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Synthetic studies in dihydroindole and indole alkaloids De Souza, Joao Pedro 1973

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f; c SYNTHETIC STUDIES IN DIHYDROINDOLE AND INDOLE ALKALOIDS BY JOAO PEDRO B.Sc, Federal U n i v e r s i t y of M.Sc, Federal University of DE SOUZA Rio de Janeiro, B r a z i l , 1966 Rio de Janeiro, B r a z i l , 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s thesis as conforming to the required sta^dar^l /j THE UNIVERSITY OF BRITISH COLUMBIA December, 1973 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. L Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada - i i - > ABSTRACT A synthetic approach toward the synthesis of vindoline (3) and a reinvestigation of the total synthesis of vincaminoridine (4) and epivincaminoridine (4a) i s described. The synthetic sequence involves alkylation with benzyl chloride of the monosodium salt of propane-l,3-diol to give y-benzyloxypropanol (197). Treatment of 197 with thionyl chloride afforded benzyl-y-chloropropyl ether (198). Alkylation of ethyl diethyl malonate with 198 provided diethyl Y ~ D e n z y l o x y P r o P y l e t n y l malonate (134). Basic hydrolysis of 134 gave y-benzyloxypropylethyl malonic acid (199), which upon decarboxylation provided 2-(y-benzyloxypropyl)-butanoic acid (200). The monoacid (200) was esterified with ethanol to provide ethyl tx-(y-benzyloxypropyl)-butanoate (135). Alkylation of 135 with a l l y l bromide gave ethyl-a-(y-benzyloxypropyl)-a-allyl-butanoate (201), which upon treatment with osmium tetroxide and sodium periodate gave ethyl a(y-benzyloxypropyl)-a-(a-formylmethyl)-butanoate (140). Condensation of 140 with 6-methoxy tryptamine afforded the tetracyclic lactam (150) . Lithium aluminum hydride reduction of the latter, followed by hydrogenolysis of the benzyl group gave two isomeric tetracyclic alcohols (204) . These intermediates were converted via their mesylate derivatives to the quaternary salts (205), which upon treatment with potassium cyanide gave the isomeric cyanides (216). Acid hydrolysis of 216 gave the corresponding carbomethoxy derivative (151). Alkylation of 151 - i i i -with methyl iodide provided dl-vincaminoridine (4) and d l -epivincaminoridine (4a) . Transannular cyclization of the latter substances gave the pentacyclic aspidosperma-type system (195) . The degradation sequence involved acid hydrolysis of vindoline (3) to provide desacetyl vindoline (224), which upon catalytic hydrogenation gave desacetyldihydrovindoline (225) . Pyrolysis of 225 afforded the ketone (86), which upon treatment with dimethyl carbonate provided the g-ketoester (226) . Treatment of the sodium enolate of 226 with oxygen-hydrogen peroxide gave the hydroxy ketoester (227). Treatment of desacetyldihydrovindoline (225) with N,N-thiocarbonyldiimidazole gave the thiocarbonate derivative (230), which upon desulfurization with Raney nickel afforded the unsaturated ester (231) . Catalytic hydrogenation of 231 gave the saturated ester (232) , which upon treatment with lithium diisopropyl amide and oxygen-hydrogen peroxide provided the hydroxyester (234). The saturated ester 232 was converted to the alcohol derivative (237) by reduction with aluminum hydride. Oppenauer oxidation of 237 gave the aldehyde (238). Finally potassium permanganate oxidation of the unsaturated ester (231) gave 5-membered lactam (240), 6-membered lactam (241), N -formyl-5-membered lactam (242), ct and NQ-formyl-6-membered lactam (243) . - i v -TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES . v ACKNOWLEDGEMENTS i x INTRODUCTION 1 1. General » 1 2. Indole A l k a l o i d s Biogenesis 5 3. S t r u c t u r e and stereochemistry of v i n d o l i n e ... 26 4. Synthesis of the Aspidosperma system 28 DISCUSSION PART I 4 8 EXPERIMENTAL I 9 1 DISCUSSION PART I I 1 0 8 EXPERIMENTAL I I 146 BIBLIOGRAPHY 160 - v -LIST OF FIGURES Figure Page 1 Proposed relationship between three main classes of indole alkaloids 7 2 Barger-Hahn-Robinson-Woodward hypothesis 9 3 Wenkert's prephenic acid hypothesis 1 1 4 Schlittler-Taylor-Leete hypothesis 1 2 5 Incorporation of acetate into ajamaline 1 3 6 Wenkert-Thomas monoterpene hypothesis . . 1 5 7 Biogenesis of the C ^ - C ^ Q unit of indole alkaloids 1 6 8 Proposed biogenesis of the Strychnos family .... 2 0 9 Wenkert's postulate for the biogenesis of the Aspidosperma and Iboga alkaloids 2 1 1 0 Biogenesis of the Corynanthe family 2 2 1 1 Biogenesis of the Strichnos, Aspidosperma and Iboga systems 24 1 2 Stork's total synthesis of dl-aspidospermine ... 2 9 1 3 Ban's total synthesis of dl-aspidospermine ..... 3 1 1 4 Kuehne's synthesis of the t r i c y c l i c amino ketone 3 3 1 5 Kutney's transannular cyclization of the cleavamine and quebrachamine systems 3 6 1 6 Kutney's total synthesis of dl-quebrachamine ... 3 7 1 7 Kutney's total synthesis of some monomeric vinca alkaloids 3 9 1 8 Harley-Mason's total synthesis of dl-aspidosper-midine 4 0 1 9 Ziegler's synthesis of quebrachamine 4 2 2 0 Ziegler's synthesis of dl-minovine 4 3 2 1 Wenkert's approach to Aspidosperma alkaloid skeleton 4 3 - v i -Figure Page 22 Ziegler's total synthesis of dl-tabersonine ... 44 23 Steven's synthesis of the hydrolulolidone 45 24 Buchi's total synthesis of dl-vindorosine 46 25 Preparation of the monoester (135) 50 26 Preparation of the aldehydoester (140) 51 27 Preparation of the mesylate (205) 53 28 NMR spectrum of the lactam ether (150) 54 29 Mass spectra of the lactam ether (150) and amino ether (203) 56 30 Fragmentation of the lactam ether (150) 57 31 NMR spectrum of the amino ether (203) 58 32 The two diastereoisomeric dl-pairs of alcohols 60 33 Conformation of the isomeric alcohols I (206a, 208a) and II (207a, 209a) 62 34 NMR of alcohol I (206a, 208a) 65 35 Mass spectra of alcohol I and alcohol II . .. .. . 66 36 NMR of alcohol II (207a, 209a) 67 37 Preparation of dl-vincaminoridine (4) and i t s epimer (4a) 69 38 High pressure liquid chromatography of cyanide I and cyanide II 73 39 NMR spectrum of 16-methoxy cyanide I (220) .... 74 40 Mass spectra of 16-methoxy cyanide I and II ... 75 i 41 Fragmentation of cyanide I and cyanide II 76 42 NMR spectrum of 16-methoxy cyanide II (221) ... 77 43 NMR spectrum of 16-methoxy-dl-vincadine (151a) 80 44 Mass spectra of 16-methoxy vincadine and i t s epimer 82 - v i i -Figure Page 45 Fragmentation of 16-methoxyvincadine and i t s epimer upon electron impact 83 46 NMR spectrum of 16-methoxy-dl-epivincadine (151b) 84 47 NMR spectrum of dl-epivincaminoridine (4a) ..... 86 48 Mass spectra of dl-vincaminoridine and i t s epimer 87 49 NMR spectrum of dl-vincaminoridine (4) 88 50 Gorman's preparation of the ketone (86) 109 51 Preparation of the ketone (86) 109 52 NMR spectrum of desacetylvindoline (224) I l l 53 Mass spectra of desacetylvindoline (224) and desacetyldihydrovindoline (225) 112 54 NMR spectrum of desacetyldihydrovindoline (225) 113 55 NMR spectrum of the ketone (86) 115 56 Mass, spectra of the ketone (86) and 8-ketoester (226) 116 57 Partial synthesis of dihydrovindoline (222) 117 58 NMR spectrum of B-ketoester (226) 119 59 Fragmentation of 8-ketoester upon electron impact 120 60 NMR spectrum of hydroxy ketoester (227) 121 61 Mass spectra of hydroxy ketoester (227) and thiocarbonate derivative 123 62 Partial synthesis of vindoline (3) 124 63 Preparation of unsaturated ester (231) 125 64 NMR spectrum of thiocarbonate derivative (230) .. 126 65 NMR spectrum of unsaturated ester (231) 128 66 Mass spectra of unsaturated ester (231) and saturated ester (232) 129 - v i i i -Figure Page 67 NMR spectrum of saturated ester (232) 130 68 NMR spectrum of saturated ester (233) 132 69 Mass spectra of saturated ester (233) and a-hydroxy ester (234) .... 133 70 Preparation of a-hydroxy ester (234) 134 71 NMR spectrum of a-hydroxy ester (234) 135 72 Preparation of aldehyde derivative (238) 137 73 NMR spectrum of amino alcohol (237) 138 74 Mass spectra of amino alcohol (237) and aldehyde derivative (238) 139 .75 NMR spectrum of aldehyde derivative (238) ...... 140 76 Proposal for the synthesis of intermediate 195 143 77 Alternative synthesis of intermediate 195 144 78 Summary of reactions which relate to the synthesis of vindoline (3) 145 - i x -ACKNOWLEDGEMENTS It i s my sincere pleasure to thank Professor James P. Kutney. His guidance, both as a teacher and a s c i e n t i s t throughout the course of th i s research have made t h i s thesis p o s s i b l e . I would also l i k e to thank Miss G. Bebault for proof reading the e n t i r e thesis and Mrs. Diane Gray f o r her very capable typing. S p e c i a l thanks are due to other members of the group f o r h e l p f u l discussions and suggestions. I am g r a t e f u l f o r scholarships from the National Research Council of B r a z i l and National Research Council of Canada which I have received during my study. F i n a n c i a l support of t h i s project by National I n s t i t u t e of Health i s greatly appreciated. - 1 -INTRODUCTION 1. General In a broad sense, alkaloids are nitrogeneous bases which occur naturally in plants. They nearly always contain their nitrogen as part of a heterocyclic system and are often quite complex in structure. A particular alkaloid is usually restricted to certain genera and families of the plant kingdom, rarely being present i n large groups of plants. In addition, the alkaloids usually show specific pharmacological activity. Alkaloids are most widely distributed among flowering plants, and rarely occur in animals, simple vascular plants, mosses, ferns, fungi and algae. 1 More than three hundred thousand plant species are known, however, fewer than 35,000 have been examined for the presence of alkaloids. About 5,000 alkaloids have been isolated but many have been only 2 3 p a r t i a l l y characterized. ' The curiosity of man regarding his natural world is evident in his earliest records, and i t is no surprise, that his f i r s t exploration of the substances we now c a l l natural products is lost in the mists of time. The exploration of the plants for chemical compounds of medicinal value has been going on for many centuries, herbalism and folk medicine, ancient and modern, have been the source of much useful therapy. A - 2 -new impetus was given to the search f o r medicinal plant p r i n c i p l e s by the discovery of the c l i n i c a l usefulness of a l k a l o i d s of Rauwolfia species. This provided fresh stimulus f o r an enlarged and concentrated attack on the s t i l l unexplored b o t a n i c a l resources of the world. A great number of academic and i n d u s t r i a l screening and evaluation programmes are now i n existence, and many thousands of new plants are being studied. In recent years many new a l k a l o i d s have been discovered, and most of them have pharmacological a c t i v i t y . However, very few have s u c c e s s f u l l y passed the screening and c l i n i c a l t e s t i n g i n order to be accepted as us e f u l drugs. In the 1950's, the periwinkle plant Vinca rosea Linn, was studied because of i t s reputation as an o r a l hypoglycemic agent, but t h i s has not survived s c i e n t i f i c examination. However, extracts of t h i s plant caused leukopenia rather than hypoglycemia i n animals. Several research groups subsequently found such extracts to be a c t i v e against experimental leukemia i n mice. Because of the i n t e r e s t i n g pharmacological a c t i v i t y of i t s a l k a l o i d a l f r a c t i o n , Vinca rosea Linn, has been subjected to an examination as intense as has been recorded f o r the analgesics of the opium poppy, the muscle relaxants of curare, and the hypotensive-sedative agents of Rauwolfia. The importance of the a l k a l o i d s of Vinca rosea Linn, l i e s i n potent antileukemic drugs, such as the dimeric a l k a l o i d s v i n b l a s t i n e (1) and v i n c r i s t i n e (2). One of these, v i n c r i s t i n e , i s now being used c l i n i c a l l y . " * While no known drug, e i t h e r n atural or synthetic, i s as e f f e c t i v e as desired i n the treatment of cancer, each new o n c o l y t i c drug, which has some p o s i t i v e e f f e c t i n the treatment of c l i n i c a l neoplasms, i s another step of encouraging - 3 -progress. It has been shown that vinblastine and vincristine possess activity against lymphomas, Hodgkin's disease, lymphosarcoma, reticulum c e l l carcinoma, monocytic leukemia, and carcinomas of the breast.^ However their application in c l i n i c a l medicine is presently R2 CO 2CH 3 limited since v i t a l information concerning the mode of action, the metabolism, and the structure-activity relationship of these drugs i s lacking. The reasons for this situation are easily understood when one considers the present av a i l a b i l i t y of these alkaloids. Vinca rosea Linn., from which these alkaloids are presently obtained, represents the only source of these drugs. The plant extract i s extremely complex, with more than sixty alkaloids as natural constituents. The d i f f i c u l t separation and the extremely low natural abundance of the above dimers makes these compounds available in minute amounts. This situation has prevented any detailed biological evaluation. In fact even for vinblastine (1) and vincristine (2) which are c l i n i c a l drugs, there i s virtu a l l y no information on the structural requirement for anti-tumor activity, specific mechanism of action, and metabolism. Another unanswered question i s to what extent can the structure of the - 4 -dimeric system be modified either stereochemically or f r a c t i o n a l l y to provide novel drugs with increased pharmacological a c t i v i t y without side effects. I t i s clear that solutions for these problems, require a laboratory synthesis. A general and v e r s a t i l e laboratory synthesis of the monomeric indole and dihydro indole units and the i r r e l a t i v e s i s e s sential for the dimer synthesis. Thus, the work described i n this thesis i s concerned with the chemistry of the indole and dihydroindole alkaloids of the Aspidosperma group, and with t h e i r biogenesis since this i s relevant to the chemistry of the synthetic pathway employed. I t i s an approach toward the t o t a l synthesis of the dihydroindole unit present i n vinblastine (1), namely vindoline (3), and related derivatives. As w e l l , the t o t a l synthesis of dl-vincaminoridine (4) i s presented, and represents an improvement over the sequence previously described.^ - 5 -Although a new numbering system, e.g. (5), has been proposed ' fo r these a l k a l o i d s f o r the sake of c l a r i t y and consistency with previous p u b l i c a t i o n s we r e t a i n the numbering system o r i g i n a l l y employed i n these f a m i l i e s rather than adopting the more recent proposal. 2. Indole A l k a l o i d s Biogenesis A l k a l o i d s are an extremely heterogeneous c l a s s of natural products. The s t r u c t u r a l types found i n d i f f e r e n t classes of a l k a l o i d s are so diverse that i t has been impossible to develop a s i n g l e biogenetic hypothesis to include a l l a l k a l o i d s . A l k a l o i d s have not been proven \ to have any d e f i n i t e function i n plant metabolism, although several suggestions have been made. Some attempts were made to r e l a t e a l k a l o i d formation to p r o t e i n synthesis or to carbohydrate metabolism. Others have suggested, that they might serve as protecting agents against herbivorous a n i m a l s . ^ ' " ^ The biogenesis of indole a l k a l o i d s , which are p a r t i a l l y derived from the combination of tryptamine (6) and a C < J - C ^ Q u n i t , has been the subject of ingenious speculation. Not only the biochemical o r i g i n of the C Q - C I N u n i t but i t s appearance i n the well-known Corynanthe-Strychnos pattern (7) has provoked stimulating comment ever since Barger drew att e n t i o n to a possible biogenesis of yohimbine i n 1934.^ Recent s t r u c t u r a l studies have increased the number of these a l k a l o i d s to more than 800, and two further main groups can be discerned i n which the C Q - C ^ Q unit (7) conforms to the Aspidosperma (8) and Iboga (9) skeletons.'''''^ T y p i c a l examples of these categories are ajmalicine (Corynanthe) (10), akuamicine (Strychnos) (11), v i n d o l i n e (Aspidosperma) (3), and - 6 -catharanthine (Iboga) ( 1 2 ) . In those alkaloids where only nine carbon atoms are present in addition to the tryptamine residue, i t is invariably the carbon atom attached to C, , (see dotted line in structure l o 7, Figure 1) that i s lost. The tryptamine portion of these molecules is derived in vivo from tryptophan, 1^ and recent work has demonstrated that tryptamine i s also an effective precursor of these 21-23 alkaloids. The remaining nine or ten skeletal carbon atoms appear i n what at f i r s t sight seems a bewildering variety of different arrangements, but closer inspection allows the postulation that three main building units (7-9) are really involved to account for the vast majority of indole alkaloids (see Figure 1 ) . The biochemical building blocks for the C^-C^Q unit, prior to I960, were obscure but a vigorous research program i n several laboratories has provided considerable information i n this direction. A brief summary of these experiments i s now provided. Origin of the C g - C 1 0 Unit In contrast to the general agreement by different workers with regard to the "tryptophan" portion of the indole alkaloids, the biogenetic origin of the "non-tryptophan" or C^-C^Q unit, has been the subject of much controversy. Several theories have been proposed 15 25 over the years. Barger and Hahn suggested in the early thirties that the indole alkaloids such as yohimbine (13, Figure 2) are formed by a Mannich reaction between tryptamine and 3,4-dihydroxyphenyl-acetaldehyde, or equivalent, and the condensed product (15) then undergoes a second Mannich reaction with formaldehyde to yield the yohimbinoid skeleton (16). To account for the carbomethoxy group in Figure 1. Proposed relationship between three main classes of indole alkaloids. Figure 2. Barger-Hahn-Robinson-Woodward hypothesis. - 10 -26 ring E (13), i t was suggested by Robinson that i t i s also derived from formaldehyde through a tropolone intermediate (17). The Barger-Hahn hypothesis found support as a consequence of the ingenious 27 28 suggestion by Woodward ' in which instead of an a-condensation, there was condensation at the 6-position with subsequent fission of the catechol type ring (18) to give strychnine (21) as indicated in Figure 2. This concept was applied to other indole alkaloids such as ajmalicine (10) and corynantheine (22). That hypothesis had a CH3o2C 22 23 29-30 number of deficiencies and i n 1959 Wenkert proposed an alternative, the prephenic acid hypothesis. In this hypothesis he suggested that the Cg-C^g unit i s derived from carbohydrates via a pathway involving prephenic acid (24). Thus, the latter rearranges according to the scheme shown i n Figure 3 to yield the key intermediate, the seco-prephenateformaldehyde (SPF) unit (29). This SPF unit can be I incorporated into yohimibinoid alkaloids such as ajmalicine (10), corynantheine (22), and ajmaline (23). Although this hypothesis could account for the carboxyl group at the C, position, and as the 16 result of the stereospecific migration of the pyruvate side chain i n compound (24), i t also rationalizes the a-configuration of the hydrogen - 11 -ure 3. Wenkert's prephenic acid hypothesis. - 12 -atom at C^ <. found in yohimbine (13), and in almost a l l natural alkaloids, i t did not stand up to experimental tests. Thus, feeding 14 experiments with alanine-2- C to Rauwolfia serpentina plants, expected to convert the amino acid to prephenic acid and so on, showed that incorporation into ajmaline (23) was extremely poor. A third 31~~33 theory was then proposed by Schlitter and Taylor, and Leete. They suggested that three molecules of acetyl-coenzyme A condense to form a poly-g-keto chain (31) which by further condensation with formaldehyde and with carbonyl-coenzyme A, as indicated i n Figure 4, forms an intermediate (32) very similar to Wenkert's SPF unit (29). This intermediate would then condense with tryptamine to give the various alkaloids i n a manner similar to the latter part of Wenkert's hypothesis. CH„0 32 Figure 4. Schlittler-Taylor-Leete hypothesis. - 13 -The experimental support for this hypothesis ' came by feeding 14 14 14 sodium acetate-1- C (33), mevalonic-2- C acid (34), and tyrosine-2- C (35) to Rauwolfia serpentina plants. By administering acetate, radioactive ajmaline (23) equally labelled at C-3 and C-19 was isolated (Figure 5), but upon feeding mevalonic acid (34) or tyrosine (35), the ajmaline (23) isolated was completely inactive. Battersby also studied the biosynthesis of ajmaline (23) and found results i n conflict to those of Leete, namely, incorporation of mevalonate took place. OH 34 35 Figure 5. Incorporation of acetate into ajmaline. 