The infrared spectrum was again most informative since i t showed, in addition to the 3475 cm band (N-II) , absorptions in the 3400-2500 cm * region assigned to the stretching vibrations of chelated N-H and 0-H groupings. The displaced C-l ketone band, observed at 1697 cm \ is indicative of the loss of conjugation between the C-l carbonyl and the exocyclic vinylogous amide in complete agreement with the changes expected in going from the original triketone to the new unsaturated g-diketoenamine system. Moreover, the carbonyl absorption for the chelated unsaturated vinylogous amide >1« 247,249 (1570 cm *) shows the effect of both unsaturation and strong intramolecular hydrogen bonding on the normal absorption range of vinylogous amides (1642-1615 cm * ) . The chelated aromatic acetyl grouping was observed as expected at 1630 cm *. Its pmr spectrum had characteristic three-proton singlets at 62.59 assigned to the C^-methyl group (enamine methyl group) and 62.65 for the aromatic acetyl grouping, while the ol e f i n i c proton appeared at 65.41. The low f i e l d region was in agreement with the B-diketoenamine system showing a broad one-proton signal at 69.56 and a sharp one-proton singlet at 612.05 assigned to the two enamine protons, while one-proton singlets at 611.72 and 612.05 were characteristic for the C_- and C - phenolic hydroxyl protons. 9 / 204 The mass spectrum of this product was typical for the series, the base peak arising from a retro Diels-Alder cleavage of the B-diketoenamine system, giving fragment a_ (m/e 260) (Figures 38 and 39) whose subsequent fragmen-tations have been previously discussed for (+)-usnic acid (35) (vide supra). An important feature of this spectrum is the low relative intensity for - 601 -- 110 -HO C H 260C10O) C H 3 -CH 3-[ C , 8 H , 7 ° 6 N ] rn/e 3<»7 (61 %) Tc H o T -[_ 13 12 4 J f 232 (60) -CH, [ C ^ H M ° 6 N ] H 328 CIO) [~C H oV 217(18) Figure 39. The Postulated Fragmentation Pattern f o r (-)-A ' -Enaminousnic I Acid ( 1 3 2 ) . - I l l -fragments arising from the alternate 8-iminoketoenolic tautomer which produces ion b_ (m/e 233). Further loss of water from the latter gives £ (m/e 215) and loss of methyl radical from the molecular ion (m/e 343) produces ion d_ (m/e 328). Based on the previously discussed chemical background on amination of a l i c y c l i c 6-triketones and supported by a l l the relevant 2 11 spectroscopic characteristics we postulated structure 132 for (-)-A ' -enaminousnic acid the condensation product of (+)-usnic acid with aqueous ammonia. Since compound 132 was to prove of pivotal importance for the future synthetic sequences based on isoxazole formation (vide infra) and i t shows a novel modification on the characteristic ring C of usnic acid i t was decided to further substantiate the postulated structure by means of an X-ray study. The X-ray diffraction pattern study was performed by Professor J. Clardy of the Chemistry Department, Iowa State University. Preliminary X-ray photographs revealed 2/m Laue symmetry and the systematic extinction hkl (missing for h + k =. 2n + 1) with diffractometer measured c e l l constants a = 20.29 (6), b = 9.74 (2), c = 12.50 (3) A and B_ = 137.0 (3)° . A calculated density of 1.35 g/cc was interpreted to mean t = 4 2 11 or one molecule of A ' -enaminousnic acid (132) per asymmetric unit - 112 -C(I9) Figure 40. Computer Generated Drawing of the X-Ray Model of (-)-A i : Enaminousnic Acid (132) . - 113 -i n the c h i r a l space group Figure 40 shows a computer generated drawing of the f i n a l X-ray model. The p o s i t i o n of the nitrogen atom, the most s i g n i f i c a n t chemical aspect of structure 132, was o r i g i n a l l y determined by c a r e f u l refinement and analysis of the thermal parameters and completely corroborated by both bond distances and H atom l o c a t i o n . Furthermore, the bond distances suggest that the best s i n g l e 2 11 representation of A ' -enaminousnic acid i s that shown i n 132 with important contributions from 133. The absolute configuration shown i n Figure 40 i s an assumed one. (133 ) The second member of the s e r i e s was the condensation product of (+)-usnic a c i d (35) with methylamine (as a 40% aqueous s o l u t i o n ) . This 26 material ([<*]D -344°) was obtained i n 73% y i e l d by r e f l u x i n g both components in absolute e t h y l a l c ohol under nitrogen atmosphere for 1.5 hours. Thus, 2 11 (-)-N-methyl-A ' -enaminousnic acid (134) showed an u l t r a v i o l e t spectrum (A 345, 293 and 223 nm) very s i m i l a r to that of 132. I t s i n f r a r e d spectrum max showed the absoption corresponding to the C - l ketone at 1700 cm *, the chelated aromatic a c e t y l grouping at 1630 cm * and the unsaturated chelated vinylogous amide (3-ketoenamine system) at 1560 cm *. The pmr spectrum of .134 (Figure 41) showed c h a r a c t e r i s t i c three-proton s i n g l e t s at 62.64 and i 62.66 assigned to the C -methyl (ethylidene methyl) group and the chelated Figure 41. Proton Magnetic Resonance Spectrum of (-)-N-Methyl-A ' -Enaminousnic Acid (134). - 115 -aromatic a c e t y l grouping, r e s p e c t i v e l y , as well as a tin.ue-proton doublet with coupling constant J = 5 Hz at 63.16 corresponding to the N-methyl substituent and a one-proton s i n g l e t at 65.77 for the o l e f i n i c proton. The low f i e l d region shows two one-proton s i n g l e t s at 611.90 and 613.30 assigned to the Cg- and C^-phenolic hydroxyl protons and a broad one-proton s i g n a l at 613.20 corresponding to the proton on the nitrogen atom. This N-methyl d e r i v a t i v e showed no [M-0H] + ions i n i t s mass spectrum, although such ions are known to increase r e p i d l y i n r e l a t i v e abundance as the , , , . 250 basic enammo nitrogen i s changed from primary to secondary to t e r t i a r y . The fragmentation [M-0] + produced i n some cases both by aromatic B-di-251 250 ketones and t h e i r corresponding enamines has not been found i n t h i s s e r i e s of compounds. The base peak was furnished by the molecular ion (m/e 357) with s i g n i f i c a n t fragments f o r the [M-15] and re t r o D i e l s - A l d e r processes at m/e 342 and 260 r e s p e c t i v e l y . Furthermore, preparative layer chromatography p u r i f i c a t i o n of the r e s i d u a l mother l i q u o r s produced a second component, (+)-N'-methyl-2 11 imino-N-methyl-A ' -enaminousnic acid (135), the equivalent to (+)-usnic 2 6 acid bisphenylhydrazone monoanhydride (120).. This material ( [ a ] D + 94°) was obtained in only 2% y i e l d and showed a large hypsochromic s h i f t i n i t s (134 ) ( 135) - 116 -CH CN u l t r a v i o l e t spectrum (A 3 300, 250 and 234 nm). In the i n f r a r e d spectrum max the absorptions i n the 3600-3100 cm * region were assigned to chelated am ino and hydroxyl functions, whereas the C - l carbonyl absorption appeared at 1700 cm * and the unsaturated chelated vinylogous amide (8-ketoenamine system) was seen at 1560 cm \ while the chelated imine function absorbed at 1630 cm The pmr spectrum of 135 showed three-proton s i n g l e t s at 62.56 for the C^-methyl group and at 62.66 f o r the C^-methyl group. The three-proton doublet, J = 5 Hz, observed at 63.18 was assigned to the a l i c y c l i c N-methyl-enamine system, while a three-proton s i n g l e t at 63.30 corresponded to the N-methyl-imino group. The low f i e l d region proved very informative as w e l l since the two one-proton s i n g l e t s at 611.40 and 613.32 were charac-t e r i s t i c of the Cg- and C^-phenolic hydroxyl protons, r e s p e c t i v e l y , while a broad one-proton s i g n a l : at 616.92 was assigned to the chelated N-methyl-enamine proton. I t s mass spectrum showed prominent peaks for the molecular ion (m/e 370), the [M-15] fragment (m/e 355) and the r e t r o D i e l s - A l d e r process (m/e 273, base peak). The condensation of (+)-usnic a c i d with a 70% aqueous s o l u t i o n of ethylamine furnished, under s i m i l a r conditions, only one product, (+)-2 11 26 N-ethyl-A ' -enaminousnic acid (136). This material ( [ « ] Q + 365°) was obtained as pale yellow plates from chloroform-methanol, mp 133-134°. Its CH CN u l t r a v i o l e t spectrum (A 3 348, 294 nm) was c h a r a c t e r i s t i c f or the max monocondensation products 132 and 134 (vide supra). In the i n f r a r e d spectrum prominent absorptions were observed for the C - l carbonyl (1705 cm * ) , the chelated aromatic a c e t y l grouping (1630 cm *) and the unsaturated chelated 8-ketoenamine (vinylogous amide) system (1570 cm * ) . The pmr spectrum of .134 showed a three-proton t r i p l e t corresponding to the A part of an A.^H^X - .117 -252 253 system ' at 61.38 assigned to the terminal methyl group of the -NH-CH^ -CH^  grouping w i t h coupling constants = 7.5 Hz and = 0 Hz, and three-proton singlets at 62.59 (C^-CH^) a n c* 62.61 (aromatic acetyl grouping). Furthermore, the A MX octet, corresponding to the M part of such a system, observed at 63.52 with coupling constants J . w = 7.5 Hz and J , = 5 Hz was assigned to the methylene group of the N-AM MX ethyl-enamino system, while the N-H proton, the X part of the A^ M^ X system, was observed as a broad one-proton signal at 613.26. One-proton singlets at 65.72, 11.91 and 13.30 correspond to the olefinic proton and the Cg- and C_,-phenolic hydroxyl protons. The mass spectrum showed, as expected, fragments corresponding to the molecular ion (m/e 371), the [M-15] fragment at m/e 356 and the retro Diels-Alder process (m/e 260, base peak), The f i n a l member of this series of condensation products, (+)-? 11 N'-benzylimino-N-benzyl-A"' -enaminousnic acid (137), was prepared by reaction of (+)-usnic a c i d (35) with 78% benzylamine in absolute ethyl 2 6 alcohol. This m a t e r i a l ( f c l ^ + 262°) was obtained as bright yellow needles, mp 197-198°. I t s ultraviolet spectrum showed an intense absorption at 301 nm, whereas the infrared spectrum had absorptions at 1695, 1620 and 1560 cm _ 1 corresponding to the C-l carbonyl, the chelated C-13 N-benzylimino functionality and the unsaturated chelated B-ketoenamine. system, respectively. (136) (137 ) - 118 -The pmr spectrum of 137 showed at 64.65 a two-proton doublet, J = 6 Hz, assigned to'the N-benzylenamino system and at 64.76 a two-proton s i n g l e t f o r the N-benzylimino moiety with a ten-proton s i n g l e t f o r the two phenyl rings at 67.37. The low f i e l d region of the spectrum proved very s i m i l a r to the other condensation product 135, showing one-proton signals at 611.35 and 11.92 corresponding to the Cg- and C^-phenolic hydroxyl protons, whereas the enamino proton was observed as a broad s i g n a l at 616.85. In the mass spectrum prominent peaks were observed for the molecular ion (m/e 522), the re t r o D i e l s - A l d e r process (m/e 349, base peak), and t r o -2 5 A pylium ion (m/e 91) a r i s i n g from the N-benzyl cleavage. Table 8 provides a summary of selected pmr data on the a l i p h a t i c amine-usnic acid condensation products. I t reveals that throughout the s e r i e s the chemical s h i f t f o r the C^-methyl group appears c o n s i s t e n t l y at higher f i e l d compared to the C^-methyl group f o r the various condensation products. The products i n v o l v i n g condensation with two moles of amine show as well a noticeable l o w - f i e l d s h i f t f o r the enamino proton as compared to the monocondensed compounds. Thus we f e e l that these r e s u l t s w i l l prove very h e l p f u l f or the i n i t i a l p o s t u l a t i o n . of the structures obtained through condensation of (+)-usnic a c i d with a v a r i e t y of amines. Furthermore, these c h a r a c t e r i s t i c spectroscopic data coupled with the important re t r o D i e l s -Alder fragmentation process observed i n the mass spectrometer w i l l allow the f a c i l e t o t a l c h a r a c t e r i z a t i o n of such d e r i v a t i v e s . The next step of t h i s i n i t i a l study was to evaluate the behavior of (+)-usnic acid (35) when treated with aromatic amines. Although the work performed i n t h i s area has been somewhat more extensive, i n i t i a l reports on the condensation of 35 with aromatic amines by Virtanen and Amine Structure C 9 b - C H 3 c i r C H 3 C 1 3-CH 3 N-CH2~R N'-CH-R N-H C9-OH C?-OH C.-H 4 Ammonia 132 1.66 2.59 2.65 - - 9.56 11.72 13.35 5.75 Methylamine 134 1.70 2.64 2.66 3.16 (R=H) - 13.20 11.90 13.30 5.77 Ethylamine 136 1.65 2.59 2.61 3.52 (R=CH3) - 13.26 11.91 13.30 5.72 Methylamine 135 (di) 1.70 2.56 2.66 3.18 (R=H) 3.30 (R=H) 16.92 11.40 13.32 5.80 Benzylamine 137 (di) 1.68 2.55 2.65 4.65 (R=C 6H 5) 4.76 (R=C 6H5) 16.85 11.35 11.95 5.72 Table 8. Summary of Selected Proton Magnetic Resonance Data for the A l i p h a t i c Amine-Usnic Acid Condensation Products ( i n 6 u n i t s ) . - 120 -coworkers^' ' were directed exclusively towards the analysis of the antimicrobial and antimycotic activity of the crude materials, thus furnishing no conclusive structural evidence or spectroscopic characteristics. However, 257 Seshadr.i and coworkers prepared condensation products of (+)-usnic acid with simple aromatic amino compounds such as _p_-aminoacetophenone, p_-amino-benzoic acid, ethyl p_-aminobenzoate and o-phenylenediamine in order to test their antitubercular activity. In fact they confirmed Barton's proposed 159 structure for (+)-usnic acid anhydrophenylhydrazone (77b) by analysis of i t s proton magnetic resonance spectrum and using this as the starting point these workers postulated structures for other condensation products. Although their analysis of the data was correct in assigning the C-ring as the active site of condensation they failed to recognize the 8-diketoenamine system present in the series, thus showing imino structures for a l l the condensation products, i.e. structure 138. One major mistake which these authors made mainly due to the lack of mass spectrometry data was the assumption that the condensation of a second mole of the amino compound would also take place on ring C, as seen for structure 139. C 0 C H 3 OH 0 I ( 138 ) Nevertheless, with the data previously obtained for the series of aliphatic amines on hand, a closer scrutiny of the reported pmr characteristics - 121 -for the aromatic amine serie s allowed us to deduce the correct structures 140 - 142 for the monocondensation products and 143 for the dicondensation material. These data are reproduced on Table 9. S tructure C 9 b " C H 3 C n - C H 3 C 1 3 " C H 3 N-H Cg-OH C y-0H C.-H 4 140 1.75 2.62 2.62 15.40 11.63 13.38 5.92 141 1.74 2.55 2.58 - 11.78 13.38 5.96 142 1.78 2.60 2.65 15.20 11.76 13.40 5.70 143 1.75 2.42 " 2.66 _ 10.83 13.23 5.83 (di) Table 9. Selected Proton Magnetic Resonance Data f o r the Aromatic Amine-257 Usnic Acid Condensation Products ( i n 6 u n i t s ) . COCH (140) R = A c (U1 ) R=C00H (142) R = C00Et COCH. (143 ) 122 -Of the compounds reported by Seshadri and coworkers'"" two of them seemed particularly interesting and i t was then decided to study them in more detail. Thus (+)-usnic acid (35) was condensed with o-phenylene-diamine under the general reaction conditions described for the aliphatic 2 11 series to produce (+)-N-l'-(2'-aminophenyl)-A ' -enaminousnic acid (144) 2 7 in 94% yield. This material ([a] (CH^CN) + 266°) was obtained as pale o 257 o yellow crystals, mp 185-186° ( l i t . mp 185-186°) and showed the ul t r a -CI! CN violet spectrum (A "3 349, 293, 228 nm) characteristic of a l l the mono-max condensation products (vide supra). In the infrared spectrum i t presented absorptions at 3500 and 3415 cm typical for aromatic amines as well as bands in the 3300-2400 cm 1 region characteristic for the stretching vibrations of chelated amino and hydroxyl groupings. Moreover,.the C-l carbonyl was observed at 1695 cm a very similar value to those recorded for the aliphatic series, the chelated aromatic acetyl was seen at 1625 cm and the unsaturated chelated vinylogous amide (ketoenamine system) at 1540 cm"1. The pmr spectrum of compound 144 showed three-proton singlets at 62.56 and 2.65 corresponding to the C ^-methyl and the C^-methyl group (aromatic methyl ketone), respectively, with a two-proton broad absorption - 123 -at 64.09 assigned to the amino portion of the o-phenylenediamine molecule. The l o w f i e l d region of the spectrum showed two one-proton s i n g l e t s at 611.62 and 11.30 t y p i c a l of the G g- and C^-phenolie hydroxyl protons and a one-proton broad absorption at 614.39 corresponding to the enamine proton 258 (chelated). As previously observed f o r other 8-triketones and t h e i r d e r i v a t i v e s these products appear i n s o l u t i o n as only one tautomeric form. In i t s mass spectrum fragments corresponding to the molecular ion (m/e 434), the [M-15] cleavage (m/e 419) and the retr o D i e l s - A l d e r process (m/e 260) were observed. 257 Although the Indian workers report that under these conditions (hot e t h y l alcohol) a biscondensation product, mp 175-176°, i s formed p r e f e r e n t i a l l y over the monocondensation product 144, which they were able to obtain by carrying out the r e a c t i o n i n dimethylaniline at room temperature, we were unable to detect i t i n our crude r e a c t i o n mixture and a l l the attempts to produce such m a t e r i a l by e i t h e r changing the rea c t i o n time, water content, molar r a t i o of the reactants, etc., proved unsuccessful. The same workers proposed structure 145 f o r such a compound based on the s i m i l a r i t y of i t s pmr spectrum to that of (+)-usnic a c i d anhydrophenylhydrazone (77b) and the f a c t that they d i d not observe any amino or imino protons. ( 145) - ! .> ; -Nevertheless, the i n a b i l i t y of the Indian workers to observe the relevant C^-methine proton in the pmr spectrum of th i s product (see 259 formula 145) coupled to the novel 1,5-benzodiazepine structure prompted us to pursue further the synthesis of such d e r i v a t i v e s of usnic a c i d . Thus, 2,3-dihydro-l,4-diazepine (146) and 1,5-benzodiazepine (147). represent parent compounds of f a i r l y complete s e r i e s , with a v a r i e t y of 260 s u b s t i t u t i o n products as well as reduced and oxo forms, which among the seven-membered systems most nearly resemble the t y p i c a l large f a m i l i e s of f i v e - and six-membered heterocycles. The two s e r i e s are obtained from ethylenediamine and o-phenylenediamine, r e s p e c t i v e l y , by condensation with a d i f u n c t i o n a l three-carbon u n i t . H (146) (147) Compounds of type 146 and 147 are strong monoacidic bases that are usually i s o l a t e d as the hydrochlorides, s u l f a t e s or perchlorates. The s h i f t of the u l t r a v i o l e t absorption maxima i n going from the c o l o r l e s s 261 base (A 265 nm) to the deep v i o l e t s a l t s (148) (A 500 nm) has max max 262 been u t i l i z e d as a c o l o r i m e t r i c assay method for acetylacetone. B a s i f i c a t i o n of the 1,5-benzodiazepinium s a l t s gives i n i t i a l l y a yellow s o l u t i o n , presumably due to the 5H-isomer 149 which then reverts to 2 63 147, whereas i n concentrated s u l f u r i c acid, both 146 and 147 give r i s e to c o l o r l e s s d i c a t i o n s of type 150. Furthermore, on heating solutions of 1,5-benzodiazepinium chlorides, ring contraction and cleavage occur to give - 1 2 5 -( U 9 ) (150) benzimidazoles, / r" t' i""' with the latter having been obtained also a s by-products in the formation of the diazepines. In the presence of biacetyl, 2 6 2,4-dimethyl-l,5-benzodiazepine (151) is converted to the quinoxaline 152, whereas reaction with phenylhydrazine affords pyrazoles corresponding to the 265 diketone moiety. The former reaction, namely displacement by biacetyl, (152 ) - 126 -can be of synthetic u t i l i t y in the series as a way to achieve the non-alkaline cleavage of 1,5-benzodiazepines with regeneration of the $-diketonic material. In addition to 8-ketoesters the reactions of B-diketones and 260 266 B-diesters with 1,2-diamines have been extensively studied. ' The condensations are generally carried out in ethyl alcohol-acetic acid solution and an optimum pH of 5.8 - 7.8 has been observed in most instances. At pH values somewhat below and above 8.0 bisenamines, obtained from a 1:2 condensation of amine and diketone, are the major products both for 267 268 aliphatic (ethylenediamine) and aromatic (phenylenediamine) amines. ' Nevertheless, the intermediate open-chain 1:1 adducts can be isolated in the absence of acid, with cyclization being brought about upon addition aromatic 1,2-diamines. 2 ^ However, when the condensation was attempted under the recommended conditions (ethyl alcohol-acetic acid) only N-acetylated products were obtained. The reaction was f i n a l l y carried out in ethyl alcohol containing concentrated hydrochloric acid to produce in 82% yield the desired material 26 (+)-lH-[l,5]-benzodiazepino[3,4-b]usnic acid (153). This material (tctl^ (CH3CN) + 920°) with molecular formula C 24 H20°5 N2' o b t a i n e d b y h i 8 h resolution mass spectrometry and elemental analysis, was obtained as bright red needles from ethyl acetate-ethyl alcohol and decomposed without melting at 180* ( l i t . 2 5 7 mp 175-176°). Its ultraviolet spectrum ( A ^ S ^ 370, 305, • ^ max 288 and 226 nm) was similar to the reported uv spectra for other 1,5-benzo-diazepines ' ( A 310, 260 nm). The infrared spectrum of 153 showed the r max presence of a secondary amine group27*"* by a weak absorption at 3340 cm "*" - 127 -together with the absorption of chelated hydroxyl (phenolic) groupings (3050-2800 cm 1 ) . The C-l carbonyl group was observed at 1700 cm 1 and the chelated aromatic acetyl grouping at 1630 cm ^. The pmr spectrum of 153 showed three-proton singlets at 62.36 and 2.66 corresponding to the C^-methyl and aromatic acetyl groups, respectively, with a one-proton singlet at 66.16 assigned to the ole f i n i c proton. Moreover, a broad one-proton signal at 69.72 indicated the presence of the enamine proton on the 1,5-benzodiazepine system, while sharp one-proton singlets at 612.25 and 13.35 were c h a r a c t e r i s t i c of the C^- and C^-phenolic hydroxyl protons, respectively. In the mass spectrum fragments corresponding to the molecular ion (m/e 416, base peak) and the [M-15] fragment (m/e 401) were observed together with peaks at m/e 387, 373 ([M-43]), 284, 241, 133, 83, 77 and 43. Therefore, based on these spectroscopic data we feel that the correct structure for this interesting product should be as follows. (153) A last point of interest was to see whether analogous condensations could be carried out on the dihydro series. Thus, (-)-dihydrousnic acid 102 (154) was Lirst per pared by Schopf and Heuck and by Asahina and co-workers144'"'^''" who found that although (+)-usnic acid (35) resists direct catalytic hydrogenation, i t s dihydro derivative could be prepared through - 128 -(+)-usnic acid diacetate (68b) which i s e a s i l y hydrogenated to (+)-dihydro-usnic acid diacetate (155) and subsequently hydrolyzed with cold concentrated s u l f u r i c a c i d to give 154. (35) (154) 272 273 Further studies by Yanagita and l a t e r on by Shoji helped determine i t s structure based on the formula for usnic acid advanced by 1A6 1A2 273 Robertson and Schopf. Moreover, i t was Shoji who recognized the necessity for a cis-B/C r i n g f u sion for 154 through the analysis of i t s pmr spectrum, despite the fact that the absolute configuration of (+)-usnic 274 acid (35) was not known at the time. Later on Shibata and coworkers concerned themselves with the c a t a l y t i c hydrogenation of (+)-usnic acid - 129 -and found that when using tetrahydrofuran as the solvent and palladium-black as c a t a l y s t 154 could indeed be d i r e c t l y obtained i n f a i r y i e l d . 274 A minor modification to Shibata s procedure allowed us to prepare (-)-dihydrousnic acid (154) i n e s s e n t i a l l y q u a n t i t a t i v e y i e l d 27 o d i r e c t l y from usnic a c i d . This material ( [a ]^ (CH^CN) -83°) was obtained 274 as pale yellow c r y s t a l s , mp 151-152° ( l i t . 149°). I t s u l t r a v i o l e t spectrum (A 337, 283 and 227 nm) proved very s i m i l a r to that of 35, the max J parent molecule, whereas i t s i n f r a r e d spectrum showed bands at 1680 cm 1 (enone system) and 1635 cm 1 (chelated aromatic a c e t y l grouping). The pmr spectrum of 154 showed three-proton s i n g l e t s at 62.60 and 2.64 c h a r a c t e r i s t i c of r i n g C chelated methyl ketone of the ( 3-tricarbonyl system and the chelated aromatic a c e t y l grouping, r e s p e c t i v e l y . The two-proton doublet at 63.10 (J = 5 Hz) was assigned to the C^-methylene, while the one-proton t r i p l e t at 64.86 with coupling constant J = 5 Hz corresponds to the C. -methine 4a proton of ct-configuration. Indeed, t h i s spin-spin coupling constant does suggest a geometrical r e l a t i o n s h i p i n which the C, -methine proton b i s e c t s 4a the C^-methylene protons. Such a r e l a t i o n s h i p can only be attained through a cis-B/C r i n g junction independently of the absolute configuration about the C_, p o s i t i o n . 9b Therefore, since X-ray determinations on (+)-usnic acid-derived materials (see Part B) have shown an a-configuration f o r the angular methyl group (C Q,-methyl group), the same should be expected for the C. -methine 9b 4a proton i n order to f u l f i l the geometry requirements imposed by the observed coupling constant. Moreover, the existence of a trans-B/C r i n g j u n c t i o n would necessitate a p r i o r i ? i n a conformationally " f i x e d " system,the appearance of two d i f f e r e n t coupling constants i n accord with the observed - 130 -V 7 5 , ? 