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Studies on the biosynthesis, degradation and synthesis of olivacine-ellipticine type indole alkaloids Grierson, David Scott 1975

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STUDIES ON THE BIOSYNTHESIS, DEGRADATION AND SYNTHESIS OF OLIVACINE-ELLIPTICINE TYPE INDOLE ALKALOIDS  BY  DAVID SCOTT GRIERSON B.Sc.  The University o f B r i t i s h Columbia (1970)  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 thesis as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1975  In presenting this thesis  in partial fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this  thesis  for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  CAfl»MIS4/•  The University of B r i t i s h Columbia 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1WS  Date  ABSTRACT Part I o f t h i s thesis describes the i s o l a t i o n of representatives of a c l a s s o f indole a l k a l o i d s , lacking the 3-S-ethyla^ino side chain, from two p l a n t sources Aspidospema a u s t r a l e , and Aspidosperna v a r g a s i i .  A preliminary  i n v e s t i g a t i o n o f the b i o s y n t h e s i s o f several of these compounds was i n Aspidosperca v a r g a s i i .  From crude extracts of Aspicosperma  conducted  australe the  pyridocarbazole a l k a l o i d s o l i v a c i n e (16) and guatambuine (25) were i s o l a t e d . From Aspidosperra v a r g a s i i uleine  (18), apparicine (19), desmethyluleine (85 )  and the pyridocarbazoles 9-methoxyolivacine isolated.  (82) and guatanbuine  Aromatic t r i t i u m l a b e l l e d tryptophan  shown to be incorporated i n t o 9-methoxyolivacine a l s o incorporated i n t o guatanbuine  (25) were  (27) and stemoadenine (13) were (82) and tryptophan (27) was  (25) i n A^p^djpsjjejr^a v a r g a s i i .  Neither  precursor was incorporated into uleine (18). In p a r t II a degradation scheme was developed f o r the i s o l a t i o n of the C - l methyl, C-2 methyl(N-methyl) and C-3 methylene groups o f the "D" r i n g of the o l i v a c i n e (16) and e l l i p t i c i n e (17) systems.  Both e l l i p t i c i n e  (17) and  o l i v a c i n e (16) were converted to t h e i r N-methyl tetrahydro d e r i v a t i v e s guatambuine (25) and N-inethyltetrahydroellipticine (26) v i a formation o f the methiodide s a l t s o f 16 and 17 followed by reduction with sodium borohydride.  Compounds 25  and 26 were converted t o t h e i r corresponding methiodides 86 and 95 and reacted under Hofmann r e a c t i o n conditions. tambuine methiodide  O l e f i n s 88 and 97 were obtained from gua-  (86) and o l e f i n 102 was obtained from 95.  102 were reacted with ozone and the formaldehyde bisdimedone  derivative.  O l e f i n s 88 and  produced was i s o l a t e d as the  iii  The C-2 v i n y l compound 97 was elaborated i n t o the C-3 v i n y l compound 112 by hydrogenation of 97 to 103, formation of the nethiodide 111 and r e a c t i o n of 111 with sodium hydride i n dinethy1formamideo The nethiodides 86 and 95 were a l s o r i n g opened to 89 and 107 by r e a c t i o n with l i t h i u n aluminum hydride.  These cocpounds were i n turn converted to t h e i r  methiodides 90 and 108 and reacted with potassium t-butoxide i n t-butanol. trimethylaaine produced during the r e a c t i o n s was amaonium iodide s a l t .  i s o l a t e d as the tetramethyl-  The e f f i c i e n c y of the N-nethyl group i s o l a t i o n  determined by degrading (N-^C  The  methyl)-guatambuine methiodide  was  (86) and N-nethyl-  t e t r a h y d r o e l l i p t i c i n e nethiodide (95) v i a the l i t h i u m aluminum hydride r i n g opening sequence. Guatambuine (25) was also ring-opened t o a C-3 v i n y l d e r i v a t i v e 125 by r e a c t i o n with a c e t i c anhydride and sodium acetate. Part I I I was concerned with the synthesis o f o l i v a c i n e  (16).  Two  approaches  were developed; i n sequence A the r e a c t i o n o f t r y p t o p h y l brocide (207) with methylacetoacetate  (205) gave 3-carboaethoxy-5-(3-indolyl)-2- pentanone (204).  C y c l i z a t i o n o f 204 l e d to an equal mixture o f  l-oethyl-2-carbomethoxycarbazole  (134) and l-methyl-2-carboaethoxy-l,2,3,4-tetrahydrocarbazole (209) formed by d i s p r o p o r t i o n a t i o n of the i n i t i a l l y forried 208. ture o f 134 and 209 over"Pd/C gave 134.  Dehydrogenation  The carbazole e s t e r 134 was  of the mixalso  obtained d i r e c t l y from 204 by c y c l i z a t i o n i n the presence of c h l o r a n i l as the hydrogen acceptor.  Compound 134 was  reduced to the a l c o h o l 157 with lithium  aluminum hydride and the alcohol 157 was oxidized to the aldehyde 152 with Jones reagent.  The aldehyde  152 was  converted to o l i v a c i n e (16) and  guataxabuine  iv  (25) by a known procedure. In sequence j i . , when 9-benzyltetrahydrocarbazole  (217) was reacted under  Vilsceier-Haack conditions l-methyl-3-forj2yl-9-benzylcarbazole (219) was f o r c e d . Compound 219 was elaborated to the aminoacetal 224 by two routes;  condensation  with asiinoacetaldehyde d i e t h y l a c e t a l (171) l e d t o the i n i n e a c e t a l 221 which was a l k y l a t e d with methylmagnesium c h l o r i d e to give 224. A l t e r n a t i v e l y 219' was a l k y l a t e d to give the cx-hydroxyethyl carbazole 222 which was converted t o i t s corresponding acetate 223. The acetate group was displaced by aainoacetaldehyde d i e t h y l a c e t a l (171) to give 224. The c y c l i z a t i o n o f 22'4 to 6-benzoo l i v a c i n e (225) followed by debenzylation to o l i v a c i n e (16) was not attempted, however the conditions necessary f o r the c y c l i z a t i o n have been worked out f o r the synthesis o f the c l o s e l y r e l a t e d molecule, e l l i p t i c i n e (17).  V  TABLE OF CONTENTS  Page TITLE PAGE. . . . ABSTRACT  •  •  •  •  . . . . . . . . •  o  «  o  «  *  o  *  «  »  <  »  »  *  «  »  »  *  °  »  <  »  *  '  »  *  i »  TABLE OF CONTENTS LIST OF FIGURES  v ' .  vi  LIST OF TABLES  ix  ACKNOWLEDGEMENTS .  •  x  PART I INTRODUCTION  . . . . . . . . . . . . . . .  1  DISCUSSION  37  EXPERIMENTAL  54  PART I I INTRODUCTION  62  DISCUSSION  68  EXPERIMENTAL  137  PART I I I INTRODUCTION DISCUSSION EXPERIMENTAL  BIBLIOGRAPHY  166 .  189 235  256  vi  LIST OF  FIGURES  Figure  Page  1  Compounds o f Major P h a r n a c o l o g i c a l  2  Some R e p r e s e n t a t i v e  3  Indole A l k a l o i d s Occurring  4  The  Barger-Hahn-Robinson-Woodward H y p o t h e s i s  9  5  The  K e n k e r t Prephenate P o s t u l a t e  10  6  The  Acetate  11  7  The  Thomas-Kenkert Monoterpene P o s t u l a t e  8  Rearrangement o f >tonoterpene U n i t i n t o the A l k a l o i d a l Skeletons  9  The as  Indole  Interest  .  2  Alkaloids  5  i n Genus A s p i d o s p e m a  6  Postulate  12 Three 15  E a r l y Stages o f I n d o l e ~ A l k a l o i d B i o s y n t h e s i s " ' Proven by Experiment  ,  .....17  10  Biogenesis  11  The  12  The K e n k e r t P o s t u l a t e f o r the B i o s y n t h e s i s o f Iboga and Aspidosperma f a m i l i e s  13  The A c r y l i c E s t e r P o s t u l a t e f o r the B i o s y n t h e s i s o f t h e Aspidospenr.a and Iboga F a m i l i e s  28  Tne K e n k e r t P o s t u l a t e f o r the B i o s y n t h e s i s O l i v a c i n e (16) E l l i p t i c i n e (17) and U l e i n e  32  14  15  16  o f t h e Cory-nan the F a m i l y . . . .  P o s t u l a t e d O r i g i n s o f the S t r y c h n o s  The D j e r a s s i P o s t u l a t e f o r t h e A p p a r i c i n e (19)  20 family  Biosynthesis  Kim and E r i c k s o n ' s i n v i t r o Study o f Biosynthesis  22 the  of (18)  24  of 33  Uleine 33  vii  Figure  Page  17  Postulated Synthesis o f Apparicine (19) v i a Fragmentation of the Tryptaraine Bridge  33  18  The Potier-Janot Postulate f o r the Biosynthesis o f the Non Tryptamine Bridged A l k a l o i d s  35  19  The N.M.R. spectrum o f Guatambuine (25)  42  20  The L a b e l l i n g Pattern i n the Biosynthesis o f the Olivacine (16) and E l l i p t i c i n e (17) Series According to the Potier-Janot Postulate.....  64  The Structure E l u c i d a t i o n o f Olivacine (16) by Ondetti and Deulofeu (1961)..  65  21 22  The Conversion of Olivacine (16) and E l l i p t i c i n e (17) to t h e i r N-Methyltetrahydro forms. 69  23  The N.M.R. Spectrum o f the Crude Reaction  Mixture  24  The N.M.R. Spectrum o f the Crude Reaction KOt-Bu/t-BuOH)  Mixture 82  25  The N.M.R. Spectrum o f l-Methyl-2-vinyl-3(a-dimethylaminoethyl)carbazole (97)  84  26  The N.M.R. Spectrum o f l-Methyl-2-(B-dimethylaminoethyl)-3-vinylcarbazole (88)  88  27  A Comparison o f the UV. Spectra f o r the O l e f i n s 88 and 97 with the Normal Carbazole Spectrum o f 98  91  28  Mass Spectrum of Carbazole  Compound 103  112  29  Mass Spectrum of Carbazole  Compound 88  112  30  Mass Spectrum of Carbazole Compound 109  112  31  P l a u s i b l e Mass Spectral Fragmentation Pattern for Compound 103  114  viii Figure Page 32  P l a u s i b l e Mass Spectral Fragmentation Pattern for Compound 88  115  P l a u s i b l e Mass Spectral Fragmentation Pattern for Compound 109  115  Approaches to the Isolation of the C - l Methyl Group Involving a C - l * Oxygen F u n c t i o n a l i t y  118  35  Acetate S u b s t i t u t i o n Approach Studied on Model Compounds  122  36  N.M.R. Spectrum o f 1,4-Dimethy1-2-(3-(N,Nmethyacetylamino) ethyl)-3-acetoxyEethylcarbazole (121)  123  37  N.M.R. Spectrum o f l-Methyl-2-(3- (N,N-methylacetylamino)ethyl-3-vinylcarbazole (125)  129  38  The UV. Spectrum o f the Iminium Cation (142), Enamine (126), and Reaction Product o f 142 Treated with Dinethylsulphate i n Base  134  Methylation o f the Anhydro Base  136  Synthesis o f Olivacine (16) and Guatambuine (25) by Schrcutz and Wittwer (I960)  168  41  Scheme A Synthesis o f O l i v a c i n e (16) According t o Wenkert and Dave  171  41  Scheme B  172  42  Synthesis o f O l i v a c i n e (16) by Mosher e t a l . (1966)....174  43  The Kametani Benzocyclobutene Analog Approach  33 34  40  (1975)  176  44  Woodward (1959) Synthesis o f E l l i p t i c i n e  45  Synthesis of E l l i p t i c i n e Saxton (1962) Synthesis of E l l i p t i c i n e (1963) Scheme A - C  46  (17)  177  (17) by Cranwell and 178 (17) by Govindachari et a l .  j  181  F i fiure  47  4S  49  Page  K i l r a i n s t o r and S a l i s b u r y (1972) S y n t h e s i s o f E l l i p t i c i n e (17) ' The S y n t h e s i s o f E l l i p t i c i n e G o y e t t c , and Ahond (1973)  1  8  5  1  8  7  (17) by Le G o f f i c ,  An Ir.proved S y n t h e s i s o f O l i v a c i n e (16) Guatanbuir.e (25), Sequence A  and 192  50  O x a l y l C h l o r i d e Route t o T r y p t o p h o l  51  N.M.R. Spectrum o f 5-Carboir.ethoxy-5-(3-indoly 1 ) 2-pentanone (204) '  197  P l a u s i b l e Mass S p e c t r a l F r a g m e n t a t i o n t h e A l k y l a t i o n P r o d u c t 204  199  52  (206)  193  Pattern f o r '  N.M.R. Spectrum o f t h e C y c l i z a t i o n R e a c t i o n  54  P l a u s i b l e Mass S p e c t r a l F r a g m e n t a t i o n Modes f o r Components 134 and 209 o f t h e C y c l i z a t i o n R e a c t i o n ?-lixture  204  The S y n t h e s i s o f O l i v a c i n e (16) f r o a c a r b a z o l e (216)  219  55.  56.  57.  58.  Mixture,  202  53  Tetrahydro-  Proposed Mechanism f o r t h e V i l s s e i e r - R a a c k F o r o y l a t i o n o f 9 - B e n z y l t e t r a h y d r o c a r b a z o l e (216)  224  N.M.R. Spectrum, o f l - M e t h y l - 3 - ( 2 , p - d i e t h o x y e t h y l i i a i n o m e t h y l ) c a r b a z o l e (221)  229  UV. Spectrum o f Compound Hydrochloric Acid  230  221 i n Water and i n D i l u t e  X  LIST OF TABLES  TABLE  I  II  III  TTT  PAGE  Column Chromatography Results on Extracts from A. v a r g a s i i .  46  Results of Incorporation of Tryptophan and Ste:madenine (13) into A. v a r g a s i i  51  (27)  C h a r a c t e r i s t i c N.M.R. Chemical S h i f t s for the Degradation Products of Guatanbuine (25) H  O  Q  . , l ^  r  A..-."  J-l-^^n  r- C \  1  r  ^ V  1  <-  f l  86 O i l  xi  ACKNOWLEDGEMENTS  I would l i k e to express my g r a t i t u d e to Dr. James P. Kutney f o r his guidance, optimism and the opportunity to l e a r n through the course of t h i s research. I would also l i k e to thank Dr. George F u l l e r and Mr. Harald Hanssen f o r t h e i r c o l l a b o r a t i o n with me in t h i s research, and also the other members of the group, past and present, f o r h e l p f u l  discussions  and suggestions. Thanks are due to Dr. P h i l i p J . S a l i s b u r y f o r his e x p e r t i s e and w i l l i n g n e s s to help me in the propogating and handling of the p l a n t s . Special thanks are due to my f a m i l y , f r i e n d s and t y p i s t s f o r t h e i r help and preserverance during the preparation of t h i s manuscript. Receipt of a National Research Council Postgraduate Scholarship i s g r a t e f u l l y acknowledged.  INTRODUCTION PART 1  The plant kingdom plays a paramount role i n both the existence maintenance of a l l animal l i f e on this planet. l i f e - s u s t a i n i n g oxygen-rich  and  The existence of the present  atmosphere i s considered i n large part to be a  consequence o f the photosynthetic production of oxygen by p r i m i t i v e plant 1,2,3 forms during the early stages o f the development of the earth.  This  same process, harnessing the power of the sun has sustained l i f e by enabling plants to produce consumable energy-containing  compounds v i t a l to the function  of the animal organism. A considerable number of these consumable plants have been found over the ages to be b e n e f i c i a l to man  i n a medicinal or maintenance manner, aiding  in h i s fight against the diseases which threaten the longevity of h i s l i f e . The use o f such herbal remedies to cure ailments i s generally considered to precede even the o r i g i n s of a g r i c u l t u r e . Accounts o f t h e i r reported use are found i n the i n s c r i p t i o n s and writings o f the ancient Egyptian, and Chinese c i v i l i z a t i o n s .  Rivalled, perhaps only by the Ayurvedic medicine  in India, Chinese herbal medicine i s the oldest continuous in their culture.  The SHANG-HAN LUN  Chung-Ching (ca A.D.  Babylonian  surviving t r a d i t i o n  ("Treatise on Fevers") written by Chang  195) i s a c l a s s i c of Chinese c l i n i c a l medicine right down  4  to the present  day.  In modern times the s c i e n t i f i c world has come to view f o l k l o r e and medicines with considerable i n t e r e s t .  Ignoring the shrouds of mystic  tribal  and  - 2 -  (3)  Figure 1.  (4)  Compounds of Major Pharmacological Interest.  considering the possible curative effects of these herbal extracts, i t has been r e a l i z e d that many possess pharmacological a c t i v i t y .  Such investigations  have often led to the i s o l a t i o n of the b i o l o g i c a l l y active component. work has been spurred on by such medically important discoveries as:  This Reserpine  (1) f o r the treatment of mental disorders, i s o l a t e d from the Indian snake root plant Rauwolfia serpentina; morphine (2) an analgesic from the opium poppy Papaver somniferum; strychnine  (3) the cardiac p r i n c i p l e and stimulant from  Strychnos nux vomica; and quinine  (4) an a n t i m a l a r i a l from Cinchona bark (Figure 1) .  These land mark discoveries have provided the incentive for the presently active and intense worldwide i n v e s t i g a t i o n of the plant kingdom for compounds of possible medicinal value.  The subsequent e f f o r t s towards the synthesis of these  -  3 -  molecules has been the foundation of the modern drug industry.  Although a  tremendous number of s y n t h e t i c a l l y prepared drugs are i n use today, i t i s estimated that approximately 50% of the medicines prescribed are 5,6 derived from natural plant sources. It was  recognized  still  early i n the investigations that some indole a l k a l o i d s  possess b e n e f i c i a l medicinal properties.  They have been found to occur  widely among flowering plants p a r t i c u l a r l y o f the Apocynaceae, Loganiaceae, 7 and Rubiaceae plant f a m i l i e s . These three families stand close together i n 8 the phylogenetic  charts of the taxonomists.  The  family Apocynaceae i s  p a r t i c u l a r l y r i c h i n a l k a l o i d s , e s p e c i a l l y the genera Rauwolfia, Vinca (Catharanthus), A l s t o n i a and Aspidosperma. The  great majority o f complex indole alkaloids can be v i s u a l i z e d to con-  s i s t of a tryptamine u n i t and a Cg-C^n unit which i s monoterpene derived. 9 Approximately 1000  indole alkaloids are known to date.  These compounds i n  general o f f e r i n g minor v a r i a t i o n s i n f u n c t i o n a l i t y , oxidation state stereochemistry according  and  lend themselves to a formal d i v i s i o n into four main groups  to t h e i r s k e l e t a l features.  These four groups, the corynanthe (5),  strychnos (5), aspidosperma (6) and iboga (7) are represented by  three  d i s t i n c t s k e l e t a l arrangements of the carbon atoms i n the CQ-C^Q unit as  illu  trated below.  (5)  (6)  (7)  i  - 4-  Examples of these groups are the r i n g closed forms: preakuammicine (9), vindoline (10) and catharanthine r i n g opened forms:  g e i s s o s c h i z i n e (8),  (11) and the corresponding  p i c r a p h y l l i n e (12), stemmadenine (13), vincadine (14) and  18-carbomethoxycleavamine (15) (Figure 2 ) . As a r e s u l t o f an intensive search o f the Apocynaceae family f o r both b i o l o g i c a l and taxonomical  purposes, i t has been found that many plants o f the  genera Aspidosperma and Ochrosia contain complex alkaloids possessing novel 7,9,10 structures.  These compounds are exemplified by the structures o f  o l i v a c i n e (16), e l l i p t i c i n e (17), uleine (18), apparicine (19) and vallesamine (20).  They are novel i n that they do not possess the 3-3-ethylamino side  chain (tryptamine bridge) indigenous indole a l k a l o i d s .  to the majority of n a t u r a l l y occurring  I t can be seen that uleine (18), apparicine (19), and  vallesamine  (20), have only a s i n g l e carbon atom separating the 3-position of (Nb) the indole nucleus from the b a s i c nitrogen, whereas, o l i v a c i n e (16), and e l l i p 11* t i c i n e (17), being s u b s t i t u t e d 6H-(4,3-b)-pyrido carbazoles  e x h i b i t a three  carbon bridge (Figure 3 ) . In terms of formality, these compounds can be considered to possess the corynanthe-strychnos  skeleton 5, however by v i r t u e o f t h e i r almost exclusive  presence i n Aspidosperma plants, they are considered to be aspidosperma alkaloids.  They are generally found to co-occur i n many o f these plant systems and  are not, therefore, simply i s o l a t e d examples o f b i o l o g i c a l p e c u l i a r i t i e s . are also found to co-occur with a small number o f normal tryptamine a l k a l o i d a l systems such as N-acetylaspidospermidine N-acetyl-ll-hydroxyaspidospermatidine 10 carpine (24) (Figure 3 ) . *  They  bridged  (21), aspidocarpine  (22),  (23), and ll-methoxy-4,19-dihydrocondyloV  Two numbering systems have been used to number the pyridocarbazole skeleton. The system used to label this system and the Uleine (18) and apparicine (19) skeletons i n t h i s thesis was that accepted by Chem. Abstracts (Ref. 11).  - 5 -  Figure 2.  Some Representative Indole A l k a l o i d s .  - 6 -  CHOOC  CH^DH  (20)  COOCH (24)  (23)  Figure 3. Indole Alkaloids Occurring i n Genus Aspidosperma.  - 7 -  In the l a t t e r two cases i t i s tempting to envisage uleine skeleton by extension of the two  the build-up of the 114  carbon tryptamine  Both o l i v a c i n e (16) and e l l i p t i c i n e  bridge.  (17) have received considerable  i n t e r e s t i n recent years as a r e s u l t of t h e i r antitumor  a c t i v i t y , however  our interest i n these compounds r e l a t e s to t h e i r b i o s y n t h e t i c build-up i n the plant system.  At the i n i t i a t i o n of the present work, the occurrence  these compounds posed the important way  questions:  What i s the mechanistic  of path-  of t h e i r i n vivo synthesis and i s there some connection between t h e i r  occurrence plant.  and that of the normal tryptamine bridged a l k a l o i d s found i n the  In other words, are these alkaloids c l o s e l y r e l a t e d i n terms of the  presently accepted indole a l k a l o i d b i o s y n t h e t i c scheme?  The answers to these  questions, i t was hoped, would be found through a study of the biosyntheses  of  these compounds i n Aspidosperma p l a n t s . A considerable number of reviews have been published concerning aspects of indole a l k a l o i d biosynthesis. state of the biosynthesis and occurrence 12 i n the recent reviews by Schmid  different  A d e t a i l e d discussion of the present of t h i s class o f compounds i s contained 13  and C o r d e l l  .  As a consequence, therefore,  only a b r i e f overview of the biosynthesis of indole a l k a l o i d s w i l l be discussed i n t h i s presentation with emphasis on the present knowledge concerning  the  biosynthesis of o l i v a c i n e (16), e l l i p t i c i n e (17), uleine (18), apparicine (19), and the N-methyltetrahydro derivatives of the pyrido carbazoles, guatambuine (21) and N-methyltetrahydroellipticine (22).  (22)  - 8 -  There i s general agreement that the tryptamine segment o f the indole alkaloids i s derived from tryptophan (27), i n accord with the postulate o f 14 Pictet i n 1906. With the development o f tracer techniques i n the l a s t 15-18 decade, this hypothesis has received ample j u s t i f i c a t i o n .  Labelled  tryptophan (27) has been found to be s u c c e s s f u l l y incorporated into a large variety of indole a l k a l o i d s .  Tryptamine, (28) however, the other  (27)  logical  (28) 18-20  precursor has been incorporated with mixed success.  The exact nature  of this r e s u l t i s d i f f i c u l t to i n t e r p r e t , i t could suggest that the decarboxylation i s delayed i n some cases, or that tryptamine (28) i s not transported to the s i t e of biosynthesis as i s the amino a c i d . The biogenetic o r i g i n o f the remaining g C^Q u n i t , i n contrast to the C  source of the 8-aminoethylindole moeity, was  -  the subject of much controversy.  The e a r l i e s t speculations as to the o r i g i n of this non-tryptophan unit were 20 based upon the s t r u c t u r a l s i m i l a r i t i e s of indole a l k a l o i d s . P r i o r to the f i r s t t r a c e r experiments concerned with the o r i g i n o f this 21-2 unit, there were three main postulates i n existence. The o r i g i n a l Barger-Hahn 23 24-6 postulate had been elaborated by Robinson and Woodward to the point where i t could accommodate the formation of a l k a l o i d s of the strychnos  and  corynanthe, as well as the o r i g i n a l yohimbe, a l k a l o i d skeletons as shown i n figure 4.  The combined hypothesis  involved the condensation of 3,4-dihydroxy-  phenylacetaldehyde (29) derived from tyrosine with tryptamine (28) e i t h e r at the 3-position o f the indole system to y i e l d strychnine  (3), or at the 2-position  - 9 -  (31)  Figure 4 .  Hie Barger - Hahn - Robinson - Woodward Hypothesis.  - 10 -  (34)  Figure 5.  The Wenkert Prephenate Postulate.  - 11 -  Figure 7.  The Thomas-Wenkert Monoterpene Postulate.  - 12 -  to y i e l d 30 which contains an aromatic r i n g E.  F i s s i o n of the  aromatic  r i n g E between the two hydroxyl groups and subsequent combination with appropriate  units leads to yohimbine ( 3 1 ) .  A number of d e f i c i e n c i e s were immediately evident i n these specula27 tions and i n 1 9 5 9 Wenkert and B r i n g i proposed an elegant a l t e r n a t i v e . They i n i t i a l l y proposed that a hydrated prephenic  acid was  the intermediate,  28  but l a t e r Wenkert  modified this hypothesis so that prephenic The  acid ( 3 2 )  i t s e l f was  the d i r e c t progenitor of the indole a l k a l o i d s .  latter  rearranges  according to the scheme shown i n f i g u r e 5 to a f f o r d a c r u c i a l  intermediate, the seco-prephenate-formaldehyde (SPF) u n i t ( 3 3 ) which can be elaborated into yohimbine ( 2 7 ) and corynantheine  (34).  This  ingenious  a l t e r n a t i v e scheme uses the a l i c y c l i c precursors of phenylalanine to account f o r the oxidation state of the E r i n g . hypothesis, the 1,2-migration of the pyruvate  directly  A key step i n t h i s  side chain o f prephenic  acid  with retention o f configuration, explains the absolute configuration at C - 1 5 29 i n corynanthe-strychnos  alkaloids.  The carbomethoxy group i s an i n t e g r a l  part o f the hydroaromatic progenitor instead of being attached whenever necessary. 31-33  30  S c h l i t t l e r and Taylor  i n 1 9 6 0 and Leete  i n 1 9 6 1 postulated that  the non-tryptophan portion o f the indole a l k a l o i d s was tate pathway.  derived v i a the ace-  The suggestion u t i l i z e d a s i x carbon chain derived from three  acetate units which condensed with malonic acid and a one carbon unit ( b i o l o g i c a l l y equivalent to formaldehyde) y i e l d i n g the desired C^Q unit (Figure 6 ) . At this time the structure e l u c i d a t i o n of a number of cyclopentane mono34-37  terpenic glucosides ( i r i d o i d s ) had been achieved,  exemplified by verbena-  l i n ( 3 5 ) , genipin ( 3 6 ) , aucubin ( 3 7 ) , and asperuloside ( 3 8 ) .  - 13 -  (39)  Realizing that these glucosides, and i n p a r t i c u l a r the seco-cyclopentane unit o f swertiamarin  (39) had the corynanthe-strychnoslike skeleton 5 as well as 29 the same stereochemistry i n the appropriate p o s i t i o n , as C-15 i n the alkaloids ,  and that the carbomethoxy function (or derivative thereof) also appeared at the 28 corresponding p o s i t i o n , led Wenkert and simultaneously and independently 38 Thomas to propose a monoterpenoid based hypothesis f o r the o r i g i n of the C  9"10  u  n  i  t  (Pig" " 1  6  7  ) •  The key intermediates i n t h e i r pathway are the cyclopentanoid  unit 40  having the corynanthe skeleton 5 and i t s c y c l i c form 41 analgous to swertiamarin (39).  The unit 41 i s derived i n t a c t without the need f o r an a d d i t i o n a l  from formaldehyde or glycine as required by the e a r l i e r  carbon  hypotheses.  The cyclopentanoid monoterpenes genipin (36) and aucubin  (37) have a  related b i o s y n t h e t i c pathway and have been shown to co-occur with the alkaloids 38 in the genus Strychnos .  - 14 -  The i n i t i a l biosynthetic experiments using radioactive  precursors 39,40  disproved  a l 1 the hypotheses concerning the genesis o f the Cg-Cjg u n i t .  However, further experiments by Battersby  and co-workers with (2-14rj)-  mevalonate afforded low incorporations o f a c t i v i t y into the ubiquitous 40,43 non-tryptophan u n i t . These r e s u l t s were r a p i d l y confirmed by two other 41,42 research groups working with the plant species Vinca rosea and Vinca major. The Thomas-Wenkert monoterpene postulate consequently became widely accepted. More experiments by these same workers u t i l i z i n g s p e c i f i c a l l y l a b e l l e d mevalonates coupled with degradative data demonstrated the i n t a c t incorporation of t h i s unit into the representative  alkaloids catharanthine (11), vindoline (10), 43-6 ajmalicine (44), serpentine (42) and perivine (43). Subsequently i t was 44,46-52 44,50 shown that geraniol pyrophosphate (45) and nerol (46) could also  (42)  serve as precursors  (43)  o f these alkaloids (Figure 9 ) . In addition, deuterium  l a b e l l e d mevalonolactone fed to V. rosea produced alkaloids whose mass s p e c t r a l 47-8 fragmentation patterns substantiated  the radioactive label f i n d i n g s .  From these experiments a pattern emerged f o r the rearrangement of the monoterpene unit into the three s k e l e t a l units 5, 6, and 7 f o r each of the a l k a l o i d a l  - 15 -  Figure 8.  (11) (10) Rearrangement of Monoterpene Unit into the Three A l k a l o i d Skeletons  families, which was  consistent with the p o s i t i o n of the r a d i o l a b e l i n the  various i s o l a t e d a l k a l o i d s , (Figure 8). skeleton was  I t appeared as though the corynanthe  formed f i r s t during the biosynthesis and i t subsequently  by the manner shown to the aspidosperma (6) and iboga (7) skeletons. known however, how  rearranged I t was  not  t h i s process occurred i n the plant or whether there were  terpenoid glycosides, as yet not i s o l a t e d , having skeleta of types 6 and 7 which independently  reacted with tryptamine (28).  With the monoterpene hypothesis  c l e a r l y substantiated, an elegant s e r i e s of  experiments by Battersby and co-workers determined the exact nature of the sought a f t e r cyclopentanoid  intermediate.  The i r i d o i d compounds genipin (36) 28 and verbenalin (35) and others proposed by Wenkert were found not to be incor50,53 porated i n t o any of the alkaloids studied.  However a very s i m i l a r monoter-  pene loganin (49) e x h i b i t i n g e s s e n t i a l l y the correct oxidation l e v e l and stereo-  - 16 -  chemistry anticipated f o r the non-tryptophan precursor was found to be s p e c i f i c a l l y incorporated i n t o the alkaloids catharantl.ine (11), vindoline 53-5 (10), ajmalicine (44), serpentine (42) and perivine (43) i n V. rosea , 55 as well as i n t o the alkaloids i n Rauwolfia serpentina , and Cephaelis 64 ipecacuanha. Furthermore, through i s o t o p i c d i l u t i o n studies i t was shown 53-4,57 that loganin (49) co-occurred with the indole a l k a l o i d s i n V.rosea 57 and Strychnos nux vomica. I t has since been shown that loganin (49) occurs 13 i n many a l k a l o i d containing p l a n t s . The isoprenoid o r i g i n o f loganin (49) was subsequently demonstrated by feeding s p e c i f i c a l l y l a b e l l e d forms o f mevalonate, geraniol, and nerol to 53-4,56-8 54-5,59-61 57-8,62 62-3 V.rosea , NL_ t r i f o l i a t a , nux vomica , and carliensis. Battersby  demonstrated also the incorporation i n  rosea o f 10-hydroxy-  geranial and 10-hydroxynerol (47) i n t o both loganin (49) and the aforementioned 65-6 alkaloids (Figure 9 ) . Randomization of the label a t the positions marked 2,6 indicated that oxidation at both o f these carbons i s a necessary part o f the sequence to deoxyloganin (48). Deoxyloganin (48) was found to be a constituent of  rosea and  nux vomica  and was shown to be a precursor o f loganin (49) and the indole constituents o f the 67-8 former plant system. The cleaved monoterpene derivative o f loganin (49), secologanin  (50) was  i s o l a t e d from rosea and found once more to be s p e c i f i c a l l y incorporated into the alkaloids i n the plant. Loganin (49) was shown also, to be a precursor of t h i s 69 compound. 28 An i n t e r e s t i n g observation i s that the c e n t r a l intermediate o f Wenkert's prephenic a c i d hypothesis, stereochemistry  the SPF unit (33) i s almost i d e n t i c a l i n structure,  and oxidation l e v e l to the c r i t i c a l monoterpene  intermediate  - 17 -  Figure 9.  The Early Stages of Indole A l k a l o i d Biosynthesis.  - 18 -  secologanin  (50), although i t was  unsupported by experimental  evidence.  With the o r i g i n o f the Cg-CjQ unit firmly supported by experiment, e f f o r t s were directed towards determining  how  secologanin  (50) was  utilized  by the plant system i n the biosynthesis of indole a l k a l o i d s . Simultaneously  and independently, vincoside (51) and isovincoside (52) 70 71 were i s o l a t e d from V. rosea , and s t r i c t o s i d i n e (52) from Rhazya s t r i c t a . 72-3 X-ray analysis shown.  has proven the r e l a t i v e configurations about C-3 to be as  In addition i t has been shown that vincoside (51) i s s p e c i f i c a l l y  70,74-5 incorporated by V. rosea plants into a l l three types of indole a l k a l o i d s , 70 and that i t i s i t s e l f derived from tryptophan (27) and loganin (49) (Figure 9). 74 Isovincoside (52) was not incorporated i n t o any of the a l k a l o i d s studied  (SI)  which was  (52)  s u r p r i s i n g since i t rather than vincoside (51) exhibits the configur  ation at C-3  found i n the corynanthe a l k a l o i d s .  To date i t has been demonstrated that the hydrogen at C-3  i s epimerized  on incorporation into the alkaloids and that through incorporation of loganin (corresponding  to C-^H)  (5-%)  i n t o the alkaloids that retention of t r i t i u m 76 occurs i n the r e q u i s i t e epimerization . Much remains to be done however to  - 19 -  determine the fate of the vincoside molecules during biosynthesis. These results i n  rosea at least strongly suggest that a c r u c i a l i n t e r -  mediate, vincoside (51), i s formed by convergent pathways i n v o l v i n g separate biosystheses of tryptophan (27) and secologanin  (50), and that t h i s interme-  diate then undergoes the appropriate rearrangements to the various families of complex indole a l k a l o i d s .  Vincoside  d i v i d i n g l i n e , i t s biosynthesis may  (51), then, serves as a convenient  be thought of as the early stages  of  biosynthesis, while the subsequent rearrangements make up the l a t t e r stages o f the biosynthesis. The bioconversion of vincoside (51) into the corynanthe alkaloids e n t a i l s an unexceptional  enzymic hydrolysis of the g l u c o s i d i c residue followed by  reductive condensation of the nascent aldehyde (53) as shown i n figure 10. Corynantheine aldehyde (54), corynantheine (34) and geissochizine (8) are the immediate products of the c y c l i z a t i o n while ajmalicine (44), an abundant corynanthe compound i n V. rosea i s subsequently reached by c y c l i z a t i o n o f 8.  The  former three compounds have been detected to be a c t i v e l y involved i n the b i o Jr  synthesis through sequential feeding and deuterium l a b e l l i n g experiments i n 77-79 V. rosea  ,  thus lending support  The strychnos  to the proposed  transformations.  alkaloids d i f f e r from the corynanthe formally only i n the  p o s i t i o n of attachment of the same C^Q u n i t 5 to tryptamine. 80 81 been proposed by Wenkert  and Scott  ments involved i n transforming  respectively depicting the  the corynanthe into the strychnos  i l l u s t r a t e d i n figure 11 f o r the major strychnos stemmadenine (13).  Two pathways have rearrangealkaloids as  compounds akuammicine (59)  g e i s s o c h i z i n e (8) containing a reactive 3-aldehydo ester  and  - 20 -  Figure 10.  The Biogenesis of the Corynanthe Family.  - 21 -  function i s the central intermediate i n both pathways. Credence but not precedence i s given to the Wenkert scheme (A) by the presence of the formylstrictamine 55 along with pleiocarpamine 82,13 Rhazya s t r i c t a .  (55)  (56) i n  (56)  By the same token Scott's postulate (B) i s supported by the i s o l a t i o n of the 6-hydroxyindolenine 57, geissochizine oxindole (58) and preakuammicine 79 (9) from sequential feedings i n V. rosea and the recently reported forma83-4 tion of oxindole alkaloids i n Mitragyna p a r v i f o l i a . Also, the incorporation 79 of geissochizine oxindole (58) into akuammicine (59)  lends credence to the  postulate. Much remains to be done however, to determine whether one or both pathways are important i n the plant, and whether or not the pathway taken depends upon the i n d i v i d u a l p l a n t . Throughout the i n v e s t i g a t i o n s o f indole a l k a l o i d biosynthesis a major concern has been to determine the manner i n which the aspidosperma and  iboga  families are derived through rearrangement o f the monoterpene portion of the corynanthe skeleton 5 as depicted i n f i g u r e 8.  F i g u r e 11.  The Postulated Origins of the Strychnos  Family.  - 23 -  Through the large body of incorporation experiments dealing with the early stages o f biosynthesis, vincoside (51) has been shown to be a precursor o f both the aspidosperma and iboga a l k a l o i d s . Subsequent incorporations of 85,79 77-79 l a b e l l e d forms of geissochizine (8) corynantheine aldehyde (54) and 77 p a r t i c u l a r l y stemmadenine (13) i n t o vindoline (11) and catharanthine (10) has demonstrated that the corynanthe-strychnos of the two f a m i l i e s .  a l k a l o i d s are also precursors 77,86  Sequential feeding experiments by Scott  i n V. rosea  seedings f u r t h e r confirms that the strychnos alkaloids are the immediate precursors o f the aspidosperma and iboga and that the postulated sequence: corynanthe  strychnos -»• a s p i d o s p e r m a i b o g a i s correct. 87 The i s o l a t i o n and incorporation of stemmadenine (13) i n rosea was of p a r t i c u l a r s i g n i f i c a n c e because i t was recognized that i t occurs furthest along the strychnos pathway towards the aspidosperma and iboga bases. 28 Wenkert  i n i t i a l l y introduced the concept that a seco C3-C7 intermediate  derived from the strychnos skeleton could act as a p i v o t a l precursor to both the aspidosperma and iboga a l k a l o i d s .  The Wenkert postulate portrayed i n  figure 12 suggests that v i a intramolecular Michael and Mannich  condensations  the seco C3-C7 piperidiene 62 obtained from a 1,5-dicarbonyl stemmadeninel i k e compound 61 (analogous to the iminium intermediate 60) i s converted into the nine membered r i n g systems 63 and 64.  Subsequent transannular c y c l i z a t i o n  of these components leads to the s i x membered r i n g bases 65 and 66 from which the aspidosperma and iboga alkaloids are derived. 88,13 A s i m i l a r intermediate 67 was proposed by Levy  to account f o r the  biosynthesis o f the quebrachamine (68) as w e l l as the aspidosperma and iboga bases.  Figure 12.  The Wenkert Postulate f o r the Biosynthesis o f the Iboga and Aspidosperma Families.  - 25 -  CH|XOC  CHOH  (67) Fundamental to Wenkert's postulate was the transannular c y c l i z a t i o n step f o r conversion of the t e t r a c y c l i c nine membered r i n g systems into the pentacyclic r i n g systems o f the aspidosperma and iboga a l k a l o i d s .  In an  e f f o r t to test this transformation, i n i t i a l experiments determined i t to be 89-93 a f a c i l e i n v i t r o process  as exemplified by the conversion of quebrach-  amine (68) and 16-carbomethoxycleavamine  (15) ( v i a t h e i r N(b) iminium s a l t s )  to t h e i r pentacyclic analogs aspidospermidine (69) and catharanthine (11).  H  H  (68)  (69)  However, a l l subsequent l a b e l l i n g experiments to test the p a r a l l e l i n vivo conversion to e i t h e r the pentacyclic aspidosperma or iboga alkaloids 94-5 f a i l e d , no incorporation was found into any o f the alkaloids studied.  - 26 -  These r e s u l t s strongly suggested postulate was  that the l a t t e r p o r t i o n of Wenkert*s  i n c o r r e c t and that i f a p i v o t a l intermediate such as the seco  C3-C7 p i p e r i d i e n e i s involved i n the biosynthesis of the aspidosperma and iboga bases, then a r a t i o n a l whereby i t could account f o r the natural t e t r a c y c l i c and p e n t a c y c l i c structures would have to be Support was  determined.  given the intermediacy of a p i v o t a l seco C3-C7 precursor by  the observation that tabersonine (70) was  e f f i c i e n t l y incorporated i n t o both  the aspidosperma a l k a l o i d v i n d o l i n e (10) and the iboga a l k a l o i d catharanthine 94 77 (11) i n V. rosea plants and seedlings. This interconversion hot only involves a considerable rearrangement of the aspidosperma skeleton of tabersoninine (70) i n c l u d i n g a reversal of the transannular c y c l i z a t i o n , but i t further demonstrates the sequence corynanthe-strychnos  COOCH  -*• aspidosperma -*• iboga.  3  (70)  I t i s pertinent to p o i n t out that the reverse process, the conversion o f 77 catharanthine (11) to tabersonine (70)» was  found not to occur  suggesting that  e q u i l i b r a t i o n with a p i v o t a l intermediate l i e s only to the side of the aspidosperma representative tabersonine (70). Observations by Scott based upon the in_ v i t r o interconversions o f s e l e c t i v e  - 27 -  alkaloids i n hot a c e t i c a c i d led to the proposal that the a c r y l i c e s t e r 72 available by r e q u i s i t e rearrangement o f stemmadenine (13) may be involved as the sought a f t e r p i v o t a l intermediate i n the biosynthesis of the aspido96-7 sperma and iboga bases.  Through the transformations o u t l i n e d i n figure  13 the t e t r a c y c l i c and p e n t a c y c l i c r i n g systems can be generated  without  having to invoke the transannular c y c l i z a t i o n process. Evidence  f o r the occurrence of a seco intermediate such as the a c r y l i c  ester 72 was obtained by the i s o l a t i o n of the dimeric indole compounds, the 98-9 secamines, from Rhazya species. Of p a r t i c u l a r s i g n i f i c a n c e was the pre100-1 sence of the monomeric secodines 73, 74 and 75.  (73)  (74)  (75)  Due to the inherent i n s t a b i l i t y of the dihydropyridinium system i n the a c r y l i c e s t e r 72 since named dehydrosecodine,  the synthesis o f l a b e l l e d forms  o f this compound f o r the t e s t i n g o f the Scott proposal has not to date been possible.  In view of t h i s , the more stable reduced  form o f the a c r y l i c ester 102-3  72, secodine (76) and the hydroxy ester 77 both available by synthesis were considered as useful intermediates i n the biosynthetic i n v e s t i g a t i o n s .  - 28 -  COOCH (77)  Figure 13.  3  COOCH (76)  The A c r y l i c Ester Postulate f o r the B'iosyntheses of the Aspidosperma and Iboga Families.  3  -29-  This view received support when Battersby proposed, on the basis of i s o t o p i c d i l u t i o n experiments that 16,17-dihydrosecodin-17-ol (77) occurs n a t u r a l l y 104 in rosea and R. o r i e n t a l i s (figure 13, bottom).  105-8 Administration of 16,17-dihydrosecodin-17-ol-(Ar-^H) (77) into V. rosea , 106,109 106-8,110 V. minor, and Aspidosperma pyricollum resulted i n each case i n deterioration of the plants with no detectable incorporation into the 103,105-6,109 alkaloids studied.  Subsequent experiments with  (Ar-^H)-Secodine  (76)  have been more successful, however the r e s u l t s must remain tentative as only very low but constant incorporations of this precursor have been obtained into Vindoline (10) and catharanthine ( 11) i n  rosea, and the alkaloids of  Further studies using various forms o f doubly l a b e l l e d secodine coupled with degradation  (76)  of the alkaloids i s o l a t e d lent stronger support  conviction that the secodine skeleton i s incorporated i n t a c t into the 105-112 sperma and iboga a l k a l o i d s .  minor.  to the  aspido-  It i s evident from the secodine results that the inherent d i f f i c u l t i e s o f low incorporation and the necessity o f feeding a precursor o f the wrong oxidation state w i l l have to be overcome before the a c r y l i c ester, dehydrosecodine (72) can be f i r m l y accepted  as the p i v o t a l precursor of the aspidosperma and  iboga a l k a l o i d s . The abnormally bridged indole alkaloids u l e i n e (18), apparicine olivacine (16) and e l l i p t i c i n e  (17)  (19),  (as well as the reduced froms of the  l a t t e r ) are presently believed to be derived from late stage intermediates the biosynthetic pathway.  in  Very l i t t l e i s presently known regarding t h e i r bio-  synthesis and as a consequence a number of d i f f e r e n t postulates have been put  - 30 -  forward to r a t i o n a l i z e t h e i r presence i n the Aspidosperma and Ochrosia  plant  systems. The anomolous structures o f these compounds i n i t i a l l y led investigators to believe however, that by contrast to the normal 3-ethylamine bridged compounds they were not tryptophan derived.  In conjunction with the  proposal  of the SPF unit 33 as the progenitor of the monoterpene p o r t i o n o f the alka28 l o i d s , Wenkert  proposed that the biosynthetic pathway to uleine  o l i v a c i n e (16) and e l l i p t i c i n e  (18),  (17) involved reaction of the SPF u n i t 33 with  g l y c o s y l i d e n e a n t h r a n i l i c a c i d (78) a precursor of tryptophan (27).  