19 Leete was unable to reproduce his original results, and the failure to find radioactivity at C-15, as was expected from this hypothesis, as well as other results, would apparently exclude the acetate hypothesis. - 14 -The fourth hypothesis, the monoterpenoid hypothesis, was suggested independently by Thomas"^ "* and Wenkert. ^ ' ^ The discovery of several non-alkaloidal glycosides such as gentiopicrin (36), bakankosin (37), and swertiamarin (38), and their remarkable similarities to the seco-prephenate-formaldehyde (SPF) unit, led them to suggest that the non-tryptophan portion of the indole alkaloids was monoterpenoid in origin. Thus on structural grounds, their derivation from non-nitrogeneous cyclopentanoid monoterpenes related to verbenalin (39), genepin (40), and asperuloside (41) would be readily explicable. 39 40 41 Cleavage of the cyclopentane ring as indicated in Figure 6, would yield the carbon skeleton of the SPF unit having the required stereo-chemistry at C-15 (equivalent to C-4 in structure 45) in the indole alkaloids. - 15 -CH3C02H 42 6) 44 2 5 it 6 . ( 6 ) / 46 Iboga 2(6) 45 47 Yohimbinoid Aspidosperma Figure 6. Thomas-Wenkert monoterpene hypothesis. Many experiments published from different laboratories,particularly those of Arigoni, Battersby, Leete and Scott, provided results which gave a clear understanding of the proposed hypothesis. A recent review 14 by Scott provides a summary of these investigations. It has been proven beyond doubt that the f i r s t three hypotheses are incorrect. The only hypothesis i n accordance with their findings i s that due to Thomas and Wenkert, namely the monoterpene hypothesis. It was shown that a l l three types of the Cg-C^Q unit (7), (8), and (9) are mono-terpenoid i n origin. Two residues of mevalonate (43) are used biologically in the normal head-to-tail combination of units, and the intermediacy of geraniol (48) was established."^ The ^ C - l a b e l l i n g patterns were determined for representatives of the three types of alkaloids 14 biosynthesized from labelled mevalonates which carried specific C - 16 -Figure 7. Biogenesis of the C -C . unit of indole alkaloids. - 17 -labels at various known positions. The results were consistent with the proposal of Thomas and Wenkert, that 45 i s generated by fission of some cyclopentane monoterpene (44) with rotation about the indicated single bond as shown in Figure 6. It was further recognized that unit 45 is structurally related to 46 and 47, and can be transformed into these other types by the bond fission and bond formation either at a_ or b_, as shown in Figure 6, thus leading to the major families. The actual bio-intermediates corresponding to the cyclopentane monoterpene (44) were found to be loganin (53) and seco-39-42 loganin (54). Desoxyloganin (52), hydroxygeraniol (50) and i t s cis-isomer hydroxynerol (51), were shown to be involved i n the 43-46 biosynthetic pathway. Condensation of tryptamine with seco-loganin (54) opened the way to studies of the later stages of the biogenesis as discussed i n the next section. Later Stages of the Biogenesis Two glucosides, vincoside (55) and isovincoside (56) (epimers at 22 47 C-3) were obtained. ' The stereochemistry at C-3 was uncertain. However, i t has now been established by X-ray analysis that i n 48 49 vincoside (55) the hydrogen at C-3 has the g-orientation. ' OGlu 55 56 - 18 -3 3 Doubly labelled [ar- H,0-methyl- H]-vincoside was incorporated by 22 47 Vinca rosea plants into a l l three types of indole alkaloids. ' These results show that the main skeleton of vincoside (55) was built intact into the Corynanthe, Aspidosperma and Iboga systems. Isovinco-side (56) was biologically inert and afforded no significant incorporations into any of the alkaloids. This result i s unexpected from the point of view of the biosynthetic chemist because the configuration at of vincoside (55) is now opposite to that at the corresponding carbon of the next established intermediate, geissoschizine (57). It i s not yet clear how this centre becomes epimerized i n the biosynthesis but the following experimental facts have to be accounted for: Isovincoside (the epimer of vincoside)is not biologically a c t i v e , ^ and the hydrogen at C,. of loganin (54) (C^ of loganin corresponds to of vincoside) i s completely retained i n the biosynthesis of the three main classes of indole a l k a l o i d s , r e p r e s e n t e d in Figure 7 by vindoline (3) (Aspidosperma), catharanthine (12) (Iboga), and ajmalicine (10) (Corynanthe). Simultaneously and independently, vincoside (55) and isovincoside 22 (56) were isolated from Vinca rosea, and isovincoside (56) from Rhazya s t r i c t a . i Before discussing the biosynthetic pathway beyond vincoside (55), i t seems appropriate at this stage to consider two important points 29 36 37 based upon structural relations at the alkaloidal level. Wenkert ' ' recognized that there must be biosynthetic significance in the fact that there is almost complete stereochemical constancy at C^ ,. i n the - 19 -Corynanthe-Strychnos group, and i t was proposed that the Strychnos, Aspidosperma and Iboga alkaloids could be derived from the Corynanthe system. A possible route from the Corynanthe to the Strychnos systems was put forth as shown in Figure 8 (pathway A). Later a second 52 pathway was proposed by Scott (Figure 8, pathway B), but no direct experimental evidence has yet been established to distinguish between these two mechanistic speculations. 29 36 37 Wenkert ' ' had also proposed that the Strychnos system (67) was converted to both the Aspidosperma (71) and Iboga (72) types, as shown in Figure 9, via the cyclization of their iminium derivatives. Returning to the pathway beyond vincoside (55), formation of the Corynanthe family requires no skeletal rearrangement and is regarded as involving enzymatic cleavage of the glucosidic residue followed by reductive condensation of the nascent aldehyde (73) as shown in Figure 10, Geissoschizine (57), corynantheine (22) and i t s aldehyde 22 (75), and ajmalicine (10) could be reached via plausible steps. Further evidence came from examination of the alkaloid content of seedlings of Vinca rosea, a technique used to obtain information about 53 the sequence of alkaloidal transformations. It was found by Scott that Corynanthe-type systems appeared before detectable amounts of Aspidosperma and Iboga alkaloids were formed. Direct.?feeding experi-' 53-55 ments showed that the corynantheine aldehyde (75) was not significantly incorporated. However geissoschizine (57) was found to specifically label the Vinca rosea alkaloids including the Strychnos alkaloid akuammicine (11). These results indicated biological conversion of the corynanthe system of geissoschizine (57) into the - 20 -Pathway A Pathway B OH CH302C CH2OH 66 Figure 8. Proposed biogenesis of the Strychnos family^ Figure 9. Wenkert's postulate for the biogenesis o f the Aspidosperma and Iboga alkaloids. Figure 10. Biogenesis of the Corynanthe family. - 23 -rearranged Aspidosperma and Iboga skeletons, and that geissoschizine underwent an a,8-rearrangement to generate the Strychnos skeleton of akuammicine (11). Further evidence for the a,B-rearrangement 3 54 came from incorporation of [ar- H]-vincoside (55) into akuammicine (11). The isolation of stemmadenine (77), tabersonine (81) and preakuam-53 55 micine (76) from young Vinca rosea seedlings, ' (the f i r s t two were known from other sources, the last was new) gave further indication of the biosynthetic pathway beyond geissoschizine (57). Thus the status of the f i r s t two as late intermediates was established by showing 3 14 that [O-methyl- H , l l - C]-stemmadenine (77) was incorporated intact into tabersonine (81), vindoline ( 3 ) , and catharanthine (12). Tabersonine (81) was also incorporated into vindoline (3) and catharanthine (12). 23 3 Kutney has confirmed the latter result by feeding [ar- H]-tabersonine to Vinca rosea and isolating radioactive vindoline (3) and catharanthine (12). These results support the sequence stemmadenine (77) -»• tabersonine (81) -> catharanthine (12). At present there i s no conclusive evidence as to the relationship between stemmadenine (77), preakuamicine (76) and akuammicine (11) or as to whether stemmadenine (77) appears on the pathway as a precursor to the Strychnos skeleton. However, mechanistic considerations as well as the timing of 53 55 stemmadenine's appearance in growing seedlings ' suggest that i t follows, or i s i n equilibrium with the Strychnos system (76) as outlined in Figure 11. Having reached the Strychnos family (76), let us now follow the series of biological transformations along the biosynthetic pathway which led to the remaining Aspidosperma and Iboga families. - 24 CH302C Preakuamicine 76 CH2OH C0 2CH 3 akuamicine 11 C0 3CH 3 79 C0 2CH 3 catharanthine 12 / CH302C CH2OH stemmadenine 77 C0 2CH 3 acrylic ester 79 C0 2CH 3 tabersonine 81 C0 2CH 3 80 Figure 11. Biogenesis of the Strycnos, Aspidosperma, and Iboga systems. - 25 -A very interesting mechanism has been independently proposed by Scott^^ and Kutney"^ which involves skeletal fission and new bond formation as outlined in Figure 11. Thus isomerization of the exo-cyclic double bond of stemmadenine (77) to yield intermediate 78, could allow fragmentation to the acrylic ester (79) which can then ring close in the two indicated ways leading to tabersonine (81) and catharanthine (12). It i s appropriate to note the similarity between the acrylic ester (79) and Wenkert's acrylic ester (70) contained i n 36 his original proposal. Support for the formation of the acrylic ester (79) i n the biological cleavage process came from the isolation of tetrahydrosecodine (82) from Rhazya stricta,"^'"*^ tetrahydrosecodin-17-ol (83) and the corresponding dihydro compound (84) from Rhazya , t . 58-60 orientalis. C0 2CH 3 82; R = H 83; R = OH C0 2CH 3 C0 2CH 3 84 85 Further support for the acrylic ester (79), came from various plant investigations In Kutney's group.^ 1 Thus when [ar- 3H]secodin (85) (different from the proposed intermediate 79 only in the oxidation - 26 -level of the plperidine ring) was fed to the V. rosea, V. minor and Aspidosperma pyricollum, low but definite incorporations were observed in the appropriate alkaloids present in these plants. Furthermore, different labelled forms of secodine (85) were synthesized and fed to these plants.61>62,64 ^ e s e r e s u i t s showed that the secodine skeleton was incorporated intact and for the purpose of this discussion i t i s sufficient to indicate that the sequence 79 -*• vindoline (3) is now reasonably well established. It was the consideration of the later stages of the biosynthetic pathway, particularly the conversion 80 -*• 81 for example, which stimulated the synthetic plan which was chosen for vindoline (3). 3. Structure and Stereochemistry of Vindoline Vindoline (3), i s the major alkaloid in the leaves of the Apocynaceous plant Vinca rosea Linn., and was f i r s t isolated i n 1958 by Kamat and co-workerswho incorrectly assigned i t the empirical 66 formula C 2 7H 3 Z,N 20 6«1/2H 20. Gorman et a l . subsequently determined the correct formula of the alkaloid, C_,.HooNo0(:, and the base was 2D 52 2 O shown to be pentacyclic and to contain an isolated double bond. Five of the oxygens were found to be present as hydroxyl, carbomethoxyl 67 68 and acetoxyl functions. Careful comparison ' of the mass spectra of ketone 86 obtained by pyrolysis of the hydrochloride of dihydro-vindoline, and dihydrovindoline with that of N-methyl diacetyl-aspidospermine (87), indicates the presence of the latter ring system in vindoline (3) and i t s derivatives, since in a l l three compounds intense peaks were found at m/e 124, 174, 188 and 298. Further consideration of the nuclear magnetic resonance, mass and ultraviolet - 27 -spectra of vindoline (3) and i t s derivatives, led to the proposal of structure 88 for vindoline. Vindoline (3) and desacetylvindoline have 87 also been obtained from vincaleukoblastine,^' ^  leurosidine,^ 1 or leurosine^ upon acid cleavage (concentrated hydrochloric acid, stannous chloride, tin-metal under reflux). These dimeric indole-indoline alkaloids also present in the leaves of Vinca rosea Linn. (Catharanthus roseus G. Don), are powerful oncolytic agents whose biological properties have been thoroughly 72 reviewed. These compounds represent examples of indole-indoline alkaloids in which the indole moiety i s linked through a C-C bond to the aromatic ring of the dihydro-indole portion of the molecule. 73 Finally an X-ray analysis of vincristine methiodide (89) permitted the complete elucidation of the structure, stereochemistry and absolute configuration of vincaleukoblastine (1) and hence of 69 vindoline (3), as a result of the known relationship among these - 28 -molecules. OAc \ 4. Syntheses of the Aspidosperma System Although many alkaloids of complex structures belonging to the 74 Aspidosperma family have been described, the synthetic works are limited to several kinds of alkaloids and related compounds constituting the fundamental skeleton. The f i r s t total synthesis of the natural Aspidosperma system was reported by Stork.^ In his successful synthesis of dl-aspidospermine (99), as well as that of dl-quebrachamine (101) (Figure 12) he u t i l i z e d the Fischer indole synthesis^ in order to achieve the construction of the desired pentacyclic skeleton. The required t r i c y c l i c intermediate 97 was obtained by employing the pyrolidine enamine reaction, which had previously been developed in Stork's laboratory, in the early stages of the sequence. The stereochemistry of the various bi c y c l i c and t r i c y c l i c intermediates u t i l i z e d was l e f t undefined since the authors f e l t that this stereochemical ambiguity was not significant here. The indolenine - 29 -C0 2CH 3 r H r H r H r H 0 1) pyrrolidine C H 3 L 2 2 2)CH2=CHC02CH3 90 3) HOAc HOC 91 1) pyrrolidine 2) CH0CCH=CH. 3 I I 2 0 3) HOAc CONH, 1) LiAlH 4 2) H+,H20 3) base Figure 12. Stork's total synthesis of dl-aspidospermine. - 30 -98 being formed under equilibrating conditions would lead to equilibration at the two centers marked by asterisks via a reverse Mannich r e a c t i o n . ^ The most stable relative arrangement of the two asymmetric centres of compound 98 would thus be expected to result whatever the stereochemistry of the intermediates or the detailed course of the indolenine cyclization process. Thus this most stable arrangement should coincide with that of dihydroaspidospermine (98). The identity of the synthetic material (99) as dl-aspidospermine was established by the identity of the infrared and mass spectra with those of the natural alkaloid. Cyclization of the phenylhydrazone of 97 to a mixture containing 78 100, followed by reductive cleavage with potassium borohydride permitted extension of the synthesis to yield dl-quebrachamine (101). H 97 100 101 79 Another total synthesis of aspidospermine (99) was reported by Ban and co-workers (Figure 13). Although the later steps were identical to those of Stork's synthesis,''"' these workers developed a different pathway to a bicy c l i c intermediate (107) which has the same planar structure as the corresponding one (94) prepared by Stork. However, the physico-chemical properties for the compound available from Ban's work, were quite different from those obtained by Stork, - 31 -Figure 13. Ban's total synthesis of dl-aspidospermine. ^ - 32 -and hence the f o l l o w i n g r e s u l t s l e d the authors to propose that they were dia s t e r e o i s o m e r s . Thus conversion of 107 to 110 and the l a t t e r to dl-aspidospermine (99) i n the same manner as i n d i c a t e d p r e v i o u s l y i n the Stork s y n t h e s i s (94 to 99) provided c o n c l u s i v e evidence that a s e r i e s of intermediates was produced which were d i a s t e r e o m e r i c to those of Stork's s y n t h e s i s . Moreover conformational analyses of the intermediates 96 and 109 allowed the assignment of the conformational s t r u c t u r e s of 96a and 109a. 0 ' 96a 109a The f a c t that dl-aspidospermine (99) was sy n t h e s i z e d from e i t h e r 97 or 110, supports Stork's proposal that e q u i l i b r a t i o n 75 occurs during the F i s c h e r i n d o l e c y c l i z a t i o n to the i n d o l e n i n e . 80 Kuehne has a l s o s y n t h e s i z e d the key t r i c y c l i c i ntermediate 97 w i t h the stereochemistry as i n d i c a t e d i n 97a and 97b. - 33 -ure 14. Kuehne's synthesis of the t r i c y c l i c aminoketone. - 34 -In his approach the starting material proline ethyl ester (111) was converted into compound 116 through the sequence shown in Figure 14. The enone (116) was then reduced to the desired t r i c y c l i c intermediate (97) either with lithium aluminum hydride or by catalytic hydrogenation followed by Oppenauer oxidation of the resulting amino alcohol. 83—87 Kutney and co-workers undertook a totally different approach toward the synthesis of Aspidosperma and related alkaloids, placing emphasis on generality and v e r s a t i l i t y . The reaction selected for this purpose involves the creation of an electrophilic center (iminium salt) in the original amine, followed by reaction of the latter intermediate with a nucleophile (B:) to yield the desired product, as indicated below. I I I + B . I I R - C — N • R- C =• N— — ^ — • R - C - N • I | I I H B 88 89 90 The participation of imines as possible intermediates i n alkaloid 81 biosynthesis has long been recognized. Postulates on the possible biosynthetic pathways of indole alkaloids have proposed these imine 36 intermediates and their use in the synthesis of some indole alkaloids 82 83—87 had already been demonstrated. Thus, Kutney in his approach ut i l i z e d a transannular cyclization reaction of an appropriate nine-membered ring intermediate and was able to show that cleavamine (120) and quebrachamine (101) ring systems could be cyclized in a completely stereospecific manner to yield the necessary stereochemistry of the natural systems shown in 123 and 125. He has also shown that the - 35 -introduction of a carbomethoxy group at the appropriate position on the above systems (126 and 131) provided an alteration in the course of the cyclization process and an entry into the Aspidosperma (128) and Iboga 88 89 alkaloids (130) (Figure 15). In his total synthesis ' of dl-quebrach-amine (101) and dl-aspidospermidine (125), he generated the nine-membered ring system by means of a reductive'cleavage in the last step of the synthesis (Figure 16). In view of the previous conversion of dl-quebrach-amine (101) to dl-aspidospermidine (125) via a transannular cyclization process he has also completed the total synthesis of the latter, 90 Further modifications of the synthetic sequence in which tryptamine was condensed with the aldehydo ester (140) provided a considerable 88 improvement over the sequence previously reported. Moreover the v e r s a t i l i t y of this synthetic approach allowed the f i r s t total syntheses of a series of monomeric alkaloids in the Vinea family. The quaternary mesylate (139) was a key intermediate. Thus treatment of 139 with potassium cyanide i n dimethyl formamide afforded + H H 123 - 36 -131 132 Figure 15. Kutney's transannular cyclization of the cleavamine and quebrachamine systems. - 37 -CH,CHnI NaOEt CH 20(CH 2) 3CH(C0 2Et) 2 133 134 f H3 CH 2 CH 20(CH 2) 3C(C0 2Et) 2 LiAlH, 1)H /Pd - " m_ 1 n a *• 1 3 8 2)CH 3S0 3C1/P y ' 1 3 9 1 0 1 Figure 16. Kutney's total synthesis of dl-quebrachamine. - 38 -the nine-membered compound (142) possessing a cyano group at which upon hydrolysis followed by esterification with ethereal diazomethane provided dl-vincadine (143) and dl-epivincadine (144) as a minor component (epimers at C^). The synthesis of dl-vincadine also completes the total synthesis of dl-vincaminoreine (145) and dl-vincaminovine (146) in view of the 91 already known interconversions. The transannular cyclization approach applied to vincadine (143) and vincaminoreine (145) afforded vinca-difformine (148) and minovine (147) respectively, and hence an entry into the pentacyclic series. 