studies in Natural Products Chemistry Volume 20 Structure and Chemistry (Part F) (Including Cumulative Index Volumes 1-20)
Studies in Natural Products Chemistry edited by Atta-ur-Rahman
Vol. 1 Stereoselective Synthesis (Part A) Vol. 2 Structure Elucidation (Part A) Vol. 3 Stereoselective Synthesis (Part B) Vol. 4 Stereoselective Synthesis (Part C) Vol. 5 Structure Elucidation (Part B) Vol. 6 Stereoselective Synthesis (Part D) Vol. 7 Structure and Chennistry (Part A) Vol. 8 Stereoselective Synthesis (Part E) Vol. 9 Structure and Chemistry (Part B) Vol.10 Stereoselective Synthesis (Part F) Vol.11 Stereoselective Synthesis (Part G) Vol.12 Stereoselective Synthesis (Part H) Vol.13 Bioactive Natural Products (Part A) Vol.14 Stereoselective Synthesis (Part I) Vol.15 Structure and Chemistry (Part C) Vol.16 Stereoselective Synthesis (Part J) Vol.17 Structure and Chemistry (Part D) Vol.18 Stereoselective Synthesis (Part K) Vol.19 Structure and Chemistry (Part E) Vol.20 Structure and Chemistry (Part F)
Studies in Natural Products Chemistry Volume 20 Structure and Chemistry (Part F) (IncludingCumulative Index Volumes 1-20)
Edited by
Atta-ur-Rahman
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
1998
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FOREWORD
Natural Product Chemistry continues to grow at an increasing pace and this growth is reflected in the present volume of Studies in Natural Product Chemistry which is the 20^^ of this series. The first 20 volumes were largely devoted to structure and synthesis of various classes of natural products, irrespective of their bioactlvity. Subsequent volumes of this series will however be devoted to the chemistry of bioactive natural products and will therefore represent a departure from the earlier volumes. The present volume contains contributions from a number of eminent scientists and covers Interesting reviews on terpenes, alkaloids and other types of natural products reported from terrestrial and marine sources. It is hoped that this volume will be received with the same enthusiasm and Interest as the previous volumes of this series. Comprehensive Indexes covering all the 20 volumes, have been prepared and they include a Cumulative General Subject Index along with more focused Cumulative Indices on Organic Synthesis, Pharmacological Activity and Biological Source. This comprehensive indexing of the volumes should make the entire series much more valuable and user-friendly. I would like to express my thanks to Dr. Farzana Akhtar, Dr. Farhana Noor-e-AIn, Mr. Zaheer-ul-Haq, and Miss Rehana Shah for their assistance in the preparation of the Index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.
Atta-ur-Rahman
Ph.D. (Cantab), Sc.D. (Cantab)
July 31. 1998
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PREFACE Volume 20 of this well known series entitled: "Studies in Natural Products Chemistry", continues the high standard of the previous volumes. The 17 Chapters represent a broad spectrum of topics which serve as an excellent overview of the vast array of natural products research continuing on a worldwide basis. The areas covered do not duplicate to any significant extent material covered in previous volumes and indeed present to the interested reader, information which is useful and pertinent to those involved in the highly diverse avenues of natural products. Each of the chapters is well written and brings fonA/ard in review fashion, a particular area of natural products chemistry. The manner of presentation in the majority of chapters allows the linkage of synthetic and structural chemistry with biological applications and relevance. As the area of natural products research continues to expand into these interdisciplinary avenues, the information provided by the authors is particularly useful not only to the specialist but to the scientist wishing to develop a general knowledge of the specific field covered within the Chapter. This Volume, as the previous volumes in this series, is highly recommended as a useful summary of important information which spans the diverse area of biological activity, medicinally Important terpenes, alkaloids, steroidal lactones, carotenoids, natural colourants, peptides and the use of enzymes in the products of chiral synthons.