Condensa-  tion of the r e q u i s i t e carbonyl groups o f the SPF unit with a nitrogen source (such as methylamine) and reaction at the C-3  of indole with loss o f the  glycosyl function leads to the alkaloids (Figure  14). 113  With the structure e l u c i d a t i o n o f apparicine (19) i n 1965, proposed that Wenkert's intermediate biosynthesis.  Isomerization  Djerassi  79 could serve as a precursor i n i t s  to the exocyclic iminium species 80 followed by  c y c l i z a t i o n with the indole nucleus as shown i n figure 15 would y i e l d apparicine  (19). 28  Wenkert's postulate  invokes a separate biosynthetic pathway to these  alkaloids not i n v o l v i n g tryptophan (27) so as to extrude the two carbon chain, and as such i t i s i n discord with the opinion that these indole a l k a l o i d s are tryptophan (27) derived v i a the pathway elucidated f o r the form a l k a l o i d a l families. In order to test the b a s i c assumption behind this proposal, (Ar- H) (27) was 3  administered  to A.pyricollum,  tryptophan -  a plant system reported to  - 31 9 (19) and uleine (19) . S i g n i f i c a n t incorporation was 110 obtained into apparicine (19) which indicates i t to be tryptophan derived, 113 contrary to Djerassi's proposal. contain apparicine  I n s u f f i c i e n t quantities o f uleine were i s o l a t e d however to permit a determination  of any incorporation into i t . Despite subsequent attempts 119,120 to radiolabel this molecule i n australe, no biosynthetic data i s presently available f o r i t or f o r the pyridocarbazoles  N-methyltetrehydroellipticine  o l i v a c i n e (16), e l l i p t i c i n e (17),  (26) and guatambuine (25).  With tryptophan (27) as the precursor of the indole portion o f apparicine  (19), i t i s necessary to determine the mechanism whereby one or  both o f the carbons o f the B-ethylamine side chain are extruded.  Some i n s i g h t  was gained when (Ar-^H) and e i t h e r C-2 or C-3-14c doubly l a b e l l e d tryptophan (27) was fed to A. pyricollum.  I t was found that C-3 of tryptophan (27) i s  incorporated i n t o apparicine  (19) with retention o f the 3H/14C r a t i o whereas 108 over 97% of the label at C-2 was shown to be l o s t . Extrapolating the knowledge that tryptophan i s the l i k e l y precursor o f 114 apparicine (19) to include uleine (18), Kim and Erickson studied a 115 mechanism proposed by Joule ejt a l i s extruded.  whereby the two carbon bridge of uleine (18)  Their experimental model f o r uleine (18) biosynthesis was based  upon the co-occurrence and close s i m i l a r i t y between uleine (18) and alkaloids of the condylocarpine  type such as 24.  They attempted without success to  accomplish the i n v i t r o expulsion of the two carbon u n i t from the oxidation produce of a condylocarpine  system 81 (figure 16).  This mechanism i s probably of l i t t l e biosynthetic s i g n i f i c a n c e however as i t i s s p e c i f i c to uleine (18) only and cannot r e a d i l y be adapted to account f o r  i  Figure 14.  The Wenkert Postulate f o r the Biosyntheses of Olivacine (16), E l l i p t i c i n e (17) and Uleine (18).  - 33 -  OHC  COOCH.  OHC  COOCH  3  Figure 15.  The Djerassi Postulate f o r the Biosynthesis of Apparicine (19)  Figure 16.  Kim and Erickson's i n v i t r o Study o f Uleine Biosynthesis.  -~ ,H  / AN  (13)  (19) Figure 17.  Postulated Synthesis o f Apparicine (19) v i a Fragmentation of the Tryptamine Bridge.  - 34 -  the lack of the 3-ethylamine bridge i n the closely r e l a t e d apparicine  (19)  structure. Stemmadenine (13) known to co-occur with apparicine (19) and uleine (18) 116 i n A. pyrico1lum was found to be e f f i c i e n t l y incorporated i n t o apparicine 108 (19).  This important  r e s u l t places apparicine (19) and very l i k e l y uleine  (18) and the pyridocarbazole alkaloids of the o l i v a c i n e (16) and  ellipticine  (17) at a late stage i n the biosynthetic scheme. 108 The subsequent low incorporation o f vallesamine  (20)  an a l k a l o i d  closely reminiscent o f Stemmadenine (13) but having only a methylene bridge suggests but does not prove that the extrusion of the carbon from the  tryptophan  bridge and the necessary modification to the e x o c y c l i c methylene at C-3 stemmadenine may  occur as a concerted  of  process. 106-8,110  Incorporation of various forms of secodine coupled with degradation  (76) i n /u_ pyricollum  data places the rearrangement processes which lead to  the abnormal skeleton of apparicine (19) at even a l a t e r stage i n the biosynthetic scheme.  I t i s not known at the present time which i f e i t h e r o f  these two precursors secodine  (76) or stemmadenine (13) i s the immediate  precursor of the apparicine system for i t i s known that an equilibrium e x i s t s between stemmadenine (13), dehydrosecodine (72) and the aspidosperma a l k a l o i d s . A more enlightened rationale for the biosynthesis of these non-tryptamine 117 bridged a l k a l o i d s was fragmentation  deduced from a study by a French group  of the i n v i t r o  o f the tryptamine bridge under modified Polonovski reaction con-  ditions ( i . e . reaction of the N-oxide with t r i f l u o r o a c e t i c a c i d ) . suggested that a s i m i l a r fragmentation extrusion o f the one (figure 17).  I t was  mechanism could be involved i n the  carbon unit during the biosynthesis o f apparicine  (19)  - 35 -  This proposal  encompases a large portion of the known incorporation  data, i n c l u d i n g the involvement o f stemmadenine (13) as a c r u c i a l and the extrusion of the C-2  and not C-3  precursor  of tryptophan i n the formation of 108  apparicine  (19).  A minor discrepancy exists however, for i t has been shown  that the hydroxymethylene and not the carbomethoxy f u n c t i o n a l i t y at C-16  is  lost during the rearrangement process. 118 Subsequently this mechanistic proposal has been extended for biosynthesis  to account  of uleine (18) and the oxidized and reduced members of the  o l i v a c i n e (16) and e l l i p t i c i n e  (17) series (figure 18).  As such, i t represents  the abnormal alkaloids to be derived from the convergent indole biosynthetic 28 113 pathway and i s thus a very viable a l t e r n a t i v e to the Wenkert  and  Djerassi  postulates. Also, the generation  of uleine (18), apparicine  alkaloids 16, 17 25 and 26 from common intermediates  (19) and the  pyridocarbazole  suggesting the involvement  of s i m i l a r enzymic processes i n the biosynthetic pathway i s i n accord with the observed frequent  co-occurrence of these compounds i n Aspidosperma and Ochrosia  plants. As mentioned however, no biosynthetic data exists at the moment to prove 118 that the u n i f i e d hypothesis as proposed by the French group for olivacine (16), e l l i p t i c i n e (17), uleine (18), guatambuine (25), or N-methyltetrahydro28 ellipticine  (26) i s correct (figure 18), or whether the Wenkert postulate  can be substantiated  (figure 14).  18.  The Potier-Janot Postulate for the Biosynthesis of the Tryptamine Bridged A l k a l o i d s .  Non-  - 37 -  DISCUSSION - PROLOGUE  The work presented i n t h i s thesis represents  part o f a long-range  program i n our laboratory for the i n v e s t i g a t i o n of the biosynthesis o f representative carbazole  alkaloids o f the uleine (18), apparicine  systems i n a v a r i e t y of Aspidosperma plants.  (19), and pyridoPrevious work on  t h i s project has concerned i t s e l f p r i m a r i l y with the biosynthesis o f 106, 108, 110, 119 apparicine (19) and uleine (18) i n A. pyricollum. Some preliminary  feeding experiments have also been conducted on the biosynthesis 119, 120  of uleine (18), o l i v a c i n e (16) and guatambuine (25) i n A. a u s t r a l e . The emphasis i n the present study however has been on developing the background work necessary f o r a detailed study o f the biosynthesis o f the pyridocarbazole systems represented by o l i v a c i n e (16), guatambuine (25), e l l i p t i c i n e (17) and N-methyltetrahydroellipticine  (26).  Four b a s i c requirements were considered  i n i t i a l l y to be e s s e n t i a l to this  d e t a i l e d biosynthetic study: 1.  a suitable plant source that contains  the compounds o f i n t e r e s t and  i s a c t i v e l y biosynthesizing them. 2.  a synthetic scheme for the s p e c i f i c l a b e l l i n g of proposed precursors, .  3.  an e f f i c i e n t degradation scheme f o r the i s o l a t i o n o f s p e c i f i c radiol a b e l l e d atoms.  4.  an adequate source of unlabelled compounds f o r purposes o f comparison, for d i l u t i o n studies, and f o r chemical  transformation.  - 38 -  Different aspects o f these requirements were developed f o r biosynthetic work i n two plant systems A. australe, and A. v a r g a s i i as described i n the three parts o f t h i s t h e s i s .  Part 1 i s concerned with plant extractions so as  to obtain the required alkaloids from a crude A. australe extract, and to determine the f e a s i b i l i t y o f plant feeding experiments i n A. v a r g a s i i .  Part 11  describes the development o f a degradation scheme f o r both the o l i v a c i n e (16) and e l l i p t i c i n e  (17) s e r i e s , and Part 111 describes the synthesis o f o l i v a c i n e  (16) and guatambuine (25) f o r the continuation o f future biosynthetic work.  - 39 -  DISCUSSION - PART 1  Section A, Plant Extractions:  Aspidosperma Australe  (Mull. Argov.) 121-3  A. australe has been reported by Ondetti  and Deulofeu  to contain  as t h e i r p r i n c i p l e a l k a l o i d a l components o l i v a c i n e (16) and both enantiomers of guatambuine (25).  The a v a i l a b i l i t y of approximately one hundred grams of 124  a crude methanolic extract from a large scale extraction of A.  australe  therefore prompted the development of an i s o l a t i o n procedure whereby convenient but  l i m i t e d supplies of these two alkaloids could be obtained f o r  degradation work. A general a l k a l o i d extraction procedure described by G i l b e r t  1 0  was  used.  This method involved treatment of the methanolic extract with 15% a c e t i c a c i d and extraction with petroleum ether to remove neutral and a c i d i c plant m a t e r i a l . By subsequent b a s i f i c a t i o n and extraction with chloroform the basic a l k a l o i d a l f r a c t i o n was  isolated.  Thin layer chromatography (TLC) on this extract showed the presence o f two major components, a v i s i b l e bright yellow spot which fluoresced under UV and sprayed brown with e e r i e sulphate which corresponded to o l i v a c i n e (16)  and  a dark blue spot under UV which sprayed turquoise with eerie sulphate corresponding to guatambuine  (25).  For t h e i r i s o l a t i o n , advantage was  taken of the observation  on TLC  that  both compounds possessed a s i m i l a r retention time on alumina whereas they exhibited very d i f f e r e n t Rf  values on s i l i c a g e l .  On s i l i c a chromatoplates  - 40 -  guatambuine (25) occurred at very low Rf whereas o l i v a c i n e (16) moved with the solvent front, this behavior r e f l e c t i n g the considerable 125 t h e i r known b a s i c i t i e s .  differences i n  This c h a r a c t e r i s t i c difference made i t possible  to f i r s t separate both components from the small amounts of other present  materials  i n the extract by column chromatography on alumina and subsequently  to separate them from each other by chromatography on s i l i c a g e l . Considerable  d i f f i c u l t i e s were encountered, however, i n the a p p l i c a t i o n  of the a l k a l o i d a l extract onto the column.  Olivacine (16) and guatambuine  (25) are soluble only i n polarsolvents, f o r example methanol, a c e t o n i t r i l e and pyridine.  I t was  found, however, that a chloroform  solution containing  minimum amounts of methanol and pyridine would s o l u b i l i z e the extract without i n t e r f e r i n g with the  separations.  Upon r e c r y s t a l l i z a t i o n from methanol, i t was determined that o l i v a c i n e (16) and  ( - ) - guatambuine (25) were present i n the crude extract to the extent o f  1.8  and 1.6%  respectively.  Olivacine (16), obtained c r y s t a l l i n e as yellow needles was characterized by i t s N.M.R. and mass spectra, as well as the superimposability 126 IR. spectra with the reported spectra. s i n g l e t s at 62.80 and 63.15  of i t s UV. and  The N.M.R. spectrum exhibited two  f o r the C-5 and C-l methyl hydrogens r e s p e c t i v e l y .  The mass spectrum exhibited a parent peak,at m/e = 246 and very l i t t l e fragment a t i o n due to the aromatic nature o f the molecule. ( - ) - guatambuine (25), obtained as cream coloured cubes was also charac126 t e r i z e d by i t s s p e c t r a l data. The mass spectrum exhibited a parent peak at  - 41 -  m/e  = 264 and c h a r a c t e r i s t i c fragments a t m/e = 249, 233, 221-218, 204.  The N.M.R. spectrum i s shown i n f i g u r e 19 because i t p o s s e s s e s s e v e r a l important f e a t u r e s which w i l l discussions.  be a l l u d e d t o r e p e a t e d l y i n subsequent  Of n o t e i s the p r e s e n c e o f a d o u b l e t a t  1.52 ( J = 7 Hz) f o r  the  C - l methyl group and the p r e s e n c e o f a q u a r t e t a t  3.90 ( J = 7 Hz) f o r  the  methine hydrogen a t the same p o s i t i o n .  The c h e m i c a l s h i f t  o f the  methine hydrogen i s v e r y c h a r a c t e r i s t i c as i t i s i n f l u e n c e d by the f a c t it  i s b o t h b e n z y l i c and a d j a c e n t t o t h e b a s i c n i t r o g e n atom.  Also present  i n the N.M.R. spectrum i s a s i n g l e t a t 2.40 (N-CH^) and a s i n g l e t a t 2.54  (C-5,CH ). 3  that  0  '  1  •  1  i  1  1  •  ' i '  1  ' ' i i i i  M  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 r  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  j. (25)  H  .•If*"  1111  1 1 1 1 1 1 1 1 1  I  i  i  i  i -I  r i  i  i  I  i  i  i  i  1  i  i  i  i  I  i  i  i  i  I  i  i  i 'i  i  i  I  i  i  I  i  10 Figure 19.  The N.M.R. Spectrum of Guatambuine (25)  i  i  i  I  i  i  i  i  r  i  i  .  i  I  i  i  i  i  I I  i  i  j [  i  i  - 43 -  Aspidosperma v a r g a s i i (A.DC.) A. v a r g a s i i plants when mature are trees standing up to twenty meters 127 i n height with r e l a t i v e l y slender branches and a close t h i n bark.  They  are indigenous to rocky arid slopes and t r a n s i t i o n f o r e s t i n Venezuala, and adjacent  Columbia and Guiana.  The bark of A. v a r g a s i i has been studied 128  previously by Burnell and Delia Casa (1967)  and has been shown to contain  as i t s p r i n c i p l e constituents three pyridocarbazole (25), N-methyltetrahydroellipticine  a l k a l o i d s , guatambuine  (26) and 9-methoxyolivacine (82).  (82) A. v a r g a s i i represents, therefore, an i d e a l plant i n which to further extend our biosynthetic program f o r the two components 9-methoxyolivacine and N-methyltetrahydroellipticine  (26) are not present  (82)  i n the previously studied  plants A. pyricollum and A. australe. P r i o r to any radioactive feeding experiments the s u i t a b i l i t y o f A. v a r g a s i i as a plant source f o r biosynthetic study had to be determined. f i r s t l y the determination  This involved  of the presence of these constituents i n the  live  plants i n quantities that could be i s o l a t e d and secondly the development of a f e a s i b l e i s o l a t i o n scheme whereby the desired constituents could be obtained i n pure c r y s t a l l i n e form f o r radioactive  counting.  -  A t y p i c a l masceration-extraction  44 -  procedure was  a crude methanolic extract of the plant. 128 Burnell and Delia Casa  the alkaloids present  U t i l i z i n g the observation  that guatambuine (25) was  a c i d i c s o l u t i o n with chloroform  u t i l i z e d to obtain by  extractable from aqueous  an extraction procedure was  devised to p a r t i t i o n  i n the crude methanolic extract into acidic-chloroform  extractable and basic-chloroform  extractable f r a c t i o n s .  Suspending the crude  methanolic extract i n 10% hydrochloric a c i d and extracting f i r s t with petroleum ether to remove non-polar material and then with chloroform  (acidic-chloroform  extract) afforded a mixture of three major a l k a l o i d a l components.  One  of these  components provided an i d e n t i c a l retention time and colour development with e i t h e r guatambuine (25) or N-methyltetrahydroellipticine t i c s o f these two a l k a l o i d s being very s i m i l a r . aqueous phase and extraction with chloroform two  (26), TLC characteris-  Subsequent b a s i f i c a t i o n of the  (basic-chloroform extract) y i e l d e d  further major components and one minor component the least p o l a r of which  corresponded to an o l i v a c i n e - l i k e compound by comparison of i t s Rf value  and  fluorescent response to UV with authentic o l i v a c i n e (16). By means of thin layer chromatography i t appeared as though the p a r t i tioning was  highly e f f i c i e n t as very l i t t l e of e i t h e r o f the  extract components could be detected i n the acid-chloroform  basic-chloroform extract, and  s i m i l a r l y only small amounts o f the guatambuine-like (25) component could be detected i n the basic-chloroform  extract.  Of p a r t i c u l a r s i g n i f i c a n c e i n the above extraction was  the detection of  three previously unreported components i n r e l a t i v e l y large amounts.  - 45 -  The separation of the various components i n each extract was plished by column chromatography on alumina.  accom-  Table 1 provides a summary of  the r e s u l t s obtained during t h i s separation. In most instances the column chromatography had to be repeated on the various combined fractions i n order to obtain s u f f i c i e n t l y pure material f o r recrystallization.  This was p a r t i c u l a r l y true f o r the separation of Uleine  (18), guatambuine (25) and the minor "uleine l i k e " component. the chromatography fractions were monitored by TLC, UV.  In each case  and, where necessary,  Fourier transform N.M.R. spectroscopy. The UV.  spectrum was  a p a r t i c u l a r l y important  diagnostic tool i n  revealing the presence of the minor "uleine l i k e " component on chromatography of the combined guatambuine (25) f r a c t i o n s .  This component was present i n  very small q u a n t i t i e s and could be detected only on TLC as a l i g h t blue colouration i n the t a i l of the turquoise-green the basis of the UV.  spot f o r guatambuine (25) .  spectrum, t h i s component i n the extract mixture was  shown to possess the chromophoric system c h a r a c t e r i s t i c of Uleine (18) apparicine (19).  On  No subsequent data was  and  obtained to further determine i t s  structure. The Fourier transform N.M.R. spectrum o f the guatambuine (25) f r a c t i o n demonstrated i t to be homogeneous and not containing any N-methyltetrahydroe l l i p t i c i n e (26) which possesses almost i d e n t i c a l properties. l a t t e r component was of i t was  Although t h i s 128 anticipated to be present i n the plant extract, no trace  ever detected i n any of the several plant extraction experiments conducted  - 46 Table 1 - Column Chromatography Results on Extracts from A. v a r g a s i i  A)  Acid Chloroform Extract:  x 10 % of Total Plant z  Frac. No.  Solvent  Wt.(mg.)  1-8  CHC1,  105  Structure  3.88 apparicine  10 - 20  CHCl :EtOAc 3  33  (19)  1.22  20:60% uleine (18)  21 - 44  CHCl :EtOAc 3  47  1.74  Et0Ac:Me0H 8% guatambuine (25) From rechromatography o f f r a c s . 21 - 44  B)  "Uleine l i k e component"  Base Chloroform Extract:  1-15  CHC1 to 3  100  3.70  CHCl :EtOAc 20%  9-Methoxyolivacine (82)  3  17 - 20  CHCl :EtOAc 3  40% dihydroolivacine (141)  21 - 35  CHCl :EtOAc 3  40% to EtOAc: MeOH 5%  46  1.73 Desmethyluleine (84)  - 47 -  The absence of N-methyltetrahydroellipticine was disappointing i t would have been desirable to evaluate  the biosynthesis  of these alkaloids i n the same plant species.  since  o f the whole series  On the other hand, i t s presence  would have complicated the i s o l a t i o n procedure greatly, for even t r i a l separations  of a mixture of pure guatambuine (25) and N-methyltetrahydro-  e l l i p t i c i n e (26) proved to be very d i f f i c u l t on a small s c a l e . The  i d e n t i t i e s o f apparicine  (19), uleine (18) and desmethyluleine (84)  were determined by a comparison of mass s p e c t r a l , N.M.R. and UV. 129,113 that already reported.  data with  A c h a r a c t e r i s t i c feature i n the N.M.R. spectra  of a l l three compounds i s a p a i r of s i n g l e t s i n the o l e f i n i c region <54-5 f o r the two protons of the exocyclic methylene group. at 6 4.11  The presence o f a doublet  i n the spectrum of the uleine compounds and assigned  to the C-1H (see  table 1) which i s both benzylic and adjacent to the basic nitrogen, also provided a f a c i l e d i f f e r e n t i a t i o n from apparicine  (19) since the l a t t e r possesses  geminal hydrogens at the C - l p o s i t i o n and exhibits an AB quartet i n the same region.  Of p a r t i c u l a r note also was the absence of the N-methyl s i g n a l i n the  spectrum of desmethyluleine  (84).  The structure of guatambuine (25) was based upon the  superimposability  of both N.M.R. and IR spectra with those of an authentic sample. The structure of 9-methoxyolivacine (82) was determined from i t s N.M.R. spectrum which exhibited two s i n g l e t s at 6 2.80 methyl protons and a s i n g l e t at 6 4.00 that the p o s i t i o n o f these absorptions  and 3.16  f o r the aromatic  for the methoxy group.  I t i s noteworthy  i s close i n value to those obtained f o r  o l i v a c i n e (16), and markedly d i f f e r e n t to the p o s i t i o n of the s i n g l e t peaks i n  - 48 -  e l l i p t i c i n e (17).  The UV.  spectrum was  almost i d e n t i c a l to that  reported  f o r 9-methoxyolivacine (82) and d i s t i n c t l y d i f f e r e n t than for o l i v a c i n e (16).  The mass spectrum possessed a parent peak at m/e  with the required molecular formula and  consistent  l i k e o l i v a c i n e (16) i t possessed  an almost n e g l i g i b l e fragmentation pattern. basic chloroform  = 276  The minor component i n the  extract (fractions 17 - 20, Table 1) o f which only several  milligrams were i s o l a t e d was  t e n t a t i v e l y determined to be  (83) on the basis of the TLC and UV comparisons with (83) available by synthesis  (See sequence A, page  3,4-dihydroolivacine  3,4-dihydroolivacine  190).  It i s c l e a r l y evident from the i s o l a t i o n r e s u l t s that A. v a r g a s i i i s a very suitable plant source for biosynthetic i n v e s t i g a t i o n s . i t contain representatives o f uleine (18), apparicine  Not only does  (19), and the o l i v a c i n e  series o f compounds, but each of the f i v e major components are present i n 30-100 mg.  quantities and can r e a d i l y be p u r i f i e d by e i t h e r c r y s t a l l i z a t i o n or  sublimation I t was  techniques. observed however that the quantities of uleine (18)  obtained  depended markedly upon the s i z e ( i . e . age) o f the plant used i n the experiment.  Only large and thick woody stemmed plants y i e l d e d r e l a t i v e l y large  quantities o f uleine (18)  (230 mg.).  of this valuable a l k a l o i d (5-10  Smaller plants y i e l d e d considerably less  mg.).  I t i s of i n t e r e s t to note that the A. v a r g a s i i r e s u l t s further demonstrate the frequent  co-occurrence of the d i f f e r e n t types of non-tryptamine alkaloids  with each other i n Aspidosperma plants.  - 49  S e c t i o n B,  Incorporation  -  Experiments i n A.  v a r g a s i i (A.DC.)  O n l y a p r e l i m i n a r y i n v e s t i g a t i o n o f the i n c o r p o r a t i o n o f i n t o the a l k a l o i d s o f A. v a r g a s i i has o b j e c t i v e b e h i n d the experiments was was  radioactivity  been conducted a t t h i s t i m e .  to determine whether a c t i v e b i o s y n t h e s i s  o c c u r r i n g i n the p l a n t and not n e c e s s a r i l y to determine any  regarding  The  the rearrangements t h a t take p l a c e on c o n v e r s i o n  information  of precursor  mole-  c u l e s i n t o the a l k a l o i d s . With r e g a r d s  t o the p r e s e n t  as o u t l i n e d i n the  knowledge o f i n d o l e a l k a l o i d  I n t r o d u c t i o n , the p r e c u r s o r s  f o r f e e d i n g experiments were a r o m a t i c t r i t i u m stemmadenine The  biosynthesis  t h a t were l o g i c a l l y  l a b e l l e d tryptophan  chosen  (27)  and  (13).  (Ar-3H)-tryptophan  (27)  experiment was  o f p r i m a r y importance f o r i t  would demonstrate t h a t o l i v a c i n e (16), guatambuine  (25), and  uleine  (18)  are d e r i v e d from the e l u c i d a t e d i n d o l e a l k a l o i d pathway, thus i n v a l i d a t i n g the Wenkert  28  and  Djerassi  Stemmadenine (13) was  113  proposals.  The  incorporation of  a l s o o f major importance because by  the  (Ar-3H)Potier-Janot  118 postulate  i t represents  the immediate p r e c u r s o r  o f the whole s e r i e s o f  non-tryptamine b r i d g e d a l k a l o i d s . Both p r e c u r s o r s  were l a b e l l e d w i t h t r i t i u m i n the a r o m a t i c r i n g f o r  reasons t h a t the t r i t i u m exchange s y n t h e s i s o f the p r e c u r s o r s simple  and  there  i s no  during biosynthesis. 119 i n our  laboratory,  significant Also u n t i l 120 130  '  '  l o s s o f l a b e l by  transformations  s y n t h e t i c stemmadenine  Stemmadenine (13)  is relatively  (13)  occurring  becomes a v a i l a b l e  l a b e l l e d i n the  r i n g i s the o n l y r a d i o a c t i v e form o f t h i s p r e c u r s o r a v a i l a b l e .  aromatic  - 50  The  aromatically labelled precursors  reaction in t r i t i a t e d a c t i v i t y and  technique  i n c o r p o r a t e d i n 1-3  and  were p r e p a r e d  by an  trifluoroacetic acid, crystallized  o l d A. v a r g a s i i p l a n t s .  The  -  The  mg.  were a d m i n i s t e r e d  allowed  p l a n t s were then mascerated and  constant  q u a n t i t i e s to approximately  precursors  the b i o s y n t h e s i s was  to  exchange  five  by the c o t t o n wick  t o p r o c e e d f o r a one-week p e r i o d .  the a l k a l o i d a l  components i s o l a t e d i n  an i d e n t i c a l manner t o t h a t d e s c r i b e d i n s e c t i o n A f o r A. v a r g a s i i . compounds s t u d i e d were r e c r y s t a l l i z e d t o c o n s t a n t d i f f e r e n c e between s u c c e s s i v e c o u n t i n g s  was  year  The  a c t i v i t y meaning t h a t  l e s s than 5%.  The  effects  the of  134 c o l o u r quenching  on the e f f i c i e n c y o f s c i n t i l l a t i o n  methoxyolivacine  were s t a n d a r d i z e d by p r e p a r i n g  counting  i d e n t i c a l c o n c e n t r a t i o n a f t e r each r e c r y s t a l l i z a t i o n . incorporation r e s u l t s i s presented The  r e s u l t s show t h a t t h e r e has  tryptophan  (27)  tryptophan  has  and  stemmadenine  v a r g a s i i was  two  (18).  The  (82)  (25).  and  Both  that  precursors  radioactive  initial  experiments.  i n c o r p o r a t i o n i n t o u l e i n e (18) was  frustrating  f o r t h i s molecule  i n c o r p o r a t i o n experiments i n o t h e r p l a n t systems. the n a t u r e  the  compounds i n t o t h e o t h e r c o n s t i t u e n t s o f  because s i m i l a r r e s u l t s have been o b t a i n e d  known c o n c e r n i n g  of  II.  i n t o methoxyolivacine  not determined d u r i n g t h e s e  absence o f any  samples  been a d e f i n i t e i n c o r p o r a t i o n o f b o t h  incorporated into uleine  i n c o r p o r a t i o n o f these  The  (13)  of  A summary o f  a l s o been i n c o r p o r a t e d i n t o guatambuine  however, were not  A*  i n Table  counting  during  Nothing i s p r e s e n t l y  o f the enzymic p r o c e s s e s  t h a t produce  compounds w i t h i n the p l a n t or what c o n d i t i o n s a r e n e c e s s a r y  to  these  stimulate  Table I I . Results o f Incorporation of Tryptophan (27) and Stemmadenine  , Compound  Activity ^ ^ '  Isolated  (DPM)  n  Experiment I  9-Methoxyolivacine (82)  Guatambuine (25)  U 1 r  ^"  c 5  (Ar-3H)-Tryptophan (27).  0  0  ,  _ 5  8  Q  ,  8  1 A 2 X  }?j?  e  0  1  0  O  i r  2 x  .ll  5.82 x 1 0  1 1  % incorporation  DPM/mmole)  3.17 x 10  5  91  0.0049  1.71 x 10  5  40  0.0018  , '  -, 10  o r i  6  0.00  1 1  6'  (Ar-3H)-Stemmadenine (13). (3.21 x 1 0  Experiment II  U  11  1  (2.50 x 1 0  Weight isolated (mg.)  i  e  9-Methoxyolivacine (82)  Specific Activity isolated (DPM/mmole)  (13) into A. v a r g a s i i .  2 9X  5.74 x 10  rt  1  0  6.29 x 1 0  1 0  0.00  4  1 0  —  DPM/mmole)  85  10  0.0077  —  - 52 -  these p r o c e s s e s .  I t i s only p o s s i b l e therefore  as t o t h e n a t u r e o f the d i f f i c u l t i e s biosynthesis.  t o put forward  a s s o c i a t e d w i t h u l e i n e (18)  I t c o u l d be t h a t a t t h e time o f y e a r a t which t h e f e e d i n g  experiments were conducted t h a t u l e i n e biosynthesized,  (18) was n o t b e i n g  o r t h a t some e n v i r o n m e n t a l o r e x t e r n a l  b e i n g met which would s t i m u l a t e  i t s production.  actively  c o n d i t i o n was n o t  I t c o u l d a l s o be t h a t t h e  time s c a l e o f t h e experiments was t o o l o n g o r o f i n s u f f i c i e n t i t s biosynthesis The i n part  t o be  can a l s o be s u g g e s t e d t o a c c o u n t  f o r t h e low i n c o r p o r a t i o n o f t r y p t o p h a n  i n t o t h e a l k a l o i d s which d i d p o s s e s s a c t i v i t y . t h a t the p r e c u r s o r s  the  e s p e c i a l l y tryptophan  than a l k a l o i d p r o d u c t i o n  low a c t i v i t i e s o b t a i n e d .  (27) and stemmadenine (13) I t i s undoubtedly probable  (27) were a l s o u t i l i z e d f o r  which would a l s o c o n t r i b u t e t o  Due t o t h e n a t u r e o f t h e f e e d i n g  however, i t was h i g h l y p r o b a b l e t h a t t h e p r o p o s e d p r e c u r s o r s absorbed i n t o t h e p l a n t b e f o r e place  (absorption The  length f o r  detected.  problem o f membrane t r a n s p o r t  purposes o t h e r  speculations  technique  were  s i g n i f i c a n t external decomposition could  time 2-5 h r . ) .  o n l y s i g n i f i c a n c e t h a t can be a t t r i b u t e d t o t h e s e  i n c o r p o r a t i o n r e s u l t s i s t h a t both stemmadenine  preliminary  (13) and t r y p t o p h a n (27)  have been u t i l i z e d by t h e p l a n t system t o produce m e t h o x y o l i v a c i n e (82) and  guatambuine (25).  T h i s does n o t i m p l y i n t h e absence o f enzymic  s t u d i e s t h a t t h e s e two compounds a r e n e c e s s a r i l y t h e p r e c u r s o r s naturally within  the p l a n t .  existing  I t a l s o cannot be s a i d t h a t because  stemmadenine (13) was i n c o r p o r a t e d  into methoxyolivacine  (82) t h a t t h e  take  - 53 -  transformations  that occurred  proceeded a c c o r d i n g  t o the  Potier-Janot  postulate. The b i o s y n t h e t i c r e s u l t s p r e s e n t e d here a r e however t h e f i r s t obtained  f o r the p y r i d o c a r b a z o l e  as t o i n t e r m e d i a c y biosynthesis.  o f tryptophan  Further  t o be  a l k a l o i d system and do g i v e some i n d i c a t i o n (27) and stemmadenine  (13) i n t h e i r  b i o s y n t h e t i c i n v e s t i g a t i o n s are c u r r e n t l y  being  conducted i n our l a b o r a t o r y t o e l u c i d a t e t h e pathway l e a d i n g t o t h i s c l a s s of indole a l k a l o i d s .  - 54 -  EXPERIMENTAL - PART I  Melting points were determined on a Kofler block and are uncorrected. A l l u l t r a v i o l e t (UV.) spectra were recorded i n methanol using a Cary 15 recording spectrophotometer.  The i n f r a r e d (IR.) spectra were recorded with  a Perkin-Elmer Model 457 spectrophotometer either i n chloroform solution (cavity c e l l s 0.5 mm.) as indicated.  or as a nujol mull between sodium chloride plates  A l l measurements were made i n cm l and c a l i b r a t i o n o f the _  spectra was achieved using the 1604 a i r * absorption band of polystyrene. Nuclear magnetic resonance spectra (N.M.R.) were obtained i n deuterochloroform solutions (unless otherwise indicated) at 100 MHz on a Varian HA-100 or a Varian XL-100 nuclear magnetic resonance spectrometer. obtained v i a the Fourier  A l l N.M.R. spectra  Transform technique (F.T.) w i l l be so noted and were  obtained with the Varian XL-100 instrument.  Chemical s h i f t s were given i n  (ppm) with reference to tetramethylsilane as the i n t e r n a l standard. m u l t i p l i c i t y , integrated areas, and proton assignments theses.  The  are given i n paren-  Mass spectra were determined on an AEI-MS-902 or an Atlas CH-4B mass  spectrometer, with high resolution mass spectra determined with the former. Woelm neutral alumina and EM Reagents GF254 s i l i c a gel were used f o r thin and preparative layer chromatography.  In part I, unless otherwise s p e c i f i e d ,  ethyl acetate was used as the solvent f o r development o f the chromatoplates. Woelm neutral alumina (Act. I l l ) and Merck s i l i c a g e l (Act. 2-3) were used f o r column chromatography. Radioactivity was measured with a Nuclear-Chicago Mark II l i q u i d s c i n tillation  counter i n counts per minute (cpm).  The r a d i o a c t i v i t y of the sample  - 55 -  in disintegrations per minute (dpm) was subsequently determined by the 133a,b external standard technique gamma r a d i a t i o n .  using a b u i l t - i n Barium-133 source o f  S c i n t i l l a t i o n cocktails used were either a prepared s o l u t i o n  of toluene and a Nuclear-Chicago PPO-POPOP concentrate or a premixed of Nuclear-Chicago PCS c o c k t a i l .  solution  A s c i n t i l l a t i o n counting sample consisted  of a solution o f the sample dissolved i n 1 ml. of methanol and 14 ml. o f the appropriate c o c k t a i l ( t o t a l volume 15 ml.).  For each sample the background  of the v i a l was predetermined and subsequently subtracted from the measured dpm's f o r the sample i n subsequent c a l c u l a t i o n s .  Each sample was counted f o r  a time period long enough for the t o t a l counts f o r the sample, less the t o t a l counts f o r the background  to exceed ten thousand counts.  The A. v a r g a s i i plants used i n this study were grown i n the H o r t i c u l t u r e Department greenhouse,  the University o f B r i t i s h  Columbia.  Extraction of A. australe (Mull. Argov.) 124 The Crude methanolic extract  (20 gm.) was suspended i n 15% a c e t i c  acid (350 ml.) and s t i r r e d f o r 1.5 hr.  The aqueous suspension was then extracted  with petroleum ether (3 x 200 ml.) and insoluble p a r t i c l e s that remained i n the aqueous phase were removed by suction f i l t r a t i o n .  The aqueous phase was b a s i -  f i e d with 10% sodium hydroxide solution to pH 10 and extracted with chloroform (3 x 200 ml.).  The chloroform layer was dried and concentrated to give a s o l i d  a l k a l o i d extract (1.26 gm.).  The very polar components were removed by rapid  - 56 -  filtration  through  an alumina  column  (Act I I I ) .  The o l i v a c i n e - g u a t a m b u i n e  f r a c t i o n o b t a i n e d from t h e f i l t r a t i o n was d i s s o l v e d i n c h l o r o f o r m c o n t a i n i n g a s m a l l amount o f methanol and p y r i d i n e (1 ml.) and then a p p l i e d t o a s i l i c a gel  column  (100 gm., A c t 2-3).  I n i t i a l , e l u t i o n w i t h c h l o r o f o r m , f o l l o w e d by  e t h y l : a c e t a t e and e t h y l acetate-methanol 1.8%).  (10%) gave o l i v a c i n e  Subsequent e l u t i o n w i t h methanol y i e l d e d guatambuine  (16) (360 mg., (25) (320 mgs.,  1.6%). The  olivacine  (16) was r e c r y s t a l l i z e d from methanol t o g i v e y e l l o w  n e e d l e s , m.p. 318-325° ( l i t . ,  m.p. 3 1 8 - 3 2 4 ° ) .  1 2 6  UV.;  Amax ( l o g e ) :  374  (3.56), 325 (3.76), 291 (4.84), 284 (4.88), 275 (4.72), 265 (4.57), 235 (4.33), 222  (4.40).  N.M.R. ( F T . ) :  C-5  C H ) . Mass spectrum: 3  Guatambuine  3.15 ( s i n g l e t ,  3H, C - l C H ) , 2.80 ( s i n g l e t , 3H, 3  M , m/e = 246. +  (25) was o b t a i n e d as cream c o l o u r e d cubes by r e c r y s t a l l i z a t i o n  from methanol, m.p. 242-250° ( l i t . ,  248-250°).  1 2 6  UV.;  Amax ( l o g e ) :  (3.48), 327 (3.63), 297 (4.26), 288(sh) (4.08), 262 (4.36), (4.64).  N.M.R. (FT.) ( F i g u r e 19):  (singlet, C-l  341  250 (4.50), 240  3.90 ( q u a r t e t , IH, J = .7 Hz, C - l H ) , 2.54  3H, C-5CH ), 2.40 ( s i n g l e t , 3H, N-CHj), 1.52 ( d o u b l e t , 3H, J = 7 Hz, 3  C H ) . Mass spectrum: 3  M , m/e = 264; +  main peaks:  249, 233, 221-218,  204.  E x t r a c t i o n o f A. V a r g a s i i  (A.DC.)  Two woody A. v a r g a s i i p l a n t s (270 gm.) ( a p p r o x i m a t e l y 5 y e a r s o l d , and c o n t a i n i n g an e x t e n s i v e r o o t system) were ground up i n a W i l e y m i l l (not predried).  The c o l l e c t e d p u l p was suspended i n methanol  (3 x 300 ml.) and  - 57 -  f u r t h e r mascerated i n a Waring b l e n d o r  (x3).  s o l i d p l a n t r e s i d u e s were s e p a r a t e d by  suction f i l t r a t i o n  with methanol.  The  A f t e r each m a s c e r a t i o n  the  and washed  combined methanol f i l t r a t e s were c o n c e n t r a t e d  liberal  to  dryness. The  crude methanol e x t r a c t was  (500 ml.)  and  suspended i n 10% h y d r o c h l o r i c a c i d  s t i r r e d a t room temperature f o r 0.5  s u c c e s s i v e l y with petroleum (3 x 250 m l . ) .  The p e t r o l e u m  e t h e r e x t r a c t was  concentrated to give a s o l i d mixture The  aqueous phase was  ammonium h y d r o x i d e chloroform s o l u t i o n  and  (500  then b a s i f i e d t o pH  (30 gm.).  The  immediately  11 w i t h  (3 x 250 m l . ) .  The  (10% increment  F r a c t i o n s 1-8  (9:1)  (300  x 10"  (C. 1.0  2  % ) , m.p.  i n CHC1 ), 3  column chromatographed on  column was  e l u t e d (5 ml.  alumina  fractions)  (See T a b l e  192-194° ( l i t . ,  finally  1).  [a]  2 7  y i e l d e d c o l o u r l e s s needles m.p.  by  chloroform:ethyl  i n c r e a s e s o f e t h y l a c e t a t e ) and  from acetone  (lit.,  sulphate  mg.).  e l u t e d w i t h c h l o r o f o r m c o n t a i n e d a p p a r i c i n e (19)  after recrystallization 3.88  The  d r i e d o v e r sodium  g r a d i e n t e l u t i o n s t a r t i n g w i t h c h l o r o f o r m f o l l o w e d by  e t h y l acetate-methanol  concentrated  d i s s o l v e d i n hot c h l o r o f o r m and a p p l i e d  t o t h e column.  acetate mixtures  the  mg.)  (Base c h l o r o f o r m e x t r a c t ) was  e x t r a c t was  chloroform  d r i e d o v e r sodium s u l p h a t e ,  extracted with chloroform  a c i d c h l o r o f o r m e x t r a c t was  and  then e x t r a c t e d  discarded while  and c o n c e n t r a t e d t o g i v e a y e l l o w s o l i d m i x t u r e The  I t was  e t h e r 65-110° (3 x 250 ml.)  c h l o r o f o r m e x t r a c t ( A c i d c h l o r o f o r m e x t r a c t ) was and  hr.  192-194°),  = ± 177° i(C. 2.16  [a]  2 6  (105  mg.,  = -165.2°  in CHC1 ). 3  which  1 1 3  UV.;  Xmax  - 58 -  (log e): singlet, 1H,  303 (4.50);  Xmin ( l o g e ) :  265 (3.70).  1H, N-H), 5.42, 5.28 (two s i n g l e t s ,  1H each,  =CH ), 5.30 ( m u l t i p l e t , 2  J = 1.5 Hz,  =CH-CH,j), mass spectrum:  222, 208.  C, 81.78;  Found:  H, 7.63;  C, 81.51;  d o u b l e t , 3H, J = 8 Hz,  M , m/e = 264;  main peaks: 249,  +  H, 7.63;  N, 10.45%.  Calc. f o r C  uleine  H_ N„: n  (20-60%) c o n t a i n e d  (18) which a f t e r r e c r y s t a l l i z a t i o n from methanol y i e l d e d  cubes (33 mg., 1.22 x 10 (broad s i n g l e t ,  (two s i n g l e t s ,  1 0  N, 10.60%.  F r a c t i o n s 10-20 e l u t e d w i t h c h l o r o f o r m : e t h y l a c e t a t e  % ) . UV.; Amax ( l o g e ) :  colourless  301 (4.50).  N.M.R.  :  1H, N-H), 7.6-7.1 ( m u l t i p l e t , 4H, a r o m a t i c ) , 5.28, 5.00  1H each,  ( s i n g l e t , 3H, N-CH^). 223,  4.28, 4.58  3  2  8.22  7.90 (broad  =CH-CH , p a r t l y o b s c u r r e d by e x o c y c l i c methylene s i n g l e t ) ,  (AB q u a r t e t , 2H, J = 18 Hz, C - l C H ) , 1.47 ( s p l i t  235,  N.M.R.:  =CH ), 4.11 ( d o u b l e t , 1H, J = 2 Hz, C - l H ) , 2  Mass spectrum:  222, 209, 208, 207, 194,  M , m/e = 266; +  main peaks:  2.30  251,  237,  180.  F r a c t i o n s 21-44 e l u t e d w i t h c h l o r o f o r m : e t h y l a c e t a t e 1:1 up t o e t h y l acetate:methanol  8% c o n t a i n e d guatambuine  (25) which a f t e r  recrystallization -2  from methanol y i e l d e d cream c o l o u r e d cubes 250-252°, 297 3.86  (lit.,  248-250°).  1 2 8  .  UV.;  (47 mg., 1.74 x 10  Xmax ( l o g E ) :  % ) , m.p.  340 (3.50), 325 (3.63),  (4.25), 287(sh) (4.08), 260 (4.36), 248 (4.50), 239 (4.64).  N.M.R. ( F T . ) :  ( q u a r t e t , 1H, J = 6 Hz, C - l H ) , 2.46 ( s i n g l e t , 3H, C-5 C H ) , 2.34 3  (singlet,  3H, N-CH ), 1.45 ( d o u b l e t , 3H, 3=1 3  M , m/e = 264; +  H, 5.62;  main peaks:  N, 6.73%.  Hz, C - l C H ) . Mass spectrum: 3  249, 233, 221-218, 204.  Calc. for C  i g  H  2 3  N I: 2  By p r e p a r a t i v e l a y e r chromatography  C, 56.17; (alumina,  Found:  C, 55.85;  H, 5.71;  N, 6.89%.  1.0 mm., EtOAc) o f a  - 59 -  s m a l l p o r t i o n o f the F r a c t i o n 21-44 m a t e r i a l t h e b l u e c o l o u r a t i o n a t t h e t a i l o f t h e guatambuine UV.;  Amax:  (25) s p o t  ( t u r q u o i s e , e e r i e s u l p h a t e ) was i s o l a t e d .  303; Amin: 265.  The base c h l o r o f o r m e x t r a c t was chromatographed on alumina i n an i d e n t i c a l  fashion.  9-methoxyolivacine yellow needles UV.;  (singlet,  261,  245.  C,  (100 mg., 3.70 x 1 0 % ) , _ 2  74.02;  m.p. 290-292° ( l i t . ,  291-293°).  3  3  C, 74.50;  H, 6.49;  H, 6.35;  3  M , m/e = 276; +  N, 9.02.  main peaks:  Calc. f o r C H N 0(CH 0H): l g  1 6  2  3  N, 9.09.  F r a c t i o n s 17-20 e l u t e d w i t h c h l o r o f o r m : e t h y l a c e t a t e  (20-40%) c o n t a i n e d  a y e l l o w i s h s o l i d which was f u r t h e r p u r i f i e d by p r e p a r a t i v e l a y e r chromatography on alumina  (1.0 mm., EtOAc:MeOH 20%).  A s m a l l undetermined  amount o f a y e l l o w powder c o r r e s p o n d i n g t o 3 , 4 - d i h y d r o o l i v a c i n e (141) by TLC  (alumina, EtOAc) was o b t a i n e d .  UV.;  Amax:  367, 310, 294, 280, 275,  235. F r a c t i o n s 21-35 e l u t e d w i t h c h l o r o f o r m : e t h y l a c e t a t e acetate:methanol  (5%) c o n t a i n e d d e s m e t h y l u l e i n e  (46 mg., 1.73 x 1 0 " % ) . 2  5.00  (two s i n g l e t s ,  spectrum:  UV.;  IH each,  M , m/e = 252; +  1 2  4.00 ( s i n g l e t , 3H, 0 C H ) , 3.16 ( s i n g l e t , 3H, C - l C H ) ,  3H, C-5 C H ) . Mass spectrum:  Found:  m.p.  yielded  330 (3.80), 295 (4.70), 269 (4.59), 239 (4.37), 222  N.M.R. ( F T . ) :  2.80  F r a c t i o n s 1-15 e l u t e d w i t h c h l o r o f o r m c o n t a i n e d  (82) which a f t e r r e c r y s t a l l i z a t i o n from methanol  Amax ( l o g e ) :  (4.32).  (20 gm.)  Amax: 303;  (84) as a y e l l o w  Amin: 265.  =CH ), 4.34 ( m u l t i p l e t , 2  (40%) t o e t h y l film  N.M.R. ( F T . ) : 5.30, IH, C - l H ) .  main peaks: 235, 233, 209-8, 194-5, 180.  Mass  - 60 -  (Ar-3H)-Stemmadenine (13) Tritiated  t r i f l u o r o a c e t i c a c i d was p r e p a r e d by d i s t i l l a t i o n o f  t r i f l u o r o a c e t i c a n h y d r i d e (0.80 ml., 5.55 x 10  mole) i n t o t r i t i a t e d  water  -3 (100 y l . , 5.55 x 10 tritiated  mole, 100 mCi) u s i n g a vacuum t r a n s f e r system.  t r i f l u o r o a c e t i c a c i d was t h e n combined  (25 mg., 7.0 x 10  The  w i t h stemmadenine (13)  mole) by means o f t h e vacuum t r a n s f e r system and t h e  r e s u l t i n g a c i d s o l u t i o n was m a i n t a i n e d under a d r y n i t r o g e n atmosphere a t room temperature distilled  f o r 48 h r .  