90 Kutney and co-workers were also able to show that the trans-annular cyclization approach could be extended to the synthesis of alkaloids bearing oxygen functions, particularly methoxy groups on the aromatic ring. Condensation of 6-methoxytryptamine (149) with the aldehyde ester (140) afforded the tetracyclic lactone (150). Further elaboration of this intermediate i n the same manner as indicated for compound 139 (Figure 17), afforded the total synthesis of 16-methoxy-vincadine (151) and 16-methoxyepivincadine (152). 92 Harley-Mason and co-workers reported another interesting synthesis of dl-aspidospermidine (125). The key step in this synthetic sequence was the acid catalyzed rearrangement of the tetracyclic hydroxy-lactam (155) to the pentacyclic aspidosperma type indolenine-lactam (156), which upon reduction with lithium aluminum hydride gave dl-aspidospermidine (125). Another synthesis of quebrachamine (101) and 3,4-dihydro-93 quebrachamine (164) has been achieved by Ziegler and co-workers. The approach employs the alkylation of l-benzyl-3-ethyl-l,4,5,6-tetrahydro-- 39 -C02CH3 148 Figure 17. Kutney's total synthesis of some monomeric vinca alkaloids. - 40 -Figure 18. Harley-Mason's total synthesis of dl-aspidospermidine. - 41 -pyridine (160) with methyl haloacetates and subsequent cyclization to a nine-membered ring in high yield with polyphosphoric acid, as indicated in Figure 19. However the last step of the sequence was a very low yielding reaction, and his attempts toward the conversion of the compound (164) to quebrachamine (101) proved to be unsuccessful. 94 A similar approach has been u t i l i z e d by Ziegler i n the total synthesis of dl-minovine (147). The most interesting aspect of the synthetic sequence involves the alkylation of the acrylic ester (167) with the enamine (160) followed by cyclization of the resultant iminium intermediate to afford the tetracyclic indole derivative (168). Hydrogenolysis of 168 gave the secondary amine 169, and the ethylene bridge necessary to complete the synthesis was introduced in one operation by alkylation with ethylene dibromide. Wenkert and 95 co-workers reported an approach toward the construction of the Aspidosperma alkaloid skeleton. Sodium borohydride reduction of 170 followed by hydrolysis of the resultant n i t r i l e with alkaline hydrogen peroxide yielded the amide (171). Catalytic hydrogenation of the latter with simultaneous cyclization at the g-position of the indole nucleus afforded the tetracyclic intermediate (172) similar 94 in structure to that obtained by Ziegler. The construction of the ethanamino-bridge (173) was performed by an intramolecular indole B-alkylation. A synthesis of dl-tabersonine (81) was reported by Z i e g l e r ^ i n 97 which he u t i l i z e s the previously developed approach for the synthesis of the key intermediate 146. The remaining steps of the synthetic sequence, were basically the same approach that has been - 42 -H 164 Figure 19. Ziegler's synthesis of quebrachamine. - 43 -Figure 21. Wenkert's approach to aspidosperma alkaloid skeleton. - 44 -taken by Kutney in his synthesis of the monomeric Vinca alkaloids. ^ ' ^ ' ^ N' KCN 1) base  N / \ 2 ) C H 2 N 2 ^ 1 1 3)Pt,0, 176 ^ 81 Figure 22. Ziegler's total synthesis of dl-tabersonine. C0 2CH 3 75 79 A new method for the synthesis of an established ' hydro-lulolidone Aspidosperma alkaloid precursor (187) has been reported 99 by Stevens. It involves the acid-catalyzed thermal rearrangement of a cyclopropyl imine (179) to a 2-pyrroline (180) as a key step. Buchi and co-workers 1^ have reported the total synthesis of vindorosine (194), a highly functionalized Aspidosperma alkaloid. The f i r s t step of his synthetic sequence involves an interesting boron trifluo r i d e catalyzed cyclization of compound 188 to yield the tetracyclic indoline (189) as a major product. The intermediate 189 was then converted to the pentacyclic keto ester (192) via the synthetic operations indicated in Figure 24. The required stereo-chemistry and functionalization of ring C was then achieved via an interesting hydroxylation reaction. - 45 -186 187 Figure 23. Steven's synthesis of the hydrolulolidone. - 46 -Figure 24. Buchi's total synthesis of dl-vindor-osine. - 47 -Thus treatment of 192 with oxygen-hydrogen peroxide in the presence of base gave the hydroxy keto ester (193) which upon reduction followed by acetylation yielded dl-vindorosine (194). - 48 -DISCUSSION PART I 1. The Total Synthesis of dl-Vincaminoridine (4) and i t s Epimer 73 On inspection of the structural formula presented for vindoline (3), i t i s obvious that the most d i f f i c u l t part of any synthetic approach to this interesting alkaloid would involve the construction of ring C, containing the six asymmetric centres. It was decided that from the number of possible pathways which might be employed in the construction of these f u n c t i o n a l i t y , the conversion of compound 4 to the pentacyclic intermediate 195, via a transannular cyclization reaction was attractive arid potentially e f f i c i e n t . The use of nine-membered compounds to generate the desired pentacyclic aspidosperma skeleton has been proposed i n biogenetic hypotheses, and Kutney and - 49 -. . co-workers were able to carry out such a conversion. Having obtained the aspidosperma skeleton (195) the remaining f u n c t i o n a l i t y would then be elaborated v i a appropriate synthetic operations. The s t a r t i n g material which was chosen f o r the synthesis of the required c r u c i a l intermediate (4) was eth y l a-(Y-benzyloxypropyl)butanoate (135) 88 103 which was prepared according to known procedures. ' The r e a c t i o n sequence i s outlined i n Figure 25 and only a b r i e f mention of some experimental d e t a i l s are' made here. Thus Y-benzyloxypropanol (197) 101 was prepared i n 66% y i e l d by condensation of the monosodium s a l t of propane-1,3-diol (196) with benzyl c h l o r i d e i n xylene. Treatment of 197 with t h i o n y l c h l o r i d e i n dimethylaniline gave r i s e to benzyl Y-chloro-102 propyl ether (198) i n 80% y i e l d . A l k y l a t i o n of e t h y l d i e t h y l malonate with 193 i n absolute ethanol i n Lhe presence of sodium ethoxide gave d i e t h y l y-benzyloxypropylethyl malonate (134) i n 54% y i e l d . Hydrolysis of the malonic ester d e r i v a t i v e (134) with aqueous potassium hydroxide i n ethanol, gave the desired y-benzyloxypropylethyl malonic acid (199) as a viscous o i l , which was c r y s t a l l i z e d from n-hexane-ether to provide a c o l o r l e s s s o l i d i n 81% y i e l d . In order to complete the synthesis of the desired monoester d e r i v a t i v e (135), we now proceeded as follows; y-benzyloxypropylethyl malonic acid (199) was smoothly decarboxylated at 160°C to provide the 2-(Y-benzyloxypropyl)-butanoic. acid (200) as a yellow viscous o i l , which was used f o r the subsequent r e a c t i o n without further p u r i f i c a t i o n . The crude monoacid (200) was e s t e r i f i e d with ethanol and s u l f u r i c a c i d to provide e t h y l 2-(Y-benzyloxypropyl)-butanoate (135) as a clear o i l i n 76% y i e l d (b.p. 135°/1.5 mm). JKOH 135 Figure 25. Preparation of the monoester (135). - 51 -Having obtained the monoester derivative (135), i t became necessary at this point, bearing in mind the i n i t i a l l y proposed synthetic approach, to direct our efforts toward the synthesis of the equivalent to the "non-tryptophan" unit of the aspidosperma skeleton, 7 90 namely the aldehydoester (140). The sequence followed ' i s shown in Figure 26. Figure 26. Preparation of aldehydoester (140). Alkylation of 135 with a l l y l bromide in ether in the presence of triphenylmethyl sodium gave a yellow o i l . After purification by i fractional d i s t i l l a t i o n under reduced pressure, the corresponding alkylated product 201 was obtained i n 83% yield (b.p. 132-134°/0.15 mm) The one step preparation of aldehydes from the corresponding a l l y l compounds is a well known reaction. Thus treatment of 201 with osmium tetroxide and sodium periodate, added successively to the reaction - 52 -mixture at room temperature, gave the desired aldehydoester (140), a f t e r p u r i f i c a t i o n by vacuum d i s t i l l a t i o n i n 70% y i e l d (b.p. 174-1760/0.75 mm). It i s perhaps worthwhile to mention that once t h i s aldehydoester (140) has been prepared, i t was immediately u t i l i z e d i n the next ' condensation reaction due to i t s known i n s t a b i l i t y . Having obtained the synthetic intermediate (140) we next considered 104 a Pictet-Spengler reaction, namely the condensation of 140 with 6~methoxy tryptamine (149) to provide the required tetrahydro-3-carboline r i n g system (150) (Figure 27). The tryptamine d e r i v a t i v e (149) was a v a i l a b l e from previous experiments and had been prepared according to a known procedure. When 140 was refluxed with 6-methoxy tryptamine (149) i n g l a c i a l a c e t i c a c i d for 1.5 hours followed by the conventional workup of the re a c t i o n mixture, a yellow residue was obtained:^ This material was p u r i f i e d by column chromatography on alumina to give, i n 98% y i e l d , the lactam (150) as a yellowish g l a s s . This intermediate (150) was a mixture of the two expected diastereoisomers, but no attempts to separate them at t h i s stage were necessary f o r our purpose. The structure assigned to compound 150, was f u l l y substantiated by s p e c t r a l d a t a. 7 Even though the s p e c t r a l data have already been presented and discussed,'' i t i s necessary f o r c l a r i t y i n discussing further work to b r i e f l y mention i t again from t h i s stage of the sequence. The presence of the lactam was evident from the strong carbonyl absorption at 1670 cm ^. The presence of the methoxy indole was evident i n the u l t r a v i o l e t spectrum (335, 321, 295, 272, 264,-227 nm) . The mixture of the expected diastereoisomers ( c i s and trans with respect to C-3 and C-15) was submitted to nmr spectroscopy (Figure 28). The most c h a r a c t e r i s t i c features were a t r i p l e t at x 5.25 (J = 8 cps) which was assigned to the - 53 -Figure 27. Preparation of the mesylate (205). - 55 -C-3 proton, and the absence of signals for the proton on the indole ring, which indicated that the cyclization at the a position of the indole ring had taken place. In the mass spectrum (Figure 29) i n addition to the molecular peak at m/e 432 and the base peak at m/e 91 (tropylium ion fragment), the other important peaks were at m/e 341, 281, 263 and 149. Tentative assignment for these fragments are indicated in Figure 30. The molecular formula, ^27^32^3^2' W a S established by elemental analysis and high resolution mass spectrometry. The next step was the conversion of 150 to the desired benzylether amine (203), which was achieved by lithium aluminum hydride reduction in refluxing tetrahydrofuran. The isolated crude product was purified by column chromatography on alumina to afford a mixture of the two diastereoisomers as a yellow amorphous material in 92% yield. The ultraviolet spectrum was that of a typical methoxy indole (230, 263, 270 and 300 nm), while the carbonyl absorption in the infrared spectrum was now absent. The nmr spectrum (Figure 31) showed a diamagnetic shift of the distorted t r i p l e t due to the C-3 proton now located at T 5.93. A l l other signals were also i n agreement with the proposed structure (203). The mass spectrum (Figure 29) showed significant fragments at m/e 91, 149, 240, 327 and 418 (M ) according to our expectations. The molecular formula, ^yH^O^^, was established by high-resolution mass spectrometry and elemental analysis. Having prepared the benzylether amine (203), we now turned our attention to the preparation of a key intermediate, the tetracyclic amino alcohol 204. Thus the benzyl group was removed by catalytic hydrogenolysis (10% palladium on charcoal, ethyl acetate, concentrated 1 OCT 8Cl <7> 2Cf r>-00 00 C H 3 0 O ^ P H il I I I I I I I I I I I I li I I ' i l l ill I I I 1 I I 1 I I I I I I I 90 100 l O O p 8 0 -150 200 250 300 350 '+00 x in 6CJ / 2 Of— III III I 1_L X m/e 430 ( 5 X ) X t—s I D X r o v > 0 CM 10 CMS 1 90 100 I I I I t. I I t I 150 200 I I t I I I I I I I I i I t I I » t I ! 250 300 350 400. 430 m/e a* Figure 29. Mass spectra of the lactam ether (150) and amino ether (203). (Taken from C. Gletsos Ph.D. thesis) - 57 -m/e 263 Figure 30. Fragmentation of the lactam ether (150). Figure 31. Nmr of the aminoether (203). (Taken from C. Gletsos Ph.D. thesis) - 59 -hydrochloric acid) to provide a f t e r the conventional workup a yellowish s o l i d . Chromatography on aluminum allowed the i s o l a t i o n of the two isomeric alcohols (204) i n 64% y i e l d . E l u t i o n with e t h y l acetate-ethanol (98:2) gave the l e s s polar a l c o h o l - I (24%) while e l u t i o n with e t h y l acetate-ethanol (9:1) afforded a l c o h o l - I I (40%). At t h i s point i t i s appropriate to discuss some stereochemical r e l a t i o n -ships between these two diastereoisomeric alcohols. Since we are dealing with two d l - p a i r s i t i s not proper to r e f e r to d i f f e r e n t f u n c t i o n a l groups of the molecule as being a or 8, since the a stereo-chemistry i n one case becomes 3-oriented i n the mirror image of the same d l - p a i r . Therefore we w i l l adopt a 'fcis-trans" nomenclature as shown i n Figure 32 i n the ensuing di s c u s s i o n . For the sake of dis c u s s i o n we w i l l r e f e r to the d l - p a i r i n which the proton at C-3 i s i n a " c i s " r e l a t i o n s h i p with the e t h y l side chain (206 and 208) as " c i s " , while i n the "trans" d l - p a i r these two asymmetric centers have a "trans" o r i e n t a t i o n (207 and 209). Let us assume that r i n g C i s i n the most favored h a l f - c h a i r conformation i n both amino alcoh o l s . At t h i s point the i n f r a r e d spectrum was very h e l p f u l i n assigning the r e l a t i v e p o s i t i o n s of the C-3 proton and the lone p a i r of electrons 106 on the nitrogen atom. Bohlmann has shown that i n c e r t a i n q u i n o l i z i d i n e a l k a l o i d s , i n f r a r e d bands i n the C-H region appear when hydrogen atoms on a carbon atom adjacent to a nitrogen atom are trans and a n t i - p a r a l l e l to the unshared p a i r of electrons on the hetero atom. This technique has proven to be a us e f u l t o o l i n stereochemical assignments i n several a l k a l o i d s . ^ ® Since i n both of our amino alcohols (204) no - 60 -Figure 32. The two diastereoisomeric dl-pairs of alcohols. Bohlmann bands between 2700 and 2800 cm ^ were apparent in the infrared spectra, this would support the situation i n which the C-3 proton i s not i n a co-planar and trans relationship with the unshared pair of electrons on the nitrogen atom of the heterocyclic ring. It i s necessary to recognize that in either the " c i s " or "trans" dl-pairs, one of the members must have a 3a-H orientation, while the enantiomer- w i l l have the 33-H stereochemistry. In other words we can have a 3a-"cis" isomer and a 3a-"trans" isomer, or a 3g-"cis" isomer and a 3g-"trans" one. Consequently any given environment of the C-3 proton with respect to the indole ring and the nitrogen atom is available in either the " c i s " or "trans" dl-pairs. On this basis - 61 -any special shielding or deshielding of the C-3 proton by the indole ring or adjacent nitrogen atom is similar in both series. It is of interest to note however that the chemical shift of the C-3 proton in the two isolated alcohols-I and II is not identical. Therefore we must consider the " c i s " or "trans" relationship of the C-3 proton in conjunction with the ethyl side chain i n order to be able to explain the observed nmr spectra. Molecular models provided some information about the stereochemistry of these amino-alcohols. When the C-3 proton is cis to the lone pair of electrons on the nitrogen atom, the ethyl chain of the trans isomer (207a and 209a) w i l l be lying above the indole ring and far away from the unshared pair of electrons on the nitrogen. As a result of that, one can expect the C-3 proton to be deshielded by the nitrogen atom, while the ethyl group in turn i s shielded by the indole ring. Therefore the "trans" dl-pair has the C-3 proton multiplet at low f i e l d (x 5.82) and the methyl t r i p l e t at high f i e l d (x 9.28). However in the " c i s " isomers (206a and 208a), the ethyl group i s not lying over the indole ring, but i s i n reasonably close proximity to the N-electrons and to the C-3 proton. Therefore we feel that the C-3 proton would be shielded by the ethyl group while the latter in turn i s deshielded by the nitrogen atom. As a result the " c i s " dl-pair has the C-3 proton l multiplet at higher f i e l d (x 5.90) and the methyl t r i p l e t at lower f i e l d (x 9.14). Support for these assignments was available from the literature. Rosen and S c h o o l e r y i n their work on Rauwolfia alkaloids assumed that axial protons absorb at higher f i e l d than the corresponding equatorial ones and on this basis they made their - 62 -OH Figure 33. Conformation of the isomeric alcohols I (206a,208a) and II (207a,209a). assignments. For i n t h e i r study, an a l k a l o i d family having the p a r t i a l structure 210 was considered. In t h i s case the C-3 proton i s i n a d i a x i a l a n t i - p a r a l l e l r e l a t i o n s h i p to the unshared p a i r of electrons on the nitrogen atom, the C-3 proton s i g n a l was always found above x 6.2. On the other hand, when the C-3 proton i s i n an e q u a t o r i a l c i s r e l a t i o n s h i p ( p a r t i a l s tructure 211), i t resonates i around T 5.6. By analogy with these r e s u l t s we may expect i n our case, both isomers to have C-3 protons of the l a t t e r type (T 5.82 and 5.90). Furthermore these authors"'""''"'" have also accounted for the lack of resolved f i n e structure i n the C-3 proton s i g n a l i n cases where the - 63 -lone pair on the nitrogen atom i s in close proximity to i t as, "due to spin coupling to the nitrogen atom, which often smears the peak due to quadrapole relaxation of the nitrogen". The same argument has been used by Wenkert et al.