7 6 dihedral angles, as seen i n Figure 42. Figure 42. Dihedral Angles Observed f o r the Dihydrousnic Acid S e r i e s . The low-field region of the pmr spectrum indicated the presence of three chelated hydroxyl protons. The corresponding absorptions were observed at o9.50 (C g-0H), 13.45 ( C -OH) and 18.25 (C 3~0H). The mass spectrum of 154 showed the molecular ion, m/e 346, as the base peak, with other important fragments at m/e 318, 233, 220, 215 and 177. As expected, ( - )-dihydrousnic acid (154) reacted with o-phenylene-diamine in alcoholic solution to produce i n 95% y i e l d the desired mono-2 11 condensation product, (-)-N-l'-(2'-aminophenyl)-A ' "-enaminodihydrousnic - 131 -acid (156). This material ( [ « ] D (CH CN) -219°) was obtained as pale yellow prisms, mp 179-180°. Its u l t r a v i o l e t spectrum (A 342, 293 and 230 nm) was once more identical to those obtained for other monocondensation products (vide supra). In the i n f r a r e d spectrum c h a r a c t e r i s t i c absorptions for aromatic amines (3500, 3400 cm "*") and chelated amino group of a $-977 -1 amino-a,3-unsaturated ketone (3000-2890 cm ) were observed together with the carbonyl absorptions of the enone carbonyl (1660 cm """), the chelated aromatic a c e t y l grouping (1620 cm "*") and the unsaturated vinylogous amide (1545 cm "*") . The pmr spectrum of 156 was again very s i m i l a r to that of other monocondensation products, showing one three-proton s i n g l e t at 62.48, corresponding to the C ^-methyl group, and another at 62.60 assigned to the chelated aromatic methyl ketone. As expected, the two-proton doublet at 63.01, J = 5 Hz, corresponded to the C^-methylene protons, while the one-proton t r i p l e t , J - 5 Hz, at 64.86 was c h a r a c t e r i s t i c f o r the C^-methine proton. A broad two-proton s i g n a l at 63.80 was assigned to the free aromatic amine protons on the o-phenylenediamine moiety, while the Cg- and C^-phenolic hydroxyl protons absorbed at 610.30 and 13.40 r e s p e c t i v e l y . The chelated amino proton of the vinylogous amide (6-ketoenamine) was seen as a broad s i g n a l at 614.20. Its mass spectrum (m/e 436, 421, 276, 233, 203, 133, 131 and 93) was typical of the series. ( 1 5 6 ) - .132 -Thus, having determined the p o s i t i o n at which condensation with a s e r i e s of a l i p h a t i c and aromatic amines takes place, namely the carbonyl group of the C^-acetyl grouping, (Figure 43), we can now proceed to study the formation of the isoxazole r i n g that w i l l serve as a s u i t a b l e protecting group for the r e a c t i v e r i n g C 8-triketone system. The synthesis of 1,2-oxazoles (isoxazoles) from a v a r i e t y of s t a r t i n g materials i s well documented i n the l i t e r a t u r e and several reviews 233 237 238 on the subject are now a v a i l a b l e . ' ' Recently, other methods have 278 been developed i n order to avoid the production of the isomeric materials which may a r i s e during the usual isoxazole synthesis from 3-diketones and hydroxylamine s a l t s . 85,102,128,142 _ , , Since the e a r l i e r workers reported the formation of only one product we decided to begin our study by u t i l i z i n g the o r i g i n a l conditions. However, to our s u r p r i s e we found that (+)-usnic a c i d (35) would not react with hydroxylamine hydrochloride i n a l c o h o l i c s o l u t i o n 85 128 (Widman ' reported the use of hydroxylamine acetate i n e t h y l a l c ohol without the a d d i t i o n of base). Therefore, we decided to attempt f i r s t u * • * i . ,. . . ... , • t, • 279,280 the formation of the corresponding oxime by conventional techniques followed by thermal c y c l i z a t i o n to the desired isoxazole. Thus, compound 35 was allowed to react with hydroxylamine hydro-chloride (approximately 1.5 equivalents) i n pyridine s o l u t i o n at room temperature for one hour, d i l u t e d with absolute e t h y l a l c ohol and heated to r e f l u x under nitrogen atmosphere for another hour. Thin layer chroma-tography i n v e s t i g a t i o n of the crude reaction mixture revealed the presence of not one but four products a l l d i f f e r e n t from the s t a r t i n g material. Therefore, we proceeded to i s o l a t e (column chromatography on s i l i c a gel) - 133 -(35) C O C H OH (132) R = H ( 1 3 M R = Me (136) R = Et (1U) R = N H . (135) R=Me (137) R = C H _ C C H C C O C H (153) Figure 43. Summary on the Condensation of (+)-Usnic Acid (35) and (-)-I) i hydiousnic Acid (154) with Aliphatic and Aromatic Amines. - 134 -and characterize these materials. The f i r s t component, i n order of p o l a r i t y and beginning with the l e a s t polar, had the molecular formula C 1 oH 1 i r0^N, indic a t e d by l o ± 3 o elemental analysis and high r e s o l u t i o n mass spectrometry, and was obtained as bright yellow needles from ethyl acetate, mp 230-231°. This material 128 indeed corresponded to the reported product ( l i t . mp 230°). Although the o r i g i n a l product was obtained from racemic usnic acid we have found 2 6 that an o p t i c a l l y a c t i v e isomer, [alp + 554°, i s obtained when the s t a r t i n g m a t erial i s (+)-usnic a c i d (35). I t s u l t r a v i o l e t spectrum (A ° max 374, 329, 281 and 219 nm) showed the expected absorption for a polysubstituted isoxazole. A l k y l - s u b s t i t u t e d isoxazoles and t h e i r corresponding 4-carboxylic acid analogues absorb i n the 210-230 nm region (log e 3.3-3.9) and i t i s w e l l known that whereas unsaturated substituents ( i e . phenyl) at the 5-p o s i t i o n show a complete fusion of the respective chromophoric systems r e s u l t i n g i n a strong bathochromic s h i f t and a considerable hyperchromic e f f e c t on the absorption maximum, the same s u b s t i t u t i o n at the 3-position 281 produces much l e s s noticeable changes. In the i n f r a r e d spectrum, absorptions corresponding to the enone system (1680 cm "*") and the chelated aromatic a c e t y l grouping (1625 cm "*") where observed. I t s pmr spectrum (Figure 44) was most informative and showed that while the angular ( C ^ - p o s i t i o n ) and aromatic methyl groups remained approximately at t h e i r usual pos i t i o n s 61.80 and 2.08, r e s p e c t i v e l y , the isoxazole methyl group (C^-methyl group) appeared as a three-proton s i n g l e t at 62.51. The chelated aromatic methyl ketone was observed at 52.70, whereas a s h i f t to lower f i e l d was experienced by the o l e f i n i c proton (66.45). As expected, no s i g n a l was found f o r the C^-enolic hydroxyl proton, with SOOO I 3S00 i J o o I 500 I HO i l o •7 •'t • 1 " r COCH I ' I I ' I I I t I I I I • I I I I I I I I I I I j I I I I I I I I I I I I I I I I I I I i f i f ! I I ' l l 1 ' l l • I . - . . • i I i ' • • I • • i i I < i i I i i i i I i i > i I < i i i I i i I i I I I I i I I i ; I I I -i > » I < < • i I • i » • I i i i . I • i i ! T 10 pprn(8) Figure 44. Proton Magnetic Resonance Spectrum of (+)-Isoxazolo[4,5-b]usnic Acid (158). - 136 -the phenolic hydroxyl protons occurring at 610.55 (C g-0H) and 13.30 (C^-OH). The mass spectrum of t h i s product was very complex (m/e 341, 326, 313, 312, 298, 285, 273, 272, 260, 257, 244, 232, 217) and showed the c h a r a c t e r i s t i c fragments f o r the expected electron impact-induced isoxazole -> a z i r i n e -+• oxazole rearrangements postulated for the s e r i e s . Moreover, the basic fragmentation processes and severe reorganizations which occur i n the mass spectra of isoxazoles are reminiscent of t h e i r 287 photochemical behavior. The sample showed the following CD character-i s t i c s : ( A E ) 373 (+6.88), 334 (+13.23), 285 (+9.26), 255 (-11.41) and 229 nm (-15.77). Based on these spectroscopic data and supported by further chemical evidence obtained upon a l k a l i treatment (vide i n f r a ) we postulated rfLiuctui-e 158»ior, L u i s , material, (T)-isoxazoio[4,3-bjusnic a c i d . C O C H 3 26 The second component ( [ a l p (CH^CN) +244°) had the same molecular formula C. QH. C0,.N, and was obtained as c o l o r l e s s prismatic needles from l o 15 o et h y l acetate, mp 260-261°. This product, apparently isomeric with the t4,5-b]isoxazole 15?, had not been i d e n t i f i e d by the e a r l i e r workers. Its u l t r a v i o l e t spectrum (A 326, 281 and 233 nm) proved s i m i l a r to those of max other amine-monocondensation products, while i n the i n f r a r e d spectrum - 137 -absorptions i n the 3200-2900 cm x regionwere i n d i c a t i v e of both chelated phenolic hydroxyl groupings and the amino group of a chelated (3-amino-277 288 —1 a ,B-unsaturated ketone, ' together with bands at 1660 cm (carbonyl of a B-diketoenamine system) and 1620 cm 1 (chelated aromatic a c e t y l ) . Although these f a c t s were t y p i c a l for r i n g C monocondensation materials i t was the proton magnetic resonance spectrum of t h i s compound, the one that yielded d e f i n i t i v e evidence as to the type of oxygen-nitrogen bond present i n t h i s isomer. The pmr spectrum (Figure 45) was run i n a 10:1 mixture of hexadeuteroacetone-hexadeuterodimethylsulfoxide and showed the signals corresponding to the angular and aromatic methyl groups at the expected chemical s h i f t s , 61.87 and 2.12, r e s p e c t i v e l y . The higher f i e l d absorption observed f o r the C^-methyl group (62.48) was compatible with the expected nitrogen condensation to form, i n i t i a l l y , the corresponding oxime followed by tautomerization to a v i n y l i c hydroxylamine, as seen for a l l the amino compounds previously studied where isomerization to the enamine appears to be a favorable process. To complement the previous assignment the absorptions f o r the chelated aromatic a c e t y l grouping (62.74) and the o l e f i n i c proton (66.13) were c e r t a i n l y i n d i c a t i v e that r i n g C had been involved i n some kind of c y c l i z a t i o n . Since the p o s s i b i l i t y of i n i t i a l hydroxylamine condensation at a p o s i t i o n other than the C^-acetyl grouping could be excluded on two counts, namely the observed chemical s h i f t f o r the C^-methyl group and the r e s u l t s obtained during the previous study on the condensation of a l i p h a t i c and aromatic amines with (+)-usnic a c i d , i t became necessary to consider other hydroxyl functions that would be able to produce, upon i n t e r a c t i o n with the v i n y l i c hydroxylamine function, c y c l i c intermediates. The only two such groupings within range for c y c l i z a t i o n were the C 1 carbonyl - 138 -- 139 -group (as enol) and the C -phenolic hydroxyl moiety. The l o w - f i e l d region y of the pmr spectrum proved very informative since no absorption corresponding to the C -phenolic hydroxyl was observed i n the 610-12 region, whereas the y c h a r a c t e r i s t i c s i g n a l f o r the C^-phenol was seen, as expected at 613.61. Moreover, the amine proton of the substituted hydroxylamine appeared as a broad one-proton s i g n a l at 64.38, well within the absorption range f o r ^ • i • . t. j , 289 other v m y l i c or aromatxc hydroxylammes. Its mass spectrum (m/e 341, 326, 308, 298, 280, 260, 232, 217) was s i m i l a r to that of the isoxazole 158. Although the behavior of oxazocines i n the mass spectrometer has not been as yet studied, i t i s quite p o s s i b l e that electron impact-induced r i n g opening-recyclization to isoxazole d e r i v a t i v e s are important c h a r a c t e r i s t i c s of the s e r i e s . Moreover, the sample presented the following c i r c u l a r dichroism properties: A (Ae) & r r m a x 345 (+9.20), 332 (+9.40), 320 (+9.60), 268 (-7.44), 235 (-5.09) and 222 nm (-11.75). Other oxazocines have been prepared by thermal rearrangement of 290 azepine-N-oxides, and i t was found that whereas loss of the oxygen atom occurs when heated at 290-300°, to produce isomeric azepines, reduction with zinc and a c e t i c acid cleaves the N-0 bond to give the corresponding amino-291 a l c o h o l . Moreover, Quin and Roof have indicated that i n the i n f r a r e d spectrum a moderately strong absorption at about 940 cm may be character-i s t i c of the N-0 bond. Based on the spectroscopic evidence so f a r presented, as well as other chemical evidence obtained during a l k a l i treatment of t h i s material (vide i n f r a ) , we postulated structure 159 for (+)-2H-[l,2]oxazocinousnic acid, a novel and i n t e r e s t i n g compound without precedent i n the s e r i e s . - 140 -2 6 The t h i r d component, [ a ] Q (CR^CN) +851°, proved to be the f i n a l isomeric m a t e r i a l , the [5,4-a]isoxazole 160. Thus, a n a l y t i c a l and high r e s o l u t i o n mass spectrometry data provided the molecular formula C^gH^i-O^N, and the product was obtained as yellow c r y s t a l s from ethyl acetate, mp 178-179°. Its u l t r a v i o l e t spectrum (X 390, 336, 282, 258 and 222 nm) was c max quite s i m i l a r , as expected, to that of the isomeric isoxazole 158, the differences a r i s i n g mainly due to the change i n s u b s t i t u t i o n pattern, a 281 fac t observed throughout the isoxazole s e r i e s . Its i n f r a r e d spectrum 213 -1 showed the c h a r a c t e r i s t i c absorptions f o r an enone system (1675 cm ) and the chelated aromatic a c e t y l grouping (1620 cm ^ ) . The d i f f e r e n c e i n s u b s t i t u t i o n was also f e l t i n the pmr spectrum, showing the isoxazole methyl group at 62.60, while the chelated aromatic methyl ketone remained constant at 62.68. Other signals observed (Figure 46) were one-proton s i n g l e t s at 66.36 ( o l e f i n i c proton), 10.89 (C--0H) and 13.40 (C -OH). The mass spectrum of"compound 160 (m/e 341, 326, 313, 298, 272, 256, 244, 217) was again s i m i l a r to that of 158. The fourth and f i n a l component was shown to be i d e n t i c a l i n a l l 2 11 respects with ( - ) - A ' -enaminousnic acid (132), the product of the conden-Figure 46. Proton Magnetic Resonance Spectrum of (+)-Isoxazolo[5,4-a]usnic Acid (160). - 142 -satzion of (+)-usnic acid (35) with concentrated ammonium hydroxide in alcoholic solution (vide supra), and i t s formation is probably due to traces of ammonia present as an impurity in the hydroxylamine hydrochloride. Under the described conditions, the formation of such isomeric materials can be rationalized as arising from the syn-methyl oxime 161a, the i n i t i a l condensation product of hydroxylamine hydrochloride with (+)-usnic acid in pyridine solution, which subsequently equilibrates with the isomeric syn-methyl oxime 161b and the vi n y l i c hydroxylamine 161c (Figure 47). Thermal cyclization of such intermediates produces the observed f i n a l products. Interesting to mention is the fact that on the "crude oxime reaction" variable amounts of the f i n a l products can already be seen by thin layer chromatography, thus indicating an inherently f a c i l e cyclization process. Based on this postulate, we decided to study tha modification of relative yields and product distributions caused by changes on the parameters controlling the thermal cyclization of intermediate 161. The results of such experiments are summarized in Table 10. These results indicate that i f the cyclization is carried out without isolation of the intermediate "oxime" (two-step process) in a pyridine-ethyl alcohol mixture (experiment I) the main product is. (.+)-2H-[l,2]oxazocinousnic acid (159), indicating that 161c (Figure 46) is the predominant species under such conditions (ie. basic solvent). However, i f protonation of the anionic intermediates occur (ie. acidic work-up followed by isolation of the crude "oxime mixture"), the mixture obtained upon cyclization produced as the main component, regardless of the solvent used, and the difference in temperature, (experiments II and III), the - 143 -[5,4-a]isoxazole 160. whereas the yield of the isomeric [4,5-b]isoxazole 158 was seen to remain more or less constant in both experiments, the yield of oxazocine 159 dropped tremendously when the reaction was carried out in absolute ethyl alcohol, indicating that the formation of such material occurs from the Cg-phenolate anion. An increase on the yield of 158 was also observed when the condensation was carried out as a one-step reaction, namely oxime formation and thermal cyclization of the intermediates were simultaneous (experiment IV). This experiment which attempted to cyclize - 144 -% Y i e l d s of Isolated Materials Experiment Conditions (158) (159) (160) % T o t a l Y i e l d I a, c 21.10 60.52 5.54 87.16 II a, b, c 20.17 2.01 56.49 78.67 III a, b, d 16.14 21.10 57.70 94.94 IV e 30.50 t t t a = oxime formation i n p y r i d i n e ( l h , r t ) b = work-up and i s o l a t i o n of the "oxime" c = thermal c y c l i z a t i o n i n absolute e t h y l alcohol ( l h , 90°) d = thermal c y c l i z a t i o n i n anhydrous pyridine ( l h , 110°) e = oxime formation-thermal c y c l i z a t i o n (one-step reaction) i n 1:1 dry pyridine-absolute e t h y l alcohol (1.5h, 80°) t = not determined Table 10. The Thermal C y c l i z a t i o n of "Usnic Acid Oxime". the "oxime" as soon as i t formed, added some proof to the o r i g i n a l postulate that the i n i t i a l condensation product must be the syn-methyl oxime 161a, which r e a d i l y e q u i l i b r a t e s under basic conditions with the isomeric oxime 161b and hydroxylamine 161c. Nevertheless, the e q u i l i b r a t i o n rate must be f a s t e r than the rate of c y c l i z a t i o n i n order to explain the observed r e s u l t s . We should also mention the f a c t that a l l attempts to further increase the y i e l d of the [4,5-b]isoxazole 158 through the use of protected species such as (+)-usnic acid diacetate (68b) were unsuccessful, r e s u l t i n g - 145 -only i n p a r t i a l deacetylation to (+)-usnic acid monoacetate (68a). Since the expected product r e s u l t i n g from the hydrogenolysis 2 11 of the [4,5-b]isoxazole 158 i s (-)-A ' -enaminousnic acid, the vinylogous amide 132, i t was of i n t e r e s t to study i n d e t a i l the h y d r o l y t i c regeneration of the rin g C 3-triketone system c h a r a c t e r i s t i c of metabolite 107. I t i s 292 293 294 well known that enamines as well as vinylogous amides ' can be e a s i l y hydrolyzed back to the parent ketone both by aqueous a l k a l i and by acids. Although S c h i f f bases and other r e l a t e d qarbonyl-amine adducts are 295 best hydrolyzed i n acid s o l u t i o n , the use of strong acids can rearrange f f M f . . . . 296 some enamines to stable ternary lmmium ions. Thus i t was decided to begin t h i s study by using the mild a c i d i c 2 11 conditions provided by aqueous a c e t i c a c i d . Indeed when (-)-A ' -enamino-usnic acid (132) was refluxed i n 80% aqueous a c e t i c a c i d f o r 8 hours, a f a i r y i e l d of usnic a c i d was obtained. However, i t was found that t h i s product had suffered complete racemization. This r e s u l t i s i n complete 85 accord with previous observations made by Widman about the f a c i l e race-mization of o p t i c a l l y a c t i v e usnic a c i d by prolonged heating i n a c e t i c 297 a c i d . Moreover, MacKenzie observed that the rate of racemization of (+)-usnic acid (35) increased accordingly to the b a s i c i t y of the solvent for eight d i f f e r e n t solvents (Table 11) and indicated that the p r o p o r t i o n a l i t y between the c a l c u l a t e d energies of a c t i v a t i o n and entropies suggested a constancy i n the mechanism of racemization throughout the range of solvents 298 studied. 297 299 Furthermore, MacKenzie and l a t e r on B e r t i l s s o n and Wachtmeister . found enhanced racemization rates, f o r 9-0-substituted materials (Table 12) as compared to those of (+)-usnic acid (35) or i t s 7-0-substituted d e r i v a t i v e s . - 146 -Solvent Temperature (°) • Time (min) a / a o (%) Dioxane 96.5 30 10 A c e t o n i t r i l e 115.8 30 13 Methyl E t h y l Ketone 94.0 30 17 Anisole 139.9 30 18 Toluene 115.0 30 27 Chlorobenzene 114.9 . 30 28 Decalin 94.1 30 53 Bicyclohexyl 94.0 30 57 Table 11. The Racemization of (+)-Usnic Acid (35) i n Various Solvents. Compound Temperature (°) Time (hr) /ao (%) (+)-Usnic Acid (35) 79.5 10 94.6 115.0 3 49.0 (+)-7-0-Acetylusnic Acid (68c) 79.5 10 97.7 (+)-9-0-Acetylusnic Acid (68a) 60.5 6 35.9 79.5 2 11.2 (+)-7,9-Di-0-Acetylusnic Acid (68b) 60.5 5 74.5 79.5 3 16.3 (+)-Sodium Usnate 98.9 2 0.0 114.8 1.5 96±5 Table 12. The Racemization of (+)-Usn.ic Acid (35) and some Derivatives . n . 297,299 in Dioxan. - 147 -Whereas the ease f o r thermal racemization shown by the former products explains the formation of a racemate on the acid-catalyzed a c e t y l a t i o n of , . , „ „ o 152b . , , ._ , ,. 102 (+)-usnic acid at 90 , i t is worthwhile noting that sodium usnate 297 i s not racemized under the described conditions. Although the p o s s i b i l i t y that racemization was caused by the u n l i k e l y disrupture of the C„ -C_, bond i n usnic acid was discussed by J 9a 9b 297 300 MacKenzie, i t was Stork who proposed an homolysis of the Cg^ -*^ bond to .the d i r a d i c a l species at for which a s e r i e s of resonance forms may be written, i n c l u d i n g the ketene 162. Such a suggestion i s i n agreement with the known reactions of usnic a c i d , such as the formation of decarbousnic acid (57,R=R'=H), eth y l acetusnetate (71), etc. - 148 -It i s i n t h i s regard that the necessity of forming a c y c l i c d e r i v a t i v e of usnic a c i d such as the [4,5-b]isoxazole 158 acquired v i t a l importance, since i t w i l l be useful not only i n protecting the highly r e a c t i v e r i n g C B-triketone moiety, but also' to s t a b i l i z e the system 159 against racemization, since i t was found that an analogous material, the anhydrophenylhydrazone 77b, would not su f f e r racemization under the experimental conditions by which usnic a c i d i s completely racemized ( i e . r e f l u x i n g toluene). This f a c t i s i n perfect agreement with Stork's p o s t u l a t e , s i n c e formation of a ketene intermediate by homolysis of the Cg^-C^ bond would require disrupture of the aromatic pyrazole (or isoxazole) r i n g system, thus being an e n e r g e t i c a l l y unfavorable process. Since other a c i d i c conditions ( i e . concentrated hydrochloric a c i d i n tetrahydrofuran) f a i l e d to produce the desired o p t i c a l l y a c t i v e m a t e r i a l , we decided to attempt a base-catalyzed h y d r o l y s i s of the B-diketoenamine system, and i t was found that upon treatment with 1.2 N aqueous sodium 2 11 hydroxide at room temperature (-)-A ' -enaminousnic acid (132) furnished (+)-usnic acid (35) i n 84% y i e l d . This mild h y d r o l y t i c condition could i n 2 11 f a c t be applied to other members of the s e r i e s , such as (-)-N-methyl-A ' enaminousnic acid (134), etc., with no detrimental e f f e c t on the y i e l d of i s o l a t e d o p t i c a l l y a c t i v e material. At t h i s point we f e l t i t would be of i n t e r e s t to investigate the behavior of the benzodiazepine d e r i v a t i v e 153 upon base treatment, since i t has been reported that (1,5)-benzodiazepines are stable towards moderately 269 301 strong a c i d and basic conditions. ' Thus when compound 153 was heated with potassium hydroxide i n dry isopropyl alcohol or methanol (Barton's 159 cleavage conditions ) only polar decomposition materials were obtained, - 149 -suggesting that the st a b i l i t y of the heterocyclic system had been altered upon condensation with usnic acid. In continuing with our study on the [4,5-b]isoxazole 158, i t only remained necessary to study the reductive cleavage of the isoxazole ring. Although the attempted ring openings with sodium wire in wet ethyl ether-301 302 ethyl alcohol solution, ' and stannous chloride in concentrated hydro-303 chloric acid both failed, a mild high yielding platinum oxide-catalyzed 2 11 hydrogenolysis of the N-0 bond, to produce (-)-A ' -enaminousnic acid (132), was found to occur in ethyl alcohol solution, a method that had been success-f u l l y applied to compounds showing a marked s t a b i l i t y towards reduction, such , . , 233,234 . . 304 , . as some styrylisoxazoles, as well as ammoisoxazoles, and lsoxazo-305 lones, whereas systems bearing only saturated substituents or heterocyclic rings that are very resistant towards reduction (ie. pyrazolylisoxazoles) are • i i j -« . . , 306,307 easily cleaved by Raney nickel.. Furthermore, the same conditions can be applied CO the isomeric isoxazole 160 and the oxazocine 159, the other products of the condensation of (+)-usnic acid with hydroxylamine hydrochloride (Figure 48). In a l l three cases, the observed yields of vinylogous amide 132 are in the 87-96% range, with the lower yield being due mainly to solubility problems. Moreover, this reductive cleavage served to interrelate such isomeric materials as arising from a common "oxime" intermediate. Having solved the synthetic problems presented by the regeneration of the 0-triketone moiety, i t was then time to look into the base cleavage of the aromatic acetyl grouping. However, when the isoxazole 158 was allowed to react 159 under Barton's conditions (refluxing 20% methanolic potassium hydroxide) mostly decomposition products were observed, although a very low yield (less than 5%)i of a deacylated material showing methanol addition across the C.-C. 4 4a - 150 -double bond was part; La 11y i d e n t i f i e d a f t e r i s o l a t i o n of the d i f f e r e n t r eaction products present i n the mixture, thus i n d i c a t i n g that the rea c t i o n was indeed proceeding. COCH ( 158) COCH ( 1 5 9 ) COCH COCH Pt02 / H2 HO EtOH I 160 ) Figure 48. The I n t e r r e l a t i o n of (+)-Isoxazolo[4,5-bJusnic Acid (158), (+)-2H-]l,2]oxazocinousnic Acid (159) and (+)-Isoxazolo[4,5-a] usnic Acid (160). Af t e r a new set of conditions was sought, we found that the reaction could be s u c c e s s f u l l y c a r r i e d out i n a water-ethyl alcohol mixture at 70° for 25 minutes. However, before we begin the discussion of the spectroscopic properties shown by the products obtained during the base treatment of the - 151 -COCH ( 158) Compound Yield, % C O C H 3 Q H -0~~ C O C H 3 g E t (165) COOH ( 167 ) 2.2 3.1 23.0 26.8 36.0 Figure 49. The Base Treatment of (+)-Isoxazolo[4,5-b]usnic Acid (158). I - 152 -isoxazole 158 (Figure 49) i t i s convenient to mention that we have assigned the normal-series structure shown by formulas 163, 164 and 167 to those compounds r e t a i n i n g the aromatic a c e t y l grouping. These assignments 147 148 149 were based on the observations of Asahina, ' Dean and Robertson, 308—310 and Shibata and coworkers," who showed during t h e i r studies leading , ., . . ,. 