The t r i t i a t e d  t r i f l u o r o a c e t i c a c i d was then  o f f and t h e r e s i d u e was taken up i n d i l u t e ammonium h y d r o x i d e  (75 ml.) and e x t r a c t e d w i t h methylene  chloride  (3 x 30 m l . ) .  c h l o r i d e f r a c t i o n s were combined, d r i e d o v e r sodium t r a t e d t o a c o l o u r l e s s foam (17 mg., 68%).  from methanol 3.21  x 10  1 0  The methylene  s u l p h a t e , and concen-  After six recrystallizations 7  t h e measured r a d i o a c t i v i t y was c o n s t a n t (8.98 x 10  DPM/mg.,  DPM/mmole.) (PP0-P0P0P c o c k t a i l ) .  Plant Incorporation  Experiment:  The A d m i n i s t r a t i o n o f (Ar-3H)-Tryptophan (Ar-3H)-Tryptophan  (27) t o A. v a r g a s i i  (27) (2.33 mg., 2.50 x 1 0  i n a s o l u t i o n o f methanol:water:acetic a c i d a d m i n i s t e r e d t o an A. v a r g a s i i p l a n t  1 1  DPM/mmole) was d i s s o l v e d  (1 ml.:4 ml.:  1 drop) and  (^130 gm.) by t h e c o t t o n wick t e c h n i q u e .  T h i s method r e q u i r e d t h e t h r e a d i n g o f a c o t t o n s t r i n g through t h e stem o f the  growing p l a n t a t a p o i n t above t h e ground, b u t below t h e b r a n c h i n g  point.  The i n t e r t w i n e d ends o f t h e wick were p l a c e d i n a 18 x 75 mm.  test  - 61 -  tube c o n t a i n i n g  the precursor  s o l u t i o n l o c a t e d a t t h e base o f t h e p l a n t .  For t h e r e m a i n i n g p e r i o d o f t h e i n c u b a t i o n absorbed  a f t e r the precursor  had been  (2-5 h r . ) t h e t e s t tube was kept f u l l by r e p e a t e d a d d i t i o n s o f  d i s t i l l e d water.  A f t e r 7 days under i n t e r m i t t e n t f l u o r e s c e n t  i l l u m i n a t i o n t h e p l a n t s were e x t r a c t e d procedure p r e v i o u s l y described. and u l e i n e  f o r t h e i r a l k a l o i d c o n t e n t by t h e  Methoxyolivacine  (82), guatambuine  (25)  (18) were i s o l a t e d by column chromatography on alumina and  r e c r y s t a l l i z e d to constant isolated,  lamp  activity  (PPO-POPOP c o c k t a i l ) .  s p e c i f i c a c t i v i t i e s and p e r c e n t a g e i n c o r p o r a t i o n s  The q u a n t i t i e s f o r these  compounds a r e p r e s e n t e d i n T a b l e I I , page 51.  The A d m i n i s t r a t i o n  o f (Ar-3H)-Stemmadenine  (Ar-3H)-Stemmadenine  (13) (1.96 mg.,  d i s s o l v e d i n methanol:water plant  (13) t o A. v a r g a s i i  3.21 x 1 0  fluorescent  previously described:  DPM/mmole) was  (1:4) (5 ml.) and a d m i n i s t e r e d  (^130 gm.) by t h e c o t t o n wick t e c h n i q u e .  intermittent  1 0  t o an A. v a r g a s i i  A f t e r 7 days under  lamp i l l u m i n a t i o n t h e p l a n t s were e x t r a c t e d as  Acid chloroform  extract  •7  (312 mg. 5.13 x 10 DPM);  7 Base c h l o r o f o r m and U l e i n e  extract  (247 mg. 1.07 x 10 DPM).  (18) were i s o l a t e d by column chromatography on alumina and  r e c r y s t a l l i z e d to constant isolated,  Methoxyolivacine (82),  activity  (PPO-POPOP c o c k t a i l ) .  s p e c i f i c a c t i v i t i e s and p e r c e n t a g e i n c o r p o r a t i o n s  compounds a r e p r e s e n t e d i n T a b l e I I , page 51.  The q u a n t i t i e s f o r these  - 62 INTRODUCTION - PART II  The elucidation of the transformations  that take place i n the biosynthetic  pathway leading to the non-tryptamine bridged indole a l k a l o i d s by the radioactive l a b e l l i n g technique requires both the synthesis of s p e c i f i c a l l y l a b e l l e d molecules that are postulated to be t h e i r precursors  and the development of a degra-  dation scheme f o r determining the p o s i t i o n of the incorporated radiolabels into the a l k a l o i d a l products. To p a r a l l e l , therefore, the biosynthetic feeding experiments that have 119 been i n i t i a t e d i n our laboratory on o l i v a c i n e (16) and guatambuine (25) i n A. australe, a degradation scheme has been developed f o r these molecules. Further, 131 as a consequence of the a v a i l a b i l i t y of large amounts of e l l i p t i c i n e  (17)  and  i n i t i a l l y of only l i m i t e d quantities of o l i v a c i n e (16) and guatambuine (17) from natural sources,  reactions studied i n the e l l i p t i c i n e (17) s e r i e s provided both  a model system and an extension  of the degradation scheme to include t h i s s e r i e s .  I n i t i a l experiments i n A. australe involved feeding l a b e l l e d forms of secodine (76), however, i t i s currently thought that secodine (76) occurs to far along the pathway to be a precursor of the non-tryptamine bridged a l k a l o i d s . Stemmadenine (13) i s considered and i t i s also considered  to be the d i r e c t progenetor of these compounds  that the equilibrium between dehydrosecodine (72)  and  stemmadenine (13) i s responsible f o r the observed low incorporations of secodine (76) into o l i v a c i n e (16) and guatambuine (25).  As a r e s u l t , stemmadenine 119,120,130 (13) i s currently i n the stages o f being synthesized i n our laboratory with the objective i n mind of being able to unambiguously introduce l a b e l l e d  - 63 -  atoms into the positions indicated i n figure 20. 118 In accord with the Potier-Janot postulate, the l a b e l l e d atoms i n stemmadenine (13) would end up i n the positions indicated i n o l i v a c i n e (16), guatambuine (25), e l l i p t i c i n e  (17) and N-methyltetrahydroellipticine  (26).  The arrow connecting tryptophan (27) and stemmadenine (13) indicates where a label i n the C-2  p o s i t i o n of tryptophan (27) would occur i n stemmadenine  and subsequently i n the a l k a l o i d s .  As can be seen, with the exception  l a b e l l e d carbomethoxy grouping which may 108 i n the "C" r i n g ,  be incorporated  into the C-5  the l a b e l l e d atoms a l l occur i n the "D"  The degradation scheme developed, therefore, was i s o l a t i o n of the C-l methyl, C-2  atom i n the e l l i p t i c i n e  of the position  ring.  devised to permit the  methyl (N-methyl), and the C-3  i n the o l i v a c i n e (16) series and s i m i l a r l y the C-2  (13)  carbon atom  methyl and the C-3  carbon  (17) s e r i e s .  This scheme necessarily had to take into account that generally only amounts of alkaloids (20-50 mg.)  with low s p e c i f i c a c t i v i t i e s were available  from plant incorporation experiments.  The demands of having to work on a small  scale without the option of d i l u t i n g with unlabelled materials placed upon the developed sequence.  small  The reactions involved had  constraints  to be simple i . e . not  i n v o l v i n g any complex work-ups, etc., very e f f i c i e n t and reproducible  i n terms o f  y i e l d s , and also, the o v e r a l l sequence should involve a minimum number of reactions. A further and important s t i p u l a t i o n was  that the end products had to be  e a s i l y p u r i f i e d and i n c r y s t a l l i n e form either i n t h e i r natural state, or as a suitable derivative f o r radioactive  counting.  F i g u r e 20.  The Labelling Pattern f o r the Biosynthesis of the Olivacine (16) E l l i p t i c i n e (17) Series According to the Potier-Janot Postulate.  and  - 65 -  (92)  Figure 21.  (91)  The Structure Elucidation of Olivacine (16) by Ondetti Deulofeu (1961).  and  - 66 -  Very l i t t l e chemistry of the o l i v a c i n e (16)-guatambuine (25) system has 121-3 been studied to date, however, the work by Ondetti and Deulofeu  (1961)  was p a r t i c u l a r l y applicable to the desired degradation o f the o l i v a c i n e (16) and e l l i p t i c i n e (17) "D" rings (Figure 21).  These workers elucidated the  structure of o l i v a c i n e (16) by i t s conversion v i a i t s methiodide guatambuine (25) and a subsequent  comparison  ducts obtained from guatambuine methiodide (18) v i a i t s methiodide  (87).  (85) to  of the Hofmann degradation pro-  (86) with those obtained from uleine  I t was found that guatambuine methiodide (86)  undergoes r i n g opening under Hofmann conditions (NaOH/EtOH) to give two major products.  The main component A (32%) was not characterized, but i t was  determined by lack o f hydrogen uptake to be a n o n - o l e f i n i c material, and the microanalysis o f i t s methiodide correlated with the composition C2QH25N2I. The second component (22%) a f t e r hydrogenation to 89 and conversion to i t s methiodide 90 was proposed to have the o l e f i n i c structure 88 by comparison o f IR., UV., melting point, and mixed melting point with methiodide 90 shown by 132 Schmutz et al_. to be obtained from uleine (18) . Support for t h e i r assignments were further strengthened by a comparison  o f the IR., UV., and melting points o f  the second Hofmann product 91. 135 Buchi et_ a l .  working concurrently i n t h i s area cleaved the s t y r y l side  chain o f 91 under osmium tetroxide-periodate conditions to give the carbazole aldehyde 92. The work o f these authors was however incomplete i n many respects. The correlation o f the degradation products o f the two alkaloids led to the correct assignment o f the structure o f o l i v a c i n e (16) and guatambuine (25), however i t i s  - 67 -  d i f f i c u l t to i n d i c a t e with certainty that the comparisons  arc v a l i d based  upon the IR., UV. and rudimentary p h y s i c a l data. No data were obtained f o r the alternate llofmann product 97 that could a r i s e from guatambuine (25), a compound whose s p e c t r a l data, and quite possibly whose melting point would bear a close resemblance  to compounds 88 and 89.  Only through the applica-  t i o n o f N.M.R. and mass spectroscopic techniques (which were not a v a i l a b l e to these authors) could the structure o f o l e f i n 88 be unambiguously  determined.  However, this sequence p o t e n t i a l l y permits the i s o l a t i o n o f a l l three desired centers i n o l i v a c i n e (16) and guatambuine (25) and consequently i t provided the b a s i s on which the degradation of as the e l l i p t i c i n e s  (17) and 26 was developed.  these two a l k a l o i d s as w e l l  - 68  -  DISCUSSION - PART II  1_.  Interconversions i n the Olivacine (16)-Guatambuine (25) and (17)-N-Methyltetrahydroellipticine (26) S e r i e s . The conversion  of o l i v a c i n e (16) and e l l i p t i c i n e  ing N-Methyltetrahydro counterparts ellipticine  (26) was  necessary  Ellipticine  (17) to t h e i r correspond-  guatambuine (25) and N-methyltetrahydro-  for the t o t a l l y aromatic structures were not  i n a suitable form to undergo reactions that would e f f e c t s c i s s i o n of the pyrido "D" r i n g . 121-3 Ohdetti and Deulofeu by formation  have converted o l i v a c i n e (16) to guatambuine  (25)  o f the methiodide of o l i v a c i n e 85 followed by c a t a l y t i c hydrogena-  tion under lew pressure (Figure 22). In a s i m i l a r fashion Goodwin, Smith, and 136 Horning converted e l l i p t i c i n e (17) to N-Methyltetrahydroellipticine (26) by formation o f i t s methiodide 93 and subsequent reduction o f the 137 s a l t with sodium borohydride.  Buchi et a l .  an alternate method o f transforming found that e l l i p t i c i n e pressure  the "D"  pyridinium  developed the f i r s t portion of r i n g of e l l i p t i c i n e (17).  They  (17) underwent f a c i l e hydrogenation at atmospheric  to give the tetrahydroderivative 94.  By subsequent simple  methylation  i t would be possible to obtain the methiodide of N-methyltetrahydroellipticine (95) . The transformation  of e l l i p t i c i n e  part presented no d i f f i c u l t i e s .  (17) to i t s N-methyltetrahydro counter-  Both routes were investigated but i t was  found  - 69 -  (94)  Figure 22.  (95)  The Conversion o f Olivacine (16) and E l l i p t i c i n e to t h e i r N-Methyltetrahydro Forms. 136  that the sequence of Goodwin et^ al_,  was  the simplest to follow.  The formation of e l l i p t i c i n e methiodide 93 never went t o t a l l y to despite the use of a great excess of methyl iodide.  completion  This necessitated the  removal of the small amount of r e s i d u a l s t a r t i n g material before reduction because i t s presence made subsequent p u r i f i c a t i o n by e i t h e r r e c r y s t a l l i z a t i o n or column chromatography d i f f i c u l t .  This was  rapid column chromatography on alumina.  most e a s i l y accomplished by a  The methiodide was  from the column with methanol:ammonium hydroxide crystals a f t e r concentration  (77%).  e f f i c i e n t l y eluted  (1%) to give bright orange  - 70 -  Reduction of the pyridinium system with sodium borohydride gave N-methylt e t r a h y d r o e l l i p t i c i n e (26) as cream coloured c r y s t a l s .  It was observed that  the crude reaction mixture began to yellow on standing, even i n a vacuum dessicator.  This decomposition was checked by r e c r y s t a l l i z a t i o n from methanol  to give cream coloured needles (91%). The s p e c t r a l data (UV., N.M.R.) f o r 26 agreed closely with that previously 126, 128 reported.  The N.M.R. spectrum possessed two singlets at 62.56 and 2.70  f o r the aromatically substituted methyl groups at C-5 and C - l l r e s p e c t i v e l y . Two other singlets were also observed, one f o r the N-methyl group (62.38), and the other f o r the i s o l a t e d methylene group at C - l (63.74).  A t r i p l e t occurred  at 62.98 (J = 5Hz) f o r the C-4 methylene protons while the corresponding t r i p l e t for the C-3 methylene protons (62.76) was p a r t l y obscured by the s i n g l e t (62.70) f o r the C - l l methyl  group.  The conversion o f 26 to i t s methiodide 95, i n preparation f o r Hofmann degradation, by reaction with methyl iodide i n methanol proceeded i n quantitative y i e l d to give colourless needles. The c a t a l y t i c hydrogenation o f e l l i p t i c i n e (17) with platinum oxide de137 veloped by Buchi et a l .  was s l i g h t l y modified by s u b s t i t u t i n g a c e t i c acid  for ethanol as the solvent. product was obtained.  I t was found that with a c e t i c acid a cleaner  The hydrogenation was complete  a f t e r several hours at  atmospheric pressure to give a highly fluorescent pale blue s o l u t i o n which after work-up yielded a colourless c r y s t a l l i n e product 94.  As reported, the  tetrahydro compound 94 was considerably s e n s i t i v e to a i r , turning yellow on standing.  For this reason a small portion o f i t was characterized as i t s  - 71 -  N-acetyl derivative 96 and the remainder was  converted d i r e c t l y to N-methyl-  t e t r a h y d r o e l l i p t i c i n e methiodide 95 by reaction with methyl iodide.  (96) The e f f i c i e n t conversion of o l i v a c i n e (16) to guatambuine (25) unlike the transformation of e l l i p t i c i n e (17) was unexpectedly difficult.  found to be very  The major hurdle i n the synthesis proved to be the methylation  of o l i v a c i n e (16) to i t s methiodide  85.  Reaction of o l i v a c i n e (16) with methyl iodide i n ethanol under a wide range of conditions from room temperature  to a sealed tube at 150° resulted  consistently i n only a 30% conversion to i t s methiodide. with the reaction also was  A p e r s i s t e n t problem  the presence of considerable quantities of uniden-  t i f i e d dark coloured material which hampered work-up. i n low y i e l d as a yellow m i c r o c r y s t a l l i n e s o l i d . the y i e l d of the methiodide was  The s a l t 85 was  obtained  For purposes of degradation  increased to 80% by repeated r e c y c l i n g of  reaction mother liquors which contained unreacted o l i v a c i n e (16). The problem o f formation of methiodide  85 was eventually overcome by 138 using methylfluorosulphonate as the a l k y l a t i n g agent and a c e t o n i t r i l e as  - 72 -  the solvent medium.  The reaction proceeded p r a c t i c a l l y instantaneously with  methofluorosulphate formation.  The s a l t was not i s o l a t e d (due to the presence  of flurosulphuric acid as a high b o i l i n g contaminant) but reduced d i r e c t l y to give guatambuine (25) as described below. In a s i m i l a r fashion to methiodide formation i t was found that c a t a l y t i c hydrogenation o f olivacine methiodide  (85) with platinum oxide at low pressure  (5 atm., 80°) proceeded i n poor or more correctly inconsistent y i e l d . On the other hand reduction o f the pyridinium r i n g o f 85 (methiodide or fluorosulphate) proceeded readily and i n excellent y i e l d by reaction with sodium borohydride.  In this manner guatambuine (25) was obtained as a pure  cream coloured c r y s t a l l i n e s o l i d (93%).  The s p e c t r a l data f o r this compound  was i d e n t i c a l to that previously obtained (Part I ) . The subsequent  conversion of guatambuine (25) t o i t s methiodide 86 pro-  ceeded i n quantitative y i e l d by reaction with methyl iodide. A preliminary attempt was made to by-pass o l i v a c i n e methiodide  (85) forma-  tion by d i r e c t hydrogenation o f o l i v a c i n e (16) i n an analogous fashion to that developed f o r e l l i p t i c i n e  (17).  Even a f t e r prolonged hydrogenation under  pressure (2.5 atm.) considerable s t a r t i n g material remained  and the product that  was formed, as previously observed, proved to be unstable i n a i r thereby adding complications to i t s i s o l a t i o n and p u r i f i c a t i o n . abandoned.  This approach was consequently  - 73 -  2_.  Hofmann Elimination: From a consideration of the mechanism of the Hofmann elimination reaction  two s t r u c t u r a l l y isomeric o l e f i n i c products 88 and 97 can be anticipated to be formed during the base induced (Hofmann conditions) r i n g opening of the methiodide of guatambuine (25).  (§6)  (88)  (97)  139 In accord with the Hofmann rule  i t could be anticipated that i n an  unsymmetrical c y c l i c amine l i k e guatambuine (25) there would be a p r e f e r e n t i a l formation of one o l e f i n i c product over the other.  In the vast majority  of  cases the; hydrogen removed during elimination derives from the least s u b s t i tuted carbon atom, this leading i n turn to the least substituted o l e f i n . the guatambuine system elimination i n either d i r e c t i o n leads to an  In  unsubstituted  o l e f i n i c product, however i t might be expected on the above consideration that elimination i n the d i r e c t i o n of the C-l methyl group leading to the C-3  olefin  88 would be the preferred course. However, an overriding influence to this general trend results from the  - 74 -  presence of a r e l a t i v e l y a c i d i c benzylic hydrogen on the C-4 carbon atom. In s i t u a t i o n s where a benzylic hydrogen can become involved i n the 6-elimination process, elimination i n i t s d i r e c t i o n tends to predominate.  The predominant  elimination product anticipated f o r guatambuine (25) would therefore be the C-2 o l e f i n 97. For purposes o f degrading guatambuine (25) the production  of both o l e f i n i c  products by Hofmann elimination would be i d e a l for then a simple oxidative cleavage o f both double bonds would complete the i s o l a t i o n o f the two desired carbon atoms.  In the s i t u a t i o n where only one of the two possible o l e f i n s i s  i s o l a t e d oxidative cleavage followed by a second Hofmann reaction again completes the sequence. 121-3 From the r e s u l t s of Ondetti and Deulofeu  i t appeared f e a s i b l e that  the C-3 o l e f i n 88 proposed by them to be the only unsaturated product formed by reaction with sodium hydroxide i n r e f l u x i n g ethanol quantities large enough to degrade. scale (50 mg.)  could be i s o l a t e d i n  On repeating t h e i r reaction on a small  two products were again observed, however i t was determined  from the measured integration o f the N.M.R. signals that the product r a t i o altered from that reported  (Figure 23), only 5-10%  had  o f the product mixture corres-  ponded to o l e f i n i c material. The major n o n - o l e f i n i c component i s o l a t e d i n 60%  y i e l d exhibited s p e c t r a l  data consistent with structure 98. The N.M.R. spectrum had a quartet at 64.85 (J = 6Hz) doublet at 61.53  (J = 6Hz)  are substituted at the C - l  and a corresponding  f o r the methine hydrogen and the methyl group which 1  carbon atom (see 97 f o r numbering system) and  are  Figure 23.  The N.M.R. Spectrum o f the Crude Reaction Mixture (NaOH/EtOH).  - 76 -  adjacent to the oxygen  atom.  The increased deshielding e f f e c t of the oxygen  compared to nitrogen resulted i n the substantial downfield s h i f t of the methine quartet with respect to the corresponding quartet i n the spectrum for guatambuine  (25) (Figure 19., Table I I I ) .  Two s i n g l e t s occurring at  62.49 and 2.40 were attributed to the C - l methyl group and the dimethylamino group respectively.  A multiplet centered at 63.04 was a t t r i b u t e d to the  C-3' methylene protons whereas the multiplet f o r the C-4' methylene protons was obscured by the singlets for the above mentioned methyl groups.  The  quartet (63.38, J = 7Hz) and t r i p l e t (61.18, J = 7Hz) absorptions were assigned to the methylene and methyl group respectively of the substituted ethoxy group.  (98)  The mass spectrum possessed a parent peak at m/e major fragmentations at m/e i n both possible d i r e c t i o n s . m/e  = 324 and only two  = 295, 278 f o r the a-cleavage of the ether linkage The high resolution mass spectrum parent peak at  = 324.2207 was within acceptable l i m i t s of the value 324.2200 calculated  for C H N 0 . 21  2g  2  For purposes o f comparison with the unassigned component A i s o l a t e d by 121-3 Ondetti and Deulofeu, compound 98 also possesses a normal carbazole UV.  - 77 -  spectrum.  I t i s highly probable that the same product has been i s o l a t e d i n  both instances.  This assumption i s strongly supported by the observation  that  t h e i r microanalytical data i s within experimental error for both the molecular composition  C20H25N2I  for the composition  proposed by them for component A and the calculated values  C22H31N2OI  f°  r  the methiodide derivative of compound 98.  I t i s apparent that 98 i s formed by s u b s t i t u t i o n of the quaternary n i t r o gen of guatambuine methiodide by ethoxide ion generated i n the reaction medium. Substitution reactions are always a competing process with elimination and w i l l become more evident i n l a t e r discussion, the benzylic center  as  (C-l) onto 140  which the nitrogen i s attached  in guatambuine (25) as well as i n uleine  (18)  113 and apparicine  (19)  i s considerably  activated towards s u b s t i t u t i o n processes.  This i s to a major extent a consequence o f s t a b i l i z a t i o n o f the b e n z y l i c carbonium ion that i s formed during reaction by the lone p a i r of electrons  on  the indole nitrogen oriented para to i t .  The intermediacy of such a s t a b i l i z e d benzylic carbonium ion implies the s u b s t i t u t i o n reaction occurs by a SNj^ type mechanism.  that  This further implies  that on formation of substitution products' the o p t i c a l a c t i v i t y of the asymmetric b e n z y l i c center i n guatambuine (25) would be l o s t .  Such has been found  78  t o be the  case when the  basic reaction  (-)-methiodide  86 was t r e a t e d under t h e  strongly  c o n d i t i o n s employed by O n d e t t i and Deulofeu-.  I t has been demonstrated a l s o i n c l o s e l y r e l a t e d systems e x e m p l i f i e d by the a r o m a t i c a l l y oxygenated t e t r a h y d r o i s o q u i n o l i n e a l k a l o i d s such as emetine  (99)  (99) t h a t t h e c y c l i c six-membered r i n g s t r u c t u r e  does n o t undergo s u b s t i t u t i o n  r e a c t i o n u n l e s s s t r o n g b a s i c c o n d i t i o n s n o r m a l l y a s s o c i a t e d wx-vh-  olv^-i'coti.iwo  139,141-3 are employed.  The p r o b a b l e r a t i o n a l e  f o r t h i s requirement i s  s u f f i c i e n t d r i v i n g f o r c e must be p r o v i d e d t o overcome t h e tendency o f nitrogenous  l e a v i n g group t o  r e v e r t back t o the  that the  quaternary amine by i n t e r n a l  return. C o n s i d e r a b l e e f f o r t was made t o i s o l a t e the crude m i x t u r e , however even by r e p e t i t i v e enriched fractions  the minor o l e f i n i c p r o d u c t from column chromatography o f  olefin  i t was not p o s s i b l e t o a d e q u a t e l y e l i m i n a t e the p r e s e n c e  of  the s u b s t i t u t i o n p r o d u c t 98 as c o n t a m i n a n t . • I t was e v e n t u a l l y p o s s i b l e though t o d e t e r m i n e j w i t h a good degree o f c o n f i d e n c e t h e i d e n t i t y o f t h e o l e f i n p r o duced d u r i n g t h e r e a c t i o n . trum ( F i g u r e 23)  T h i s assignment was i n f e r r e d from the N . M . R .  by a comparison w i t h the c h e m i c a l s h i f t s f o r  the  spec-  - 79 -  components of the a- and 3-dimethylaminoethyl, and v i n y l side chains found i n the o l e f i n i c and saturated forms of the ring-opened products of guatambuine (25) (Table I I I ) . As w i l l be alluded to again i n subsequent  discussion the two major d i s -  tinguishing features between the two possible elimination products are: r e l a t i v e positions o f the s i n g l e t f o r the protons of the dimethylamino  the group  as i t occurs i n the C-2 and C-3 side chains and i i ) the chemical shifts for the c i s and trans - coupled o l e f i n i c  hydrogens.  It can be seen from Table III that the s i n g l e t f o r the  dimethylamino  group i n the C-3 side chain of compounds 97, 103, and 98 occurs at higher f i e l d than does the s i n g l e t f o r the same group i n the C-2 pounds 88 and 89.  side chain o f com-  The absence therefore of a peak at <v 62.25, f o r the C-3  side chain, integrating for 10% o f the dimethylamino  group s i n g l e t at 62.40  would suggest that the elimination product possesses a 3-dimethylaminoethyl side chain at C-2 and that i t has structure 88.  This assignment was  confirmed  as a r e s u l t of the occurrence o f the trans coupled hydrogen of the double bond to lowerfield (65.68, J p ^ = 11Hz).  1  =  17Hz)  t  n  a  n  the c i s coupled hydrogen  (65.29,  Jii2»  This justaposition of the o l e f i n i c signals i s c h a r a c t e r i s t i c of the  C-3 v i n y l substituent. The N.M.R. data substantiated the production of the ring-opened o l e f i n 88 121-3 under Hofmann conditions as predicted by Ondetti and Deulofeu. I t was somewhat s u r p r i s i n g that i t was  the only unsaturated product produced by the  reaction since the majority of s t r u c t u r a l l y s i m i l a r molecules such as the t e t r a 144 145 hydroisoquinolines s a l s o l i d i n e (100) and lophocerine (101) r i n g open so  - 80 -  as to give the alternate Hofmann product which i s predicted by the Hofmann rule to be the predominant product.  (101)  I t would appear evident from the observed formation o f the s u b s t i t u t i o n product 98 with the o l e f i n 88 as the minor side product that the benzylic carbonium ion mechanism plays a central role i n the r e a c t i v i t y of guatambuine methiodide (86) under sodium hydroxide i n ethanol  conditions.  The  formation  of the small amount of the elimination product 88 during the reaction can be envisaged to r e s u l t from a competing n e u t r a l i z a t i o n of the carbonium ion by loss of a proton from the adjacent methyl center The  (Ej e l i m i n a t i o n ) .  lack of substantial quantities of the elimination product 88 in the  reaction mixture necessitated the study of other reaction conditions to  increase  - 81 -  i t s o v e r a l l y i e l d or that of the alternate elimination product 97.  The  s u b s t i t u t i o n product formed i n 60% y i e l d wasn't, but could have been used f o r subsequent  i s o l a t i o n s of the N-methyl group (see Section 3.)  because the ether  f u n c t i o n a l i t y would be e s s e n t i a l l y i n e r t to the basic reaction conditions used and i t s presence therefore would not lead to any complications. To t r y and enhance the formation of o l e f i n i c product during the Hofmann reaction, potassium t-butoxide i n t-butanol was  selected as the base.  The  reason for t h i s choice being that potassium t-butoxide i s a stronger base and weaker nucleophile than sodium ethoxide and i t would be less prone therefore to produce undesirable substitution products during reaction. The N.M.R. spectrum f o r the crude reaction mixture d i d indicate that the product r a t i o s had a l t e r e d s i g n i f i c a n t l y .  A small percentage o f s u b s t i t u t i o n  product was produced as deduced from the presence of a s i n g l e t at 6 1.28 f o r the hydrogens of the t-butyl group, however the predominant portion of the reaction mixture consisted of a mixture of two o l e f i n i c products (Figure 24). From the measured integration of the two s i n g l e t s for the dimethylamino o l e f i n i c product r a t i o was  determined to be 60:40 (compound 97:88).  During i n i t i a l experiments the reaction mixture was  groups the  (30 mg.  scale) i s o l a t i o n o f the components of  accomplished by preparative layer chromatography.  Only one product corresponding to the major spot on TLC could be i s o l a t e d i n y i e l d s (30%) which enabled s p e c t r a l data to be obtained. From the s p e c t r a l data the i d e n t i t y of the o l e f i n i c compound was  consis-  tent with the structure 97 which i s the expected product on the basis of the 121-3 Hofmann r u l e , but which i s opposite to that reported by Ondetti and Deulofeu .  Figure 24.  The N.M.R. Spectrum o f the Crude Reaction Mixture  (KOt-Bu/t-BuQH).  - 83 -  /  N\ N H (97)  Identifying features i n the N.M.R. were the doublet  (61.39, J = 6Hz),  quartet (63.75, J = 6Hz), and s i n g l e t (62.25 f o r the methyl group, methine hydrogen and dimethylamino group respectively f o r the side chain at C-3 of the carbazole system (Figure 25). A s i n g l e t was also present at 62.50 f o r the protons o f the C - l methyl group.  The signals f o r the three hydrogens  of the v i n y l side chain at C-2 portrayed a t y p i c a l ABX system.  A pair of  doublet of doublets occurred i n the o l e f i n i c region, one at 65.20 17Hz,  ^'141  = 2Hz) f o r the trans C-4' hydrogen, the other at 5.65  11Hz,  J4i 4i  = 2Hz) f o r the c i s C-4' hydrogen.  >  (J31^41 ^31^41  = =  A series o f four lines was  also observed downfield at 66.98 ( J 3 1 41 = 17Hz, ^ 1 ^ 4 1 = 11Hz) f o r the C-3' hydrogen o f the double bond. The difference i n the magnitude o f the coupling constant enabled the c i s and trans hydrogens to be distinguished and i t i s o f i n t e r e s t that the chemical s h i f t positions o f these resonances i n o l e f i n 97 are reversed from that observed f o r styrene.  This c h a r a c t e r i s t i c feature may be a consequence o f  s t e r i c or e l e c t r o n i c interactions o f the N-dimethylamino group with the v i n y l system which r e s u l t s i n a reversal o f the influence that the aromatic r i n g has on the two terminal hydrogen atoms.  - 84 -  - 85 -  A parent peak was observed at m/e = 278 i n the mass spectrum o f 97, and major fragmentation peaks came at m/e = 263, 234, 233, 219, 218, 204. The fragmentation reactions that occur f o r the series o f C-2 and C-3 unsaturated and saturated ring-opened products are very c h a r a c t e r i s t i c and f o r t h i s reason a discussion of the mass spectrum w i l l be reserved till  l a t e r (Section 5 ) . The parent peak at m/e = 278.1780 i n the high  resolution spectrum was within acceptable l i m i t s of the calculated value m/e = 278.1782 f o r the composition  C^r^^.  The i n t e r a c t i o n o f the v i n y l system with the carbazole back bone caused only a s l i g h t modification o f the normal carbazole UV. spectrum. There was a bathochromic absorption.  s h i f t of 10mm to A  max  2 4 7 f o r the predominant  The spectrum obtained f o r o l e f i n 97 bore no resemblance to 121-3  the highly modified carbazole UV. spectrum reported  f o r o l e f i n 88.  P r i o r to the eventual i s o l a t i o n o f the second elimination product  from  the reaction by column chromatography i t s i d e n t i t y was also deduced by i n ference from the N.M.R. spectrum o f the crude reaction (Figure 24).  As  previously discussed, i t has been shown from the data presented i n Table I I I that the two d i f f e r e n t nitrogen containing side chains f o r the ring-opened products could be distinguished by the p o s i t i o n o f the chemical s h i f t f o r the respective dimethylamino group. Two s i n g l e t s f o r the dimethylamino group o f both side chains are present i n the spectrum of the crude reaction mixture.  The s i n g l e t at 62.32 corres-  ponded to the C-3 side chain o f the major o l e f i n i c product 97 i s o l a t e d from  Table I I I : C h a r a c t e r i s t i c N.M.R. Chemical S h i f t s f o r the Degradation Products o f Guatambuine (25)  Chemical S h i f t :  Structure:  la 6C-1 CH  3  6N(CH )  lb  6C-1'H  SC-l'O^  2.40  3.90  1.52  3  2  (25)  2.54  (97)  2.50  2.25  3.75  1.39  (103)  2.49  2.29  3.62  1.42  f  lc 6 = CH (trans) 2  Id 6 = CH (cis) 2  5.20  5.65  00  ON  2.49  2.40  2.38  <  (88)  2.50  N  ( (89)  2.64  2.54  (89) from LAH on (25)  2.52  2.46  (89) from (18) degradation  2.48  2.38  4.85  1.53  5.29  5.68  le  l a . (q,J = 6Hz), b. (d,J = 6Hz), c. ( J ^ - 17 H z , J  B C  = -2.0Hz) d. ( J  e. (FTl N.M.R.. verv low concentration.  x c  = 11 Hz)  f i n guatambuine This carbon i s C-_5_  - 87 -  the reaction mixture.  I t was  very probable therefore from the correlations  that the remaining s i n g l e t peak (62.40) corresponded to the C-2 present i n o l e f i n 88. (65.70 (J = 17Hz,  side chain  A p a i r of doublet of doublets i n the o l e f i n i c  2Hz);  65.32 (J = 11Hz,  these a t t r i b u t e d to o l e f i n 97.  2Hz))  region  could be distinguished from  From the measured coupling constants i t was  determined that the chemical s h i f t s of the c i s and trans-oriented  terminal  hydrogens coincided with those f o r styrene but were reversed with respect the resonances f o r the elimination product 97.  to  This would be expected f o r  compound 88 f o r no s t e r i c or e l e c t r o n i c i n t e r a c t i o n would be anticipated between the C-3 v i n y l group and the dimethylamino group. I t was  not possible to conclusively prove the structure of the  unisolated  o l e f i n from the N.M.R. spectrum of the crude mixture, however the data are consistent with the postulated structure During the i n i t i a l experiments i t was  88. thought that only the predominant  Hofmann product 97 could be i s o l a t e d i n adequate y i e l d and that i t would not be possible to i s o l a t e the C-3 degradation approach.  o l e f i n i c product 88 d i r e c t l y by the Hofmann  I t became necessary i n order to determine any  activity  at the C-l methyl of guatambuine (25) i n subsequent incorporation experiments to f i n d an alternate method which would generate the C-3 v i n y l side chain i n a ring-opened compound. To this, end the C-2  o l e f i n 97 became of p a r t i c u l a r importance f o r not  only could i t be o x i d a t i v e l y degraded so as to i s o l a t e the C-3  carbon atom of  guatambuine (25), but i t s p o t e n t i a l second Hofmann elimination product would contain the desired C-3  (carbazole numbering) v i n y l side chain.  The  - 88 -  -  89 -  development o f this l a t t e r chemistry and the study of a number o f alternate methods aimed at producing a ring-opened compound with the C - 3 v i n y l side chain comprises a considerable portion of the work on the degradation sequence described i n subsequent sections. I t has been found however from further work that by p u r i f y i n g the crude reaction mixture by column chromatography on alumina that i t became possible to i s o l a t e the C - 3 o l e f i n 88 i n 18% y i e l d ( o l e f i n 9 7 was i s o l a t e d i n 36% yield).  (88)  The N.M.R. spectrum (Figure 2 6 . ) as a n t i c i p a t e d possessed a s i n g l e t a t 62.38  which was i n close proximity ( A 6 0 . 1 2 ) to the s i n g l e t f o r the C - l methyl  group ( 6 2 . 5 0 ) .  A m u l t i p l e t centered a t 6 3 . 0 8 integrated f o r the b e n z y l i c  methylene protons o f the 3-dimethylamino side chain while the multiplet f o r the remaining two protons was buried beneath the two s i n g l e t absorptions f o r the respective methyl groups.  Two p a i r s o f m u l t i p l e t s (doublets o f doublets)  occurred i n the o l e f i n i c region f o r the terminal C - 2 hydrogen atoms. The 1  s h i f t o f the trans coupled C - 2 '  hydrogen 6 5 . 6 8 ( J i » , 2 »  =  1 7 H z  >  J  2',2"  = 2 . 0 Hz)  to lower f i e l d with respect to the one which i s coupled i n a c i s fashion 65.29  (Jj, 2 i  =  11  H z  >  2'  =  H z  ^  W  a  S s  n  o  w  n  t  o  ^  e  characteristic for  the C - 3 o l e f i n i c side chain as suspected from the N.M.R. spectrum o f the crude  -90-  r e a c t i o n mixture. The  mass spectrum o f 88 p o s s e s s e d a p a r e n t peak a t m/e  major f r a g m e n t a t i o n s found at m/e (see  = 263,  249,  section 5 for a detailed discussion).  234,  The  p o s s e s s e d a p a r e n t peak a t 278.1780 which was c o m p o s i t i o n ^1^22^2 The  UV.  ^  o r  c  o  m  P  o  u  n  c  spectrum o r the  27).  gross d i s s i m i l a r i t i e s  close coincidence  high  220,  with  219-8,  204  r e s o l u t i o n spectrum  consistent with  the  88.  spectrum o f 88 b o r e no  carbazole The  i  233,  = 278  resemblance t o e i t h e r the  spectrum o b t a i n e d f o r the C-2 i n the UV.  o l e f i n 97  s p e c t r a o f 88  o f the v a l u e s o b t a i n e d i n the  normal  and  97  (Figure  and  the  spectrum o f 88 w i t h  the  121-3 reported  v a l u e s by O n d e t t i  and  Deulofeu  t h e y had  proposed the c o r r e c t s t r u c t u r e  isolated  i n t h e i r study.  I t would have been d i f f i c u l t shape o f the a b s o r p t i o n 97 o r the  patterns  f o r t h e two  f a c t t h a t the d i f f e r e n c e i n C-2,  these data are  system and  f o r the  elimination  postulate  that  resonance c o n t r i b u t i o n s  C-3  f o r the two  compounds.  the d i f f e r e n c e s may  be  the  e x c i t e d s t a t e s o f 88  1  would  that  interactions  t o the  due  Now  carbazole  the and  s u b s t i t u t i o n patterns  p o s i t i o n s with respect  i n the  product  e l i m i n a t i o n p r o d u c t s 88  a v a i l a b l e i t i s p o s s i b l e to consider  groups i n the two  that  t o have p r e d i c t e d b e f o r e h a n d e i t h e r  r e s u l t i n completely d i f f e r e n t spectra  the v i n y l  proved c o n c l u s i v e l y  of ring  t o the d i f f e r e n t and  97  (as  shown).  I  220  Figure 27.  1  250  1  300  1  1  350  400  A Comparison of the UV. Spectra f o r the Olefins 88 and 97 with the Normal Carbazole Spectrum of 98.  - 92 -  N  <  (88)  < (97) F u r t h e r confirmation for the production of o l e f i n 88 was  provided  by reducing i t to i t s saturated d e r i v a t i v e 89, and comparing i t s s p e c t r a l data with 89 obtained both by u l e i n e degradation and lithium aluminum hydride ring opening of guatambuine methiodide (86) (Section 3).  < (89) The product d i s t r i b u t i o n ( 2:1 by weight) for the r i n g opening o f ;  guatambuine methiodide (86) using potassium r e f l e c t s the enhancement of the E  2  t-butoxide i n t-butanol  elimination mechanism over the  mechanism observed with the previous conditions. The i s o l a t i o n of the C-3 o l e f i n 88 d i r e c t l y from the Hofmann reaction was  an important  r e s u l t because as discussed i n l a t e r sections i t i s ques-  tionable whether or not i t would be f e a s i b l e to i s o l a t e the C - l methyl group of guatambuine (25) by the alternate routes developed.  However, even the  - 93 -  y i e l d of 88 obtained under these conditions  (18%) becomes of marginal prac-  t i c a l i t y when the reaction i s repeated on a projected plant i s o l a t i o n scale (30-40  mg.).  To see i f the product d i s t r i b u t i o n could be s h i f t e d or the o v e r a l l y i e l d of the two elimination products could be increased, hot aqueous hydroxide studied as the basic medium. not a l t e r s i g n i f i c a n t l y y i e l d was  I t was  was  found however that the product r a t i o did  (N.M. R, integration) and that the o v e r a l l reaction  lower (60%) .  The use of sodium hydride  as the base i n hot dimethylformamide  was  considered i n the hope that by using a very powerful base the r e l a t i v e a c i d i t i e s of the B-hydrogens i n guatambuine might become the sole determining factor i n the reaction and as a consequence the product r a t i o might be s h i f t e d completely towards the C-2 I t was  olefin  97.  found quite unexpectedly a f t e r reaction, however, that exactly  the opposite had occurred.  The C-3  only reaction product i s o l a t e d .  o l e f i n 88 obtained i n 70% y i e l d was  The s p e c t r a l data was  obtained f o r 88 above using potassium t-butoxide In terms of the mechanistic o l e f i n 88 was  rationale i t was  i d e n t i c a l to that  as the base. not e n t i r e l y c l e a r why  the exclusive reaction product, however, the p r a c t i c a l  of this r e s u l t was  that the C-3  the  aspect  o l e f i n 88 became available i n adequate quan-  t i t i e s such that subsequent oxidative cleavage (see Section 3) enabled the C-l methyl groups o f guatambuine to be unambiguously i s o l a t e d . The a p p l i c a t i o n of the Hofmann elimination reaction to the degradation of N-methyltetrahydroellipticine methiodide (95) was there i s only one  s t r a i g h t forward f o r  d i r e c t i o n i n which B-elimination can occur.  - 94 -  < (102)  (95)  To reduce the occurrence of the competing reaction was  s u b s t i t u t i o n reaction the  conducted using potassium t-butoxide i n r e f l u x i n g t-butanol.  A single product was spectral data was  formed during the reaction. (TLC) i n 96% y i e l d and the  consistent with the o l e f i n i c product  102.  The N.M. R, possessed two s i n g l e t s at 62.50 and 2.90  f o r the protons  of the aromatically substituted methyl groups at C - l and C-4 A t h i r d s i n g l e t occurred at 62.24 f o r the dimethylamino  respectively.  group.  As i n the  guatambuine (25) series the proximity of t h i s s i g n a l r e f l e c t e d the presence of the dimethylamino was  group on the C-3 p o s i t i o n .  Another s i n g l e t (63.64)  a t t r i b u t e d to the methylene protons both b e n z y l i c (C-l) and adjacent to  the nitrogen atom. 65.20 (J2',3' =  1 7 H z  Two pairs of multiplets (doublets of doublets) came at » 3«,3» = J  2 H z  )  a n d  6 5  -  6 4  ( 2',3' J  =  1 1  Hz  >  J  3',3'  =  2  H z  )  and once again the u p f i e l d p o s i t i o n of the trans coupled hydrogen with respect to the c i s hydrogen was  c h a r a c t e r i s t i c o f the C-2 v i n y l side chain.  The mass spectrum possessed a parent peak at m/e tations at 263, 234, 233, 219, 218, 204.  = 278 and major fragmen-  The high resolution spectrum possessed  a parent peak at 278.1780 which was within acceptable l i m i t s of the value 278.1782 calculated f o r the composition C j g r ^ ^ .  - 95 -  The UV . spectrum bore a d i s t i n c t resemblance to that obtained f o r carbazole, this behavior again being c h a r a c t e r i s t i c of the presence of the C-2 v i n y l side chain i n the elimination product. The oxidative degradation of the Hofmann product i s discussed i n section 3.  - 96 -  3.  N-Methyl, C-3 Methylene and C - l Methyl Group I s o l a t i o n s . Due to the scale o f the biosynthetic experiments, i t was accepted that f o r  the guatambuine system (25) a l l three carbon centers could not be i s o l a t e d from the same experiment.  With the Hofmann elimination reaction developed to the  point where the ring-opened o l e f i n s 88 and 97 from guatambuine (25) and 102 from N-methyltetrahydroellipticine  (26) were available i n adequate y i e l d s , the two  carbon centers ( C - l and C-3) could then be i s o l a t e d i n d i v i d u a l l y by ozonolytic cleavage.  