^" 1"^ in the stereo-chemical elucidation of the isomeric alkaloids ajmalicine (10) and tetrahydroalstonine. Again the equatorial and cis-oriented C-3 proton had a lower f i e l d resonance ( T 5.55). During the structural elucidation of corynantheidine type alkaloids using i r , nmr, ord and 113 cd, Beckett et a l . have used extensively the already proposed argument for the C-3 proton and i t s electronic environment. They have also discussed the relative effect of the unshared pair of electrons on the nitrogen atom upon the methyl group i n some quinolizidine compounds. For instance i n 212 the methyl signal was found at x 8.9, whereas in 213 i t was at x 9.16. This result indicated that the methyl signal shifted downfield (x 0.26), by changing i t from the " c i s " 1,3-diaxial to 1,3-axial-equatorial orientation. i 82 Further support for our proposed assignment came from Wenkert's nmr studies concerning the conformational implications i n several flavopereirine derivatives, where again arguments pertinent to the C-3 proton and the methyl group of the ethyl side chain were made. These authors accept even larger limits between an axial and an equatorial - 64 -C-3 proton, anticipating a difference as large as x 1.26. Partial structure (214) and (215) are given for these alkaloids. Based on what we have discussed, we f e l t that alcohol-I i s the " c i s " dl-pair (206a) and (208a), whereas alcohol-II i s the "trans'/ dl-pair (207a) and (209a) i n Figure 33. Alcohol-I was crystallized from methylene chloride-hexane, mp 154-155°. The ultraviolet spectrum had a typical indole chromophore (227.5, 268, 297 nm), while the nmr spectrum (Figure 34) showed a multiplet at x 5.90 due to the C-3 proton. The methyl protons of the ethyl side chain resonated as a t r i p l e t at x 9.14, and the aromatic region had only three protons instead i of eight as noted in the starting material 203. The mass spectrum (Figure 35) had fragments at m/e 149, 186, 199 and 328 (M+) as expected. The molecular formula for this compound, C.-H-QO-N,, , was established 2U 2O 2 2 by elemental analysis and high resolution mass spectrometry. The more polar isomer alcohol II, was more easily crystallized from - 65 -- 99 - 67 -- 68 -methylene chloride to afford an analytical sample, mp 168-169°. The isomeric nature of both alcohols was established beyond doubt by elemental analysis as well as mass spectrometry. The infrared spectrum of this compound was almost superimposable with that of alcohol I. The nmr spectrum (Figure 36) was consistent with that of alcohol I but now the C-3 proton was seen as a multiplet at x 5.82 while the methyl protons of the ethyl group resonated at higher f i e l d (x 9.28). The mass spectrum (Figure 35) had the same pattern of fragmentation as was found for alcohol I, therefore each isomer exhibited spectral data i n complete accord with the assigned structures. Having obtained the required intermediate (204), our next aim was the generation of the nine-membered ring compound bearing the appropriate functional group at C-3, which would provide the basic Aspidosperma skeleton. The synthetic approach i n which the piperidine ring was formed via intramolecular nucleophilic displacement of a mesyl group, i s outlined i n Figures 27 and 37. The resulting quaternary ammonium salt (205) upon nucleophilic attack at the carbon atom adjacent to the quaternary center, underwent a ring cleavage reaction with simultaneous introduction of the necessary functionality at C-3 to generate the vincaminoridine ring system (206) (Figure 37). Thus treatment of each of the isomeric alcohols (204) with methane sulfonyl chloride i n the presence of triethylamine at 0°, provided a quantitative yield of the corresponding mesylates (205). These compounds were not completely characterized. When either of the mesylates of alcohol-I or alcohol-II was treated with potassium cyanide i n dimethylformamide at 150° (bath temperature) followed by - 69 -Figure 37. Preparation of dl-vincaminoridine (4) and i t s epimer. conventional workup of the reaction mixture,a dark gummy product was obtained. Investigation of the product mixture from either mesylate by means of t i c showed them to be identical. Chromatography of the crude product gave some starting material (205), and the two desired isomeric cyanides possessing.the formula (216) but in unsatisfactory i yield. I n i t i a l attempts to prepare these cyanides (216) failed or gave other products when the reaction was carried out In solvents such as diethylene glycol, dimethyl sulfoxide and dimethyl acetamide. In most of the cases, starting material (205) was recovered unreacted. A wide variation of reaction conditions (temperature, time, concentra-tion, solvent) was attempted. A summary of these preliminary - 70 -investigations using 100 mg of the starting material (205) is given in Table I. Table I. Solvent Temperature (°C) Time (hr) mg of CN containing product DMSO 60 48 3 DMSO 60-110 48 20 DMSO reflux 48 20 DMF 150 6 26 DMF reflux 24 20 DMF/10% CH30H reflux 24 15 HMPT 180 24 50 Perhaps the reason for the low yield of the reaction i s due to the steric interference of the ethyl side chain. However i t was obvious that the solvent effect plays a decisive role. It was clear from other investigations i n our laboratory^ that one of the most serious side reactions in this type of reaction i s the ab i l i t y of the i cyanide ion to act as a base, and to perform a Hofmann elimination. That i s , the intermediate 3-vinylindole derivative (217) resulting from the Hofmann reaction undergoes addition of cyanide to yield compounds of type 218. In a parallel series of investigations^ on intermediates lacking the methoxyl group in the indole ring, such reactions did compete and the resulting product (219) was completely - 71 -H characterized. On the basis of these results, i t was clear that an increase i n the yield of the conversion of 205 to 216 (Figure 37), was possible i f we were to increase the nucleophilicity of the cyanide ion. Since dimethyl formamide showed some encouraging i results we decided to turn our attention to other dipolar aprotic solvents. Hexamethylphosphoramide (HMPT) i s a member of this class of reagents. To understand the special properties of HMPT as a reaction medium, one must be aware of the distinction between protic and dipolar 114 aprotic solvents, which i s extensively reviewed in the literature. Protic solvents are proton donors such as water, alcohols, and formamide. - 72 -D i p o l a r a p r o t i c s o l v e n t s are c h a r a c t e r i z e d by d i e l e c t r i c c o n s t a n t s 1 5 5 e 7 , d i p o l e moments at 7.3 D, and an i n a b i l i t y to act as proton 114 donors. To a f i r s t approximation, c a t i o n s are so l v a t e d by both c l a s s e s of sol v e n t s f a i r l y w e l l by i o n - d i p o l a r i n t e r a c t i o n s . However, anions are best s o l v a t e d by molecules that can form H bonds and HMPT, l i k e a l l d i p o l a r a p r o t i c s o l v e n t s , l a c k s t h i s c h a r a c t e r i z a t i o n . Hence, s o l v a t i o n of anions i n these s o l v e n t s i s g r e a t l y reduced; not only do anions have a higher a c t i v i t y i n HMPT"''''"'' but bim o l e c u l a r r e a c t i o n s w i l l be con s i d e r a b l y a c c e l e r a t e d . On the b a s i s of the above argument we decided on the use of HMPT as a so l v e n t i n order to in c r e a s e the n u c l e o p h i l i c i t y of the cyanide i o n . Indeed when, intermediate 205 was t r e a t e d w i t h potassium cyanide i n HMPT at 165° f o r 7 hours, f o l l o w e d by con v e n t i o n a l workup, we were able to i s o l a t e a f t e r column chromatography two cyanides (216) i n 49% y i e l d , as w e l l as some unreacted s t a r t i n g m a t e r i a l (205). The i s o m e r i c nature of these cyanides was e s t a b l i s h e d by elemental a n a l y s i s and high r e s o l u t i o n mass spectrometry which i n d i c a t e d the formula C^^H^^ON^. An a n a l y t i c a l sample was obtained by high pressure l i q u i d chromatography (Figure 38). Cyanide I was c r y s t a l l i z e d from n-hexane-acetone, then methylene c h l o r i d e (19%), mp 186-187°. The i n f r a r e d spectrum showed the n i t r i l e band at 2225 cm \ w h i l e the u l t r a v i o l e t spectrum had maxima, at 226, 277 and 298 nm. The nmr spectrum (Figure 39) showed a qua r t e t a t x 6.12 (J^g = 10 cps, J = 3 cps) due to the. C-3 proton (-CHCN), and a t r i p l e t a t x 9.07 ( J = 7 cps) assigned to the methyl group of the e t h y l s i d e chain. The main fragments i n the mass spectrum (Figure 40) were found at m/e 124, - 73 -min. Figure 38. High pressure li q u i d chromatography of cyanide I and II. 2. Cyanide-I (20 min); 3. Cyanide-II (29 min). - 74 -a o a. 16-METHOXY CYANIDE-I UJ LU s ' o o ' " V o o ' . o ' ' '.i5o:o,"'"'2flo:o'"rnao:o' ' ' a b p . o ' 350.0 400.0 450.0 s o o . o . s s o . o e o o . o : M/E. 16-METHOXY CYRNIDE-II i i i i i i ' i 1 1 i I i i i i i i 1 1 1 — i — 1 1 1 1 i (Do 1—in —J LU OH CN 4 50 i i • |—f i • i i | ' i i ' i — i — | — — i i | ' '—< 1 I 1r 0 100.0 150.0 200.0 250.0 300 P M/t - i — i — i i | 350.0 400.0 450.0 Figure 40. Mass spectra of 16-methoxy cyanide I and II. s o o . o 550.0 600.0 - 76 -126, 177, 212, and 337 (M ). These fragments are depicted in Figure •I . . . . . 117,118 41, and are in agreement with expectation. a m/e 337 (M ) \ N m /e 126 m/e 212 H 2 C < N + X N m/e 177 m/e 124 Figure 41. Fragmentation of cyanide I and cyanide I I , The cyanide I I was crystallized from methanol (30%), mp 191-192°. The infrared spectrum was almost superimosable with that of cyanide I. The nmr spectrum (Figure 42) showed a quartet at x 4.03 ( J A T > = 10 cps, J = 3 cps) assigned to the C-3 proton, while the methyl protons of the ethyl side chain resonated as a t r i p l e t at x 9.34 (J = 7 cps). The mass spectrum (Figure 40) of this compound showed the same pattern of fragmentation as the less polar cyanide I . The chemical shift of the C-3 proton for the cyanides I and I I can be explained by considering several conformations similar to those - 78 -proposed by Rompis.""""1^ According to h i s proposal, the C-3 proton i s away from the lone p a i r of electrons on the nitrogen i n the case of cyanide I r e s u l t i n g i n no s p e c i a l e f f e c t by the nitrogen atom and a more normal p o s i t i o n for t h i s type of proton (T 6.12, structure 220), while i n the case of cyanide II the C-3 proton i s close to the nitrogen r e s u l t i n g i n magnetic deshielding of t h i s proton (x 4.03, structure 221). . H 151a,R = C0 2CH 3 151b, R = C0 2CH 3 Since i t was known from previous experiments^ that the a l k a l i n e h y d r o l y s i s of cyanide-I and cyanide-II gave the desired carbomethoxy d e r i v a t i v e (151) i n only 30% y i e l d and decarboxylation may be a serious side reaction, i t was f e l t that h y d r o l y s i s under a c i d i c conditions might provide the ester d e r i v a t i v e (151) i n better y i e l d . I t would also provide us with some information about the stereochemistry at C-3 under these conditions. We f i r s t sought to carry out such a transformation by using a l i m i t e d amount of hydrochloric acid, that had been generated i n s i t u by s o l v o l y s i s of a c e t y l c h l o r i d e i n i anhydrous methanol. Thus a mixture of the epimeric cyanides (216) was treated as indicated above, and s t i r r e d f or 94 hours, followed by the conventional workup. The r e a c t i o n product was i s o l a t e d e i t h e r by preparative t i c or high pressure l i q u i d chromatography. However we found that t h i s - 79 -approachhad been unsuccessful as far as the conversion of the cyano to the carbomethoxy group was concerned. Instead we had promoted an epime-rization of cyanide-II to cyanide-I, completely reversing the ratio cyanide-I/cyanide-II i n i t i a l l y present i n the starting material (see. Figure 38). We next increased the acid concentration and were able after conventional workup to isolate the desired carbomethoxy derivative (151) in 20% yield. Finally we decided to perform the hydrolysis i n methanol that had been saturated with anhydrous hydrogen chloride. Thus when the mixture of epimeric cyanides (216) was treated according to the above conditions we were able to isolate 16-methoxy-dl-vincadine (151a) and i t s C-3 epimer i n 26% and 9% yield respectively. We observed that purification on alumina or s i l i c a gel resulted in poor recovery and decomposition of the desired esters. Perhaps i t i s appropriate to note that 16-methoxy-dl-vincadine (the major isomer) had the stereochemical features of cyanide-I (the minor isomer in the previous step of the synthetic sequence), as can be seen in the nmr spectra (Figures 39 and 43). It had a typical methoxy indole ultraviolet spectrum (228, 278 and 301 nm) and i t s infrared spectrum showed a strong carbonyl absorption at 1725 cm The nmr spectrum (Figure 43) was most informative. A new three-proton singlet due to the protons of the carbomethoxy group could be seen at x 6.31, while an unresolved quartet at x 6.35 was assigned to the C-3 proton. The methyl group of the ethyl side chain was found at t 9.18. - 80 -- 81 -The main fragments in the mass spectrum (Figure 44) were present at + m/e 124, 138, 210, 245, and 370 (M ). A rationalization for some of the fragments is presented in Figure 45. High resolution mass spectrometry established the formula 2^2^ 39^ 3^ 2 ^ o u n < ^ : 370.225; c a l c : 370.225). The minor isomer had also a methoxy indole chromophore and an infrared spectrum which was almost superimposable with that of the major isomer. However the nmr spectrum (Figure 46) now had the C-3 proton quartet ( J A R I = 12 cps, J = 2 cps) at T 4.49. The three-A B A C proton singlet due to the carbomethoxy group was found at x 6.38, while the methyl protons of the ethyl side chain were found as a t r i p l e t at x 9.36. The mass spectrum (Figure 44) was similar to that of 16-methoxy vincadine. The isomeric nature of these two diastereo-isomers was confirmed by high resolution mass spectrometry. The next step in the synthetic sequence in order to achieve the required intermediate 4, was the methylation of the indole nitrogen atom of the ester derivatives (151). This was done via the previously 120 published procedure. Thus treatment of 151 with sodium amide in liq u i d ammonia, followed by alkylation of the resulting anion with methyl iodide, gave the N-methyl compound (4) in good yield. The reaction product after conventional workup and purification by preparative t i c on s i l i c a gel gave the dl-epimers in 62% yield. The more polar of these dl-pairs on s i l i c a gel chromatoplates, had an infrared spectrum with no NH absorption and a strong carbonyl absorption (CC^CH^) was present at 1735 cm"1. The ultraviolet spectrum (232, 288, 300 nm) was in good agreement with that reported in the l i t e r a t u r e . ^ ' T h e nmr >-00 100 80 60 f_ u 4 0 L-5 20 '!!! ,1!!!:! 41 CM 16-METHOXY-dl-VINCADINE I;'!;! 0 I—.':.!iii!ii!.i)ii!!i;iiii:>ii!i .:i6u;ii;!iiil,!iiiiL;!ii!i!l!LJii,iil J-in in IT) o •—i CM 1 4 ill CM O ro II t 1 * t » I » I I » i i i i i i I M | t , t i " i - i i 90 100 150 200 250 -300 100 _ m/e I I I I 350 400 >-H oo 2C 80 60 16-METHOXY-dl-EPIVINCADINE (71 40 r~ 20 U U i l l CM II in m r - i i n r o I—t CM + I S o ro C0,CH t > y i » » i » i i i i »• i t i i t i i i i i i i j , , 9C 100 150 200 250 300 350 I I I I 400 m/e CO Figure 44. Mass spectra of 16-methoxyvincadine and i t s epimer. - 83 -Figure 45. Fragmentation of 16-methoxyvincadine and i t s epimer upon electron impact. Figure 46. Nmr spectrum of 16-methoxy-dl-epivincadine (151b). (Taken from C. Gletsos Ph.D. thesis) - 85 -spectrum (Figure 47) showed the absence of the signal due to the N-H proton, and a new singlet at x 6.56 was assigned to the N-methyl group. The C-3 proton was found at x 6.20 as a multiplet overlapping with one of the methoxy signals. The formula ^23H32^3N2 t ^ s c o m P o u n d was established by high resolution mass spectrometry (found: 384.239, Calcd.: 384.241). The mass spectrum (Figure 48) showed the expected fragments at m/e 124, 210, 259, and 384 (M+). The less polar dl-pair had an infrared spectrum almost superimposable to that of the above compound. The ultraviolet spectrum was also a typical indole chromophore (232, 288, 298 nm). The nmr spectrum (Figure 49) showed the absence of signals due to the NH proton, and the N-methyl protons were found at x 6.46. The C-3 proton was present as a multiplet at x 3.90. The mass spectrum (Figure 48) had the expected peaks at + 121 m/e 124, 210, 259 and 384 (M ). Comparison of the spectral data of the natural vincaminoridine, an alkaloid isolated from Vinca rosea 119 Linn, which has been assigned the structure 4, with those of the above diastereoisomers confirmed the identity of our less polar isomer (C-3 proton at x 3.90) with the natural alkaloid. Having obtained the required nine-membered ring intermediate (4), the next obvious step i n order to achieve the synthesis of the pentacyclic aspidosperma-type system (195) would be the transannular cyclization reaction. Although the pentacyclic structure (195) has not yet been isolated from natural sources, i t was f e l t to be a valuable intermediate for the total synthesis of vindoline (3), and therefore would provide an entry into the dimeric series as well. However, the transannular cyclization process (4 to 195) via the RELATIVE INTENSITY RELATIVE INTENSITY -F CT> CO O N) -P CD o o o o o o o o o o o o o - LS -dl-VINCAMINORIDINE (100 Mc/s) J 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Figure 49. Nmr spectrum of dl-vincaminoridine (4). (Taken from C. Gletsos Ph.D. thesis) - 89 -iminium intermediate has important stereochemical implications. The necessary "folding" of the nine-membered ring system (4) requires a definite stereochemistry in the end product (195). Although this process has received considerable application particularly in our laboratory, and to a lesser extent i n other groups, the overall importance of the stereochemistry at C-3 has not been ascertained. Preliminary investigation of the conversion of 4 to 195 using mercuric acetate oxidation in glacial acetic acid at room temperature on a mixture of the epimeric carbomethoxy derivatives gave a reaction product which was purified by preparative t i c on alumina. Two semi-crystalline materials in 11% (less polar component) and 24.5% (more polar component) yields were obtained. The infrared spectra of both of these compounds were superimposable. Mass spectra of this material had fragments at m/e 149, 202, 263, and 382 (M+). High resolution mass spectrometry established the formula, C^jR^®^! ^ o u n c ^ : 382.224; C a l c : 382.255). The ultraviolet spectrum of the less polar compound had maxima at 225, 310 ( i n f l . ) and 335 nm, while the more polar had maxima at 225, 310 ( i n f l . ) and 340 nm. The above results suggested that they were the desired cyclization product (195), but the small amount of material available did not allow us to take an nmr spectrum. Since our synthetic approach led predominantly to one epimer (4) i and we were not sure which stereoisomer would undergo the transannular cyclization reaction in better yield, we decided to investigate the possibility of epimerization of the major isomer. Indeed dl-epivinca-minoridine upon treatment with sodium methoxide in refluxing methanol for 48 hours, followed by the conventional workup and isolation by - 90 -thin layer chromatography, provided dl-vincaminoridine and dl-epivinca-minoridine in a 2:3 ratio. At this point i t was decided that we should turn our efforts toward another series of investigations