James P. Kutney
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CONTENTS Foreword
V
Preface
vii
Contributors
xi
Terpenes and their Biological Relevance G.G. HABERMEHL AND W. FLIEGNER
3
Crinitol, an Acyclic Diterpene Diol from Marine Algae I. KUBO AND L.R. SMITH
25
Stereoselective Synthesis of Methylcyclopentanoid Monoterpenes A. BIANCO
41
Chemical Constituents of Taxus Species V.S. PARMAR AND A. JHA
79
Withanolides, Biologically Active Natural Steroidal Lactones : A Review A.S.R. ANJANEYULU, D. S. RAO, P.W. LEQUESNE
135
Natural Products by Oxidative Phenolic Coupling Phytochemistry, Biosynthesis and Synthesis 263 G.M. KESERU AND M. NOGRADI
Narcissus Alkaloids J. BASTIDA, F. VlLADOMAT AND C. CODINA
323
Total Synthesis of Naphthylisoquinoline Alkaloids M.A. RIZZACASA
407
DNA - Damaging Natural Products with Potential Anticancer Activity A.A. LESILE GUNATILAKA AND DAVID G.I. KINGSTON
457
In Vitro Models of Human Disease States J.M. PEZZUTO, C.K. ANGERHOFER AND H. MEHDI
507
Synthesis of Carotenoids H. PFANDER, M. LANZ AND B. TRABER
561
Structure Elucidation of Phenylpropanoid Wood Extractives S. OZAWA, T. SASAYA AND S. NlSHIBE
613
Chemical and Biological Investigations of Salvia Species Growing in Turkey A. ULUBELEN AND G. TOPCU
659
The Chemistry of Some Natural Colourants J.H.P. TYMAN
719
Bioactive Natural and Synthetic Acronycine Derivatives Modified at the Pyran Ring S. MICHEL AND F. TILLEQUIN
789
Chiral Synthons by Selective Redox Reactions Catalysed by Hitherto Unknown Enzymes Present in Resting Microbial Cells H. SIMON AND H. GUNTHER
817
Microcystins and Nodularins Hepatotoxic Cyclic Peptides of Cyanobacterial Origin L. MORODER AND S. RUDOLPH-BOHNER
887
Subject Index
921
CONTRIBUTORS A.S.R. Angerhofer
Professor of Organic Chemistry, School of Chemistry, Andhra University, Visakhapatnam-530 003, India
Cindy Kay Angerhofer
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, U.S.A.
A.S.R. Anjaneyulu
School of Chemistry, Andhra University, Visakhapatnan 530 003, India
Jaume Bastida
Department of Natural Products, Faculty of Pharmacy, University of Barcelona, Avda., Diagonal 643-08028 Barcelona, Catalonia, Spain
Armandodoriano Bianco
Centro di Studio CNR per la Chimica delle Sostanze Organiche, Naturali Dipartimento di Chimica-Universita La Sapienza-Roma, Italy
Carles Codina
Department of Natural Products, Faculty of Pharmacy, University of Barcelona, Avda., Diagonal 643-08028 Barcelona, Catalonia, Spain
W. Fliegner
Chemisches Institut der Tieraztichen, Hochschule Hannover, Lehrgebiet fur Organische Chemie, Bischofsholer Damm 15, HausNr. 123, 30173 Hannover, Germany
Abeysinghe Arachchige Leslie Gunatilaka
Bioresources Research Facility, The University of Arizona, Tucson Arizona, 250 East Valencia Road, Tucson, Arizona 85706, USA
Helmut Giinther
Institut fur Organische Chemie und Biochemie, Universitat Munchen, D-8 5 747 Garching, Germany
G.G. Habermehl
Chemisches Institut der Tieraztichen, Hochschule Hannover, Lehrgebiet fur Organische Chemie, Bischofsholer Damm 15, HausNr. 123, 30173 Hannover, Germany
Amitabh Jha
Department of Chemistry, University of Delhi, Delhi-110 007, India
G.M. Keserti
Research Group for Alkaloid, Chemistry of the Hungarian, Academy of Sciences, P.O. Box 91, H-1521 Budapest, Hungary
Gyorgy Miklos Keserii
Department of Chemical Information Technology, Technical University of Budapest, POB 91, H-1521 Budapest, Hungary
Technische
David George Ian Kingston
Bioresources Research Facility, The University of Arizona, Tucson Arizona, 250 East Valencia Road, Tucson, Arizona 85706, USA
Isao Kubo
Department of Environmental Science, Policy and Management, University of California, 201 Wellman Hall, Berkely California 94720-3112, U.S.A.
Marc Lanz
Department fiir Chemie und Biochemie, University of Berne Freiestr. 3, CH-3012 Berne, Switzerland
P.W. LeQuesne
Department of Chemistry, North Eastern University, Boston, MA 02115, USA
Haider Mehdi
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, U.S.A.