Determination of the N-methyl group would then be a separate experi-  ment involving a Hofmann reaction from the methine methiodides of any o f the above o l e f i n s or from t h e i r saturated derivatives 89, 103 or 104.  (89)  (103)  To gain the most information  (104)  from the biosynthetic experiments u t i l i z i n g  the Hofmann reaction chemistry, the i s o l a t i o n experiments were divided i n t o two catagories.  In the f i r s t , the C - l methyl group was unique to guatambuine (25)  and i t s i s o l a t i o n v i a the ozonolytic cleavage o f C-3 o l e f i n 88 was considered as a separate experiment.  In the second, the N-methyl and C-3 methylene  were common to both 25 and N-methyltetrahydroellipticine  groups  (26) and e f f o r t s were  therefore directed at developing a combined experiment whereby both centers could be i s o l a t e d sequentially.  - 97 Before attempting the ozonolysis o f the valuable C-3 o l e f i n 88, the reaction conditions were worked out f o r compound 102 derived from N-methyltetrahydroellipt 146 cine (26).  The procedure u t i l i z e d by Battersby and Harper  was adopted, being  modified only i n that methylene chloride was used as the solvent.  The formalde-  hyde produced (C-3 carbon) on reductive work-up was i s o l a t e d as the bisdimedone 2/0 derivative  by steam d i s t i l l a t i o n o f the reaction mixture into a saturated  dimedone s o l u t i o n .  (102)  (105)  +  CHO  2  No attempt was made to i s o l a t e the carbazole aldehyde residue 105 and i t was thus not known whether ozone also attacked the benzylic dimethylamino group. The formaldehyde dimedone was i s o l a t e d as long c r y s t a l l i n e needles i n 87% y i e l d . The ease with which the C-3 carbon could be i s o l a t e d by ozonolysis indicated that the technique would be generally applicable within the series o f C-2 and C-3 v i n y l carbazole d e r i v a t i v e s . C-3 v i n y l system o f O l e f i n 88 was s i m i l a r l y r e a d i l y cleaved on ozonolysis. The formaldehyde dimedone (C-l methyl) was i s o l a t e d as long needles i n 60% y i e l d . Again, no attempt was made to i s o l a t e the aldehyde 106 also proposed to be formed during the reaction.  This then completed the degradation o f guatambuine  i s o l a t e the C - l methyl group.  so as to  - 98 -  (88)  (106)  For the i s o l a t i o n of the remaining two centers i n 25 and 26, i t was sidered that the most e f f i c i e n t order i n which the degradation could be  conconducted  would be by sequential Hofmann reaction which would eliminate the N-methyl group and produce the C-2 v i n y l system that would subsequently be cleaved o x i d a t i v e l y . ^  + (CH^N  —  \  C  H  Q  +  CHO  By conducting the reaction i n the reverse sequence i . e . oxidative cleavage, f i r s t i t was thought that problems would a r i s e , f o r i t was known that i n general, 145-6 the techniques used f o r i s o l a t i n g the one carbon unit as the dimedone d e r i v a t i v e lead to poor recovery of the remainder of the molecule. It was necessary to u t i l i z e the B-dimethylaminoethyl  side chain f o r t h i s  degradation because as w i l l become clearer i n Section 4, considerable d i f f i c u l t i e s a r i s e during methiodide formation of compound 103 containing the b e n z y l i c dimethylamino group. To produce a suitable derivative i n both the guatambuine (25) and N-Methylt e t r a h y d r o e l l i p t i c i n e (26) advantage was taken of the r e a c t i v i t y of the C - l ben140 z y l i c center towards nucleophiles. I t has been shown that i n both uleine and 113 apparicine that reaction of the quaternary s a l t s with lithium aluminum hydride  - 99  -  e f f e c t e d displacement o f the n i t r o g e n center by hydride.  In a s i m i l a r manner  i t was found that the methiodides o f 25 and 26 were attacked by l i t h i u m aluminum hydride t o g i v e i n good y i e l d s compound 89 and  107.  Compound 89 was prepared a l s o f o r the purpose o f comparing i t s p h y s i c a l and 132 s p e c t r a l p r o p e r t i e s w i t h those f o r the degradation product o f u l e i n e and w i t h the hydrogenated Hofmann product o f O l e f i n 88 prepared both i n the present work 121-3 and p r e v i o u s l y  (Table I I I ) .  The N.M.R. spectrum of compound 89 possessed a m u l t i p l e t 53.00, and a t r i p l e t (61.36, J = 7 Hz) f o r the newly formed e t h y l side c h a i n .  Present a l s o were the  s i n g l e t s a t 62.52 and 2.46 f o r the C - l methyl and dimethylamino groups r e s p e c t i v e l y . As mentioned p r e v i o u s l y the chemical s h i f t f o r the l a t t e r group was  characteristic  f o r the presence o f the B-dimethylaminoethyl s i d e chain a t the C-2 p o s i t i o n . The mass spectrum possessed a parent peak a t m/e m/e  = 266, 236, 222, 207, 206, 204 (Section 5 ) .  parent peak at m/e f o r the composition  = 280 and major fragments at  The h i g h r e s o l u t i o n mass spectrum  = 280.1975 was c o n s i s t e n t w i t h that c a l c u l a t e d (m/e = 280.1938) C1QH24N2.  For f u r t h e r comparison and p r o o f o f s t r u c t u r e 89 the methiodide o f u l e i n e 87 120,132 was ring-opened under Hofmann c o n d i t i o n s  to give the compound 89.  The  s p e c t r a l data f o r 89 prepared by the two d i f f e r e n t routes were almost i d e n t i c a l . An added f e a t u r e t o conversion o f 87 •*• 89 was t h a t the subsequent i s o l a t i o n o f the N-methyl and C-3 methylene groups from compound 89 c o n s t i t u t e d a degradation o f  - 100 -  both uleine (18) and guatambuine (25). The N.M.R. spectrum of compound 107 exhibited three s i n g l e t s at 62.75, 2.42,  and 2.36  f o r the C-4, C - l and C-3 methyl groups r e s p e c t i v e l y .  s i n g l e t was present at 62.32 f o r the dimethylamino group.  A fourth  The mass spectrum  possessed a parent peak at m/e = 280 with major fragments at m/e = 266, 249, 236, 222, 207-6, 204,  191 (Section 5).  The parent peak i n the high r e s o l u t i o n  mass spectrum m/e = 280.1903 was again consistent with the calculated value m/e  = 280.1938 f o r C-^rL^N^ Both compounds 89 and 107 were converted to t h e i r methiodides 90 and 108  i n quantitative y i e l d s i n preparation f o r the Hofmann r e a c t i o n .  The  procedure  used f o r i s o l a t i n g the N-methyl group w i l l be described below, a t the present time attention w i l l be focussed on the formation of the o l e f i n s 91 and 109.  (90)  (91)  1  V  H  1  + N(CH ) 3  (110)  4  +N(CH ) 3  (108)  (109)  4 H  The N-methyltetrahydroellipticine derived methiodide  (111) 108 was reacted with  potassium t-butoxide i n t-butanol i n the same manner as described i n Section 2, The desired o l e f i n 109 was obtained as a brown o i l i n 85% y i e l d . The N.M.R. spectrum f o r the product showed a doublet of doublets at 65.68 ( J j , 4 , = 12 Hz, J^, 4 , = 2 Hz) f o r the c i s C-4H'  and a complimentary doublet  - 101 of doublets at 65.24 ( J 3 ' , 4 • =  1 8  H z  »  J  4',4« =  2  H z  )  f o r  t h e  trans C-4*H.  The s i n g l e t s f o r the three methyl groups occurred at 62.84 (C-4), 2.52 and 2.40  (C-3).  The mass spectrum possessed a parent peak at m/e  main peaks at m/e  = 220, and 205  possessed a parent peak at m/e f o r that calculated (m/e  (Section 5).  (C-l),  = 235 with  The high resolution spectrum  = 235.1325 which was within acceptable l i m i t s  = 235,1360) f o r C H N . 17  The reaction of the methiodide  17  90 derived from the guatambuine system  with potassium t-butoxide i n t-butanol did not proceed as well as a n t i c i p a t e d , a dark brown product containing considerable q u a n t i t i e s of material was obtained.  The o l e f i n 91 was obtained as a brown o i l i n 57%  a f t e r p u r i f i c a t i o n by preparative layer chromatography on A cleaner product was  90 with  However i n t h i s instance as well, the  low (40%).  The N.M.R. spectrum f o r t h i s product  (of poor q u a l i t y ) showed the p a i r o f  doublets of doublets, 65.65 ( J , , = 12 Hz, J , , 3  and 65.29 (J31  yield  alumina.  obtained by reaction of the methiodide  sodium hydride i n dimethylformamide. product y i e l d was  decomposition  41  = 18 Hz, J 4 1  J 4  4  I  4  4  = 2 Hz) f o r the cis_ C-4'  = 2 Hz) f o r the trans C-4' H.  The  H  singlet  for the C - l methyl group was r e a d i l y discerned at 62.54 but the signals f o r ethyl groups occurred as complex m u l t i p l e t s due to the presence o f i m p u r i t i e s . The mass spectrum f o r 91 possessed a parent peak at m/e fragments at m/e  = 220 and 205.  parent peak at m/e  = 235 and major  The high r e s o l u t i o n spectrum possessed a  = 235.1384 within acceptable l i m i t s of the value 235.1360  calculated f o r CjyHjyN. The N-methyl group of guatambuine (25) and N-methyltetrahydroellipticine (26) was i s o l a t e d during the Hofmann reactions by mine that was  converting the trimethyla-  formed to i t s tetramethylammonium iodide d e r i v a t i v e .  This was  - 102 -  done by passing the gases produced during the reaction through a s o l u t i o n of methyl iodide. To determine the e f f i c i e n c y of the trapping technique, a mock/biosynthetic experiment was  conducted where (N-C^  methyl) guatambuine (25) and N-Methyltetra-  h y d r o e l l i p t i c i n e (26) were converted to t h e i r ring-opened methiodides 90 and and subjected  to the Hofmann reaction.  present i n 25 and 26 was  1  108,  A t o t a l of 51 % and 72% of the a c t i v i t y  recovered as the tetramethylammonium iodide s a l t .  These  r e s u l t s demonstrated that the N-methyl group could be r e a d i l y i s o l a t e d and  that  the i s o l a t i o n technique would be suitable to future biosynthetic experiments. The subsequent ozonolyses of o l e f i n s 91 and methylene group of 25 and 26 was  unfortunately  109 so as to i s o l a t e the  C-3  not investigated at t h i s time. In  order f o r the ozonolysis reaction to be f e a s i b l e i n the guatambuine series however, i t would be f i r s t necessary to further develop the Hofmann r e a c t i o n of so as to obtain a higher y i e l d of the o l e f i n 109.  Considering  108  the success of  the ozonolysis reaction i n the cleavage of the C-3 v i n y l side chain of o l e f i n s 88 and  102,  i t would be anticipated that no major d i f f i c u l t i e s would be encoun-  tered during ozonolysis of 91 and  109.  An o v e r a l l view of the degradation scheme f o r the o l i v a c i n e (16) system i n v o l v i n g the Hofmann reaction followed by ozonolytic cleavage reactions i s presented i n below.  Considerable work was  also done to develop alternate  routes whereby the N-methyl, C-l methyl and C-3 methylene groups could be isolated.  This work i s presented i n subsequent sections.  ?  103 -  - 104 -  4.  The Synthesis  o f l-Methyl-2-ethyl-3  vinylcarbazole  (114) from the C-2  O l e f i n 97. To r e i t e r a t e f o r a moment, the synthetic schemes described i n the following sections were aimed at devising a f e a s i b l e a l t e r n a t i v e to the d i r e c t Hofmann reaction o f guatambuine methiodide (86) f o r creating a C-3 v i n y l carbazole  derivative f o r the subsequent degradative i s o l a t i o n  of the C - l methyl carbon o f guatambuine  (25). This work was prompted  by the i n i t i a l b e l i e f that the C-3 o l e f i n 88 would be unavailable the potassium t-butoxide-t-butanol  from  reactions.  Two d i f f e r e n t approaches were developed, an alternate r i n g opening procedure (Section 6) and the second Hofmann reaction approach s t a r t i n g from the r e l a t i v e l y abundant C-2 o l e f i n 97.  This l a t t e r approach to be  discussed i n this section i s i l l u s t r a t e d below.  (112)  In preparation  (111)  f o r the second Hofmann reaction the C-2 v i n y l side  chain o f 97 was reduced to compound 103 i n near quantitative y i e l d by  - 105 -  c a t a l y t i c hydrogenation over platinum oxide.  This compound l i k e i t s  saturated Hofmann counterpart 89 was also prepared as an a i d f o r i n t e r p r e t i n g the N.M.R. of the crude Hofmann elimination reaction mixtures (Table I I I , section 2.). The N.M. R. spectrum f o r the reduced compound 103 showed a t r i p l e t (61.20, J = 6 Hz) and a quartet (62.94, J = 6 Hz) f o r the methyl and methylene hydrogens respectively of the newly formed C-2 ethyl side chain.  Present also was a doublet (61.42, J = 6 Hz) and a quartet  (63.62,  J = 6 Hz) for the C - l ' methyl group and the methine hydrogen substituted both b e n z y l i c and adjacent to nitrogen i n the C-3 side chain.  Two  singlets  occurred at 61.48 and 1.28 f o r the C-l methyl and dimethylamino group, the p o s i t i o n o f the l a t e r absorption being c h a r a c t e r i s t i c f o r the C-3 nitrogen bearing side chain. The mass spectrum possessed a parent peak at m/e fragmentations occurred at m/e  = 280 and major  = 265, 236, 235, 220, 207-5, 204 (see d i s -  cussion section 5 ) . Compound 103 exhibited a normal carbazole UV . spectrum as expected. L i t t l e information was obtained from the I P. spectrum other than i t resembled very closely the spectrum obtained f o r the saturated component 89. The subsequent formation of the methiodide 111 scale proved to be extremely d i f f i c u l t .  i n pure form on a small  Reaction of 103 with methyl iodide  as a neat solution or i n a v a r i e t y o f solvents such as ether, chloroform, and methanol resulted i n the co-formation o f several non-polar products.  This  was a consequence of the i n s t a b i l i t y of the newly formed quaternary nitrogen  - 106 with respect to n u c l e o p h i l i c displacement, which i s inherent to i t s 147 gramine  l i k e structure.  Purification  of the methiodide 111 by column chromatography  attempted as i t was thought that the molecule would decompose.  was not Re c r y s t a l l i -  zation from polar solvents l i k e methanol was unsuccessful since TLC showed that with increasing time i n methanol the concentration of non-polar side product increased. The methiodide was s u f f i c i e n t l y  purified  f o r subsequent reaction by  simply washing the concentrated reaction product with chloroform which removed the soluble non-polar materials and l e f t behind the insoluble methiodide 111. In the case where the methiodide 111 was formed by reaction with methyl iodide i n methanol i t was shown by N.M. R. that the major non-polar component corresponded as anticipated to the 0-methyl ether 113, formed by 143,  147  displacement.  (113)  Unfortunately the i n a b i l i t y to obtain 111 i n pure form made i t impossible 121-3 to compare i t s physical properties with those previously reported.  - 107 -  The choice of reaction conditions  f o r the subsequent  elimination  reaction to 112 had to take into account the l a b i l e nature of the quaternary nitrogen group, substituted at the b e n z y l i c C - l ' center, towards displacement reaction. 143 Openshaw  I t has been shown by Norcross and  i n a study of the c y c l i c and a c y c l i c methiodides of model  compounds r e l a t e d to the emitene system  (99) that i n the a c y c l i c case  exemplified by 114, the b e n z y l i c a l l y substituted quaternary amino group, which has an electron donating group oriented para to i t , possesses an exceptional r e a c t i v i t y towards n u c l e o p h i l i c s u b s t i t u t i o n . was a t t r i b u t e d to the a b i l i t y of a s t a b i l i z e d carbonium i n the t r a n s i t i o n state of the reaction (SNj mechanism).  This r e a c t i v i t y  ion to be formed  - 108 -  The close p a r a l l e l between these systems and the methiodide 111 derived from guatambuine (25) was 148 this and other studies  s e l f evident.  I t was  apparent from  that the b a s i c aqueous or a l c o h o l i c condi-  tions normally employed f o r the Hofmann degradation would f a i l to produce the desired o l e f i n i c product 112 . To circumvent t h i s same problem i n the emetine (99) s e r i e s , Openshaw 142-3 et a l .  found that p y r o l y s i s of the a c y c l i c methiodides i n an a p r o t i c  solvent l i k e d i e t h y l ketone resulted i n o l e f i n formation.  I t was  also  observed, however, that i n accord with the carbonium ion mechanism, the y i e l d of the reaction depended markedly on the mesomeric influence of the a l k y l substituent ( R) on the side chain.  As the s i z e of the  R group  decreased, so did the y i e l d and i t could be anticipated from t h i s trend that the side chain where R = H indiginous to 111 may y i e l d of o l e f i n  r e s u l t i n a poor  112.  With this p o s s i b i l i t y i n mind, i t was  decided to study the  of the p y r o l y t i c elimination reaction on a model compound (117) from salsolidine. methiodide (115)  feasibility derived  instead of using valuable quantities o f  HI.  (115) (118)  - 109 -  I t was found that prolonged heating of 117 i n d i e t h y l ketone  resulted  only i n the i s o l a t i o n of small amounts o f u n i d e n t i f i e d materials. None o f the desired o l e f i n 118 was  detected, and this approach was consequently  abandoned. The C-3 o l e f i n 112 was eventually obtained i n 70% y i e l d from 111 by reaction with sodium hydride as the base i n dimethylformamide.  This  reaction had previously proven successful i n r i n g opening guatambuine methiodide  (86) to the analogous o l e f i n 88.  To avoid confusion at this point i t must be mentioned that the successful completion of the sequence t o the C-3 vinylcarbazole 112 (the compound that was supposed t o take the place of 88 i n the degradation sequence) came a f t e r the conditions were found to obtain 88 d i r e c t l y . The s p e c t r a l data f o r the o l e f i n i c product was consistent with i t s structure 112 .  The presence of the v i n y l grouping at C-3 was r e a d i l y  (112)  determined by i t s c h a r a c t e r i s t i c UV . spectrum and the p o s i t i o n of the j o l e f i n i c absorptions (5.69 (Jj i  2  i  =  I  7 H z  » ^2' 2'  J , , = 2 Hz) ) i n the N.M.R. spectrum. 2  2  =  2  , ( 5 5  .28(Jp  2  » = 11 H  - 110 -  The mass spectrum possessed a parent peak at m/e fragmentations at m/e tion spectrum  = 220 and 205.  = 235 and major  The parent peak i n the high resolu-  235.1380 was consistent with the composition CjyH^yN.  The considerable d i f f i c u l t y i n preparing the methiodide  113 makes  the sequence to compound 112 u n r e l i a b l e and low y i e l d i n g i n terms o f the quantities of o l e f i n obtained.  I t would be doubtful i f s u f f i c i e n t  quantities of formaldehyde (as i t s dimedone d e r i v a t i v e ) would be obtained from the oxidation of 112 to substantiate any incorporation experiments by t h i s route. 6 was  I t was  initiated.  f o r t h i s reason that the work presented i n section  - Ill -  5.  Mass Spectral Correlations of Ring "D" (25) and E l l i p t i c i n e (17).  Opened Derivatives o f Guatambuine  A discussion of the mass spectra of the o l e f i n i c (88, 97, reduced (89, 103, 107)  102)  and  ring-opened products of guatambuine (25) and  (17) as well as t h e i r second Hofmann products (91, 112,  109)  ellipticine  has been reserved  a separate section because close s i m i l a r i t i e s existed i n the fragmentation patterns of these derivatives.  I t appears as though the occurrence of several  c h a r a c t e r i s t i c fragments i n the spectra i s i n t e r n a l l y consistent with t h e i r t r i - and t e t r a - substituted carbazole  structures.  C h a r a c t e r i s t i c features i n the spectra o f the r i n g opened products are a substantial parent peak e i t h e r at m/e  = 278  (unsaturated) or m/e  (saturated) and a fragmentation pattern consisting of successive  =  280  losses o f 14  and 15 mass units which corresponds to the formal loss of successive methylene and methyl groups (Figure 28 $ 29). These c h a r a c t e r i s t i c fragments occur at m/e 234,  = ^ 2 8 0 ) , 265,  205 and m/e  =(M+:278), 263,  249,  Weaker fragments are also observed i n the region m/e  = 191-193, 180,  and  233,  220,  219,  236, 218,  235, 205,  167 f o r further losses of 13-15 represents  the carbazole  222,  220,  207,  204.  mass u n i t s .  The m/e  = 167 fragment presumably  ion.  Except i n the spectra of the o l e f i n i c compounds the peak at m/e corresponding to loss of 29 mass units occurred with the exception side chain the ^ - 1 5  o f the compounds 97 and peak was  presence of a substantial M*-15  = 249  only to a minor extent, and  103 with the a-dimethylaminoethyl  also not intense.  In the l a t t e r instance,  the  i s probably a consequence of a f a c i l e cleavage  for  '2-  -i  JJl 200  F i g u r e 28.  »°  240  2S0  260  .  » °  » °  230  240  250  260  Mass Spectrum of C a r b a z o l e Compound  200  F i g u r e 30.  230  •Ill  -11111 210  220  4J  L  L  220  Mass Spectrum of C a r b a z o l e Compound  l  F i g u r e 29.  210  270  210  (]03)  270  in  (88).  . .HI.  230  240  Mass Spectrum of C a r b a z o l e Compound ( ' 0 9 ) ,  - 113 -  of the C - l methyl group so as to form an even electron system (Figure 28). 1  This same cleavage i s an important fragment i n the mass spectrum of guatambuine (25) and i s only o f minor occurrence i n the spectrum of N-methyltetrah y d r o e l l i p t i c i n e (26). The f i r s t major fragment after the parent occurs at m/e  = 266, 265  (264,  263) f o r loss of 44 and 45 mass units which corresponds to the cleavage o f the dimethylamino group plus one hydrogen (equivalent to three methyl groups). Plausible fragmentation pathways f o r compounds 97 and 88 i n the guatambuine s e r i e s which are consistent with the presence o f the observed fragments and the compositions  for these fragments determined from the high resolution  spectrum are presented i n figures 31 § 32.  Pathways involving s i m i l a r pro-  cesses can be formulated f o r t h e i r isomeric ring-opened counterparts 103 and 89.  I t should be emphasized however that i n the absence of any s e l e c t i v e  l a b e l l i n g and other experiments  to v a l i d a t e the structures of the fragment  ions the proposed structures and fragmentation patterns are purely hypothetical. Several comments can be made concerning the postulated breakdown o f compound 103 (M , m/e +  = 280).  Only a s i n g l e metastable at m/e  = 206 corres-  ponding to the fragmentation 235 •*• 220 was evident i n i t s spectrum.  Considerably  more information was obtained from the spectrum o f i t s unsaturated analog 97 (M*, m/e  = 278) where metastables were present f o r the analogous M", m/e 1  = 234 f o r m/e  = 263  248  205  234 -»- 219  191  218  184  263 + 220  204  fragmentations:  - 114 -  (C H N) 16  14  220 Figure 31.  Plausible Mass Spectral Fragmentation Pattern f o r Compound 103.  - 115 -  263  (C H N) 12  16  218 Figure 32.  Figure 33.  (C H N) 1 5  U  205  Plausable Mass Spectral Fragmentation Pattern f o r Compound 88.  Plausable Mass Spectral Fragmentation Pattern f o r Compound 109.  - 116 -  The fragmentation m/e = 220 •> 205 involved cleavage o f the C - l methyl from the aromatic r i n g .  Such processes are well known, and i t i s currently  believed that the species obtained i s not a phenyl type r a d i c a l but a r i n g 149 opened species containing an acetylenic linkage. Cleavage o f the v i n y l side chain to l i b e r a t e an acetylenic system was not postulated since i t s involvement i n the fragmentation processes would lead to a more complex spectrum than was observed. Precedent f o r the formulation o f the structures o f a number o f the fragments was available from a d e t a i l e d study of the mass spectrum o f 140 uleine (18) and i t s derivatives.  Uleine fragments i n an expected manner  to the ring-opened t r i s u b s t i t u t e d carbazole derivative 119 which i s very s i m i l a r to compound 89.  The mass spectrum o f this synthesized compound  exhibits many o f the fragments observed f o r the guatambuine (25) derived components.  (119)  The fragmentation processes postulated f o r the elimination product 88 are straightforward, being governed by the a- and 8- cleavage o f the C-2 side chain.  On the other hand the fragmentation processes occurring f o r the  - 117 -  N-methyltetrahydroellipticine derived compounds 102 and 107 are more d i f f i c u l t to postulate structures f o r because the fragmentation process involves successive losses o f methyl groups from the carbazole r i n g . The second Hofmann products  (91 , 112 and 109 ) possess structures which  p o t e n t i a l l y correspond to the postulated fragments at m/e = 235 f o r the f i r s t Hofmann products.  I t i s not too s u r p r i s i n g , therefore, that t h e i r spectra  also possesses dominant fragments at ^ - 1 5 (m/e = 220) and M^-30 (Figure  (m/e = 205)  3 0 ) . This does not necessitate however that the fragmentation pro-  cesses are the same i n both cases. In the spectrum o f 109 metastables are present at m/e = 206 f o r the f r a g mentation m/e = 235  220 and m/e = 191 f o r the process m/e = 220  205.  The  second metastable i s very weak and the l a t e r fragmentation process may not be of major importance  (Figure 33) .  The general features o f the mass spectra o f these compounds are also found i n the spectra o f many o f the other C-2 and C-3 substituted derivatives discussed i n this t h e s i s .  1-methylcarbazole  - 118 -  6.  Alternate Rj ng " D' Opening Re actions of Guatambuine (25). This section i s concerned with the work that was  d i r e c t e d toward r i n g -  opening guatambuine (25) i n a manner which would s p e c i f i c a l l y cleave the C - l carbon-nitrogen  bond and enable the C-l methyl group to be i s o l a t e d .  approaches to the problem were considered : i )  Two  the d i r e c t opening of the  ring by p y r o l y t i c elimination to give the C-3 v i n y l system, and i i )  "D"  the  development of s u b s t i t u t i o n and oxidation reactions which would introduce  an  oxygen bearing f u n c t i o n a l i t y i n t o the C - l ' p o s i t i o n (carbazole numbering). This f u n c t i o n a l i t y could subsequently be elaborated i n t o a derivative s u i t a b l e for cleavage by one of three routes depicted i n figure 34 . Of these d i f f e r e n t routes the dehydration-ozonolysis  was  the most s u i t a b l e 118  because i t would be anticipated from the Potier-Janot postulate that radioactive stemmadenine (13) would label the C-l methyl group with t r i t i u m only.  The Baeyer-  V i l l i ger-acetate hydrolysis approach would permit the i s o l a t i o n of the t o t a l  Figure 34.  Approaches to the I s o l a t i o n of the C - l Methyl Group Involving a C - l ' Oxygen F u n c t i o n a l i t y .  - 119 -  s p e c i f i c a c t i v i t y , however, considerable d i f f i c u l t i e s would be associated with i s o l a t i n g the acetate s a l t .  The Haloform reaction would be the least useful  since a l l three of the radioactive hydrogens would be A.  lost.  Pyrolytic Elimination ; The p y r o l y s i s of guatambuine methiodide  (86) as with the base induced  Hofmann reaction can lead to formation of e i t h e r / o r both o l e f i n s 88 and 97. I t was hoped, however, that p y r o l y t i c cleavage would lead to a preference f o r the production of the C-3 o l e f i n 88. buine methiodide  I t was  found that p y r o l y s i s of guatam-  (86) i n d i e t h y l ketone at 100° did not induce cleavage of the  quaternized tetrahydropyridine r i n g system.  This r e s u l t p a r r a l l e l s the obser-  vation by Norcross and Openshaw 143 that the 2-phenylpiperidinium r i n g system of 120 also f a i l e d to open under these conditions. This they a t t r i b u t e d to the s t a b i l i t y of the s i x membered r i n g since the anologeous ring-opened compound!^ cleaved r e a d i l y on p y r o l y s i s i n d i e t h y l ketone.  (120)  Pyrolysis of the methiodide 86 i n the absence of solvent at 200° also f a i l e d to induce o l e f i n formation.  I t was  determined  from the N.M.R. spectrum  that instead of ring-opening under these more d r a s t i c conditions, the nitrogen dequaternized with reformation of guatambuine (25). unexpected  This was not a t o t a l l y  r e s u l t since some dequaternization generally occurs during Hofmann  - 120 -  139 elimination,  and dequaternization  of methoacetates by p y r o l y s i s i s a 150 known s y n t h e t i c a l l y applicable procedure. The p y r o l y s i s of the quaternary ammonium hydroxide ( c l a s s i c a l Hofmann reaction) was not t r i e d although i t was probably that elimination would have occurred under these conditions.  This was due to technical d i f f i c u l t i e s  that were encountered during the p y r o l y s i s o f the methiodide 86. Very l i t t l e product was formed and i t was always contaminated by considerable  quantities  of decomposition products. Total s e l e c t i v i t y towards formation o f the C-3 o l e f i n could have been 151 achieved by p y r o l y s i s o f the amine oxide o f guatambuine (25).  This reaction  would have been l i m i t e d to elimination i n the d i r e c t i o n o f the C-l methyl group because unlike the 8-hydrogens i n the s i x membered r i n g , the hydrogens of the methyl group are able to adopt the cis-coplanar o r i e n t a t i o n i n the t r a n s i t i o n state, a condition which i s necessary f o r e l i m i n a t i o n . has  The reaction  the major drawback however, i n that elimination can occur from the c i s  (a,e) configuration only, t h i s l i m i t s the maximum y i e l d to 50%. F o r t h i s reason and due to the d i f f i c u l t i e s previously encountered i n the p y r o l y s i s technique the amine oxide reaction was not attempted and the p y r o l y s i s approach to the ring-opening o f guatambuine was abandoned. Acetate Substitution Ring-Opening : The s u s c e p t i b i l i t y of the C - l b e n z y l i c center o f guatambuine towards attack by a nucleophile provided  an opportunity  to introduce  an oxygen f u n c t i o n a l i t y  into t h i s p o s i t i o n . From a consideration o f the possible degradation routes  - 121 -  presented i n figure 34  the introduction of a hydroxyl  group to give  the  alcohol derivative became the goal. This could not be achieved  d i r e c t l y because i t was  known that reaction  of guatambuine methiodide (86) with aqueous sodium hydroxide resulted i n elimination rather than s u b s t i t u t i o n , and s o l v o l y s i s with water f a i l e d to e f f e c t reaction o f the s i x membered r i n g .  I t has been shown, however, that  reaction o f the closely r e l a t e d gramine system under acetylation conditions 147 resulted i n a f a c i l e displacement of the t e r t i a r y nitrogen by  acetate.  Application of this reaction to the cleavage of guatambuine (25)  followed  by subsequent hydrolysis of the acetate presented an e n t i r e l y f e a s i b l e route to the formation  of the desired a-hydroxyethyl side chain.  Due to the l i m i t e d resources  of guatambuine, t h i s sequence and  the  subsequent dehydration were developed beforehand using N-methyltetrahydroe l l i p t i c i n e (26) and l-methyl-3-(a-acetoxyethyl)-9-benzylcarbazole  £23 )  (available from the synthesis presented i n sequence B, part III) as model systems f i g u r e  35).  The acetylation ring-opening  conditions were worked out  using compound 26 and the conditions necessary f o r the hydrolysis were developed using both compounds 26 and the carbazole  223.  The dehydration  reaction was  developed using  alcohol 222 .  140 Joule and  Djerassi  observed that when uleine was  anhydride i n pyridine, i t was t i a l l y formed  N (b)-acyl +  treated with a c e t i c  pyridine and not acetate that displaced the  ion.  This somewhat s u r p r i s i n g r e s u l t was  ini-  probably  unique to the uleine system because treatment of N-methyltetrahydroellipticine  - 122 -  Ac 0 2  /  NaOAc (26)  (121)  7  t-BuOH/aq.NaOM  /  O  (122)  OH  OAc  (223)  (222)  (123)  Figure 35. Acetate Substitution Approach Studied on Model Compounds, under the same conditions led to the formation o f the expected N-acetyl acetate 121.  Singlets i n the N.M.R. spectrum f o r the a c e t y l methyl  groups  were r e a d i l y discernable i n the region o f 62.1 as was the c h a r a c t e r i s t i c downfield p o s i t i o n of the C - l ' methylene s i n g l e t . The crude product mixture was dark i n colour which was t y p i c a l f o r acetylation reaction i n r e f l u x i n g p y r i d i n e .  A cleaner product mixture which  spontaneously c r y s t a l l i z e d i n benzene was obtained by reaction with acetic anhydride and sodium acetate (76% y i e l d ) .  The s p e c t r a l data f o r the acetate  formed under these conditions was i d e n t i c a l to that obtained by reaction i n pyridine. The N.M. R. spectrum o f 121 was considerably more complex than anticipated (Figure 36).  A mixture o f two separate conformers o f 121 (a,b) (about 2:1 by  Figure 36.  N.M.R. Spectrum of 1,4- Dimethyl-2-($-(N,N-methylacetylamino)ethyJ)-3-acetoxymethylcarbazole  (121).  - 124 -  integration) could be distinguished which most probably arose from a com152-4 bination of hindered i n t e r n a l r o t a t i o n about the amide linkage  and  s t e r i c crowding of the C-3 side chain by the bulky N-acetyl group.  These  two conformers influenced the environments of the C - l methyl (62.60,  2.52)  and N-methyl groups (63.05, 3.00) as well as the N-acetyl methyl (62.08,  i (121a)  1.98)  (121b)  and the C - l * methylene group (65.50, 5.46). peaks being present f o r each o f these groups.  This resulted i n twin sets of Singlet resonances occurred  at 62.88 and 2.03 f o r the C-4 methyl and the acetate methyl groups, respectively.  These groups were not influenced by the existence o f the molecule i n  two d i s t i n c t conformation. The IR. spectrum possessed absorptions at 1730 cm"* the  and 1635 enf* f o r  carbonyls of the acetate and N-acetyl groups, r e s p e c t i v e l y .  carbazole spectrum was obtained i n the UV. o f compound 121 .  A normal  The mass spectrum  possessed a parent peak at m/e = 366 with major fragmentations at m/e (M* - HOAc), 280 (M+ - C H NO), 263, 233, 222-1. 4  8  = 306  The parent peak at 366.1956  was within acceptable l i m i t s of the calculated value 366.1943 f o r the composition  C H26N203)« 22  - 125 -  The hydrolysis o f the acetate group of 121 to the corresponding alcohol 122 proved to be more d i f f i c u l t than expected.  The N-acetyl group was com-  p l e t e l y i n e r t to the b a s i c media whereas the acetate group was r e a d i l y d i s placed by alkoxide when either aqueous methanol or ethanol was used as s o l vent.  A two phase system o f t-butanol and aqueous sodium hydroxide was  found to e f f e c t hydrolysis without any competing n u c l e o p h i l i c s u b s t i t u t i o n by t-butoxide i o n . The reaction proceeded slowly at room temperature. 24 hr. unreacted s t a r t i n g material was detected.  After  When the temperature was  raised to 100°, the acetate group o f 121 was completely hydrolyzed a f t e r 1 h r . Only a s i n g l e peak at 1635 cm  -1  f o r the carbonyl of the N-acetyl group  was present i n the IR. spectrum which was consistent with the hydrolysis to the alcohol 122 .  The UV . spectrum remained e s s e n t i a l l y unchanged as expected.  The mass spectrum possessed a weak parent peak at m/e = 324 and a major fragmentation at m/e = 308 f o r loss o f 16 mass u n i t s .  The parent peak at m/e =  324.1814 i n the high r e s o l u t i o n mass spectrum was consistent with the composition C2 H24N O2. 0  2  The N.M.R. spectrum f o r 122 was too complex to properly analyze, again as a r e s u l t o f the presence o f conformers.  The complexity o f the spectrum may  also have been increased by the a b i l i t y o f the hydroxyl group to hydrogen bond to the amide carbonyl oxygen.  A p a i r of multiplets were discernable at 66.20  and 5.00 f o r the methylene hydrogens at C - l * .  The remaining s i n g l e t s f o r the  various methyl groups were s p l i t i n a complex manner which made t h e i r assignment ambiguous.  - 126 -  The hydrolysis of the 3-(pi -acetoxyethyl) carbazole derivative 223 the same two phase b a s i c media also proceeded 100° ( i s o l a t e d y i e l d 79%). duct alcohol 222  was  using  to completion after 1 hr. at  A l l the s p e c t r a l and a n a l y t i c a l data for the pro-  i d e n t i c a l to that obtained f o r the same compound 222  thesized i n sequence B, part III (page 225)* loss of the acetate carbonyl peak at 1720 Basic media was  Pertinent at t h i s point was  cm-1  synthe  i n the IR spectrum.  required f o r the subsequent dehydration o f the alcohol 222  to the o l e f i n 123;. because a c i d i c media ( f o r example 20% r^SO^) would r e s u l t i n concomitant h y d r o l y s i s o f the amide group.  The dehydration was  effected by  taking advantage of the known i n s t a b i l i t y of benzylic tosylates with respect to elimination.  By r e f l u x i n g the alcohol 222  sulphonyl chloride the o l e f i n 123  was  i n p y r i d i n e containing p-toluene,155  obtained i n 60% y i e l d .  The N.M.R. spectrum possessed a doublet of doublets at 65.78 ( J j , i 2  17 Hz, <J ,2' ,  **z) ^  =  2  b l e t s at 65.21  (Jii^'  =  ^  o r  1 1  H z  »  e  t  J  r  a  n  C-2*  s  2',2'  =  1 > 5  =  Hydrogen and a second doublet o f douH z  )  f  o  r  t h e  cis_ C-2» hydrogen.  The  corresponding doublet o f doublets f o r the C - l hydrogen was p a r t l y obscured by 1  the aromatic s i g n a l s .  The chemical s h i f t s for the c i s and trans C-2*  hydrogens  coincided closely with those found f o r o l e f i n 88 which also possesses the vinyl  side chain.  C-3  S i n g l e t s occurred at 65.72 and 2.60 r e s p e c t i v e l y f o r the  methylene and C-l methyl group. The UV . spectrum also bore a close resemblance to that obtained f o r o l e f i n 88 as expected.  The mass spectrum possessed a parent peak at m/e  = 297  and  - 127 -  almost no fragment peaks.  The parent peak at 297.1472 i n the high  resolution spectrum was within acceptable l i m i t s o f the calculated value f o r the composition C22H19N. Having worked out the conditions necessary f o r formation of the r i n g opened acetate, i t s hydrolysis, and dehydration of the a l c o h o l f o r the model compounds, attention was directed towards applying these reactions to guatambuine  (25).  The acetylation reaction was  anhydride i n p y r i d i n e .  i n i t i a l l y conducted using a c e t i c  Under these conditions the reaction proceeded as a n t i -  cipated to give the N-acetyl acetate 124.  (124) The presence of two conformers of 124  was again observed i n the N.M.R.  The N-acetyl and C - l methyl groups and the C - l ' methine hydrogen were influenced the most by the two separate conformations of the molecule.  Shoulders were  observed on the s i n g l e t peak f o r the N-methyl group (63.00) and on the doublet peak f o r the C - l  1  methyl group (61.64, J = 6 Hz).  Also, a multiplet instead of  the anticipated quartet was obtained for the C - l ' methine hydrogen (66.34). resonances for the C - l methyl (62.48, 2.40) 1.98)  The  and N-acetyl methyl groups (62.08,  were present as a d i s t i n c t l y separated p a i r of s i n g l e t s .  The s i n g l e t f o r  - 128 -  the acetate methyl group (62.08) was superimposed upon the singlet f o r Nacetyl methyl group of the major conformer of 124.  A single peak was thus  obtained for both groups. The mass spectrum possessed a weak parent peak at m/e  = 366 and a major  fragment a t m/e = 306 f o r loss of the elements o f a c e t i c a c i d .  Two  peaks  were present at 1710 and 1620 cm"*  i n the IR. f o r the carbonyls of the acetate  and N-acetyl groups respectively.  A carbazole UV. spectrum was obtained f o r  124 as expected. When sodium acetate i n a c e t i c anhydride was used f o r the acetylation r i n g opening, i t was observed that considerable quantities o f o l e f i n i c material was present i n the reaction mixture. ture f o r 10 hr. the o l e f i n 125  By conducting the reaction at r e f l u x tempera-  was obtained as the major product i n 64% y i e l d .  (125) The spectral data f o r 125 was consistent with i t s assigned structure. The occurrence of two d i s t i n c t conformers of 125 r e s u l t i n g from hindered r o t a t i o n about the N-methylamide linkage was again observed i n the N.M.R. spectrum (Figure 37).  Twin sets of peaks were observed f o r the N-methyl  (63.00, 2.92),  C - l methyl (62.58, 2.52) and N-acetyl methyl groups (62.10, 1.96), and the C-2' c i s oriented v i n y l proton (65.37, 5.34). 65.72 f o r the trans C-2' hydrogen.  A doublet of doublets was present at  This proton was f a r enough removed from the  - 129 -  - 130 -  amide group to be uninfluenced by i t . The mass spectrum possessed a parent peak at m/e = 306 and major fragments at 234, 220 and 204.  The parent peak i n the high resolution spectrum  at m/e = 306.1736 was within acceptable l i m i t s of the calculated value m/e = 306.1732 f o r C o 2 2 2 ° * H  N  A  s i n  2  g  l e  carbonyl absorption at itCO cm  i n the IR. spectrum f o r the N-acetyl group.  -1  was present  The UV. spectrum f o r compound 125  was s i m i l a r to that f o r the C-3 o l e f i n 88 i n that the absorption maxima consisted of two broad humps i n the region o f 280 mm. and 240 mm. It was apparent from these r e s u l t s that reaction with a c e t i c anhydride i n sodium acetate induced the p y r o l y t i c elimination o f the i n i t i a l l y formed acetate to occur.  Identical r e a c t i v i t y has been observed f o r a - a r y l a l k y l quaternary 143  ammonium s a l t s on p y r o l y s i s .  This property was of considerable value since  i t meant that guatambuine (25) could be transformed i n one step and i n good y i e l d to the desired C-3 v i n y l d e r i v a t i v e . In terms o f the development o f the degradation sequence f o r the i s o l a t i o n of the C - l methyl group e i t h e r the acetate ring-opening reaction o r the Hofmann reaction using sodium hydride i n dimethylformamide could be used.  The only advan-  tage that the Hofmann reaction offered was that the y i e l d was somewhat higher and the r e a c t i o n time was considerably shorter. C.  Oxidation Reactions This part of the discussion i s concerned with a p a r t i a l review o f the possible  a p p l i c a t i o n o f several d i f f e r e n t types o f oxidation-hydrolysis reactions towards the i s o l a t i o n o f the C - l methyl group of guatambuine (25). Very l i t t l e work was done i n t h i s area however, due to the successes achieved with the other approaches.  - 131 One area that was not studied at a l l was  the p o s s i b i l i t y of the d i r e c t  o x i d a t i v e cleavage o f the C - l methyl from the parent aromatic, compound o l i v a c i n e (16).  The p y r i d i n e nitrogen imparts a r e a c t i v i t y to the a - s u b s t i t u t e d C - l  methyl which i s not possessed by the C-5 methyl group.  I t should be p o s s i b l e  therefore through the choice of the proper o x i d i z i n g c o n d i t i o n s to s e l e c t i v e l y o x i d i z e the C - l methyl group p r e f e r e n t i a l l y to the C-5 methyl group. IV II 156-8 Both Ce and Ag o x i d i z i n g reagents have been shown to s e l e c t i v e l y o x i d i z e a s i n g l e a l k y l group of a p o l y a l k y l benzene system.  I t has been f u r t h e r  shown that i t was p o s s i b l e to conduct a stepwise o x i d a t i o n of that methyl group from the a l c o h o l to the benzoic a c i d d e r i v a t i v e . The c o n t r o l over the oxidat i o n process e x h i b i t e d by these compounds makes t h e i r a p p l i c a t i o n t o the oxidat i o n o f the C - l methyl o f o l i v a c i n e (16) e n t i r e l y p o s s i b l e .  