that involved the degradation of vindoline (3). The degradation sequence could provide a feasible high yielding route to the pentacyclic intermediate 195. It would also give us an opportunity to compare our totally synthetic materials, presently in racemic form, with the appropriate optically active templates. These latter substances would not only help us i n the identification and characterization of the synthetic products, but they might be ut i l i z e d as relays for further synthetic work. Furthermore the degradation sequence would also provide, i n optically active form, appropriate dihydroindole units that would be invaluable in other synthetic areas. Those modified templates could perhaps lead to the synthesis of new dimers related to vinblastine (1) and vincristine (2),but with different pharmacological activity. A discussion of these experiments i s present in Part II of this thesis. - 91 -EXPERIMENTAL I Melting points were determined on a Kofler block and are uncorrected. Ultraviolet spectra were measured in 95% ethanol or methanol on a Cary model 15 spectrophotometer. Infrared spectra were recorded on a Perkin-Elmer 21 or 137 spectrophotometer. Nuclear magnetic resonance spectra (nmr) were taken in deuterochloroform solution on Varian Associates spectrometers, models T-60, HA-100 or XL-100. Line positions are given in the Tiers T scale, with tetramethylsilane as an internal standard. The types of protons, integrated areas, multiplicity and spin coupling constant J are indicated i n parentheses. Mass spectra were recorded on an Atlas CH-4B or Associated E l e c t r i c a l Industries MS-902 spectro-meter, high resolution measurements being determined on the latter instrument. S i l i c a gel G and alumina Woelm containing 2% by weight of a fluorescent indicator were used for thin-layer chromatoplates. As spraying reagent a solution 1:2 of antimony pentachloride in carbon tetrachloride, or a solution of eerie sulfate in aqueous sulfuric acid were used extensively. Unless otherwise specified column chromatography was performed using either Woelm grade s i l i c a or neutral alumina, and deactivated as required with the correct amount of water. D i s t i l l e d solvents were used. High pressure liquid chromatography was performed either on a Waters ALC-100 or ALC-202 instrument. - 92 -Elemental analyses were performed by Mr. P. Borda of the Micro-a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia. Synthesis of Aldehydo Ester (140) The i n i t i a l experiments i n v o l v i n g the preparation of the aldehydo ester (140) were performed according to the previously established procedure.7»88,103 D e t a i l s of the q u a n t i t i e s used and the y i e l d s obtained i n t h i s work, f o r the various steps are given below. Also where small differences i n conditions and/or y i e l d s were observed these are i n d i c a t e d . y-Benzyloxy propanol (197) The monosodium s a l t of propane-1,3-diol (500 g) i n xylene was treated with benzyl c h l o r i d e under the conditions o u t l i n e d i n the l i t e r a -ture."^"'' The product was obtained as a c l e a r o i l (254.4 g, 66% y i e l d ) , bp 95-100°/l-2 mm ( l i t . 145-150°/13 mm). Benzyl y-Chloropropyl Ether (198) y-Benzyloxy propanol (197) (234 g) was treated with t h i o n y l c h l o r i d e 102 (190 g) i n dimethyl a n i l i n e , according to the known procedure. The product was obtained as a c o l o r l e s s o i l (200 g) i n 80% y i e l d , bp 95-i 100°/1 mm ( l i t . 129*716 mm). Di e t h y l y-Benzyloxypropylethyl Malonate (134) Ethy l d i e t h y l malonate (81 g) was a l k y l a t e d with benzyl y-chloro-propyl ether (198) (81 g) i n the presence of sodium ethoxide i n absolute 88 ethanol according to the l i t e r a t u r e procedure. The desired product.. - 93 -was obtained as a c o l o r l e s s o i l (76 g) i n 54% y i e l d , bp 140-150°/0.1 mm. y-Benzyloxypropylethyl Malonic Acid (.199) Di e t h y l Y~benzyloxypropylethyl malonate (134) 976 g) was treated with potassium hydroxide i n ethanol/water according to the l i t e r a t u r e 88 procedure. The product was c r y s t a l l i z e d from n-hexane-ether as c o l o r l e s s mass i n (51 g) 81% y i e l d , mp 117-120°. 2-(Y-Benzyloxypropyl)-butanoic Acid (200) Y-Benzyloxypropylethyl malonic acid (51 g) (199) was decarboxylated to give the desired product as a yellow viscous o i l (43 g). The product was not furth e r p u r i f i e d but subjected to the next react i o n . E t h y l q-(Y-benzyloxypropyl)-butanoate (135) A s o l u t i o n of 2-(Y-benzyloxypropyl)-butanoic acid (40 g) (200) i n 88 absolute ethanol was e s t e r i f i e d according to the l i t e r a t u r e procedure. The desired product was obtained as a c l e a r o i l (38 g) i n 76% y i e l d , bp 135°/1.5 mm. Ethyl q-(Y-Benzyloxypropy.l)-cx-allylbutanoate (201) Ethy l a-(Y~benzyloxypropyl)-butanoate (24 g) (135) i n ether, was al k y l a t e d with a l l y l bromide (11.6 g) i n the presence of sodium 7 90 tr i p h e n y l methane as in d i c a t e d i n the l i t e r a t u r e . ' The desired a l k y l a t e d product was obtained as a c o l o r l e s s o i l (22 g) i n 83% y i e l d , bp 132-134°/0.15 mm. - 94 -Ethyl g-(y-benzyloxypropyl)-g-(g-formylmethyl)-butanoate (140) A solution of the a l l y l compound (24 g) (201) in tetrahydrofuran was treated with osmium tetroxide and sodium metaperiodate according 7 90 to the literature procedure. ' Purification by vacuum d i s t i l l a t i o n , gave the desired aldehydo ester (168) in 70% yield as a colorless o i l , bp 174-176°/0.75 mm. Preparation of 11-methoxycyclic lactam (150) 6-Methoxy tryptamine (149) (7 g, 36.8 mmole) and the aldehyde ester (140) (15 g, 49 mmole) i n glacial acetic acid (25 ml) were refluxed for 1.5 hr, under an atmosphere of oxygen-free nitrogen. The acetic acid was removed under reduced pressure to give a yellow residue. Purification on a basic alumina column (Shawnigan, 400 g) and elution with pet. ether-ethyl acetate (2:3) gave the pure lactam (150) (16 g, 98%). This product-was a mixture of the two expected diastereo-isomers but no attempts to separate them at this stage were necessary for our purpose. Infrared ( l i q . film) 3250 (broad, hydrogen bonded NH), 1735 (small) and 1670 (strong, lactam) cm 1 . Ultraviolet; A (log e): D13.X 227 (4.12), 264 (sh, 3.73), 272 (sh, 3.72), 295 (3.75), 321 (3.47), 335 (3.39) nm. Nmr signals (100 MHz): x 1.56 (broad singlet, IH, NH), 2.75 (multiplet, 6H, aromatic), 3.20 (singlet, IH, C-12 proton), 3.25 (quartet, J . = 8 cps, J =2 cps, IH, C-10 proton), 5.25 ortho r meta (broad t r i p l e t , J = 8 cps, IH, C-3 proton), 5.55 and 5.66 (singlets, 2H, C,HcCHo0-), 6.23 (singlet, 3H, CH_0-), 9.05 and 9.28 (triplets, J = 7 b 5 2 J and 7 cps, 3H, CH^CH^-). Mass spectrum, main peaks: m/e 91, 149, 188, 263, 341, 432 (M +). Molecular weight: 432.241. Calc. for C ^ H ^ O ^ : 432.241. Found: C, 75.21; H, 75.21; H, 7.51; N, 6.52. Calcd. for - 95 -C 2 7 H 3 2 ° 3 N 2 : C ' 7 4 ' 9 7 ; H ' 7 , 4 6 ; N ' 6 - A 8 ' Preparation of 11-methoxycyclic amine (203) The lactam (150) (25 g, 0.051 mole) was dissolved i n anhydrous tetrahydrofuran (150 ml, d i s t i l l e d from L i A l H ^ and stored over Cal-^) and slowly added with s t i r r i n g to a s o l u t i o n of L i A l H ^ (16 g, 0.420 mole) i n anhydrous tetrahydrofuran (450 ml). The reaction was performed under dry conditions and an atmosphere of oxygen-free nitrogen. Refluxing with adequate s t i r r i n g f o r 72 hr followed. The r e a c t i o n mixture was cooled to room temperature and then i n an ice-water bath. Wet tetrahydro-furan (water i n THF, 1:3) was added c a r e f u l l y with vigorous s t i r r i n g to decompose the complex and excess-'-LIAIH^. The white sludge was s t i r r e d f o r 20 minutes more and i t was'• f i l t e r e d through a bed of c e l i t e . The cake was washed three times with hot tetrahydrofuran. The f i l t r a t e was d r i e d over anhydrous magnesium s u l f a t e . F i l t r a t i o n and removal of the solvent under reduced pressure gave a l i g h t yellow gum (24.5 g). This gum was chromatographed on a basic alumina column (Shawinigan, 400 g). E l u t i o n with benzene-ethyl acetate (20-100%) gave the desired product, a pure mixture of the two dl-epimers (22 g, 92%). Infrared (neat): no carbonyl absorption. U l t r a v i o l e t ; A (log e): 230 (4.18), 263 (3.68), • max 270 (sh, 3.66), 300 (3.74) mm. Nmr signals (100 MHz): T 2.04 (broad s i n g l e t , IH, NH), 2.70 (multiplet, 6H, aromatic), 3.25 ( s i n g l e t , IH, C-12 proton), 3.30 (quartet, J , = 8 cps, J = 2 cps, IH, C-10 proton), ortho meta 1 5.53 and 5.65 ( s i n g l e t s , 2H, Cgl^CH^O-), 5.93 (broad t r i p l e t , J = 6 cps, IH, C-3 proton), 6.26 ( s i n g l e t , 3H, CII-jO-), 9.16 and 9.30 ( t r i p l e t , J = 7 cps and 7 cps, 3H, CH^CH^-). Mass spectrum; main peaks: m/e 91, 149, 214, 260, 327 and 418 (M +). Molecular weight: 418.265. Calc. for - 96 -C o-,H. w0„N„: 418.262. Found: C, 77.61; H, 8.21; N, 6.62. Calc. for C 2 7 H 3 4 ° 2 N 2 : C ' 7 7 , 4 7 ; H» 8 ' 1 9 ; N> 6 ' 6 9 ' Debenzylation of the Mixture of dl-Epimeric Amines (203) To the mixture of amines (203) (20 g, 47.8 mmole) dissolved i n e t h y l a l c ohol (250 ml) and concentrated HC1 (8 ml), 10% palladium on charcoal (2 g) was added. The mixture was s t i r r e d and hydrogenated at room temperature and 1 atm. pressure f o r 6.5 hr. When no more uptake of hydrogen was noted the r e a c t i o n was stopped. The c a t a l y s t was removed by f i l t r a t i o n , through a bed of c e l i t e and the cake was washed several times with methanol. The f i l t r a t e was concentrated under reduced pressure at room temperatre and then b a s i f i e d by c a r e f u l a d d i t i o n of a saturated s o l u t i o n of sodium carbonate. To the r e s u l t i n g basic s o l u t i o n (litmus paper), water (150 ml) was added and e x t r a c t i o n with methylene chl o r i d e (6 x 100 ml) followed. The combined organic layers were washed with water (2 x 200 ml) and dried over anhydroxis sodium s u l f a t e , f i l t r a t i o n and removal of the solvent under reduced pressure afforded a yellowish s o l i d (15 g). This m a t e r i a l was dissolved i n a minimum amount of e t h y l acetate. To the s o l u t i o n was added a few grams of alumina and the e t h y l acetate was evaporated o f f from the s l u r r y . The alumina coated with the. same was then transferred to the top of a column f i l l e d with alumina (Shawinigan, 650 g) i n benzene. Gradient e l u t i o n with e t h y l acetate-ethanol (98:2) gave alco h o l - I (less polar) and with e t h y l acetate-ethanol (9:1) afforded a l c o h o l - I I . 11-Methoxy Alcohol-I - Amorphous s o l i d (3.73 g, 24%). It was c r y s t a l l i z e d from methylene chloride-n-hexane (3:1), washed with cold - 97 -acetone and recrystallized once more from wet methanol, mp 154-155°. Infrared (KBr): absence of strong benzylic bands between 770-690 cm \ Ultraviolet: X (log e): 227.5 (4.42), 268 (3.66), 297 (3.73) mm. max Nmr signals (100 MHz): T 2.28 (broad singlet, IH, NH) , 2.71 (doublet, J , = 9 cps, IH, C-9 proton), 3.22 (doublet, J _ = 2 cps, IH, C-12 ortho meta proton), 3.30 (quartet, J =2 cps, J ,^ = 9 cps, IH, C-10 proton), r meta r ortho 5.90 (multiplet, IH, C-3 proton), 6.22 (singlet, 3H, CH^O-), 6.55 (tri p l e t , 2H, -CH20H), 9.14 (tr i p l e t , 3H, CH_3CH2-). Mass spectrum; main peaks: m/e 149, 186, 199, 214 and 328 (M +). Molecular weight: 328.215. Calc. for C o nH o o0„N.: 328.215. Found: C, 72.89; H, 8.69; N, zU zo 2 2 8.60. Calcd. for Co_HOQ0.N_: C, 73.13; H, 8.59; N, 8.53. Z U Z o 2 2 11-Methoxy Alcohol-II - Amorphous sol i d (6.31 g, 40%). Crystallized easier than alcohol-I from methylene chloride, mp 168-169°. Infrared (KBr): absence of strong benzylic bands between 770-690 cm ^  and similar with that of alcohol-I. Ultraviolet: X (log e): 227 max (4.50), 269 (3.71), 297 (3.79) mm. Nmr signals (100 MHz): T 2.16 (distorted singlet, IH, NH), 2.70 (doublet, J o r t h o = 9 cps, IH, C-9 proton), 3121 (doublet, J m e t a = 2 cps, IH, C-12 proton), 3.30 (quartet, J =2 cps, J . — 9 cps, IH, C-10 proton), 5.82 (multiplet, IH, meta ortho C-3, proton), 6.23 (singlet, 3H, CH 0-), 6.38 (t r i p l e t , 2H, -CH OH), 9.28 (tr i p l e t , 3H, CH^CH^). Mass spectrum; main peaks: m/e 199, 214 and 328 (M+). Molecular weight: 328.216. Calc. for C O AH o o0 oN o: 328.215. ZU 2o 2 2 Found: C, 72.82; H, 8.85; N, 8.63. Calc. for C o r iH. o0 oN o: C, 73.13; zU z o 2 2 H, 8.59; N, 8.53. - 98 -Preparation of ll-Methoxy Mesylates (205) (a) ll-Methoxy alcohol-I (204) (600 rag, 1.82 mmole) was dissolved in a mixture of dry triethylamine (10 ml, d i s t i l l e d over sodium hydroxide) and chloroform (26 ml) and cooled to -10-0°C (ice-rock salt bath). Keeping anhydrous conditions, freshly d i s t i l l e d methane sulfonyl chloride (500 mg, 4.37 mmole, d i s t i l l e d over I ^ s ^ w a s a c^ed dropwise with efficient s t i r r i n g . The reaction mixture was allowed to come slowly to room temperature and l e t stand for 44 hours. The solvent was removed at room temperature and reduced pressure to give a deep red gum. This gum was dissolved in chloroform (20 ml) and extracted with aqueous ammonium hydroxide (4 N, 4 x 15 ml) and once with water (15 ml). The combined aqueous layers were washed with a l i t t l e chloroform (5 ml) and the water was removed under reduced pressure and moderate heating. Any remaining water in the resulting yellow solid was azeotroped several times with dry benzene. The residue was extracted with dry hot chloroform (4 x 10 ml) which dissolved only the mesylate but not the inorganic material. The chloroform solution was f i l t e r e d and the f i l t r a t e was evaporated to dryness under reduced pressure to give the pure mesylate (723 mg, 98%) as a light yellow foam, and i t was used for the subsequent steps without further purification. Infrared (neat): 3448 (NH), 1639 (C=C) cm"1. Nmr signals (100 MHz): T 2.82 (doublet, J , = 8 cps, IH, ortno i C-9 proton), 2.93 (doublet, J =2 cps, IH, C-12 proton), 3.31 (quartet, meta J ^ = 2 cps, J , = 8 cps, IH, C-10 proton), 4.95 (multiplet, IH, meta r ortho r C-3 proton), 6.24 (singlet, 3H, CH^O-), 9.15 (t r i p l e t , 3H, CH^CH^). (b) ll-Methoxy alcohol-II (204) was mesylated exactly as i s described above to give the pure mesylate (205) in quantitative yield. - 99 -Again this material was used for the next step without further purification Infrared (neat): 3448 (NH), 1645 (C=C) cm"1. Nmr signals (100 MHz): -0.84 (singlet, IH, NH), 2.78 (doublet, J o r t h o = 8 cps, IH, C-9 proton), 3.03 (doublet, J =2 cps, IH, C-12 proton), 3.36 (quartet, J _ = meta r r n meta 2 cps, J o r t h o = 8 cps, IH, C-10 proton), 5.08 (multiplet, IH, C-3 proton), 6.28 (singlet, 3H, CH_30-), 9.24 (t r i p l e t , 3H, CH_3CH2-) . Preparation of 16-Methoxy Cyanides (216) (a) To a solution of mesylate alcohol-I (450 mg) i n dry dimethyl-formamide (25 ml) pulverized potassium cyanide (330 mg) was added. The reaction was performed i n dry nitrogen atmosphere. The reaction mixture was heated at 155° (bath temperature) and stirred for 6 hours. The dark reaction mixture was cooled to room temperature and 6 N aqueous ammonium hydroxide (40 ml) was added to i t under s t i r r i n g . The resulting basic solution was extracted with benzene (5 x 35 ml). The combined benzene extracts were washed with brine (2 x 15 ml). The organic layer was dried over anhydrous sodium sulfate, f i l t r a t i o n and removal of the solvent under reduced pressure at room temperature gave 190 mg of a brown gum. Chromatography of the crude product on alumina neutral Woelm, activity III, using benzene as eluent gave 120 mg of a mixture of the epimeric cyanides. i (b) To a solution of the mesylate of alcohol-II (205) (1.265 g, 3.11 mmole) i n dry hexamethylphosphoamide (40 ml), pulverized potassium cyanide (1.170 g, 18 mmole) was added. The resulting mixture was heated at 175° (bath temperature) and stirred for 7.5 hr under an atmosphere of oxygen-free nitrogen. The dark reaction mixture was cooled to room temperature and aqueous ammonium hydroxide (80 ml, 5 N) was added to i t - 100 -with s t i r r i n g . The resulting basic solution was extracted with ethyl ether (6 x 40 ml). The combined ethereal extracts were washed with water (3 x 15 ml). The organic layer was dried over anhydrous sodium sulfate, f i l t e r e d and the solvent removed under reduced pressure at room temperature to afford 600 mg of a yellowish foam. (c) The mesylate of alcohol-I (205) (2.276 g, 5.60 mmole) was treated exactly as indicated above to afford 1.000 g of a yellowish foam and checking the reaction products ( t i c , i r , nmr information) i t was shown that both mesylates gave the same mixture of epimeric cyanides. The crude reaction products (600 mg) were chromatographed on alumina neutral column (40 g, Woelm, activity III). Elution with pet. ether-benzene (3:2) gave cyanide-I and further elution with the same solvent system (1:1) afforded cyanide-II. 16-Methoxy Cyanide-I - Amosphous solid (199 mg, 19%). Recrystallized from n-hexane-acetone and methylene chloride, mp 186-187°C. Infrared (KBr): 3346 (strong, NH) and 2252 (medium, -CN) cm"1. Ultraviolet; A (log e): 227 (4.48), 275 (3.69), 300 (3.81) mm. Nmr signals (100 MHz): T 1.80 (broad multiplet, IH, NH), 2.69 (doublet, J o r t h o = 8 cps, IH, C-14 proton), 3.20 (singlet, IH, C-17 proton), 3.27 (quartet, J , ornto 8 cps, J _ = 2 cps, IH, C-15 proton), 6.12 (quartet, J,„ = 4 cps, J._ = meta r n AB AC 2 cps, IH, C-3 proton), 6.21 (singlet, 3H, CH^O-), and 9.07 ( t r i p l e t , J = 7 cps, 3H, CH-jQ^-)- Mass spectrum; main peaks: m/e 124, 126, 177, 212 and 337 (M +). Molecular weight: 337.218. Calc. for C 2 1 H 2 7 ° N 3 : 337.215. Found: C, 74.61; H, 8.52; N, 12.39. Calc. for C^U^OUy C, 74, H, 8.07; N, 12.45. - 101 -16-Methoxy Cyanide-II - Amorphous solid (320 mg, 30%). Recrystallized from methanol and n-hexane, mp 191-192°. Infrared (KBr): 3356 (strong, NH), 2232 (medium, -CN) cm"1. Ultraviolet; A (log e ) : 226 (4.45), max 277 (3.37), 298 (3.83) mm. Nmr signals (100 MHz): 1.94 (broad singlet, IH, NH), 2.64 (doublet, J , = 8 cps, IH, C-14 proton), 3.15 (doublet, — ortho J =2 cps, IH, C-17 proton), 3.23 (quartet, J =2 cps, J , = meta meta ortho 8 cps, IH, C-15 proton), 4.03 (quartet, J._ = 10 cps, J = 3 cps, IH, AB AC C-3 proton), 6.18 (singlet, 3H, CH_30-), 9.34 (t r i p l e t , J = 7 cps, 3H, CH3CH2-). Mass spectrum; main peaks: m/e 124, 126, 177, 212, 337 (M+). Molecular weight: 337.216. Calc. for C^H^ON.