142,148,311 to the i d e n t i f i c a t i o n and synthesis of the a-coumaranone diacetate 309 310 70 and i t s i s o - s e r i e s analog ' 168, the ozonolytic products of (+)-usnic acid diacetate (68b) and (+)-isousnic acid diacetate (169), r e s p e c t i v e l y , that compounds possessing the aromatic a c e t y l grouping occur p r e f e r e n t i a l l y as the normal-series isomers. AcO AcO - 153 -309 310 Thus i t v;us found ' that the isocoumaranone diacetate 168 reverts to the normal-series isomer 170 simply by an acid-catalyzed deacetyl-ation, and (+)-isodi.hydrousnic acid (171), a product resulting from the 273 27A 310 thermal isomerization ' ' of (-)-dihydrousnic acid (154), converts 310 back to 154 even during the process of recrystallization. (154) (17D Added spectroscopic support to our conclusion was obtained from the proton magnetic resonance data of various usnic and isousnic acid derivatives regarding the typical chemical shift for the aromatic acetyl grouping (Table 13). It is from these data that we were able to conclude that, regardless of any modification present in ring C , the observed chemical shift for the ring A acetyl grouping consistently occurs at lower f i e l d (A60.05-0.16 ppm) in compounds belonging to the iso-series as compared to those of the normal-series. The various products resulting from the base cleavage of the [4,5-b]isoxazole 158 (Figure 49) were isolated by column chromatography on s i l i c a gel, and will.be discussed in order of increasing polarity (ie. order of elution off the column) beginning with the least polar. The f i r s t Chemical Shift, ppm (6 units) Compound C,-C0CHo 6 3 Cg-COCH3 C2-COCH 3 C n - C H 3 Normal-Series -(+)-usnic acid (35) (+)-isoxazole 4,5-b usnic acid (158) (-)-dihydrousnic acid (154) (+)-usnic acid anhydrophenylhydrazone (77b) cleavage product 164 cleavage product 167 2.70 2.70 2.64 2.63 2.55 2.66 2.70 2.60 2.51 2.60 2.42 2.40 Iso-Series (+)-isousnic acid (112) (+)-isodihydrousnic a c i d 2 7 3 ' 3 0 9 (171) 310 (+)-isousnic acid anhydrophenylhydrazone (17.-) 312 (-)-placodiolic acid (173) 2.77 2.69 2.79 2.76 2.68 2.58 2.69 2.60 Table 13. The Observed Chemical Shift for the Acetyl Groupings in the Usnic and Isousnic Acid Series. - 155 -component (+)-a-hydroxyisoxazolo[4,5-b]usnic acid (163), c o l o r l e s s prisms from chloroform-ethyl ether, mp 202-204°, was obtained i n very low y i e l d (2.2%). Its u l t r a v i o l e t spectrum (X 330, 286 and 223 nm) was very max s i m i l a r to that of the parent isoxazole 158, showing only a reduction on the e x t i n c t i o n c o e f f i c i e n t s f o r the various maxima, thus i n d i c a t i n g the loss of conjugation between the two aromatic chromophores upon the base-catalyzed hydration of the C^-C^ double bond. Its i n f r a r e d spectrum showed the expected bands for the C - l carbonyl (1680 cm "*") and the chelated aromatic a c e t y l grouping (1620 cm ^ ) . Although no pmr spectrum could be obtained at the time, due both to the very small amount of a v a i l a b l e m a terial and i t s higli i n s o l u b i l i t y i n most common deuterated solvents, a n a l y t i c a l and high r e s o l u t i o n mass spectrometry data supported the molecular formula C. 0H -,0_N, a r i s i n g from a d d i t i o n of water to the o r i g i n a l isoxazole 158 lo 1 / / (C^gH^^O^N) . The stereochemical assignment for the C^-hydroxyl group followed from a comparison of i t s c i r c u l a r dichroism properties with those of the cleavage products 164 and 166 (Table 14), for which such assignment was based on the analysis of t h e i r proton magnetic resonance spectra. - 156 -Compound C i r c u l a r Dichroism Properties, A (Ae) • . • max 163 < V -aOH) 280 .(+ 6.08) 251 (+8.42) 225 (-14.51) 164 <C4a" -otOEt) 279 (+11.11) 250 (+14.10) 226 (-26.87) 165 « V -BOH) 330 (+ 2.64) 276 (- 0.61) 236 (+11.89) 166 <C4a--aOH) 295 (+ 4.21) 267 (+ 9.92) 239 (- 1.97). Table 14. The C i r c u l a r Dichroism Properties of the Cleavage Products of (+)-Isoxazole[4,5-b]Usnic Acid (158). The second component, (+)-a-ethoxyisoxazolo[4,5-b]usnic acid (164) was, ob.Lained. as. c o i o r i e s s nexagonal prisms, mp 2i9-z20". Wnne. i t s u l t r a -v i o l e t spectrum (A 330, 285 and 223 nm) was e s s e n t i a l l y i d e n t i c a l to that v max of the former m a t e r i a l , i n d i c a t i n g r e t e n t i o n of the r i n g A a c e t y l grouping, i t s i n f r a r e d spectrum had absorptions c h a r a c t e r i s t i c of the C - l ketone (1695 cm •*") and the chelated aromatic a c e t y l grouping (1620 cm ^ ) . The proton mag-n e t i c resonance spectrum of 164 (Figure 50) was very informative and showed a three proton t r i p l e t , J = 7 Hz, at 61.18 f o r the terminal methyl group of the C. -0CH„CH„ substituent. Moreover, the two three-proton s i n g l e t s at 62.42 and 4a I 3 2.55 were assigned to the C^-methyl group (isoxazole methyl) and the aromatic methyl ketone, r e s p e c t i v e l y . Whereas the AB quartet (J = 18 Hz) at 53.17 co r r -esponded to the C^-methylene protons, a two proton quartet at 63.74, J = 7 Hz, was t y p i c a l of the methylene protons of the C. -ethoxy group. The low f i e l d 10 p p m ( 6 ) Figure 50. Proton Magnetic Resonance Spectrum of (+)-a-Ethoxyisoxazolo[4,5-b]usnic Acid (164). - 158 -region of the spectrum had a broad absorption at 66.58 and a one-proton singlet at 613.46 that were assigned to the Cg- and C^-phenolic hydroxyl protons, respectively. Analytical and high resolution mass spectrometry data indicated the molecular formula C ^ Q H ^ ^ O ^ N , arising from a base-213 catalyzed homo-Michael-type addition of ethyl alcohol to the C^-C^ double bond on the parent isoxazole 158. The third product, isolated in 23% yield, showed a molecular formula ^ - ^ H ^ O ^ N , compatible with both cleavage of the ring A aromatic acetyl and hydration of the double bond. Its ultraviolet spectrum (X max 329, 260 and 223 nm) showed the expected hypsochromic shift for a deacylated species, and in the infrared spectrum in addition to the C-l enone band (1665 cm the 1620 cm ^ (C=C, aromatic ring) absorption appeared with reduced intensity, thus adding strong support to the previous postulate. Its pmr spectrum showed only the three-proton singlet corresponding to the C^-methyl group (isoxazole methyl) at 62.39. Thus the lack of an absorption for the chelated aromatic methyl ketone coupled with the appear-ance of a one-proton singlet in the low-field region of the spectrum (66.06) confirmed beyond doubt that deacylation of the ring A chelated acetyl grouping had taken place. The C^-methylene protons were observed as an AB quartet, J = 18 Hz, at 6 3.53, whereas the C -g-hydroxyl group proton was seen as a broad one-proton band at 67.10 (Figure 51). This material is represented by structure 165 in Figure 49. The chemical shift of the C^-methylene protons in the ^^ a~ a~ a n d 8-series can be used as characteristic in assigning the stereochemistry of the C, -substituent, since i t has been observed that for the a-series such 4a chemical shift occurs consistently at higher f i e l d when compared to the one 400 p p m ( 6 ) Figure 51. Proton Magnetic Resonance Spectrum of (+)-p-Hydroxydesacetylisoxazolo[4,5-b]usnic Acid (165). - 160 -for compounds of the C. -S-series (Table 15). Moreover, i t was found that Chemical S h i f t , ppm (6 units) Compound C. -Methylene Group C Q, -Methyl Group (-)-dihydrousnic acid (154) 3.10 1.70 cleavage product 164 (ct-OEt) 3.17 1.94 cleavage product 166 (ct-OH) 3.08 1.88 cleavage product 165 (6-OH) 3.53 1.68 312 (- ) - p l a c o d i o l i c ac i d (173) 3.37 1.46 273 309 (+)-isodihydrousnic acid ' (171) 3.10 1.70 Table 15. . The Chemical S h i f t of the C^-Methylene Protons and the C_, -Methyl 9b Group f o r the C. -a- and t 4a S-Hydroxyisoxazole Series. the angular methyl group (C^-methyl) i - s also responsive to the change i n conformation brought about by the introduction of the C ^ - s u b s t i t u e n t . Thus the observed chemical s h i f t for t h i s group i n the ct-series (cis-B/C junction) occurs c o n s i s t e n t l y at lower f i e l d as compared to compounds belonging to the 8-series (trans-B/C j u n c t i o n ) , not only f o r the normal-series ( e n t r i e s 1 and 2 i n Table 15) but also for the i s o - s e r i e s (entries 3, 4 and 6). This r e l a t i o n holds equally well for compounds possessing a C^-a-methyl group (entries 1-4 and 6), i . e . those materials derived from (+)-usnic acid (35), than f o r those having the C_, -3-methvl group (entry 5), derived from (-)-9 b usnic acid (111). Entry 5, ( - ) - p l a c o d i o l i c ac i d (173), a product i s o l a t e d - 161 -from the l i c h e n Lecanora rubina ( V i l l . ) Ach., i s quite important, since 312 from an analysis of i t s pmr spectrum Huneck was able to deduce a trans-B/C junction, assigning an a-configuration to the C. -substituent. Thus in materials derived from (-)-usnic a c i d (111), the C. -a-configuration 4a i s equivalent to the C. -8-configuration observed for the enantiomeric na s e r i e s derived from (+)-usnic a c i d (35), both giving r i s e to a trans-B/C junc t i o n . I t must be mentioned, however, that the assignment of such chemical s h i f t s as being representative of the C^-a- o r B-series was done a f t e r a c a r e f u l a n a l y s i s of t h e i r molecular models, which showed that only for the C^-a-conf ig u r a t i o n the B-proton of the C^-methylene group i s allowed to l i e above the plane of the aromatic system of r i n g A, thus 253 experiencing the s o - c a l l e d "ring-current e f f e c t " r e s u l t i n g i n a s h i e l d i n g ( i e . a s h i f t to higher f i e l d ) of the relevant methylene protons. The fourth component, c o l o r l e s s prisms mp 224-226°, was i s o l a t e d i n 27% y i e l d and shown to be a r i n g A deacylated material by i t s molecular formula, C. ,H, ..O-N. Its u l t r a v i o l e t spectrum (A 286, 262 and 221 cm"1) 16 15 6 max was again compatible with aromatic deacylation, while i n the i n f r a r e d spectrum absorptions corresponding to the C - l carbonyl (1690 cm "*"), a t r i s u b s t i t u t e d isoxazole ring^"*"^ (1640 cm ^) and the aromatic r i n g (1600 cm ^) were observed. From i t s pmr spectrum i t became quite c l e a r that t h i s material was the C^ a~ a-hydroxy analog of the previous product, showing a six-proton s i n g l e t at 61.88 that was assigned to both the angular and aromatic methyl groups, and a three-proton s i n g l e t at 62.37 corresponding to the C^-methyl group. The C^-methylene protons appeared as an AB quartet, J = 18 Hz, at 63.08, and the aromatic C Q-proton as a one-proton s i n g l e t at 66.00 (Figure 52). I • • • • • • • • • I . . . . I . . . • I. . I . . . . I . . . . I . . . . I . . . . I . . . . I . . 10 p p rr. (6) Figure 52. Proton Magnetic Resonance Spectrum of (+)-<x-Hydroxydesacetylisoxazolo[4,5-b]isousnic Acid (166). - 163 -Although at t h i s time the i s o - s e r i e s structure now c h a r a c t e r i s t i c of the cleavage products 165 and 166 could not be deduced from the observed spectroscopic properties, the correct assignment followed from t h e i r chemical conversion into (+)-isousnic acid (112) (vide i n f r a ) , thus i n d i c a t i n g that, under the cleavage conditions, hydration of the double bond i s accompanied by concomitant r i n g opening and isomerization to an isousnic acid-derived structure. This observation suggests that f o r the r i n g A deacylated materials, isomerization to the i s o - s e r i e s i s a favorable process, therefore showing an opposite behavior to that observed f o r the a'cylated analogs (vide supra) . The f i n a l product, the carboxylic a c i d 167, was obtained i n 36% y i e l d as long f l u f f y needles mp 258-259°, and proved instrumental in assigning structure 158 to the isoxazole used as s t a r t i n g material, since only t h i s product and not the isomeric isoxazole 160 would produce the o p t i c a l l y i n a c t i v e carboxylic acid 167 upon base cleavage of the "weak" C_, -C. bond. 9b 1 Thus, the ac i d 167, whose molecular formula C^gH^O^N was indicated by a n a l y t i c a l and high r e s o l u t i o n mass spectrometry data, showed i n the i n f r a r e d spectrum the expected absorption (1720 cm "*") c h a r a c t e r i s t i c of a 3 , 5 - d i a l k y l -- 164 -isoxazole-4-carboxylic a c i d . I t s pmr spectrum confirmed the assigned structure 167, with three-proton s i n g l e t s at 62.08 and 2.35 corresponding to the C,.- and C^-methyl groups on the benzofuran system, r e s p e c t i v e l y , and the chelated methyl ketone appearing at 62.66. Furthermore, the methylene bridge protons appeared as a two-proton s i n g l e t at 64.50, and the isoxazole methyl group at 62.40. Further spectroscopic data were obtained upon conversion to the methyl ester 174 by rea c t i o n with excess ethereal diazomethane i n methanolic s o l u t i o n . Thus the methyl ester 174, s i l k y needles mp 217-218°, showed an u l t r a v i o l e t spectrum (A 349, 303 and 242 nm) i d e n t i c a l to that of the max parent acid 167. In i t s i n f r a r e d spectrum the carbonyl of the ester group was found to occur at 1720 cm \ while the proton magnetic resonance spectrum had a new three-proton s i n g l e t at 63.90 corresponding to the carbomethoxy grouping. COOH COOMe (167 ) (174) The next step i n our synthetic scheme was the dehydration of the C. -a- and B-hydroxy d e r i v a t i v e s 166 and 165 (Figure 53). Although t h i s 4a reaction was attempted with a serie s of known dehydrating agents ( i e . POCl^/ Py, S0Cl 9/Py, etc.) i t was found that the best experimental conditions were - 165 -- 166 -e s s e n t i a l l y those used by Barton and coworkers^"" during t h e i r synthetic e f f o r t s leading to the laboratory preparation of racemic usnic a c i d , namely a 0.5% s o l u t i o n of concentrated s u l f u r i c acid i n a c e t i c anhydride at 50° under nitrogen atmosphere. Thus under these conditions the ct-and the B-hydroxyisoxazole, and a 1:1 mixture of both isomers were success-f u l l y dehydrated, i n over 90% y i e l d , to produce the diacetate 175 (Figure 53), (+)-desacetylisoxazolo[4,5-b]isousnic acid diacetate. I t was indeed that upon c a r e f u l analysis of the spectroscopic properties shown by the diacetate 175 the f i r s t clues as to the i s o - s e r i e s structure shown for 165 and 166 came about. While i t s u l t r a v i o l e t spectrum showed the batho- and hyperchromic s h i f t s (A 300, 245 and 216 nm) expected max upon r e s t o r a t i o n of the conjugation between the two chromophoric systems, the i n f r a r e d spectrum contained the c h a r a c t e r i s t i c absorptions f o r phenolic acetates (1770 cm "*"), the enone carbonyl (1675 cm ^ ) , and-the isoxazole r i n g (1620 cm "*") . It s pmr spectrum showed, besides the isoxazole methyl group (62.53), two three-proton s i n g l e t s at 62.32 and 2.49 that were assigned, r e s p e c t i v e l y , to the C y- and Cg-phenolic acetate groupings. The o l e f i n i c proton was seen at 66.00, while the aromatic proton appeared as a one-proton s i n g l e t at 66.72. When t h i s absorption was compared to that of the aromatic proton i n (+)-6-deacetylusnic acid diacetate (113), which i s observed at 66.81,.the l a c k o f correspondence was immediately r e a l i z e d . However, at t h i s time i t was decided to complete the synthetic sequence and compare the r e s u l t i n g deacylated material with (+)-6-desacetylusnic acid (107), the metabolite obtained during the b a c t e r i a l biodegradation of (+)-usnic a c i d . F i n a l l y , the diacetate 175, with molecular formula C^U^O^N presented the following CD c h a r a c t e r i s t i c s : A (Ae) 330 (+18.45), 265 (-5.88), 225 nm • max -167 -(-15.73). Whereas t h e a t t e m p t e d d i r e c t a c y l a t i o n o f t h e d i a c e t a t e 175 under e i t h e r F r i e d e l - C r a f t s 3 1 5 ' 3 1 6 ( A l C l 3 / A c C l / A ) o r F r i e s 3 1 7 , 3 1 8 ( A c C l / a m b e r l i t e IR-120 ( H + ) r e s i n / A ) c o n d i t i o n s , b o t h f a i l e d , p r o d u c i n g a n u n i d e n t i f i e d o p t i c a l l y i n a c t i v e m a t e r i a l i n t h e f o r m e r c a s e ( s e e e x p e r i -m e n t a l ) and s i m p l y unchanged s t a r t i n g m a t e r i a l i n t h e l a t t e r i n s t a n c e , -a s u c c e s s f u l b a s i c h y d r o l y s i s o f t h e p h e n o l i c a c e t a t e g r o u p i n g s was c a r r i e d o u t i n 86% y i e l d i n t e t r a h y d r o f u r a n s o l u t i o n by u s i n g f r e s h l y p r e p a r e d IN aqueous sodium h y d r o x i d e a t room t e m p e r a t u r e , t h u s a f f o r d i n g ( + ) - d e s a c e t y l -i s o x a z o l o [ 4 , 5 - b ] i s o u s n i c a c i d (176). T h i s r e a c t i o n had l a r g e s y n t h e t i c i m p l i c a t i o n s s i n c e i t a l l o w e d t h e f a c i l e h y d r o l y s i s o f t h e a c e t a t e s w i t h o u t d i s t u r b i n g t h e C^-C^ a d o u b l e bond, w h i c h i n t h e i s o x a z o l e s e r i e s we have f o u n d i s v e r y s u s c e p t i b l e t o a t t a c k under b a s i c c o n d i t i o n s , and s e c o n d l y i t e l i m i n a t e s t h e need o f p e r f o r m i n g t h e same r e a c t i o n i n s t r o n g a c i d i c m edia ( i e . s u l f u r i c a c i d ) a s recommended f o r u s n i c a c i d i t s e l f . 8 ^ ' 1 6 6 The d i p h e n o l 176 had an u l t r a v i o l e t s p e c t r u m (X 301, 354, 222 max nm) v e r y s i m i l a r t o t h a t o f t h e c o r r e s p o n d i n g d i a c e t a t e 175, s h o w i n g t h e e x p e c t e d h y p s o c h r o m i c s h i f t c a u s e d by d e a c y l a t i o n when compared t o t h a t o f t h e C - a c y l a t e d a n a l o g 158. I n t h e i n f r a r e d s p e c t r u m t h e c h a r a c t e r i s t i c a b s o r p t i o n s f o r t h e enone c a r b o n y l (1660 cm 1 ) and t h e i s o x a z o l e r i n g (1630 cm 1 ) were p r e d o m i n a n t , w h i l e t h e pmr s p e c t r u m showed t h e i s o x a z o l e m e t h y l group a t 62.46, t h e o l e f i n i c p r o t o n a t 65.95, and t h e a r o m a t i c p r o t o n a t 66.31. T h i s a b s o r p t i o n p r o v i d e d a n o t h e r c l u e r e g a r d i n g t h e t y p e o f s e r i e s we were now d e a l i n g w i t h , s i n c e a g a i n t h e r e was no co m p l e t e c o r r e s p o n d e n c e w i t h t h e o b s e r v e d s i g n a l f o r ( + ) - 6-desacetylusnic a c i d (107). I t s mass s p e c t r u m , a n a l y t i c a l d a t a , and h i g h r e s o l u t i o n m o l e c u l a r w e i g h t d e t e r m i n a t i o n - 168 -indicated the molecular formula C, .H., „0,_N. 16 13 5 Attempted Hoesch a c y l a t i o n 1 6 9 ' 1 7 0 (CH 3CN/ZnCl 2/Et 20) of t h i s material under s i m i l a r conditions to those employed during the synthesis of methylphloroacetophenone (85) from methylphloroglucinol (41) produced an o p t i c a l l y a c t i v e material, mp 148-150°, as yet u n i d e n t i f i e d , showing chlorine incorporation. Its pmr spectrum had a new AB quartet, J = 18 Hz, at 63.16 i n t e g r a t i n g f o r two protons, and two one-proton s i n g l e t s at 65.49 and 5.69. Moreover, the attempted a c y l a t i o n with a c e t y l chloride-boron 319 t r i f l u o r i d e etherate produced i n 87% y i e l d e x c l u s i v e l y the corresponding diacetate 175. Since most of the a c y l a t i o n reactions t r i e d on the diphenol 176 were unsuccessful, we decided to carry out the remaining synthetic trans-formations (Figure 53) and repeat the acylations on the unprotected m a t e r i a l . Thus the isoxazole diacetate 175 was hydrogenolyzed i n e t h y l alcohol s o l u t i o n over platinum oxide at atmospheric pressure to produce, i n 95% y i e l d , (+)-2 11 desacetyl-A ' -enaminoisousnic a c i d diacetate (177), the material r e s u l t i n g from reductive cleavage of the isoxazole N-0 bond. Its u l t r a v i o l e t spectrum 2 11 (A 328, 284, 268, 204 nm) when compared to that of (-)-A ' -enaminousnic max acid (132) showed the e f f e c t s of deacylation, namely a blue s h i f t on the absorption maxima, while i t s i n f r a r e d spectrum was i n d i c a t i v e of the assigned 2 A 9 277 vinylogous amide ' structure (chelated B-diketoenamine) with absorptions at 3400-3200, 1630 and 1580 cm"1, and c h a r a c t e r i s t i c bands for the phenolic acetates (1770 cm "*") and the C - l carbonyl group (1700 cm ^ ) . The pmr spectrum of 177 showed a three-proton s i n g l e t at 62.28 assigned to the C^-phenolic acetate and a six-proton s i n g l e t at 62.35 corresponding both to the isoxazole methyl group and the C.-phenolic acetate. The o l e f i n i c and aromatic protons 9 - 169 -were observed as one-proton s i n g l e t s at 65.65 and 6.57, r e s p e c t i v e l y , while the enamine protons appeared as broad signals at 66.40 and 10.56. Moreover, i t s mass spectrum supported the postulated ketoenamine diacetate structure. The f i n a l step, the hydrolysis of both the phenolic acetates and the diketoenamine system with regeneration of the r i n g C 8-triketone c h a r a c t e r i s t i c of usnic a c i d , had been previously sorted out during the 2 11 studies on (-)-A ' -enaminousnic acid (132) and the desacetylisoxazole diacetate 175, therefore, when the r e a c t i o n was c a r r i e d out on compound 177, under the same conditions, a 75% y i e l d of (+)-8-desacetylisousnic a c i d (178) 2 6 r e s u l t e d . This material, [ a]-^ + 605°, was obtained as bright yellow needles from chloroform-petroleum ether, having mp 210-211° (melts p a r t i a l l y at 183-184° with r e s o l i d i f i c a t i o n and change i n c r y s t a l l i n e form to thick prismatic needles), and showed a melting point depression .when mixed with authentic (+)-6-desacetylusnic a c i d (107) of mp 134.5-136°. Its u l t r a v i o l e t spectrum (A 330, 268, 244 nm) was only s l i g h t l y d i f f e r e n t from that of max the metabolite 107, and i t s i n f r a r e d spectrum showed the expected absorptions t y p i c a l of the enone system (1680 cm ^) and the chelated enolized 1,3-di-ketone system (1540 cm ^ ) . This spectrum (Figure 54) was shown not to be superimposable with the one of metabolite 107. Although i t s proton magnetic resonance spectrum (Figure 55) proved very s i m i l a r to that of 107 (Table 16), small differences were observed on the o l e f i n i c proton (65.80) and the C^-phenolic hydroxyl proton (68.85). However, the l a t t e r chemical s h i f t i s not very useful due to i t s marked concentration and solvent dependance, as seen by the changes introduced i n the spectrum when recorded in deutero-benzene, s o l u t i o n (Table 16). The mass spectrum of this material, (m/e 302, t Figure 54. The Infrared Spectrum of: A) (+)-8-Desacetylisousnic Acid (178) and B) Desacetylusnic Acid (107). Compound Fu n c t i o n a l i t y 178 C 9 b - C » 3 V % C 8-CH 3 C^OCOCH C9-OCOCH_3 C9-C0CH3 C,-H 6 — C„-H C7-OH C3-OH CDCl, C 6D 6-CDC1 3 107 CDCl, 1. 70 2.07 2.59 5.80 6.23 8.85 9.71 18.71 1.72 2.13 2.65 5.89 6.22 5.90 9.95 18.65 1.70 2.11 2.61 5.62 6.26 5.67 10.23 18.64 179 CDCl, 1.80 2.13 2.31 2.40 2.54 5.87 6.69 113 CDCl, 1.78 1.97 2.31 2.43 2.52 5.81 6.81 18.25 18.41 Table 16. Proton Magnetic Resonance Data of ( +)-8-Desacetylisousnic A c i d (178) and ( +)-6-Desacetylusnic Acid (107) and t h e i r Corresponding Diacetates. - 173 -218, 206, 191, 105, 78,A3) although very helpful in determining that ring A was the site where deacylation had taken place could not be used to determine the actual position, and was essentially identical to that of the normal-series isomer. By the same token, the fragmentation pathways observed for both series should be considered equivalent and w i l l not be discussed again (see Figures 34 and 35). However, a very significant difference was observed in the Circular Dichroism spectra of these compounds (Table 17) when recorded in methanol solution containing 100 ul of a 5% methanolic potassium hydroxide solution. Thus i t was found that the 285 nm maximum exhibited a positive Cotton effect for the normal-series isomer 107, that changed into a negative one for the iso-series isomer 178. Both maxima have approximately the same intensity. This i s indeed a very important characteristic since i t allows the faci l e differentiation between the two series while requiring only minute amounts of material (0.05 mg of compound dissolved in 5 ml of the appropriate solvent). Compound Solvent 107 A (Ac) max CH30H 328 (+ 9.99) 284 (- 6.66) 262 (- 3.53) 241 (+13.11) CH OH+KOH 345 (+ 8.11) 310 (+ 6.86) 285 (+ 1.66) 264 (-12.90) 236 (+10.19) 178 CH30H 325 (+ 9.43) 282 (- 6.73) 264 (- 5.66) 238 (+12.40) CH3OH+KOH 432 (+ 7.81) 309 (+ 6.20) 285 (- 1.61) 266 (-13.74) Table 17. Circular Dichroism Data for (+)-6-Decateylusnic Acid (107) and (+)-8-Dcsacetylusnic Acid (178). 174 -Further r.p. <• i r o : . f e data on the iso-series deacylated material 178 were obtained through t h e corresponding diacetate 179. This product was obtained i n 87% yi e l d by an acid-catalyzed acetylation ( i e . acetic anhydride-s u l f u r i c acid), as recommended for usnic acid i t s e l f , although a 35% y i e l d of the same diacetate was produced by using a 1:1 mixture of pyridine-acetic anhydride. Thus (+) - 8- d o s a c e t. y 1 i sou sn i c acid diacetate (179) was obtained 2 6 as pale yellow c r y s t a l s , mp 150-152°, [a] + 275°. While i t s infrared spectrum showed the absorptions characteristic of the phenolic acetates (1770 cm "*") , the enone carbonyl (1690 cm "*") and the chelated ring C diketone system (1540 cm ^ ) , the proton magnetic resonance spectrum had absorptions at 62.31 and 2.40 corresponding to the C^- and Cg-phenolic acetate groupings, respectively. A very important difference with the normal series was recog-nized i n the chemical s h i f t of the aromatic proton, which i n the iso-series diacetates appears at higher f i e l d (A60.12). Such a d i s t i n c t i o n i s not so clear when referred to the free phenolic materials (Table 17). The f i n a l proof required to confirm the postulated isomerization can be obtained by the chemical transformation of (+)-8-desacetylisousnic acid (178) into (+)-isousnic acid (112) (Figure 56), an important trans-formation since i t w i l l in fact constitute not only the f i r s t synthesis of the naturally occurring isomer of (+)-usnic acid, but also a chemical i n t e r -conversion between the. two series i n which the f u l l usnic acid c h a r a c t e r i s t i c s have been retained. The only other such interconversion that has so far been _ ,273,300,310 , , . r / \ , •, j reported dealt with the thermal transformation of (-)-dihydrousnic acid (154) into (+)-isodih y drousnic acid (171). 