Permanganate i o n  would a l s o be a s e l e c t i v e o x i d i z i n g agent since i t i s known that  2-methylpyridine  i s r e a d i l y oxidized to pyridine-2-carboxylic acid. 159 A number o f methods i n c l u d i n g the use o f permanganate i o n , manganese 160 II 161-2 dioxide, and Ag salts are a v a i l a b l e f o r the o x i d a t i o n of amines to the corresponding carbonyl compound.  They are g e n e r a l l y a p p l i c a b l e ' o n l y to primary  or secondary amines however s i n c e the intermediate imines which are formed are h y d r o l y t i c a l l y unstable with respect to the aldehydes or ketones. 163 phoxide  Dimethyl s u l -  has been shown t o be a r e l a t i v e l y good o x i d i z i n g agent f o r t e r t i a r y  amine hydrochlorides  and quaternary ammonium s a l t s .  However, the o x i d a t i o n o f  1-phenylethylamine s a l t s which were c l o s e l y r e l a t e d t o guatambuine (25) f a i l e d due to the i n s t a b i l i t y o f the k e t o n i c products formed i n the r e a c t i o n media. These types o f reactions were therefore not attempted on the guatambuine system. The majority o f reactions f o r o x i d i z i n g amines t o the carbonyl compounds  - 132 -  involve formation o f the imine o r imminium cation as the s t a r t i n g material 164 or as the reaction intermediate.  Mercuric acetate  has been u t i l i z e d  successfully f o r the oxidation of t e r t i a r y amines to t h e i r carbonyl counterparts.  The reaction involves p r i o r formation o f the iminium cation which  r e a d i l y hydrolyzes i n aqueous base.  The a p p l i c a t i o n o f t h i s reaction t o c y c l i c  systems such as guatambuine (25) however does not r e s u l t i n ketone formation, b a s i f i c a t i o n r e s u l t s i n the formation of the ot,B-enamine.  The subject of ena-  mine formation w i l l be returned t o s h o r t l y .  CH=N—  CH,I 3  +/ OH" CH^rM^—  CH=0  165 Ozonolysis  has been used to cleave the carbon-nitrogen double bond of  S c h i f f s bases and nitrones t o the corresponding aldehydes o r ketones.  Oxaziranes  and amides are generally formed also during the r e a c t i o n and i n comparable y i e l d s to the carbonyl component.  This would be a major drawback t o i t s application t o  small scale degradation work. A method which has been developed s p e c i f i c a l l y f o r the oxidation o f t e r t i a r y amines involves treatment of the amine o r the iminium s a l t with warm  166 buffered permanganate followed by rapid product removal.  I t would be necessary  - 133 however f o r the degradation of 142  to modify the i s o l a t i o n procedure  f o r the  r e a c t i o n to accommodate working on a small s c a l e with n o n - v o l i t i l e carbonyl compounds. Some p r e l i m i n a r y experiments  t h a t were t r i e d i n v o l v e d an attempt t o  hydrolyze the iminium s a l t of guatambuine 142 i n a c i d i c and b a s i c media. iminium s a l t 142  The  was a v a i l a b l e as a s t a b l e c r y s t a l l i n e s o l i d from the synthesis  presented i n sequence A, p a r t I I I . Ketone formation could not be detected under either conditions.  In aqueous base however, an intense yellow coloured s o l u t i o n  was produced and an amorphous yellow p r e c i p i t a t e developed.  The formation o f  t h i s intense c o l o u r a t i o n has been a s c r i b e d i n r e l a t e d systems t o the formation 136,167-8 of the a,8-enamine (anhydro base).  ' In comparison to the iminium c a t i o n  142 only minor s h i f t s i n the UV. spectrum were observed t o accompany the formation o f the enamine 126 (Figure 38).  (142)  (126)  Attempts to i s o l a t e the enamine 126 (anhydro base) by e x t r a c t i o n i n t o e t h e r failed.  This was not a t o t a l l y unexpected r e s u l t s i n c e they are known to be  unstable w i t h respect to the quaternary i o n . A ring-opening of the B-carboline r i n g system has been developed by Gupta 167-8 and Spenser  during t h e i r i n v e s t i g a t i o n o f the methylation o f the anhydro  bases o f Harman d e r i v a t i v e s (Figure 39).  Figure 38..  The UV. Spectrum of the iminium Cation (142) Enamine (126), and Reaction Product of 142 Treated with Dimethylsulphate i n Strong Base.  - 135 -  They found that the enamine o f Harmaline 128 could be i s o l a t e d a f t e r base treatment of the iminium s a l t 127.  When the enamine was subsequently treated  with methyl iodide, the quaternary ammonium iodide produced was hydroxide to give the r i n g opened ketone 129.  displaced by  A Hofmann elimination of the  amine function of the C-3 side chain led to the C-3 v i n y l product 130.  This  same process has also been shown to occur f o r the l-methyl-3,4 dihydroisoquino169 line systems by treatment with dimethylsulphate i n concentrated base. It i s e n t i r e l y f e a s i b l e that t h i s sequence of reactions could be c a r r i e d out on guatambuine (25).  The enamine 126 could be made a v a i l a b l e by mercuric 164  acetate oxidation of guatambuine (25).  The reaction with dimethyl sulphate  i n 20% aqueous sodium hydroxide has been conducted on a small s c a l e .  Within 5  min. a f t e r heating the reaction mixture at 100° the yellow colouration f o r the enamine 126 disappeared. available (Figure 38).  At the present time, however, only the UV data  was  The absorption curve f o r the reaction mixture was quite  reminiscent of both the imine (141) and the C-3 o l e f i n 88 (Figure 27) which gives some i n d i c a t i o n at least that the s t a r t i n g material 142 a l t e r s i n the reaction media and that perhaps the k e t o - o l e f i n (131) was produced. If the chemistry o f t h i s ring-opening reaction could be developed to the point where good y i e l d s were obtained, then i t could serve as a f e a s i b l e a l t e r n a t i v e to the Hofmann pr acetate s u b s t i t u t i o n approaches previously d i s cussed.  By a proper choice of reactions, the s e l e c t i v e i s o l a t i o n of the C - l  methyl, N-methyl and C-3 carbons of guatambuine could be achieved, i . e . borohydrive reduction of the ketone to the a l c o h o l , then ozonolysis of the double 170-171 bond followed by a Haloform reaction on the secondary a l c o h o l .  -136-  (129) OH-  (130)  Figure 39.  Ring Opening of the 3-Carboline Ring System by Methylation of the Anhydro Base.  - 137 -  EXPERIMENTAL - PART I I  For a description o f the general experimental information, see Experimental part 1. A l l T.L.C. plates were developed i n chloroform ( C H C I 3 ) or e t h y l acetate unless otherwise indicated, and the alumina f o r column chromatography was deactivated to A c t i v i t y III by the addition o f water ( 6 % ) .  N-Methyl-1,2,3,4-tetrahydroellipticine  (26)  A s o l u t i o n o f e l l i p t i c i n e (17) (500 mg., 2.03 x 10"^ mole) dissolved i n methanol (400 ml.) containing an excess o f methyl iodide (5 ml.) was s t i r r e d at room temperature f o r 24 hr.  The reaction mixture was then  concentrated to dryness, taken up i n methanol (200 ml.) and preadsorbed onto alumina (10 gm.), and applied to the top o f an alumina column (40 gm.). Traces o f unreacted s t a r t i n g material were eluted with ethyl acetate:methanol 5% and the desired methiodide product 93 was subsequently eluted with methanol: ammonium hydroxide 1%.  The methiodide 93 was obtained as a bright orange  c r y s t a l l i n e s o l i d (630 mg., 77%). -3 The methiodide 93 (500 mg. 1.24 x 10 mole) was dissolved i n aqueous  - 138 -  ethanol (250 ml.) and reacted with an excess o f sodium borohydride at room temperature  f o r 15 hr.  The reaction mixture was then concentrated, taken up  i n water (150 ml.) and extracted with chloroform ( 3 x 50 ml.).  The combined  chloroform fractions were dried over sodium sulphate and concentrated to give 26 as a pure colourless c r y s t a l l i n e s o l i d (300 mg., 91%) which turned yellow on prolonged standing.  The product 26 was s t a b i l i z e d by r e c r y s t a l l i z a t i o n  from methanol to give cream coloured needles, m.p. 217-220° ( l i t . , m.p. 215126 220°). UV.; 260 (4.49),  m  a  v  ( l o g e ) : 340 (3.49), 326 (3.62), 296 (4.25), 286 (sh)(4.02),  248 (4.60), 238 (4.74).  N.M.R. (F.T.):  8.20 (doublet, J = 7Hz,  C-10H), 7.84 (broad s i n g l e t , N-H), 3.76 ( s i n g l e t , C - l CH ), 3.00 ( t r i p l e t , J • 2  5Hz, C-4 CH ), 2  C-3 CH ), 2  N-CH ).  2.76 ( t r i p l e t , J = SHz, (partly obscured by a s i n g l e t at 3.70),  3.70 ( s i n g l e t , C - l l CH ), 2.56 ( s i n g l e t , C-5 CH ), 2.38 ( s i n g l e t , 3  3  Mass spectrum: M+; m/e = 264; main peaks:  3  221, 204-5.  Found: C, 81.67; H, 7.64; N, 10.30.  263 (base peak), 249, 233, Calc. f o r C H p N 18  2  2  : C,  81.78; H, 7.63; N, 10.60.  N-Methyl-l,2,3,4-tetrahydroellipticine methiodide  (95).  A s o l u t i o n o f 26 (265 mg. 1.00 x 10~ mole) i n methanol (25 ml.) con3  taining an excess o f methyl iodide was allowed t o stand at 0° f o r 24 hr.  The  colourless c r y s t a l s o f 95 that p r e c i p i t a t e d were c o l l e c t e d by suction f i l t r a t i o n , washed with methanol and dried under vacuum (396 mg. 98%), m.p.307 - 308° Found: C  19 23 2 H  N  I :  C  '  5 6  -  2 9  J  H  » 5  6 9  J  N  C, 55.91; H, 5.87; N, 6.50. Calc. f o r  » « 1' 6  9  - 139 -  1,2,3,4-Tetrahydroellipticine Ellipticine  (94).  (17) (100 mg., 4.06 x 10~* mole) was dissolved i n g l a c i a l  a c e t i c a c i d (20 ml.) and the solution was hydrogenated at room temperature and atmospheric pressure over Adams catalyst ( P t 0 , 85 mg.) f o r 12 h r . The 2  mixture was f i l t e r e d to remove the catalyst which was c a r e f u l l y washed with a d d i t i o n a l a c e t i c a c i d (10 ml.).  The combined f i l t r a t e s were concentrated,  taken up i n water (75 ml.), b a s i f i e d with 10% sodium hydroxide and the r e s u l t i n g white suspension was extracted with chloroform (3 x 30 ml.).  The  combined chloroform f r a c t i o n s were d r i e d over sodium sulphate, and concentrated to give 94 as colourless c r y s t a l s (95 yellow on standing.  UV.; X  m a x  :  mg.,93 % ) , which r a p i d l y turned  340, 324, 293, 284, 261, 250, 241 ( l o g (e)  values were not obtained, however, the r e l a t i v e 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 absorption maxima were v i r t u a l l y i d e n t i c a l t o those observed f o r N-methylt e t r a h y d r o e l l i p t i c i n e (17)). derivative f o r further  Compound 94 was converted to i t s N-acetyl  characterization.  The preparation o f the methiodide 95 o f compound 94 was c a r r i e d out i n an i d e n t i c a l manner to the reaction of (17).  N-methyltetrahydroellipticine  The melting point and microanalytical  with the values previously  data agreed s a t i s f a c t o r i l y  obtained.  137 N - A c e t y l - l ^ S ^ - t e t r a h y d r o e l l i p t i c i n e (96). Compound 94 (35 mg. 1.40 x 10~* mole) was dissolved i n a mixture o f a c e t i c anhydride (3 ml.) and pyridine  (3 ml.), and s t i r r e d at 70° f o r 3 h r .  - 140 The reaction mixture was  then concentrated, taken up i n water (75 ml.),  b a s i f i e d with 10% sodium hydroxide s o l u t i o n , and extracted with chloroform (3 x 30 ml.).  The combined chloroform f r a c t i o n s were washed with water,  d r i e d over sodium sulphate, and concentrated to give crude 96 (40 mg.,  97%)  Several r e c r y s t a l l i z a t i o n s from methanol afforded 96 as colourless p l a t e s , m.p.  262-265° ( l i t . m.p.  Calc. f o r C H N O : ig  20  2  272.5-273°).  C, 78.05; H, 6.89;  Olivacine methofluorosulphate  N,  C, 78.01; H, 7.02;  N,  9.30.  5.47.  (85) and i t s reduction to Guatambuine (25).  Olivacine (16) (20mg., 8.13 (25 ml.) was  Found:  x 10"  5  mole) dissolved i n a c e t o n i t r i l e  treated with methylfluorosulphate (30ul, 3 equiv.)  d i s t i l l e d ) at room temperature.  (freshly  A yellow p r e c i p i t a t e formed within minutes  and a f t e r 15 min. the reaction mixture was  concentrated to give the  fluorosulphate s a l t 85 as a yellow paste. The crude product 85 was suspended i n aqueous ethanol (100  ml.)  and reacted with an excess of sodium borohydride at room temperature, 15 hr.  for  The yellow colouration f o r the o l i v a c i n e s a l t disappeared almost  instantaneously on addition o f the reducing agent.  The reaction mixture  was then concentrated to a paste, taken up i n water (75 ml.), and extracted with chloroform (3 x 30 ml.).  The combined chloroform fractions were d r i e d  over sodium sulphate, and concentrated to give guatambuine (25) as a cream coloured s o l i d (20 mg., less needles, m.p.  93%).  R e c r y s t a l l i z a t i o n from methanol gave colour-  248° ( l i t . , m.p.  248-250°).  The UV. and N.M.R. and low  resolution mass spectra f o r the reaction product was previously obtained f o r guatambuine (25). Calc. f o r C. H N : lo Z\j I  264.1626.  Found:  consistent with that  High resolution mass spectrum:  264.1663.  - 141 Guatambuine methiodide  (86).  The methiodide 86 was prepared i n quantitative y i e l d by reaction o f 25 with an excess of methyl iodide at 0° f o r 24 hr.  Colourless needles  were obtained which were r e c r y s t a l l i z e d from methanol, m.p. 121-3 301°).  N.M.R. (DMS0-d6):  ( s i n g l e t , IH, C - l l H), 4.96  8.10  299° ( l i t . ,  (doublet, IH, J = 8Hz, C-10 H),  (quartet, 1 H, J = 6Hz, C - l H), 3.80  2.90  (broad  multiplet, 2 H, C-4CH ), 3.30  (multiplet, obscured by s i n g l e t absorption  at 3.18,  (two s i n g l e t s , 3 H, N(CH )2), 1.74  2  C-3 CH ),  3.18,  2  3H, J = 6Hz, C - l CH ). 19 23 2 H  N  I :  C  »  5 6  -  2 9  J  Olivacine methiodide  H  »  3  Found:  3  C  3.12  5  -  6 8  J  N  »  C, 56.34; H, 5.69; 6  N, 6.63.  299-  (doublet,  Calc. f o r  -80.  (85) and i t s Reduction to Guatambuine (25).  Olivacine (16) (15 mg.,  6.09  x 10"^ mole) dissolved i n ethanol  (25 ml.) was treated with an excess of methyl iodide and refluxed f o r 0.5 hr.  The c r y s t a l l i n g p r e c i p i t a t e was  c o l l e c t e d and the mother liquors  were concentrated, taken up i n a minimum amount of methanol and treated again with methyliodide.  The second crop o f c r y s t a l l i n e material was  c o l l e c t e d and the mother liquors were again concentrated and rereacted with methyl iodide.  By r e c y c l i n g the mother liquors i n t h i s fashion four*  times, an o v e r a l l 80% y i e l d of impure o l i v a c i n e methiodide was  (85) (18.4  mg.)  obtained. The crude product 85 (14 mg.,  3.49  x 10"^ mole) was  reduced with  sodium borohydride i n an i d e n t i c a l manner to the methofluorosulphate 85 described i n the previous experiment.  salt  The s p e c t r a l data was consistent  - 142 with that previously obtained f o r guatambuine (25).  Hofmann Degradation i)  o f (-)-Guatambuine Methiodide  (86).  Preparation o f  l-Methyl-2-(6-dimethylarainoethyl)-3-(a-ethoxyethyl) 121-3 carbazole (98) and C-3 o l e f i n 8 8 . (-)-guatambuine methiodide ( 8 6 ) (40.2 mg., 9.92 x 10"  5  mole) was  suspended i n a 10% sodium hydroxide i n 9 5 % ethanol s o l u t i o n (60 and refluxed f o r 2 . 5 hr.  The reaction mixture was  to a paste, d i l u t e d with water ( 7 5 ml.) 30 ml.).  then  ml.)  concentrated  and extracted with ether (3 x  The combined ether f r a c t i o n s were d r i e d over sodium sulphate,  and concentrated to give a l i g h t brown o i l containing a mixture o f compounds 98 and 8 8 (40 mg.,>100%) N.M.R.:  (See Figure 23).  Compound 98 was i s o l a t e d as a transparent o i l by successive preparative layer chromatography on alumina (1 mm., EtOAc) (24 mg., s i l i c a gel (19 mg., 60%).  An a n a l y t i c a l sample was obtained by d i s 25  t i l l a t i o n of 98 at 160-180° at 0.07-0.01 mm. ( a ) D  = -112°).  UV.; X  (Figure 23):  m a x  4.85  :  340, 326,  297, 287 (sh), 261, 250,  2.40  3  CH ), 3  3  H  N  0 :  3 2 4  2 . 4 9 ( s i n g l e t , 3 H,  2  1.20 ( t r i p l e t , 3 H, J = 7 H z , O - C H 2 - C H 3 ) .  21 28 2  N.M.R.  (singlet, 6 H, N ( C H ) ) , 1.53 (doublet, 3 H, J = 6 H z , C - l '  324; main peaks: C  240.  (quartet, 1 H, J = 6 H z , C-l'H), 3.42 (quartet, 2 H, 2  CH ),  25 = 0° (before: ( a ) n  J = 7 H z , O C H 2 C H 3 ) , 3.05 (multiplet, 2 H, C-3» CH ), C-l  7 5 % ) and  -  295, 278.  2 2 0 0  -  F  o  u  n  d  Mass spectrum:  High r e s o l u t i o n mass spectrum: :  324.2207.  Found:  M*, m/e =  Calc. f o r  C, 56.85; H, 6.60; N,  - 143 -  5.75.  Calc. f o r C H N I O : 22  31  C, 56.80; H, 6.67; N, 6.0.  2  ) Preparation o f l-Methyl-2-vinyl-3-("-dimethylaminoethyl)carbazole (97), and l-Methyl-2-(o-dimethylaminoethyl)-3-vinylcarbazole A)  (88).  Guatambuine methiodide (86) (20 mg., 4.93 x 10"^ mole) was suspended  i n t-butanol (12 ml.) containing potassium at r e f l u x for 1.5 hr.  t-butoxide  (30 mg.) and heated  The s o l u t i o n was then concentrated to a paste,  d i l u t e d with water (75 ml.) and extracted with ether ( 3 x 30 ml.). The combined ether fractions were dried over sodium sulphate, and concentrated to give a l i g h t brown o i l containing a mixture o f compound 97 and 88 (10 mg., 72%). J  r  2'  =  1  N.M.R. (Figure 24):  7 H z  » 2 ' 2' ~ J  2 H z  * " ' C  2  H  5.70 ( d i s t o r t e d doublet o f doublets, 1 H, t  r  a  n  (  s  8 8  ) overlapped with C-4  c i s (97) ) 5.68 (doublet o f doublets, 1 H, J 3 1 ^, C-4  H  = 11 Hz, ^ 1 ^ 4 1 = 2 Hz,  H c i s (97) ) , 5.23 (doublet o f doublets, 1 H, J 3 1  1  1  < 4  i  = 17 Hz,  J41  >  4  t=  2 Hz, C-4 trans (97) ), 5.32 (doublet o f doublets, 1 H, J j , , = 11 Hz, 1  2  J , , = 2 H z , C-2 H cis_ (88) ), 3.82 (quartet, J = 6 H z , C - l ' H), 2.52 1  2  2  ( s i n g l e t , C - I C H 3 ) , 2.40 ( s i n g l e t , N ( C H ) ) , 2.32 ( s i n g l e t , N ( C H ) ) , 1.44 3  (doublet, J = 6 Hz, C - l  1  2  3  CH ), 1.28 ( s i n g l e t , 3  2  0(CH ) ). 3  3  The major compound 97 was i s o l a t e d by preparative layer chromotography on alumina (1 mm., ethyl acetate) (4 mg., 30%).  UV. (figure 27); A  :  HI CLA.  340, 326, 297, 248. N.M.R. (Figure 25): 6.98 (doublet o f doublets, 1 H, J ,  > 4  5.65  4  3  8.08 (multiplet, 214, C-4, C-5 H),  , = 11 Hz, J  3  ^  4  , = 17 Hz, C-3'H),  (doublet o f doublets, 1 H, J , , = 11 Hz, J , , = 2 Hz, C-4' H c i s ) , 3  4  4  - 144 5.20  (doublet of doublets, 1 H, J i 4 i 3  trans), 3.75 2.25  = 17 Hz, ^ . ^ 4 1 = 2 Hz, C-4' H  #  (quartet, 1 H, J = 6Hz, C - l ' H), 2.50  ( s i n g l e t , 6 H, N ( C H ) ) , 3  1.39  2  ( s i n g l e t , 3 II, C - l CH ), 3  (doublet, 3 H, J = 6 Hz, C-1»CH ). 3  0  Mass spectrum:  M*, m/e  220-217, 205, 204. 278.1782. 0  B) 10~  High r e s o l u t i o n mass spectrum:  Found:  for C2 H N I: 25  = 278 (base peak); main peaks:  278.1780.  Found:  Calc. f o r C  1 9  H  N : 2  Calc.  6.68.  In a second experiment, guatambuine methiodide (86) (80 mg., 4  2 2  C, 56.92; H, 5.71; N, 6.63.  C, 57.27; N, 5.96; N,  2  263, 249, 235-3,  1.97 x  mole) was suspended i n t-butanol (30 ml.) containing potassium  t-butoxide (80 mg.)  and heated at r e f l u x f o r 2.5 h r .  of o l e f i n s 88 and 97 was obtained (40 mg., by column chromatography  72%).  on alumina (5 gm.).  On work-up a mixture  The mixture was separated  By e l u t i o n with benzene:  chloroform 1:1 o l e f i n 97 was obtained (20 mg.,  36%).  The s p e c t r a l data  f o r 97 was i d e n t i c a l to that obtained i n experiment A.  By increasing  the solvent p o l a r i t y to benzene:chloroform 70%, o l e f i n 88 was eluted to give a l i g h t brown o i l (broad hump), 295  (10 mg.,  18%).  1 H, C-l'H (obscured by CHC1 l '  2'  =  1 7  H z  1 H, J i ^ i 2  »  J  2 ' 2'  =  2  H z  = 11 Hz, J t  C-3'CH ), 2.50 2  Mass spectrum:  (Figure 27); X .: mov  (sh), 280, 267 (sh), 241.  (multiplet, 3 H, C-4, C-5 H, N-H),  J  UV.  2  » j 2  c  i  " ' 2  n  t r a n s  )»  5  -  = 2 Hz, C-2'H  +  m/e  = 278  8.00  (doublet of doublets,  (doublet o f doublets, 1 H, (doublet of doublets,  2 9  c i s ) , 3.10  ( s i n g l e t , 3 H, C - I C H 3 ) , 2.38 M,  N.M.R. (Figure 26):  approximately 7.2  peak) ), 5.68  325  (multiplet, 2 H,  ( s i n g l e t , 6 H,  N(CH ) ). 3  2  (base peak); main peaks; 263, 249, 234,  233,  - 145 -  220, 219-19, 204. High resolution mass spectrum: 278.1782. for C  H  Found:  278.1780.  Found:  Calc. f o r CigH22 2 N  C, 56.86; H, 6.27; N, 6.22. Calc.  N I: C,57.15; H, 5.99; N, 6.66, For the methiodide  280-282°. UV.;A  mQV  :  (loge) : 330(3.25), 296(sh)(3.98),  o f 88. m.p.  278(4.23), 240(4.41).  i ) Preparation o f Olefins 88 and 97. Guatambuine methiodide  (86) (15 mg., 3.70 x 10" mole) was sus5  pended i n a 40% aqueous sodium hydroxide s o l u t i o n (20 ml.) and heated at r e f l u x temperature f o r 3 hr.  The cooled reaction mixture was d i l u t e d  with water (50 ml.) and extracted with chloroform (3 x 30 ml.).  The  .combined chloroform extracts were washed with water, dried over sodium sulphate, and concentrated t o give a brown o i l (6mg., 60%).  The s p e c t r a l  data f o r the crude reaction mixture was i d e n t i c a l to that using potassium t-butoxide as the base i n t-butanol (reaction  )  ii).  Preparation of l-Methyl-2-(8^dimethylaminoethyl)-3-vinylcarbazole  (88).  -4 Guatambuine methiodide  (86) (76 mg., 2.42 x 10  mole) and sodium  hydride (50 mg.) i n dry dimethylformamide was heated at 100° f o r 2 min. a f t e r which time the excess hydride was destroyed by the careful addition of water.  The reaction mixture was then d i l u t e d with water (75 ml.) and  the r e s u l t a n t white suspension was extracted with ether (4 x 30 ml.).  The  combined ether layers were washed with water (3 x 30 ml.), dried over sodium sulphate and concentrated to give o l e f i n 88 as a colourless o i l which slowly s o l i d i f i e s to an amorphous white s o l i d under vacuum (37 mg., 70%).  UV. (Figure 27); X  m a v  : 325 (broad hump), 295 (sh), 280, 267 (sh),  - 146 -  (Figure 26) 241.  N.M.R.:  8.00 (multiplet, 3 H, C-4,  doublets, 1 H, J  J  2 f  C-5, N-H),  5.68 (doublet o f  , = 17 Hz, J , , = 2 Hz, C-2'H trans), 5.29 (doublet 2  of doublets, 1 H, J i » 2 '  =  1  1 H z  t  2  » 2',2' J  =  2 H z  »  C  ~ ' 2  H  c i s ) , 3.10 (multiplet  2 H, C-3'CH2), 2.50 ( s i n g l e t , 3 H, C - I C H 3 ) , 2.38 ( s i n g l e t , 6 H, N ( C H ) ) . 3  2  Hofmann Degradation of N-Methyltetrahydroellipticine methodide (95).  Preparation o f l,4-Dimethyl-2-vinyl-3-dimethylaminomethylcarbazole N-Methyltetrahydroellipticine methiodide  (102).  (95) (50 mg., 1.23 x 10~  4  mole)  was suspended i n t-butanol (25 ml.) containing potassium t-butoxide (40 mg.) and heated at r e f l u x f o r 4 hr.  The s o l u t i o n was concentrated to a paste,  d i l u t e d with water (75 ml.) and extracted with ether (3 x 30 ml.).  The com-  bined ether fractions were d r i e d over sodium sulphate, and concentrated to give 102 as a tan coloured o i l (33 mg., 96%). 250.  N.M.R.:  1 H, N-H),  UV.: A  ra  : 335, 322, 293,  8.25 (broad doublet, 1 H, J = 6 Hz, C-10 H), 8.02 (broad hump,  7.46-6.96 (multiplet, 5 H, 4 aromatic H, C-3'H), 5.64 (doublet o f  doublets, 1 H, J j , , = 11 Hz, J , , = 2 Hz, C-4'H, c i s ) , 5.20 (doublet o f 4  4  4  doublets, 1 H, J j ,,,."= 17 Hz, J , , = 2 Hz, C-4'H, t r a n s ) , 4  4  4  3.64 ( s i n g l e t ,  2 H, C-l'CH ), 2.90 ( s i n g l e t , 3 H,C-4 CH_), 2.50 ( s i n g l e t , 3 H, C-l C H ) , £  2.24  O  ( s i n g l e t , 6 H, N ( C H ) ) . 3  2  Mass spectrum:  «3  M*, m/e = 278; main peaks:  263, 249, 248, 234, 233 (base peak), 219, 218, 204. Calc. f o r C H „ N , : 1 Q  278.1782.  Found:  278.1780.  High r e s o l u t i o n spectrum:  - 147 l-Methyl-2-(3-dimethylaminoethyl)-3-ethylcarbazole O l e f i n 88 (2 mg., 7.19  x 10"  6  (89) from (88).  mole) dissolved i n methanol (3 ml.) was  hydrogenated at room temperature and atmospheric pressure over Adams catalyst (Pt0 , 5 mg.) f o r 0.5 hr. 2  The mixture was f i l t e r e d to remove the  catalyst which was c a r e f u l l y washed with a d d i t i o n a l methanol (10 ml.). The combined f i l t r a t e s were concentrated under vacuum to give 89 as a colourless oil  (2 mg.).  The s p e c t r a l data f o r the reaction product 89 was consistent  with that f o r the same product 89 obtained through reaction o f guatambuine methiodide  (86) with l i t h i u m aluminum hydride and through Hofmann degradation o f  Uleine (18) (to be subsequently described). CH ), 3  N.M.R. (FT):  2.64 ( s i n g l e t , 3H, C - l  2.54 (singlet, 6 H, N ( C H ) ) . 3  2  l-Methyl-2-(B-dimethylaminoethyl)-3-ethylcarbazole Guatambuine methiodide hydride (50 mg.)  (89) from (86).  (86) (50 mg., 1.23 x 10~* mole) and lithium aluminum  suspended i n dry tetrahydrofuran (25 ml.) was heated at r e f l u x  temperature f o r 4 hr.  The excess hydride reagent was destroyed by the successive  addition o f water (1 ml.), 10% aqueous sodium hydroxide  (2 ml.), and water (1 ml.).  The resultant p r e c i p i t a t e was removed by suction f i l t r a t i o n , and washed with methanol.  The combined f i l t r a t e s were concentrated, taken up i n ether (75  and washed with water (3 x 30 ml.).  The ether layer was then dried over sodium  sulphate, and concentrated to give 89 as a transparent o i l (30 mg., X  max  :  3 4 0  »  3 2 6  »  296,  2  8  5  ml.)  (sh), 260, 247, 239.  N.M.R.:  88%).  UV:  8.03 (doublet, 1 H, J =  7 Hz, C-5 H), 7.78 ( s i n g l e t , 1 H, C-4H), 3.2-2.6 (multiplet, 6 H, Ar-CH -CH , 2  CH2-CH -N(CH ) ), 2.52 ( s i n g l e t , 3 H, C - l CH ), 2  3  2  3  2.46 ( s i n g l e t , 6 H,  3  - 148 -  N ( C H ) ) , 1.36 ( t r i p l e t , 3 H, J = 7 Hz, Ar-CH -CH ). 3  2  2  m/e = 280 (base peak); main peaks: r e s o l u t i o n mass spectrum:  3  Mass spectrum:  M, +  266, 236, 222, 207, 206, 204. High  Calc. f o r I Q 2 4 2 c  H  N  :  280.1938.  Found:  280.1975  Compound 89 obtained from guatambuine methiodide (86) was further characterized as i t s methiodide s a l t 113 which was formed i n quantitative y i e l d by reaction with an excess o f methyl iodide i n methanol at 0°C f o r 121-3 24 h r . m.p. 291-293° ( l i t . , m.p. 287-8 ) . N, 6.42.  Calc. f o r C  2fJ  H N I: 27  2  Found:  C, 56.72; H, 6.40;  C, 56.88; H, 6.44; N, 6.63. UV.; A  m a x  340 (3.32), 325 (3.40), 297 (4.12), 285 (3.88), 260 (4.09), 239 (4.52).  (loge)  - 149 -  l-Methyl-2-(B-dimethylaminoethyl)-3-ethylcarbazole (89) from the Hofmann Degradation o f Uleine Methiodide (87). 132 Uleine methiodide (87) was prepared according to Schmutz et_ a l .  ixo and a modification o f t h e i r procedure Uleine methiodide (87) (60 mg.,  was used to degrade t h i s compound. 1.47 x 10~  4  mole) was suspended i n a  s o l u t i o n of 5% sodium hydroxide i n 95% ethanol (25 ml.) and refluxed f o r 2 hr.  The reaction mixture was then concentrated to a paste, d i l u t e d with  water (75 ml.) and extracted with ether (3 x 30 ml.).  The combined ether  f r a c t i o n s were dried over sodium sulphate, and concentrated to give a brown oil.  Polar contaminants were removed by column chromatography  on alumina  (5 gm.). E l u t i o n with chloroform y i e l d e d 89 as a l i g h t brown o i l (31 mg. 75%) UV; X : 337, 324, 296, 285 (sh), 260, 245, 237. N.M.R.: 7.74 ( s i n g l e t , max 1H, C-4H), 3.2-2.7 (multiplet, 6 H, Ar-CH CH , -CH -CH -N(CH )2)» 2.48 2  3H, C - l CH ), 2.36  ( s i n g l e t , 6 H, N ( C H ) ) ,  3  Ar-CH CH ). 2  3  Mass spectrum:  3  236, 222, 207, 206, 204. C  19 24 2 H  N  :  2 8 0  «  1 9 3 8  -  M+, m/e  2  3  2  1.30  2  ( t r i p l e t , 3H, J = 7 Hz,  = 280 (base peak); main peaks:  High r e s o l u t i o n mass spectrum:  Found:  (singlet  3  266,  calc. for  280.1915.  Compound 89 obtained from uleine methiodide (87) was further charact e r i z e d as i t s methiodide s a l t 113, which was  formed i n quantitative y i e l d  by reaction with an excess of methyl iodide i n methanol at 0°C f o r 24 hr., 121-3 m.p.  285° ( l i t . m.p.  Calc. f o r C  2()  H N I: 27  2  287-8°).  Found:  C, 56.88; H, 6.44;  N,  C, 56.89; N, 6.47; 6.63.  N,  6.70.  - 150 -  l,3,4-Trimethyl-2-(8-dimethylaminoethyl)carbazole N-Methyltetrahydroellipticine methiodide  CI07) .  -4 (95) (50 mg., 1.23 x 10 mole)  and lithium aluminum hydride (50 mg.) suspended i n dry tetrahydrofuran (25 ml.) was heated at r e f l u x temperature f o r 4 hr.  The excess hydride reagent was  destroyed by the successive addition o f water (1 ml.), 10% aqueous sodium hydroxide  (3 ml.), and water (1 ml.).  The resultant p r e c i p i t a t e was removed  by suction f i l t r a t i o n , and washed with methanol.  The combined f i l t r a t e s  were concentrated, taken up i n ether (75 ml.) and washed with water (3 x 30 ml.).  The ether layer was then dried over sodium sulphate, and concentrated  to give 107 as an opaque s o l i d (30 mg., 88%).  Ifecrystallization from  chloroform yielded 107 as colourless c r y s t a l s , m.p. 302-304°.  UV.; \  i a x  '• 340,  326,  297, 284 (sh), 262, 241. N.M.R  : 8.18 (doublet, 1 H, J = 7Hz, C-5 H),  7.90  (broad hump, 1 H, N-H), 3.04 (multiplet, 4 H, C H 2 - C H 2 - N ( C H ) ) , 2.75 3  2  ( s i n g l e t , 3 H, C-4 CH ), 2.42 (singlet, 3 H, C - l CH ), 2.36 ( s i n g l e t , 3 H, 3  C-3 CH ), 3  3  2.32 (singlet, 6 H, N ( C H ) ) . 3  2  Mass spectrum : M*, m/e = 280 (base  peak); main peaks : 266, 249, 236, 222, 207, 206, 204, 191. High resolution mass spectrum : Calc. f o r C  i g  H  2 4  N  2  : 280.1938. F o u n d : 280.1903  Compound 107 obtained from N-methyltetrahydroellipticine methiodide (95) was  f u r t h e r characterized as i t s methiodide  s a l t 108, which was formed i n  quantitative y i e l d by reaction with an excess o f methyl iodide i n methanol at 0° f o r 24 hr. C  20 27 2 H  N  I :  C  '  Found : C, 56.93; H, 6.36; N, 6.36. Calc. f o r 5 6  -  8 8  ; H» 6  4  4  i  N  »  6  *  6 3  *  -151  l,3 4-Trimethyl-2-vinylcarbazole  -  (109).  )  Methiodide 108 (25 mg., 5.93 x 10~ mole) was suspended i n t-butanol 5  (20 ml.) containing potassium t-butoxide (30 mg.) and heated at r e f l u x f o r 2.5 hr.  The reaction mixture was concentrated to a paste, d i l u t e d with water  (75 ml.) and extracted with ether (3 x 30 ml.).  The combined ether fractions,  were dried over sodium sulphate, and concentrated to give a dark coloured oil.  Polar contaminants  were removed from the crude product mixture by  column chromatography on alumina.  By e l u t i o n with chloroform the desired  o l e f i n 109. was obtained as a tan coloured o i l (12 mg., 85%). 322,  = 18 Hz, J i , 4 i  3  3  1 H, J , , = 12 Hz, J I 3  2.52  : 335,  m a x  293, 250. N.M.R. : 8.26 (doublet, 1 H, J = 7 Hz, C-5H), 6.94 (doublet o f  doublets, 1 H, J i , 4 i  J31  UV.; *  4  4  41 = 18 Hz, J I 4  > 4  I  J 4  I  = 12 Hz, C-3'H), 5.68 (doublet o f doublets  = 2 Hz, C-4«H, c i s ) , 5.24 (doublet o f doublets, 1 H  ?  = 2 Hz, G-4'H, trans), 2.84 ( s i n g l e t , 3 H, C-4 CH ), 3  ( s i n g l e t , 3 H, C - l CH ), 2.40 ( s i n g l e t , 3 H, C-3 CH ) . 3  3  Mass spectrum:  M , m/e = 235; main peaks : 220, 205. High r e s o l u t i o n spectrum : C a l c . f o r +  C H N : 1 7  1 7  235.1360. Found : 235.1325. :  -  -  -  -  -  -  '.'  1.  l-Methyl-2-vinyl-5-ethylcarbazole (91). A.  Methiodide 90 (25 mg., 5.93 x 10" mole) was suspended i n t-butanol (IS 5  ml.) containing potassium t-butoxide (40 mg.) and heated at r e f l u x f o r 2.5 h r . The reaction mixture was then concentrated to a paste, d i l u t e d with water (75 mi.) and extracted with ether (3 x 30 ml.).  The combined ether f r a c t i o n s  were dried over sodium sulphate, and concentrated t o give a dark coloured o i l (17 mg.).  Column chromatography on alumina  (3 gm.) (elution with  chloroform)  - 152 -  removed a portion of the dark coloured contaminants  (14 mg.).  duct was further p u r i f i e d by preparative layer chromatography Bz:CHCl UV.:  3  1:1).  1 H, ^ 1 ^ 4 1 = 12 Hz, J 4 1 41 = 2 Hz, C-4'H J 3 1 41 = 18 Hz, ^ ^ 4 1 M*, m/e  Calc. f o r C H N : 17  B_.  on alumina (1  Compound 91 was obtained as a dark brown o i l (about 8 mg.,  Indicated the presence of contaminants.  spectrum:  The crude pro-  17  = 2 Hz, C-4'H  Methiodide 90 (40 mg.,  220, 205.  Found:  9.50  c i s ) , 5.29  trans), 2.54  = 235; main peaks: 235.1360.  N.M.R.:  x 10~  5.65  mm.,  57%).  (doublet of doublets,  (doublet of doublets, 1 H,  (singlet, 3 H, C - l CH ).  Mass  3  High r e s o l u t i o n mass spectrum:  235.1384. 5  mole) was dissolved i n dime thy lformamide  (5 ml.) containing sodium hydride (10 mg.)  and heated at 100° f o r 2 min.  The  reaction mixture was then d i l u t e d c a r e f u l l y with water (70 ml.) and extracted with ether (3 x 30 ml.). The combined ether f r a c t i o n s were washed with water (3 x 30 ml.), dried over sodium hydride, and concentrated to give 91 as an opaque oil  (9 mg.,  40%).  The spectral data f o r compound 91 was described i n experiment A .  (N-* C methyl) Guatambuine methiodide (86). 4  Guatambuine (5 mg., reacted with C-methyl 14  temperature.  1.89 x 10"^ mole) dissolved i n methanol iodide (1 ml.,  /.ox  (2 ml.)  10 DPM/ml.) f o r 15 hr. at room ?  The solvent was removed and the crude methiodide 86 was  with unlabelled 86 (50 mg.) ( / . t f x lO^DPM/m mole).  was  and r e c r y s t a l l i z e d from methanol  (36 mg.),  diluted m.p.  299°  - 153 -  (N-* C methyl) N-methyltetrahydroellipticine methiodide (95). 4  N-methyltetrahydroellipticine (5 mg., 1.89 x 10" mole) dissolved i n 5  methanol (2 ml.) was reacted with C-methyliodide (1 ml., 14  for 15 hr. at room temperature.  1  ,Q x 10 DPM/ml.) ?  The solvent was removed and the crude  methiodide 95 was d i l u t e d with unlabelled 95 (50 mg.) and r e c r y s t a l l i z e d from methanol (47 mg.), ( 3 .8? x 10 DPM/m mole).  Lithium Aluminum Hydride Ring-Opening o f (86), Hofmann Degradation o f (90), and Isolation o f the N-Methyl Group o f (25). Methiodide 86 (36 mg., . 4«.x 10*DPM) was suspended i n tetrahydrofuran (25 ml.) and reacted with lithium aluminum hydride (50 mg.) at r e f l u x temperature f o r 4 hr.  The product 89 was i s o l a t e d and converted t o i t s methiodide  90 (19 mg., 51%) as previously described. The methiodide 90 (19 mg., 4.51 x 1 0  - 5  mole) was reacted with potassium  t-butoxide (30 mg.) i n t-butanol (20 ml.) at r e f l u x temperature f o r 2.5 h r . A slow stream of nitrogen gas was passed through the solution and the e f f l u e n t gas containing the trimethylamine was passed through methyl iodide s o l u t i o n . The methyl iodide solution was concentrated and the (N-* C methyl) tetramethyl4  5  ammonium iodide was r e c r y s t a l l i z e d from methanol (2.5 mg.,2 .fe1x 10 DPM) PCS c o c k t a i l ) .  Isolation E f f i c i e n c y : 5? %.  - 154 -  Lithium Aluminum Hydride Ring-Opening of (95), Hofmann Degradation of (108), and Isolation of the N-Methyl Group of (26). Methiodide 95 (47 mg.,  «9 , / a 10 DPM)  was suspended i n tetrahydrofuran  (25 ml.) and reacted with lithium aluminum hydride (50 mg.) ture f o r 4 hr. 108 (25 mg.,  at r e f l u x  tempera-  The product 107 was i s o l a t e d and converted to i t s methiodide  51%) as previously described.  The methiodide 108 (25 mg., t-butoxide (30 mg.)  5.93 x 10"  5  mole) was reacted with potassium  i n t-butanol (20 ml.) at r e f l u x temperature f o r 2.5 hr.  A slow stream o f nitrogen gas was passed through the solution and the e f f l u e n t gas containing the trimethylamine was passed through methyl iodide solution This methyl iodide s o l u t i o n was concentrated and the (N-* C methyl)tetramethyl4  ammonium iodide was r e c r y s t a l l i z e d from methanol cocktail).  Isolation E f f i c i e n c y :  ( 3 mg.  Z.io x 10 DPM)  (PCS  17. %.  Ozonolysis of (102), I s o l a t i o n of the C-3 Methylene Group of (26). 146 The procedure followed was e s s e n t i a l l y that by Battersby and Harper. Ozonized oxygen (200 bubbles per minute) was passed through a solution of o l e f i n 102 (44 mg.,  1.58 x 10"  4  mole) i n methylene  chloride (10 ml.) at -78°  and then through a c i d i f i e d potassium iodide s o l u t i o n .  The gas was passed f o r two  times the i n t e r v a l required to produce the f i r s t colour of iodine i n the potassium iodide s o l u t i o n (3-4 min.).  After the methylene  chloride had been evaporated  the ozonide was decomposed by being heated under r e f l u x f o r 0.5 hr. with water (20 ml.), zinc dust (200 mg.),  and s i l v e r n i t r a t e (10 mg.).  Half the water  was  - 155 then d i s t i l l e d at atmospheric pressure into a solution of dimedone (300 mg.) in water (20 ml.), and ethanol (8 ml.), water (10 ml.) was added to the d i s t i l l i n g f l a s k and the d i s t i l l a t i o n to h a l f volume was repeated into the same dimedone s o l u t i o n .  M i c r o c r y s t a l l i n e needles of the dimedone derivative began  to separate out during d i s t i l l a t i o n . c o l l e c t e d by centrifugation  A f t e r 15 h r . a t 0° the c r y s t a l s were  (27 mg., 87%) and r e c r y s t a l l i z e d from 50% aqueous  ethanol to a f f o r t the pure derivative,  \ ;  :  Ozonolysis o f (88), Isolation of the C - l Methyl Group of (25). O l e f i n 88 (50 mg., to compound 102.  1.18 x 10~ mole) was ozonolyzed i n an i d e n t i c a l manner 4  M i c r o c r y s t a l l i n e needles of the dimedone derivative began to  separate out during d i s t i l l a t i o n . by centrifugation  After 15 hr. at 0° the c r y s t a l s were c o l l e c t e d  (20 mg., 60%) and r e c r y s t a l l i z e d from 50% aqueous ethanol to  afford the pure derivative.,  \  - 156 -  l-Methyl-2-ethyl-3-(g-dimethylaminoethyl)carbazole O l e f i n 97 (18 mg.,  6.47  (103).  x 10"*"' mole) dissolved i n methanol (10 ml.)  was  hydrogenated at room temperature and atmospheric pressure over Adams c a t a l y s t (Pt0 , 10 mg.) 2  which was  f o r 0.5 hr.  The mixture was  f i l t e r e d to remove the c a t a l y s t  c a r e f u l l y washed with additional methanol (10 ml.).  The combined  f i l t r a t e s were concentrated under vacuum to give 103 as a colourless f i l m (18 mg.,  99%).  UV.;  \  m a x  : 337, 324, 297, 286  3.62  (quartet, 1 H, J = 6 Hz, C-1'H), 2.94  2.50  (singlet, 3 H, C - l CH ), 3  3 H, J = 6 Hz, C-1'CH ), 1.20 3  M*", m/e  2.38  (sh), 261, 248, 239.  (quartet, 2 H, J = 7 Hz,  ( s i n g l e t , 6 H, N ( C H ) ) , 1.34 3  2  ( t r i p l e t , 3 H, J = 7 Hz, C-4'CH ). 3  = 280; main peaks : 265, 236, 235  resolution mass spectrum : Calc. f o r C  i g  H  N.M.R. : C-3»CH ),  (doublet, Mass spectrum  (base peak), 220, 207-204. 2 4  N  2  2  High  : 280.1938. Found: 280.1923.  Attempts were made to further characterize compound 103 as i t s methiodide  113,  (see following experiment).  l-Methyl-2-ethyl-3-(a-trimethylammonioethyl)carbazole The hydrogenated compound 103 chloroform methanol 1:1 was for 12 hr. (27 mg.)  (18 mg.,  6.4 x 10"  iodide (111). 5  mole) dissolved i n  reacted with an excess of methyl iodide at  The reaction mixture was  0°  concentrated to give a granular o i l  consisting of three products.  The desired methiodide  111 was  obtained  i n p a r t i a l l y pure form by washing the crude product mixture with chloroform (2 x 10 ml.).  The p a r t u a l l y p u r i f i e d methiodide i l l  s o l i d (15 mg.,  55%).  No p h y s i c a l data was  was obtained as an opaque  obtained f o r this compound.  - 157 -  The chloroform washings were combined and concentrated to a yellow  oil.  The major non-polar component 113 was i s o l a t e d (12 mg.) by e l u t i o n of an alumina column (5 gm.) with chloroform.  N.M.R. (60 MHz) : 3.72 (quartet,  1 H, J = 6 Hz, C-l'H), 3.26 ( s i n g l e t , 3 H, 0CH ), 2.50 ( s i n g l e t , 3 H, C - l CH ), 3  1.52  3  (doublet, 3 H, J = 6 Hz, C-1'CH_).  l-Methyl-2-ethyl-3-vinylcarbazole (112) . Methiodide 111  (15 mg., 3.60 x 10"  5  mole) and sodium hydride (15 mg.)  in dry dimethylformamide was heated at 100° f o r 2 min. a f t e r which time the excess hydride was destroyed by the c a r e f u l addition o f water.  The reaction  mixture was then d i l u t e d with water (75 ml.) and the resultant white was extracted with ether (4 x 30 ml.).  suspension  The combined ether layers were washed  with water (3 x 30 ml.), dried over sodium sulphate, and concentrated to give o l e f i n 112  as a brown o i l (7 mg.).  tography on alumina. •brown o i l ( 4 mg.). 5.69(J J  2',2'  m/e C  , 1  l j 2  = 2 H z  The crude product was p u r i f i e d by chroma-  By e l u t i o n with benzene 112 UV.; X ^ :  3 2 5 <  2  was obtained as a l i g h t  95(sh), 280, 267{sh), 241. N.M.R. :  = 17 Hz, J » » = 2Hz., C-2' H trans>, 5.28( J-j, ,= 11 Hz, 2  » " ' C  2  H  c i s  )'  > 2  2  -  4 6  2  (  s i n  gl t, e  C-l CH ). Mass spectrum: M , +  3  = 235; main peaks : 220, 204. High resolution mass spectrum: Calc. f o r H N : 235.1360. Found : 235.1380. 17  - 158 -  S a l s o l i d i n e methiodide  (115). 144  A mixture o f s a l s o l i d i n e iodide (28 gm.),  (100)  (3.0 gm.,  and aqueous sodium carbonate  under gentle r e f l u x overnight.  1.71  (6 gm.  x 10"  mole), methyl  2  i n 40 ml.) was warmed  The reaction mixture was  then concentrated  to dryness and taken up i n water (50 ml.) whereupon tan coloured c r y s t a l s immediately p r e c i p i t a t e d (2.3 gm.).  The aqueous mixture was b a s i f i e d with  5% sodium hydroxide and extracted with chloroform (3 x 30 ml.),  The  com-:  bined chloroform fractions were d r i e d over sodium sulphate, and concentrated to an amber coloured foam.  By r e c r y s t a l l i z a t i o n from methanol-ether  crystals of 115 were obtained (2.20 gm., Found: 6.07;  N,  C, 46.13; H, 6.10;  N, 3.59.  35%), m.p.  205° ( l i t . , m.p.  beige 229-235*4,)  Calc. f o r C H 0 N I : C, 46.41; H, 1 4  2 2  2  3.87,  l-(a-dimethylaminoethyl)-2-vinyl-4,5-dimethoxybenzene S a l s o l i d i n e methiodide  (115)  (50 mg.,  1.38  (116), ,  x 10"  4  mole) was  i n a 10% sodium hydroxide i n 95% ethanol s o l u t i o n (30 ml.) f o r 1.5 hr.  The reaction mixture was  suspended  and refluxed  then concentrated to a paste, d i l u t e d  with water (75 ml.) and extracted with ether (3 x 30 ml.).  The combined  ether f r a c t i o n s were d r i e d oyer sodium sulphate, and concentrated to give a l i g h t brown o i l (20 mg,,  61%).  UV.;  : 310  7.23  (doublet o f doublets, 1 H, J J I ^ I  6.94  (2 s i n g l e t s , 1 H each, C-3, C-6 H), 5.50  J 3 1 41 = 16 Hz, J 4 i 4 i j  = 16 Hz, J ,  = 2 Hz, C-4'H), 5.16  J j , , «= 10 Hz, J , , - 2 Hz, C-4'M), 3.96 4  4  4  (sh), 290, 262, 3  < 4  N.M.R,:  , = 10 Hz, C-3'H)  f  7,02,  (doublet of doublets, 1 H,  (doublet of doublets, 1 H, ( s i n g l e t , 6 H, 0CH ), 3.50 (quartet, 3  - 159 -  1 H, J = 7 Hz, C-l'H), 2.33 ( s i n g l e t , 6 H, N ( C H ) ) , 3  2  1-35 (doublet, 3 H,  J = 6 Hz, C - l * CH ), 3  Reduction o f (116) to 1-(a-dimethylaminoethyl) -2-ethyl-4,5-dimethoxybenzene and formation of methiodide 117 . O l e f i n 116 (64 mg., 2.72 x 10" mole) dissolved i n methanol 4  (10 ml.) was  hydrogenated at room temperature and atmospheric pressure over Adams c a t a l y s t (Pt02, 10 mg.) f o r 0.5 h r .  The mixture was f i l t e r e d to remove the c a t a l y s t  which was c a r e f u l l y washed with additional methanol  (10 ml.).  The combined  f i l t r a t e s were concentrated under vacuum to give 117 as a colourless o i l (64 mg. 98%).  UV; \  : 280.  v  N.M.R.: 7.10 ( s i n g l e t , I I I , C-6H),6.64  (singlet, 1 H ,  C-3 H), 3.90 ( s p l i t s i n g l e t , 6 H, 0CH ), 3.43 (quartet, 1 H, J = 7 Hz, C-l'H), 3  2.65 (quartet, 2 H, J = 7 Hz, Ar-CH CH ), 2.33 (singlet, 6 H, N ( C H ) ) , 2  (doublet, 3 H, J = 6 Hz, C-1'CH  3  3  3  2  (overlapped by t r i p l e t at 1.13) ), 1.13  1.40 (tri-  p l e t , 3 H , J = 6 Hz, ArO^CHj (overlapped by doublet at 1.40) ) . The hydrogenated compound (64 ml.) was converted to i t s corresponding methiodide 117 by reaction with an excess of methyl iodide i n methanol. Recrys t a l l i z a t i o n from methanol, m.p. 151-153°.  Found : C, 47.97; H, 6.20; N, 3.29.  Calc. f o r C H N 0 I : C, 47.59; H, 6.88; N, 3.70 (analysis u n s a t i s f a c t o r y ) . 1 5  2 6  2  Attempted formation of 1-V inyl-2-ethyl-4,5-dimethoxybenzene ( H 8 ) . The methiodide  (30 mg., 8.24  x 10" ^ mole) was refluxed i n d i e t h y l  ketone (10 ml.) f o r 2 h r . The solvent was then removed under vacuum to give  - 160 -  an amber o i l .  The crude product was d i s t i l l e d at 120-180° at 0.05  mm.  A yellow f i l m was obtained which was dissolved i n ether (25 ml.) and washed with 10% sodium thiosulphate solution to give a colourless f i l m o f product ( y i e l d undetermined).  