^: 337.215. Found; C, 74.57; H, 8.31; N, 12.41. Calc. for C 2 1H 2 7ON 3: C, 74.74; H, 8.07; N, 12.45. High Pressure Liquid Chromatography of the Epimeric Cyanides (216) INSTRUMENT: Waters' ALC-202. SOLVENT: Pet. ether (30-60°)-chloroform (1:1). FLOW RATE: 0.6 ml/min. COLUMN: Alumina neutral Woelm 6 f t . x 2 mm O.D. SAMPLE LOAD: 10 mg, total. RETENTION TIME: Cyanide-I (20 min), cyanide-II (29 min). i v  Epimerization of the Isomeric Cyanides (216) To a cooled solution (ice-water bath) of the isomeric cyanides (120 mg, 0.356 mmole) in anhydrous methanol, acetyl chloride (0.2 ml) was added under s t i r r i n g . After 10 minutes d i s t i l l e d water (0.1 ml) was added and the reaction mixture was allowed to come slowly to room temperature. Stirring was continued for 94 hours. The solvent was - 102 -then removed under reduced pressure without heating. The residue was taken up in ether (10 ml) and neutralized with saturated sodium bicarbonate solution, and extracted with ether (3 x 10 ml). The combined organic extracts were washed with brine (10 ml) and dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent gave 175 mg of a yellowish solid. Purification of the crude reaction mixture either by column chromatography on alumina neutral Woelm (10 g, activity III) using pet. ether-benzene (1:1) as eluent or high pressure liquid chromatography (Waters' ALC 100, pet. ether-chloroform (3:1), 9 ml/min, alumina neutral Woelm, 4 f t x 3/8" O.D.), afforded 62 mg of cyanide-I (15 min) and 12 mg of cyanide-II (33 min). Preparation of 16-Methoxy-dl-vincadine (151a) and i t s Epimer (a) To a cooled solution of the epimeric cyanides (216) (18 mg) in anhydrous methanol (10 ml), acetyl chloride (7 ml) was added. The reaction mixture was allowed to come slowly to room temperature and let stand for 48 hours, when d i s t i l l e d water (0.1 ml) was added. After an additional 24 hours, the solvent was removed under reduced pressure without heating. The residue was taken up in ether, neutralized with saturated sodium bicarbonate solution, and extracted with ether (3 x 15 ml). The combined organic extracts were dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent gave 16 mg of a gum. Purification of this crude reaction product by high pressure liquid chromatography (Waters' ALC-100, pet. ether-chloroform (3:1), 9 ml/min, alumina neutral Woelm 4 f t . x 3/8" O.D., 16-methoxy-dl-vincadine, 5 min; 16-methoxy-dl-epivincadine, 8 min) gave (6 mg) of the desired carbomethoxy derivative (151) in 20% yield. - 103 -(b) To a solution of 16-methoxy cyanide-II (216) (280 mg, 0.83 mmole) in anhydrous methanol (20 ml), water (0.2 ml) was added. The solution was cooled (ice-water bath) and saturated with anhydrous hydrogen chloride (20 min). The reaction mixture was then allowed to come slowly to room temperature and l e f t standing for 96 hours. The solvent was removed under reduced pressure without heating and the residue was taken up in a small volume of dichloromethane and neutralized by the addition of a saturated solution of sodium bicarbonate. The resulting solution was then extracted with methylene chloride (3 x 10 ml). The combined organic layers were dried over sodium sulfate. F i l t r a t i o n and removal of the solvent gave 222 mg of a brown gum. The above reaction was repeated with a mixture of both cyanides and an identical mixture of diastereoisomeric carbomethoxy derivatives was obtained. Separation was obtained by preparative t i c on s i l i c a gel Woelm, developed with a mixture of benzene-ethyl acetate (4:1). 16-Methoxy dl-epivincadine: amorphous solid (9%), resisted crystallization, leading to decomposition products. Infrared (CHCl^) '• 3370 (small, NH), 1725 (strong, C09CH ) cm"1. Ultraviolet: X (log e): 228 (4.43), 278 (3.65), 300 (3.76) mm. Nmr signals (100 MHz): 1.49 (singlet, IH, NH), 2.70 (doublet, IH, J . = 8 cps, IH, C-14), 3.20 (doublet, J = ortho r meta 2.5 cps, IH, C-17 proton), 3.30 (quartet, J =8 cps, J k = * * * * ^ ortho meta 2.5 cps, IH, C-15 proton), 4.49 (quartet, J A t > = 11 cps, 3 = 2 cps, ArS A C IH, C-3 proton), 6.23 (singlet, 3H, CH^O-), 6.38 (singlet, 3H, C0 2CH 3), 9.36 ( t r i p l e t , J = 7 cps, 3H, CH^CH^). Mass spectrum; main peaks: m/e 124, 126, 210, 245, and 370 (M +). Molecular weight: 370.224. Calc. for C 2 2 H 0 N : 370.225. - 104 -16-Methoxy dl-vincadine - amorphous solid (25%), resisted crystallization, leading to decomposition products. It was more polar on s i l i c a gel chromatographic plates developed with benzene-ethyl acetate (4:1). Infrared (CHC13): 3370 (medium sharp, NH), 1730 (strong, CO CH_) cm"1. Ultraviolet; X (log e): 228 (4.43), j 1113. X 278 (3.65), 301 (3.76) mm. Nmr signals (100 MHz): 1.16 (broad singlet, NH), 2.70 (doublet, J o r t h o = 8 cps, IH, C-14), 3.21 (doublet, J =2 cps, IH, C-17 proton), 3.30 (quartet, J = 8 meta f > > r / >.i ortho cps, J = 2 cps, IH, C-15 proton), 6.22 (singlet, 3H, CH„0-), 6.31 (singlet, 3H, C02CH_3) , 6.35 (multiplet, IH, C-3 proton), and 9.18 (tr i p l e t , 3H, CH^C^-). Mass spectrum; main peaks: m/e 124, 138, 210, 245 and 370 (M +). Molecular weight: 370.225. Calc. for C 2 2 H 3 0 ° 3 N 2 : 370.225. N„ -Methylation of 16-Methoxy dl-vincadine (151a) and i t s  Epimer (151b) Sodium amide (0.25 mmole) was prepared from r e d i s t i l l e d liquid ammonia (4-5 ml) and freshly cut sodium metal (5.85 mg, 0.25 mmole). A trace of f e r r i c nitrate was added as a catalyst to the solution of sodium amide in liquid ammonia, kept under highly purified nitrogen and efficient s t i r r i n g . A solution of epimeric esters (151) (58 mg, 0.15 mmole) in dry tetrahydrofuran (1 ml) was added with a syringe. The dark solution was kept in a dry ice-acetone bath and s t i r r i n g continued for 30 minutes more. Methyl iodide (16 ml, 0.25 mmole) in a few drops of tetrahydrofuran was added with a syringe. The reaction mixture was kept cold and stirred for 25 minutes more and then the ammonia allowed - 105 -to evaporate slowly under a stream of nitrogen. The removal of ammonia was enhanced by blowing a stream of warm a i r around the r e a c t i o n v e s s e l . The dark residue was taken into a mixture of aqueous ammonium ch l o r i d e - e t h y l ether (10 ml, 1:1) and extracted several times with e t h y l ether. The combined organic laye r s , a f t e r washing with water were dried over anhydrous sodium s u l f a t e . F i l t r a t i o n of the inorganic agent and removal of the solvent under reduced pressure at room tempera-ture gave 55 mg of a yellow gum. Preparative t i c on s i l i c a gel chromatoplates developed with benzene-ethyl acetate (4:1) gave the two K-methylated epimers (62%). dl-Vincaminoridine (4): As an amorphous s o l i d (9 mg). Infrared -1 (CHC1 3): No NH absorption and 1735 (strong, C0 2CH 3) cm . U l t r a v i o l e t ; X (log e): 232 (4.47), 2SS (3.78), 299 (3.82) nm. Nmr signals (100 MHz): 2.65 (doublet, J , = 9 cps, IH, C-14 proton), 3.25 ortho (quartet, J = 8 cps, J = 2 cps, IH, C-l7 proton), 3.90 (quartet. ^ ortho meta J.„ = 10 cps, J.„ = 2 cps, IH, C-3 proton), 6.16 ( s i n g l e t , 3H, CH 0-), AB AC J 6.38 ( s i n g l e t , 3H, C02CH_3), 6.46 ( s i n g l e t , 3H, CH_3N), and 9.33 ( t r i p l e t , J = 6 cps, 3H, CH 3CH 2-). Mass spectrum; main peaks: m/e 124, 210, 259 and 384 (M +). Molecular weight: 384.240. Calc. for C 2 3 H 3 2 ° 3 N 2 : 384.241. dl-Epivincaminoridine (4a): Amorphous s o l i d (32 g). This was the more polar epimer i n s i l i c a g e l chromatoplates developed with benzene-ethyl acetate (4:1). Infrared (CHC1 3): No NH absorption and 1725 (strong, CO CH ) cm"1. U l t r a v i o l e t ; X (log e): 232 (4.53), 288 (3.78), and 300 (3.81) nm. Nmr s i g n a l s (100 MHz): 2.66 (doublet, J = 9 cps, 1H,. C-14 proton), 3.30 (quartet, J , = 9 cps, J ^ = ortho 1 ' 1 ' ortho meta - 106 -2 cps, IH, C-15 proton), 3.31 (doublet, J =2 cps, IH, C-17 proton), meta 6.15 (singlet, 3H, CH^O-), 6.20 (distorted quartet, IH, C-3 proton), 6.34 (singlet, 3H, CC^CH ), 6.56 (singlet, 3H, CH N), and 9.08 (t r i p l e t , J = 7 cps, 3H, CH^C^-). Mass spectrum; main peaks: m/e 124, 210, 259 and 384 (M +). Molecular weight: 384.239. Calc. for C 2 3 H 3 2 ° 3 N 2 : 384.241. Epimerization of dl-Epivincaminoridine (4a) to dl-Vincaminoridine (4) To a stirred freshly prepared solution of sodium methoxide in absolute methanol dl-epivincaminoridine (23 mg) was added. The solution of sodium methoxide was made by addition of 14 mg of freshly cut sodium i n absolute methanol (12 ml) under a dry nitrogen atmosphere and efficient s t i r r i n g . After the addition of dl-epivincaminoridine (4a) the reaction mixture was refluxed under dry nitrogen for 48 hours. The cooled solution was concentrated under reduced pressure and the residue taken up i n chloroform. F i l t r a t i o n and removal of the solvent gave 32 mg of a yellowish gum. Preparative t i c on s i l i c a gel chromatoplates developed with benzene-ethyl acetate (4:1) gave 7.3 mg of dl-vincaminoridine (4) and 11.5 mg of dl-epivincaminoridine (4a). Transannular Cyclization of dl-Vincaminoridine (4a) and i t s Epimer A mixture of dl-vincaminoridine and i t s epimer (22 mg, 5.75 mmole) in gl a c i a l acetic acid (11 ml), and mercuric acetate (96 mg, 30.25 mmole) was stirred for 43 hours at room temperature under an atmosphere of nitrogen. The formed mercurous acetate was f i l t e r e d off and the f i l t r a t e was basified by careful addition of a 10% aqueous solution of sodium bicarbonate. The resulting basic solution was - 107 -extracted with methylene chloride and the combined organic layers were washed with-water, dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent gave 17 mg of an amorphous solid. Preparative t i c on alumina neutral Woelm, developed with chloroform-ethyl acetate (1:1) gave two isomeric products. The less polar of them (2.0 mg, 11%) was obtained as an amorphous solid. Infrared (CHCl^): 1715 (strong, a,6-unsaturated ester), 1660 (strong, C=C i n conjugation) cm 1. Ultraviolet; X : 225, 310 ( i n f l . ) and 335 nm. The more polar max r compound (4.5 mg, 24%) was also obtained as an amorphous solid. Infrared (CHCl^): 1715 (strong, a,g-conjugated ester), 1660 (strong, C=C in conjugation) cm 1 . Ultraviolet, \ 225, 310 ( i n f l . ) and 340 nm. The max mass spectra of both compounds had main peaks at m/e 124, 149, 263 and 382 (M +). Molecular weight: 382.224. Calc. for C 2 3 H 3 0 ° 3 N 2 : 3 8 2 • 2 2 5 • - 108 -DISCUSSION PART II 1. Partial Synthesis of Appropriate Dihydroindole Units For the reasons presented in the end of the discussion of Part I we directed our efforts toward the degradation sequence which could provide the relay compounds 225, 226 and 195. One of the required 67 intermediates, ketone 86, had been previously obtained by Gorman via either a soda lime d i s t i l l a t i o n of vindoline (3) at 325°, or in better yield by another process. Hydrogenation of vindoline yields dihydro-vindoline (222) which could then be converted to an amorphous hygroscopic hydrochloride. Pyrolysis of this salt at 195-200° in vacuum gave a d i s t i l l a t e from which the ketone 86 was obtained in 15% overall yield (Fig. 50). Since desacetyldihydrovindoline (225) was the required intermediate that would eventually lead us to the synthesis of the relay compound 195, we decided to obtain the ketone 86, via a slightly modified sequence of reactions (Fig. 51). Thus the natural alkaloid vindoline (3) was refluxed in concentrated hydrochloric acid for a short period of time on a pre-heated bath, followed, by conventional workup of the reaction mixture. Desacetylvindoline (224) was obtained in quantitative yield as a foam, which crystallized upon addition of ether. Recrystallization from methanol gave colorless plates, m.p. 160-162°. The nmr spectrum - 109 -C0 2CH 3 Pt02/EtOH-HCl 3 C0 2CH 3 222 H CH3O 223 C0 2CH 3 Fig. 50. Gorman's preparation of ketone 86. HC1 , 8 min CH„ C0 2CH 3 86 HMPT Pt02/H2/95% EtOH C0 2CH 3 Fig. 51. Preparation of ketone 86. 225 - 110 -( F i g . 52) showed c l e a r l y that only the acetate group had been removed ( s i n g l e t at x 7.93 i n the s t a r t i n g m a t e r i a l ) . The mass spectrum (Fig. 53) had s i g n i f i c a n t and expected fragments at m/e 107, 121, 135, 174, 188, and 414 ( M + ) . 1 2 2 The molecular formula, ^-JH^QO^^ , w a s c o n r : ] ' - r m e d by elemental analysis and high r e s o l u t i o n mass spectrometry. The next step was the c a t a l y t i c hydrogenation of the double bond present i n r i n g D. When desacetylvindoline (224) was hydrogenated i n 95% ethanol i n the presence of Adam's c a t a l y s t (PtC^), desacetyldihydrovindoline (225) was obtained as an o i l i n 95% y i e l d . C r y s t a l l i z a t i o n from ether, and r e c r y s t a l l i z a t i o n from methanol gave an a n a l y t i c a l sample, m.p. 181-183°. In the nmr spectrum ( F i g . 54) the signals due to the v i n y l i c protons (x 4.22, F i g . 52) were now absent. The mass spectrum ( F i g . 53) had the main fragments at m/e 124, 188, 242, 298, and 416 (M+) , which are 122 c h a r a c t e r i s t i c of the aspidospermine type skeleton. The formula, ^23^32^5^2' w a s c o n f i r m e ( l by elemental a n a l y s i s and high r e s o l u t i o n mass spectrometry. We next attempted the p y r o l y s i s of desacetyldihydrovindoline i n a pyrex tube heated at 280-290°. Af t e r p u r i f i c a t i o n of the crude product by chromatography on n e u t r a l alumina the desired ketone 86 was obtained i n 30% y i e l d (based on-recovered s t a r t i n g m a t e r i a l ) . However when we t r i e d to increase the amount of the compound i n the p y r o l y s i s the y i e l d was lower. At t h i s point we decided to t r y a more e f f i c i e n t way to perform such a conversion, without having to l i m i t the amount of 123 compound to be pyrolyzed i n each run. Monson reported that primary and secondary alcohols were dehydrated by treatment with hexamethyl-124 phosphoric triamide at 220-240 . Lomas et a l . , " have also shown that - I l l -DESRCETYL VINDOLINE a 5s-LU H 3 C0 2CH 3 i **[' T i " i 1—| I'I i r—| i i—i—r""j—i i i ' i i i i I I i i i i—r 100.0 150.0 200.0 250.0 300.0 350.0 400.0 M/E . . . . '• DESRCETYL DIHYDRQVINDQLI.NE i i i i— i i i i—| i i—i i | 500.0 550.0 600.0 50.0 450.0 o a LU *—* . I—in. a: 4-C H 3 C0 2 C H 3 i i D [ — i —i — — | — i — I ' I i ) ' i I " I *I I —r— i — i — ! * - 1 — i — i—i— r— " — i — i — I— i—p r—i— i—r — |— i J i i—i—|—i—r—i—i—|—i—i i i—|—i—i—i—i—| 50.0 100.0 15Q.0 200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 500.0 M/E Figure 53. Mass spectra of desacetylvindoline (224) and desacetyldihydrovindoline (225) - 113 -- 114 -t e r t i a r y alcohols can be dehydrated by t h i s method. On t h i s basis we decided to apply t h i s procedure hoping to be able to prepare the g-keto ester (226, F i g . 55) d i r e c t l y from desacetyldihydrovindoline (225) without having to go v i a the intermediate ketone 86. However, when preliminary i n v e s t i g a t i o n s were performed at d i f f e r e n t temperatures, they showed that no conversion at a l l had taken place below 170°, and that the ketone 86 was formed at 170° without production of any detectable amount (by t i c ) of the 8-ketoester (226). Since the re a c t i o n proceeded slowly at 170°, we therefore raised the temperature to 220°C. We were g r a t i f i e d to f i n d that the ketone 86 was obtained, a f t e r conventional work-up and p u r i f i c a t i o n by chromatography on n e u t r a l alumina, i n 40% y i e l d . This method was better than the previous one u t i l i z e d by Gorman*^ (15% y i e l d ) . R e c r y s t a l l i z a t i o n from methanol gave an a n a l y t i c a l sample, m.p. 135-137°. The s p e c t r a l properties were i n complete accord with the assigned s t r u c t u r e . ^ Thus, the i n f r a r e d spectrum showed a strong carbonyl absorption at 1701 cm \ while the u l t r a v i o l e t spectrum (213, 252, and 305 nm) had a t y p i c a l 16-methoxy dihydroindole chromophobe. In the nmr spectrum ( F i g . 55) the absorptions due to the C-4 proton, and the methyl protons of the carbomethoxy group (T 5.66 and 6.27, F i g . 54) were now absent. The mass spectrum ( F i g . 56) had the expected fragments at m/e 124, 174, 188, 298, and 340 (M +). The molecular formula, c 2 i H 2 8 ° 2 N 2 ' i was confirmed by elemental a n a l y s i s and high r e s o l u t i o n mass spectrometry, (found: 340.211, ca l c u l a t e d for c 2 i H 2 8 ° 2 N 2 : 3 4 0 - 2 1 5 ) - Having obtained the ketone 86, we concerned ourselves with the int r o d u c t i o n of the carbomethoxy group which would provide us with the required g-ketoester (226, F i g . 57). This was s u c c e s s f u l l y accomplished by condensation of 125 the ketone 86 with dimethyl carbonate i n the presence of sodium hydride. F i g u r e 55. Nmr spectrum of ketone 86. KETONE If) a UJ _ l UJ cr _LL ~ i —i i i — i — | — i — i — i — i — p T 50.0 100.0 i i | l i t i | i r i I | i i i 150.0 200.0 250.0 300.0 350.0 M/E KETOESTER 400.0 450.0 500.0 550.0 600.0 a 2 cr UJ ce fl S WW - i — i — i — P - J — i — i — i — r — p — | — r — i — i — i — | S50.0 600.0 ' I ' 1 100.0 ] — i ' i 1 i i | i — i i i | — i — i — i i ' | — i — r 150.0 200.0 250.0 300.0 M/E • ' I 50.0 lnil.O 150.0 200.0 25Q.0 3UU.U 350.0 400.0 450.0 500.0 Figure 56. Mass spectra of the ketone 86 and 8-ketoester 226. - 117 -Figure 57. Partial synthesis of dihydrovindoline (222). - 118 -A mixture of the g-ketoester (226) and i t s enol tautomer was obtained in 71% yield after conventional work-up and purification by chromato-graphy on s i l i c a gel. No attempts were made to isolate the tautomeric forms since i n the next step the stereochemistry, whatever i t might be, is going to be destroyed by generation of the enolate. The infrared spectrum had two strong carbonyl absorptions at 1725 and 1700 cm \ while the ultraviolet spectrum had maxima at 212 (4.39), 252 (3.88), and 304 (3.58) nm. The mixture of keto and enol tautomeric forms was evident in the nmr spectrum (Fig. 58). The most characteristic features were a singlet at T -3.62 which was assigned to the C-3 proton of the enol form, a singlet at T 5.7 due to C-2 proton of the enol form, and two doublets at x 5.82 and 6.00 (J.„ = 4 and 6 cps) resulting from AD the C-3 proton of the keto form. The mass spectrum (Fig. 56) had the expected fragmentation pattern, and the main fragments were found at m/e 124, 174, 188, 298, and 398 (M +). An assignment for these fragments i s outlined in Fig. 59. The molecular formula, ^^H^gO^^, was established by high resolution mass spectrometry (Found: 398.220, calculated for C ^ H ^ O ^ : 398.218). Having obtained the g-ketoester derivative (226) in satisfactory yield, we now directed out efforts toward the introductipn of the hydroxy group at the C-3 position. At this time Buchi and co-workers 1^ published their total synthesis of vindorosine (194) in which they applied an interesting hydroxylation reaction, we therefore decided to u t i l i z e t h e i r procedure. When a solution of the g-ketoester (226) in tert-butyl alcohol-l,2-dimethoxyethane containing potassium tert-butoxide was treated with 98% hydrogen peroxide and molecular oxygen at -35°, we were able to isolate after conventional work-up and - 119 -- 120 -+ m/e 188 m/e 174 m/e 124 Fig. 59. Fragmentation of g-ketoester upon electron impact. purification by t i c chromatography on s i l i c a gel, the desired hydroxy ketoester (227) in 59% yield. The infrared spectrum had two strong carbonyl absorptions at 1750 and 1712 cm \ while the ultraviolet spectrum had maxima at 213 (4.49), 248 (3.81), and 303 (3.67) nm. The nmr spectrum (Fig. 60) was consistent with the proposed structure i (227). The methyl protons of the carbomethoxy and methoxy groups were found at x 6.17 and 6.26, while the N-methyl group resonated at x 7.37. In addition, the doublet due to the C-3 proton (present in the starting material 226) was now absent. The indicated stereochemistry (g-OH) was based upon comparison with an authentic sample obtained from Moffatt - 122 126 oxidation of dihydrodesacetylvindoline (225) , and k i n d l y provided to us by Dr. Bunzli-Trepp of t h i s laboratory. Both products were shown to be i d e n t i c a l by s p e c t r a l data and t i c chromatography. The mass spectrum ( F i g . 61) had main fragments at m/e 124, 174, 188, 298, and 414 (M +), which also locates the hydroxy group at the C-3 p o s i t i o n . The molecular formula, ^23^30^5^2' W a S e s t a b l i s h e d by high r e s o l u t i o n mass spectrometry (found: 414.214, ca l c u l a t e d f or ^^H^nO^^: 414.215). We have therefore completed the p a r t i a l synthesis of dihydrovindole (222), since the l a s t two remaining steps, namely the reduction of the carbonyl function to an a l c o h o l having the required stereochemistry, 126 followed by a c e t y l a t i o n has already been accomplished i n our laboratory (see F i g . 