316 320 Although a direct Fr i d o l Crafts acylation using acetyl chloride-alum i m i n t t r i c l i lor i d e in carbon d i s u l f i d e produced exclusively the - 1 7 5 -( 112 ) 56. The Synthesis of (+)-Isousnie Acid (112). - 1.76 -corresponding diacetate 178 in 42% yield, an aluminum trichloride-catalyzed 445 Fries Rearrangement, followed by basic work-up furnished (+)-isousnic acid (112) in 62% yield. This material, [ a ] ^ 6 + 505°(lit. 8 3 [ a ] 2 5 + 500°), 83 had mp 152-153° ( l i t . mp 152-153°), undepressed by admixture with authentic (+)-isousnic acid (supplied by Professor S. Shibata), and showed superimpos-able untraviolet (A 336, 285, 232 nm) and infrared (1690, 1630, 1540 cm - 1) max ' spectra with those of the naturally occurring material. Its pmr spectrum (Figure 57) is very characteristic and has been reproduced in Table 18, together with that of (+)-usnic acid (35). Compound (+)-Usnic Acid (35) (+)-Isousnic Acid (112) Functionality C9b- C»3 1.76 1.85 V % — 2.10 C8-CH3 2.10 C2-COCH3 2.70 2.68 C.-COCH 6 —3 2.70 __ Cg-C0CH3 — 2.77 C.-H 4 — 5.96 5.96 C9-0H 11.01 11.28 C7-0H 13.31 14.35 C3-0H 18.83 18.81 Table 18. Proton Magnetic Resonance Data of (+)-Usnic Acid (35) and (+)-Isousnic Acid (112). Figure 5 7 . Proton Magnetic Resonance Spectrum of (+)-Isousnic Acid (112) - 178 -The next point of interest was to determine at which stage did the isomerization occur. Working backwards in the synthetic sequences previously outlined (Figures 53 and 56) we decided to look f i r s t at the base-catalyzed (pyridine-acetic anhydride) acetylation of (+)-8-desacetylisousnic acid (178), a reaction that by thin layer chromatography had revealed several minor components besides the desired diacetate 179. Therefore, the diacetate 179, prepared under such conditions, was hydrolyzed back to 178 in tetra-hydrofuran solution by using freshly prepared IN aqueous sodium hydroxide, and i t was found that the phenolic product obtained was identical in a l l respects (spectroscopically and chromatographically) with (+)-8-desacetyl-isousnic acid (178), thus indicating that this was not the reaction where isomerization had taken place. Similar results were obtained for the acid-catalyzed acetylation. Next we decided to look into the dehydration step. This reaction, performed essentially under those conditions previously described by Barton 166 and coworkers, was tested in the following manner. A sample of the C^a~a-hydroxydesacetylisoxazole 166 was acetylated under very mild conditions (pyridine-acetic anhydride) to produce the corresponding C -a-acetoxydiace-4a tate 180, as shown by the phenolic acetate (1775 cm ^) absorption seen in its infrared spectrum. The pmr spectrum of 180 was very characteristic, showing the C^-ct-acetoxy grouping as a three-proton singlet at 52.11, whereas the C^- and Cg-phenolic acetates were observed at 62.30 and 2.43, respectively. Furthermore, the C^-methylene protons, as expected, suffered the deshielding effect of the vi c i n a l C^-a-acetoxy grouping, thus appearing at 63.83 as an AB quartet with coupling constant J =18 Hz. The aromatic proton was seen as a one-proton singlet centered at <H6.75, a chemical shift almost identical to - 179 -the observed one for the dehydrated diacetate 175. The molecular formula C 2 2 H 2 1 ° 9 N ' s u8gested by analytical data, was fully supported by high resolution mass spectrometry molecular weight determination. (175) Having prepared the triacetate 180 we were now in a position to test the following postulate: Tf isomerization was to occur at the dehydration stage, then the required ring opening-recyclization of the normal series isomer into the iso-series had to be a faster reaction relative to acetylation of the phenolic hydroxyl groupings, since once the corresponding acetates have formed no cyclization can occur even in a ring-open intermediate. Furthermore, such intermediate would be susceptible for acetylation, thus yielding a phenolic triacetate, a fact that has been observed for similar compounds.*^ Therefore, - 1 8 0 -i f the a-hydroxyisoxazole 166 s t i l l belonged to the normal-series and isomerization indeed occurred during the acid-catalyzed reaction, dehydra-tion of 180 should furnish an olefinic diacetate showing different character-i s t i c s to those observed for compound 175, that is a diacetate belonging to the normal usnic acid series. However, when the reaction was carried out a 70% yield of diacetate 175 (ie. iso-series) was obtained, thus indicating that acid-catalyzed dehydration is not the step causing the isomerization, but that such change must have occurred during the base cleavage. This result, when coupled to the known st a b i l i t y of 0-alkyl derivatives of methylphloroacetophenone (87) towards both C-alkylation 169 and base cleavage, does suggest that, whereas removal of the aromatic acetyl grouping should occur in a ring-open intermediate that allows free rotation about the C - C ^ bond, the carboxylic acid 167 arises from hydroxide ion attack at the C-l position of the starting isoxazole with subsequent cleavage of the C^-Ci bond and formation of a benzofuran system (Figure 58). The two i n i t i a l reactions (ie. Michael addition to the C.-C, double 4 4a bond and the opening of ring C) are of a competitive nature, since i t was found that under similar conditions formation of the carboxylic acid 167 does not occur when the C^-methoxy isoxazole 192 is used as starting material (vide infra). ( 19?.) - 181. -( 158) KOH-ROH 58. Proposed Mechanism for the Base Cleavage of (+)-Isoxazol [4,5-b]usnic Acid (158). - 182 -The importance of the carboxylir acid 167 (Figure 49) in determining the isomeric relationship between isoxazoles 158 and 160 was stressed when, as expected, no analogous acid was isolated during the attempted base cleavage of compound 160. Keeping in mind that both materials are derived from the same oxime intermediate, as shown by hydro-genolysis experiments, an analysis of the postulated structures (Figure 48) clearly indicates that only 158 and not 160 would produce, upon cleavage of the Cyb'C^ bond, an isoxazole-4-carboxylic acid such as 167. The compound isolated from the base cleavage of isoxazole 160 apparently resulted from hydroxide ion attack at the carbon-nitrogen double bond, a reaction that has been observed for some 3-substituted isoxazoles having a free 5-position. Such an addition is strongly influenced by the 281 321 nature of the substituent at the 3-position. ' Thus, while the ul t r a -violet spectrum O M 327, 289, 222 nm) of (+)-hydrated is'jxazolo [5,4-a] usnic acid (181) lacked the absorption at 258 nm observed for the parent isoxazole 160, i t s infrared spectrum showed the characteristic bands of the unsaturated carbonyl system (1670 cm "*") and the chelated aromatic acetyl grouping (1635 cm ^) . In i t s pmr spectrum the absorption assigned to the isoxazole methyl group (62.28) was found to occur at higher field than the corresponding methyl group signal on the parent isoxazole (62.60), a fact that is consistent with the proposed loss of aromaticity upon hydroxide ion addition to the isoxazole C-N double bond. Moreover, whereas the required N-H proton appeared as a broad one-proton signal at 63.63, the characteristic absorptions corresponding to the chelated aromatic methyl ketone (62.67) and the olefinic proton (65.89) suggested that no further changes had been introduced in the molecule. The molecular formula C ^ l l 0 N, derived from analytical data and high resolution - 183 -mass spectrometry measurements, as well as i t s mass spectrum (m/e 359, 344, 341, 43) supported the postulated hydration of the isoxazole r i n g . H ( 1 81 ) Having previously determined that the isomerization step occurs during the base treatment, the necessity of preparing modified s t a r t i n g materials became obvious. Upon analysis of the isomerization mechanism postulated i n Figure 58, three alternatives were considered: i ) direct cleavage of the acetyl grouping without solvent addition to the double bond; i i ) preparation of intermediates having a blocked Cg-phenolic hydroxyl group, and i i i ) synthesis of compounds possessing a C^-substituent such that, without allowing the opening of the five-mernbered r i n g , would permit, the f a c i l e regeneration of the C.-C. double bond. 4 4a The f i r s t alternative had already been tested i n the anhydrophenyl-hydrazone case (vide supra) where i t was found that although Michael addition to the double bond can be avoided by a judicious choice of solvent, the main cleavage product i s a carboxylic acid s t i l l possessing the aromatic acetyl grouping. However, the oxazocine 159 presents an i d e a l l y suited system to test our second a l t e r n a t i v e , namely the C -hydroxy group has been protected by formation of an eight-membered ring, the only unknown parameter being, the 184 -s t a b i l i t y of tin- 0-N bond in such a system towards severe a l k a l i n e treatment. Thus, when (+)-2H-[ 1,2]oxazocinousnic acid (159) was treated with 20% aqueous potassium hydroxide at room temperature for 1.5 hours, the form-ation of several products wis observed. After p u r i f i c a t i o n by column chromatography on s i l i c a gel the following components were isolat e d (Figure 59): The least polar product, isolated i n 10% y i e l d , was characterized as (+)-a-hydroxy-2!I-[1,2]oxa?oeinousnic acid (182). A n a l y t i c a l data and high resolution mass spectrometry indicated the molecular formula C , CH 0 N lo 17 7 for this material, mp 211.5-212.5°, [ a l ^ (CH CN) +158°. Its u l t r a v i o l e t spectrum 3'30, 285, 223 nm) was very si m i l a r to that of the previously isolated analog (+)-rt-hydroxyisoxazolo [4,5-b]usnic acid (163), while i t s infrared spectrum had absorptions c h a r a c t e r i s t i c of the C-l carbonyl group (1685 cm ^) and the chelated aromatic acetyl grouping (1620 cm 1 ) . As usual the pmr spectrum of 182 proved most informative showing the expected signals for the C^-methyl group and the aromatic methyl ketone at 6 2.37 and 2.53, respectively. Whereas the enamine proton was seen as a broad one-proton signal at 6 3.10, the C^-methylene group appeared as an AB quartet centered at 63.12 with coupling constant J = 18 Hz. This c h a r a c t e r i s t i c chemical s h i f t , when compared to the data presented i n Table 15, c l e a r l y indicated that the angular hydroxy group must exist i n the a-configuration. Such an assignment was confirmed by examination of the corresponding molecular models from which i t can be seen that a nucleophile w i l l approach the C^-C^ double bond exclusively from the a-side. Moreover, once the C, -a-config-4a uration has been established, one of the protons on the C^-methylene group w i l l l i e direct ly above the plane of the aromatic A r i n g , thus experiencing C O C H ( 159 ) C o m p o u n d COCH (182 ) ( 183 ) COCH (184) Y i e l d , 10.1 60.9 28 M gure 59. The Base Cleavage of (+)-211-[ 1, 2]oxazocinousnic Acid (159) 18f> -a shielding that results in the observed shift to higher f i e l d . The circular dichroism properties displayed by this compound: A ( A F ) max 276 (+3.02), 250 (+3.45) a n d 226 nm (-7.32), could be directly correlated w i t h those presented i n Table 14 for the [4,5-b]isoxazole 158, thus demon-strating the general a p p l i c a b i l i t y of our conclusions. The main component, isolated in 61% yield, was shown to be (+)-ct-hydroxydesacetyl-2H-\1.2]oxazocinoisousnic acid (183) and was obtained as colorless prisms, mp 227-229°, [ct] D (CH^CN) +284°. Its ultraviolet spectrum (^ m a x 287, 263 and 217 nm) proved very similar to that of (+)-ct-hydroxydesacetylisoxazolo[4,5-b]isousnic acid (166) and showed the expected hypsochromic s h i f t t y p i c a l , of the. deacylated materials, while in the infrared spectrum absorptions at 1700, 1630 and 1600 cm 1 were assigned to the ring C 0-diketoenamine system. The pmr spectrum of 183 showed the characteristic three-proton singlet, at 62.40 corresponding to the C ^-methyl group (the oxazocine methyl group) and a two-proton AB quartet centered at 63.10 with coupling constant J = 18 Hz that was assigned to the C^-methylene group. As before, this chemical s h i f t , indicates an cc-configuration for the angular hydroxyl group. Other r e l e v a n t signals are the one-proton singlet at 66.08 corresponding to the C -aromatic proton and a broad one-proton absorption at 68.16 assigned to the enamine proton. Further support for the C -ct-configuration was obtained from the CD spectrum of 183, which showed maxima of similar i n t e n s i t y and displayed identical Cotton effects to those observed for compound 1.66 (Table 14): A (Af:) 300 (+4.54), 268 (+10.26), 239 (-3.50) max and 218 nm (-6.01) . The f i n a l product, the optically inactive unsaturated acid 184, proved to be very important since it war. instrumental in determining the 1 8 7 -presence of a free carbonyl group at the one-position (ie. usnic acid numbering system), similarly to the analogous carboxylic acid 167. In the ultraviolet spectrum i t showed absorptions at 356, 287 and 237 nm. Its infrared spectrum presented at. 1685 cm 1 the characteristic absorption for a dimeric u.C-unsaturated carboxylic acid, while the bands at 1640, 1620 and 1590 cm 1 were assigned to the chelated aromatic acetyl grouping, 277 the carbonyl group oi a B-amino-a,B-unsaturated ketone system (vinyl-ogous amide) and the enone system double bond, respectively, since i t is well known that in such systems the carbonyl band appears at unusually 249,277 low frequencies. ' Furthermore, the pmr spectrum of 184 indicated the formation of a benzofuran system quite clearly, showing absorptions at 62.11, 2.33 and 2.73 assigned to the aromatic methyl group, the benzofuran methyl group and the aromatic acetyl grouping, respectively, whereas the olefinic methyl substituent appeared at 52.46, the C -methylene group was 6 seen as a two-proton singlet at 64.28. It is important to bear in mind that, as in the [4,5-b]isoxazole case, the isomeric nature of compound 183 (Figure 59) was not realized at this stage, since, direct comparison with analogous materials belonging to the normal series was not available. However, the isomerism was recognized when, upon dehydration, w e isolated an 0,N-diacetyl derivative which on alkaline hydrolysis furnished a phenolic oxazocine whose spectroscopic and chromatographic pr i>perties were different to those previously observed for the starting material 159. The r i • f o re , (+)-(• ~hyd roxydesace ty 1.-2H- [ 1, 2 ] oxazocinoisousnic acid (183) upon dehydration with 0.5% concentrated sulfuric acid in acetic anhydride at 55° produced the following pure components (Figure 60): - 188 Compound Yield, % ( 187 ) Figure 60. The Acid-Catalyzed Dehydration of (+)-a-Hydroxyde.sacetyl-211- [ 1, 2 ]oxazocinoisousnic Acid (183). .1.89 -The main product., isolated in 80% yield, was characterized as (+)-N-acetyl-drsacetyl-?!l-[1,2]oxazocinoisousnic acid acetate (185), colorless prisms from ethyl alcohol, mp 154-155°, [u}^ (CH^CN) +205°. It has been pr«wJ.ou«iy shown that the spectroscopic properties displayed by compounds belonging to the oxazocine series are indeed very similar to those observed for the isoxazole series, and compound 185 was no exception to this rule. Its ultraviolet spectrum (^max 300, 243, 216 nm) proved to be identical to that of (+)-desacetylisoxazole[4,5-b]isousnic acid diacetate (175), while in its infrared spectrum absorptions corresponding to the phenolic acetate ( J 760 cm 1 ) , the enone system (1675 cm "*") and the ct,6-unsaturated tertiary amide (1660 cm "*") were observed. The pmr spectrum of 185 (Figure 61.) showed two new three-proton singlets at 62.32 and 2.50 assigned, respectively, to the C -phenolic acetate grouping and to the N-acetyl moiety. Whereas the olefinic proton was seen as a one-proton singlet at 66.02, the aromatic proton, observed at 66.76 due to the deshielding influence of the C -phenolic acetate grouping, appeared at a very similar chemical shift to that found for the analogous ol e f i n i c isodiacetate 175, thus suggesting that the isomerization step had not been at a l l eliminated. '1 i V: i sooc I 2500 10O0 sL Jo 1 ( 185 ) N A c -l_J_J__l_i_l_l-| I I | | | | , | | T l I i , I , , , ; | , ppm (6) 0 Figure 61. Proton Magnetic Resonance Spectrum of (+)-N-Acetyl-desacetyl-2H-[l,2]oxaZocinoisousnic Acid Acetate (185). - 191 -Such a result can only be explained if we consider that the oxazocine 0-N bond exists, under strong alkaline conditions, in an equilibrium with the corresponding phenolic hydroxylamine. However, upon acidification such equilibrium must be shifted towards the cyclic oxazocine intermediate. As expected, the CD properties displayed by this sample, A (Ae) 328 (+17.02), 265 (-7.10), 225 nm (-16.41), were in good agreement with the previously recorded data for the isoxazole diacetate 175. The second component isolated from the dehydration mixture (+)-N-acetyl-[1,2]oxazocinoisousnic acid acetate (186), resulted from an acid-catalyzed acylation of the aromatic ring. This material was obtained in 6% yield as a colorless foam that could not be induced to cr y s t a l l i z e . Its ultraviolet spectrum had absorptions at 385, 310, 277, 241 and 227 nm, and in the infrared spectrum the characteristic bands of the phenolic acetate (1775 cm "*"), the aromatic acetyl (1690 cm ^ ) , the ^none system (1670 cm "*"), and the tertiary amide (1660 cm 1) were observed. In agree-ment with the molecular formula C„0H., 0oN, derived from analytical and high 22 19 o resolution mass spectrometry measurements, i t s pmr spectrum showed three-proton singlets at 62.33, 2.45 and 2.50 corresponding to the C^-phenolic acetate, the aromatic acetyl grouping and the N-acetyl moiety, respectively, in addition to the one-proton singlet at 65.90 assigned to the C^-olefinic pro ton. The f i n a l component, (+)-N-acetyl-a-ac.etoxydesacetyl [1, 2 ]oxazocino-isousnic acid acetate (187), was isolated in 5% yield as colorless prismatic needles, mp 182-183°, [a] 2, 6 (CIKCN) +112°. Its ultraviolet spectrum (A D 3 max 280, 238, 223 nm) proved quite similar to that of the u-acetoxyisoxazole 180, while i t s infrared spectrum presented the expected absorptions for the acetate - .192 -groupings (1761 cm x) and the C-l carbonyl group (1695 cm X ) . Its pmr spectrum was very informative showing signals for the C^-a-acetoxy grouping (62.04), the phenolic acetate (62.24), and the unsaturated N-acetyl moiety (62.38). The C^-methylene protons appeared as an AB quartet, centered at 63.65, with coupling constant J = 18 Hz, whereas the aromatic proton was seen as a one-proton singlet at 66.70. Both these values are in agreement with the observed chemical shifts for the analogous a-acetoxyisoxazole diacetate 180 (vide supra). Alkaline hydrolysis of the 0,N-diacetyl-C-acylated oxazocine 186 furnished the f i r s t iso-series intermediate available for direct comparison with the normal series, (+)-2H-[l,2]oxazocinoisousnic acid (188). The hydrolysis was performed at room temperature in tetrahydrofuran solution by using freshly prepared 0.8% aqueous sodium hydroxide. Compound 188 was isolated as colorless crystals, mp 170-172°, [a] (CH CN) +357°, and showed an mp depression when mixed with authentic (+)-2H-[1,2joxazocino-usnic acid (159). Although i t s ultraviolet spectrum (X 325, 281 and 233 max nm) and infrared spectrum (1655, 1625 cm ^) proved very similar to those of 159, the main differences were derived from i t s pmr spectrum (Table 19), especially for the absorptions corresponding to the oxazocine methyl group (62.58), the C^-olefinic proton (66.01) and the enamine proton (67.45). Although at this point i t was certain that we were dealing once more with iso-series derivatives, we decided to complete the necessary steps - hydrogenolytic cleavage of the oxazocine 0-N bond followed by alkaline regeneration of the ring C 3-triketone system - in order to complete the synthetic sequence outlined in Figure 62. Thus, (+)-N-acetyl-[1,2]oxazocino-isousnic acid (185) was dissolved in absolute ethyl alcohol and hydrogenolyzed Functionality Compound, ppm (6 units) ( 1 59) (188) 1.87 1.82 V ™ 3 2.12 — Cg-CH3 — 2.15 C n - C H 3 2.48 2.58 C.-COCH 6. —3 2.74 — Cg-COCH — 2.75 C.-H 4 — 6.13 6.01 N-H 4.38 7.45 C -OH 13.61 13.63 Table 19. Proton Magnetic Resonance Data of (+)-2H-[1,2]Oxazocinousnic Acid (159) and (+)-2H-[l,2]Oxazocinoisousnic Acid (188). COCH NAc ( 1 8 8 ) ( 1 8 6 ) ( 1 7 8 ) Figure 62. The Preparation of (l-)-8-l>esacetylisousnic Acid (178) from (+)-2H[l,2]oxazocinousnic Acid (159). over Platinum Oxide (PtO^) at room temperature. The resulting intermediate, the N-acetyl (3-diketoenamine 189, was not isolated since thin layer chroma-tography analysis of the crude reaction mixture indicated essentially a quantitative conversion. Therefore, this material was dissolved, without further purification, in tetrahydrofuran and then treated with freshly prepared IN aqueous sodium hydroxide at room temperature. Usual work-up produced in 80% yield the expected (+)-8-desacetylisousnic acid (178), identical in a l l respects with the material isolated from the isoxazole route (Figure 56). However, from the preparative point of view i t is important to note that i f the preparation of either (+)-8-desacetylisousnic acid (178) or (+)-isousnic acid (112) is the f i n a l synthetic goal, by a l l means the oxazocine sequence (Figure 62) should be preferred since in general fewer intermediates and higher yields are involved. A f i n a l attempt to overcome the isomerization stsp, and therefore render the previous approaches useful synthetic sequences leading to the normal series, was carried out bearing in mind that perhaps a second base-catalyzed rearrangement was feasible. Thus, when (+)-N-acetyl-desacetyl-[1,2]oxazocinoisousnic acid acetate (186) was submitted to the original cleavage conditions, namely 20% aqueous potassium hydroxide at room tempera-ture, two products were obtained. The least; polar one was shown to be (+)-desacetyl-2H-[1,2]oxazocinoisousnic acid (190), a compound whose isoxazole analog had been previously isolated (ie. (+)-desacetylisoxazole[4,5-b]iso-usnic acid (176)). Analytical data and high resolution mass spectrometry measurements provided the molecular formula C, J-l ON. Whereas i t s ultra-16 13 5 violet spectrum ( A m 309, 226, 206 nm) proved very similar to that of 176, it:; infrared spectrum presented the absorptions characteristic of the fl-- 196 -322 -1 dikctoenamine system (3205, 1650, 1631 cm ). The pmr spectrum of 190 showed a three-proton singlet at 62.54 corresponding to the oxazocine methyl group in addition to two one-proton singlets at 55.93 and 6.27 that were assigned to the C.-olefinic- and C„-aromatic protons, respec-4 o tively. The second material, obtained in 57% yield proved to be identical with the previously isolated (+)-a-hydroxydesacetyl-2H-[1,2]oxazocino-isousnic acid (183), thus showing that a second base-catalyzed isomeriza-tion cannot be performed. Moreover, acetylation of (+)-desacetyl-2H-[1,2]oxazocinoisousnic acid (190) with acetic anhydride-pyridine furnished exclusively the corresponding 0,N- diacetyl derivative 185, which can be converted back to 190 by mild alkaline hydrolysis (1% aqueous sodium hydroxide at room temperature). 0 NH (190) - 197 -Going back to the previously considered a l t e r n a t i v e s (vide supra), the l a s t proposal consisted on the preparation of intermediates possessing a C ^ - s u b s t i t u e n t such that, without allowing opening of the five-membered r i n g , would permit the f a c i l e regeneration of the C.-C, 4 4a double bond. One such substituent could very w e l l be a methoxy group, as indicated by the researches of B a r t o n 1 5 ^ and T a k a h a s h i , 1 6 2 as well as by our own experience i n e l i m i n a t i o n of the angular methoxy group i n (-)-usnic acid isomethoxide monoacetate'''6"'''162 (82) by the use of boron tribromide i n dry dichloromethane, therefore, we decided to turn our a t t e n t i o n towards the preparation of C^ a-raethoxy[4,5-b]isoxazoles. When the isoxazole 158 was treated with 20% methanolic potassium hydroxide at 50°, a mixture of the epimeric C^-cx- and B-methoxy d e r i v a t i v e s 192 and 191 was i s o l a t e d i n approximately 60% y i e l d . Both materials showed the same molecular formula C QH 0 N and presented very s i m i l a r u l t r a v i o l e t i y l y / spectra (X 328, 283, 228 nm and X 333, 283, 228 nm, r e s p e c t i v e l y ) . However, i n t h e i r i n f r a r e d spectra some diff e r e n c e s were encountered; whereas 26 the l e s s polar m a t e r i a l , the C^-B-methoxyisoxazole 191, mp 152-154°, [°i]D (CH^CN) +120°, had absorptions at 1660 and 1620 cm 1 corresponding to the i s o x a z o l y l ketone and the chelated aromatic a c e t y l grouping, the more polar 26 C. -a-methoxyisoxazole 192, mp 222-224°, [ a ] n (CH„CN) +188°, showed bands 4a u 3 at 1690 and 1620 cm ^. Based on the preceding observations on the character-i s t i c chemical s h i f t s of the C^-methylene group protons, we were able once more to derive a stereochemical assignment f o r the C ^ - s u b s t i t u e n t . The pmr spectra of compounds 191 and 192 i s shown i n Table 20. Furthermore, the r e l a t i v e amounts of i s o l a t e d materials indicated an 80:20 r a t i o of C, -a- to 4a C, -g-substituted m a t e r i a l s . 4a 198 -C O C H , MeOH ( 158) Functionality C9b- C» 3 C8-CH3 C I R C H 3 C,-C0CHo 6 —3 V»2 C. -aOCH-4a —3 C9-OH C7-OH 90CH3 OMe ( 191 ) COCH 3 OMe ( 1 9 2 ) Compound, ppm (6 units) (191) (192) 1.75 2.06 2.46 2.60 3.66 (J = 18 Hz) 3.55 8.61 13.33 1.95 2.06 2.45 2.63 3.18 (J = 18 Hz) 3.50 6.61 13.45 Table 20. Proton Magnetic Resonance Spectra of (+) - p.