Sylvie Michel
Universite Rene Descartes-Paris V, Faculte De Pharmacie, Laboratoire de Pharmacognosie, U.R.A. au C.N.R.S.N. 1310 4, Avenue de I'Observatoire, 75270 Paris Cedex 06, France
Luis Moroder
Max-Plank-Institut far Biochemie, Am Klopferspitz 18a, D-82152, Martinsried, Germany
Sansei Nishibe
Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-02, Japan
Mihaly Nogradi
Research Group for Alkaloid, Chemistry of the Hungarian, Academy of Sciences, P.O. Box 91, H-1521 Budapest, Hungary
Shuji Ozawa
Department of Forest Science, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Virinder Singh Parmar
Department of Chemistry, University of Delhi, Delhi-110 007, India
John Michael Pezzuto
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, U.S.A.
Hanspeter Pfander
Department fiir Chemie und Biochemie, University of Berne Freiestr. 3, CH-3012 Berne, Switzerland
D. Satyanarayana Rao
School of Chemistry, Andhra University, Visakhapatnan 530 003, India
Mark A. Rizzacasa
School of Chemistry, The University of Melbourne, Parkville, Victoria 3052, Australia
Sabine Rudolph-Bohner
Max-Plank-Institut fur Biochemie, Am Klopferspitz 18a, D-82152, Martinsried, Germany
Takashi Sasaya
Department of Forest Science, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Helmut Simon
Institut fur Organische Chemie und Biochemie, Universitat Munchen, D-85747 Garching, Germany
Leverett R. Smith
Department of Environmental Science, Policy and Management, University of California, 201 Wellman Hall, Berkely California 94720-3112, U.S.A.
Francois Tillequin
Universite Rene Descartes-Paris V, Faculte De Pharmacie, Laboratoire de Pharmacognosie, U.R.A. au C.N.R.S.N. 1310 4, Avenue de I'Observatoire, 75270 Paris Cedex 06, France
Giilacti Topcu
Department of Chemistry, Tubitak, Marmara Research Center, P.O. Box 21, 41470, Gebze-Kocaeli, Turkey
Bruno Traber
Department fur Chemie und Biochemie, University of Berne Freiestr. 3, CH-3012 Berne, Sv^itzerland
John Henry Paul Tyman
Department of Chemistry, Brunei University West London, Uxbridge, Middlesex UB8 3PH, U.K.
Ayhan Ulubelen
Department of Chemistry, Faculty of Pharmacy, University of Istanbul, 34452 Istanbul, Turkey
Francese Viladomat
Department of Natural Products, Faculty of Pharmacy, University of Barcelona, Avda., Diagonal 643-08028 Barcelona, Catalonia, Spain
Technische
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Structure and Chemistry
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
Terpenes and their Biological Relevance G.G. Habermehl and W. Fliegner 1.
INTRODUCTION Terpenes are a large group of natural products, occurring mainly in higher
evolved plants as secondary metabolites. The occurrence in higher evoluted plants only may be understood as a consequence of phylogenesis. Under the pressure of selection from herbivorous animals, the synthesis of substances with repellent properties has been necessary in order to make possible a coexistence of different plant and animal species. The first chemical deterrence mechanisms were of an unspecific kind. The synthesis proceeded via the shikimic acid pathway which was already completely developed in gymnosperms. One example of this is the synthesis of lignin. Lignin is one of the most abundand contents of wood, varying between 18 and 30 %. Although its main property is the formation of the stroma, it possesses some antifungal and insectizide properties. By the shikimic acid pathway phenols and tannins are formed, too, to prevent an attack from microorganisms and animals. During the following evolution this pathway has been left stepwise, until in the angiosperms the acetate-mevalonate pathway dominated. This pathway in angiosperms has opened an immense chemical source to specifically acting poisons and repellents, many of them being terpenes. This invention was followed by intense evolutionary interactions between plants and animals, yielding that high diversity of angiosperms, and at the same time, animal species since about 100 million years ago. About 65 million years ago this development of plants was perfect, and the angiosperms predominant. This evolution of poisonous substances in plants coincides with the extinction of the dinosaurs. If one looks closer to this event, one may observe that there has been no complete extinction; many of the saurians survived in one way or another. For example, our birds come from flying saurians; the close connection between the birds and the reptiles of today can be seen in their closely related physiology, leading to the zoological classification of "Sauropsidae", enclosing both, reptiles and birds. And if you have a look at the saurians of today, nearly all of them are carnivorous. All these facts may lead to the conclusion that the herbivorous saurians only became extinct. This is supported by the fact that the dinosaurs did not disappear within a short period of time, as one should accept if this was due to a cosmic event, or a big vulcanic eruption, but within a million of years or so. All these observations fit together, if one accepts that the complete change in
the plant kingdom, together with the newly developed substances led to the end of the dinosaurs. The new physiology with the new acetate-mevalonate pathway gave the opportunity to the plants to produce a tremendous amount of substances acting as protecting agents. 2.