From separate experiments the N.M.R. spectrum  for the crude and p u r i f i e d reaction product showed that the s t a r t i n g  material  had t o t a l l y decomposed.  Thermal Pyrolysis of Guatambuine Methiodide (86). Guatambuine methiodide (about 10 mg.) hr. at 0.05 mm. and 200°C.  was heated under vacuum f o r 12  A small y i e l d o f yellowish coloured  sublimed during t h i s period, m.p. 200-210°, to that obtained f o r guatambuine (25) .  crystals  The N.M.R. spectrum was  identical  - 161 -  l-Methyl-3-(ot-hydroxyethyl)-9-benzylcarbazole ( 2 2 2 ) . Carbazole acetate 223  (100 mg.,  2,80  x 10~  4  mole) was  between a two phase medium consisting of t-butanol (5 ml.) sodium hydroxide hr.  partitioned and 20% aqueous  (5 ml.), and heated at 100° with vigorous s t i r r i n g f o r 1  The reaction mixture was  then concentrated to remove the t-butanol,  d i l u t e d with water (75 ml.) and extracted with ether (3 x 30 ml.).  The  combined ether layers were washed with water, d r i e d over sodium sulphate and concentrated to give a colourless foam.  The crude product was  by column chromatography on alumina (10 gm.). carbazole acetate  purified  Trace amounts of unreacted  and other non p o l a r contaminants were removed by  e l u t i o n with benzene.  By subsequent e l u t i o n with chloroform and concen-  t r a t i o n , the desired alcohol 222 was obtained as a colourless foam (70 79%).  UV.;  \ „  : 342, 327, 293, 287, 283, 263, 237,  1 H, J = 2 Hz, C-5H), 7.98 9 H, aromatic), 5.66 ArCH(0H)CH ), 2.54 3  (multiplet, 1 H, C-4H), 7.2,  ( s i n g l e t , 2 H, N-CH -C H ), 5.00 2  ( s i n g l e t , 3 H, C-l CH ), 3  (doublet, 3 H, ArCH(OH)CH ). 3  main peaks : 300, 297.  N.M.R. : 8.08  6  5  1,97  6.95  (doublet  (2 m u l t i p l e t s ,  (quartet, 1 H, J = 6 Hz  ( s i n g l e t , 1 H, OH),  Mass spectrum ; M , m/e  mg.,  = 315  1.54  (base peak);  High r e s o l u t i o n mass spectrum : c a l c .  for  C22H21NO :  315.1623. F o u n d : 315.1632.  l-Methyl-3-vinyl-9-benzylcarbazole ft23 )• Carbazole alcohol 222  (75 mg.,  2,38  x 10"  4  mole) and toluenesulphonyl  chloride (58 mg.,.1.3 equiv.) i n pyridine (8 ml.) was heated at r e f l u x temperature  f o r 6 hr.  The pyridine was  then removed under vacuum and the  - 162 -  residue was dissolved i n chloroform (75 ml.) and washed successively with d i l u t e hydrochloric acid, 5% sodium hydroxide and water. layer was oil  dried over sodium sulphate, and concentrated to give a dark coloured  (54 mg.).  of alumina  The crude product was p u r i f i e d by f i l t r a t i o n through a column  (3 gm.).  By e l u t i o n with chloroform the o l e f i n 123  as an amber coloured o i l (42 mg., 60%). 241.  The chloroform  UV.; ^  was obtained  : 350, 336, 279, 270 (sh),  N.M.R.: 8.14 (multiplet, 1 H, C-5H), 8.05 (multiplet ( s p l i t s i n g l e t ) ,  1 H, C-4 H), 6.90 (doublet of doublets, C-l'H, obscured by aromatic s i g n a l s ) , 5.78  (doublet of doublets, 1 H,  2  , = 17 Hz, J , , = 1.5 Hz, C-2'H 2  (partly obscured by 5.72  s i n g l e t ) , 5.72  of doublets, 1 H, J  = 11 Hz, J ,  3 H, C - l CH ). 3  V t 2 1  2  > 2  2  trans  ( s i n g l e t , 2 H, Ar-CH -N), 5.21 (doublet 2  , = 1.5 Hz, C-2'H  c i s ) , 2.60 ( s i n g l e t ,  - 163 -  (12l)  1,4-Dimethyl-2-(B-(N,N-methylacetylamino)ethyl)-3-acetoxymethylcarbazole  N-Methyltetrahydroellipticine (26) (50 mg. 1.89 x 10~ mole) and anhydrous 4  sodium acetate (20 mg.) i n a c e t i c anhydride ture f o r 8 hr.  (3 ml.) was heated at r e f l u x tempera-  The cooled reaction mixture was then d i l u t e d with 5% aqueous  sodium hydroxide solution (75 ml.) and s t i r r e d f o r 15 min. a f t e r which time the resultant suspension was extracted with chloroform (3 x 30 ml.).  The combined  chloroform f r a c t i o n s were dried over sodium sulphate, and concentrated to a tan coloured glass which r e a d i l y c r y s t a l l i z e d from benzene to give colourless crystals of i'2-i (53 mg., 76%).  UV.; A  m a x  :  335 (3.50), 321 (3.56), 293 (4.13), 285  (3.95), 263 (4.49), 250 (4.70), 243 (4.74). IR. ( C H C I 3 ) : 5.50,  1730, 1635 cm" . N.M.R.: 1  5.46 (2 x s i n g l e t , 2 H, ArCH OAc), 3.50 (multiplet, 2 H, C-2'CH ), 3.05, 3.00 2  2  (2 x s i n g l e t , 3 H, NCH ), 2.88 ( s i n g l e t , 3 H, C - 4 C H ) , 2.60, 2.52 (2 x s i n g l e t , 3  3 H, C - l CH ), 3  Mass spectrum:  3  2.08, 1.98 ( s i n g l e t , 3 H, C - l N C 0 C H ) , 2.03 ( s i n g l e t , 3 H, N C O C H 3 ) . 1  3  M , m/e = 366; main peaks:  306, 280, 263, 233, 222-1.  +  l u t i o n mass spectrum:  Calc. f o r C H N 0 : 2 2  2 6  2  3  366.1943.  C, 72.20; H, 7.11; N, 7.20. Calc. f o r C H N 0 : 2 2  2 6  2  3  Found:  High reso-  366.1956.  Found:  C, 72.11; H, 7.18; N, 7.64.  1,4-Dimethyl-2- (8 -(N,N-methylacetylamino)ethyl)-3-hydroxymethylcarbazole  (122)  Carbazole acetate 121 (50 mg., 1.36 x 10" mole) was p a r t i t i o n e d between a 4  two phase medium consisting of t-butanol (5 ml.) and 20% aqueous sodium hydroxide (5 ml.), and heated at 100°C with vigorous s t i r r i n g f o r 1 hr.  The reaction mix-  ture was then concentrated to remove the t-butanol, d i l u t e d with water (75 ml.), and extracted with ether (3 x 30 ml.).  The combined ether fractions were washed  with water, dried over sodium sulphate and concentrated t o give 122 as a colourless s o l i d .  The crude product was p u r i f i e d by preparative layer chromatography  - 164 -  on alumina (1 mm., EtOAc:MeOH 20%). 243.  IR.: 1635 cm , N.M.R.:  6.20,  -1  spectrum:  UV.; J j ^ :  M*, m/e = 324; main peak:  for C 2 0 H 2 4 N 2 O 2 :  324.1837 .  Found:  335, 321, 293, 285, 263, 250,  5.00 (multiplets, 2 H, -CH OH). 2  Mass  308. High r e s o l u t i o n mass spectrum:  Calc.  324.1814.  l-Methyl-2-(B -(N,N-methylacetylamino)ethyl)-3-(ct -acetoxyethyl) carbazole(124) • Guatambuine (60 mg.,  2.27 x 10~  4  mole) was dissolved i n pyridine (4 ml.)  containing a c e t i c anhydride (2 ml.) and Heated at r e f l u x temperature f o r 2 hr. The solvent was then removed under high vacuum to give a dark red o i l . The crude product was p a r t i a l l y p u r i f i e d by column chromatography  on alumina (3 gm.).  By e l u t i o n with chloroform 124 was obtained as an amber coloured o i l (58 mg., 70%). 6.34  UV.; X^y.: 335, 323, 295, 285, 260, 247, 239.  1620.  N.M.R.:  (multiplet, 1 H, C-l'H), 3.50 (multiplet, 2 H, C-3' CH ), 3.00 ( s i n g l e t , 3 H 2  N-CH ), 2.48, 3  1.98  IR.: 1710,  2.40 (2 x s i n g l e t , 3 H, C - l CH ), 3  (2 x s i n g l e t , 3 H, N C O C H 3 ) .  Mass spectrum:  2.08 (Singlet, 3 H, O C O C H 3 ) , 2.08 M , m/e = 366, main peaks: 306 +  234, 233, 221, 220 (base peak), 205,204.  1-Methy 1-2-(B-(N,N-methylacetyland.no)ethyl) -3-vinylcarbazole (125 ) . Guatambuine (25) (60 mg.,  2.27 x 10~  4  mole) and anhydrous sodium acetate i n  a c e t i c anhydride was heated at r e f l u x temperature f o r 10 hr.  The cooled reaction  mixture was then d i l u t e d with 5% aqueous sodium hydroxide solution (75 ml.) and s t i r r e d f o r 15 min. a f t e r which time the resultant suspension was extracted with chloroform (3 x 30 ml.).  The combined chloroform f r a c t i o n s were dried over  sodium sulphate, and concentrated to a l i g h t brown o i l . ture was p u r i f i e d by column chromatography  The crude reaction mix-  on alumina (5 gm.).  By e l u t i o n with  - 165 -  chloroform the o l e f i n 125 was obtained as a f a i n t l y coloured o i l (45 mg., 64%), UV; Xj^xt  325 (broad hump), 295 (sh), 285-275 (broad peak), 262, 240, IR,:  1630 cm" .  N.M.R.:  1  5.72 (doublet o f doublets, 1 H, J i i »  =  1  7  H z  )2  » 2',2' J  2 Hz, C-2'H trans.), 5.37, 5.34 (2 x doublet o f doublets, 1 H, J i i ^ i  =  1  1  H  z  J t 2 « = 2 Hz, C-2' H c i s ) , 2.50 (multiplet, 2 H, C-3' CH ), 2.20 (multiplet, 2  >  2  2 H, C-4' CH ), 3.00, 2.92 (2 x s i n g l e t , 3 H, NCH ), 2.58, 2.52 (2 x s i n g l e t , 2  3  3 H, C - l CH ), 2.10, 1.96 (doublet(J 3  spectrum:  = 2 Hz) and s i n g l e t , 3 H, NC0CH ), 3  M , m/e = 306; main peaks:  234, 220 (base peak), 204, High  +  tion mass spectrum:  Calc, f o r C  2()  H N 0: 22  2  306,1732,  Found:  Mass, resolu-  306,1736  Formation o f the Enamine (126), and Reaction with Dimethylsulphate. In a small scale experiment  the enamine 126 was found to be p r e c i p i t a t e d  as an amorphous yellow s o l i d a f t e r the addition of 20% aq, sodium hydroxide solution (10 ml.) to a water solution (10 ml.) o f the iminium cation 3 mg.). UV. (20% aq. NaOH) (Figure 38); A,^:  (about  365, 335, 310, 294, 280, 270  (sh), 243, 237, Addition o f dimethylsulphate (1 ml.) t o the basic s o l u t i o n o f the enamine and heating at 100 yellow colouration,  f o r 10 min, r e s u l t e d i n the t o t a l disappearance o f the  UV, (Figure  38)l^^:  278, 245 (sh), 235,  - 166 -  INTRODUCTION: (PART III)  During the course of experiments on the biosynthesis and degradation of the pyridocarbazole  a l k a l o i d s o l i v a c i n e (16) and guatambuine (25), i t  became apparent that for the continuation of the work that larger quantities of these compounds would be required than were a v a i l a b l e from plant extracts 124 of Aspidosperma a u s t r a l e .  Attention was,  therefore, directed toward the  development of an e f f i c i e n t synthesis of these two a l k a l o i d s adaptable to large scale preparation (1 - 10 gm.). Both the o l i v a c i n e (16) and e l l i p t i c i n e  (17) systems have been previously  synthesized as a consequence of two separate i n t e r e s t s . 172-6 synthetic corroboration  was  various Aspidosperma and Ochrosia t e t r a c y c l i c structure.  In the early s i x t i e s  necessary following t h e i r i s o l a t i o n from plants, and the e l u c i d a t i o n of t h e i r novel  The subsequent determination  that o l i v a c i n e (16),  ellipticine  (17) and t h e i r analogs exhibited anti-tumor a c t i v i t y , led again to 177-180 the development of syntheses of these a l k a l o i d s . The approach adopted i n the present synthesis of o l i v a c i n e (16) guatambuine (25) was these molecules.  designed i n view o f what was  and  known about the chemistry of  I t i s , therefore, necessary to b r i e f l y review the relevant  d e t a i l s of the established syntheses. 172 For purposes o f structure e l u c i d a t i o n Schmutz and Wittwer (1960) were the f i r s t to publish a synthesis o f o l i v a c i n e (16).  They attacked  the  problem o f construction of the complex t e t r a c y c l i c r i n g system by d i v i d i n g the synthesis i n t o two d i s t i n c t stages.  The f i r s t stage was  the synthesis of  - 167 an appropriately substituted t r i c y c l i c carbazole intermediate containing the "A, B, and C" rings of o l i v a c i n e (16) .  The second stage was 11  construction of the fourth or (4,3-b) fused pyrido "D"  the  ring.  The f i r s t stage carbazole intermediate required a methyl group i n the 1-position and a v e r s a t i l e group i n e i t h e r the 2- or 3- p o s i t i o n which could subsequently be elaborated to give the remaining "D" r i n g .  Bearing i n mind  the proposed design of the "D" r i n g synthetic e f f o r t s were aimed at producing as the intermediate, l-methylcarbazole-2-carboxylic a c i d chloride (135). This compound was prepared i n s i x steps from 2-methyl-3-aminobenzonitile (Figure  40).  (132)  By e i t h e r o f two well known condensation reactions, the modified  Bischler or the Borsche synthesis, the s t a r t i n g material 132 was 7-cyano-8-methyl-l,2,3,4-tetrahydrocarbazole  (133).  Hydrolysis of the cyano  group and subsequent e s t e r i f i c a t i o n , followed by dehydrogenation r i n g gave the carbazole ester 134. carbazole ester 134 was  converted to  of the saturated  By reaction with thionyl chloride the  transformed into the desired a c i d chloride 135.  Wolff rearrangement of the diazoketone 136  formed by reaction of the a c i d  chloride 135 with diazomethane produced the corresponding homologous amide 137 This amide 137 was  .  transformed i n t o the N-acetyl derivative 140 by dehydration  to the n i t r i l e 138 with phosphorus oxychloride followed by reduction with Raney n i c k e l to the amine 139  and subsequent acetylation with a c e t i c anhydride i n  pyridine. Bischler c y c l i z a t i o n o f the amide 140 (141), imine 141 s a l t 142  completed  to the imine, 3,4-dihydro  olivacine  the synthesis of the t e t r a c y c l i c o l i v a c i n e skeleton.  The  was e a s i l y converted to guatambuine (25) by formation o f the methiodide and reduction over platinum oxide.  A l t e r n a t i v e l y the imine 1 4 1  was  - 168 -  (25)  gure 40.  (16)  Synthesis of Olivacine (16) and Guatambuine (25) by Schmutz and Wittwer (1960).  - 169 -  readily dehydrogenated with palladium The  c y c l i z a t i o n of 140 was  on charcoal to give o l i v a c i n e (16).  aided by the e l e c t r o n i c influence o f the  indole nitrogen para to the C-3 p o s i t i o n i n the "C" r i n g .  A stable  resonance contributing structure can be drawn depicting increased  electron  density centered at the C-3 p o s i t i o n which enhances the attack on  the  carbonyl  carbon o f the amide.  (140) The synthesis has  the advantage that i t can f o r the most part be  adapted to large scale preparation  of e i t h e r guatambuine (25) or o l i v a c i n e (16),  The  construction o f the "D"  r i n g however suffered from several drawbacks, i t  was  both i n d i r e c t and necessitated the use of large quantities of diazomethane,  The major drawback to the o v e r a l l sequence was  however, i t s excessive  seventeen steps i n t o t a l are involved when i t i s considered necessary to f i r s t synthesize the s t a r t i n g material 132  length,  that i t was  from o-toluidine  (143),*  - 170 -  A second synthesis of o l i v a c i n e (16) was published by Wenkert and Dave 173 (1962)  shortly a f t e r the work by the Swiss group.  As with t h e i r  predecessors  they aimed at the synthesis of an intermediate carbazole 152 which possessed  an  aldehyde f u n c t i o n a l i t y at the C-2 p o s i t i o n required for the elaboration of the "D" r i n g .  A considerably d i f f e r e n t approach f o r the synthesis of t h i s  intermediate was  chosen however, u t i l i z i n g 1-ketotetrahydrocarbazole  (144) as  the s t a r t i n g material, this compound being r e a d i l y a v a i l a b l e by Fisher indole synthesis.  The ketone group of t h i s molecule served the dual r o l e o f d i r e c t i n g  the course of f u n c t i o n a l i z a t i o n onto the C-2 p o s i t i o n and subsequently p r e f e r e n t i a l methylation onto the C-l p o s i t i o n . developed  the  The two schemes that were  are presented i n figure 41.  Considering Scheme A, sodium ethoxide induced acylation of 1-ketotetrahydrocarbazole  (144) with e t h y l oxalate gave the ethoxalyl d e r i v a t i v e  (R=H), which on a c e t y l a t i o n y i e l d e d the d i e s t e r 145 acid reduction of e i t h e r the d i e s t e r 145 ester 146  (R=0Ac).  Zinc and a c e t i c  (R=0Ac) or the a,3-unsaturated  (obtained by p a r t i a l hydrogenation  145  keto-  of the diester) produced the  saturated keto-ester  147.  Reaction of the keto-ester 147 with methyllithium  gave two products 148  and  149, which on palladium on charcoal dehydrogenation  led to the corresponding carbazoles 150 side chain o f compound 150  and 151.  Ozonolysis of the o l e f i n i c  gave the carbazole aldehyde  intermediate f o r the t o t a l synthesis.  152,  the v i t a l  In the study of the haloform reaction  on the acetone side chain of carbazole 151 i t was hoped that the presence of the C-methyl group would provide s u f f i c i e n t s t e r i c hindrance to prevent reaction at the b e n z y l i c carbon over reaction at the methyl carbon thus g i v i n g r i s e to the  - 171 -  (152)  Figure 41.  (153)  Scheme A, Synthesis o f Olivacine (16) According to Wenkert and Dave (1962).  - 172 CH Li  HCOOEt  3  OR  (154)  (144)  CHO  72% (155)  R = H = i-pr  Pd/C  o  78%  Base NH OH  CHO  79%  2  (158) (152) PCI* pyr (16) (141)  (159)  Figure 41. Scheme B, Synthesis of Olivacine According to Wenkert § Dave (1962)  acetic acid derivative.  However i n analogy with phenylacetone  carbazole carbocylic a c i d 153 was  obtained.  the corresponding  In view of the fact that carbazoles  with only one carbon side chains were available from the above sequence, i t was decided to study the same reaction with a one-carbon a c y l a t i n g agent, Scheme B_. Condensation  of 1-ketotetrahydrocarbazole (144) with ethyl formate gave the  formyl derivative 154 ether 154  (=H) .  Reaction with i-propyl iodide produced the i-propyl  (R=i-pr), which on treatment with methyllithium y i e l d e d the  dihydrocarbazole aldehyde 155  .  Upon dehydrogenation  of t h i s aldehyde  was s u r p r i s i n g to observe that the major i s o l a t e d product was zole (156).  Equally s u r p r i s i n g was  a t i o n could be c a r r i e d out, i t was  the extreme ease with  1,2155, i t  1,2-dimethylcarba-  which the dehydrogen-  found that by merely heating a benzene solution  - 173 of the 1,2-dihydrocarbazole aldehyde 155  i n the presence of palladium on  charcoal f o r one-half hour gave the desired carbazole aldehyde 152 . However, even i n this extremely mild case, small amounts o f the 1,2-dimethylcarbazole (156) was produced.  I t i s probable that the f a c i l i t y o f the reaction was  to a rapid disproportionation of the 1,2-dihydrocarbazole aldehyde 155 carbazole alcohol 157  p r i o r to i t s reduction to the 1,2-dimethyl  due  to the.  compound 156  or reoxidation to the carbazole aldehyde 152 ( i s o l a t e d i n small q u a n t i t i e s ) . 182 Such disproportionation reactions have been observed previously  although  as yet not much i s recorded regarding the energetics o f the process. CHO  CHO  (155) (156) The "D" r i n g was constructed by base catalyzed condensation of the i n t e r mediate aldehyde 152 with acetone to give the chalcone 158. the l a t t e r and reaction with hydroxylamine 159.  Beckmann rearrangement  Hydrogenation o f  gave the oxime of the dihydrochalcone  of the oxime 28 occurred with the concomitant  c y c l i z a t i o n to give the imine, 3,4-dihydroolivacine (141).  Dehydrogenation  of  the imine 141 yielded o l i v a c i n e (16). The synthesis developed by Wenkert's group was  e f f i c i e n t i n that o l i v a c i n e  (16) could be obtained i n only nine steps from s t a r t i n g material.  However, i t  was adapted to only small scale preparations. A further synthesis of o l i v a c i n e and i t s analogs has been developed by 177 Mosher et a l (1966) as part of a program on cancer chemotherapy. This synthesis represents a refinement of the l a t t e r stages of the scheme developed 172 by Schmutz and Wittwer (1960) . Instead o f u t i l i z i n g the Wolff rearrangement  - 174 -  COOCH.  (132)  (134)  LAH 92% ( yr) Cr0 P  2  3  (157)  IHH  2  A c  2°  (139)  (160)  POC1 -toluene 3  Pd/C  (16) Figure 42.  (141)  Synthesis o f Olivacine (16) by Mosher ejt al_. (1966).  they used the nitromethane the e l l i p t i c i n e  176 condensation, developed by Govindachari et^ a l  in  (17) series as a more d i r e c t means o f constructing the "D" r i n g ,  In essence the synthesis represents the better halves of two other syntheses united together to give the f i r s t synthesis o f o l i v a c i n e (16) adaptable to large scale preparation (Figure 42). The intermediate carbazole aldehyde 152 was obtained by reduction o f the ester 134 followed by Sarett oxidation o f the alcohol 157 .  Condensation  - 175 -  of the aldehyde 152  with nitre-methane led to the nitrostyrene 160  i n high  y i e l d , which on lithium aluminum hydride reduction produced the amine 139 , again i n high y i e l d .  141 was  Conversion  of the corresponding  amide 140  to the imine 172  again carried out according to the scheme of Schmutz and Wittwer,  Olivacine (16) was immediately a v a i l a b l e from this imine Recently some exploratory work on the synthesis of the pyridocarbazole 184-5 skeleton has been published by T. Kametani et al_ (1974) . For several years they have been u t i l i z i n g benzocyclobutene derivatives as precursors f o r the 183 synthesis of isoquinoline alkaloids  , and are at present attempting  to expand  the scope of t h e i r benzocyclobutene approach to include the synthesis of indole alkaloids of the o l i v a c i n e (16) and e l l i p t i c i n e  (17) type (Figure 43).  I t has  184 been found  that on intermolecular cycloaddition of indole (161) with the  benzocyclobutene analog 4,5-dibromomethyl-3- hydroxy-2-methylpyridine  hydrobroraii  (162) i n dimethylformamide f o r four hours, followed by a c e t y l a t i o n gave the expected dihydropyridocarbazole isomer 164  i n 15% y i e l d .  type compound 165  derivative 163  i n 4% y i e l d and i t s s t r u c t u r a l  Subsequent dehydrogenation produced the o l i v a c i n e  and i t s corresponding  isomer 166 .  The various syntheses that have been described represent the current state of the synthesis of the o l i v a c i n e (16) skeleton.  The design of the present  synthesis of t h i s system was not however derived s o l e l y from the chemistry  of  olivacine (16) but to a considerable extent from a study of the synthesis of the closely r e l a t e d pyridocarbazole, e l l i p t i c i n e  (17).  I t i s relevant, therefore,  to discuss the chemistry o f the e l l i p t i c i n e (17) s e r i e s since the difference i n the p o s i t i o n of one methyl group has r e s u l t e d i n a number of quite d i f f e r e n t approaches to the synthesis of t h i s system.  - 176 -  (165)  Figure  43 .  The Kametani Benzocyclobutene Analog Approach  (1975)  - 177 -  (17)  Figure 44 .  Woodward (1959) Synthesis of E l l i p t i c i n e  The f i r s t synthesis o f e l l i p t i c i n e  (17) was  (17),  achieved i n a somewhat 174  unusual and elegant manner by Woodward and co-workers (1959) structure e l u c i d a t i o n work on Aspidosperma a l k a l o i d s .  as part o f  They were able to  incorporate both the desired methyl f u n c t i o n a l i t y i n the C-5 p o s i t i o n of the "C" ring and the complete pyrido - "D" r i n g i n one step through the condensation of indole (161) with 3-acetyl pyridine to give 1,1-bis-(3_indolyl)1-1 (3-pyridyl)ethane (167) as i l l u s t r a t e d this dimeric product was  i n f i g u r e 44.  The pyridine r i n g of  reduced with zinc i n a c e t i c anhydride to the  - 178 -  Figure 45 .  (173) The Synthesis o f E l l i p t i c i n e (17) by Cranwell and Saxton (1962)  1,4-diacetylpyridine derivative 168 the 4-acetyl group o f which was i d e a l l y set-up f o r condensation with the electron r i c h 2-position of the indole molecule i n a manner i d e n t i c a l to the i n i t i a l condensation with pyridine.  3-acetyl  The condensation was effected by p y r o l y s i s under vacuum at 200°C.  Unfortunately  however, even though the process was intramolecular,  (17) was i s o l a t e d i n only a 2% y i e l d .  ellipticine  This approach was, therefore,  incompatible  with any scheme f o r large scale preparation of the a l k a l o i d . A d i f f e r e n t and more elementary approach was devised several years l a t e r 175 by Cranwell and Saxton (1962)  .  Following the theme developed f o r the  synthesis o f o l i v a c i n e (16) they considered a two stage synthesis o f e l l i p t i c i n e (17) to avoid the necessity o f a lengthy  Fisher indole synthesis.  By  - 179 -  reaction o f  indole (161)  with hexane-2,5-dione i n ethanol-HCl  carbazole (169) was obtained i n 36% y i e l d .  1,4-dimethyl-  Subsequent formylation under con-  t r o l l e d conditions gave the desired l,4-dimethyl-3- formyl carbazole (170) as the predominant product.  The d i r e c t i o n of formylation was  influenced by the  presence of the methyl groups i n the "C" r i n g (Figure 45). The construction of the "D" r i n g from the aldehyde 170 required the d i r e c t condensation of nitrogen onto the carbonyl f u n c t i o n a l i t y with subsequent provision f o r a r i n g closure.  Such requirements form the basis  of a well known Pomeranz-Fritsch reaction between an aldehyde and an amino acetal.  Reaction of the aldehyde 170 with aminoacetaldehyde  (171) y i e l d e d i n 85% y i e l d the r e q u i s i t e imine acetal 172.  diethylacetal I t was  found  however, that reaction of t h i s imine acetal 172 under any of the normally employed a c i d i c conditions, i . e . sulphuric acid, polyphosphoric acid, boron t r i f l u o r i d e etherate, or arsenic pentoxide f a i l e d to e f f e c t c y c l i z a t i o n . C y c l i z a t i o n under the above conditions was  also t r i e d without success on the  amino acetal 173 and on the corresponding acid derived from condensation of the aldehyde 170 with glycine. low y i e l d on the amino acetal 173  C y c l i z a t i o n was  eventually achieved i n  by r e f l u x i n g i n dry ethanol - HCl.  The  proposed product obtained 3,4-dihydroellipticine (191) was not p u r i f i e d but immediately dehydrogenated  with palladium on charcoal to give e l l i p t i c i n e  (17) i n very low y i e l d . The f a i l u r e of the c y c l i z a t i o n process to proceed i n a f a c i l e manner can be r a t i o n a l i z e d to be a consequence of the poor n u c l e o p h i l i c character  - 180 -  of the C-2 p o s i t i o n of the carbazole skeleton.  I t may be seen that resonance  structures invoking p a r t i c i p a t i o n of the indole nitrogen i n the b u i l d up of e l e c t i o n density at C-2  are unfavourable  the aromatic resonance of the "A"  due to the required disruption of  ring.  On the basis of i t s design this synthesis was  very p r a c t i c a l , however  the d i f f i c u l t i e s encountered i n the c y c l i z a t i o n step t o t a l l y deplete i t s value, Almost simultaneously with the p u b l i c a t i o n of the above work, Govindachari, 176 Rajappa and Sudarsanam (1963)  reported a new  synthesis o f e l l i p t i c i n e  (17).  They u t i l i z e d the c l a s s i c a l Fisher indole approach f o r the synthesis of appro^ priate carbazole intermediates.  D i f f i c u l t i e s were encountered during two  key  stages of t h e i r sequence r e q u i r i n g i n each instance complete redesign of the  i approach.  The three sequences^are~presented  In scheme A, 2,5-dimethyl-4-nitrobenzoic  i n figure 46, schemes A to C_. a c i d (174) was  converted to the  tetrahydrocarbazole 175 which on dehydrogenation y i e l d e d the desired dimethyl carbazole-3-ester (176). ester 176  The subsequent objective was  1,4^  to convert the  i n t o the corresponding aldehyde 178 by lithium aluminum hydride  reduction and oxidation o f the alcohol 177. aluminum hydride resulted unexpectedly 1,3,4-trimethylcarbazole  (179).  However, reaction with lithium  i n t o t a l reduction of the e s t e r 176  to  S i m i l a r t o t a l reduction has been observed  on  - 181 -  - 182 -  - 183 -  186 reaction o f 3-acetyl and 3-formylindole with lithium aluminum hydride. This r e s u l t can be r a t i o n a l i z e d by proposing that i n the basic  solution  i t i s the conjugate base that i s the reactive species, this intermediate being hydrogenolyzed.  O"  (176) Attempting to modify the synthesis, i n order to a l l e v i a t e the necessity of having to reduce a C-3 ester, the Scheme B_ was undertaken. approach 5-cyano-2-nitro-p-xylene (180 ) was  In t h i s  converted v i a the appropriate  arylhydrazine to 6-cyano-l,2,3,4-tetrahydro-5,8-dimethylcarbazole (181). Dehydrogenation  of t h i s compound and subsequent Raney n i c k e l reduction o f  the n i t r i l e 182  gave the amine 183.  i t was thought that condensation o f the  amine 183 with glyoxyl diethoxyacetal followed by Pomeranz-Fritsch c y c l i z a t i o n of 184 would then y i e l d e l l i p t i c i n e (17) d i r e c t l y .  However the same d i f f i -  c u l t i e s were encountered with this conversion as were experienced by Cranwell 175 and Saxton  and the approach was  abandoned.  Scheme C_ involves the conversion o f 3,6-dimethyl-2-nitrobenzaldehyde (185) to an intermediate, 6 -amino-2-cyano-p-xylene (186) very reminiscent o f 172 the s t a r t i n g material u t i l i z e d by Schmutz 5 Wittwer (1960)  .  (Figure 40)  Fisher i n d o l i z a t i o n to the tetrahydrocarbazole 187 , hydrolysis of the cyano group, and subsequent carbazole (188).  dehydrogenation afforded  I t was  l,4-dimethyl-2-carbomethoxy-  found that t h i s C-2 ester could e a s i l y be reduced to  - 184 -  the corresponding alcohol 189 without the complication experienced i n Scheme A, t h i s again probably being a r e s u l t of low electron density at C-2.  Sarett oxidation o f the alcohol 189 yielded the aldehyde intermediate  190.. The u i - n i t r o s t y r y l approach to the synthesis o f the "D" r i n g developed by these workers has been discussed previously i n conjunction with Mosher's 177 synthesis o f o l i v a c i n e (16)  .  The dehydrogenation o f the imine, 3,4-  d i h y d r o e l l i p t i c i n e Q.91 ) obtained i n four steps from the aldehyde provided e l l i p t i c i n e (17). The f i r s t stage o f the work portrayed i n Scheme C p a r a l l e l s 172 that developed by Schmutz § Wittwer (1960), the same drawback, i t s excessive length.  closely  and consequently i t suffers from The nitromethane condensation reaction  177  however as Mosher for  r e a l i z e d was a very d i r e c t and highly e f f i c i e n t method  construction o f the "D" r i n g .  178 Subsequently Dalton and co-workers (1967) were able to overcome the problem o f the c y c l i z a t i o n o f the imine acetal 172 to produce e l l i p t i c i n e (17). 175 The synthetic scheme o f Cranwell and Saxton's was adopted (Figure 45 ) with improvement of the i n i t i a l condensation step between indole (161) and hexane-2, 5-dione.  By s u b s t i t u t i n g p-toluenesulphonic acid-ethanol f o r t h i s condensation  i t was found that the y i e l d could be increased to 51%, compared with the 36% previously obtained.  I t was found that the c r u c i a l c y c l i z a t i o n o f the imine  acetal 172 could then be effected i n 33-40% y i e l d s using o-phosphoric a c i d . 175 Contrary to the r e s u l t s o f Cranwell and Saxton  who claimed that  ethanol - HC1 c y c l i z a t i o n o f the amino acetal 173 produced  3,4-dihydroellipticine  (191) (not characterized) i t was found that when the amino acetal 173 was heated  - 185 -  in o-phosphoric acid that e l l i p t i c i n e known that 1,2-dihydroisoquinolines  (17) was i s o l a t e d d i r e c t l y .  It is  produced by s i m i l a r c y c l i z a t i o n s undergo  187 disproportionation  and oxidation to isoquinolines, so that the previous  authors may have produced e l l i p t i c i n e  (17) d i r e c t l y and t h e i r dehydrogenation  step was probably unnecessary. This synthesis proved to be o f considerable value f o r large scale preparation o f e l l i p t i c i n e  (17) due to i t s b r e v i t y although the y i e l d s were  uniformly low. Recently  there has been a r e v i v a l o f i n t e r e s t i n the chemistry and  biological activity of ellipticine  (17).  Two new syntheses have been reported 1 *7 A  and both adopt the approach, f i r s t devised by Woodward (1959)  , involving  the condensation o f an appropriate unit containing a preformed pyrido r i n g onto the indole nucleus.  The general view taken with t h i s strategy i s that r i n g  closure o f the C " r i n g would require fewer conversions M  than are involved i n  the b u i l d up o f the pyridine r i n g . 179 Kilminster § Sainsbury (1972)  reinvestigated Woodward's condensation  of 3-acetylpyridine onto indole (161).  They preferred however to study the  condensation into respective indolin-2 and -3-ones 192 and 193 .  I t was found  that although i n i t i a l condensation products would form with ease the subsequent r i n g closure reaction could not be induced, presumably due to the i n e r t character o f the carbonyl groups.  From these observations  successful i n synthesizing e l l i p t i c i n e  they were, however,  (17) through condensation o f a substituted  3-acetylpyridine with l - a c e t y l i n d o l - 3 - y l acetate  (196) (Figure 47 ) .  The diacetyldihydropyridine 194 was prepared, oxidized to the acetyl  - 186 -  H  H  (192)  (193)  (17) Figure 47 .  (198)  Kilminster and Sainsbury  pyridine 195,  (1972) Synthesis of E l l i p t i c i n e ,  and condensed with the indole 196 a f f o r d i n g a mixture of both  geometrical isomers 197.  Reduction of e i t h e r isomer with sodium borohydride  followed by a c i d i f i c a t i o n  gave the compound 198 .  This material when heated i n aqueous hydrogen bromide, followed by l i z a t i o n and absorptions onto s i l i c a gel afforded e l l i p t i c i n e This synthesis o f f e r s both a short and e f f i c i e n t  neutra-  (17) i n 40%  route to e l l i p t i c i n e  yield.  (17),  180 In the second synthesis, Le G o f f i c , Gouyette, and Ahond (1973) proposed to unite the preformed "D"  ,  r i n g through a piperidone enamine conden-  sation with an appropriately substituted gramine molecule.  - 187 -  (17) Figure 48.  (200)  The Synthesis o f E l l i p t i c i n e Ahond (1973).  Consideration was  (17) by Le G o f f i c , Goyette, and  i n i t i a l l y given to the condensation  p y r r o l i d i n e enamine of l-benzyl-4-piperidone (Figure 48). ellipticine  between the  (199) and 2-ethyl gramine  (200)  This would i d e a l l y set the stage f o r a two-step synthesis of (17).  However i t was  found that the attempted  condensation  reaction gave only dimeric pxoducts and none of the desired m a t e r i a l . To circumvent t h i s problem the ethyl group was ring "D" instead. product 201 isolated.  This was  incorporated onto the  accomplished by reaction of the  condensation  with sodium a c e t y l i d e , epimeric t e r t i a r y alcohols 202  being  The alcohols were reacted i n the presence of formic anhydride to  - 188 -  give the desired t e t r a c y c l i c carbazole product 203 possessing the proper o r i e n t a t i o n o f the two methyl groups.  Debenzylation  § dehydrogenation over  palladium on charcoal produced e l l i p t i c i n e (17) i n 24% o v e r a l l y i e l d from indole  (161).  The excellent work done by the French group represents the culmination of the work to date i n the f i e l d o f the synthesis o f the pyridocarbazoles o l i v a c i n e (16) and e l l i p t i c i n e (17).  - 189 -  DISCUSSION - PART I I I  Two d i s t i n c t synthetic themes presided i n the many syntheses o f olivacine (16) and e l l i p t i c i n e (17) presented i n the preceding introduction, the two-stage approach where a suitable t r i c y c l i c carbazole intermediate was f i r s t formed and subsequently elaborated i n t o the t e t r a c y c l i c pyridocarbazole system, and the approach i n v o l v i n g the condensation o f a preformed "D" r i n g onto the indole nucleus, followed by r i n g closure t o give the " C " r i n g and the complete t e t r a c y c l i c structure. The former, two-stage approach was adopted i n the design o f the present syntheses o f o l i v a c i n e (16) and guatambuine (25).  In this design consideration was taken o f the f a c t that 177  the w - n i t r o s t y r y l approach u t i l i z e d by Mosher  provided an e f f i c i e n t means  of constructing the pyrido "D" r i n g o f o l i v a c i n e (16), whereas The PomeranzF r i t s c h approach adopted i n the e l l i p t i c i n e (17) series provided a considerably shorter but lower y i e l d i n g method o f pyrido r i n g construction. An e f f i c i e n t synthesis o f these two pyridocarbazole a l k a l o i d s could be obtained, therefore, i f a short 5 high y i e l d i n g synthesis o f e i t h e r o f the two formylcarbazole compounds l-methyl-2-formylcarbazole  0-52) o r 1-methy 1-3-formylcarbazole (219)  could be devised. These considerations formed the basis behind the developed syntheses o f o l i v a c i n e (16) and guatambuine (25) presented i n Sequences A § B.  - 190 -  Sequence A:  175  By analogy with Cranwell and Saxton's  synthesis of e l l i p t i c i n e  (17)  the condensation reaction between a methyl ketone f u n c t i o n a l i t y and indole was  adopted f o r the synthesis of the carbazole-2-aldehyde 152.  ellipticine  Unlike  (17) however, the absence of a symmetrical methyl group s u b s t i -  tution pattern i n o l i v a c i n e (16) necessitated the p r i o r construction o f an appropriately f u n c t i o n a l i z e d compound 204 condensation with C-2  where a subsequent methyl ketone  of the indole moeity would comprise the second step  i n the construction of the carbazole  skeleton.  R =  CHO  = COOCH,  (204)  (205)  An inspection o f the structure of compound 204  immediately indicated that  i t could be synthesized by the a l k y l a t i o n reaction o f an appropriate  1,3-dicar-  bonyl with a 3-substituted indole d e r i v a t i v e l i k e tryptophyl bromide (207). Methyl acetoacetate  (205, R = COOCHj) was  chosen as the 1,3-dicarbonyl component  instead of the corresponding aldehydo compound (205, R = CHO)  for reasons that  the carbonyl o f the carbomethoxy group i s less r e a c t i v e towards n u c l e o p h i l i c attack than the carbonyl of the ketone, thus eliminating the competition between these two  centers during the subsequent condensation r e a c t i o n .  Also, the trans-  formation o f the carbomethoxygroup i n t o the desired aldehyde f u n c t i o n a l i t y could be effected v i a a published procedure.  - 191 -  The synthesis of compound (204, tryptophol (206)  R = COOCH^) from commerically available  and i t s c y c l i z a t i o n with subsequent dehydrogenation to the  carbazole ester 134  or i t s c y c l i z a t i o n i n the presence of a hydrogen acceptor  reagent to give the carbazole ester 134 The reduction of the ester 134  d i r e c t l y i s presented i n figure 49.  to the carbazole alcohol 157  oxidation to the desired l-methyl-2-formylcarbazole Elaboration of this aldehyde 152  and i t s subsequent  (152 ) i s also depicted.  i n t o both o l i v a c i n e (16) and guatambuine  (25)  v i a the t*>-nitrostyryl approach proceeded i n an anologous fashion to that 177 reported by Mosher e_t al_ (1966) The present synthesis of the carbazole ester 134  i n three high y i e l d i n g  steps from the r e a d i l y available s t a r t i n g material tryptophol  (206 )  a considerable improvement i n e f f i c i e n c y over the alternate nine-step i n d o l i z a t i o n approach previously employed.  represents Fisher  The o v e r a l l synthesis c o n s i s t i n g  of ten steps to o l i v a c i n e (16) i s thus more e f f i c i e n t than those previously reported  172-3,177 . „ , , and i s e a s i l y adaptable to the large-scale preparation of both  188 o l i v a c i n e (16) and guatambuine (25).  - 192 -  Figure 49 .  An Improved Synthesis of Olivacine (16) and Guatambuine (25), Sequence A.  - 193 -  Tryptophol  (206)  Tryptophol  (206) was  obtained both commercially and v i a a simple  synthesis.  Before entering into a discussion of the synthesis of o l i v a c i n e (16) from t h i s s t a r t i n g material, i t i s perhaps advantageous to b r i e f l y discuss i t s synthesis 189,190-1 Several methods are reported,  but for the purposes o f the present work the  reaction of indole (161) with o x a l y l chloride (210) was most adaptable to large-scale preparation  CP  H  considered to be  the  (Figure 50 ) .  cy^o  93% (210) (211) Me OH 92%  (161)  LAH 60% (206)  Figure  50.  (212)  Oxalyl Chloride Route to Tryptophol  (206).  This synthesis follows quite c l o s e l y i n experimental 190 p a r a l l e l synthesis o f tryptamines by the same method 211 was  .  d e t a i l with  The glyoxyl chloride  obtained as e a s i l y i s o l a t e d b r i g h t yellow c r y s t a l s i n 93%  R e c r y s t a l l i z a t i o n proved unnecessary since the microanalysis was with the anticipated structure.  the  yield. i n agreement  The product proved to be moderately stable,  not reacting perceptibly with water, and discolouring on standing i n a i r only  - 194 -  a f t e r a period of several days. Reaction o f the glyoxyl chloride 211 with methanol gave the corresponding ester 212  i n 92% y i e l d .  s t r u c t u r a l assignment.  Again, the microanalytical data supported the Reduction o f the ester 212  i n the presence o f an  excess o f lithium aluminum hydride gave tryptophol C206 ) i n 60% y i e l d . chromatography on alumina was s u f f i c i e n t to remove base l i n e present i n the crude product mixture.  Column  contaminants  Tryptophol £06 ) was i d e n t i f i e d by i t s  c h a r a c t e r i s t i c N.M.R. spectrum consisting of a p a i r o f well separated t r i p l e t s centered at 62.96 (J = 6Hz) and 3.86 (J = 6Hz) (CH ) protons r e s p e c t i v e l y . 2  f o r the C-l (CH 0H) and C-2 2  A sharp s i n g l e t was present a t 61.53  f o r the  hydroxy 1 proton..:. Tryptophol (206) could r e a d i l y be converted to the bromide 207 by reaction 192 with phosphorus tribromide. P u r i f i c a t i o n by r e c r y s t a l l i z a t i o n proved to be more d i f f i c u l t than expected, and the product was subsequently p u r i f i e d by column chromatography on alumina.  The mass spectrum exhibited the expected doublet  for the parent i o n at m/e = 223 and 225 due to the equal abundance o f the two bromine isotopes. centered at 43.6  The N.M.R. spectrum possessed a single complex multiplet for the four methylene hydrogens.  The t r i p l e t at 6 3.0 f o r the  methylene protons adjacent to the oxygen atom i n tryptophol (206 ) has s h i f t e d u p f i e l d i n the spectrum of the bromide. 3-Carbomethoxy-5-(3-indolyl) -2-pentanone (204 ) Several d i f f e r e n t reaction conditions were studied f o r the a l k y l a t i o n reaction i n order to obtain a reproducible r e s u l t .  A 2:1 r a t i o of the sodium  - 195 -  s a l t o f methyl acetoacetate (205) to tryptophol bromide (207) was normally employed to compensate f o r any n e u t r a l i z a t i o n o f the anion that might occur as a r e s u l t of removal of the i n d o l i c hydrogen atom. Conducting the condensation i n r e f l u x i n g tetrahydrofuran proved to be generally unsuccessful.  Varying periods of r e f l u x from three to f i f t e e n  hours were t r i e d i n an e f f o r t to complete  the r e a c t i o n , however, the N.M.R.  and mass s p e c t r a l data i n d i c a t e d that normally only a mixture o f s t a r t i n g materials was obtained. 193  Zaugg e_t al_ (1960)  i n d i c a t e d dimethylformamide  to be a s u p e r i o r  solvent f o r the a l k y l a t i o n o f the anion o f e t h y l acetoacetate with a s e r i e s o f primary and secondary a l i p h a t i c h a l i d e s .  Usual y i e l d s of about 70% were  obtained when the r e a c t i o n was conducted at 100°-  f o r three to four hours.  In a p r e l i m i n a r y experiment with tryptophol bromide (207) using dimethylformamide hours.  the r e a c t i o n was permitted to remain at 100°  for f i f t e e n  The N.M.R. spectrum of the i s o l a t e d product showed several expected  absorptions; a s i n g l e t due to the ketone methyl a t 52.20 and a group o f d i s t o r t e d t r i p l e t s at 6 2.3 and 2.8 due t o the methylene hydrogen atoms. However, concomitant with these observations was the absence of a s i n g l e t f o r the methyl hydrogens o f the carbomethoxy group, i n d i c a t i n g that decarboxylation had accompanied the c y c l i z a t i o n process.  The mass spectrum supported t h i s  i n t e r p r e t a t i o n , having a parent peak at m/fe product lacking the ester f u n c t i o n a l i t y .  = 201 consistent with an a l k y l a t i o n  This phenomenon was thought to be a  r e s u l t of excessive reaction time and that by shortening the reaction p e r i o d the undesirable side reaction might be e l i m i n a t e d .  - 196 -  Thus, i t was at 100°  f o r three to four hours gave one predominant product as  from T.L.C. was  found that conducting the reaction i n dimethylformamide depicted  By column chromatography on alumina the desired product  i s o l a t e d as a pure yellow o i l .  