-Me thoxy isoxa zolo[4,5-b] usnic Acid (191) and (+)-a-MetboxyJsoxazolo[4,5-b1usnic Acid (192) - 199 -However, due to low yields obtained in the preparation of the [4,5-b]isoxazole 158 (see Table 10) we were forced to investigate other synthetic routes leading to the C. -methoxy derivatives 191 and 192. In 4 a 1953, Takahashi"'"^1 was able to prepare in good yield (+)-usnic acid iso-methoxide monoacetate bisphenylhydrazone monoanhydride (85) by refluxing an 161 ethanolic solution of (-)-usnic acid isomethoxide monoacetate (82) with phenylhydrazine hydrochloride-sodium acetate. Moreover, compound 85 can be easily converted to the phenylhydrazone monoanhydride 86 by treatment 162 with methanolic hydrochloric acid. Based on these results, we decided ( 8 6 ) • - 2 0 0 -to attempt the laboratory preparation of an analogous oximeanhydride (ie. isoxazole) derivative possessing the relevant C^-methoxy substituent. (-)-Usnic acid isomethoxide monoacetate (82) was originally obtained by Takahashi 1 6 1 by an acid-catalyzed methanol addition to the C^-C^^ double bond of (+)-usnic acid diacetate (68b) with concomitant cleavage of the C^-phenolic acetate. However, i t s preparation was consider-ably improved by the use of 10% anhydrous methanolic hydrogen chloride, prepared by careful addition of an appropriate amount of acetyl chloride to cold anhydrous methanol,and a shorter reaction time. Thus compound 82 has the molecular formula C2i H22°9' 3 S s n o w n analytical data and high resolution mass spectrometry measurements, and was obtained as pale yellow prisms, mp 167-168° ( l i t . 1 6 1 mp 167-168°), [ a ] ^ 6 (CI^CN) -54° ( l i t . 1 6 1 [ ' i ] ^ (CHCl^) -83°). Its ultraviolet spectrum showed absorptions at 346, 267 and 216 nm, while i t s infrared spectrum presented the characteristic bands corresponding to the phenolic acetate (1760 cm 1 ) , the enone system (1670 cm 1 ) , the aromatic acetyl grouping (1630 cm *) and the chelated 201 -triketonesystem (1560 cm L). The proton magnetic resonance data observed for the epimeric a n c' a-methoxyisoxazoles 191 and 192 (Table 20) shed some light on the structural problem presented by the sp l i t t i n g observed in the pmr spectrum of compound 82 (Figure 63). Although 162 Takahashi and coworkers originally proposed that such a spli t t i n g was due to the existence in solution of the tautomeric structures 82a and 82b ( 68) Q) R = H; R = A c b) R, R'= A c we have now demonstrated that it is indeed caused by the epimeric nature of the C. -methoxy group. That is, methanol addition to the C -C, double bond 4a 4 4a takes place from both sides of the essentially planar diacetate molecule 68b. In fact, the proton magnetic resonance spectrum of (-)-usnic acid isomethoxide monoacetate (82), when examined accordingly to our previous results, clearly - 20') -supports the presence of epimerio C^-substituents (Table 21) in a 60:40 relative ratio with the a-methoxy derivative being the predominant epimer. Functionality Compound 82, cx-0CH3 ppm (<5 units) B-OCH C9b- C«3 L.68 1.79 C8-CH3 1.91 1.94 C9-OCOCH3 2.36 2.40 C2-COCH3 2.44 2.58 C.-C0CHo 6 —3 2.64 2.64 3.16 3.09 (J = 16 Hz) (J = 16 Hz) C4a- O Cii3 3.47 3.42 C7-0H 13.22 13.25 C3-OH 17.64 18.25 Proton Magnetic Resonance Spectrum of (-)-Usnic Acid Isomethoxide Monoacetate (82) . Although the chemical shift of the C^-methylene groups seems to be reversed when compared to what, had been previously observed, i t must be remembered, however, that we are now dealing with O-acetylated species for which apparently the inverse re I a 1 i < mshLp holds. Nevertheless, our assign-- 204 -ments were made only after the synthetic modifications leading to the methoxy derivatives 191 and 192, previously isolated from the base-catalyzed methanol addition to the [4,5-b]isoxazole 158, indicated an identical 60:40 relative ratio of -a- to 3-epimers in the final products. Moreover, i t ( 1 9 1 ) ( 1 9 2 ) R e l a t i v e R a t i o : 40 :60 is interesting to note that whereas the base-catalyzed methanol addition (vide supra)produces an 80:20 epimeric ratio, the acid-catalyzed reaction furnishes a 60:40 distribution with the m-methoxy component (ie. cis-B/C junction) being predominant in both instances. Thus compound 82 was f i r s t treated with hydroxylamine hydro-chloride in dry pyridine at room temperature in order to form the corres-ponding oxime, which without further purification was thermally cyclized in methanolic solution to produce in 98% yield (+)-methoxyisoxazolo[4,5-2 6 bjusnic acid monoacetate (193), pale yellow prisms, mp 190-191°, [ a J n (CH^CN) +105°. It showed in i t s ultraviolet spectrum absorptions at 346 and 266 nm, while bands at 1780 cm 1 (phenolic acetate), 1700 cm - 1 (C-l carbonyl group) and 1640 cm 1 (chelated aromatic acetyl grouping) were observed in i t s infrared spectrum. Unexpectedly i t s pmr spectrum con-sisted in only one set of lines with the C -methoxy group appearing as 4-a a three-proton singlet at 63.50 and the C^-methylene group as an AB quartet centered at 63.63 with coupling constant J = 18 Hz. The molecular formula C 0 1H 0 N resulted from both analytical and high resolution mass spectro-21 21 o metry data. The monoacetate 193 was then hydrolyzed under mild alkaline conditions (IN sodium hydroxide at room temperature) to produce a 64:36 ratio of a-methoxy and B_methoxyisoxazolo[4,5-b]usnic acid (192 and 191) respectively, in addition to a small amount of unreacted starting material. Two other points of interest were investigated at this time. One of them consisted in the study of alternate ways for the removal of the angular methoxy group based on i t s acetal properties. However, when the acid-catalyzed hydrolysis (60% perchloric acid) of (-)-usnic acid isomethoxide monoacetate (82) was attempted, the resulting product proved to be the C -4a 2 6 a-methoxy isomer 194, mp 171-172°, [ ( * l n (CH CN) -63°, indicating that only epimerization had taken place under such conditions. This epimerization must be the result of selective protonation on the dihydrofuran ring oxygen - 206 -atom with concomitant ring opening and recyclization to the more stable a-isomer (ie. cis-B/C junction). 3 The second study, related to the hydrolytic cleavage of the acetate moiety in compound 193, showed that even under strong alkaline conditions the C^-methoxy derivatives do not rearrange to the iso-series for as long as the; aroin.il- it: acetyl grouping forms part of the molecule. Thus treatment of the inonoueotate 82 with 16% aqueous potassium hydroxide at room temperature for one hour produced, as expected, (-)-usnic acid isomethoxide (195), i d e n t i c a l in a l l respects to the material isol a t e d from the acid-eatniy/.ed hydrolysis of the Cg-phenolic a c e t a t e . 1 6 1 ' Moreover, compound 195 was easily converted to the known normal-series derivative decarbousnic ac id"* ' "* ^ ' (57) by hot 98% formic acid."'"6"'" OR" (57 ) R.h? =H However, the attempted cleavage of the aromatic acetyl grouping in compound 19'! produced not only the expected deacylation reaction but removal of the a n g u l a r methoxy group w i t h accompanying isomerization as we The reaction pr.vluel, i s o I at od in 86% y i e l d , was shown to be i d e n t i c a l to - 208 -the iso-series derivative (+)-a-hydroxydesacetylisoxazolo[4,5-b]isousnic acid (166), a material previously obtained during the base cleavage of the [4,5-b]isoxazole 158, by comparison of their spectroscopic and chromatographic properties and by its acid-catalyzed dehydration to the known (+)-desacetyl-isoxazolo[4,5-b]isousnic acid diacetate (175) (see Figures 49 and 56). Worthwhile noting is the fact that the formation of the carboxylic acid 167 was not observed during the base cleavage of the C^-methoxyisoxa-zole 193, thus adding support to the proposed competitive and mutually excluding nature of the attack at the C-l position with accompanying cleavage of the C -C. bond versus solvent addition to the C.-C. double bond (see 9b 1 4 4a 209 -Figure 58). Since a l l the previous synthetic routes failed to produce the required normal-series deacylated materials, i t became quite clear that the only way to stop the isomerization was through the use of stable 9-0-substituted compounds such as the corresponding ether derivatives, obtain-able by reacting the C-9 phenolate anion with a suitable alkyl halide. The preparation of usnic acid-derived "salts" was reported as 323 early as 1866 by Hesse, although i t is now certain that the drastic conditions utilized in some instances (ie. refluxing aqueous barium hydroxide) caused ring cleavage as well. Therefore, the careful analysis of the described experimental conditions and postulated structural formulas is 196 recommended, since we found that the so-called "ammonium usnate" (ie. the ammonium salt of usnic acid), prepared by bubbling dry ammonia gas 197 through a suspension of (+)-usnic acid (35) in absolute ethyl alcohol, 2 11 is no other than the previously isolated (-)-A ' -enaminousnic acid (132). - 210 -In 1953, Robertson and coworkers attempted the f i r s t preparation of an enol ether of usnic acid and assigned structure 197 to the material obtained upon methylation with dimethyl sulfate in aqueous sodium hydroxide. HO-(197) ( 198 ) 197 Although infrared spectroscopy investigations led Sharma to support the 324 previous assignment, Takahashi and coworkers demonstrated beyond any doubt by means of several degradation experiments, that the observed product, (+)-methylusnic acid (198), results from C-alkylation on the aromatic A ring rather than the expected ring C enol methylation. Moreover, the same 325-330 workers showed the generality of the reaction by preparing, under similar, conditions, (+)-methyldihydrousnic acid (199) as well as other derivatives. In general, O-alkylation competes significantly with C-alkylation only when in the active methylene compound involved the equilibrium concen-tration of the enol is relatively high (ie. 1,3-dicarbonyl systems and 331 phenols). However, the C-alkylation of phenols, a reaction mode observed 332333 169 for a number of phcnoxide ions ' including those of phloroglucinol and phloroacetophenone:,'^^ is a a well documented process. The choice of the appropriate reaction conditions is of prime importance in determining - 211 -the r a t i o of O-alkylated to C-alkylated products obtained, and a survey of 3 3 A ~~ 3 3 7 some of the abundant material a v a i l a b l e i n t h i s area shows r e a d i l y that i f a l k y l a t i o n at the more electronegative atom of an ambident anion (at oxygen rather than at carbon for an enolate or phenolate anion) i s desired, then whenever possible the reaction should be c a r r i e d out i n 335 s o l u t i o n ( i e . homogeneous mixture), using polar a p r o t i c solvents such as 336 338 339 3A0 dimethylsulfoxide ' " (DMSO), or hexamethylphosphoramide ' (HMPA). Their advantage over p r o t i c solvents ( i e . alcohols, water) l i e s i n the fact that they do not solvate the phenolate anion and, consequently, do not diminish i t s r e a c t i v i t y as a nucleophile. On the other hand, they do have i - 212 -the ability to solvate the cation, thus separating it from the cation-phenolate anion pair, and generating a relatively free anion which being more nucleophilic than the ion pair produces very substantial increases in 341 • the corresponding rates of alkylation. Since the chemical shift of an aromatic hydrogen is related to the electron density at the position of 342 the ring, recent proton nuclear magnetic resonance studies have shown that the upfield shift of the phenoxide hydrogens reflects the ionic character of the metal-oxygen bond (in aprotic solvents) and hence the solvating ability of the medium. Such shifts become quite apparent (AS 0.3-0.8 ppm) when solvents such as DMSO or HMPA are utilized, indicating the presence of largely ionic metal-oxygen bonds. ' 336 338 Moreover, i t has been demonstrated ' that the yields of O-alkylation increase in the enolate counterion series L i + < Na+ < K + < Cs + < R,N + 4 in correspondence with the augmented ionic character of the bond. Thus, 343 whereas large cation generally show enhanced rates of alkylation, dications such as the magnesium cation seem particularly effective in 344 supressing O-alkylation. The structure of the alkylating agent is also a factor of great importance, and i t has been found that relatively high 338 340 O-alkylation rates correlate with a low SN2 reactivity, ' with the 337 following leaving groups being preferred. _ _ _ _ BF 4~, CK>4 , TsO >C1 >Br~ >I In summary, present evidence suggests that in order to promote alkylation at the more electronegative atom of an ambident anion one should use a polar aprotic medium, a large counterion, low concentration of the - 213 -anion, a "hard" leaving group and an a l k y l a t i n g agent of low S^2 r e a c t i v i t y . 299. We must mention the fa c t that B e r t i l s s o n and Wachtmeister were able to i s o l a t e i n low y i e l d the f i r s t ether d e r i v a t i v e of (+)-usnic acid, (+)-7-0-methylusnic acid (200), by prolonged treatment with excess methyl iodide and potassium carbonate i n r e f l u x i n g acetone. Although the same workers were unable to prepare the corresponding diether, i t was 346 subsequently found that reaction of (+)-usnic acid (35) with ethereal diazomethane i n dichloromethane affords no simple O-methylated components but the t e t r a c y c l i c furan d e r i v a t i v e 201 i n 14% y i e l d . (200) ( 201 ) However, since the formation of such materials was very poor from a preparative point of view, preliminary experiments performed i n our 347 laboratory furnished the monoether 200 i n 34% y i e l d by reaction of ,. 196 . , , -, . 348,349 . , disodium usnate with trimethyloxonium tetratluoroborate i n dry hexamethylphosphoramide (HMPA) at room temperature. 350 In 1966, Sharma and Jannke reported t h e i r observations on the a c i d i c character of usnic acid as shown by spectrophotometric and non-aqueous t i t r a t i o n measurements. They found that whereas the former technique allows - 214 -the calculation of three pK values (4.4, 8.8 and 10.7), thus accounting for a l l three hydroxy! groups, the non-aqueous titration furnishes only two. Furthermore, when the two phenolic hydroxyl groupings are blocked, such as in the diacetate 68b, usnic acid s t i l l behaves as a monobasic acid, a fact that allows the assignment of the pK value of 4.4 to the highly acidic enol functionality at the 3-position. These authors assigned the pK values of 10.7 and 8.8 to the C -a 7 and Cg-phenolic hydroxyl groupings respectively, without any further experi-mental evidence, and based solely on the para relationship between the Cg-hydroxyl group and the chelated acetyl moiety. However, we feel that such results are not in complete agreement with the alkylation experiments 299 347 previously discussed, since i t has been found ' that ether formation occurs preferentially at the 7-position, regardless of the alkylating agent and/or base utilized, thus suggesting that the former assignment should indeed be reversed. Based on the experimental evidence thus far presented, we began the study of new high yielding methods to prepare mono- and diether derivatives of usnic acid. Therefore (+)—usnic acid (35) was f i r s t treated with tetra— 351 methylammonium hydroxide pentahydrate in dry hexamethylphosphoramide at 0° for one hour, and the resulting phenoxide ion alkylated with a-bromotoluene (benzyl bromide) to produce, after purification by column chromatography on s i l i c a gel, (+)-7-0-benzylusnic acid (202) in 75% yield. ' This material was 2 6 obtained as yellow prisms, mp 131-133°, [a] (CH^CN) +208°, and presented in i t s ultraviolet spectrum absorptions at 346, 266 and 239 nm. Its infrared spectrum i ne i ; ! ed the loss of chelation between the aromatic acetyl (1672 cm "*") and the C -piuT.o.'l.i.-e hydroxyl group, while the ring C chelated triketone system - 215 -remained unchanged (1672, .1.540 cm ^ ) . The pmr spectrum of the monoether 202, of molecular formula (25I"22^>7' P o s s e s s e c ' a ^ t n e information required in order to assign conclusively the site at which ether formation had taken place (Figure 64). As expected, the absorption corresponding to the C^-phenolic hydroxyl proton was not observed and, whereas the ring C acetyl grouping remained at its usual position (62.64), an upfield shift was seen for the aromatic methyl ketone (62.55); the methylene group of the C^-O-benzyl substituent appeared as a two-proton singlet at 64.86. Its mass spectrum (m/e 434, 416, 392, 343, 323, 308, 301, 281, 273, 260, 259, 241, 233, 215, 203 and 90) supported the postulated substitution as well. Although the minor component, isolated in 19% yield for an overall 92% yield of exclusively O-alkylation, had suffered complete racemization (probably during the isolation and/or recr.ystallization processes,), and was shown' to- be~the f i r s t diether derivative of usnic acid ever prepared. This result is in complete accord with previous observations (vide supra) that the 7,9-di-0-substituted derivatives of usnic acid (ie. the diacetate 68b) racemize very readily. Thus (±)-7,9-di-0-benzylusnic acid (203), mp 145-146°, has the molecular formula C ^ ^ g O ^ a n d shows in i t s ultraviolet spectrum absorptions at 368, 325, 259 and 226 nm, while in i t s infrared spectrum the bands corresponding to the aromatic methyl ketone (1680 cm ^) and the chelated triketone system (1680, 1560 cm 1) appeared somewhat shifted. From the chemical shifts of the angular (61.82) and aromatic (62.27) methyl groups i t can be deduced that the second benzyl group was not introduced on carbon, as observed for (•» -)-mot:hylusnic acid (198), but that the alkylation had taken place at the. oxygen atom of the Cg-hydroxyl group. Moreover, this assumption was thoroughly confirmed when, although no absorptions were seen for the - 216 -- 217 -phenolic hydroxyl groupings (610-14) a sharp one-proton singlet at 617.99 indicated the presence of the enolic functionality at the, 3-position (Figure 65). Whereas the rings A and C acetyl moieties absorbed at 62.50 and 2.58, respectively, the methylene group of the C^-O-benzyl substituent appeared as a two-proton singlet at 64.94, with the one corresponding to the C n-substituent seen as a partially obscured AB quartet y showing a geminal coupling constant J = 10 Hz. Such spl i t t i n g arises quite probably from a hindered rotation of the benzyl substituent at the 9-position, thus exposing the methylene protons to a diasteromeric environment. (203) The generality of our experimental procedure was tested by the alkylation of (+)-usnic acid (35) with a, p_-dibromotoluene (pj-bromobenzyl bromide). Tims (+)-7-0-p_-bromobenzylusnic acid (204), a compound required for X-ray studies regarding the determination of the absolute configuration of the angular methyl group on (+)-usnic acid, was isolated in 82% yield. This material, f " ] ^ (Cii.^ CN) +245°, produced pale yellow-green plates, mp 122-123°, when recrystal1ized from ethyl alcohol,and showed the molecular - 218 -- 2]9 -formula C. rH„.. 0_,Br. Its u l t r a v i o l e t spectrum (A 266, 229 nm) can be 25 21 7 max considered t y p i c a l f o r a l l the 7-0-alkyl ( a l k y l = methyl, benzyl) d e r i v a -tives of usnic a c i d ; whereas i t s i n f r a r e d spectrum presented the absorp-tions c h a r a c t e r i s t i c of the aromatic a c e t y l grouping (1670 cm "*") and the r i n g C chelated triketone system (1530 cm "*"), which again are t y p i c a l f o r the s e r i e s . I t s pmr spectrum (Figure 66) was most informative and showed the expected u p f i e l d s h i f t f o r the aromatic methyl ketone (62.56), while the triketone system a c e t y l grouping remained at i t s usual p o s i t i o n (62.66). Moreover, the methylene group of the C^-pj-bromobenzyl substituent was observed as a two-proton s i n g l e t at 64.86, with the aromatic r i n g protons appearing, under normal r e s o l u t i o n , as an "apparent" AB system (in r e a l i t y 253 they form part of a very complex AA'XX' system ) centered at 67.45 with coupling constant J = 8 Hz. F i n a l l y , two one-proton s i n g l e t s at 610.81 and 18.82 indicated the presence of the C -phenolic hydroxyl group and of the enol f u n c t i o n a l i t y at the 3-position, r e s p e c t i v e l y . Our postulated s u b s t i t u t i o n was supported by low and high r e s o l u t i o n mass spectrometry measurements. Although only low y i e l d s of diether d e r i v a t i v e s have been obtained by our f i r s t method, we have found that somewhat more vigorous conditions (sodium ' hydride i n dry tetrahydrofuran at 60°) produce good y i e l d s of d i -O-alkylated materials. This experimental procedure may also be used for the preparation of mixed-diethers. Thus, when (+)-7-0-benzylusnic acid (202) was alkylated under such conditions with dimethyl s u l f a t e , a 72% y i e l d of (±)-7-0-benzyl-9-0-methylusnic acid (205) was obtained. This compound, bright yellow a c i c u l a r prisms, mp 112-113°, was completely racemic, as expected, and showed the u l t r a v i o l e t spectrum (A 370, 320, 259, 226 nm) i max i - 220 -- 221 -c h a r a c t e r i s t i c of the diether s e r i e s (compare with compound 203). While i t s i n f r a r e d spectrum showed bands at 1680 cm 1 (aromatic, a c e t y l grouping) and 1670, 1540 cm 1 (chelated g-triketone system), i t s pmr spectrum (Figure 67) indicated the introduction of the new Cg-methoxy group with a sharp three-proton s i n g l e t at 64.06. The molecular formula C„,H„.0 zo 24 / was derived from a n a l y t i c a l data and high r e s o l u t i o n mass spectrometry. Moreover, t h i s route did allow the preparation of the f i r s t 9-0-a l k y l ether d e r i v a t i v e of usnic a c i d . Although i t was f e l t that hydrogen-i ,• i 352,353 T , „ , , , i_. 354 „. , o l y t i c cleavage or removal by other known reductive methods were not desirable, s e l e c t i v e debenzylations can be c a r r i e d out by the use of 355 356 boron tribromide i n dichloromethane, neat t r i f l u o r o a c e t i c acid (TFA), 357 or even better, t r i f l u o r o a c e t i c acid i n dichloromethane (used p r i o r to our experiments for the removal of 9-anthrylmethyl ethers). Thus, when compound 205 was treated with neat TFA or with t r i f l u o r o a c e t i c acid i n dichloromethane (10 equivalents TFA per benzyl group) at room temperature, y i e l d s of over 90% of the required (±)-9-0-methylusnic acid (206) were obtained. This material, b r i g h t yellow thick prisms, mp 195-196°, showed i n i t s u l t r a v i o l e t spectrum absorptions at 345, 274 and 228 nm, which we might consider representative of the 9-0-alkyl s e r i e s , while i n i t s i n f r a r e d spectrum bands c h a r a c t e r i s t i c of the C - l carbonyl group (1680 cm ^ ) , the chelated aromatic a c e t y l grouping (1625 cm ^ ) , and the r i n g C triketone system (1560 cm "*") were observed. I t s pmr spectrum (Figure 68) showed the disappearance of the signals corresponding to the benzyl group (see Table 22), while the three proton s i n g l e t assigned to the 9-0-methyl substituent was seen at 64.14. Moreover, the molecular formula C, nH o0_, was obtained by 19 18 7 a n a l y t i c a l and high r e s o l u t i o n mass spectrometry, and i t s mass spectrum Figure 68. Proton Magnetic Resonance Spectrum of (±)-9-0-Methylusnic Acid (206). - 224 -(m/e 358, 343, 274, 247, 246, 229) was t y p i c a l for a usnic acid derived molecule bearing a methoxy group i n the aromatic A r i n g . F u n c t i o n a l i t y Compound, ppm (6 units) 200 202 203 204 205 206 C9b- C^3 1.88 1.76 1.82 1.78 1.83 1.86 C 8-CH 3 2.20 2.18 2.32 2.18 2.25 2.24 C,-C0CHo 6 —3 2.62 2.55 2.50 2.56 2.54 2.74 C 2-COCH 3 2.66 2.64 2.58 2.66 2.54 2.56 C ?-OCH 3 3.80 — — — — — V O C H 3 — — — 4.06 4.14 C-,-OCH„C,Hc / —/ b 5 — 4.86 4.94 4.86 4.89 — C 9-OCH 2C 6H 5 5.36 (J = 10 Hz) C.-H 4 — 5.99 5.95 5.86 5.99 5.83 5.91 C?-OH 10.80 — — — — 13.22 — 10.78 — 10.81 — — C3-OH 18.80 18.80 17.99 18.82 18.15 18.00 Table 22. Proton Magnetic Resonance Data of Several Ether Derivatives of Usnic Acid. - 225 -' However, treatment of the racemic monoether 206 with hydroxylamine hydrochloride i n pyridine followed by an attempted thermal c y c l i z a t i o n of the "crude" oxime resulted i n a very complex reaction mixture that was not further investigated. We then decided to i s o l a t e and characterize the oxime intermediate, and i t was found that a d e r i v a t i v e showing the required molecular formula ^gH^gO^N could be i s o l a t e d i n 94% y i e l d by re a c t i n g (±)-9-0-methylusnic acid (206) with hydroxylamine hydrochloride i n a 1:1 mixture of e t h y l a l c ohol - 4% aqueous sodium hydroxide at room temperature. This material, (±)-9-0-methylusnic acid oxime (207), obtained as yellow prisms, mp 149.5-150°, presented i n i t s u l t r a v i o l e t spectrum maxima at 325, 268 and 228 nm, while i t s i n f r a r e d spectrum showed at 1660 cm 1 the band corresponding to an oxime C-N double bond, and at 1680, 1585 cm 1 the absorptions character-i s t i c of the r i n g C 8-triketone system. The pmr spectrum of 207 produced the evidence that allowed the determination of the p o s i t i o n at which condensation had taken place. Thus, whereas the chemical s h i f t s of the C2~acetyl grouping and the corresponding C-3 eno l i c proton remained constant at 52.55 and 18.08, - 226 -respectively, upfiold shifts were detected for both the aromatic acetyl grouping (52.45). now as the oxime derivative, and the C^-phenolic hydroxyl proton (512.26). Moreover, this spectrum showed that the methoxy substituent at the. 9-position (64.05) and the C^-C,^ double bond (65.83) were not affected under the reaction conditions. The mass spectrum (m/e 373, 358, 357, 328, 289, 273, 262, 246, 244, 44, 43) of 207 was again in complete agreement with the postulated structure. Therefore the introduction of a substituent blocking the Cg-phenolic hydroxyl group has completely altered the up-to-now observed reactivity of usnic acid towards the condensation with aliphatic or aromatic amines. The present reactivity is such that condensation reactions take place exclusively at the aromatic acetyl grouping. These observations offer now an explanation for the unsuccessful preparation of an isoxazole intermediate from (+)-usnic acid diacetate (68b), a reaction that resulted exclusively in par t i a l deacetyl-ation (vide supra). OH n3 ( 207 ) (208) - 227 -Furthermore, the existence of a ring A oxime derivative was demonstrated by the ring C opening of compound 207 in pyridine at 90°, thus producing 4-0-methyldecarbousnic acid oxime (208) in 47% yield. Its pmr spectrum was very similar to that of decarbousnic acid (57) i t s e l f . It showed the expected absorptions corresponding to the furan methyl group (62.30), the aromatic acetyl grouping (62.58), as the oxime deriva-tive, the methoxy group (A3.83) and a two-proton singlet at 63.68 assigned to the methylene bridge between the aliphatic 8-diketone and the benzo-furan ring. The molecular formula C. DH 0 N was derived from analytical lo 2.L o and high resolution molecular weight determinations. 