TERPENES AND THEIR ACTIVITIES 2.1 Terpenes as Repellents In a few cases only, the topical situation (spines, thorns) is of protective
value against herbivores. The most potent and effective chemical repellents against these animals are those producing pain or discomfort because of a bad taste or smell. Typical examples are the irritating diterpene-esters from Euphorbiaceae or from Thymelaeaceae (1), or the toxic bitter principles from the Cucurbitaceae, e.g. the Cucurbitacines (2). There are also numerous terpenes acting as contact allergens with serious skin irritations, like the sesquiterpenelactones from Asteraceae (3). Sometimes substances may be effective even after consumption, provided that the animal can recognize the connection between feed and result. In many cases this is not true and, therefore, not a protection. It is well known that cattle or horses like to eat outside the fence, even if they are on a good meadow, either for curiosity or instinct of play. Good examples are here andromedotoxin from Rhododendron
and Andromeda
species, or the
terpene-alkaloids from Aconitum, belonging to the most potent plant toxins. 2.2 Resistance Formation Microorganisms are able to establish resistances because of their great genetic flexibility, the short generation times, and intraspecific as well as interspecific gen exchange. A generally known example of this capability is the resistance against therapeutically used antibiotics. But insects, too, are able to adapt to chemical repellents, even using such substances for their own defense, as is well known from ants and beetles.In such cases a substance which should protect its producer (allomone) becomes a substance useful for the attacker (kairomone), (4, 9). 2.3 Underground Competition Not only animals but also plants are competing in the struggle for the best conditions of life, and they use chemical defense mechanisms, too. Some plants are able to set free germicides directly from the roots. Another mechanism is to exsude from the surface of the leaves such substances which come into the earth by the rain (5). Typical examples are Salvia
leucophylla
and
Artemisia
californica. These shrubs suppress the growth of any plants within a radius of 1 meter completely, by excretion of 1.8-cineol and campher; these substances
inhibit the growth up to a distance of 9 meters (6). 2.4 Terpenes as Signal Substances Terpenes are also used as signal substances, especially with ants but also with other insects. Farnesol is used as male attractrant (7), Citral, geraniol, nerolic acid, and geranic acid are used to transmit information on feed sources (8). These substances, mostly monoterpenes, and sesquiterpenes are produced by plants and taken up by the insects who store them in special glands from which they are released according to their needs. These mechanisms have been developed in a most sophisticated way by ants, butterflies, and beetles (9). Using mixtures of terpenes in different concentrations, they have established a real "chemical language". 2.5 Activities within the Plants. Some terpenes act as plant hormones, e.g. as growth regulators. Such regulators are the brassinolides, steroids with a seven-membered lacton ring B as well as two vicinal diol functions in ring A, and in the side chain (10). The brassinosteroids show in submicromolar concentrations a complex physiologic activity spectrum, e.g. a raise in resistance against stress factors like heat, cold, drought or phytopathogenic infection. The biochemistry of these effects has been studied in detail (11).
3.
TERPENES AS PLANT POISONS AND REPELLENTS. 3.1 General aspects Among all the activities of terpenes from plants, their toxicity is most
important for several reasons. One aspects is their role in ecology as protection of the plants, the other is the economic aspect and also the health aspect, as terpenes are involved in quite a number of envenomations. Envenomations in humans are not rare; mostly children are involved who eat or suck berries or leaves. However, in an increasing number, also adults are involved. On the "green wave" they collect herbs and roots by themselves, considering them "more healthy" than the commercial ones, but confounding them with dangerous species. In the same way therapeutics from plants are estimated harmless for being "natural remedies", and envenomation occurs, frequently due to an overdose.But in most of the cases animals are involved, not only grazing animals but also small animals at home. In the following section the most important plants with terpenes as toxins will be presented together with their chemistry and the symptoms of poisoning.