The y i e l d s obtained were found  to  be very dependent on the scale of the chromatography, the y i e l d varying from 75% to 50% on proceeding from a small scale (1-2 gm.)  to large scale (10-20  gm.)  purification. No attempts were made to further optimize the a k l y l a t i o n reaction i n terms o f time or temperature of reaction, although i t was  known that the  r e a c t i v i t y of the corresponding tryptophyl tosylate (213)  towards n u c l e o p h i l i c  displacement i s considerably greater than that of simple primary a l i p h a t i c 194 tosylates. This rate enhancement has been demonstrated to be the r e s u l t of a unique a b i l i t y of the 3-position of the indole r i n g to p a r t i c i p a t e i n the displacement of the leaving group through formation  o f the spiro-indolenine  214.  c  n  f H  (213)  % V  ~ .  a / H  —  -  Ocn H  (214)  Subsequent s o l v o l y s i s or reaction of the cyclopropyl intermediate with a nucleophile being a f a c i l e step, thereby providing the substituted tryptophol derivative.  ,  - 197 -  - 198 -  Under reaction conditions where an appropriate nucleophile i s either 111 very weak or absent, the s p i r o indolenine 214 has been i s o l a t e d .  The reaction  conditions employed for the present a l k y l a t i o n were such, however, that i t s presence would not be detected as a reaction product. Conclusive i d e n t i f i c a t i o n o f the a l k y l a t i o n product 204 was made on the basis o f i t s s p e c t r a l data. characteristic. 6 2.12  The N.M.R. spectrum (Figure 51) was very  Two s i n g l e t s f o r methyl protons were r e a d i l y assigned, one at  for the ketone methyl, the other at 63.66 for the carbomethoxy methyl  group.  Both signals were s h i f t e d s l i g h t l y u p f i e l d from those obtained f o r the  corresponding groups (6 2.20  and 3.76) i n methyl acetoacetate.  Three d i s t i n c t  sets o f d i s t o r t e d t r i p l e t s were assignable, one f o r each o f the two methylene groups 6 2.28,  (C-4) and 62.76 ( J = 7Hz,C-5), and the t h i r d f o r the methine  hydrogen at 6 3.48 spike at 6 6.94, the indole r i n g .  ( J = 7Hz,C-3).  Of p a r t i c u l a r note was the presence o f a  i n t e g r a t i n g f o r the one hydrogen atom at the C-2 p o s i t i o n o f This s i g n a l i n d i c a t e d that under the reaction condition the alky-  l a t i o n product had remained i n tact without subsequent c y c l i z a t i o n . Further confirmation that c y c l i z a t i o n had not occurred was derived from the U.V. spectrum which showed a t y p i c a l indole absorption.  The I.R. spectrum con-  tained two carbonyl absorptions at 1750 and 1725 cm l f o r the ester and ketone _  carbonyls r e s p e c t i v e l y . In the low r e s o l u t i o n mass spectrum the parent peak at m/e = 259 was r e l a t i v e l y intense (25%) .  Important fragmentations  were seen at  m/e = 228, 186, 184, 144, 143, and 130. A possible mode o f fragmentation i s presented i n Figure 52. The high r e s o l u t i o n spectrum was within accepted and corroborates the parent peak at m/fe = 259.1212 ( c a l c . 259.1207) f o r  limits  C15H17O3N.  - 199 -  It further corroborates the formulation o f the fragment peaks giving the expected compositions f o r each fragment.  CXif H CC H N) 9  8  H 130.0656  (C H N) 10  10  144.0812  (C H 0 N) 15  17  3  259.1207 (C H N) 10  9  143.0734  ^OCH C 14 14°2 C  H  N)  228.0918  0 ^ H  (C H N) U  1 0  (C H ON) 12  1S6.0812  gure 52.  12  186.0918  Plausable Mass Spectral Fragmentation Pattern f o r the A l k y l a t i o n Product 204.  - 200 -  C y c l i z a t i o n Reaction 178 The conditions employed by Dalton et_ al_  i n the s i m i l a r condensation  of a methyl ketone center with the C-2 p o s i t i o n o f indole presented an obvious f i r s t choice f o r the c y c l i z a t i o n o f the a l k y l a t i o n product 204. It was consequently found that a reaction d i d occur with complete  consumption  of the s t a r t i n g material when the a l k y l a t i o n product 204 was treated with toluene s u l f o n i c acid i n r e f l u x i n g ethanol.  T.L.C, however, indicated the  presence o f an equal mixture o f two products.  These r e s u l t s were unexpected  i n that the s p e c t r a l data f o r the products were inconsistent with the a n t i c i pated product, l-methyl-2-carbomethoxy-3,4-dihydrocarbazole  (208) •  Alternate conditions were sought i n an e f f o r t to control the course o f the c y c l i z a t i o n so as to produce the desired compound 208 as the exclusive product.  B i s c h l e r - N a p i e r a l s k i c y c l i z a t i o n conditions, phosphorous oxychloride  i n toluene were thus studied.  Reaction conditions ranging from r e f l u x i n g i n  toluene f o r one hour to s t i r r i n g at room temperature  f o r f i f t e e n minutes were  a l l found to e f f e c t quantitative conversion of the a l k y l a t i o n product 204 . T.L.C. and N.M.R. indicated that the product composition obtained was i d e n t i c a l with that observed using Dalton's conditions. 175 Methanol-HCl,  conditions u t i l i z e d by Cranwell and Saxton  resulted once  again i n a quantitative conversion to a mixture o f the same two products. T.L.C. demonstrated reactants.  that the reaction had occurred instantaneously on mixing the I t was evident from these r e s u l t s that the c y c l i z a t i o n under a  variety o f a c i d i c conditions leads to the formation o f a mixture o f two products.  - 201 -  A detailed examination o f the s p e c t r a l data revealed the nature o f the processes that occurred.  The N.M.R. spectrum, (Figure 5 3 ) portrayed the  presence o f two carbomethoxy methyl group s i n g l e t s at 5 3.84 and 3.68 as w e l l as a p a i r of doublets at 6 1.00 and 1.15 (J = 7Hz) i n d i c a t i v e o f two d i f f e r e n t methyl groups attached to a saturated carbon atom bearing a methine  hydrogen.  These two methyl doublets integrated f o r a t o t a l of three hydrogen atoms thereby suggesting that they are due to the presence o f epimers.  The  methylene  hydrogens were spread out over the region 6 1.5 to 3.5 and the integration o f these signals was d i f f i c u l t to ascertain with any degree o f accuracy. The presence o f a s i n g l e t at 6 2.67, i n t e g r a t i n g f o r three hydrogens was i n d i c a t i v e of a methyl attached to an unsaturated center.  The multiplet i n the aromatic  region centered at 6 7.2 integrated f o r ten hydrogens, two more than twice the number o f hydrogens expected i n the desired substance.  This f a c t , coupled with  the presence of two signals f o r carbomethoxy methyl and methyl groups suggested that the product mixture consisted of two components each containing the indole moeity, a methyl group and a carbomethoxy group. 5 6.94  The absence of a peak a t  f o r the indole C-2 hydrogen was important i n that i t meant that c y c l i z a t i o n  onto the C-2 p o s i t i o n had occurred as expected. Based on a comparison o f the r e l a t i v e i n t e n s i t i e s o f the carbomethoxy signals i t was deduced that the reaction mixture consisted o f a 1:1 mixture o f the two products.  The i d e n t i t y o f the two components could not be deduced  however, from the N.M.R. spectrum alone, but with the added support o f the mass s p e c t r a l data, t h e i r structures were determined.  Ii  sooo  I 'III  i)  II  i  I  II  i | i  II  I i i  I  I i i  1(1  i |  t  i i i  f  i i i f i i i i' i i i  I'M  i  i' i i i i'  I l|  I  .1  JJOO  iJgo so J  0  lie  Figure 53.  N.M.R. Spectrum of the C y c l i z a t i o n Reaction Mixture.  l ,i  I I l'I  I |I  I  i  i  I  i  I  i  i  i  i  |  i  i  'i  i  I  i  'i  i  i  M  i  i  i  I  i  i  - 203 -  The region o f the mass spectrum corresponding to the expected molecular ion was characterized by the presence of two intense peaks at m/e and m/e  = 243 (50%), and an almost n e g l i g i b l e peak at m/e  molecular ion f o r the proposed 3,4-dihydrocarbazole 208.  = 239 (100%)  = 241, the expected These two peaks were  considered to be the parent peaks f o r each o f the two i s o l a t e d components. That the m/e  = 239 peak did not a r i s e v i a fragmentation of the m/e  = 243 peak  was strongly supported by the presence o f fragmentations f o r loss o f C H 3 , O C H 3 , and C O O C H 3 from each o f these parent peaks, figure 54  i l l u s t r a t e s the  c h a r a c t e r i s t i c fragmentations observed i n the low resolution spectrum. Of great importance was the presence o f fragmentation peaks at m/e  = 169, 168,  157,  143, and 130 which could only a r i s e through disruptions of a saturated carbazole  C" r i n g . Such an appropriately methyl and carbomethoxy substituted  tetrahydro carbazole would give r i s e to the observed m/e m/e  = 243 peak. The  =239 peak can thus be considered to a r i s e from loss of four hydrogens i n  the former to give the f u l l y aromatized carbazole compound. To determine whether or not the species observed i n the mass spectrum arose from a d i s p r o p o r t i o n a t e o f the 3,4-dihydrocarbazole 208  during c y c l i z a t i o i  or was simply an event i n the spectrometer, experiments were conducted where low i o n i z i n g voltages o f 53eV and low temperatures f o r v o l a t i l i z a t i o n were employed.  In a l l cases the spectra were i d e n t i c a l to that obtained under  normal operating conditions ( i . e . 70eV)  COOCH(134) M^m/e  S  = 239  CO  COOCH. (209) M,m/e - 243 +  8 8 T—i—r  100.0  50.0  gure 54.  mmif ,  150.0  200.0  M/E  T r—i—r—i—|—i—i—r—i—r—i—i—i—r 1 250.0 300.0 350.0 400.0  Mass Spectral Fragmentation Patterns f o r Cimponents 134 and 209 of the C y c l i z a t i o n feaction  Mixture.  - 205 -  On the basis of the N.M.R. and mass spectral analysis i t was  proposed  that the c y c l i z a t i o n reaction produced the f u l l y unsaturated compound, l-methyl-2-carbomethoxy carbazole (134) and i t s f u l l y saturated counterpart l-methyl-2-carbomethoxy-l,2,3,4-tetrahydrocarbazole  (209) .  To accommodate the existence of the saturated methyl group i n two separate environments, two configurations o f the methyl with respect to the carbomethoxy groups were i n f e r r e d .  (134)  (209a)  (209b)  Conclusive proof of the above assignments was not possible however without separation of the two components and p r o v i s i o n of appropriate c h a r a c t e r i z a t i o n data.  Considering that the carbomethoxy f u n c t i o n a l i t y i s e s s e n t i a l l y n e u t r a l ,  i t was not s u r p r i s i n g to f i n d that the T.L.C. c h a r a c t e r i s t i c s for the c y c l i z a tion product mixture were i d e n t i c a l to those obtained f o r an equal mixture carbazole and tetrahydrocarbazole.  I  N  of  both cases two c l o s e l y overlapping  spots were obtained giving i d e n t i c a l colour reactions when sprayed with eerie sulphate. Separation of the two reaction components proved to be very d i f f i c u l t . On column chromatography an enrichment of the impure tetrahydro compound . was  obtained, whereas, pure carbazole component 134  (about one h a l f of the material a p p l i e d ) . spontaneously  was  obtained i n good y i e l d  This l a t t e r material c r y s t a l l i z e d  from highly concentrated chloroform solution to give colourless  - 206 -  c r y s t a l s , M.P. = 138-140°. Very poor weight recovery o f the tetrahydrocar-^ bazole 209 from the column was achieved. This s i t u a t i o n also prevailed when this material was subsequently further p u r i f i e d by preparative layer chroma^ tography.  A f t e r several p u r i f i c a t i o n s small amounts o f carbazole 134 were  s t i l l detected. Complete a n a l y t i c a l and s p e c t r a l data were obtained f o r the c r y s t a l l i n e carbazole 134 and these were i n compliance with i t s structure.  The N,M,R,  spectrum exhibited the anticipated s i n g l e t s at 63.84 and 2.70 f o r the hydrogens of the two methyl groups, while signals f o r methylene protons observed i n the above mixture were now t o t a l l y absent. The U.V. spectrum was s u r p r i s i n g i n that i t was i d e n t i c a l i n shape to that f o r the crude reaction mixture.  The measured e x t i n c t i o n c o e f f i c i e n t s  compared favourably with those f o r the corresponding ethyl ester prepared by 172 Schmutz and Wittwer (I960) o f the maximum.  , however a discrepancy arose as t o the p o s i t i o n  The maximum f o r the methyl ester (measured i n methanol) was  reported at 303 nm and that f o r the ethyl ester (measured i n ethanol) i s observed at 308 nm. The fragmentation pattern i n the low r e s o l u t i o n mas spectrum  corresponded  to one h a l f o f the observed fragments i n the composite spectrum f o r the crude reaction mixture. was  The parent peak i n the high r e s o l u t i o n spectrum m/e = 239.0946  found to be consistent with the correct molecular composition C 1 5 H 1 3 O 2 N , as  were the compositions f o r the fragment ions corresponding to the loss o f C H 3 , O C H 3 and C O O C H 3 .  - 207 -  The spectral data determined f o r the tetrahydrocarbazole 209 was i n that inadequate  quantities of the compound could be obtained i n pure form.  C r y s t a l l i z a t i o n could not be induced, consequently oil. 290,  The U.V.  poor  i t was  studied as a viscous  spectrum exhibited a t y p i c a l indole absorption having maxima at  282, and 275 nm,  t h i s being expected f o r a tetrahydrocarbazole.  The  extinction c o e f f i c i e n t s f o r the indole chromophore are low, compared to a carbazole chromophore, and they may  represent only a small contribution to the  composite spectrum which may possibly explain the s i m i l a r i t y of the spectra of the carbazole 134 and the crude mixture. Fourier transform N.M.R. portrayed the presence of the carbomethoxy f u n c t i o n a l i t y and the saturated methyl as a s i n g l e t at 6 3.68  and a p a i r of  high f i e l d doublets r e s p e c t i v e l y (the l a t t e r p a r t l y obscured by impurities) . P a r t i c u l a r l y s i g n i f i c a n t were the small set of multiplets centered at 6 3.38 and 3.96  attributed to the methine protons i n t h e i r two respective configurations  (209 a and b ) .  The absorption f o r the methine hydrogen geminal with the  carbomethoxy group was masked by the s i n g l e t peak at 6 3.68 The parent peak i n the mass spectrum at m/e  = 243  f o r this group.  (85%) was  clearly  evident, as were the fragments corresponding to loss of C H 3 , O C H 3 and C O O C H 3 respectively.  Those fragments corresponding to the disruption of the saturated  "C" r i n g were also evident. Thus the c y c l i z a t i o n of the a l k y l a t i o n product^04 did not give the  expected  3,4-dihydrocarbazole 208 , but proceeded further to y i e l d the carbazole 134 tetrahydrocarbazoles (709  a and b ) .  Their formation can be mechanistically  and  - 208 -r  envisaged to r e s u l t from a t o t a l disproportionation of the i n i t i a l l y formed 3,4-dihydrocarbazole 208  under the reaction conditions employed (figure 49 )  f  (204) -»• |(208)| -+ (134) + (209). Such a disproportionation r e f l e c t s the inherent i n s t a b i l i t y o f the dihydrosystem with respect to i t s anomatic counterpart, Other examples of t h i s occurrence e x i s t , p a r t i c u l a r l y i n cases where potential  dihydropyridine systems are involved such as the 3,4-dihydroiso-r 195 168 quinolines and 3,4-dihydro-8-carbolines . I t i s o f i n t e r e s t to note that the 3,4-dihydrocarbazole aldehyde 155 synthesized by Wenkert and Dave 173 (1962)  did not undergo disproportionation on i t s formation.  Whether t h i s  r e f l e c t s the difference i n the method of preparation of t h i s compound or i t s  (155) s t a b i l i t y i s not known.  The aldehyde 155  did disproportionate (see page  172)  however even on mild dehydrogenation treatment with palladium on charcoal. Methanol - HC1  (2%) conditions were employed f o r a l l large scale c y c l i r  zations due to the s i m p l i c i t y of the reaction and the quantitative y i e l d s of the two products.  Column chromatography was not used to separate the two  components during preparative work because subsequent pure carbazole 134.  dehydrogenation gave  The o v e r a l l y i e l d f o r the subsequent  dehydrogenation  could be increased by p r i o r s e l e c t i v e r e c r y s t a l l i z a t i o n of the carbazole  - 209 -  component 134  from a highly concentrated chloroform solution of the mixture.  Dehydrogenation  was,  therefore, conducted only on the mother liquors from the  c u p t a l l i z a t i o n of 13^. l-Methyl-2-carbomethoxycarbazole  (134).  The use of palladium on charcoal f o r the dehydrogenation  of tetrahydro-  carbazole and other dihydro compounds has considerable precedence  particularly  for the systems dealt with i n the present work (see introduction part I I I ) . Palladium on charcoal was,  therefore, the obvious choice f o r the conver-  sion of the c y c l i z a t i o n reaction mixture into the carbazole 134 . reaction conditions, however, only p a r t i a l dehydrogenation was  observed.  Changing to platinum oxide as the c a t a l y s t enabled complete i n a l l experiments  conducted.  Under t y p i c a l  dehydrogenation  However, the large quantities necessary made i t s  use p r o h i b i t i v e on a preparative s c a l e . Attention was agents.  thus turned towards the use of quinone  dehydrogenating  Quinones, e s p e c i a l l y c h l o r a n i l and dicyanodichlorobenzoquinone  (DDQ)  have been u t i l i z e d with great success i n the dehydrogenation of a wide range 196-7 of carbazole compounds. 134  With c h l o r a n i l a homogeneous carbazole ester  reaction product mixture was obtained.  Traces of coloured impurities were  removed from the crude product e i t h e r by r e c r y s t a l l i z a t i o n from chloroform or by column chromatography.  The product was  eluted with benzene as a yellow o i l  which subsequently c r y s t a l l i z e d to y i e l d colourless c r y s t a l s . quite variable ranging normally between 70-80%  The y i e l d s were  although occasionally dropping  as low as 60%. To prevent manipulative d i f f i c u l t i e s during chromatography due to the nonpolar nature of both the carbazole compound and the coloured contaminants  1  - 210 -  p u r i f i c a t i o n was normally postponed u n t i l a f t e r reduction to the corresponding alcohol 157 .  The reduction was  found not to be affected by the presence of  these impurities and the alcohol 157  being more polar was more e a s i l y separated  from them. The spectral and a n a l y t i c a l data obtained f o r the dehydrogenation  product  were i d e n t i c a l to that obtained f o r the carbazole component 134 of the c y c l i z a tion mixture, thus confirming the above conversion, Cyclization -  Dehydrogenation  Advantage was taken of the disproportionation reaction to achieve the d i r e c t conversion of the a l k y l a t i o n product 204  i n t o the carbazole 134  i n one  step by conducting the c y c l i z a t i o n reaction i n the presence of an excess of chloranil  as the hydrogen acceptor.  that the 3,4-dihydrocarbazole208;would pared with c h l o r a n i l .  The r a t i o n a l e behind t h i s reaction  act as a poor hydrogen acceptor com*  On t h i s basis the a l k y l a t i o n product 204  dissolved i n  benzene containing excess c h l o r a n i l was treated with methanol - HC1 i s o l a t e d product, formed i n 84% y i e l d , was  The  step was eliminated,  (157) .  The conversion of the ester group of the carbazole 134 alcohol 157 was  (2%) .  indeed the desired carbazole 134.  Thus the necessity f o r a d i s t i n c t dehydrogenation 1-Methy1-2-hydroxymethylcarbazole  was  to the corresponding  r e a d i l y accomplished by making use of lithium aluminum hydride 172  reduction conditions developed by Schmutz and Wittwer carbazole ester 134  .  gave the desired carbazole alcohol 157  Reaction of pure as a colourless cry-  s t a l l i n e s o l i d i n 95% y i e l d . The corresponding reduction of impure carbazole ester 134 obtained v i a  211  c h l o r a n i l dehydrogenation was  conducted i n an i d e n t i c a l manner.  Chromatogra-  phic p u r i f i c a t i o n of the product provided an o v e r a l l 60% y i e l d of the alcohol 157.* The s p e c t r a l data were consistent with the structure 157.  The mass spectrum  +  showed a parent peak at m/e  = 211 as well as an M -17 f o r loss of hydroxyl.  absence of the carbonyl absorption at 1710 The U.V.  cm"*  i n the I.R.  spectrum was  The  noted.  spectrum exhibited a t y p i c a l carbazole absorption as expected due  to  loss of conjugation with the carbomethoxy f u n c t i o n a l i t y . The s i n g l e t at 6 3.84 i n the N.M.R. f o r the e s t e r methyl group was s i n g l e t at 64.88  (CH2OH)  absent, having been replaced by a  and a s i n g l e t at 61.50  (OH).  l-Methyl-2-formylcarbazole (152) The conversion of the b e n z y l i c alcohol 157 to the aldehyde 152 proved to be 177 a low y i e l d i n g step. (Ci^-py^)  Mosher et_ al_  used bispyridine-chromium  trioxide  i n pyridine, i . e . Sarett conditions to oxidize this alcohol  reporting a y i e l d of about 70% f o r a large scale reaction (16 gm). et a l report a higher y i e l d of 84% f o r a small scale (1 gm) the closely r e l a t e d alcohol 189 i n the e l l i p t i c i n e I t was  157,  Govindachari  Sarett oxidation of  (17) synthesis.  1 7r»  found during the present work, however, that consistently lower  y i e l d s ( 60%) were obtained f o r the Sarett oxidation of the alcohol 157.  In an  attempt to improve upon this y i e l d a considerable number of oxidation conditions were studied during the present synthesis.  •footnote:  For ease of manipulation  out p u r i f i c a t i o n at each step.  A tabulation of the various oxidation  the entire sequence could be conducted with-  In terms o f o v e r a l l y i e l d s t a r t i n g from trypto-  phyl bromide, a comparison of both procedures showed there to be no advantage i n not p u r i f y i n g each product (see  experimental).  212  methods including the Sarett, the conditions under which the reactions were conducted and y i e l d s or comments on the reaction outcome i s presented i n Table I I I . From t h i s study i t was determined that an e f f i c i e n t oxidation procedure depended upon two c r i t e r i a , media and  i ) The s o l u b i l i t y of the alcohol i n the reaction  i i ) An e f f i c i e n t uncomplicated work-up. 198  C o l l i n s conditions with y i e l d s of  were an obvious a l t e r n a t i v e to the Sarett reaction  90% being reported f o r the r e l a t e d conversion  alcohol to benzaldehyde.  of benzyl  S i m i l a r l y high y i e l d s are obtained from S i l v e r ^ * * )  oxide*"'*' or C e ^ ^ o x i d a t i o n * " of the b e n z y l i c alcohols.  Hov/ever i n a l l three  cases poor s o l u b i l i t y of the alcohol 157 i n the reaction solvent hindered o x i dation and under the conditions quoted f o r the reaction no detectible formation of aldehyde 152 was observed. Considerable d i f f i c u l t i e s during work-up  r e s u l t i n g from mutual s o l u b i l i -  t i e s o f the extracting solvents and reaction reagents were encountered f o r the pyridine-based  oxidation methods and this was one of the major drawbacks to  the Sarett oxidation.  The influence of the work-up  on product y i e l d was  p a r t i c u l a r l y d r a s t i c i n the case o f the HOAc/Cr03-pyr2 o x i d a t i o n  2 0 0  where a drop  from 85% to 35% y i e l d accompanied the s c a l i n g of the reaction to larger quantities. The pyridine-S03/DMS0-Et3N oxidation method developed by Doering and co201  workers  f o r work i n the s t e r o i d f i e l d gave a mixture o f three products on  work-up .  Separation  of the aldehyde component from t h i s mixture by e i t h e r  213  Table IV. Results f o r Oxidation  o f Alcohol (157)  Method  Conditions  Y i e l d or Comment  Sarett  Cr03«pyr2 i n pyr  60-65%  d  24 hrs., RT.  HOAc/Cr0 .pyr  i n HOAc -5 to +16° 15 min.  85%, 35%  Collins  Cr0 *pyr  no product  3  3  i n CH Cl2  2  2  d  detected  RT., 0.5 h r .  Ag 0  1 M H P0 , 25  IX  3  l  no reaction  4  a,b  3-4 h r .  Ce™  H0Ac/H 0 90°  no r e a c t i o n  2  3  12 h r .  Pyr'SOj/DMSO  RT., 24 hr.  inseparable  Pb(0Ac) /pyr  RT., 24 h r  50-60%  Jones  Cr0 , H S0 (aq.) i n  70-75%  mixture  1  Et N 3  4  3  2  Acetone  a.  4  1 min., RT,  TLC detection  ( A 1 0 , CHC1 ), b.  300 mg.,  large scale > 2.0 gm.  d.  2  3  d  UV detection,  c.  small scale $  3  - 214  alumina or s i l i c a chromatography was  -  unsuccessful  as was  attempted recry-  s t a l l i z a t i o n from chloroform. The problems associated with the presence of pyridine during extraction 202 were eliminated by using the lead tetraacetate in pyridine conditions the completion of reaction the solvent was tion of the product.  The  aldehyde 152 was  i n this reaction.  T.L.C. with authentic  The  At  f i r s t removed i n vacuo before extracobtained as the major product i . e .  (50-60% y i e l d ) a f t e r column chromatography. detected  .  Several minor by-products were  of a non-polar component coinciding on  l-methylcarbazole-2-acetate (215), t h i s material being  an expected by-product.  (215)  Jones oxidation^ " 0  5  proved to be a viable a l t e r n a t i v e to the Sarett  reaction y i e l d i n g the aldehyde 152 as the sole product. leads d i r e c t l y to the carboxylic a c i d stopped at the aldehyde stage.  , but i n this instance the reaction  The reaction did not appear to be dependent  on either the time span or the amount of oxidant present. oxidation o f the aldehyde 152  produced the y i e l d s were not  (about 70%), t h i s again r e f l e c t i n g the i n e f f i c i e n c y o f  product extraction during chromium (VI) oxidations. i f any,  Attempted further  resulted i n the r e - i s o l a t i o n of s t a r t i n g m a t e r i a l .  Although a s i n g l e reaction product was exceptionally high  This reaction often  Despite the only moderate,  improvement i n y i e l d s , the Jones conditions were considerably more  -  215  -  e f f i c i e n t than the Sarett i n terms of reaction time and ease o f work-up. Proof of the aldehyde structure 1 5 2 was derived from a comparison of the melting point and quantitative U.V. spectrum with that reported by 177  Mosher et^ a_l,  as well as from the other spectral and a n a l y t i c a l data.  The  N.M.R. spectrum exhibited two s i n g l e t s , one at 62.87 due to the aromatic methyl, and the other at 610.39 f o r the aldehydic hydrogen.  The I.R. spectrum indicated  the expected aldehyde carbonyl band (1665 cm"*) and i n the mass spectrum parent -  peak was at m/e m/e  = 209.  High resolution mass measurement provided the value  = 209.083] which established the desired molecular formula (C^H^NO  requires:209.0842). "D" Ring Synthesis The construction of the "D" r i n g of o l i v a c i n e through formation o f the imine, 3,4-dihydro o l i v a c i n e ( 1 4 1 ) from the aldehyde intermediate 1 5 2 was 177  accomplished by following the procedure published by Mosher e_t al_.  No  unexpected complications were encountered during the interconversions and a l l a n a l y t i c a l and s p e c t r a l data were i n accord with the proposed structures and compared within acceptable l i m i t s with l i t e r a t u r e values where quoted. Condensation o f the aldehyde 1 5 2 with nitromethane i n the presence o f ammonium acetate produced the nitrostyrene 1 6 0 product i n 94% y i e l d .  as the sole reaction  R e c r y s t a l l i z a t i o n to crimson needles was possible from  large volumes o f ethanol.  The nitrostyrene 1 6 0 was reduced to the amine 1 3 9 by  lithium aluminum hydride reduction i n tetrahydrofuran at room temperature. product i s o l a t e d as a white c r y s t a l l i n e s o l i d was obtained i n 95% y i e l d .  The The  amine 1 3 9 was converted without p u r i f i c a t i o n to the N-Acetylamine by reaction  - 216 -  with a c e t i c anhydride i n p y r i d i n e . s o l i d obtained i n 95% y i e l d .  The product was a white c r y s t a l l i n e  The Bischler-Napieralski c y c l i z a t i o n o f the  l a t t e r to 3,4-dihydroolivacine 141  was accomplished by r e f l u x i n g the  amide 140 with phosphorous oxychloride i n toluene.  The u l t r a v i o l e t  spectrum  of 141 run i n water and d i l u t e hydrochloric a c i d showed the expected batho125 chromic s h i f t  accompanying the transformation C = N •*• C = NH*.  Reaction o f the imine 141 with methyl iodide i n methanol produced the methiodide s a l t 142 i n quantitative y i e l d .  The s h i f t s observed i n the UV.  spectrum o f t h i s iminium cation i n base r e f l e c t e d i t s rearrangement corresponding enamine (anhydro base)  to i t s  (Section 6., part I I ) . Reduction  142  of the methiodide  e i t h e r by sodium borohydride i n aqueous ethanol or by  c a t a l y t i c hydrogenation over platinum oxide gave i n high y i e l d the t e t r a c y c l i c a l k a l o i d (±)-guatambuine (25). Proof o f the structure o f guatambuine (25) was derived from the superimposibility o f the UV. and IR. spectra with the 126 reported spectra  and by the close comparison o f the N.M.R. and mass spec-  trum with that obtained f o r guatambuine (25) i s o l a t e d from  australe (Figure  20). Dehydrogenation o f the imine 141 by reaction over palladium on charcoal i n r e f l u x i n g decalin produced the t e t r a c y c l i c a l k a l o i d o l i v a c i n e (16) i n 70% yield.  Total characterization of t h i s product was also derived from a com-  parison o f the N.M.R. and mass s p e c t r a l data with that obtained f o r o l i v a c i n e i s o l a t e d from A. australe. at 62.82 and 3.16  The N.M.R. spectrum displayed a p a i r o f s i n g l e t s  f o r the C - l and C-5 methyl groups r e s p e c t i v e l y .  The mass  spectrum possessed a parent peak at m/e = 246 corroborated by the high resolut i o n spectrum parent peak at m/e = 246.1165 ( C i 7 H N 14  2  requires m/e = 246.1156).  - 217 -  Sequence B A short route to the synthesis of the carbazole-3-aldehyde  219  was  provided from the reported o b s e r v a t i o n ^ that the Vilsmeier-Haack formy2  lation o f 9-benzyl-I,2,3,4-tetrahydrocarbazole  (217) instead o f leading to 205  the expected 7-formyl-9-benzyl-l,2,3,4-tetrahydrocarbazole l-methyl-3- formyl-9-benzylcarbazole (219). this conversion was  (218) gives  The y i e l d of about 50% f o r  acceptable since this provided a one step synthesis of  this desired intermediate aldehyde from a r e a d i l y available s t a r t i n g material (217).  N  OHC-  Bz (218)  : II  N  I  Bz CHO (217)  (219)  In the application of the Pomeranz-Fritsch  reaction f o r the synthesis  of the pyrido ring the necessity of incorporating a methyl group at C-l of the o l i v a c i n e system (16) created complications.  I t i s known that the con206  densation of amino acetals with ketones i s f a r less e f f i c i e n t  than the  - 218 -  corresponding reaction with aldehydes.  Hence the most direct route to  olivacine (16) by reaction of aminoacetaldehyde  d i e t h y l a c e t a l (171) with  the 3-acetylcarbazole 220 had l i t t l e chance of being successful.  (220)  Two  alternatives to t h i s condensation were a v a i l a b l e .  the l-methyl-3-formylcarbazole intermediate (219) was aminoacetaldehyde 221 was  On the one hand  f i r s t condensed with  d i e t h y l a c e t a l (171) and the r e s u l t i n g iminomethyl  acetal  then methylated with methylmagnesium chloride to give the aminoethyl  acetal 224.  A l t e r n a t i v e l y , the 3-formylcarbazole compound 219 was  methylmagnesium chloride to give the a-carbazolyl converted to the corresponding acetate 223.  ethanol system 222 which was  Substitution of the acetate func-  t i o n a l i t y by the aminogroup of aminoacetaldehyde produced the desired aminoethyl acetal 224  reacted with  d i e t h y l a c e t a l (171) again  (Figure  55) .  C y c l i z a t i o n of t h i s aminoethyl acetal 224 to 6-benzoolivacine (225) lowed by debenzylation to o l i v a c i n e (16) was not attempted.  fol-  However, the con-  d i t i o n s necessary f o r the c y c l i z a t i o n have been worked out f o r the synthesis of the c l o s e l y related molecule, e l l i p t i c i n e (17).  - 219 -  R  I P=Bz =H  Figure 55.  (225) (16)  Synthesis of Olivacine (16) from Tetrahydrocarbazole  (216).  This synthesis as i t presently stands i s generally low y i e l d i n g i n each of the i n d i v i d u a l steps, however, i t i s p o t e n t i a l l y a short and  simple  route to o l i v a c i n e (16) s t a r t i n g from r e a d i l y a v a i l a b l e s t a r t i n g material. It could e a s i l y be adapted to large scale preparation and towards the preparation of various forms of radioactive o l i v a c i n e (16) CH^  group) which may  ( p a r t i c u l a r l y the C-l  be required i n future biosynthetic experiments.  - 220 -  9-Benzyltetrahydrocarbazole (217) Tetrahydrocarbazole  (216) was r e a d i l y available i n large quantities as  a s t a r t i n g material through the condensation of phenylhydrazine with cyclo207 hexanone i n the presence o f a c i d .  The indole nitrogen (Na) o f tetrahydro-  carbazole (216) was converted to i t s benzyl derivative 217 by reaction o f benzyl bromide and sodium hydride.  A colourless o i l obtained i n 77% y i e l d  possessed the expected s p e c t r a l c h a r a c t e r i s t i c s .  Thus the mass spectrum showed  the expected parent peak at m/e = 261 while the N.M.R. spectrum exhibited the presence of the methylene o f the benzyl group (6 5.10,  s i n g l e t ) and the methylenes  of the c a r b o c y c l i c r i n g system (pair o f multiplets centered at 62.60 and 1.86). The absence of the N-H stretch s i g n a l i n the region o f 3500 cm  -1  i n the IR  spectrum was also i n d i c a t i v e o f reaction at the indole nitrogen. The 9-benzyl  derivative 217 was formed f o r three reasons.  sequent Vilsmeier-Haack  F i r s t l y , the sub-  formylation to be c a r r i e d out has been shown to proceed  to the desired l-methyl-3-formylcarbazole compound 219 only when the indole nitrogen i s protected by an a l k y l group i . e . methyl, benzyl, e t c . Secondly, having the indole nitrogen protected eliminates any undesirable side reactions that may occur during methylation o f the formyl or iminomethyl group under Grignard conditions.  T h i r d l y , the benzyl group i s p r a c t i c a l l y the  only a l k y l p r o t e c t i n g group which can be e f f e c t i v e l y removed i n the l a s t step to y i e l d o l i v a c i n e (16).  - 221 -  l-Methyl-3-formyl-9-benzylcarbazole  (219) .  Reaction o f 9-benzyltetrahydrocarbazole  (217) with 1.3 equivalents  of phosphorus oxychloride i n dimethylformamide (Vilsmeier-Haack conditions) proceeded as r e p o r t e d ^ 2  (219) .  4  to give the desired  l-methyl-3-formyl-9benzylcarbazole  The reaction mixture was p u r i f i e d by column chromatography on  Whereupon the aldehyde 219 was which was  subsequently  alumina.  recovered i n 48% y i e l d as a pure yellow o i l  c r y s t a l l i z e d to give pale yellow rosettes.  The N.M.R. spectrum of the product 219 possessed a s i n g l e t at 6 1 0 . 0 0 f o r the aldehyde proton as well as s i n g l e t s at 6 5.72  and 2.60  f o r the methylene  of the benzyl group and the C-l methyl groups r e s p e c t i v e l y .  The correct j u s t a -  p o s i t i o n of the methyl (C-l) and formyl (C-3) groups to each other was  clear  from the presence o f two d i s t o r t e d s i n g l e t s (meta coupling not resolved) a t 68.46 and 7.66  f o r the C-4 and C-2 hydrogens r e s p e c t i v e l y .  The absence of  any multiplet absorptions f o r the methylene protons c h a r a c t e r i s t i c of a t e t r a hydrocarbazole system confirmed the aromatic nature of the molecule. The UV. spectrum was  altered considerably from the normal carbazole  spectrum as a consequence of the extension of the carbazole chromophore by the formyl group. cm"*  The IR spectrum possessed a carbonyl absorption at  1670  for the aldehyde carbonyl i n conjugation with the aromatic r i n g .  The  mass spectrum displayed a very dominant parent peak at m/e very l i t t l e fragmentation.  = 299  The p o s i t i o n of the parent peak was  by the high resolution spectrum which had a peak at m/e within accepted l i m i t s of the calculated mass at m/e  (100%)  and  corroborated  = 299.1309 which was  = 299.1305 f o r  C21H17NO.  - 222 -  A s l i g h t l y less polar component eluted during column chromatography was  the only detectable side product of the r e a c t i o n .  i t completely  from the carbazole aldehyde 219 f a i l e d , however i t s N.M.R.  spectrum gave every i n d i c a t i o n that i t corresponded tetrahydrocarbazole (226). at  5.13  to l-formyl-9- benzyl-  Thus present i n the N.M.R. spectrum was a s i n g l e t  f o r the benzyl methylene and two multiplets at 6 2.60  methylenes of the tetrahydro r i n g . at 6 9.83  Attempts to separate  and 1.80 for the  The aldehyde proton absorption occurred  s l i g h t l y up f i e l d from the aldehyde absorption i n 219.  C H O  (226)  The presence o f the formyl tetrahydro compound 226 was  o f considerable  s i g n i f i c a n c e f o r although l i t t l e i s presently known about the mechanism o f 208 the alkylation-formylation-dehydration process, i t has been shown Vilsmeier-Haack  formylation of 6-chloro-9-methyltetrahydrocarbazole  under mild conditions produces as the reaction product methyltetrahydrocarbazole  (227)  l-formyl-6-chloro-9-  (228) and that subsequent further reaction of this  compound 228 at elevated temperature r e s u l t e d i n conversion to the formyl carbazole 229.  that  l-methyl-3-  - 223 -  Cl  Cl  ClN CHO  (226)  A  (227)  (228)  It i s apparent from this observation that i n the present reaction of 9-benzyltetrahydrocarbazole  (217) that the C - l methyl of the product aldehyde  219 i s also derived v i a an i n i t i a l C-l formylation of On mechanistic grounds i t was  anticipated that  217. l-methyl-6-formyl-9-  benzylcarbazole (230) would also be produced during the formylation r e a c t i o n . However a d e t a i l e d examination  of the aromatic region of the N.M.R. spectra  of respective column fractions gave no i n d i c a t i o n of i t s presence.  (230)  In l i g h t of these results a p l a u s i b l e mechanism can be put forward which suggests that both a l k y l a t i o n and formylation takes place before aromatization of the tetrahydro r i n g (Figure 56).  I f aromatization occurred f i r s t , then a  mixture of the 3- and 6-formyl carbazole compounds would have been  observed.  - 224 -  Figure 56.  Proposed Mechanism f o r the Vilsmeier Haack Formylation of 9Benzyltetrahydrocarbazole  (216).  - 225 -  l-Methyl-3-(g-hydroxyethyl) carbazole  (222)  To explore the route whereby the methyl group i n the p o t e n t i a l C - l p o s i t i o n of o l i v a c i n e (16) i s introduced before reaction with  aminoacetalde-  hyde d i e t h y l a c e t a l (171) the 3-formyl group of 219 was methylated with methylmagnesium chloride i n tetrahydrofuran.  The reaction proceeded  p l e t i o n (TLC) within minutes at room temperature the predominant product.  to com-  to y i e l d the alcohol 222 as  P u r i f i c a t i o n by column chromatography provided a  64% y i e l d of the desired product. The methylation reaction was  also accomplished using methyllithium instead  of the Grignard reagent, however i t offered no advantages i n terms of reaction time, y i e l d s , or ease of handling so i t was not employed r o u t i n e l y . Minor impurities were removed from the crude reaction mixture by rapid chromatography on alumina.  The alcohol 222 was obtained as a colourless foam  which subsequently proved to be extremely d i f f i c u l t to r e c r y s t a l l i z e .  I t was  eventually necessary to convert the a l c o h o l to i t s corresponding acetate i n order to obtain a s a t i s f a c t o r y microanalysis. The N.M.R. spectrum of this product exhibited a doublet at 6 1.55  (J *  6 H z ) f o r the b e n z y l i c methyl group and a corresponding quartet at 6 5.03  (J =  6 H z ) f o r the methine proton which i s both b e n z y l i c and adjacent to the oxygen of the hydroxyl group. ( C - I C H 3 ) , and  5.70  a parent peak at m/e (M - C H 3 ) and m/e  Three s i n g l e t s were also evident at 6 1.83  (N-CH2-C5H5) respectively. = 315  The mass spectrum  (OHJ),  possessed  (100%) and only two major fragments at m/e  = 297 f o r (M -H 0). 2  2.60  = 300 f o r  The high resolution mass spectrum  parent  - 226 -  peak at 315.1632 was within acceptable l i m i t s of the calculated mass o f 315.1623 f o r C H2iNO. 22  The absence of a carbonyl absorption at 1670 cm"^ i n the IR. spectrum was consistent with the proposed structure as was the UV. spectrum which exhibited a t y p i c a l carbazole absorption,  l-Methyl-3-(a-acetoxyethyl)  carbazole (223)  The acetate derivative 223 was prepared by reaction of the alcohol 222 with a c e t i c anhydride i n p y r i d i n e .  I t was obtained as a colourless c r y s t a l l i n e  s o l i d i n high y i e l d a f t e r c a r e f u l r e c r y s t a l l i z a t i o n from methanol.  The acetate  group proved to be somewhat l a b i l e however, as an overexposure to hot methanol resulted i n a substantial conversion o f the acetate to the methyl ether by solvent displacement  of the acetoxy group.  I t was also observed  (N.M.R.) that  small amounts of o l e f i n i c products accompanied formation and subsequent heating of the carbazole acetate  (section 6, part I I ) ,  The spectral data was very c h a r a c t e r i s t i c f o r the acetate derivative 223, The N.M.R. spectrum showed a c h a r a c t e r i s t i c downfield s h i f t i n the p o s i t i o n of the b e n z y l i c methine quarter to 66.00 ( J = 7Hz) due to the added deshielding influence of the carbonyl group.  The corresponding b e n z y l i c methyl group  removed from the influence of the acetate function absorbed at 61.56 ( J = 7Hz), Singlets were observed at 6 1.98 and 2.52 f o r the protons of the acetate methyl and the methyl group at C - l respectively.  A s i n g l e t was also observed at 6 5.68  f o r the methylene of the benzyl group substituted at the indole nitrogen. change i n the e l e c t i o n density o f the "C" r i n g i n the conversion o f the  The  - 227 -  conjugated aldehyde 219 to the non conjugated acetate 223 r e f l e c t e d i t s e l f i n the position of the absorptions f o r the C-2 ring.  and C-4 hydrogens of the  "C"  Both of these signals s h i f t e d u p f i e l d , the C-4 proton s i g n a l to 6 7.98  while that of the C-2 hydrogen had s h i f t e d to the region occupied by the large aromatic multiplet (centered at 6 7.10). The mass spectrum possessed a strong parent peak at m/e and a major fragment at m/e mentation was  = 357 (100%)  = 298 f o r loss of the acetate group.  accompanied by the presence of a metastable at m/e  corresponding to m/e  = 357 —• m/e  = 298.  Ibis frag= 249  The high resolution spectrum parent  peak at 357.1676 was within accepted l i m i t s of the value 357.1729 calculated for C 2 4 H 2 3 N O 2 . The UV spectrum portrayed a t y p i c a l carbazole absorption as anticipated and the IR spectrum has a carbonyl absorption f o r the acetate group at 1720  cm" . 1  l-Methyl-3-(a-(N-2* 2'-diethoxyethylamino)ethyl)carbazole >  (224) from acetate  223  In an attempt to synthesize the aminoethyl acetal 224 from the carbazole acetate 223 i t was  determined  that high temperature and prolonged heating  times were necessary i n order to induce displacement of the acetate function with the amino group of aminoacetaldehyde d i e t h y l a c e t a l (171).  