347 At this point i t is important to mention that we also prepared the ultimate derivative, (+)-methoxy-9-0-methylisoxazolo[4,5-b]usnic acid (209). Thi.s racemic material, although not very useful for the f i n a l synthesis of the optically active metabolite 107 since i t would require additional optical resolution, was prepared via an acid-catalyzed methanol addition to the monoether 206 (racemic) followed by regular isoxazole formation, namely, in spite of the blocked 9-position solvent addition to the C^-C^a double bond regenerates the "normal" behavior of usnic acid towards conden-sation with amino compounds. However, this material proved altogether resistant towards alkaline cleavage of the ring A acetyl moiety even under quite drastic conditions (ie. refluxing 40% potassium hydroxide in a methanol-water mixture). These last, results marked the completion of a l l the synthetic sequences bavin®, n« pivotal step the removal of the aromatic acetyl grouping upon base treatment, since we were unable to achieve such cleavage while preventing, -thg accompany in); isomerization. - 228 -R = H or Me Of a l l the new approaches that were investigated, the most interesting one turned out to be that necessitating the transformation of the ring A acetyl function into another suitable, easy to remove, functional group. Such requirements were thoroughly fulfi l led by an amide grouping 358-360 (ie. an acetanilide derivative), since i t is well known that amide hydrolysis can be accomplished under a variety of conditions, and the resulting products, upon deamination, lead to the replacement of the amine c • i • i_ , i J 361—366 functionality by hydrogen. Thus treatment of the racemic monoether 206 under the conditions 367 described by Chrochet and Kovacic for the facile and high yielding conversion of o-hydroxyaldehydes and ketones into o-hydroxyanilides by 368 369 the use of monochloramine,' ' which is prepared by the reaction of cold - 229 -370 373 aqueous ammonia with sodium hypochlorite, ' ' produced the f i r s t r i n g -aminated d e r i v a t i v e of usnic a c i d , (±)-N-acetyl-6-amino-9-0-methylusnic acid (210) i n over 90% y i e l d . This material, which showed the molecular formula C^gH 0 N, was obtained as bright yellow prisms, mp 118-119°. In i t s u l t r a v i o l e t spectrum absorption maxima was observed at 305, 262 and 221 nm, while i t s i n f r a r e d spectrum indicated the existence of an associated secondary amide grouping (3300, 1660, 1550, 1280 cm ^ ) . The pmr spectrum of 210 (Figure 69) showed as well the presence of a secondary amide with absorptions at 62.34 and 7.70, assigned to the N-acetyl substituent and to the proton on the nitrogen atom, r e s p e c t i v e l y . Moreover, i t s mass spectrum (m/e 373, base peak, 331, 316, 289, 262, 260, 247, 246, 245, 232, 220, 115, 83) was t y p i c a l of the usnic acid s e r i e s (vide supra) with important fragments a r i s i n g from both the N-acylated and unsubstituted amine moieties v i a the important re t r o D i e l s - A l d e r and Hydrogen Transfer processes. The amide deacylation step was shown to proceed by el i m i n a t i o n of ketene. 372 373 Although i t has been demonstrated ' that i n o_-hydroxyacet-a n i l i d e s a c e t y l group migration occurs p r e f e r e n t i a l l y from oxygen to nitrogen, therefore precluding the formation of the corresponding acetates which would be much more susceptible towards hy d r o l y s i s , a v a r i e t y of new methods are a v a i l a b l e for breaking the amide bond. Amongst them there are examples of 374 . . . 375 . . 376 base cleavage, ammolysis, conversion to isothiocyanates, treatment 377 378 with a l k a l i n e metals or with aromatic anion r a d i c a l s i n a v a r i e t y of 379 solvents, as well as cleavage by e l e c t r o l y t i c methods, and ion exchange 380 r e s i n s . However, some of the milder methods require the formation of . . . 381 , . u , , . 382 lmido esters p r i o r to the actual h y d r o l y s i s . sooo I 3500 Figure 69. Proton Magnetic Resonance Spectrum of (±)-N-Acetyl-6-amino-9-0-methylusnic Acid (210). - 231 -However, attempted hyd r o l y s i s of the racemic methoxyamide 210 by formation of the corresponding a l k y l imidate 211 followed by a c i d i c 383—385 work-up, e i t h e r by use of triethyloxonium tetrafluoroborate, or 386 phosphorous pentachloride i n pyrid i n e , both f a i l e d producing only recovered s t a r t i n g material or very complex reaction mixtures that were not investigated, r e s p e c t i v e l y . Similar unsuccessful cleavages re s u l t e d from the acid-catalyzed hydrolysis (6N hydrochloric acid i n tetrahydrofuran) 387 388 and the attempted boron t r i f l u o r i d e etherate-catalyzed methanolysis. ' Therefore, i t was decided to remove the methoxy group at the 9-position before hydrolyzing the amide f u n c t i o n a l i t y , since previous experiments ( i e . r i n g opening to the decarbousnic acid d e r i v a t i v e 208) demonstrated the inherent u n s t a b i l i t y of compounds possessing a Cg-substituent. Thus, treatment of the methoxyamide 210 with boron tribromide under the 389 390 conditions described by McOmie and coworkers and by Tyman and Durrani produced i n 60% y i e l d an o p t i c a l l y i n a c t i v e compound for which we have postulated the dibenzofuran structure 212, which although r e t a i n i n g the amide - 232 -group as shown by i t s i n f r a r e d spectrum (3280, 1655, 1550, 1270 cm ), i t s pmr spectrum indicated the absence of the angular methyl group with concomitant cleavage of the methoxy substituent at the 9-position. More-over, the molecular formula C^H^O^N and i t s mass spectrum (m/e 345, 327, 303, base peak , 286, 285, 44, 43) seem to substantiate t h i s proposal. (213 ) Preparative layer chromatography of the mother l i q u o r s produced i n 12% y i e l d the desired component (±)-N-acetyl-6-aminousnic acid (213). Furthermore, whereas compound 213 was obtained i n 34% y i e l d by the use of 391 392 anhydrous aluminium t r i c h l o r i d e i n dichloromethane, ' the use of boron i 355 393-395 t r i c h l o r i d e allowed the i s o l a t i o n of the same material i n 81% y i e l d . Thus the racemic amide 213 formed bright yellow prisms, mp 228-230°, and showed i n i t s u l t r a v i o l e t spectrum absorption maxima at 335, 265 and 230 nm, while i t s i n f r a r e d spectrum indicated the presence of an amide grouping (3460, 1685, 1520, 1260 cm - 1). Its pmr spectrum (Figure 70) supported the r e t e n t i o n of the N-acetyl grouping (62.19), while the accompanying demethylation was demonstrated by the appearance of the one-proton s i n g l e t c h a r a c t e r i s t i c of the Cg-phenolic hydroxyl proton (69-97). I t s mass spectrum, s i m i l a r l y to that of the monoether 210, was t y p i c a l of the usnic acid s e r i e s . However, i f t h i s s e r i e s was to be used towards the preparation of metabolite 107, the synthesis of o p t i c a l l y a c t i v e amido compounds was of prime importance, and i t was decided to study the formation of o p t i c a l l y a c t i v e amides d i r e c t l y from (+)-usnic acid (35). Although treatment of 35 under the conditions of the Schmidt 396 reaction (sodium azide-conceutrated s u l f u r i c acid i n chloroform) resulted only i n recovered s t a r t i n g material, the r e a c t i o n with monochloramine 3 6^ i n 2% methanolic sodium hydroxide or even better i n 0.6% aqueous sodium hydroxide produced i n 52% y i e l d the o p t i c a l l y a c t i v e amide 214, yellow prisms, mp 230-232°, [a] (Ch^CN) +437°, which was further c y c l i z e d with o n 7 t h i o n y l c h l o r i d e to produce the corresponding (+)-oxazolo[4,5-h]usnic acid (215) i n 56% y i e l d . This material, molecular formula C „H A N , was (214 ) (215 ) - 234 -- 235 -26 obtained as yellow prisms, mp 173-174°, [«l n (CH^CN) +538°, and showed in i t s u l t r a v i o l e t spectrum maxima at 335, 268 and 218 nm, whereas i t s in f r a r e d spectrum presented the absorptions c h a r a c t e r i s t i c of the rin g C triketone system (1680, 1540 cm ^ ) . The pmr spectrum of 215 confirmed the presence of an i n t a c t C r i n g , with the six-proton s i n g l e t at 62.60 being assigned to both the C^-acetyl grouping and the oxazole methyl group (previously the methyl group of the N-acetyl f u n c t i o n a l i t y ) , Some complications began to surface when i t was found that protection of the r e a c t i v e r i n g C 3-triketone system by means of an isoxazole-group could not, be achieved from the amide 214 nor by a mono-chloramine re a c t i o n performed on the [4,5-b]isoxazole 158, Furthermore, i t was demonstrated that the attempted d i r e c t O-alkylation of the isoxazole 398 399 158 with either a-bromotoluene (benzyl bromide) or chloromethylmethylether, ' under the conditions previously described for usnic a c i d , resulted only i n complex product mixtures. A l k y l a t i o n under such conditions i s probably complicated even further because of the enhanced s u s c e p t i b i l i t y of the isoxazole i t s e l f and of a l l the ether d e r i v a t i v e s towards n u c l e o p h i l i c attack on the C^-C^a double bond, a fac t that was observed by means of proton magnetic resonance spectroscopy for the benzyl bromide r e a c t i o n . However, i f diether d e r i v a t i v e s of the isoxazole 158 could be prepared, a very i n t e r e s t i n g a l t e r n a t i v e remained yet to be tested. It involved the formation of amide f u n c t i o n a l i t i e s by means of the Beckmann 400 . . , , 401 . rearrangement, since i t has been shown that a n i l i t i e s are the mam product i s o l a t e d i n most reactions i n v o l v i n g aliphatic-aromatic ketoximes. ' 402 Moreover, a number of inv e s t i g a t o r s have observed that d i - o -a l k y l - s u b s t i t u t e d acetophenone oximes s u f f e r spontaneous rearrangement to - 236 -the corresponding acetaniLides under the strenuous conditions required to form such oxime intermediates from the parent ketones and hydroxylamine salts. This fac i l e reaction has been interpreted in terms of the so-called "Ortho-effect", which is attributed to the steric interaction between the ortho-substituents and the oxime functionality, resulting in the loss of coplanarity of the latter with the aromatic ring, and whereas the rate of rearrangement is roughly proportional to the dielectric constant of the solvent, n u c i g o p h i i i c solvents such as water, amines and alcohols tend to compete for the resulting imine intermediate, thus producing imino ethers, amidmes, etc.. Although we were aware of the d i f f i c u l t i e s previously encountered . , . , „ , , . , . , 402a,d,405 , in the oximation of 2,6-disubstituted acetophenone systems, the necessity of preparing ether derivatives of the isoxazole 158 resulted from the obervation that oximes of oMiydroxyacetophenones, or i t s hydrochlorides, yield benzoxazoles when subjected to the conditions of the Beckmann rearrange-ment. Therefore, we decided to test this new and interesting alternative with the racemic dibenzyl ether 203, and although i t was soon discovered that oxime formation under the usual conditions (ie. hydroxylamine hydrochloride-pyridine) did not occur readily, good yields of the desired oxime intermediate could be accompli sod by performing the reaction in 5% methanolic potassium hydroxide-water (2:1) at 50°. Moreover, a f a c i l e Beckmann rearrangement was observed when the crude oxime was treated with phosphorous pentachloride in anhydrous ethyl ether at room temperature j ^ " * " 3 a system that, favoring stereo-specific rearrangement, has been recommended for determining the configuration f . . , . r . 407,408 , t, . . or oximes on ttie basis of anti migration, however, this is not an 409 unequivocal method since Lansbury and Mancuso were able to demonstrate the - 237 -authentic n o n s t e r e o s p e c i f i c i t y of some Beckmann rearrangements. In t h i s manner (±)-N-acetyl-6-amino-7,9-di-0-benzylusnic acid (217) was obtained i n 48% y i e l d , as f i n e yellow needles, mp 168-170°, from the rearrangement of the syn-methyl oxime 216. I t s u l t r a v i o l e t spectrum ^max 3 3 ^ ' 265, 215 nm) proved to be very s i m i l a r to that of (+)-N-acetyl-6-aminousnic acid (214), obtained by the monochloramine rearrangement of HO (216) (217) (+)-usnic acid (35), while i t s i n f r a r e d spectrum indicated the presence of both an N-acetyl f u n c t i o n a l i t y (3430, 1680, 1520, 1250 cm"1) and the rin g C 3 - t r i -ketone system (1680, 1540 cm ^ ) . Furthermore, i t s pmr spectrum showed the expected three-proton s i n g l e t at 62.27 and a broad one-proton s i g n a l at 66.87 assigned to the Cg-N-acetyl grouping and to the secondary amide proton, r e s p e c t i v e l y . As previously observed f o r the diether 203, the benzyl ether substituents could be e a s i l y d i f f e r e n t i a t e d by t h e i r chemical s h i f t . Thus, - 238 -whereas the methylene protons of the benzyl group at the 7-position were seen as a two-proton AB quartet centered at 64.82, with coupling constant J = 10 Hz, the ones corresponding to the C-9 substituent appeared as an AB quartet (J = 10 Hz) centered at 65.25. The molecular formula C H 0 7N was derived 32 29 / from a n a l y t i c a l and high r e s o l u t i o n mass spectrometry data. Moreover, the same compound was obtained i n 62% y i e l d by the f a c i l e t h i o n y l c h l o r i d e -410 catalyzed Beckmann rearrangement of intermediate 216, thus suggesting the general a p p l i c a b i l i t y of such a r e a c t i o n i n the usnic acid s e r i e s . However, at t h i s time we were s t i l l confronted with the preparation of o p t i c a l l y a c t i v e r i n g C-protected diether d e r i v a t i v e s , and a f t e r several unsuccessful attempts (vide supra) i t was found that predominant O-alkylation of the [4,5-b]isoxazole 158 was achieved by the use of methyl i o d i d e - s i l v e r oxide i n chloroform, a method o r i g i n a l l y introduced by Purdie^"'" for the exhaustive methylation of carbohydrates and s u c c e s s f u l l y applied by Garden and 412 Thomson for the a l k y l a t i o n of the strongly chelated peri-hydroxyl group of juglone (218) ( 218 ) (219) 2 3 9 -The methyl ied ide-r>Liver oxide method complements adequately the alkylation c o n d i t i o n s previously developed for usnic acid (ie. tetramethyl-ammonium hydroxide pentaliydrate in dry hexamethylphosphoramide), since i t is applicable to the r i n g C protected derivatives such as the isoxazole 158. However, even under a v a r i e t y of experimental conditions (ie. by using different s o l v e n t s such as methanol,^ 1 1 chloroform,^ 1 2 dimethylformamide,^ 1 3'^ 1^ , i r -i 415,416 . c , „ 415,417s dimethylsulioxide, or mixtures or the last two solvents ) i t proved unsuccessful when dealing with usnic acid i t s e l f . Thus the isoxazole 158 was dissolved in dry chloroform, treated with an excess methyl iodide and silver oxide and mechanically stirred at 50-55° for 20 hours. Purification of the crude reaction mixture by column chroma-tography on . s i l i c a gel produced two main components. The least polar of the two proved to be the des i r e d (+)-7,9-di-0-methylisoxazolo[4,5-b]usnic acid (220). This material was i s o l a t e d in 71% yield as a bright yellow foam that could not be induced to crystallize, \<i]^ (CH CN) +335°. Absorption maxima at 360, 305, 250 and 205 nm were observed in i t s ultraviolet spectrum, while the. presence of the methoxy ethers (2940, 2850 cm 1 ) , the aromatic acetyl group (1690 cm 1) and the C-l isoxazoly1 ketone (1660 cm 1) were confirmed by i t s infrared spectrum. As expected, i t s pmr spectrum (Figure 71) allowed the clear identification of the 0-methyl s u b s t i t u e n t s which were observed as two three-proton singlets, c3.74 and 4.02, assigned to the C^- and C^-methyl ethers, respectively. While the isoxazole methyl group was seen at 62.44, the chelated aromatic acetyl grouping appeared as a threu-proton singlet at 62.57. The proposed structure was further supported by its mass spectrum (m/e 369, base peak, 354, 341, 326, 301, 285, 243, 43) and molecular formula (0,o H O N). 20 19 o Ana 1yt ieal data and high resolution molecular weight determination Figure 71. Proton Magnetic Resonance Spectrum of (+)-7,9-Di-0-methylisoxazolo[4,5-b]usnic Acid (220). - 241 -indicated for the minor component, the isoxazole 221, the molecular formula C„„H_O^N. This material, isomeric with the diether 220, was i s o l a t e d i n 20 19 6 9 £ 16% y i e l d as thick prismatic needles, mp 185-187°, [ot] D (CH CN) +354°, and resul t e d from the competitive d i - C - a l k y l a t i o n (methylation) of the aromatic ring 1,3-diphenolic system. I t s u l t r a v i o l e t spectrum showed absorptions at 318, 264, 220 and 214 nm, while i t s i n f r a r e d spectrum presented carbonyl bands at 1730, 1690 and 1675 cm I t s proton magnetic resonance spectrum 324 (Figure 72) appeared somewhat s i m i l a r to that of (+)-methylusnic acid (198) and contained two three-proton s i n g l e t s at 61.49 and 1.64 that were assigned to the C„-a- and g-methyl groups together with three-proton s i n g l e t s at o 61.68 and 1.78 corresponding to the C.n-a- and C: -a-methyl groups, respect-10 9b i v e l y . In add i t i o n , two other three-proton s i n g l e t s were observed at 62.43 and 2.45, corresponding to the C^Q-g-acetyl grouping and to the isoxazole methyl group, and a one-proton s i n g l e t at 66.62 c h a r a c t e r i s t i c of the C^-o l e f i n i c proton. On the basis of these spectroscopic data, we have postulated structure 221, i n which not only a l l the various i n t e r a c t i o n s seem to have been minimized, but the a c e t y l grouping has acquired a pseudoequatorial ( i e . stable) conformation, f o r the minor product r e s u l t i n g from the a l k y l a t i o n experiment. Moreover, s i m i l a r compounds have been obtained by Riedl and 418 coworkers by the two-stage nuclear methylation of phloroacetophenone (91) with methyl iodide-potassium methoxide i n l i q u i d ammonia. The r e s u l t i n g 419 product, tetra-C-methylphloroacetophenone (222), was further alkylated with methyl i o d i d e - s i l v e r oxide i n the absence of solvent to produce 1,3,3,5,5-pentamethyl-l-acetyl-2,4,6-cyclohexatrione (223) i n 12% y i e l d together with the corresponding O-alkylated d e r i v a t i v e 224 (23% y i e l d ) . I n t e r e s t i n g l y 243 -enough, treatment, of compound 222 with ethereal diazomethane furnished in 66% yield the hydroxydihydrofuran 225, which was later on thermally dehy-drated (90% yield) to the furan 226 (Figure 73). These series of compounds, 420 prepared by model reactions leading to the synthesis of tasmanone (227), resemble quite closely the behavior of usnic acid and i t s derivatives upon alkylation. The next step in our proposed synthetic sequence leading to the preparation of (+)-6-desacetylusnic acid (107) (Figure 74) required the transformation of the dimethoxyacetylisoxazole 220 into an acetanilide derivative by means of a Beckmann rearrangement. However, because of the enhanced susceptibility of the dimethyl ethers of the isoxazole 158 towards Michael-type solvent addition to the C.-C. double bond, the original conditions 4 4a » o used for the racemie dibenzyl ether 203 could not be successfully applied to compound 220, although i t was soon realized that proper reaction could be - 244 -( 227 ) Figure 73. Riedl's Synthesis of C-Alkylated Derivatives of Phloroaceto-| phenone (91). - 245 -(107) Figure 74. The Proposed Synthesis of (+)-6-Desacetylusnic Acid (107). - 246 -accomplished by t r e a t i n g 220 with hydroxylamine hydrochloride i n dry pyridine at 55° for 2.75 hours. The actual rearrangement step, c a r r i e d out by using t h i o n y l c h l o r i d e i n anhydrous et h y l ether at room temperature for 12.5 minutes, was followed by quenching with a 1:1 mixture of e t h y l acetate-water. The r e a c t i o n product, (+)-N-acetyl-6-amino-7,9-di-0-methyl-isoxazolo[4,5-b]usnic acid (228), was i s o l a t e d i n 70% y i e l d as f i n e yellow 9 ft needles, mp 188-189°, [ a ] D (CH^CN) +354°, and presented i n i t s u l t r a v i o l e t spectrum absorption maxima at 364, 315, 281, 253 and 208 nm. I t s i n f r a r e d spectrum indicated the presence of both free (3460, 1685, 1535, 1265 cm ^) and associated (3320, 1650, 1550, 1300 cm "*") amide f u n c t i o n a l i t i e s , while i t s pmr spectrum (Figure 75) showed .retention of C^- and C^-methoxy groups (63.78 and 3.96, r e s p e c t i v e l y ) as well as the newly formed N-acetylamino grouping, whose s i g n a l coalesced with that of the aromatic methyl group producing a six-proton s i n g l e t at 62.19. The molecular formula C20^20^6 N2 was obtained from a n a l y t i c a l data and high r e s o l u t i o n mass spectrometry measurements. Table 23 presents a summary of selected pmr data for the various amide d e r i v a t i v e s prepared. Due to the severe d i f f i c u l t i e s encountered during the attempted cleavage of the amide C-N bond (vide supra), i t became necessary to f i n d a r e a c t i o n whose net r e s u l t was the s u b s t i t u t i o n of an aromatic N-acetyl f u n c t i o n a l i t y by hydrogen, without being of h y d r o l y t i c nature. At this time 421 came to our a t t e n t i o n the early works of Bamberger on the use of a c y l -arylnitrosamines as a r y l a t i n g agents, as well as the more recent k i n e t i c studies on the decomposition of N-nitrosoacetanilide (231) by Hey and 442 coworkers whom, by measuring the rate at which nitrogen was evolved, determined that the r e a c t i o n was of f i r s t - o r d e r and s u b s t a n t i a l l y independent Figure 75. Proton Magnetic Resonance Spectrum of (+)-N-Acetyl-6-amino-7,9-di-0-methylisoxazolo [4,5-b]usnic Acid (228). F u n c t i o n a l i t y (210) Compound, ppm (6units) (213) (214) (217) (228) C--NHC0CH, 2.34 2.31 2.30 2.27 2.19 6 —3 C i r C H 3 - - - - 2.43 C 2-C0CH 3 2.54 2.65 2.65 2.46 C^-NH-COCH, 7.70 9.18 9.15 6.87 7.28 o ~ 3 C 7-OCH 3 — -- — — 3.78 C g-0CH 3 3.99 -- — — 3.96 C -0CH oC.H c — -- -- 4.82 — / ~l 6 5 C 9-OCH 2C 6H 5 -- - - 5.25 C,-H 5.62 5.88 5.86 5.77 6.14 4 — C y-0H 9.43 9.93 9.90 C -OH — 10.61 10.62 / ~ 9 — » Table 23. Selected Proton Magnetic Resonance lata for the Various Amide Derivatives of Usnic Acid. 249 -of the nature of both the solvent and nuclear substituents. Later on Hey 423 and coworkers carried out studies on the effects of aryl radicals on various aromatic compounds, chlorinated materials, and aliphatic esters derived from acetic acid that resulted in the evaluation of the different 424 mechanistic p o s s i b i l i t i e s involved. 425 Simultaneously, DeTar performed a quantitative study of the orienting influence of the nitro group in the formation of nitrobiphenyls, leading to a mechanistic proposal for the decomposition of N-nitroacetanilide (231) in m e t h a n o l . S i m i l a r studies were conducted by White and coworkers^^ in the aliphatic and a l i c y c l i c amide series, dealing as well with the analogous N-nitroamides and carbamates. 429 Furthermore, Kirmse carried out investigations not only on the reductive sodium borohydride-induced deamination of N-nitrosoureas, but extended his studies to include the ring opening reactions of bicyclo[n.1.0] alkyldiazonium ions (n = 3,5), and the alkaline cleavage of N-nitrosocarbamates, 430 whereas in the heterocyclic f i e l d , Newman studied the products obtained from the alkaline treatment of various 3-nitroso-2-oxazolidones. However, Suschitzky and coworkers observed that the decomposition of acylarylnitrosamines in benzene produces not only free radicals, but also intermediate ion pairs, and experimentally demonstrated the generality of this dual mechanism by the use of fluorinated amides. Further mechanistic studies on the decomposition of N-nitrosoacetanilide (231) in ethyl ether, which is k n o w n to give benzene, 1-ethoxyethyl acetate and acetaldehyde, was 432 433 performed by Denny, Gershman and Appelbaum and by Cadogan and coworkers, whom by using e s . r . measurements demonstrated that a phenyl radical, obtained hy electron transfer between the diazonium cation (II) and the - 2 5 0 -R R R a-ethoxymethyl r a d i c a l (IV), i s the actual chain c a r r i e r i n t h i s case. PhNO- Ph« I PhNAc < PhN (N0)Ac • PhN + AcO (I) (ID - 251 -I 3 Ph- + Et 0 • PhH + CH.CHOEt > CH-N(NAcPh)0 • 3 j EtO (IV) (III) CH3CHOEt + A r N 2 + »• Ph- + N 2 + CH CHOEt (IV) (II) CH^HOEt + AcO y CH 3CH(OEt)OAc Ph- + E t 2 0 y (IV) y et seq. CH3CHOEt y CH CHO + Et-(IV) Although the general preparation of a l i p h a t i c N-nitrosoamides by the use of sodium n i t r i t e i n a mixture of a c e t i c a c i d - a c e t i c anhydride has 434 been recommended by White, aromatic amides are commonly nit r o s a t e d by 424 435 the use of reagents such, as n i t r o s y l c h l o r i d e (N0C1), ' n i t r o s y l s u l f u r i c 436 437 acid (NOHSO^) , dinitrogen tetroxide (^0^) , and various nitrosonium — — — — 438 s a l t s (NOX, X = PF 6 , BF^ , SbF 6 , AsF 6 ). Moreover, the d i r e c t a r y l a t i o n 439 440 of aromatic amines and amides, v i a the intermediate diazonium s a l t s , has been performed by the use of a l k y l n i t r i t e s i n r e f l u x i n g benzene and molten biphenyl (85°), r e s p e c t i v e l y . From the analysis of the vast number of studies performed on the products r e s u l t i n g from the decomposition of N-n i t r o s o a c e t a n i l i d e (221) - 252 -i n various solvents (Table 24), i t was r e a l i z e d that i f the decomposition of acylarylnitrosamines i s c a r r i e d out under neutral conditions i n solvents able to donate hydrogen atoms, f a i r y i e l d s of the corresponding s u b s t i t u t i o n products can be obtained. Although the attempts to prepare the desired N-n i t r o s o d e r i v a t i v e s by the usual methods (vide supra) were.all unsuccessful, encouraging r e s u l t s were obtained by the use of a l k y l n i t r i t e s ( i e . isoamyl n i t r i t e ) i n b o i l i n g c y c l i c ethers. Thus, treatment of the di-O-methyl amide 22.8 with isoamyl n i t r i t e i n r e f l u x i n g tetrahydrofuran, a method previously used 16 5 for the one-step deamination of aromatic amines by Cadogan and coworkers, met only with l i m i t e d success due probably to the slow decomposition of the relevant N-nitroso intermediate at t h i s temperature, conducting the r e a c t i o n i n dry dioxan at 88° produced up to 78% y i e l d s of the desired product, (+)-6-desacetyl-7,9-di-0-methylisoxazole[4,5-b]usnic acid (229). This material showed the expected formula C^gH^O^N and was obtained as b r i g h t yellow prisms, mp 156.5-158°, [a]^ (CE^CN) +300°. I t s u l t r a v i o l e t spectrum showed absorption maxima at 368, 305, 281, 254 and 211 nm, and i t s i n f r a r e d spectrum indicated the presence of the methyl ethers (2940, 2850 cm "*"), the C - l carbonyl (1680 cm 1 ) and a t r i s u b s t i t u t e d isoxazole r i n g (1650 cm 1 ) , which was fur t h e r supported by the corresponding proton magnetic resonance spectrum (Figure 76). Thus the pmr spectrum of 229 showed a three-proton s i n g l e t at 62.44 that was c h a r a c t e r i s t i c of the isoxazole r i n g methyl group, and two more at 63.78 and 3.96 assigned to the C ?