3.2 Family: Apicaceae Sium latifoliurriy Water parsnip. The plant is abundand in Central Europe but occurs also in the rest of the continent, and also in parts of Africa. The fruit contains (6-7%) of a mixture of limonen (80 %), perillaaldehyde (6%),oc-, and B-pinene, 6-bisabolon, and c?<.-curcumene. Poisoning has been observed in cattle and in sheep with gastroenteritis which led to sudden death. In humans having eaten roots, vomiting, diarrhea, bradycardia as well as muscle paralysis have been observed (14). 3.3 Family: Aquifoliaceae Ilex aquifolium, Holly. The family includes 440 species distributed all over the world with centres in China (112 species) and Brasil (75 species). Ilex aquifolium
is the most
abundand species in Central Europe where it may be found in forests, but it is also very popular as ornamental plant in houses and gardens. The poisonous parts are the berries, and to a lesser extent also the leaves; they contain ursolic acid, uvaol, o^-amyrin, and a bisnor-monoterpene, Ilex-lactone (12), as well as 27-p-cumaroxy-ursolic acid (13). The South American
Ilex
paraguariensis
contains also coffein and theobromin, and is therefore used as tea. Symptoms of poisoning have been observed in children after consumption of berries, with vomiting, diarrhea and drowsyness. 3.4 Family: Araliaceae Hedera helixy Ivy Ivy is far distributed in Europe, and in South Africa; it has been introduced to North America. Preferred locations are forests with old trees, bushes, and walls. It is a creeping plant which may go up about 30 meters. It must not be confounded with poison ivy ! The poisonous parts are the leaves, and even more the blue-black pea-size berries. The active terpenes are triterpene saponins, about 2 - 6 % of the weight. The main saponins are oc-hederin which on hydrolysis yields hederagenin, arabinose and rhamnose, and 6-hederin yielding oleanolic acid, arabinose and rhamnose on hydrolysis. Furthermore the sesquiterpenes germacren, 6-elemen, and elixen are found (14). The hederins share with other saponins a strong hemolytic activity (15). Typical symptoms after ingestion are irritation of mucous membranes, vomiting, headache, drowsyness, and irregular pulse. Milk fever like disease has been observed in cattle (16), and poisoning of horses is also described (17). Allergic contact dermatidis in humans has been described extensively (18, 19, 20). The hederosaponins possess antibacterial as well as antifungal activities (20).
Perilla aldehyde
Limonen
Ursolic acid, R=COOH -Amyrin, R=CH3
Ilex lactone
;ooH
Hederagenin, R-j = CH2OH R2= Rhamnose-Arabinose
Germacen
B-Elemen
Oleanolic acid, R-| = CH3
Elixen
3.5 Family: Asteraceae The Asteraceae (Compositae) comprise about 15,000 species, and thus belong to the biggest plant families. They include herbs, shrubs, and trees, and they are divided into two subfamilies: The Asteroideae distributed worldwide, and the Cichorioideae, distributed mainly on the Northern Hemisphere, and characterized by containing a milky juice. Lactuca virosa, Prickleylettuce Prickleylettuce is endemic to the Mediterranean area, but, due to its former use as medicinal plant has been distributed over Europe and Western Asia. It is no more used in medicine due to its toxicity which in humans leads to an increase of the respiration connected with sweating and drowsyness, and eventually exhaustion which explains its former use as narcotic, and as antaphrodisiacum. In animals damage of the heart, and hypotension have been described (14). The toxicity of the main components lactucin, and lactucopicrin is relatively low. The main protection of the plants is due to its bitter taste connected with both of these compounds. Xanthium species, Cocklebur The toxic compounds are atractyloside and carboxyatractyloside (21). The aglycones of these glycosides are of the kauran type (22). These compounds had earlier been isolated from Atractylis gummifera, endemic to the Mediterranean Area (23, 24). The action mechanism of the poisoning is a specific inhibition of the ATP-ADP carrier of the inner mitochondria membranes, thus preventing the transport of the nucleotides to the place of cell respiration within the mitochondria. The result is a deficiency of ADP there, and finally stop of the respiration chain. The toxicity of carboxyatractyloside is 10.7mg/kg body weight (mouse, i.p.) (23). Typical symptoms in animals are faintness, ataxia interrupted by agitation, koma, and finally death. The hematologic examination showed an increase in the neutrophil granulocytes, extreme hypoglycaemia, and a significant rise of the liver enzymes (25). Carboxyatractyloside is a substance inhibiting the growth of other plants; it may be compared in its inhibiting activity with abscissic acid. Wedelia species. Wedelia glauca is a shrub causing losses in cattle in Argentina, Brasil, and Uruguay (26). The toxic substance again is atractyloside (27) and related kauran type acids (26). Wedelia asperrima is responsible for considerable losses in grazing animals, especially of sheep in Australia. The toxin responsible is wedeloside (28) with some unusual features in its structure: The occurrence of an amino sugar is rare in plants; the distribution of the hydroxy groups in the kauran system is quite