Even a f t e r  f i f t e e n hours at 160° considerable s t a r t i n g material was present i n the reaction mixture which by t h i s time had turned very dark. By column chromatography on alumina a small quantity of material was  - 228 -  i s o l a t e d whose s p e c t r a l properties corresponded with the anticipated aminoethyl acetal 224 (see conversion (221) — > (224) f o r a description of spectral data). It was concluded i n view of the d r a s t i c reaction conditions that were necessary to provide a small percent conversion to product that the acetate f u n c t i o n a l i t y was not a s u f f i c i e n t l y good leaving group f o r t h i s purpose. However, instead of exploring the properties o f the corresponding bromide as a leaving group, i t was decided to study the methylation o f the iminomethyl acetal 221 as a means o f obtaining the same aminoethyl acetal  l-Methyl-3-(3,g-diethoxyethyliminomethyl)carbazole  224.  (221)  The aldehyde condensation reaction had already been developed i n the 175 synthesis of e l l i p t i c i n e (17)  and i t s application to the 3-formyl carbazole  intermediate 219 lacking one methyl group at the C-4 p o s i t i o n presented no difficulties.  Reaction of the aldehyde 219 with an equimolar quantity o f  aminoacetaldehyde  d i e t h y l a c e t a l (171) with the azeotropic removal o f the water  formed gave the iminomethyl acetal i n 85% y i e l d . The product was p u r i f i e d by r e c r y s t a l l i z a t i o n from a petroleum combination.  ether-benzene  An attempt to p u r i f y the product by column chromatography on  alumina resulted i n hydrolysis on the column of the imine bond to give back the s t a r t i n g aldehyde. The N.M.R. spectrum (Figure 57) o f the c r y s t a l l i n e product 221 showed a t r i p l e t at 64.81  (J = 5Hz) f o r the hydrogen of the acetal carbon (CH(OEt) ), a  second t r i p l e t at 6 1.14  2  ( J = 7Hz) f o r the two methyls o f the d i e t h y l a c e t a l and  a complex multiplet centered at 6 3.70  f o r the methylenes o f the acetal and f o r  - 229 -  o  neutral  220  Figure 58.  250  300  350  UV. Spectrum o f Compound 221 i n Water and i n Dilute Hydrochloric Acid.  nm 400  - 231 -  the methylene adjacent to the imine nitrogen. at 65.72 and 2.58  Two  s i n g l e t s were also present  f o r the benzyl methylene and the C - l methyl group r e s p e c t i v e l y .  A distorted singlet at 68.32 was  a t t r i b u t e d to the proton on the imine  while the singlets at 68.40 and 7.60  carbon  having moved downfield with respect to the  acetate 223 were assigned to the C-4 and C-2 hydrogens r e s p e c t i v e l y . The mass spectrum exhibited a parent peak at m/e mentations at m/e  = 369, 339, 311, 298, 284.  the major fragmentation processes, m/e  = 414 and important  frag-  Metastable peaks were observed f o r  = 414 -»• m/e  = 369 and m/e  = 311  m/e  =  284.  The high resolution mass spectrum corroborated the parent peak at 414.2250 and the molecular compositions f o r the postulated  fragments.  The presence of the conjugated imine system was detected by an absorption at 1690 cm-1  i n the IR. spectrum.  The u l t r a v i o l e t spectrum of 221 run i n water  and d i l u t e hydrochloric acid showed the expected bathochromic s h i f t accompanying the transformation C = N -> C =  NH . +  l-Methyl-3-(a-(N-B,B-diethoxyethylamino)ethyl)carbazole  (224) •  Methylation of the imine 221 as with the formyl compound 219 could be effected through reaction with methylmagnesium chloride i n tetrahydrofuran.  The  less  reactive nature of the imine system however, necessitated reaction at r e f l u x temperature f o r twenty-four hours. remained.  Even a f t e r prolonged heating some s t a r t i n g material  Three major products were observed on TLC with the amino acetal  being the most polar.  224  The three components were e f f i c i e n t l y separated from each  other by column chromatography on  alumina.  - 232 -  The least polar component (bright orange spot on TLC) eluted with benzene remains uncharacterized.  The N.M.R. spectrum possessed a quartet (63.93, J =  6 Hz) and doublet (61.36, J = 6 Hz) f o r the C - l * methine and C - l methyl group 1  which indicated that the molecule was a 3-(a-substituted ethyl)carbazole derivative.  The nature o f the substituted group could not be discerned from the  N.M.R. o r the IR spectrum.  The mass spectrum possessed a parent peak at m/e = 368  with a major fragment at m/e = 299 f o r the loss of the unknown substituent. It was not known which f u n c t i o n a l i t y was substituted onto the C - l ' p o s i t i o n of the unknown carbazole component with a mass o f 69 and no c h a r a c t e r i s t i c absorpt i o n s i n the N.M.R. The second component i s o l a t e d by e l u t i o n with benzene corresponded t o the aldehyde 219.  The i s o l a t i o n of t h i s compound resulted from the presence o f  unreacted s t a r t i n g material i n the crude reaction jnixture which hydrolyzed during the column chromatography. The desired amino-acetal 224 was i s o l a t e d i n 20% y i e l d by elution with benzene:chloroform mixtures and chloroform.  The N.M.R. spectrum f o r 224 possessed  a multiplet at 68.14 f o r the C=5H and a s i n g l e t at 67.98 f o r the C-4 H. The s i n g l e t f o r the C-2 H had moved u p f i e l d i n t o the region o f the aromatic multiplet compared to the p o s i t i o n o f the C-2 H i n the imino a c e t a l 221.  A quartet was  present at 63.95 (J = 6 Hz) f o r the methine hydrogen on the alkylated C - l ' carbon. The doublet f o r the corresponding C - l ' methyl group occurred at 61.47 6 Hz).  (J »  The doublet (62.69, J = 6 Hz) f o r the methylene group of the diethoxy-  ethylamino group i n 224 had s h i f t e d u p f i e l d from i t s p o s i t i o n under the multiplet for the methylenes o f the 0-ethyl groups i n compound 221.  The signals f o r the  - 233 -  two methyls of the O-ethyl groups were very complex compared to the t r i p l e t for these groups i n the imino acetal 221. The mass spectrum possessed a parent peak at m/e mentation peaks at m/e  = 415, 369, 298, 288.  possessed a parent peak at m/e of the value m/e  = 430 with major f r a g -  The high resolution mass spectrum  = 430.2633 which was within acceptable l i m i t s  = 430.2620 calculated f o r C 2 8 H 3 4 N 2 O 2 .  No attempt was made to improve the reaction y i e l d e i t h e r by changing the solvent, or the Grignard reagent or by conducting the reaction f o r a longer period of time. The c y c l i z a t i o n of the amino acetal 224 to 6-benzoolivacine (225) was not attempted at t h i s time. the analogous  I t was known however that o-phosphoric acid e f f e c t e d  c y c l i z a t i o n i n the synthesis o f e l l i p t i c i n e  (30% y i e l d ) .  I t would  be anticipated therefore that the c y c l i z a t i o n reaction would also work f o r the o l i v a c i n e system  (16).  There i s the p o s s i b i l i t y however, which does not e x i s t  for e l l i p t i c i n e , that c y c l i z a t i o n can occur i n either of two directions to give compounds 225 or 231. determined.  Whether or not t h i s w i l l be a problem remains to be  The subsequent  debenzylation of 225 to o l i v a c i n e (16) would be a  t r i v i a l step.  (16)  (231)  - 234 -  This synthesis has the p o t e n t i a l of being a short and simple route to o l i v a c i n e (16) which could r e a d i l y be adapted to a large scale preparation of t h i s material f o r future biosynthetic work.  By minor modifications i t would be  possible to obtain quantities of guatambuine  (25) a l s o .  - 235  -  EXPERIMENTAL - PART III  For a description of the general experimental information, Experimental part  see  1.  A l l T.L.C. plates were developed i n chloroform (CHCI3) unless otherwise indicated, and the alumina for column chromatography deactivated  was  to A c t i v i t y III by the addition of water (6%).  Sequence A Indole -3-glyoxylic acid chloride  (21l)  To a s t i r r e d solution of Indole (25 gm., ether (400 ml) at 0° was (31.2 was  ml, 3.64  x 10  1  2.14  x 10"  1  mole) i n anhydrous  added an ether solution of o x a l y l chloride  mole) over a 0.5  hr period.  A bright yellow p r e c i p i t a t e  produced almost immediately, however the reaction was  for 0.5 hr a f t e r addition was  completed.  (210)  The product was  allowed to continue c o l l e c t e d by vacuum  f i l t r a t i o n , and washed l i b e r a l l y with dry ether to give the a c i d chloride as yellow crystals (41 gm. H,3.10,N,6.38%.  93%), m.p  Calc. f o r C H 0 NC1: 10  Methyl indole-3-glyoxylate  dry methanol.  2  Found:  C,58.13;  C,58.40; H,2.92; N,6.81%  (212)  To the acid chloride 211 hydrofuran (500 ml)  6  123-126*  (39gm.,1.88 x 10"  1  mole) dissolved i n dry t e t r a -  and s t i r r e d at room temperature was  added a large excess of  A beigh p r e c i p i t a t e formed almost immediately, however the  reaction  - 236 -  was  allowed  to s t i r for 0.5 hr.  f i l t r a t i o n (29 gm.)  The p r e c i p i t a t e was  c o l l e c t e d by suction  and washed l i b e r a l l y with tetrahydrofuran.  liquors were concentrated and the b r i g h t pink s o l i d obtained washed also with tetrahydrofuran.  The mother  (6 gm.)  was  The s o l i d products were combined and  r e c r y s t a l l i z e d from methanol-tetrahydrofuran  to give the ester 212 as beige  coloured crystals (35 gm.,  227-8°.  H,4.44; N,6.62%.  92% y i e l d ) , m.p.  Calc. f o r C H N 0 3 : n  9  Found:  C,64.76;  p  C.65.02; H,4.46; N.6.89%.  190 Indole-3-ethanol  (Tryptophol)  (206)  The glyoxylate ester 212 (20.7 gm.,  1.0 x 10"  1  moles) was  dissolved i n  dry tetrahydrofuran (600 ml) under gentle r e f l u x and under a nitrogen atmosphere (mechanical s t i r r e r ) . moles) was  Lithium aluminum hydride (7.6 gm.,  2.0 x 10"  added to the r e f l u x i n g solution i n small portions e i t h e r as a  powder or as a s l u r r y i n tetrahydrofuran.  After addition was  further quantity of tetrahydrofuran (200 ml) was The reaction mixture was reaction vessel was  completed, a  added, f i n a l volume (800 ml).  refluxed gently f o r 15 hr. a f t e r which time the  cooled i n i c e and the excess  lithium aluminum hydride  destroyed by the sequential addition of water (8 ml), 15% sodium hydroxide (8 ml), and water (24 ml).  The resultant granular p r e c i p i t a t e was  removed by  suction f i l t r a t i o n and washed twice with tetrahydrofuran (2 x 200 ml).  The  combined tetrahydrofuran solutions were concentrated to 50 ml and taken up i n water (600 ml). ml p o r t i o n s ) .  The aqueous mixture was  extracted repeatedly with ether  (200  The ether fractions were combined, washed with water, dried over  - 237 -  sodium sulphate, and concentrated to dryness  (benzene azeotrope) to give a  colourless c r y s t a l l i n e product (9.6 gm., 60%). p u r i f i e d by column chromatography on alumina  The crude product was  (100 gm.).  The product was  eluted with chloroform to give colourless c r y s t a l s , m.p. = N.M.R. (60 MHz): 6Hz2H,C-l,  3.86 ( t r i p l e t , J = 6Hz,2H,C-2,  CH ),  ai ), 2  0  (lit °).  2.96 ( t r i p l e t , J =  1.56 (Singlet, 1H, 'OH)  2  192 Indole-3-(2-ethylbromide)  (Tryptophyl Bromide)  (207)  A solution of phosphorous tribromide (2ml,2.07 x 10"  2  mole) i n ether  (25 ml.) was added dropwise over 0.5 hr. to a solution of tryptophol (206) (10 gm. 6.22 x 10"  2  mole) i n ether (200 ml.) at 0 ° .  The reaction mixture was  s t i r r e d at 0°C for 15 hr. after which time the supernatant was decanted, the red syrupy residue was washed with several portions of ether (100 ml), and the combined ether extracts were washed with saturated sodium bicarbonate and water. The ether layer was  dried over sodium sulphate and concentrated (benzene azeotrope)  to give a colourless crude c r y s t a l l i n e product (12.5 gm., 91%).  The crude  product was p u r i f i e d by e i t h e r r e c r y s t a l l i z a t i o n from benzene:hexane or column chromatography on alumina (200 gm.),  eluting with benzene.  obtained as colourless c r y s t a l s , m.p. = 3.40  (multiplet,4H,C-l and 2CH ). 2  0  (lit.  Mass spectrum:  3-Carbomethoxy-5-(3-indolyl)-2-pentanone  The bromide was  °) . N.M.R. (60  MHz):  M,m/e = 223,225. +  (204)  Methyl acetoacetate (205) (12.66 ml, 1.18 x 10"  1  mole) was added in four  portions to a suspension o f sodium hydride (2.88 gm., 1.20 x 10"  1  mole)  - 238 -  (prepared by washing a 57% o i l dispersion (5.05 gm.) dimethylformamide (500 ml).  with benzene) i n  The mixture was s t i r r e d at room temperature  u n t i l gas evolution had ceased and a clear solution was obtained (about 1 hr.).  Tryptophyl bromide  (207) (12.3 gm.,  5.5 x 1 0  -2  moles) was then added  i n one portion and the reaction was s t i r r e d at 100°C f o r 3.5 hours under a nitrogen atmosphere. reaction.  A white p r e c i p i t a t e was formed during the course of the  The cooled reaction mixture was d i l u t e d with water (700 ml) and  extracted repeatedly with ether.  The combined ether extracts were washed  with water, dried over sodium s u l f a t e and concentrated to give an amber coloured viscous o i l (11.5 gm. 80%).  The crude product was e i t h e r c y c l i z e d  d i r e c t l y o r p u r i f i e d by column chromatography on Alumina (500 gm.).  Elution  of the column with benzene removed a l l contaminants and subsequent e l u t i o n with benzene:chloroform 1:1 and f i n a l l y with chloroform y i e l d microanalytic a l l y pure 204 as a yellow o i l (7.90 gm., b.p. = 135+10  at 0.05 m.m.  From column chromatography on a small scale  (1-2 gm) t y p i c a l y i e l d s were 70-75%. X  m a x  ( l o g e):  8.04  50% from tryptophyl bromide (207) ),  IR. ( C H C 1 ) : 3  290 (4.30), 280(4.37), 272(4,34).  3500, 1750, 1725.  N.M.R. (see Figure 51):  (broad s i n g l e t , IH, N-H), 7.6-7.0 (2 m u l t i p l e t s , 4H, aromatic),  (Spike, IH, C-2H), 3.66 CH), 2.76  (Singlet, 3H, C O O C H 3 ) , 3.48  ( t r i p l e t , J = 7 H z , 2H, C-5 CH ), 2.28 2  (Singlet, 3H, C O C H 3 ) .  Mass spectrum:  144,143 (base peak), 130. 259.1207.  Found:  M, +  m/e  Found:  6.94  ( t r i p l e t , J = 7 H z , IH, C-3  (multiplet, 2H, C-4 CH )» 2  2.12  = 259; main peaks: 228, 186, 184,  High resolution mass spectrum:  259.1212.  UV.  calc. for C 1 5 H 1 7 O 3 N :  C, 69.36; H,6.52; N,5.20%.  Calc f o r  - 239 -  C H 0 N: 1 5  1 7  3  C, 69.48; H,6.61; N, 5.40%  C y c l i z a t i o n Reaction to (134) and (209) A  To a s o l u t i o n o f the a l k y l a t i o n product 204 (7.90 gm., 3.05 x 1 0 "  2  mole)  i n methanol  ( 1 0 0 ml.) at room temperature was added an equal volume of methanol  - HCL ( 5 % ) ,  and the reaction mixture was allowed to s t i r f o r one minute  concentration 2 . 5 % ) .  HC1  (final  On mixing, the reaction was exothermic and accompanied  by a change to a darker colour.  The solvent was then removed to give a dark  foam (7.15 gm., 98%) c o n s i s t i n g o f an equal mixture o f 1-methyl-2-carbomethoxycarbazole (134) and l-methyl-2-carbomethoxy-l,2,3,4-tetrahydrocarbazole (209)' IR.  (CHC1 ): 3  3480, 1740-1680.  (see Figure 53):  UV; A  m a x  8.44 (broad s i n g l e t ,  aromatic protons), 3.84 ( s i n g l e t ,  340, 303, 295 (shoulder), 248. N.M.R.  :  1H,  N-H),  3H, COOCH3),  8.0-7.0  (2  multiplets,  3.68 ( s i n g l e t ,  (Singlet, 3 H , CH ), 1 . 0 0 , 1.15 ( 2 doublets, J = 7 H z , 3 H , C H 3 ) . 3  M-, m/e = 243, 239 (base peak); main peaks: 169, 168, 157, 143, 130. 1 5  B_  2  2.67  Mass spectrum:  Found:  calc. for  C15H17O2N:  243.1248 and 239.0933.  The i d e n t i c a l procedure was followed f o r the c y c l i z a t i o n o f impure  a l k y l a t i o n material. 10"  1 3  3H, COOCH3),  m/e = 228, 224, 2 1 2 , 208, 184, 180,  High resolution mass spectrum:  243.1258, and f o r C H 0 N : 239.0946.  11H,  2  In t h i s manner the a l k y l a t i o n product ( 1 1 . 5 gm., 4.4 x  mole) was reacted with methanol-HCl  compounds 134 and 209  (10.12  (2.5%)  to give an equal mixture o f  gm., 88%). The spectral data were i d e n t i c a l to  those quoted i n part A.  Separation o f Compounds 134 and 209 R e c r y s t a l l i z a t i o n o f the crude mixture o f compounds 134 and 209 (7.15 gm.)  - 240 -  from concentrated chloroform solution afforded pure compound 1 3 4 as colourless crystals.  Column chromatography  o f a small portion of the mixture (500 mg.)  on alumina (20 gm.,) using benzene gave impure compound 209 i n the f i r s t and pure compound 134 i n subsequent fractions (about 200 mgs.,  40%).  fraction  The l a t t e r  component was r e c r y s t a l l i z e d from chloroform to give colourless crystals o f l-methyl-2-carbomethoxycarbazole  (134) with m.p. 138-140°, ( l i t . , m.p. 126-127°  for ethyl e s t e r ) .  3425, 1710.  IR. (nujol):  303(4.38), 246(4.69). Mass spectrum:  N.M.R. (F.T.):  (log ) :  m a x  £  m/e = 224, 208, 180,  +  c a l c . for C 1 5 H 1 3 O 2 N :  C,75.15; H,5.38; N,5.77%.  340(3.62),  3.84 (Singlet, C O O C H 3 ) , 2.70 (Singlet, C H 3 ) ,  M , m/e = 239 (base peak); main peaks:  High resolution mass spectrum: Found:  UV. A  239.0946.  Calc. f o r C H 0 N : 15  By preparative layer chromatography  13  2  Found:  239.0948,  C,75.30; H,5.48; N,5.85%.  using alumina plates ( 1 m.m.) (Benzene;  chloroform 1:1) a small quantity of l-methyl-2-carbomethoxy-l,2,3,4-tetrahydro* carbazole (209) was i s o l a t e d as an o i l i n about 90% p u r i t y . 282, 275, 3.38  N.M.R. (F.T.):  (multiplet, C - l CH).  UV.; A  m a x  : 290,  3.96 multiplet, C - l CH), 3.68 (Singlet, C O O C H 3 ) , Mass spectrum:  M*, m/e = 243; main peaks:  228, 212,  184 (base peak), 169, 168, 157, 144, 143, 130.  1-Methy1-2-carbomethoxycarbazole  (134)  The c y c l i z a t i o n mixture o f compounds 134 and 209 (7.15 gm., 2.98 x 10~  2  mole) was dissolved i n m-xylene (300 ml.) and c h l o r a n i l (7.15 gm., 2.90 x 10" mole) was added.  The resultant dark solution was refluxed for 24 hr. a f t e r  which time i t was d i l u t e d with water (300 ml) and washed repeatedly with 10% sodium hydroxide solution to remove dark coloured contaminants. The  2  - 241 -  organic layer was then washed with water (3 x 200 ml.), dried over sodium sulphate and concentrated to give a dark c r y s t a l l i n e produce (6.5  gm.),  R e c r y s t a l l i z a t i o n from chloroform gave grey coloured c r y s t a l s of ester (2.6 gm.).  Column chromatography o f the mother l i q u o r s on alumina (200  using benzene yielded the ester colourless  as a yellow o i l which c r y s t a l l i z e d to  crystals on standing (2.7 gm.), m.p.  of (5.3 gm.,  81%) was obtained.  IR. (nujol):  340 (3.62), 303 (4.38), 246 (4.69). 2.70 m/e  (Singlet, CH3). Mass spectrum: = 224, 208, 180.  C H 0 N: 1 5  1 3  2  Found:  gm.)  138-140°. 3425, 1710.  N.M.R. (F.T.): M, +  m/e  3.84  An o v e r a l l y i e l d UV.;A  m a x  (log§:  (Singlet, COOCH3),  = 239 (base peak); main peaks:  C, 75.53: H, 5.65; N, 5.48%, Calc. f o r  C, 75.30; H, 5.48; N, 5.85%.  C y c l i z a t i o n - Dehydrogenation of 204 to 1-Methyl-2-carbomethoxycarbazole (134). A solution of methanolic-HCl (2.0%) (10 ml.) was added dropwise over several minutes to a s t i r r e d solution of the a l k y l a t i o n product 204 -3 (1.0 gm.,  3.86 x 10  mole) and c h l o r a n i l (1.5 equiv.) i n benzene  (15 ml.).  The reaction mixture was s t i r r e d at room temperature f o r an additional 5 min. a f t e r which time i t was concentrated to dryness redissolved i n chloroform (50 ml.) and washed successively  with 10% aqueous sodium  hydroxide (5 x 30 ml.) and water (3 x 30 ml.).  The chloroform  layer  was dried over sodium sulphate and concentrated to give l i g h t brown  - 242 -  crystals.  R e c r y s t a l l i z a t i o n from concentrated chloroform solution  yielded 204 as colourless c r y s t a l s (774 mg., 84%), m.p. 138-140°. The spectral data obtained f o r 134 obtained from 204 was i d e n t i c a l to that previously described.  l-Methyl-2-formylcarbazole (152) 203 Jones reagent  (5M) was t i t r a t e d into a s o l u t i o n o f the carbazole -3 '  alcohol 157 (1.35 gm., 6.4 x 10  mole) dissolved i n acetone at room  temperature u n t i l a p r e s i s t e n t l y red coloured reaction mixture was obtained (about 1.5 equiv.).  The reaction was s t i r r e d f o r an additional  f i v e minutes a f t e r which time methanol was added u n t i l a green colouration was obtained.  The reaction mixture was then suction f i l t e r e d  and the residue washed with copious amounts o f methanol.  The f i l t r a t e  was concentrated, taken up i n water and exhaustively extracted with ether.  The ether extracts were dried over sodium sulphate, concentrated  and applied to an alumina column (10 gm.). The product was eluted with chloroform to y i e l d yellow c r y s t a l l i n e needles  (1.00 gm., 70%).  R e c r y s t a l l i z a t i o n from chloroform afforded an a n a l y t i c a l sample, m.p. 165166° ( l i t . , m.p. 164-164.5). 365 2.87  8  IR. (nujol):  (3.52), 317 (4.42), 255 (4.63). ( s i n g l e t , CH^).  Mass spectrum:  1665. UV.; Amax (log e):  N.M.R. (FT.):  10.39 (singlet, CHO),  M , m/e = 209 (base peak); +  180, 152. High resolution spectrum:  Calc. f o r C  Found:  H, 5.30; N, 6.39%.  209.0831.  C^H^NO:  Found:  C, 80.36;  C, 80.08;  H, 5.30; N, 6.69%.  H 1 4  N 0 : i ; l  main peaks:  209.0840. Calc. f o r  - 243 -  1-Methyl-2-hydroxymethy1carbazole (157) A  -2 The Carbazole ester 134 (6.6 gm., 2.77 x 10 mole) dissolved i n  anhydrous ether (100 ml.) was added dropwise over a period o f 0.5 hr. to a _2 s l u r r y of lithium aluminum hydride (2 gm., 5 x 10 (250 ml.) at room temperature.  mole) i n anhydrous ether  Large aggregate lumps of lithium aluminum  hydride formed during the addition.  The reaction was s t i r r e d f o r an  additional 0.5 hr. a f t e r which time excess hydride reagent was destroyed by the addition of water (2 ml.), 15% sodium hydroxide water (6 ml.).  (2 ml.) and f i n a l l y  The resultant granular p r e c i p i t a t e was removed by suction  f i l t r a t i o n and washed l i b e r a l l y with ether.  The combined ether f r a c t i o n  was washed with water (3 x 100 ml.), dried over sodium sulphate, and concentrated to give a colourless c r y s t a l l i n e s o l i d (5.26 gm., 90%). R e c r y s t a l l i z a t i o n from chloroform gave an a n a l y t i c a l sample o f the alcohol 15 7 with m.p. 184 - 186° ( l i t . , m.p. 187 - 188°). 3400-3325.  UV.;  Amax (log e):  2  3  m/e = 211 (base peak);  3420,  335 (3.63), 324 (3.68), 294 (4.36), 285  (4.16), 257 (4.33), 247 (4.56), 238 (4.73). Ar-CH 0H), 2.58 (singlet, CH ),  IR. (nujol):  N.M.R. (FT.):  4.88 ( s i n g l e t ,  1.50 ( s i n g l e t , OH). Mass spectrum:  main peaks:  M, +  194, 180, 167. High r e s o l u t i o n mass  spectrum:  Calc. f o r C^H^NO;  211.0996.  Found:  C, 79.63;  H, 6.49; N, 6.37%.  Calc. f o r C^H^NO:  211.0982. C, 79.59;  Found: H, 6.20;  N, 6.63%. -2 Impure carbazole ester 134 (9.3 gm., 3.89 x 10 alcohol 157 i n an i d e n t i c a l manner.  mole) was reduced to the  The dark c r y s t a l l i n e material (7.2 gm.)  - 244 -  t h a t was o b t a i n e d was p u r i f i e d by column chromatography on a l u m i n a E l u t i o n with benzene:chloroform yielded  the alcohol  (350 gm.).  1:1 f o l l o w e d by chloroform:methanol  157 as c o l o u r l e s s  crystals  5%  (4.9 gm., 6 0 % ) ,  209 l - M e t h y l - 2 - ( c o - n i t r o v i n y l ) c a r b a z o l e (160) _2 The  aldehyde  nitromethane was heated material  152  (2.50 gm., 1.19 x 10  (20 m l . ) , ammonium a c e t a t e (0.50 gm.) was added and t h e m i x t u r e  a t 100°C f o r 0.5 h r .  first  precipitated  dissolved  out.  During the course o f h e a t i n g the s t a r t i n g  then an orange p r e c i p i t a t e  The c o o l e d r e a c t i o n  (2.62 gm.).  with chloroform yielded  o f orange c r y s t a l l i n e p r o d u c t i z a t i o n from e t h a n o l  375  e t h a n o l and d r i e d  f i l t e r e d and t o g i v e orange  C o n c e n t r a t i o n o f t h e mother l i q u o r s , d i s s o l u t i o n i n  water and r e p e a t e d e x t r a c t i o n  (lit.,  o f product  m i x t u r e was s u c t i o n  t h e p r o d u c t o b t a i n e d was washed w i t h c o l d needles  mole) was t a k e n up i n  further  (0.230 gm., o v e r a l l y i e l d  95%).  quantities Recrystall^  (250 ml/gm.) gave l o n g c r i m s o n n e e d l e s , m.p.  m.p. 244-247°).  IR. ( n u j o l ) :  3340, 1360.  (4.39), 292 (3.87), 248 (4.50), 237 (4.46).  UV.;  Amax  N.M.R. ( F T . ) :  242-244°  (loge): 8.51  ( d o u b l e t , J = 14 Hz, -CH=CH-N0 ), 7.59 ( d o u b l e t , J = 14 Hz, -CH=CH-N0 ), 2  2.64 205  2  ( s i n g l e t , CH^). Mass spectrum: (base p e a k ) ,  252.0938. Calc.  for C  180.  Found: 1 C  H  1 0  252.0905.  N 0 : 0  High r e s o l u t i o n  0  Found:  C, 71.42;  M, +  m/e = 252;  spectrum: C, 71.70;  H, 4.79;  main peaks:  Calc,  for Cj^H^N^^  H, 4.97;  N, 11.10%.  220,  N, 10.82%,  - 245 -  l-Methyl-2-(g-aminoethyl)carbazole  (139)  This compound was prepared according to the procedure developed  by  177 Moster et ad. -2 A solution of the nitrostyrene 160 (2.85 gm., tetrahydrofuran (50 ml.) was  and the hydride was  S t i r r i n g was  mole) i n  i n tetrahydrofuran (250  continued f o r an additional 20  ml.)  min.,  then decomposed by the successive addition of water  (2 ml.), 10% aqueous sodium hydroxide resultant granular p r e c i p i t a t e was (200 ml.).  x 10  added dropwise over 0.5 hr. to a s t i r r e d  suspension of lithium aluminum hydride (2 gm.) at room temperature.  1.13  (6 ml.), water (2 ml.).  The  f i l t e r e d and washed with tetrahydrofuran  The combined f i l t r a t e s were concentrated to a paste, taken up  i n ether (75 ml.) and washed with water (3 x 30 ml.).  The ether layer  was  dried over sodium sulphate and concentrated to give 139 as a s l i g h t l y yellowed c r y s t a l l i n e s o l i d (2.52 gm.,  95%). UV.,  257,245,236.  4.76  3H, C - l CH,).  N.M.R. (for HC1 s a l t ) : Mass spectrum:  M+,  m/e  =  X^:  335,320,293,284(sh)  (singlet, 2H, NHz), 224.  2.40  (singlet,  - 246 -  l-Methyl-2-(B-(N-acetylamino)ethyl)carbazole (140) This compound was prepared following the procedure developed by 172 Schmutz and Wittwer. _2 The amine 139 (2.52 gm., 1.12 x 10  mole) was dissolved i n a  mixture of pyridine (30 ml.) and acetic anhydride (3 ml.) and s t i r r e d at 60° f o r 0.5 hr.  The solvent was removed under vacuum and the residue  was dissolved i n ether (75 ml.) and washed with d i l u t e hydrochloric acid (3 x 30 ml.) and water (3 x 30 ml.).  The ether layer was then dried  over sodium sulphate and concentrated to give 140 as a colourless c r y s t a l l i n e s o l i d (2.93 gm., 95%). 245, 238. IR. (CHCI3):  UV.; X  1660 cm" . 1  m a x  N.M.R:  :  335, 320, 294, 283 (sh), 257,  5.56 (broad hump, 1 H, NHCOCH3);  3.46  ( t r i p l e t , 2 H, J = 6 Hz, ArCH CH N ) , 3.00 (multiplet, 2 H, A r O ^ O ^ N ) ,  2.46  (singlet, 3 H, C - l CH ), 1.93 ( s i n g l e t , 3 H, COCH3).  2  3  m/e = 266; main peaks:  mass spectrum: M+,  223, 220, 207 (base peak), 193, 180, 167.  3,4-Dihydro-l,5-dimethyl-6H-pyrido-(4,3-b)carbazole  (141) .  Phosphorous oxychloride (9 ml.) was added to a solution of the amide 140 (1.45gm., 5.45 x 10" mole) i n hot toluene (250 ml.). 3  The reaction mixture was  refluxed f o r 1 hr. a f t e r which time the solvent was removed to give a green cry-  s t a l l i n e s o l i d which was extracted with hot d i l u t e hydrochloric acid (3 x 250 ml.) Insoluble residues were removed by suction f i l t r a t i o n .  The combined acid f r a c -  tions were b a s i f i e d to pH 11 with concentrated ammonium hydroxide and extracted with chloroform (4 x 300 ml.).  The combined chloroform extracts were dried over  - 247 -  sodium sulphate and concentrated to give brown c r y s t a l l i n e m a t e r i a l . The crude product was r e c r y s t a l l i z e d from chloroform or chloroform:methanol  mix-  tures to give 3,4-dihydroolivacine (141) as colourless c r y s t a l s (0.85 gm., 63%), m.p. 310°  ( l i t . , m.p. 300-315°).  UV. (H 0 $ aq HC1); 2  X  m a x  log (e): 370  (3.93), 340 (3.71), 324 (3.84), 310 (4.15), 290 (sh) (4.35), 280 (4.56), 273 (4.54), 245 (4.36), 234 (4.44). N.M.R.: 2.60  UV. (aq. NaOH); X  3.76 ( t r i p l e t , 2 H, J = 6Hz, C-4 CH ), 2  (distorted s i n g l e t , 3 H, C - l CH ), 3  spectrum: Found:  m a x  N, 10.99.  283, 272, 245, 236.  2.90 ( t r i p l e t , 2 H, C-3 CH ), 2  2.50 ( s i n g l e t , 3 H, C-5 CH ). 3  Calc. f o r C  Mass  233, 220-217, 204, 191.  M*, m/e = 248 (base peak); main peaks:  C, 82.14; H, 6.55;  :  1 7  H  1 6  N : 2  C, 82.22; H, 6.49;  N, 11.28.  The imine 141 was converted to i t s methiodide  142 i n quantitative y i e l d  by reaction with methyl iodide i n methanol:chloroform ture f o r 4-5 hr. m.p.  The pale orange methiodide  > 300° ( l i t . , m.p. 320°).  UV. (H 0)  solution at room tempera-  142 was r e c r y s t a l l i z e d from methanol,  (Figure 38); X ^ t l o g e ) :  2  308  (4.32), 300 (4.17), 279 (4.61), 265 (sh) (4.37), 225 (4.48).  X  : 365, 335, 310, 294,  m  C-ll  280, 270 (sh), 243, 237.  H), 3.98 (multiplet, 2 H, C-4 CH ), 2  2 H, C-3 CH ), 2  8.86 (singlet, 1 H, 3  3  N, 7.18.  UV. (aq. NaOH);  3.68 ( s i n g l e t , 3 H, N-CH ), 3.20 (multiplet  2.90 (singlet, 3 H, C - l CH ),  C, 55.00; H, 4.90;  N.M.R.:  365 (4.33)  2.48 (singlet, 3 H, C-5 CH ).  Calc. f o r C H N ~ I : 18  1R  3  C, 55.40; H, 4.87;  Found:  N, 7.06.  - 248 -  l,5-dimethyl-l,2,3,4-tetrahydro-6H-pyrido-4(,3-b)carbazole  (25)  (Guatambuine) A.  The methiodide  142 (100 mg.,  2.64  x 10~  mole) was dissolved i n  4  methanol (20 ml.) and the solution was hydrogenated  at room temperature  atmospheric pressure over platinum oxide (Pt02» 20 mg.) ture was f i l t e r e d to remove the catalyst which was additional methanol (10 ml.). give a pale yellow s o l i d .  f o r 2 hr.  and  The mix-  c a r e f u l l y washed with  The combined f i l t r a t e s were concentrated to  The product was  obtained as a colourless c r y s t a l l i n e  s o l i d by f i l t r a t i o n through a small alumina column (CHCl :MeOH 5%).  A l l analy-  3  t i c a l and spectral data i s presented below. B.  The methiodide  142 (250 mg.,  6.17  x 10"  mole) was dissolved i n aqueous  4  ethanol (100 ml.) and reacted with an excess o f sodium borohydride at room temperature  f o r 2 hr.  The reaction mixture was then concentrated, taken up i n  water (75 ml.) and extracted with chloroform (3 x 30 ml.).  The combined  chloroform fractions were dried over sodium sulphate, and concentrated to give colourless c r y s t a l s (160 mg.,  98%).  The product was r e c r y s t a l l i z e d from 128  methanol to give 25 as cream coloured cubes m.p. UV.;  (log e):  (4.36), 250  (4.50), 240  C-l H), 2.40  (3.48), 327 (4.64).  3  3  Mass spectrum:  High resolution mass spectrum:  264.1626.  Found:  C H N :  C, 81.78; H, 7.63;  1 8  2 0  2  264.1656.  (3.63), 297  N.M.R. (FT.):  (singlet, 3 H, C-5 CH ),  J = 7 Hz, C-l CH ). 204.  341  Found:  248-250° ( l i t . ,  248-250°).  (4.26), 288 (sh) (4.08), 3.90  (quartet, 1 H, J = 7 Hz,  ( s i n g l e t , 3 H, N-CH ), 1.52  (doublet, 3 H,  3  M+,  m/e  = 264; main peaks:  Calc. f o r C j g ^ o ^ :  249, 233, 221-218,  264.1626.  C, 81.55; H, 7173; N, 10.76.  N, 10.60.  262  Found:  Calc. f o r  - 249 -  l,5-dimethyl-6H-pyrido-(4,3-b)carbazole  (16)  (Olivacine)  Dihydroolivacine (83) (30 mg., 1.20 x 10~ mole) was suspended i n deca4  lin hr.  (4 ml.) containing 5% palladium on charcoal (50 mg.) and refluxed f o r 1.5 The reaction mixture was cooled to 0° and the solvent was removed by  suction f i l t r a t i o n .  The residue containing the o l i v a c i n e (16) was taken up  i n hot chloroform (200 ml.) and r e f i l t e r e d .  The washing procedure was repeated  (3 x 200 ml.) and the combined chloroform f i l t r a t e s were concentrated to give an orange coloured s o l i d .  The crude product mixture was r e c r y s t a l l i z e d from  methanol to give o l i v a c i n e (16) as bright yellow needles (20 mg., 70%), m.p. 126 318-324° ( l i t . , m.p. 318-326°). 291  UV.; V a x ( 2 ) lo  e  :  3  7  ( - )»  4  3  (4.84), 275 (4.72), 265 (4.57),235 (4.33), 222 (4.40).  56  3  2  5  ( - 6), 3  N.M.R. (FT.):  (singlet, 3 H, C - l CH ), 2.82 ( s i n g l e t , 3 H, C - l CH ). Mass spectrum: 3  3  m/e = 246. High resolution mass spectrum: Found:  246.1165.  Found:  Calc. f o r C-^H^^:  C, 82.46; H, 5.94; N, 11.20.  C, 82.90; H, 5.73; N, 11.37.  7  3.16  M+,  246.1156.  Calc. f o r  C J J H J ^ ^ :  - 250 -  Sequence B.  1,2,3,4-tetrahydrocarbazole  (216)  This compound was prepared according to the procedure of Rogers and 207 Corson.  Cyclohexanone (98 gm.,  1.0 mole) and phenylhydrazine (108  gm.,  1.0 mole) were reacted i n a c e t i c acid (360 ml.) at r e f l u x temperature f o r 1 hour.  The reaction mixture was then cooled to about 5° and the c r y s t a l l i n e  mass which p r e c i p i t a t e d was  c o l l e c t e d by suction f i l t r a t i o n .  The f i l t e r cake  was washed with water (100 ml.), then with 75% ethanol (100 ml.) and dried under vacuum.  The crude product was  c r y s t a l l i z e d from methanol (700 ml.)  after  treatment with activated charcoal to y i e l d colourless c r y s t a l s of tetrahydrocarbazole (216) (135 gm.,  79%), m.p.  113-115° ( l i t . , m.p.  115-116°).  9-Benzyl-l,2,3,4-tetrahydrocarbazole (217) Tetrahydrocarbazole (216) (7.10 ml., 5.84  x 10"  and s t i r r e d at 0°C.  2  (10 gm.,  5.84  x 10~  2  mole) and benzyl bromide  mole) were dissolved i n dry dimethylformamide  Sodium hydride (1.44 gm.,  four portions over one hour. hour a f t e r which time i t was with ether (3 x 200 ml.).  6.00  x 10  The reaction mixture was  -2  (100  mole) was  ml.)  added i n  s t i r r e d f o r an additional  c a r e f u l l y d i l u t e d with water (400 ml.) and extracted  The ether f r a c t i o n s were combined and washed with  water, dried over sodium sulphate and concentrated to give a colourless mobile oil.  Small amounts of s t a r t i n g material were removed by column chromatography  on alumina (400 gm.,  Act I I I ) . Addition of the crude reaction mixture to the  column i n benzene and e l u t i o n with petroleum ether y i e l d e d the desired 9-benzylated tetrahydrocarbazole 217 as a transparent o i l (11.6 gm. N.M.R.:  5.10  (Singlet, 2H, NCH^CftHQ, 2.60  77%).  (multiplet, 4H, C-2,3CH ), ?  1.86  - 251 -  (multiplet, 4H, C-1,4CH ) .  mass spectrum: M , +  1-Methy1-3-formy1-9-benzylcarbazole  m/e  = 261.  (219)  A solution of 9-benzyltetrahydrocarbazole (217)  (10 gm.,  3.83  x  10"  2  mole) i n dry dimethylformamide (50 ml.) with phosphorous oxychloride (4.55 ml., 1.3 equiv.) was  s t i r r e d at 100°C f o r eight hours.  Hydrolysis of the  reaction mixture was  then effected by addition o f 30% potassium acetate  s o l u t i o n (20 ml.) with heating f o r an a d d i t i o n a l 20 minute period. mixture was  The cooled  then d i l u t e d with water (200 ml.) and extracted with ether  (5 x 100 ml.).  The combined ether fractions were washed with water, dried  over sodium sulphate and concentrated to give a viscous dark o i l .  The  product was p u r i f i e d i n i t i a l l y by column chromatography on alumina  (250  Act I I I ) . The crude mixture was desired carbazole aldehyde  m  '»  235  max  ( 2 lo  (4.66).  8.46  )  :  3 4 0  219 was  eluted with petroleum ether:benzene 48%).  sh  IR. ( C H C I 3 ) :  (4.22), 327 (4.30), 289 1670.  N.M.R. (100 Mhz):  (distorted s i n g l e t , IH, C-4H), 8-10  m/e  (Singlet, 2H, benzyl CH )» 2  6 0  2  = 299  (base peak).  10.00  Found:  C H NO:  C,84.25; H,5.72; N,4.68.  21  17  299.1309.  Found:  (Singlet, IH,  (multiplet, IH, C-5H), 7.66  (4.60),  CHO),  (distorted  (multiplet, 2H, aromatic),  (Singlet, 3H, C - I C H 3 ) .  High resolution mass spectrum:  299.1305.  102-106°.  (4.60), 273 (4.72), 244  s i n g l e t , IH, C-2H), 7.4-7.l(multiplet, 6H, aromatic) 6.9 5.72  1:1  By subsequent r e c r y s t a l l i z a t i o n form  colourless needles were obtained, m.p.  ( )  gm.  applied to the column i n benzene and the  to give an orange glass (5.47 gm. petroleum ether-benzene  crude  Mass spectrum:  calc. for C 2 1 H 1 7 N O :  C,8405; H,5.62; N,4.57.  Calc. f o r  M  + >  - 252 -  l-Methyl-3-(a-hydroxyethyD-9-benzylcarbazole To the carbazole aldehyde 219 hydrofuran  (100 ml.) was  (222)  (112 gm.,  3.74  x 10~  mole) i n dry t e t r a -  2  added i n one portion an excess of methylmagnesium  * chloride - THF solution  (20 ml., 3.16M).  The reaction was  s t i r r e d at room  temperature for 0.5 hr. a f t e r which time the excess Grignard reagent destroyed by the c a r e f u l addition of d i l u t e hydrochloric acid. was  concentrated u n t i l a l l the tetrahydrofuran was  The  was mixture  removed, d i l u t e d with  water (250 ml.) and extracted with ether (3 x 100 ml.).  The ether fractions  were combined, washed with water, dried over sodium sulphate and then concentrated to give a pale yellow o i l (11 gm.).  The crude reaction mixture  p u r i f i e d by column chromatography on alumina (200 gm.,  Act I I I ) . By e l u t i o n  with chloroform and ethyl acetate the carbazole alcohol 222 was colourless foam (7.5 gm., 237.  N.M.R.:  7.2,  6.95  8.08  22  2  X  m a x  :  342,  1.54  ArpH_(0H) CH^,  2.54  (Singlet, 3H, C-1CH ), 1.97 3  3  315.1623.  Found:  The solvent was  M, +  High r e s o l u t i o n mass spectrum:  263,  m/e  (Singlet,  = 315  (base  calc. for  2.22  (223)  x 10"  2  mole) was  taken up i n pyridine  (10 ml.) and gently heated at 60°C for one hour.  then removed and the r e s u l t i n g residue taken up i n ether  Fisher Reagent.  5.00  315.1632.  The Carbazole alcohol 222 (7 gm., (75 ml.) and a c e t i c anhydride  287, 283,  (multiplet, 1H, C-4H),  (doublet, 5H,ArCH(OH)CH ) . Mass spectrum: 300, 297.  obtained as a  ( s i n g l e t , 2H, N - O ^ - C ^ ) ,  l-Methyl-3-("-acetoxyethyl)-9-benzylcarbazole  *  327, 293,  C-5H), 7.98  (2 m u l t i p l e t s , 9H, aromatic), 5.66  peak); main peaks: C H iNO:  UV.;  (doublet, 1H, J = 2Hz,  (quartet, 1H, J = 6Hz, 1H, OH),  64%).  was  (350  - 253 -  ml.)  and washed with 5 % sodium hydroxide s o l u t i o n and with 5 % hydrochloric  acid r e s p e c t i v e l y .  The ether layer was  concentrated to give a tan coloured gum.  then dried over sodium sulphate and The crude reaction product was  f i l t e r e d through a short alumina column and r e c r y s t a l l i z e d from methanol, m.p.  137-140 . UV; X  ( 4 . 0 4 ) , 263 ( 4 . 5 5 ) ,  (loge):  m Q V  238 ( 4 . 7 9 ) ,  (multiplet, 1 H , C - 5 H ) , 7.98  341 ( 3 . 7 5 ) , 230 ( 4 . 7 7 ) .  327 ( 3 . 7 1 ) , 293 ( 4 . 3 0 ) , 283 IR.: 1720. N.M.R.: 8.05  (distorted s i n g l e t , 1 H , C - 4 H ) , 7 . 3 - 6 . 8  (two  multiplets, 9 H , aromatic), 6 . 0 0 (quartet, 1 H , J = 7 H z , A r - C H ( C H ) 0 A ) , 5 . 6 8 3  (Singlet, 2 H , benzyl O ^ ) , - C O C H 3 ) , 1.56 m/e  2.52  (Singlet, 3 H , C - I C H 3 ) 1.98  (doublet, 3 H , J = 7 H z , A r - C H ( C H 3 ) 0 A c ) .  = 3 5 7 (base peak); main peak:  Calc. f o r C N, 3 . 9 3 .  2 4  H  2 3  N0 : 2  Calc. f o r C  357.1727. 2 4  H  2 3  N0 : 2  c  (Singlet, 3 H ,  Mass spectrum:  M, +  298. High r e s o l u t i o n mass spectrum: Found:  357.1676.  Found:  C,80.40; N, 6.38;  C, 80.64; H, 6 . 4 9 ; N, 3.92.  l-Methyl-3-(ct- (N- B ,.g -diethoxyethylamino) ethyl) -9-benzylcarbazole ( 2 Z 4 ) from ( 3 2 3 ) . The carbazole acetate 223 ( 5 0 mg. 1.40 x 1 0 " mole) was dissolved i n 4  aminoacetaldehyde d i e t h y l a c e t a l (171)  ( 1 m l . ) and heated at r e f l u x temperature  (160°) f o r 15 hr. a f t e r which time the excess amine 171 was vacuum.  The dark orange o i l which was obtained was column chromatographed on  alumina  ( 3 gm.).  A small quantity of the desired aminoethyl acetal 224 was  obtained by e l u t i o n with benzene.  N.M.R.:  8.14 (multiplet, 1 H , C - 5 H ) , 7.98  (Singlet, 1 H , C - 4 H ) , 7 . 2 (complex m u l t i p l e t , aromatic), 5.72 N-CH C H ) , 4.64 ( t r i p l e t , 1 H , J = 6 H z , C H ( 0 E ) ) , +  2  removed under high  6  5  2  (singlet, 2 H ,  3 . 9 5 (quartet, 1 H , J = 6 H z ,  - 254 -  Ar-CH(CH )-N), 3.58 (complex multiplet, 4 H ,  ffl(OCH CH ) ),  3  2  3  2  2.69 (doublet,  2 H , J = 6 H z , -N-CH -), 2.60 (Singlet, 3 H , C - 1 C H ) , 2 . 0 (broad s i n g l e t , 1 H , 2  3  N(b)H), 1 . 4 7 (doublet, 3 H , J = 6 H z , Ar-CH(CH3)-N),  1 . 2 0 (complex multiplet,  l-Methyl-3-(g,g-diethoxyethyliminomethy1)-9-benzylcarbazole l-Methyl-3-formylcarbazole  (219) ( 2 0 gm. 6.68 x 10"  acetaldehyde d i e t h y l a c e t a l (171) (1 ml., 8.26  x 10  3  3  (221) mole) and amino-  mole) were heated at 100°  for two hours, then benzene (15 ml.) was added and d i s t i l l e d o f f to remove water formed i n the reaction.  The residue c r y s t a l l i z e d from petroleum ether  (65-110°) - benzene to give a colourless c r y s t a l l i n e powder (2.34 gm.,  85%),  m.p.  (4.23),  285  92-94 . UV.  (MeOH) Amax ( l o g s ) :  3 4 5 (3.87), 330(sh) (4.07), 310  (4.62), 275 (4.61), 247 ( 4 . 5 9 ) , 237 (4.60).  280, 270, 233.  IR.:  1690.  N.M.R.:  8.40  (MeOH-HCl) A  r a a x  :  380, 313,  (Singlet, 1H, C - 4 H ) , 8.32  s i n g l e t , 1H, CH = N-), 8.10 (multiplet, 1H, C - 5 H ) , 7.60  305  (distorted  (Singlet, 1H, C - 2 H ) , 7.4  7.1 (multiplet, 6 H , aromatic), 7 . 0 (multiplet, 2 H , aromatic), 5.72 (Singlet, 2 H , benzyl H), 4.81 ( t r i p l e t , 1 H , J = 5 H z , C H ( 0 E t ) 2 ) , 3.70  (complex m u l t i p l e t , 6 H ,  C H ( 0 C H C H 3 ) 2 ) , = N-CH -), 2.58 (Singlet, 3 H , C - I C H 3 ) , 1.14 2  2  CH(OCH CH ) ).  Mass spectrum:  311, 298, 284.  High resolution mass spectrum:  2  Found:  3  414.2250.  Found:  C, 78.23; H, 7.29; N,  M, +  m/e  = 414  (base peak); main peaks: 369, 3 3 9 , Calc. f o r C 2 7 H 3 0 N 2 O 2 ; 414.2307.  C . 7 7 . 9 5 , H, 7 . 2 0 ; N, 6.56.  6.76.  (triplet, 6H, J = 7 H z  Calc. f o r C 2 7 H 3 0 N 2 O 2 :  - 255  l-Methyl-3-(g-(N- 3, g-diethoxyethylanu.no)ethyl)-9-benzylcarbazole (224) The iminomethyl  acetal 221 (1.6 gm., 3.86 x 10~ mole) i n dry tetrahy3  drofuran (50 ml.) was treated with an excess of methylmagnesium chloride  r (4.8 ml., 3.16M) at r e f l u x temperature  f o r 24 hr.  The excess Grignard  reagent was then destroyed by the addition o f water and the resultant p r e c i s p i t a t e was removed by suction f i l t r a t i o n and washed l i b e r a l l y with ether, The f i l t r a t e was  concentrated, taken up i n water (250 ml.) and extracted  with ether (3 x 100 ml.).  The ether layer was washed with water, dried over  sodium sulphate and concentrated to give an orange foam (1.52 gm.). The crude product mixture was column chromatographed on alumina  (100 gm., Act III) and  the desired aminoethyl acetal was eluted with benzene:chloroform chloroform to give a f a i n t l y  coloured o i l (330 mg. 20%).  293, 284, 263, 239, 231. N.M.R.:  UV;  ^  m  (1:1) and S  L  X  -  341, 327,  8.14 (multiplet, IH, C-5H), 7.98 (Singlet,  IH, C-4H), 7.2 (complex multiplet, aromatic ( C H C I 3 contamination)), 5.72 (Singlet, 2H, N-CH C H ) , 4.64 ( t r i p l e t , IH, J = 6Hz, CH(0Et) ), 3.95 (quartet, 2  6  5  2  IH, J = 6Hz, Ar-CH(CH.3)-N), 3.58 (complex m u l t i p l e t , 4H, OUOCH^CH^^), 2.69 (doublet, 2H, J = 6Hz, -N-CH -), 2.60 (Singlet, 3H, C - I C H 3 ) , 2.0 (broad s i n g l e t , IH, N(b)H), 1.47 (doublet, 3H, J = 6Hz, Ar-CH(CH3)-N), 1.20 (complex multiplet, 2  +  6H, CH(OCH CH3)). 2  Mass spectrum:  M , m/e = 430; main peaks:  (base peak), 288. 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U. Corson, C. U. Rogers, Org. Syn. C o l l . V o l . , 4, 884 (1972).  208.  Private communication  with Dr. Y. Murakami, our laboratory, whose  contribution i s g r a t e f u l l y acknowledged. 209.  B. A. Whittle, E. N. P. Young, J . Med. Chem., 6, 378 (1963).  210.  E. C. Horning, M. G. Horning, J . Org. Chem., 11, 95 (1946).  GRADUATE STUDIES CONT'D Seminar i n Special Topic (Natural Products)  J.P. Kutney  Seminar i n Chemistry  D.G. Clark PUBLICATIONS  I.H. Rogers, D. Grierson Can. Dep. F i s h . For., Bi-Mo. Res. Notes, 25, (4), 33 (1969). Extractives from the bark of Grand F i r [Abies grandis (Dougl.) L i n d l ] I.H. Rogers, D. Grierson Wood & Fiber, 4, (1), 33 (1972) Extractives from Grand F i r [Abies grandis (Dougl.) L i n d l ] Bark J.P. Kutney, D;S. Grierson, G.D. Knowles and N.D, Westcott Tetrahedron, 29, 13 (1973) Studies on Constituents of Abies grandis. The Structure and Absolute Stereochemistry of Cyclograndisolide and Epicyclograndis o l i d e , Novel Cyclopropane Triterpene Lactones J.P. Kutney, D.S. Grierson Heterocycles, 3 (2), 171 (1975) An Improved Synthesis of the O l i v a c i n e type Indole A l k a l o i d s  

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