- and C g-methyl ethers, r e s p e c t i v e l y . Moreover, the s u b s t i t u t i o n of the amide f u n c t i o n a l i t y was confirmed by the appearance of a new one-proton s i n g l e t at 66.43 corresponding to the aromatic C,-proton. Thus, the acylarylnitrosamine rearrangement 6 allowedj us to perform i n good y i e l d , the o v e r a l l removal of the aromatic 0 si I 250 I 100 I so CH 3 o JJ\J 0 CPS ro 10 Figure 76. pp m(6) Proton Magnetic Resonance Spectrum of (+)-6-Desacetyl-7,9-di-0-methylisoxazolo 0 [4,5-b]usnic Acid (229) - 254 -Solvent Products, % Reference CH3OH - PhH (54) PhOMe ( 6) 426 CH 3OH-Na 2C0 3 PhH (49) PhOMe ( 3) 426 CH„0H-H„S0, j 2 4 PhH ( 2) PhOMe (75) 426 AcOH — PhOAc (48) 426 E t 2 0 PhH (52) CH 3CH(OEt)OAc (22) 432 CHC1 3 PhH (20) PhCl (15) 423c CC1. 4 — PhCl (32) 423c EtOAc PhH (40) — 423d Table 24. Selected Studies-on the Decomposition of N-Nitrosoacetanilide (231) i n Various Solvents ( 229 ) I - 255 -a c e t y l grouping under non-hydrolytic conditions. This was indeed a very important r e a c t i o n for our synthetic purposes, since for the f i r s t time no isomerization to the i s o - d e r i v a t i v e s was observed. However, i t must be noted that t h i s r e a ction, when applied to (+)-usnic acid i t s e l f , produced only complex mixtures that were not investigated further. The next steps i n our synthetic sequence c a l l e d for the removal of the various protecting groups beginning with the ether f u n c t i o n a l i t i e s . Although treatment of the deacylated diether 229 with potassium iodide i n dry hexamethylphosphoramide (HMPA), a modification of the methods 441 442 previously described by Harrison and by Walker, and with pyridine 393 443 hydrochloride at 180 , ' a procedure recommended for the cleavage of ether groups derived from 1,3-diphenols, for. which the use of boron 444 tribromide r e s u l t s i n quite low y i e l d s , furnished mostly unreacted 389 390 s t a r t i n g material, the use of boron tribromide ' at -50° i n d i c h l o r o -methane produced.in 98% y i e l d the monoether d e r i v a t i v e (+)-6-desacetyl-7-0-methylisoxazolo[4,5-b]usnic acid (232), a r i s i n g from the f a c i l e cleavage of the Cg-methoxy group. A n a l y t i c a l and high r e s o l u t i o n mass spectrometry data indicated the molecular formula C^H ^0 N for t h i s material, pale yellow prismatic needles, mp 167-168°, [ a ] D (CH^N) +250°, which showed an u l t r a v i o l e t spectrum (X 372, 304, 256, 214 nm) very s i m i l a r to that of the s t a r t i n g max acylated isoxazole dimethyl ether 220. In agreement with the p a r t i a l l y demethylated structure 232, i t s i n f r a r e d spectrum presented absorptions i n the 3500-3100 cm region corresponding to the chelated phenolic hydroxyl group at the 9-position, as well as a strong band at 1660 cm ^ that was assigned to the hydrogen bonded i s o x a z o l y l ketone. Moreover, i t s pmr spectrum - 256 -secured the previous assignment, when the one-proton s i n g l e t c h a r a c t e r i s t i c of the Cg-phenolic hydroxyl group was observed at 69.67, i n ad d i t i o n to the disappearance of the lower f i e l d methoxyl s i g n a l . whereas the absorption corresponding to the ether group at the 7-position remained e s s e n t i a l l y unaltered (63.76), the C^-aromatic proton (66.25) experienced a large u p f i e l d s h i f t (Figure 77). F i n a l proof f o r the proposed cleavage was obtained through the preparation of (+)-6-desacetyl-7-0-methylisoxazolo[4,5-b]usnic acid mono-acetate (233) by a c e t y l a t i o n ( a c e t i c anhydride-sulfuric acid or even better a c e t i c anhydride-pyridine) of the monoether 232. This material, pale yellow 2 6 plat e s , mp 195-196°, [ a ] Q (CH^CN) +500°, showed i n i t s i n f r a r e d spectrum (1770 cm ^) and pmr spectrum (62.46) the absorptions c h a r a c t e r i s t i c of the newly introduced f u n c t i o n a l i t y . However, when the diether 229 was allowed to react with boron 389 390 tribromide i n dichloromethane ' at 10°, a f t e r mixing the reagents at -78°, i a complete demethylation was accomplished, thus producing (+)-6-desacetyl-isoxazolo [4 , 5-b ] usnic acid (230) i n 87% y i e l d ( a f t e r r e c y c l i n g a small amount I • I . I . I . I . I . I . I I I . . . . I . . . . I . I . . . I 10 ppm ( 5 ) 0 Figure 77. Proton Magnetic Resonance Spectrum of (+)-6-Desacetyl-7-0-methylisoxazolo[4,5-b]usnic Acid (232). - 258 -amount of unreached starting material and monoether 232) as pale yellow 2 6 crystals, mp 194.5-]96°, [ a ] D (CH CN) +608°, with molecular formula ^16^13^5^" T n : i- S material enabled the f i r s t direct comparison with synthetic intermediates belonging to the iso series (ie. with the isoxazole 176). Some of the main differences arose from their infrared spectra; hence the carbonyl group at the 1-position was observed at 1675 cm ^ for the normal-series isomer 230, but appeared at 1660 cm for the iso-series analog 176. The single most important analytical tool for the series has undoubtedly been proton magnetic resonance spectroscopy, and in this case i t proved once more i t s usefulness by allowing the f a c i l e differentiation between the two isomers (Table 25). A minor product was isolated from the crude mixture in 8% yield and was characterized as (+)-6-bromoisoxazolo[4,5—b]usnic acid (234). In fact, the nuclear halogenation (bromination) of 1,3-diphenolic materials has been previously observed during the cleavage of the corresponding diethers by 394 means of Lewis acids such as anhydrous aluminum tribromide (AlBr^). The molecular formula C^^H^^O^NBr, i n i t i a l l y suggested by elemental analysis, was later on confirmed by high resolution mass spectrometry molecular weight determination and elemental composition analysis (see experimental section). 2 6 This product was isolated as pale yellow crystals, mp 209-211° (d), [a] (CH-CN) +593°, and showed ultraviolet (X 371, 324, 255, 210 nm) and 3 max infrared (3500-3100, 1675, 1638, 1625, 1600 cm"1) spectra resembling those of the hydrogen-analog 230. In the low f i e l d region of i t s pmr spectrum, as expected, only the one-proton singlet at 66.32 corresponding to the olefinic proton was observed. Moreover, the noticeable downfield shift (from 62.07 in the Il-eompound to 62.32 for the bromo-derivative), experienced - 259 -by the aromatic methyl group i s a d i r e c t consequence of the bromine atom introduction into the aromatic A r i n g . Functional! t y (230) Compound, (236) ppm (<5 units) (176) (175) C9b"% 1.70 1.77 1.80 1.83 C 8-CH 3 2.07 1.95 — — V % — — 2.14 2.16 C7-OCOCH3 — 2.29 — 2.32 Cg-OCOCH — 2.41 — 2.49 C n - C H 3 2.44 2.47 2.46 2.53 C.-H 4 — 6.16 6.19 5.95 6.00 C,-H o — 6.28 6.78 — — Cg-H — — 6.31 6.72 C -OH 8.63 — 8.59 — C9"0H 9.62 — 8.59 — Table 25. Proton Magnetic Resonance Lata f o r (+)-6-Desacetylisoxazolo[4,5-b] Usnic Acid (230) and (+)-6-Desacetylisoxazolo[4,5-b]Isousnic Acid (176) and the i r Corresponding Acetates. The same 1,3-diphenolic intermediate 230 can be prepared by boron tribromide treatment of the monoether 232. However, much lower y i e l d s (33%) have been r e a l i z e d from t h i s process. - 260 -(231 ) Acetylation of compound 230 by means of pyridine-acetic anhydride produced two components that could be easily separated by preparative layer chromatography. The least polar of them turned out to be the C^-monoacetate derivative, (+)-6-desacetylisoxazolo[4,5-bJusnic acid monoacetate (235), 2 6 pale yellow needles, mp 168-170°, [a]^ (CH.CN) +547°, which was character-b 3 ized by means of its infrared (1760, 1675, 1640 cm-"'") and pmr spectra. The latter showed a new three-proton singlet at 62.27 corresponding to the acetate grouping at. the 7-position in addition to the typical Cg-phenolic hydroxyl proton asnd isoxazole ring methyl group absorptions at 69.99 and 2.46, respect-- 261 -ively. The postulated structure was further supported by the determination of its molecular formula ( C H , , O N ) and by mass spectrometry measurements l l ) i i b (m/e 341, base peak, 299, 284, 271, 270, 256, 243, 231, 230, 215, 202, 201, 190, 43). (236 ) The minor, more polar product was shown to have the molecular formula C 2 Q H ^ 0 ^ N , thus corresponding to the diacetylated material (+)-6-desacetylisoxazolo[4,5-b]usnic acid diacetate (236), colorless prisms, mp 2 ft 242-243°, [a] D ( C R ^ C N ) +368°, which as expected had an infrared spectrum (1765, 1685 cm "*") slightly different to that of the iso-series isoxazole diacetate 175 (1770, 1675 cm ^). Further differences were observed in the corresponding pmr spectra, which have been summarized in Table 25. - 262 -The f i n a l step of the sequence leading to the preparation of metabolite 107 had already been performed with the i s o - s e r i e s diacetate 175 (vide supra) and involved a reductive cleavage of the isoxazole N-0 bond followed by a l k a l i n e regeneration of the $-triketone system. Thus, hydrogenolysis of the diphenol 230 was c a r r i e d out i n ethyl alcohol s o l u t i o n i n the presence of platinum oxide (Pt02) at room temperature and the r e s u l t i n g $-ketoenamine 237 was immediately subjected to a l k a l i n e treatment (IN sodium hydroxide) to produce i n 97% y i e l d the desired (+)-6-desacetylusnic acid (107), f i n e yellow needles, mp 134.5-136°, mixed mp with the authentic material i s o l a t e d from the b a c t e r i a l biodegradation of 26 (+)-usnic acid 134.5-136°, [ c t ] D +689° ( i d e n t i c a l to the s p e c i f i c r o t a t i o n previously recorded for metabolite 107). Moreover, t h e i r u l t r a v i o l e t (A 335, 266, 233 nm) and i n f r a r e d (1670, 1630, 1540 cm"1) spectra were max r i d e n t i c a l as w e l l . I t s pmr spectrum (Figure 78) agrees p e r f e c t l y with the proposed structure and has been summarized i n Table 26 together with that of the natural compound and t h e i r corresponding diacetates. I ; I I I I i I I , I I I ; I I i i i I i i I i i p r i T p - n i i - t r T - i - t - i - r r - r T T - f - | - r - | - - r - r - r - r T - r i i i I I I r r p r n I I I I I I i i i i i • ' ' ' I ' ' ' ' I 1 / J 1 i i | \ i i i 1 i i i ' i i i I i i i M i | f i i i i i i i i' i i i r i i i i i i i I ' I i i I I I i I ' I i M i i i n i i I ' I i I ' I i I ' | I I I ' l I I ) I ' I i 1 1 1 I ' 1 i i I i i ' 5000 T-10 r ti_ n .j n f t * » * r*r i * ' '* i i i i i i i i i i i i i i i i i i i i i i ppm ((5) Figure 78. Proton Magnetic Resonance Spectrum of Synthetic (+)-6-Desacetylusnic Acid (107), 0 ro ON - 264 -Compound, ppm (6 units) F u n c t i o n a l i t y Natural Synthetic (107) (113) (107) (113) C9b-CV 1.70 1.78 1.71 1.78 C 8-CH 3 2.11 1.97 2.09 1.98 C 7-0C0CH 3 — 2.31 — 2.33 C 9-0C0CH 3 — 2.43. — 2.45 C2-C0CH_3 2.61 2.52 2.59 2.55 C ?-0H 5.67 — 5.28 — 5.79 5.81 5.78 5.83 VI 6.26 6.81 6.22 6.82 C 9-0H 10.23 10.22 . — C 3-0H 18.64 18.41 18.65 18.31 Table 26. Proton Magnetic Resonance Data f o r the Natural and Synthetic (+)-6-Desacetylusnic Acid (107) and t h e i r Corresponding Acetates. Further agreement was observed by the ana l y s i s of t h e i r C i r c u l a r Dichroism properties under n e u t r a l and basic conditions (Table 27), a very important c h a r a c t e r i s t i c , since we have found that t h i s technique allows the f a c i l e d i f f e r e n t i a t i o n between the normal and i s o - s e r i e s (see Table 17), and by the acid-catalyzed preparation of the corresponding diacetate 26 d e r i v a t i v e 113, pale yellow needles, mp and mixed mp 208-209°, [ a J j +125° 26 (the s p e c i f i c r o t a t i o n [ a ] n +121° was recorded for the natural material), which as expected showed u l t r a v i o l e t (X 325, 280, 261, 220 nm), in f r a r e d max (1755, 1685, 1620, 1600, 1545 cm "*") and proton magnetic resonance (see Table - 265 -26) spectra identical to those of the material obtained by acetylation of the biodegradation metabolite. Compound 107 Solvent Natural A (Ae) max CH30H 328 (+ 9.99) 284 (- 6.66) 262 (- 3.53) 241 (+13.11) CH OH + KOH 345 (+ 8.11) 310 (+ 6.86) 285 (+ 1.66) 264 (--12.90) 236 (+10.19) Synthetic CH30H 328 (+ 9.42) 283 (- 6.06) 260 (- 3.59) 239 (+13.24) CH30H + KOH 344 (+ 7.85) 308 (+ 6.28) 285 (+ 1.79) 266 (-12.57) 238 (+ 9.87) Table 27. Circular Dichroism rata for Natural and Synthetic (+)-6- Eesacetyl-usnic Acid (107). Furthermore, the synthetic diphenol 107 was converted via 445 a one-step acetylation-aluminum trichloride-catalyzed Fries rearrangement followed by basic work-up into synthet ic (+)-usnic acid, which showed identical spectroscopic (Figure 79) and chromatographic properties to those of the naturally occurring optically active lichen substance 35 (vide supra), thus allowing the completion of the structure elucidation and synthesis of (+)-6-desacetylusnic acid (107) the metabolite isolated during the biodegradation of (+)-usnic acid (35) by the bacterial isolate. - 266 -i - 268 -Part B Since i t was demonstrated i n Part A of t h i s thesis that b i o -degradation of (+)-usnic acid (35) by the Pseudomonas species i s o l a t e r e s u l t s e x c l u s i v e l y i n a r i n g A aromatic deacylation, we f e l t that an appropriate follow-up i n v e s t i g a t i o n would be a comparable study on the biodegradative t a l e n t s of s o i l f u n g i . Therefore, a fungal i s o l a t e showing the a b i l i t y to degrade (+)-usnic a c i d (35) was i s o l a t e d from surface s o i l samples c o l l e c t e d by 189 Dr. J.D. Leman i n the Whistler Mountain area, and has been characterized 446 as Mucor globosus (Fischer, 1892) by Dr. John J . E l l i s of the United States Department of A g r i c u l t u r e , A g r i c u l t u r a l Pesearch Service, Peoria, I l l i n o i s . Thus, under fermentation conditions s i m i l a r to those previously described for the b a c t e r i a l i s o l a t e (see Experimental Section, Part A), the Mucor globosus i s o l a t e , s t r a i n MU-1, was able to biodegrade samples of (+)-usnic a c i d (35) to a s i n g l e component i n up to 32% y i e l d . This material was i s o l a t e d as bright yellow prisms from ethyl acetate, mp 221-222°, 2 6 [a]^ (CH^CN) +635°. A n a l y t i c a l data and high r e s o l u t i o n mass spectrometry measurements indicated the molecular formula C,-H,.0,, i d e n t i c a l to that of 16 14 6 the product obtained during the b a c t e r i a l fermentation (Part A), and r e s u l t i n g from the removal of 42 mass un i t s , a C H.O fragment, from usnic a c i d . Although i t s u l t r a v i o l e t spectrum (X 330, 287, 218 nm) was somewhat s i m i l a r to that max of the s t a r t i n g m a t e r i a l , important d i f f e r e n c e s resulted from i t s i n f r a r e d spectrum, which showed absorptions corresponding to the s t r e t c h i n g v i b r a t i o n s of the chelated phenolic hydroxyl groupings at the 7- and 9-positions (3300-- 269 -2500 cm """), the C - l enone carbonyl (1670 cm x ) , and the aromatic methyl ketone (1625 cm . However, the band c h a r a c t e r i s t i c of the chelated r i n g C a c e t y l grouping, usually occurring i n the 1560-1540 cm 1 region, was not observed, suggesting that a deacylation reaction had taken place at that p o s i t i o n . The same conclusion was obtained upon analysis of i t s pmr spectrum (Figure 80), which showed two three-proton s i n g l e t s at 61.70 and 2.Q5, c h a r a c t e r i s t i c of the angular and aromatic methyl groups, r e s p e c t i v e l y . Whereas i n usnic acid the absorptions corresponding to the C„- and C,-acetyl 2 o groupings coalesce into a six-proton s i n g l e t at 62.70, th i s compound showed only a three-proton s i n g l e t at 62.65 that was assigned to the chelated r i n g A aromatic methyl ketone. This l a s t assignment was supported by the appear-ance of the one-proton s i n g l e t t y p i c a l of the phenolic hydroxyl group at the 7-position (613.31). Furthermore, the s i g n a l corresponding to the C^-o l e f i n i c proton was observed as a doublet centered at 65.85 with coupling constant J = 2 Hz, i n addi t i o n to a new one-proton doublet at 65.38 (J = 2 Hz). The l a t t e r absorption must correspond to the proton r e s u l t i n g from the removal of the a c e t y l grouping at the 2-position and shows the expected 253 long-range coupling with the C ^ - o l e f i n i c proton. 204 As expected, i t s mass spectrum (Figure 81) completely agreed with the postulated deacylation. The retro Diels-Alder process, a r i s i n g from cleavage of the C r i n g , resulted from an [M-42] fragmentation and was observed as the base peak at m/e 260. However, for usnic acid i t s e l f (see Figures 30 and 31) t h i s proces gives r i s e to an [M-84] l o s s , thus i n d i c a t i n g that r i n g C i s lacking the 42 mass units corresponding to the C^-acetyl grouping as a consequence of the fungal deacylation. Thus ion £ (m/e 260) - 272 -further fragments by loss of carbon monoxide to b_ (m/e 232) and then of a methyl r a d i c a l to c_ (m/e 217). Worthwhile noting i s the fact that the major fragmentation process observed for both (+)-usnic acid (35) and i t s 6-desacetyl analog 107, the one a r i s i n g v i a hydrogen transfer from the e n o l i c hydroxyl group at the 3-position, appears i n t h i s case with a sub-s t a n t i a l l y decreased r e l a t i v e i n t e n s i t y (30%) as ion d (m/e 233). I t s occurrence further establishes the presence of an a c e t y l grouping i n the 200 447 aromatic A r i n g . S i m i l a r l y to other 6-diketones ' d i r e c t l o s s of carbon monoxide i s possible and produces ion j* (m/e 246) (Figure 82). F i n a l l y , the sample presented the following C i r c u l a r Dichroism properties : X (Ae) 375 (+0.94), 333 (+6.08), 298 (-4.32), 273 (+5.27) max and 238 nm (-5.67). On the basis of a l l the previously presented spectro-scopic evidence we have assigned to the metabolite r e s u l t i n g from the fungal deacylation the (+)-2-desacetylusnic acid structure 108. (108 ) A f i n a l proof for this novel C-2 deacylated structure was obtained 199 from an X-ray d i f f r a c t i o n analysis performed by Professor J . T r o t t e r of the Chemistry Department, U n i v e r s i t y of B r i t i s h Columbia, who found that the aromatic ring i s s l i g h t l y , but s i g n i f i c a n t l y nonplanar, e x i s t i n g i n an - 273 -c=o OH J g b e 2 6 0 ( 1 0 0 ) Figure 82. The Postulated Fragmentation of (+)-Desacetylusnic Acid (108) i n the Mass Spectrometer. - 274 -asymmetric boat conformation with C and C displaced +0.015 and +0.022 9 a / o A from the mean plane of the r i n g . Although the mean angle of the r i n g i s 120.0°, s i g n i f i c a n t deviations among the i n d i v i d u a l values ( i e . the angles at C, and C Q are contracted to 114.5 and 118.5°) are the r e s u l t of fusion o o to the five-membered r i n g . The cyclohexadiene r i n g has the sofa conform-o ation with the 9b-carbon atom displaced 0.316 A from the mean plane of the o r i n g . Moreover, the C g b " c 1 0 b o n d (1-572 A) i s s i g n i f i c a n t l y longer than.a 3 3 normal C(sp )-C(sp ) bond, i t s weakening being a consequence of s t e r i c d i s t o r t i o n of the C_,-tetrahedron. The c r y s t a l structure (Figures 82-84) 9b consists of molecules l i n k e d by intermolecular hydrogen bonds (0...0, o 2.625 A) to form continuous s p i r a l s about the 2^ a x i s . The molecular geometry ind i c a t e s that there are several resonance forms which are important contributions to the structure, with the f u l l y conjugated form 238 being s l i g h t l y favored over the cross-conjugated 239. Furthermore, the absolute configuration shown i n Figures 83-85 was determined based on the anomalous 448 s c a t t e r i n g from oxygen and carbon atoms. ( 2 3 8 ) ( 2 3 9 ) I Figure 83. Computer Generated Stereo View of the Crystal Structure of (+)-2-[Jesac.ety]usnic Acid (108). - 276 -OC O O oH Figure 84. The C r y s t a l Structure of (+)-2-Desacetylusnic Acid (108) Viewed Down c (Y=b and X=a s i n B). - 277 -Figure 85. The C r y s t a l Structure of (+)-2-Desacetylusnic Acid (108) Viewed Down b. - 278 -Added chemical proof to the structure of (+)-2-desacetylusnic acid (108) was obtained by a c e t y l a t i o n and subsequent ozonolysis to the o p t i c a l l y i n a c t i v e a-coumaranone diacetate 70.previously i s o l a t e d by . ,. 147,148 _ , _ , „ 149 . '.. _ , . 308-310 Asahina, Dean and Robertson, and Shibata and coworkers, by ozonolysis of (+)-usnic acid diacetate (68b). Thus the metabolite 108 was acetylated with p y r i d i n e - a c e t i c anhydride at room temperature overnight and the crude "acetate mixture" ozonized at 0° i n carbon t e t r a c h l o r i d e (26 mg 0^/ minute) u n t i l the yellow color was discharged and the c r y s t a l l i n e ozonide began to p r e c i p i t a t e . The l a t t e r was then hydrolyzed with hot water to produce (±)-7-acetyl-3,5-dimethyl-4,6-dihydroxy-coumaran-2-one diacetate (70) i n 30% y i e l d as c o l o r l e s s c r y s t a l s , mp and mixed mp 131-132° 84 ( l i t . mp 130-132°). This product was characterized by i t s i n f r a r e d spectrum, which showed absorptions corresponding to the aromatic lactone -1 -1 (1815 cm ), the phenolic acetates (1765 cm ) and the aromatic a c e t y l grouping (1690 cm ^"). Moreover, i t s pmr spectrum (Figure 86) presented the expected three-proton doublet, J = 7.5 Hz, at 61.50 assigned to the methyl group at the 3-position, as w e l l as three-proton s i n g l e t s at 62.33, 2.39 and 2.61 corresponding to the C.-, C -phenolic acetate, and C - a c e t y l grouping, r e s p e c t i v e l y . Furthermore, (+)-2-desacetylusnic acid (108) was converted into 102 135 usnetic acid ' (51) by treatment with 75% aqueous potassium hydroxide at 9,0-95° (see Part A) and into e t h y l acetusnetate (71) by the conditions (ethyl a l c o h o l i n a sealed tube at 150°) under which usnic acid gives r i s e to the acylated analog usnic acid ethoxide (240), thus demonstrating the relevant C^-deacylated structure of metabolite 108. Although under milder conditions (EtOH, r e f l u x ) (+)-usnic acid diacetate (68b) gives r i s e to Figure 86. Proton Magnetic Resonance Spectrum of the a-Coumaranone Diacetate 70. - 280 -OH (51 ) usnic acid ethoxide diacetate (73b), treatment of usnic acid with ethanol at 190° (sealed tube) produces ethyl acetusnetate. (2^.0 ) - 281 -Thus et h y l acetusnetate (71) was i s o l a t e d as pale yellow needles mp and mixed mp 147-149°, and presented i n i t s u l t r a v i o l e t spectrum absorption maxima at 346, 301 and 243 nm, while in i t s i n f r a r e d spectrum the bands at 1725, 1700 and 1620 cm 1 corresponded to the ester group, 213 the ketone of the 6-ketoester side chain, and the chelated aromatic a c e t y l grouping, r e s p e c t i v e l y . The pmr spectrum of compound 71 showed the expected three-proton t r i p l e t at 61.22 and the two-proton quartet at 64.16, both wth coupling constant J - 7 Hz, corresponding to the e t h y l ester f u n c t i o n a l i t y , i n a d d i t i o n to a three-proton s i n g l e t at 62.70 and two two-proton s i n g l e t s at 63.49 and 4.18 that were assigned to the chelated aromatic a c e t y l grouping, the g-ketoester side chain methylene group, and the C2-methylene group, r e s p e c t i v e l y . The postulated structure was further supported by the determination of the elemental composition (C H 0 ) by l o 20 / means of high r e s o l u t i o n mass spectrometry and a n a l y t i c a l chata and by the corresponding mass spectrum (m/e 348, 260, 234, 233, base peak, 219, 215, 69). However, the treatment of (+)-usnic acid (35) with e t h y l alcohol under the same conditions r e s u l t e d only i n the i s o l a t i o n of usnic acid ethoxide (240), the C-acylated analog, i n 60% y i e l d . This material was i s o l a t e d as yellow c r y s t a l s , mp 103-105°, showing the molecular formula C„„H„„0„. Whereas i t s u l t r a v i o l e t spectrum (X 347, 296, 241 nm) was quite 20 22 o max s i m i l a r to that of e t h y l acetusnetate (71), i t s i n f r a r e d spectrum showed absorptions at 1705 and 1665 cm 1 corresponding to the e n o l i c B-ketoester side chain. Its pmr spectrum showed sig n a l s at 61.33 (three-proton t r i p l e t , J = 6 Hz) and 4.30 (two-proton quartet, J = 6 Hz) c h a r a c t e r i s t i c of the et h y l ester f u n c t i o n a l i t y , as w e l l as two three-proton s i n g l e t s at 62.38 - 282 -and 2.70 assigned to the side chain enolic methyl ketone and to the aromatic acetyl grouping, respectively. Moreover, its mass spectrum (m/e 390, 344, 260, 234, 233, base peak, 219, 217, 215, 191, 76) supported the structural formula bearing a C-acylated side chain. Although enol ethers of cyclohexane-1,3-diones have been 449 prepared by a variety of methods, i t was observed that upon treatment with excess etheral diazomethane in methanolic solution (+)-2-desacetyl-usnic acid (108) gives rise to the corresponding enol ether (+)-2-desacetyl-3-methoxyusnic acid (241) in 38% yield, whereas under the same conditions (+)-usnic acid (35) produces the tetracyclic benzofuran derivative 201 346 (see Part A). Thus, compound 241 was isolated as yellow prisms, mp 2 6 163-165°, [al (CH-CN) +622°, and showed an ultraviolet spectrum (A D 3 max 370, 330, 282, 218 nm) very similar to that of metabolite 108. Its infrared spectrum showed absorptions at 1692 and 1634 cm 1 corresponding to the C-l carbonyl group and the ring A chelated acetyl grouping, respect-ively, while its pmr spectrum presented a new three-proton singlet at 63.85 characteristic of the 0-methyl substituent at the 3-position, and two one-proton singlets at 611.19 and 13.26 assigned to the C n - and C -phenolic 9 / hydroxyl protons, respectively. Furthermore, the C 2 - and C^-olefinic protons were observed as two doublets centered at 65.30 and 5.74 with a coupling constant J = 1.8 Hz indicative of the long range coupling existing between the two positions. One of the most interesting chemical transformations of the deacylated metabolite 108 would be, no doubt, its conversion into (+)-usnic acid by the introduction of an acetyl grouping at the 2-position. Although 450 most of the C-acylation reactions attempted resulted exclusively in - 283 -(2U2) mixtures of d i - and t r i a c e t a t e s , i t was found that by the use of aluminum t r i c h l o r i d e i n a mixture of a c e t i c anhydride - g l a c i a l a c e t i c a c i d the corresponding enol acetate 242 could be prepared i n 84% y i e l d . This material showed the molecular formula C. 0H 0 and was i s o l a t e d as bri g h t yellow l o l o / 26 prisms, mp 159-160°, [a] (CH CN) +564°. The c h a r a c t e r i z a t i o n of compound D 3 242 was derived from i t s i n f r a r e d (1769, 1623, 1215 cm ^) and pmr spectra. The l a t t e r consisted i n three-proton : s i n g l e t s at 62.31 and 2.65 assigned to the enol acetate and aromatic a c e t y l groupings, r e s p e c t i v e l y , i n a d d i t i o n to a' two proton s i n g l e t at 65.89 corresponding to the C ?