Lactucin, R=H Lactucopikrin, R =CO-CH2-C6H4-(p-OH)
HOCH
Atractylosid, R = H Carboxyatractylosid, R = C O O H
PhCH2CH2(:o—J
r-^UO
OOC-^ ^COOH
Wedeiosid
OH
10
unusual. The toxicity is 1 mg/kg body weight (mouse). Death occurs within 24 hours after ingestion of the plant. Experiments showed (28) that this compound has a strong antitumor activity. Helenium species Helenium microcephalum, Smallhead sneezeweed. Helenium microcephalum
is a herbal plant distributed in the South of the
USA, and in North Mexico, where it is responsible for poisoning of cattle, sheep and goats. The toxic substance is helenalin (29, 30). 2.5 g of fresh plant material per kg body weight lead to the death of the animals. The symptoms occur early, after one hour already and include salivation, colic, diarrhea, vomiting and progressive faintness, and finally death, usually within 24 hours. Post mortem findings include changes in liver, heart, brain, kidney, and mucosa (31). Helenium amarumy Bitter sneezeweed. This plant is endemic to the South and the East of North America. Poisoning in animals is rare because of its low toxicity, and its extreme bitter taste which prevents animals from eating. Occasional uptake of small amounts of leaves, however, are sufficient to give the milk of those animals a bitter taste. The bitter principles are tenulin and isotenulin (32).. Milk with 1 ppm tenulin is undrinkable. The toxicity of tenulin is far lower that that of helenalin; in experiments sheep tolerated up to 700 mg tenulin/kg body weight without showing signs of poisoning (33). Hymenoxis odorata, Western Bitterweed. Western Bitterweed is distributed in the Southwest of the USA, and is the main reason for big losses in sheep and goats (34, 35). The responsible compound is the sesquiterpen lacton hymenovin. Sheep are extremely sensitive: 150 - 200 mg will cause death within 24 hours. Most poisonings do not occur acute but are chronic, as may be seen from the fact that sheep develop the first symptoms after being one or two weeks on grassland with Western Bitterweed. The symptoms start with loss of appetite, followed by mucous flux from the nose, and progressive faintness. Post mortem findings included edema of the mucous membranes as well as necroses in liver and kidneys; the gall bladder shows petechial bleedings. Geigeria species, Vermeerbos. Geigeria species are responsible for losses of sheep and goats, occasionally also cattle in South Africa. The poisoning
is known under the
name
"Vermeersiekte" (Vomiting disease). Just to give one example: In 1954 about 50.000 sheep died from this poisoning in the area close to Kimberley (36). As the name of this disease explains, the prominent symptom is heavy vomiting; muscle tremor is followed by immobility. The toxins are vermeerin and geigerin,
11
Isotenulin
Tenulin
xp:^ Helenalin
Vermeerin
Hymenovin
Gejgerin
12 the first one showing again the exocyclic methylene lacton group, and the second the unsaturated five membered ring keton. Structure-activity relationship. The cytotoxicity of these compounds is due to a very reactive centre in the molecule, the exocyclic oC-methylene-/^lacton group which may easily add to sulfhydryl groups of enzymes or other proteins (32, 34, 35). Therefore, cystein applied immediately has been shown to be a useful antidot in hamsters (34). Tenulin, which is less toxic than the other compounds lacks this methyl en el acton group; it possesses an <>c,6-unsaturated keto group in the cyclopentane ring, as is found in many toxins and carcinogens. Allergic Reactions. Sesquiterpene lactones are a special characteristic of the Asteraceae. In a test of more than 80 of such compounds it has been found that the exocyclic methylene lacton system mentioned above is also responsible for sesitization (37, 38, 39), and allergic reactions. Many Asteraceae have been used as teas or tinctures in traditional medicine. The abundant content of poisonous compounds or allergenes has led to less and less application. 3.6 Family: Brasicaceae Iberis amara, Wild Candytuft, Bitter Candytuft This plant, growing up to 40 cm, is endemic to the Mediterranean Area but may also be found cultivated in gardens. The whole plant is toxic especially the seeds. Poisoning by Candytuft is a synergistic effect from many compounds. As in most other Brassicaceae it contains glucosinolates, but also the cucurbitacines E, I, J, and K (40). The symptoms, therefore, depend on the dominating substances. Glucosinolates possess bacteriostatic properties, the cucurbitacines are cytotoxic. The gastrointestinal form of poisoning is connected with loss of appetite, loss of rumination, thirst, salivation, decrease of milk production, colic, and diarrhea or also constipation. The respiratory form is characterized by edema
and
emphysema of the lungs resulting in difficulties in breathing. The nervous form of the poisoning shows loss of orientation, and sometimes loss of vision. Hemoglobinuria is frequent (41). 3.7 Family: Chenopodiaceae Chenopodium ambrosioides, Wormseed, Jerusalem oak, Stinking weed. Chenopodium introduced
to
Chenopodium
abrosioides
Central
is endemic to South America, but it has been
and North
anthelminticum
further varieties, differing
America, to
Europe
and
to
Africa.