57). Furthermore, since the f u n c t i o n a l i z a t i o n of r i n g D of dihydrovindoline (222), namely the i n t r o d u c t i o n of a double bond, has 126 127 also been achieved i n our laboratory, ' as ind i c a t e d i n Figure 62, t h i s also completes the p a r t i a l synthesis of v i n d o l i n e (3) (see Figures 57 and 62). Having completed the l a t t e r stages of the t o t a l synthesis, we turn our a t t e n t i o n to the connection between the intermediate 195 and the r e l a y compound 226 i n order to complete the t o t a l synthesis of v i n d o l i n e (3). Our i n i t i a l aim was to prepare the unsaturated ester 231 ( F i g . 63) and the synthetic approach selected, was the one ! 128 developed by Corey and co-workers, namely o l e f i n synthesis from 1,2-d i o l s v i a a c y c l i c thiocarbonate d e r i v a t i v e . Desacetyldihydrovindoline (225) was refluxed with N,N-thiocarbonyldiimidazole i n butanone for 28 hours under an atmosphere of nitrogen. The crude reaction mixture a f t e r conventional work-up was p u r i f i e d by chromatography on s i l i c a g e l . Continuous e l u t i o n with e t h y l acetate gave the desired thiocarbonate HYDROXY KETOESTER in a U J i \ I* i i i i ' i i I i i ' i * 7 D | , . . . | i i " i i | i | i i i i | i i r i -| 1 1 ' 1 I 1 1 50.0 100.0 150.0 200.0 250.0 300.0 350.0 M/E I i 1 i i i I i i i i I i — i i i | • i i i | 400.0 450.0 500.0 550.0 600. a d a —l If) a U J THIQCflRBQNATE i r i i I i i r i I i i I i i i i | i i i i | i i i i | 50.0 100.0 150.0.. 200.0 250.0 300.0 350.0 400.0 4S0.0 500.0 550.0 600. M/E Figure 61. Mass spectra of hydroxy ketoester 227 and thiocarbonate derivative. - 124 -OAc CH CO CH 222 Hg(OAc) 2 dioxane,A 1) Me_0+BF'!"/CH„C19 ' 3 4 2 2 2) NaBH./EtOH n . 3) Si0 0/MeOH/H„0 OAc 2 2 C H ^ Q Figure 62. P a r t i a l synthesis of v i n d o l i n e (3), C0 2CH 3 1) <J>3CLi/THF 2) AcCl 229 d e r i v a t i v e (230, F i g . 63) i n 88% y i e l d . R e c r y s t a l l i z a t i o n from et h y l acetate gave an a n a l y t i c a l sample, m.p. 222-223°. The i n f r a r e d spectrum showed strong bands at 1739 and 1304 cm 1 which were assigned r e s p e c t i v e l y to carbonyl and thiocarbonyl groups. The u l t r a v i o l e t spectrum had the 16-methoxyindole chromophore with maxima at 208 (4.53, 233 (4.33), and 298 (3.69) nm. The nmr spectrum ( F i g . 64) was i n good agreement with the proposed structure (230). The main features were a s i n g l e t at T 4.69 assigned to the C-4 proton, a three proton s i n g l e t at T 6.07 due to the methyl protons of the carbomethoxy group, and a one proton s i n g l e t at T 6.20 a r i s i n g from the C-2 proton. I t i s perhaps appropriate to note that the above sig n a l s were found at lower - 125 -195 Figure 63. Preparation of the unsaturated ester (231). f i e l d than in the starting material (225). The reason for this s h i f t , at least in part, may be the deshielding effect of the thiocarbonate group. The mass spectrum (Fig. 61) had main fragments at m/e 124, 149, 298, 381, and 458 ( M + ) . The molecular formula, G^H^Q^OI-S, was established by elemental analysis. The second part of the two-step synthesis of the unsaturated ester 231, which comprises the elimination of the thiocarbonate group with simultaneous formation of the double bond, was performed in the following way. The thiocarbonate derivative (230) in tetrahydrofuran and in the presence of Raney nickel was refluxed for 24 hours. After Figure 64. Nmr spectrum of thiocarbonate derivative (230). - 127 -usual work-up and p u r i f i c a t i o n of the crude r e a c t i o n product by chromato-graphy on s i l i c a g e l , the desired product (231) was obtained i n 81% y i e l d . The spectra data were i n good agreement with the assigned structure. The i n f r a r e d spectrum had now a strong carbonyl absorption at 1703 cm 1 i n d i c a t i n g c l e a r l y a s h i f t caused by the conjugated double bond, while the u l t r a v i o l e t spectrum had a t y p i c a l 16-methoxydihydro-indole chromophore. The nmr spectrum ( F i g . 65) showed a one proton s i n g l e t at T 2.77 due to the o l e f i n i c proton, and a one proton s i n g l e t at T 5.73 was assigned to the C-2 proton. The molecular formula, C^^L^O^i^, was established by elemental a n a l y s i s and high r e s o l u t i o n mass spectrometry (found: 382.222, c a l c u l a t e d f o r c 2 3 H 3 o ° 3 N 2 : 3 8 2 - 2 2 5 ) -Having obtained the a,3-unsaturated ester (231) we i n i t i a l l y considered the p o s s i b i l i t y of isomerization of the double bond i n order to obtain the r e l a y compound 195 ( F i g . 63). However since preliminary i n v e s t i g a -127 tions proved unsuccessful i t was decided to attempt such a conversion v i a the saturated ester (232)„ This would provide us with an intermediate on which a good leaving group could be introduced at the a - p o s i t i o n of the carbomethoxy group and subsequent el i m i n a t i o n could eventually lead to the desired compound 195. Thus, c a t a l y t i c hydrogenation of 231 with 10% palladium on charcoal i n 95% ethanol gave a f t e r conventional work up and p u r i f i c a t i o n by chromatography on s i l i c a g e l , the i saturated ester 232 ( F i g . 63) i n 80% y i e l d . The i n f r a r e d spectrum showed a strong carbonyl absorption at 1735 cm \ while the u l t r a v i o l e t spectrum had maxima at 212 (4.35), 253 (3.69), and 305 (3.53) nm. The nmr spectrum ( F i g . 67) revealed the presence of a one proton doublet at T 6.4 (J A 1, = 2 cps) due to the C-2 proton and a one proton m u l t i p l e t AE at T. 5.9 assigned to the C-3 proton. The a-stereochemistry of the Figure 65. Nmr spectrum of the unsaturated ester (231). UNSATURATED ESTER 8 SATURATED ESTER Figure 66. Mass spectra of the unsaturated ester (231) and saturated ester (232). Figure 67. Nmr spectrum of saturated ester (232). - 131 -carbomethoxy group was based on the coupling constant of the v i c i n a l protons on C-2 and C-3. A large v i c i n a l coupling constant (8-14 cps) between protons is associated with an approximate diaxial orientation of the atoms, while smaller splittings (1-5 cps) are associated with 129 axial-equatorial or diequatorial interactions. Since the coupling constant found was 2 cps, this strongly suggests the assigned stereo-chemistry for the saturated ester (232). The mass spectrum (Fig. 66) showed the usual aspidospermine-like fragmentation and the main fragments were found at m/e 124, 210, 298, and 384 (M +). The molecular formula, ^23^32^3^2' W a S e s t a b l i s h e d by elemental analysis. Having obtain 232 we next considered the introduction of the leaving group. For this purpose an ct-hydroxy ester derivative was our i n i t i a l aim. At f i r s t we sought to perform such a synthetic operation via hydrogen peroxide oxidation of the enolate generated by treatment of intermediate 232 with potassium t-butoxide. This attempt proved to be unsuccessful; the reaction products after work up and isolation by t i c chromatography on s i l i c a gel were found to be the starting material (232, 8%) and i t s epimer at C-3 (233, 92%). The latter compound 233, was recrystallized from methanol, m.p. 162-164 . The infrared spectrum had a strong carbonyl absorption at 1725 cm \ while the ultaviolet spectrum showed a 16-methoxyindole i chromophore with maxima at 216 (4.42), 256 (3.80), and 307 (3.65) nm. The nmr spectrum (Fig. 68) showed a one proton doublet (J._ = 10 cps) AJJ at T 6.15 due to the C-2 proton. Such a large vi c i n a l coupling constant 129 may be associated with an approximate diaxial orientation and therefore is in good agreement with the assigne d structure 233. The molecular formula ^^^H^^O^^, was established by elemental analysis. Figure 68. Nmr spectrum of saturated ester (233). - CCT -- 134 -It has been reported that the proton alpha to a carboxyl 130 131 group or ester function can be abstracted with lithium d i a l k y l -amide reagents to generate the a-carbanion which on treatment with electrophilic reagents affords the corresponding a-substituted product. At this point i t was decided to attempt the use of lithium diisopropyl-amide as a base. This approach proved successful. Thus, reaction of 232 with hydrogen peroxide-molecular oxygen in the presence of lithium diisopropylamide in anhydrous tetrahydrofuran at room temperature for 18 hours, produced, in 54% yield (based on recovered starting material), the a-hydroxy ester (234). The nmr spectrum (Fig. 70) showed a one proton singlet at T 6.13 due to the C-2 proton, while the three proton singlet assigned to the methyl protons of the carbomethoxy group was found at x 6.13. There was an evident shift toward low f i e l d , as a result of the deshielding effect of the a-hydroxy substituent. The mass spectrum CH3 C02CH 234 Figure 70. Preparation of a-hydroxyester (234). - 135 -- 136 -(Fi g . 69) had the main fragments at m/e 124, 174, 188, 298 and 400 (M+) supporting the proposed structure 234. High r e s o l u t i o n mass spectrometry established the molecular formula C 2 3 H 3 2 ° 4 N 2 ( f o u n d : 400.235, c a l c u l a t e d 13? for C 2 3 H 3 2 0 4 N 2 : 400.236). At t h i s stage the work of Wieland et a l . 133 and Grdinic and co-workers i n the curane a l k a l o i d s came to our atte n t i o n . These authors were able to obtain the unsaturated aldehyde 236 v i a Oppenauer oxidation of the corresponding saturated a l c o h o l 235. We therefore decided to attempt t h i s a l t e r n a t i v e approach toward the synthesis of the intermediate 195. Thus 232 i n anhydrous tetrahydrofuran Oppenauer p-oxidation CH20H CHCH3 ~ C ' H 0 CHCH3 235 236 was refluxed with l i t h i u m aluminum hydride f o r 1 hour. Conventional work-up and p u r i f i c a t i o n of the crude r e a c t i o n mixture by chromatography gave the amino al c o h o l 237 i n 70% y i e l d . The i n f r a r e d spectrum had a broad absorption at 3500-3200 cm 1 due to the hydroxyl group, and no carbonyl absorption, while i n the nmr spectrum ( F i g . 73) the three proton s i n g l e t due to the methyl protons of the carbomethoxy group was absent. The mass spectrum ( F i g . 74) showed main fragments at m/e 124, 174, 188, 220, 298, 338, and 356 (M+) which substantiate the assigned structure 237. The molecular formula, C 2 2 H 3 2 ° 2 N 2 ' W a S e s t a b l i s n e d by high r e s o l u t i o n mass spectrometry (found: 356.246, calculated f o r C 2 2 H 3 2 ° 2 N 2 : 3 5 6 • 2 4 6 ) . Having obtained the amino alcohol (237) i n - 137 -238 239 Figure 72. Preparation of aldehyde derivative 238. satisfactory yield we then applied the Oppenauer oxidation to i t , namely potassium _t-butoxide and benzophenone in refluxing benzene. However we failed to obtain the desired a,g-unsaturated aldehyde (239). We were able to isolate only the saturated aldehyde (238) in high yield. The infrared spectrum had a strong carbonyl absorption at 1723 cm \ characteristic of a non-conjugated aldehydic carbonyl, while the u l t r a -violet spectrum had maxima at 213 (4.36), 255 (3.66), and 307 (3.54) nm, typical of the dihydroindole chromophore. The nmr spectrum (Fig. 75) showed a one proton singlet at T 0.5 due to the aldehydic proton, a 1000 0 1 2 3 4 5 6 7 8 Figure 73. Nmr spectrum of aminoalcohol 237. o a RLCOHCL LU LU CC 1 • • 1 1 CH3 CE^ OH — r 4 - r • • | — i ' J i ' 'i | i — i — i — i — | i i — i i [ — i r i I | • i — i — i — ' — I — i — I — i — r - i — i — i — i — r — | — i — i — i — i — 1 — i — i — i — i — i 50.0 If) a Ss-LU LU CC 100.0 150.0 200.0 250.0 300.0 350.0 M/E ALDEHYDE 400.0 450.0 500.0 550.0 600 —|—^ I i — I — - — r — i -200.0 250. U T — r - r - i — I — T — f I I I 1 T I 1 I I I I I I I ' ' 1 1 I ' 1 50.0. 100.0 r 150.0 300.0 M/E 350.0 400.0 450.0 500.0 550.0 Figure 74. Mass spectra of aminoalcohol 237 and aldehyde derivative 238. 600 - 141 -one proton doublet at T 6.20 assigned to the C-2 proton, and two three proton s i n g l e t s at T 6.22 and 7.28 assigned r e s p e c t i v e l y to the methoxy and the N-methyl groups. The mass spectrum ( F i g . 74) had the expected fragments at m/e 124, 188, 298, 326, and 354 (M +). The molecular formula, C^H-JQC^^J w a s established by elemental a n a l y s i s . 133 Since the proposed mechanism i n which the saturated aldehyde (238), i n i t i a l l y formed i n the Oppenauer oxidation, dehydrogenates to give the corresponding ct,8-unsaturated aldehyde (239) requires the formation of an iminium intermediate; we thought that perhaps the presence of the N-methyl group had prevented such formation. Therefore we decided to d i r e c t our e f f o r t s toward another s e r i e s of i n v e s t i g a t i o n s i n which we had two ob j e c t i v e s . Our f i r s t purpose would be to obtain the N-desmethyl d e r i v a t i v e s , that could eventually lead to the desired r e l a y compound 195. Our second aim would then be the synthesis of N-formyl d e r i v a t i v e s to be u t i l i z e d i n the synthesis of v i n c r i s t i n e (2) type compounds. Our preliminary i n v e s t i g a t i o n involved the potassium permanganate oxidation of the unsaturated ester (231), Thus, treatment of compound 231 with potassium permanganate i n r e f l u x i n g acetone followed by conventional work up and p u r i f i c a t i o n of the crude r e a c t i o n mixture by t i c chromatography, we were able to i s o l a t e four compounds, here described by increasing order of p o l a r i t y on s i l i c a g e l chromato-i plates developed with e t h y l acetate: 5-membered r i n g lactam (240), 6-membered r i n g lactam (241), N -formyl-5-membered r i n g lactam (242), and cl N -formyl-6-membered ri n g lactam (243). I t i s hoped that the N-formyl d e r i v a t i v e s can be cleaved to y i e l d the N-desraethyl intermediate since there are several precedents for t h i s i n the l i t e r a t u r e , 1 ^ ^ ' 1 ^ ^ The - 142 -242, R = 0, R x = H 2 4 3 , R = H , Rl = 0 resultant product 244 could then lead to the desired intermediate 195 v i a the rea c t i o n sequences ou t l i n e d i n Figure 76. I have also discussed previously the synthesis of the a-hydroxyester 234 and i t i s now appropriate to r e v e a l i t s usefulness as a s t a r t i n g material i n an a l t e r n a t i v e synthesis of the desired intermediate 195. There i s a v a r i e t y of methods already known i n the l i t e r a t u r e which may allow dehydration of 234 to 195 and some of them are outlined i n F i g . 77. The above a l t e r n a t i v e s are under under i n v e s t i g a t i o n i n our laboratory. In conclusion, I would l i k e to say that the synthetic sequence leading to dl-vincaminoridine (4) has been re i n v e s t i g a t e d and improved (Figures 25, 26, 27, and 37). In add i t i o n , the above i n v e s t i g a t i o n s have provided much of the chemistry e s s e n t i a l i n the completion of the highly oxygenated a l k a l o i d v i n d o l i n e (3), which forms the lower - 143 -242 NaH/DMe C0 2CH 3 Pt/RV 0 C02CH3 247 1) P 2 S 5 2) RaNi or 1) CH 30 + ~BF^ 2) NaBH, Hg(OAc) or KMnO, 0 2CH 3 CH2OH 244 245 248 ' 1) t-BuO K+/(C,H_)„CO / o o I 2) [0] 3) CH2N2 CH3 C0 2CH 3 195 Figure 76. Proposal for the synthesis of intermediate 195. - 144 -Figure 77. Alternative synthesis of intermediate 195» half of the biologically important Vinca alkaloids. A summary of the reactions as they relate to the vindoline synthesis i s provided in Figure 78. The dotted arrows indicate the conversions which are presently incomplete. ^ - 145 -135 > > 195 Ac 0/NaOAc J « — ~ C0 2CH 3 0 2CH 3 , LAH/THF ~ -78° 222 C0 2CH 3 225 226 I P H 3 CO CH 1) t-BuO~K /DME 2) H 20 2/0 2/-35° C0 2CH 3 227 Hg(OAc) j d i o x a n e / ^ 228 l ) P h CLi/THF 2) AcCl 'Ac "~ i ^ O A c '* 229 C0 2CH 3 + j 1) CH 30 BF 4/CH 2C1 2 "W 2) NaBH4/EtOH N-i ? CH, CO„CH *33 OAc 2~"3 Figure 78. Summary of r e a c t i o n s which r e l a t e to the sy n t h e s i s of v i n d o l i n e . - 146 -EXPERIMENTAL PART II For the general experimental information see page 91. Preparation of Desacetylvindoline (224) A s o l u t i o n of v i n d o l i n e (3) (959 mg) i n concentrated hydrochloric acid (30 ml) was brought to a gentle r e f l u x for 8 min i n an o i l bath (previously preheated to 95-100°), under a nitrogen atmosphere. The so l u t i o n was allowed to cool for 30 min, then cooled to 0° (ic e bath), d i l u t e d with water (30 ml), and b a s i f i e d by c a r e f u l a d d i t i o n of ammonium hydroxide (6 N). The r e s u l t i n g mixture was extracted with chloroform (3 x 30 ml), and the combined chloroform extracts washed with brine (2 x 10 ml) and drie d over anhydrous sodium s u l f a t e . F i l t r a t i o n and removal of the solvent under reduced pressure gave qu a n t i t a t i v e y i e l d of desacetylvindoline (224) as a foam, which c r y s t a l l i z e d upon add i t i o n of d i e t h y l ether. R e c r y s t a l l i z a t i o n from methanol gave white plates, m.p. 160-162°C. Infrared (CHC± 3): 3600-3500 (medium, Oil), 1727 (strong, C0 o0CH_), 1616 (medium, C=C) cm"1. U l t r a v i o l e t ; X (log e) : 214 2. 