- and C ^ - o l e f i n i c - 284 -protons as well as the two one-proton s i n g l e t s at 610.59 and 13.29 corresponding to the C - and C^-phenolic hydroxyl protons. Moreover, i t s mass spectrum (m/e 344, 302, 287, 260, base peak, 232, 217, 43) was i n complete agreement with the postulated st r u c t u r e . Since the C-acylation of metabolite 107 could not be e a s i l y achieved, a d i r e c t chemical c o r r e l a t i o n with (+)-usnic acid (35) was attempted by means of i t s laboratory preparation. The synthetic scheme 451 (Figure 87) was based on the known reduction of a-acyloxyketones to the parent carbonyl compound by the use of chromium (II) s a l t s . Thus chromium (II) c h l o r i d e has been s u c c e s s f u l l y used to hydrogenolyze acetoxyl 452 453 and lactone functions i n the s t e r o i d and g i b b e r e l l i c a c i d s e r i e s . The key step of the synthesis consisted i n the preparation of an a-acetoxy-6-diketone, a process that can i n general be accomplished by 454 means of the B a e y e r - V i l l i g e r oxidation. Although i t i s known that 1,3-diketones and 3-ketoacids do not follow the normal pattern of the Baeyer-455 V i l l i g e r r e a c t i o n , g i v i n g r i s e instead to a-hydroxylated intermediates, 456 encouraging r e s u l t s were obtained by H a s s a l l , who found that the r e a c t i o n of 3-triketones such as 2-acetylindan-l,3-dione (243) with hydrogen peroxide i n d i e t h y l ether under neutral conditions r e s u l t s i n p r e f e r e n t i a l oxidation of the acyl side chain leading to the formation of the corresponding a-acetoxy d e r i v a t i v e 244. - 285 -(108 ) Figure 87. The Synthesis of (+)-2-Desacetylusnic Acid (108). Similar transformations have been c a r r i e d out by Akhrem and 457 458 459 coworkers, who i s o l a t e d the reductone 246, dihydropyrogallol, ' by the B a e y e r - V i l l i g e r oxidation of the enolic 2-acetyl-cyclohexane-l,3-dione (245) with monoperoxyphtalic acid i n chloroform s o l u t i o n . Furthermore, Davies, Erdtman and N i l s s o n ^ 6 ^ accomplished the oxidation of the dimethyl-- 286 -phloroacetophenone dimer 247 to the diacetate 248 by means of hydrogen peroxide in pyridine solution, and were able to successfully apply their conditions to the analog dimethylphloroglucinol aldehyde dimer 249. Therefore, when (+)-usnic acid (35) was treated with 30% hydrogen peroxide in dry pyridine at room temperature for one hour an 83% yield COR OCOR COR O C O R R = ' C H 3 ( 2 * i 7 ) R = C H 3 ( 2 Z i 8 ) R = H ( 2 Z » 9 ) R = H ( 2 5 0 ) of the desired a-acetoxy compound 251 was obtained (Figure 87). This material!, (+)-2-acetoxyusnic acid (251), was obtained as a bright yellow - 287 -2 6 glass that could not be induced to c r y s t a l l i z e , [«]^ (CH^OH) +389°, showing the molecular formula C.0H\ ,0 , derived from high r e s o l u t i o n mass lo l o o spectrometry. I t s u l t r a v i o l e t spectrum (^ m a x 334, 289, 223 nm) was quite s i m i l a r to that of the deacylated metabolite 108, thus suggesting that no changes had been introduced i n the aromatic A r i n g , and i t s i n f r a r e d spectrum (1768, 1715, 1695, 1625 cm "*") confirmed the presence of both the a-acetoxy -3-diketone system and the chelated aromatic a c e t y l grouping. Its mass spectrum (m/e 360, 318, 302, 300, 272, 262, 260, 232, 217, 202, 199, 97, base peak, 84, 83) and proton magnetic resonance spectrum (Figure 88) further supported the postulated structure. Thus the l a t t e r showed the expected disappearance of the a l i c y c l i c a c e t y l group and the surging of a new three-proton s i n g l e t at 62.24 corresponding to the g-diketo-a-acetoxy f u n c t i o n a l i t y . Moreover, while the o l e f i n i c proton was seen as a one-proton s i n g l e t at 5.86, the broad two-proton s i g n a l at 69.30 and a sharp s i n g l e t at 613.28 were assigned to the e n o l i c and phenolic hydroxyl protons, r e s p e c t i v e l y . F i n a l l y , the reduction of t h i s intermediate with chromium (II) c h l o r i d e i n acetone under an atmosphere of carbon dioxide furnished the expected (+)-2-desacetylusnic a c i d (108) i n 72% y i e l d . The synthetic material, bright yellow prisms, mp 220-221°, mixed mp with the authentic material 220-222°, [ a ] ^ 6 (CH CN) +625°, proved to be i d e n t i c a l with the biodegradation metabolite by the comparison of i t s u l t r a v i o l e t (^ m a x 328, 289, 216 nm), i n f r a r e d (1670, 1630 cm - 1) and pmr (Figure 89) spectra, as w e l l as i t s C i r c u l a r Dichroism c h a r a c t e r i s t i c (A (Ae) 374 (+0.95), 333 max (+6.10), 298 (-4.29), 272 (+5.23) and 238 nm (-5.63)) with those of the natural! m a t e r i a l . Figure 89. Proton Magnetic Resonance Spectrum of Synthetic (+)-2-Desacetylusnic Acid (108). - 290 -Part C Amongst the microorganisms o r i g i n a l l y screened by Bandoni and 79 461 Towers, the fungal i s o l a t e M o r t i e r e l l a i s a b e l l i n a (Oudemans 1902), was chosen as a promising candidate for a comprehensive examination. Members of the genus M o r t i e r e l l a are usually i s o l a t e d from s o i l , where they often occur at considerable depth, associated with fragments of plant debris or with l i v i n g plant roots. The I s a b e l l i n a group • i s found c h i e f l y i n f o r e s t s , heath or sandy s o i l s with an a c i d i c r e a c t i o n between pH 3-6, and while both M o r t i e r e l l a vinacea and M. i s a b e l l i n a occur i n moist s o i l s , only the l a t t e r i s found in wet s o i l s . Moreover, 462 Turner has selected M o r t i e r e l l a i s a b e l l i n a as a type species for the genus M o r t i e r e l l a and gave thorough morphological d e s c r i p t i o n s of both the species and the genus. Thus, a sample of the i s o l a t e used for the following study, i d e n t i f i e d as M o r t i e r e l l a i s a b e l l i n a // 129, was obtained through the generosity of Professor R.J. Bandoni of the Botany Department, U n i v e r s i t y of B r i t i s h Columbia. The actual fermentation step was c a r r i e d out i n shake culture f l a s k s , each containing an appropriate amount of (+)-usnic acid (35), for 10 days at room temperature. The representative e x t r a c t i o n procedure indicated i n Figure 90 was developed i n order to separate the complex biodegradation mixtures into two main categories: i ) Neutral F r a c t i o n , containing a l l the non- or weakly a c i d i c materials, and i i ) Acid F r a c t i o n , c o n s i s t i n g i n products d i s p l a y i n g a c i d i c p r o p e r t i e s . Moreover, t h i s procedure allows the f a c i l e recovery of any unreacted s t a r t i n g material by benzene i - 291 -Medium and c e l l s C e l l s Discard (3200 ml, pH 9.2) 1) F i l t e r 2) Wash, c e l l s (3 x 100 ml water) Ce l l s -1) Air-dry 2) Extract with benzene (2 x 100 ml) 3) F i l t e r Benzene Extract F i l t r a t e (3500 ml, pH 9.2) Neutral Fraction 1) Dry 2) Evaporate 3) R e c r y s t a l l i z e Recovered Usnic Acid Acid Fraction 1) Extract with EtOAc (3 x 1000 ml) Aqueous Phase 1) Acidify.- with (+)-Tartaric Acid (to pH 3) 2) Extract with EtOAc (3 x 1000 ml) Aqueous Phase Discard Figure 90. A Representative Extraction Procedure for the Biodegradation of (+)-Usnic Acid (35) by Mortierella isabellina. - 292 -extr a c t i o n of the f i l t e r e d s o l i d s . Although the presence of several usnic acid-derived materials was observed i n both the Neutral and Acid F r a c t i o n s , at the present time only those components which have been f u l l y characterized w i l l be discussed. Moreover, i t has been observed that approximately one-third of the t o t a l biodegradation products (by weight) resid e i n the Neutral F r a c t i o n , with the remaining two-thirds appearing i n the Acid F r a c t i o n (Table 28). Neutral F r a c t i o n Acid F r a c t i o n Component Weight Component Weight FA(N) 0.940 g FA (A) 1.250 g A(N) 0.075 g A (A) 0.060 g B(N) 0.124 g B(A) 0.700 g C(N) 0.010 g C(A) 0.150 g T o t a l : 1.149 g T o t a l : 2.160 g S t a r t i n g amount of (+)-usnic a c i d (35) = 5.0 g FA = f a t t y acid ester residue A,B,C = components (according to p o l a r i t y ) Table 28. Representative Product D i s t r i b u t i o n f o r the Biodegradation of (+)-Usnic Acid (35) with M o r t i e r e l l a i s a b e l l i n a . Thus p u r i f i c a t i o n of the Neutral F r a c t i o n by column chromatography on s i l i c a g e l afforded besides the Fatty Acid Ester Residue, which w i l l not - 293 -be discussed in this thesis as it has been the subject of previous 463 investigations carried out in our laboratories, two mam components. The least polar of them has been labelled A(N), indicating that it is the first component isolated from the Neutral Fraction, and has not been fully characterized due to its inherent unstability. The second component, 26 designated B(N), was obtained as colorless plates, mp 236-238°, [a]^ (CH0CN) +187°, molecular formula C.,H_,0, . Its ultraviolet spectrum 3 lb lb b (A 324, 282, 215 nm) suggested the presence of a phloroacetophenone-max derived system, and its infrared spectrum contained absorption characteristic of chelated hydroxyl groups (3300-3000 cm )^ and o-hydroxy aryl ketones (1625 cm 1 ) , together with a band at 1658 cm 1 corresponding to an inter-molecularly hydrogen bonded a,B-unsaturated ketone. At this point it became clear that the bands assigned to the ring C B-triketone system characteristic of usnic acid (1680 and 1540 cm )^ were absent, thus providing the first firm indication as to the position where microbial attack had taken place. Its pmr spectrum (Figures 91-93) consisted in two three-proton singlets at 61.48 and 2.04 assigned to the-angular and aromatic methyl groups, respectively, although the upfield shift experienced by the former suggested that some kind of change had been introduced at the adjacent 1-position. Moreover, the three-proton singlet at 62.66 was characteristic of the chelated aromatic acetyl grouping, and the one-proton singlet at 65.75 of the olefinic proton at the a-position of an a,B-unsatura-ted ketone fully substituted (substituents other than hydrogen) at the 253 B-position. However, the low field region of the spectrum demonstrated the presence of three hydroxyl protons, two of them as a broad signal at 1 : woo Figure 91. Proton Magnetic Resonance Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252). \ ~ . 1 1 T T - l 1 | I 1 I I |V| 1 I | I I I I | I 1 I i | i i I I | I I I 1 I I I I I' I I I I 1 I I I l ' | I I I I I I I I I I I I I T—r—i—i—T—f "r- | i M I i I I ,1 { II i i I ' I i ' i I I I I | I I . I I 1 . I 1 i i ri i i i i i i COCH a » ' » » ^ l » i n i l t W « A » V » « v t ' ' l ' « » * i | » 1 * « t ^ H L « » ^ ^ V > i t » l n 1 U < < i ' i l » > a » < i * ' < W > » | l ' ' | l M » 1 ^ ' | * V > V ' ' " ' ' I I I I I I I ' I I I ' < > « * « a i » . i W « < l ' > * " > ' JJJ • 1 i ' i j I ' I : > I : . ; '! I l i i 1 I ' • • 1 . . ' i I • , . I • i i . I • 1 i i . i I •• • • I 10 ppm (6) 0 Figure 92. Proton Magnetic Resonance Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252) (Upon Addition of D20). Figure 93. Proton Magnetic Resonance Spectrum of (+)-la-Hydroxy-2-desacetylusnic Acid (252) (Recorded at 60 MHz). - 297 -68.76, and the third, the C -phenolic hydroxyl proton as a singlet at 613.32, thus suggesting that the fungal attack had resulted not only in deacylation but in the reduction of the C-l carbonyl group to the corresponding alcohol as well, as indicated by the molecular formula. Additional absorptions were observed in the 62.50-4.30 region of the spectrum. The high field one, which under normal resolution (60 MHz) resembled a two proton singlet (Figure 93), when examined under high resolution (Figure 91) appeared as the AB part of an ABX system^6^ partially obscured by the aromatic acetyl signal. However, the situation was somewhat clarified by the addition of deuterium oxide (f^ )^> t n u s allowing the assignment of chemical shifts, and coupling constants corresponding to the various protons. Nevertheless, since the chemical shift difference (Av) between the AB protons (ie. the protons of the C^-methylene group) is similar to their geminal coupling constant, J - IS Hz, directly AB measured from the spectrum, severe distortions from the typical ABX pattern are observed; thus the inner peaks are strong, the outer ones weak, and the lines overlap. An analysis of the corresponding molecular model and apparent chemical shifts of the C -methylene protons indicates that the H proton, deshielded relative to the proton by about 22 Hz as a consequence of the deshielding 465 influence of both the C r t , -C . and C -C. bonds, must exist in a pseudo-9b 1 3 4 equatorial orientation, while for the pseudoaxial 11^  proton, which is observed at 6^2.60 the opposite reasoning applies. Furthermore, the X part of the system, assigned to the C-l methine proton, was observed as a characteristic multiplet consisting on four lines of approximately equal intensity centered at 64.30 with coupling constant ^AX+^BX= - 298 -16.5 Hz. It must be remembered that the splittings between the various lines do not correspond exactly to J and J , although a good approxi-AX BX mation is realized when the frequency difference (Av) between the A and B protons is greater than 10 Hz and larger than J 2 5 2 > 2 5 3 > 4 6 6 However, since no changes were observed in the pattern due to the X proton when the spectrum was recorded at both 60 and 100 MHz (Figures 93 and 91, respect-ively), i t is probable that in our case the splittings are good approxi-252 mations to the coupling constants. Further examination of the molecular models revealed that i n order to explain the observed pmr data the C-l methine proton must exist i n an axial orientation, since the alternate equatorial orientation w i l l result in this proton nearly bisecting the dihedral angle between the C -methylene protons, thus generating coupling constants J and J of similar AX BX 252 magnitude, and i t is well known that as they become closer the inner lines of the X multiplet w i l l begin to approach each other, eventually coalescing into a tr i p l e t . In the extreme case that J = J and v = v , AX D A A O the A and B protons are magnetically equivalent and the spectrum i s simply 252 253 an A^X system with the X proton appearing as a true t r i p l e t " ' ' (ie. (-)-dihydrousnic acid (154)). F i n a l l y , i t s mass spectrum (Figures 94 and 95) completely agreed with the postulated structure for (+)-lct-hydroxy-2-desacetylusnic acid as indicated by formula 252, showing the retro Diels-Alder fragmentation as the main feature of the spectrum and o r i g i n a t i n g the base peak at m/e 260. (252 ) The f i n a l component of the Neutral F r a c t i o n , C(N), consisted mainly on dark base l i n e material and was not investigated f u r t h e r . We then turned our a t t e n t i o n to the Acid F r a c t i o n , which produced, besides the usual Fatty Acid Residue FA(A), several other components. The l e a s t polar of them, A(A), was i s o l a t e d as bright yellow prisms,, mp 221-222° [a] ° (CH.CN) + 630°, molecular formula C,_H.1/0_. This sample showed D J l o 14 o i d e n t i c a l chromatographic and spectroscopic properties to those of the material previously i s o l a t e d from the Mucor globosus biodegradation of (+)-usnic a c i d (Part B). Thus i t s u l t r a v i o l e t (X 330, 287, 219 nm), max i n f r a r e d (1670, 1630, 1600 cm ^'), pmr (Figure 96) and mass spectra were superimposable with those of natural and synthetic (+)-2-desacetylusnic a c i d (108) (vide supra). Furthermore, no depression on i t s mp was observed when mixed with e i t h e r material. • -After successive p u r i f i c a t i o n s the second component, B(A), was - ooc -- 301 -OH H 3 C OH [ C « H , 6 0 3 ] 289(51) m 274.7 - C H , C O C H HO H,C OH 3 OH [ C I 6 H , 6 0 6 ] m/e 3 0 4 (86%,). C O C H 3 OH 233 (57) -CO - C g H 4 Q OH 3 OH [ C , 4 H , 6 < \ ] 261 (72) HO CH C O C H 3 OH C H = C = 0 | C H , [ C W H , 2 0 5 ] 26O0OO),, m" 207.0 - C O [ C , 3 H , 2 ° 4 ] + " 232 (79) - C H , [c i 2 H 9 0 4 ] + 217(76) Figure 95. The Postulated Fragmentation of (+)-la-Hydroxy-2-desacetylusnic Acid (252) in the Mass Spectrometer. / ' . i -T-i-r-n s1 . . ; . I i 'i i i M iOOO I ISOO I 1000 ,1 1 ! t I 1 I I I I I I I I ' I | I I 1 i I • I I ' I ' ' ' 1 I I I I | I 1 I i | 1 I I I | . i I I f I I COCH (108 ) i i , i • ' i i, i i 10 i i i i i i i i ! i i i i i i I i i i i I i i I I I i ' 1 I I j i i i I,' : i. i . I, i, I, ',! I .' ,i ,' , ppm Figure 96. Proton Magnetic Resonance Spectra of (+)-2-Desacetylusnic Acid (108) Isolated from Mortierella isabellina. C J o N3 - 303 -obtained as an unstable bright glassy material, [ c ] ^ 0 (CH OH) +392°, whose u l t r a v i o l e t (A 335, 291 and 221 nm), infrared (1765, 1720, 1690, 1625 cm" 1), pmr (Figure 97), and mass (m/e 360, 318, 302, 290, 272, 262, 260, base peak, 232, 217) spectra were i d e n t i c a l with those of synthetic (+)-2-acetoxyusnic acid (251), a material prepared i n Part B of t h i s t h e s i s . For obvious reasons the spectroscopic properties c h a r a c t e r i s t i c of t h i s material w i l l not be f u l l y discussed at t h i s time. The f i n a l Acid F r a c t i o n component, C(A), turned out to be an unstable usnic a c i d derived material that showed spectroscopic data somewhat s i m i l a r to those of the former compound. However, i t has not 472 yet been f u l l y characterized. ( 108 ) (251) The next step on the structure e l u c i d a t i o n of the new materials was the c o l l e c t i o n of chemical evidence that w i l l further support our spectroscopi assignments. Therefore, (+)-la-hydroxy-2-desacetylusnic a c i d (252) was acetylated at room temperature overnight by means of a c e t i c anhydride-pyridine to produce the expected t r i a c e t a t e 253, (+)-la-hydroxy-2-desacetylusnic acid t r i a c e t a t e . Although this material could not be induced to c r y s t a l l i z e , upon Figure 97. Proton Magnetic Resonance Spectrum of Natural (+)-2-Acetoxyusnic Acid (251). - 305 -7 f> sublimation (190°, 0.03 mm Hg) c o l o r l e s s droplets, mp 93-95°, [ a ] Q (CH^CN) +152°, were produced. A n a l y t i c a l and high r e s o l u t i o n mass spectrometry indicated for t h i s material the molecular formula C 2 2 H 2 2 ° 9 ' a r i s i n § from the incorporation of three a c e t y l groupings (as acetates) to the parent molecule ^6^16^6* This f i n d i n g was further confirmed by examination of i t s i n f r a r e d spectrum which demonstrated the presence of both phenolic (1770 cm ^) and saturated (1720 cm "*") acetates. Furthermore, i t s pmr spectrum showed three new absorptions at 62.23, 2.33 and 2.40 corresponding to the l a - and the C - and C^-acetate groupings, r e s p e c t i v e l y . In addi t i o n the A and'B Protons (^62.51 and 2.98) of the ABX system, the X proton ( C - l methine proton) experienced a downfield s h i f t to 65.50 as a consequence of the deshielding influence of. the C^-a-acetoxy substituent, and showed a coupling constant J + J = 16.5 Hz ( i d e n t i c a l to the one observed f o r AX BX compound 252) that suggested that no major conformational changes occurred upon a c e t y l a t i o n . Furthermore, the p a r t i c u l a r coupling constants J = 10 Hz ( a x i a l - a x i a l ) and J = 6.5 Hz ( e q u a t o r i a l - a x i a l ) could be deduced D A from the an a l y s i s of the X part of the spectrum. The same constants can a l s be determined from the pmr spectrum of the phenolic material 252, thus i n d i c a t i n g that they are indeed good approximations to the r e a l values. F i n a l l y , i t s mass spectrum (Figures 98 and 99) showed a complete agreement with the postulated structure. In turning our att e n t i o n to the metabolite 251, (+)-2-acetoxy-usnic a c i d , several f a c i l e c o r r e l a t i o n s were r e a d i l y v i s u a l i z e d . Thus upon ozonization under the conditions previously described for (+)-2-desacetyl-usnic acid (108) (vide supra) a 38% y i e l d of the o p t i c a l l y i n a c t i v e a-cou-maranone diacetate 70 was obtained, i n d i c a t i n g that the aromatic r i n g A had - 306 -- 307 -[c 1 4H 1 2o 5] 2 6 0 ( 1 0 0 ) Figure 99. The Postulated Fragmentation of (+)-la-Hydroxy-2-desacetyl-usnic Acid Triacetate (253) in the Mass Spectrometer. - 308 -not suffered any modification during the microbial degradation. (254 ) Moreover, upon hydrogenation with 5% Platinum on Carbon, the expected dihydro d e r i v a t i v e 254 was produced. This material, (+)-dihydro-2-acetoxyusnic a c i d , was obtained as a c o l o r l e s s glassy material that could 2 6 not be induced to c r y s t a l l i z e , [a] (CH OH) + 45°, molecular formula - 3 0 9 -C. Q H 0 . I t s u l t r a v i o l e t s p e c t r u m (A 322, 286, 222 nm) p r o v e d s i m i l a r l o l o o max to t h a t o f t h e s t a r t i n g m a t e r i a l , w h i l e i t s i n f r a r e d s p e c t r u m (1775, 1730, 1630 cm "*") i n d i c a t e d t h e r e t e n t i o n o f the u n s a t u r a t e d a c e t a t e g r o u p i n g . The l a t t e r c o n c l u s i o n was c o n f i r m e d by i t s pmr s p e c t r u m , t h a t showed t h e e x p e c t e d a c e t a t e a b s o r p t i o n (62.20). As f o r t h e c a s e o f ( - ) - d i h y d r o u s n i c a c i d (154) the a-conf i g u r a t i o n of t h e C ^ - m e t h i n e p r o t o n i s deduced from t h e ob s e r v e d c o u p l i n g c o n s t a n t s (see P a r t A ) . Thus the C^-methylene p r o t o n s appeared as a t w o - p r o t o n d o u b l e t a t 63.06 w i t h c o u p l i n g c o n s t a n t J = 5 Hz, w h i l e t h e C^- p r o t o n was o b s e r v e d as a o n e - p r o t o n t r i p l e t a t 64.84 w i t h c o u p l i n g c o n s t a n t J = 5 Hz. As e x p e c t e d , i t s mass s p e c t r u m showed agreement w i t h t h e p o s t u l a t e d s t r u c t u r e . The f i n a l and d e f i n i t i v e e v i d e n c e f o r the a - a c e t o x y - g - d i k e t o n e s y s t e m o f m e t a b o l i t e 251 came fr o m i t s chromium ( I I ) c h l o r i d e r e d u c t i o n i n a c e t o n e s o l u t i o n to pr o d u c e i n 74% y i e l d ( + ) - 2 - d e s a c e t y l u s n i c a c i d , t h e p r o d u c t i s o l a t e d f r o m b o t h f u n g a l b i o d e g r a d a t i o n s . T h i s r e a c t i o n e s t a b l i s h e d as w e l l t h e c h e m i c a l i n t e r r e l a t i o n between t h e two m e t a b o l i t e s . ( 251 ) ( 108 ) - 310 -Finally, we decided to look into the sequential production of these metabolites according to time, as a possible way to differentiate between the several possible biodegradation pathways involved. Our results (Tables 29 and 30) indicate that after a short induction period of 1-2 days some biodegradation products already begin to appear in the fermentation broth, which grows increasingly alkaline until days 9-10 when it reaches its final pH value of ^9.22. whereas (+)-usnic acid (35) is being nicely incorporated throughout the time interval considered (20 days) (Figure 100), the first product isolated is (+)-2-acetoxyusnic acid (251), which appears as early as the second day. After the fifth day noticeable amounts of (+)-2-des-acetylusnic acid (108) can be isolated from the growth medium (Figure 101). However, due to the inherent problems associated with relative degradation rates, enzyme induction, etc., i t is not possible at this time to forward a biodegradation pathway that would adequately incorporate a l l the observations at hand. Nevertheless, regarding this point, further studies are being performed in our laboratories by means of radioactively labelled precursors (see Part D). i - 311 -Day Recovered (+)-usnic a c i d (mg) pll o f* Medium Tota l weight of Acid F r a c t i o n (mg) To t a l weight of Neutral F r a c t i o n (mg) 0 500 5.20 — --1 272 7.22 18 0.5 2 265 7.48 19 1.5 3 23