is a variety thereof, and there are a number of in their contents of terpenes. The
botanical
classification has still to be revised. Toxic parts include the fresh blooming
13
leaves, the seeds, and especially the seed oil. "Oleum chenopodii" contains about 70 % Ascaridol, and about 22% of Cymol, limonen, isolimonen, and
plant itself
but from
improper
application of
Oleum
Chenopodii in pharmacy. Due to its content of ascaridol it is used against intestinal parasites.Even with proper treatment side effects like nausea, vomiting, trembling of hands and feet, as well as deafness (paralysis of the nervus cochlearis) are not rare. After longer application liver, spleen and kidneys are also affected. 3.8 Family: Cucurbitaceae. Bryonia alba, White bryony Bryonia dioica. Red bryony Ecballium elaterium. Squirting cucumber Citrullus colocynthiSy Bitter apple These four plants are endemic to the Mediterranean Area but have been introduced to other parts of Europe; the bitter apple is also found in the savannahs of Africa. All parts of the plants are poisonous, especially roots and fruits. The compounds responsible are the cucurbitacines, a rather big family of tetracyclic triterpenes, occurring either in free form or as glycosides. They have been studied intensively (2, 43, 44, 45, 46). Envenomations in humans occur either by improper use of preparations in traditional medicine; there is a broad spectrum of indications described, so that one can hardly believe the real effectivity. Children are endangered because of the fruits. 15 berries of bryony are considered to be fatal. A general feature of the cucurbitacines is their cytotoxicity, causing irritations of the skin leading to necroses and ulcera. Most of the internal symptoms follow the same way: Diarrhea, vomiting, headache, and in severe cases convulsions and death. There is no difference of symptoms between animals and humans. Most animals would avoid the plants because of their bitter taste. But in cattle it has been observed that they eat again, after having recovered from the first poisoning. Due to the cytotoxicity of the cucurbitacines they have been investigated as potential antitumor agents. In cell cultures they decreased the protein biosynthesis remarkably, but in vivo the side effects are too severe as that there was any practical use.
14
Ascaridol
Pjnocarvon
Acetylandromedol (Andromedotoxin)
Cucurbitacin I: R=OH Cucurbitacin E: R=OAc
15
Cucurbitacin D (Elatericin A), R = =H Cucurbitacin B, R=OAc
^"3 Cucurbitacin K: RrCH(OH)-(j-OH CH3
Cucurbitacin L
^"3 Cucurbitacin J: R=lj-OH CH,
16
3.9 Family: Cupressaceae, Thuja occidentalis American arbor vitae Thuja orientaliSy Oriental arbor vitae Thuja occidentalis is, as its common name says, endemic to North America Thuja orientalis to China. Both plants, however, are widely spread now worldwide as ornamental plants. There is no difference in the composition of their terpenes. The main component, also responsible for the toxicity is thujon with more than 50 per cent of the essential oil from Thuja. Furthermore fenchone, campher, sabinene, and terpineol-4, tannins and bitter principles are found, too. Envenomation in humans was formely due to its use as abortivum. Thujon is a local irritant. Ingestion causes bleedings of the mucous membranes in the gastrointestinal system resulting in diarrhea and vomitus. In more severe cases the temperature is elevated, liver (atrophy), heart (bleeding into the muscle) and kidney (degeneration) are damaged. Tonic-clonic convulsions and finally paralysis of the Central Nervous System preceed death (47). Today Thuja oil is still used in the so-called "natural medicine" as done by non-medical practitioners. Unfortunately people have the idea that "natural medicine" is especially healthy. Poisoning in animals is rather rare as the taste of the tips of the branches is bitter and burning. Juniperus sabina. Savin Juniperns virginiana, Virgin Cedar Juniperus communis, Common Juniper Juniperus spp. are distributed worldwide. The poisonous compounds in the essential oil are sabinen, sabinol, sabinyl acetate as the main substances, and pinen, myrcen, limonen, Cymol and Thujon as byproducts. Envenomation in humans is by improper use of the essential oil in "natural medicine". Besides acute gastroenteritis, central nervous symptoms may be observed as nausea and vomiting, convulsions and respiratory arrest. Other syptoms are arrhythmia of the heart, damage of kidneys and liver, uterine cramps. Juniperus communis is much less toxic and is used as diureticum, diaphoreticum, nervicum, and emenagogum. It is also used against ectoparasites. The fruits are used as aromatics and for the preparation of liquors. Poisoning of animals has not been observed, because of the bad taste of the plant (48).