3 m3.x (4.55), 254 (3.90), 306 (3.73) nm. Nmr signals (100 MHz): 3.14 (doublet, J = 8 cps, IH, C-14 proton), 3.72 (quartet, J , = ortho ortho 8 cps, J = 2 cps, 1 H, C-15 proton), 3.95 (doublet, J = 2 cps, • meta meta IH, C-17 proton), 4.22 (multiplet, 211, -CH=CH-), 5.93 ( s i n g l e t , 111, H-C-OH), 6.20 ( s i n g l e t , 3H, CI^O-) , 6.27 ( s i n g l e t , 3H, -C02CH_3), 6.40 - 147 -(singlet, 3H, CH 0-), 6.27 (singlet, 3H, -CC^qy , 6.40 (singlet, IH, -NCH3CH-), 6.58 (multiplet, 2H, -NCH_2CH=), 7.32 (singlet, 3H, -N-CH^), 9.35 (t r i p l e t , J = 7.5 cps, 3H, -CH CH_3). Mass spectrum, main peaks: m/e 107, 121, 135, 174, 188, 240, 298 and 414 (M +). Molecular weight: 414.213. Calc. for C 23 H30°5 N2 : 4 1 2 • 2 1 5 • Found: C, 66.58; H, 7.60; N, 6.55. Calc. for c 23 H3o°5 N2 : C ' 6 6 > 6 5 5 H» 7- 3 05 N» 6.55. Preparation of Desacetyldihydrovindoline (225) Desacetylvindoline (224) (01.054 g) in 50 ml of ethanol (9.5%) was hydrogenated at room temperature and atmospheric pressure using 85 mg of platinum oxide catalyst. Absorption of hydrogen (58 ml) stopped after 1.25 h. The catalyst was removed by f i l t r a t i o n and the solution was evaporated to a light o i l . The product was crystallized from ether (1.001 g, 95%). Recrystallized from methanol, m.p. 181-183°C. Infrared (CHC13): 3570 (medium, OH), 1724 (strong, C0 2CH 3), 1613 (strong, C=C) cm"1. Ultraviolet: X (log e): 214 (4.53), 251 (3.86), 303 max (3.72) nm. Nmr signals (100 MHz): 3.13 (doublet, J t h o = 8 cps, IH, C-14 proton), 3.70 (quartet, J = 8 cps, J =2 cps, IH, C-15 r ortho r meta proton), 3.95 (doublet, J =2 cps, IH, C-17 proton), 5.66 (singlet, meta IH, HCOH), 6.18 (singlet, 3H, -0CH3), 6.27 ( singlet, 3H, -C02CH3), 6.29 (singlet, IH, H^N-CH), 7.39 (singlet, 3H, H3CN-), 9.31 (multiplet, 3H, -CH2CH_3). Mass spectrum, main peaks: m/e 124, 188, 242, 298, 416 (M+) . Molecular weight: 416.235. Calc. for C 2 3 H 3 2 0 5 N 2 : 416.231. Found: C, 66.02; H, 7.65; N, 6.73. Calc. for C 2 3 H 3 2 0 5 N 2 : C> 6 6 - 3 2 J H» 7 - 7 ^ N, 6.73. - 148 -Py r o l y s i s of Desacetyldihydrovindoline (225) (a) D i r e c t Method 477 mg of desacetyldihydrovindoline (225) i n a pyrex tube was heated i n a heating box for 3 h at 280-290°C under an atmosphere of nitrogen. The crude product was chromatographed on a neutral alumina column (Woelm, 15 g, a c t i v i t y II). E l u t i o n with benzene gave 100 mg (30% based on s t a r t i n g m a terial recovered) of the desired ketone 86. Further e l u t i o n with benzene-ether (4:1) gave 70 mg of the s t a r t i n g m a t e r i a l . The ketone 86 was then r e c r y s t a l l i z e d from methanol, m.p. 135-137°. Infrared (CHCip: 2890 (strong, CH), 1701 (strong, C=0), 1621 (strong, C=C) cm"1. U l t r a v i o l e t ; X (log E): 213 (4.48), 252 (3.81), 305 (3.73) nm. Nmr sign a l s (100 MHz): 3.02 (doublet, J o r t h o = 8 cps, IH, C-14 proton), 3.74 (quartet, J , = 8 cps, J = 2 cps, n ortho meta IH, C-15 proton), 3.97 (doublet, J m e t a = 2 cps, IH, C-17 proton), 6.24 ( s i n g l e t , 3H, CH^O-), 7.33 ( s i n g l e t , 3H, CH 3N-), 9.53 ( t r i p l e t , J = 7 cps, 3H, CH^C^-) • Mass spectrum; main peaks: n/e 124, 166, 174, 188, 298, and 340 (M +). Molecular weight: 340.211. Calc. f o r C o 1 H O Q 0 o N o : 340.215. Found: C, 74.25; H, 8.50; N, 8.09. Calc. f o r Z l Zo z Z C 2 1 H 2 8 ° 2 N 2 : C ' 7 4 - 0 8 ' H ' 8 ' 2 9 ' N> 8 ' 2 3 -(b) Using Hexamethylphosphoramide (HMPT) 530 mg of desacetyldihydrovindoline (225) i n 8 ml of hexamethyl-i o phosphoramide was s t i r r e d and heated at 224 C ( o i l bath) f o r 2 h under an atmosphere of nitrogen. The r e s u l t i n g brown s o l u t i o n was cooled to room temperature and water (15 ml) was added. The rea c t i o n mixture was extracted with ether (5 x 20 ml), and the combined ethereal extracts were washed with water ( 2 x 5 ml) to remove r e s i d u a l HMPT, and dried - 149 -over anhydrous sodium s u l f a t e . F i l t r a t i o n and removal of the solvent under reduced pressure at room temperature gave 578 mg of a yellow gum. The crude product was chromatographed on a n e u t r a l alumina column (Woelm, 20 g, a c t i v i t y I ) . Gradient e l u t i o n with hexane-ether gave 143 mg (40% base on s t a r t i n g material recovered) of the desired ketone 86. Further e l u t i o n with ether-methanol (19:1) gave 92 mg of the s t a r t i n g m a t e r i a l . Considering the d i r e c t method (30%), t h i s was s l i g h t l y superior. Preparation of the g-Ketoester (226) To a suspension of sodium hydride (300 mg of 56% dispersion) i n 20 ml of anhydrous tetrahydrofuran, a s o l u t i o n of ketone 86 (400 mg) i n anhydrous tetrahydrofuran (5 ml) was added dropwise over a period of 20 min, under an atmosphere of nitrogen. The r e a c t i o n mixture was s t i r r e d f o r 2.3 h and dimethyl carbonate (1.8 ml) was then added. Aftei the a d d i t i o n of the dimethyl carbonate, the mixture was refluxed f o r 38 h, then cooled with an ice-water bath and the excess of sodium hydride destroyed by the a d d i t i o n of several drops of g l a c i a l a c e t i c a c i d . The s o l u t i o n was taken up i n water (20 ml), and extracted with ether (4 x 20 ml). The combined organic extracts were washed with aqueous sodium bicarbonate, then with brine, and dried over sodium i s u l f a t e . F i l t r a t i o n and removal of the solvent l e f t 700 mg of a yellow gum, which was chromatographed on a s i l i c a gel column (Woelm, 20 g, a c t i v i t y I I ) . Gradient e l u t i o n with benzene-ether gave 336 mg (71%) of desired g-ketoester (226). Infrared (CHC1 3): 1725 (strong, CO^CH^), 1700 (strong, C=0), 1610 (strong, C=C) cm"1. U l t r a v i o l e t ; X (log e) - 150 -212 (4.39), 252 (3.88), and 304 (3.59) nm. Nmr s i g n a l s (100. MHz): -3.62 ( s i n g l e t , III, C-3 proton, enol form), 3.04 and 3.08 (two doublets, J = 8 cps, IH, C-14 proton, keto and enol forms), 3.74 and 3.82 ortho (two q u a r t e t s , J = 8 cps, J = 2 cps, IH, C-15 proton), 3.97 ortho meta, and 4.08 (two doublets, J = 2 cps, IH, C-17 pro t o n ) , 5.7 ( s i n g l e t , meta IH, C-2 proton, enol form), 5.82 and 6.00 (two doublets, J A T 1 = 4 and A h 6 cps, IH, C-3 proton, keto form), 6.22 and 6.24 (two s i n g l e t s , 6H, C02CH_3 and CH_30) , 7.30 and 7.32 (two s i n g l e t s , 3H, NCH 3, keto and enol forms), 9.40 and 9.50 (two t r i p l e t s , 3H, CHLjCH , keto and enol forms). Mass spectrum; main peaks: m/e 124, 174, 188, 298, and 398 (M ) and 398 (M +) . Molecular weight: 398.220. C a l c . f o r C ^ H 0 ^ : 398.218. Pr e p a r a t i o n of the.Hydroxy Ketoester.(227) To a s o l u t i o n of the. g-ketoester (226) (70 mg, 0.175 mmole) i n 1,2-dimethoxyethane (20 ml) and _t-butanol (1 m l ) , potassium t-butoxide (7 ml of a s o l u t i o n prepared w i t h 214 mg of potassium i n 25 ml of anhydrous t-butanol) was added. The r e a c t i o n mixture was s t i r r e d f o r 15 min at room temperature. The r e a c t i o n f l a s k was then cooled to -35°C (dry i c e - b e n z y l c h l o r i d e ) and 0.08 ml of a 98% hydrogen peroxide s o l u t i o n was added. Molecular oxygen was then passed through the r e a c t i o n mixture f o r a p e r i o d of 21 hours. The r e a c t i o n mixture was allowed to come slo w l y to room temperature. The s o l v e n t was removed under reduced pressure without h e a t i n g . The r e s i d u e was taken i n b r i n e (5 ml) and e x t r a c t e d w i t h e t h y l a cetate (3 x 10 ml) . The combined organic l a y e r s were d r i e d over anhydrous sodium s u l f a t e . F i l t r a t i o n and removal of the s o l v e n t gave 100 mg of a y e l l o w i s h gum. P u r i f i c a t i o n - 151 -of the crude reaction mixture by tic chromatography on s i l i c a gel developed with benzene-ethyl acetate (1:1), provide 15 mg of the starting material ( 226 ) and the desired hydroxy ketoester 227 (35 mg) in 59% yield (based on recovered starting material). Infrared (CHCl^): 3450 (medium, OH), 1750 (strong, C0 2CH 3), 1712 (strong, 0=0), 1616 (C=C) cm"1. Ultraviolet; X (log E ) : 213 (4.49), 248 (3.81), 303 (3.67) nm. Nmr signals (100 MHz): 3.14 (doublet, J . = 8 cps, IH, or tno C-14 proton), 3.68 (quartet, J = 8 cps, J =2 cps, IH, C-15 ^ ' ortho ' meta ^ proton), 3.92 (doublet, J = 2 cps, IH, C-17 proton), 6.17 (singlet, 3H, C0 2CH 3), 6.26 (singlet, 3H, CH30-), 7.37 (singlet, 3H, CH3N), 9.50 (tri p l e t , J = 7 cps, 3H, CH3CH2~). Mass spectrum; main peaks: ra/e 124, 174, 188, 298, and 414 (M +). Molecular weight: 414.214. Calc. for C 2 3H 3 ( )0 5N 2: 414.215. Preparation of Desacetyldihydrovindoline Thiocarbonate (230) To a solution of 2.1 g of desacetyldihydrovindoline (225) in 125 ml of anhydrous butanone, 5.4 g N,N'-thiocarbonyldiimidazole was added. The reaction mixture was then refluxed for 28 hours under an atmosphere of nitrogen. The solvent was removed under reduced pressure and the crude reaction product was chromatographed on s i l i c a gel column (500 g). Continuous elution with ethyl acetate gave the desired thiocarbonate i derivative (230) in 88% yield (2.057 g). Recrystallization from ethyl acetate gave an analytical sample, m.p. 222-223°. Infrared (CHC13): 1739 (strong, CO.CH.), 1304 ( C=S) cm"1. Ultraviolet; X (log e): z J max 208 (4.53), 233 (4.33), and 298 (3.69) nm. Nmr signals (100 MHz): 302 (doublet, J = 8 cps, IH, C-14 proton), 3.63 (quartet, J , = ortho f » » f / » \ M > ortho 8 cps, J _ =2.3 cps, IH, C-15 proton), 3.90 (doublet, J = 2.3 cps, meta v ' r ' ' meta r - 152 -IH, C-17 proton), 4.69 ( s i n g l e t , IH, C-4 proton), 6.07 ( s i n g l e t , 3H, C0 2CH 3), 6.20 ( s i n g l e t , 111, C-2 proton), 6.24 ( s i n g l e t , 311, CH_30) , 7.37 ( s i n g l e t , 3H, NCH 3), 9.54 ( t r i p l e t , J = 7 cps, 3H, CH CH 2-). Mass spectrum; main peaks: m/e 124, 149, 298, 381, and 458 (M +). Found: C, 63.16; H, 6.9; N, 5.97. Calc. for C 2 4 H 3 o N 2 ° 5 S : C ' 6 2 ' 8 7 ' H> 6* 6 0> N, 6.11. Preparation of the a,g-Unsaturated Ester (231) Raney n i c k e l a c t i v e c a t a l y s t (W.R. Grace Co. #28, 12 g) was washed with acetone (6 x 25 ml), decanted a f t e r s e t t l i n g and the solvent discarded. The c a t a l y s t was then refluxed f o r 4 h i n reagent actone. A f t e r cooling to room temperature, the acetone was-decanted and the c a t a l y s t was washed with tetrahydrofuran (6 x 25 ml). Washed Raney n i c k e l was suspended- in 25 ml of tetrahydrofuran and to t h i s was added a s o l u t i o n of 0.645 g of desacetyldihydrovindoline thiocarbonate (230) i n small volumes of tetrahydrofuran. The rea c t i o n mixture was refluxed with magnetic s t i r r i n g f o r 24 hours. F i l t r a t i o n of the c a t a l y s t and removal of the solvent under reduced pressure gave 453 mg of crude c t,g-unsaturated ester (84% crude). P u r i f i c a t i o n of the crude product by chromatography 'on s i l i c a g el column (500 g) eluted with e t h y l acetate-methanol (97.5:2.5) provided the desired product (231) i n 81% y i e l d . Infrared (CHC1 0): 1703 (strong, C=C-C0oCHn) cm"1. U l t r a v i o l e t ; X J . 2 3 max (log e): 212 (4.53), 253 (3.82), and 307 (3.62) nm. Nmr signals (100 MHz): 2.77 (broad s i n g l e t , IH, C-4 proton), 3.04 (doublet, J o r t h o = 8 ° p S ' 1 H ' C ~ 1 4 P r o t o n>> 3 - 7 8 (quartet, J * o r t h o = 8 cps, J m £ t a = 2 cps, 111, C-15 proton), 4.06 (doublet, J = 2 cps, III, C-17 proton), meta 5.73 ( s i n g l e t , IH, C-2 proton), 6.22 ( s i n g l e t , 3H, CC^CH^), 6.25 ( s i n g l e t , - 153 -3H, CH_30) , 7.25 ( s i n g l e t , 3H, NCH3) , 9.41 ( t r i p l e t , J = 7 cps, 3H, CH^CH^)• Mass spectrum; main peaks: m/e 124, 149, 174, 208, 263, and 382 (M +) . Molecular weight: 382.222. Cal c . f o r C 0 H ^ O ^ : 382.225. Found: C, 72.29; H, 7.89; N, 7.30. C a l c . f o r C 2 3 H 3 0 ° 3 N 2 : C ' 7 2 , 2 8 > H, 7.87; N, 7.32. P r e p a r a t i o n of 3,4-Desoxydihydrovindo 1 ine (232)_ 10% Palladium on c h a r c o a l (360 mg) was added to a s o l u t i o n of the a,3-unsaturated e s t e r 231 (438 mg) i n 95% ethanol (10 ml) at room temperature under an atmosphere of n i t r o g e n . The r e a c t i o n mixture was then hydrogenated at room temperature and one atmosphere of hydrogen w i t h continuous s t i r r i n g f o r 72 hours. The c a t a l y s t was removed by f i l t r a t i o n f h^o'tjjh >Q r e l ? t.e pad and the r>».d subsequently washed w i t h warm methanol (3 x 10 ml). Removal of the solvent under reduced pressure gave a ye l l o w v i s c o u s product which was chromatographed on a s i l i c a g e l column (50 g). E l u t i o n w i t h benzene-ethyl acetate (1:1) gave the d e s i r e d saturated e s t e r 232 (385 mg) i n 80% y i e l d . I n f r a r e d (CHC1J : 1735 ( s t r o n g , C0„CH o) cm'1. U l t r a v i o l e t ; A ( l o g e) : 3 1 5 max 212 (4.35, 253 (3.69), and 305 (3.53) nm. Nmr s i g n a l s (100 MHz): 3.08 (doublet, J o r t h o = 8 CP S> 1 H> C"1^ p r o t o n ) , 3.78 ( q u a r t e t , J o r t h o = 8 cps, J = 2 cps, 1H, C-15 p r o t o n ) , 4.00 (doublet, J = 2 cps, IH, meta meta C-17 p r o t o n ) , 5.9 ( m u l t i p l e t , IH, C-3 proton), 6.32 and 6.34 (two s i n g l e t s , 6H, C0 ?CH 3 and CH 0) , '6.4 (doublet, J ^ = 2 cps, III, C-2 pro t o n ) , 7.45 ( s i n g l e t , 3H, NCH_3), 9.5 ( t r i p l e t , J = 7 cps, 311, • CH 3CH 2) . Mass spectrum; main peaks: m/e 124, 210, 298 and 384 (M +). Found: C, 7.1.93; H, 8.58; N, 7.41. C a l c . f o r C 2 3 H 3 2 0 3 N 2 : C ' 7 1 - 8 4 ' H ' 8 ' 3 9 ; N, 7.29. - 154 -Epimerization of 3,4-Desoxydihydrovindoline (232) To a stirred solution of 3,4--desoxydihydrovindoline (232) (93 mg) in 1,2-dimethoxyethane (5 ml) and ^-butanol (1 ml), potassium _t-butoxide (53 mg) was added. The reaction mixture was stirred for a further 30 min at room temperature, cooled to -35°, and 98% hydrogen peroxide (0.03 ml) was added. Molecular oxygen was passed through the stirred solution for a period of 8 h. The reaction mixture was then allowed to come to room temperature and the solvent was removed under reduced pressure without heating. The residue was taken up in brine (5 ml) and extracted with ethyl acetate (3 x 10 ml). The combined organic layers were dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent under reduced pressure gave 91 mg of a colorless gum. Purification of the crude reaction mixture by t i c on s i l i c a gel developed with b enzene—ethyl acetate (1:1) gave 6 mg of 3,4-desoxydihydrovindoline (232) and 68 mg of epi-3,4-desoxydihydrovindoline (233). The latter was recrystallized from methanol, m.p. 162-164°. Infrared (CHCl^):. 1725 (C0„CHo) cm"1. Ultraviolet; X (log e): 212 (4.42), 256 (3.80), 2 5 max and 307 (3.65) nm. Nmr signals (100 MHz): 3.13 (doublet, J = 8 cps, ortho IH, C-14 proton), 3.84 (quartet, J , = 8 cps, J =2 cps, IH, ortho r meta C-15 proton), 4.06 (doublet, J =2 cps, IH, C-17 proton), 6.15 meta (doublet, J = 10 cps, IH, C-2 proton), 6.24 and 6.31 (two singlets, A D 6H, C02CH_3 and CH_30) , 7.29 (singlet, 3H, NCH_3) , 9.33 (t r i p l e t , J = 7 cps, 3H , CH-jC^-) . Mass spectrum; main peaks: m/e 124, 188, 298, and 384 (M +). Found: C, 71.65; H, 8.45; N, 7.04. Calc. for C 2 3 H 3 2 ° 3 N 2 : C, 71.84; H, 8.39; N, 7.29. - 155 -Preparation of Hydroxyester (234) Redistilled diisopropylamine (0.61 ml, 0.5 mmole) in anhydrous tetrahydrofuran (8 ml) was introduced into a nitrogen swept flask and cooled to 0-5°. Butyllithium in heptane solution (0.4 ml, 0.7 mmole) was introduced in a fine stream through a rubber septum with a syringe. The reaction mixture was then stirred for 15 min. A solution of 3,4-desoxydihydrovindoline (232) (95 mg, 0.25 mmole) in anhydrous tetrahydrofuran (2 ml) was added dropwise, followed by st i r r i n g for 30 min at 0-5° and then at room temperature for 1 h. Molecular oxygen was then bubbled into the solution for 18 h. Water (20 ml) was added and the resulting mixture extracted with ether (3 x 15 ml). The combined organic layers were washed with brine (10 ml) and dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent under reduced pressure gave 109 mg of a yellowish gum. Purification by t i c on s i l i c a gel developed with benzene-ethyl acetate (1:1) gave 20 mg of unreacted starting material (232), 23 mg of i t s epimer (235), and 31 mg (54% based on recovered starting material) of the hydroxy ester (234). Nmr signals (100 MHz): 3.15 (doublet, J o r t h o = 8 cps, IH, C-14 proton), 3.82 (quartet, J = 8 cps, J =2 cps, IH, C-15 proton), 4.02 (doublet, n ortho meta r J = 2 cps, IH, C-17 proton), 6.13 (singlet, IH, C-2 proton), 6.16 (singlet, 3H, C02CH_3) , 6.22 (singlet, 3H, CH_30), 7.22 (singlet, 3H, NCH_3), 9.32 (tr i p l e t , J = 7 cps, 3H, CH^Q^). Mass spectrum; main peaks: m/e 124, 174, 188, 298, and 400 (M +). Molecular weight: 400.235. Calc. for C -rio„0.No: 400.236. 23 32 4/ 2 - 156 -Reduction of 3,4-Desoxydihydrovindoline (232) with Lithium Aluminum  Hydride Anhydrous tetrahydrofuran (12 ml) and lithium aluminum hydride (100 mg) were introduced into a nitrogen swept flask, and a solution of 3,4-desoxydihydrovindoline (183 mg) in anhydrous tetrahydrofuran (13 ml) was added dropwise with s t i r r i n g . The reaction was then refluxed for 1 h under an atmosphere of nitrogen. The reaction mixture was cooled to 0° (ice bath) and the excess reagent destroyed by careful addition of a saturated aqueous solution of sodium sulfate. The precipitate was removed by f i l t r a t i o n and washed several times with hot tetrahydrofuran. The f i l t r a t e and washings were combined and dried over anhydrous sodium sulfate. F i l t r a t i o n and removal of the solvent under reduced pressure gave 181 mg of the crude product. Purification by t i c on s i l i c a gel developed with ether provided the desired alcohol 237 (118 mg), 70% yield. Infrared (CHCLj): 3500-3200 (broad, OH). Nmr signals (100 MHz): 3.15 (doublet, J _ = 8 cps, IH, C-14 proton), 3.79 (quartet, J , = ortho r r ' ^ ' ortho 8 cps, J = 2 cps, IH, C-15 proton), 3.99 (doublet, J =2 cps, IH, meta r r ' meta ^ C-17 proton), 6.27 (singlet, 3H, CH_30) , 7.21 (singlet, 3H, NCH_3) . Mass spectrum; main peaks: m/e 124, 174, 188, 220, 298, 338, and 356 (M+) . Molecular weight: 356.246. Calc. for C ^ H ^ O ^ : 356.246. I Oppenauer Oxidation of Alcohol 237 To a st irred solution of the alcohol 237 (118 mg, 0.33 mmole) and benzophenone(300 mg, 1.65 mmole) in anhydrous benzene (10 ml), potassium t_-butoxide (_t-BuOH free) was added under an atmosphere of nitrogen. The reaction mixture was then brought to reflux for 35 min. The reaction mixture was cooled to room temperature, diluted with water (20 ml) and - 157 -e x t r a c t e d w i t h ether (3 x 10 ml). The combined organic l a y e r s were ex t r a c t e d w i t h an aqueous 5% h y d r o c h l o r i c a c i d s o l u t i o n (4 x 10 ml). The combined aqueous e x t r a c t s were washed w i t h ether (2 x 10 ml) and poured i n t o a mixture of i c e and concentrated ammonium hydroxide s o l u t i o n . The r e s u l t i n g b a s i c s o l u t i o n was e x t r a c t e d w i t h ether (3 x 10 ml), and the e t h e r e a l e x t r a c t s d r i e d over anhydrous sodium s u l f a t e . F i l t r a t i o n and removal of the solvent under reduced pressure gave 103 mg of the aldehyde 238 (88% y i e l d ) . An a n a l y t i c a l sample was obtained by t i c on f l u o r i s i l chromatoplates developed w i t h ether-methanol (95:5). I n f r a r e d (CHC1J: 1723 ( s t r o n g , C=0) cm"1. U l t r a v i o l e t : X ( l o g e) : 3 max 213 (4.36), 255 (3.66), and 307 (3.54) nm. Nmr s i g n a l s (100 MHz): 0.5 ( s i n g l e t , IH, CHO), 3.11 (doublet, 1 t h o = 8 cps, IH, C-14 p r o t o n ) , 3.79 ( q u a r t e t , J L = 8 cps, J = 2 cps, IH, C-15•proton), 3.99 ortho meta (doublet, J t = 2 CPS, IH, C-17 p r o t o n ) , 6.20 (doublet, H i , C-2 p r o t o n ) , 6.22 ( s i n g l e t , 3H, CH_30), 7.28 ( s i n g l e t , 3H, NCH_3), 9.41 ( t r i p l e t , J = 7 cps, 3H, CH 3CH 2-) . Mass spectrum; main peaks: m/e .124, 188, 298, 326 and 354 (M +). Found: C, 74.80; H, 8.77; N, 7.50. C a l c . f o r C 2 2 H 3 0 ° 2 N 2 : C, 74.54; H, 8.53; N„ 7.90. Potassium Permanganate Ox i d a t i o n of the a,6-Unsaturated Est e r (231) To a r e f l u x i n g s o l u t i o n of a,8-unsaturated e s t e r 231 (53 mg) i n acetone (10 m l ) , potassium permanganate (718 mg) was added por t i o n w i s e over a 5 h p e r i o d . The r e a c t i o n mixture was cooled ( i c e - b a t h ) , i c e added to i t and s u l f u r d i o x i d e was passed through. The r e s u l t i n g s o l u t i o n was b a s i f i e d w i t h aqueous sodium bicarbonate s o l u t i o n and e x t r a c t e d w i t h ether (4 x 20 ml). The combined organic e x t r a c t s were washed w i t h water (10 m l ) , b r i n e (10 ml), and d r i e d over anhydrous sodium - 158 -sulfate. F i l t r a t i o n and removal of the solvent under reduced pressure gave 38 mg of crude reaction product. Preparative t i c chromatography of this material on s i l i c a gel developed with ethyl acetate gave 1.5 mg of the starting material (231), 2.5 mg of the lactam (240), 135 mg of the lactam N -formyl (242), 1.5 mg of the lactam (241), and 7 mg of the 3. lactam N^-formyl (243). 5- Membered ring lactam (240): Infrared (CHC13): 1710, 1680 cm"1. Mass spectrum, main peaks: m/e 149, 174, 258, 381, and 396 (M +). Molecular weight: 396.202. Calc. for C„ oH„ oN o0.: 396.204. 23 28 2 4 6- Membered ring lactam (241) : Infrared (CHCl.^): 1710, 1620 cm"1. Mass spectrum; main peaks: m/e 174, 207, 234, 297, 367, 381, and 396 (M+) . 5- Membered ring lactam N -formyl (242) : Infrared (CHC1-): 1720, • • i ......... i - i i . . . . , 3. —————————— _3 1665, 1595 cm"1. Nmr signals (100 MHz, F.T.): 1.37 (singlet, IH, N_CH0), 2.25 (broad singlet, IH, C-17 proton), 2.82 (doublet, J o r t h o = 8 cps, IH, C-14 proton), 2.97 (broad singlet,.IH, C-4 proton), 3.28 (quartet, J = 2.5 cps, J = 8' cps, IH, C-15 proton), 4.86 (singlet, IH, meta r ortho C-2 proton), 6.15 and 6.18 (two singlets, 6H, CH^ O, and C02CH_3) , 9.32 ( t r i p l e t , J = 7 cps, 3H, -CH2CH3). Mass spectrum; main peaks: m/e 78, 160, 321, 357, 382, and 410 (M +). Molecular weight: 410.174. Calc. for C o„H o,0 cN_: 410.184. 23 26 5 2 i 6- Membered ring lactam N -formyl (243): Infrared (CHC1 ): 1715, •• • • 1 3. — — — — — j 1675, 1620 cm"1. Nmr signals (100 MHz, F.T.): 1.15 (singlet, IH, NCHO), 2.35 (broad singlet, IH, C-17 proton), 2.92 (doublet, J o r t h o = 8 cps, IH, C-14 proton), 3.11 (singlet, IH, C-4 proton), 3.31 (quartet, J tho 8 cps, j " m e t a = 2.5 cps, IH, C-15 proton), 6.17 and 6.19 (two singlets, - 159 -6H, CH30, and -C02CH_3) , 9.23 (tr i p l e t , J = 7 cps, -CH2CH3) . 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