17
3.10 Family: Ericaceae Rhododendron
species
Rhododendron
ponticum. Common Rhododendron
Rhododendron
chrysanthum. Yellow Alpine Rose
Rhododendron
luteum
Rhododendron
Alpine Rose
ferrugineum,
Rhododendron
hirsutum, Alpine Rose
Rhododendron
simsii, Azalea
Rhododendron
species are far distributed on the Northern hemisphere and in
Australia. Poisoning has been observed in humans and in animals, the most important substance responsible being andromedotoxin (=Acetylandromedol) (48, 49). Usually the whole plant is toxic, including pollen and nectar which is important as honey may be poisonous from andromedotoxin. Andromedotoxin is synonymous with acetylandromedol, asebotoxin, rhodotoxin, and graynatoxin (50); its LD50 is 0.3 mg/kg body weight (dog, orally), and 2-5 mg/kg in rats (orally) (14). Rhododendron ursolic
acid,
Rhododendron
uvaol,
and
hirsutum,
simiarenol.
endemic
to
ponticum also contains the triterpenes Rhododendron Central
Europe
and
ferrugineum, does
not
possess
andromedotoxin; its toxicity is due to ursolic acid which shows the same symptoms but is much less toxic. Andromedotoxin poisoning leads to a gastroenteritis, connected with burning sensation in the mouth, nausea, vomiting, bradycardia
and
respiratory
distress.
Its
hypotensive
activity
in
low
concentrations is used therapeutically The pharmacology has been studied in detail (51). Poisoning occurs in humans and in animals. The oldest case report in the history of toxicology stems from the Greek author Xenophon: His soldiers during a campaign against Persia had eaten honey and suffered from vomitus and diarrhea; they behaved like drunk, a few of them died. Today we know that this was honey from Rhododendron
ponticum (52, 53). Occasionally, one can
read in newspapers that such poisonings with honey are still occurring in Turkey. Among animals, mainly sheep are affected; goats and cattle use to vomit immediately, and so get rid of the toxic compounds. This mechanism is also a learning effect. Horses are unable to vomit, and therefore are especially endangered; detailed reports on sheep poisoning have been published (54,55). Ledum palustre, Dutch myrte. Ledum palustre contains two not really toxic but pharmacologically active compounds, ledol, and palustrol, responsible for poisoning (56). They cause local
18
Ledol
Palustrol
P h o r b o l : R-, = R 2 = H TPA, ( 1 2 - 0 - T e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ) : R-j = C O - ( C H 2 ) i 2 - C ^ H 3 R2 = C O - C H 3
CH2OH Huratoxin: R = CH=CH-CH=CH-(CH2)6-CH3
19 ecstasy, which has been used to prepare "Porstbeer", beer with addition of a small amount of ledol, producing a "good" intoxication. The use of such herbs in brewing beer, therefore has been forbidden in Germany since 1415, by the so-called "Purity Law for Beer". 3.11 Family: Euphorbiaceae Euphorbia species, Spurge. Euphorbia spp. are distributed worldwide with about 680 species. Their outer appearance may vary considerably, from herbs and shrubs in temperate zones to cactus-like, succulent stems with or without leaves in subtropical or tropical areas. All of them have in common that they contain abundandly milky juice, containing the active compounds. Up to now about 80 species have been investigated for such compounds, and practically all of them contain substances with skin irritating activity followed by formation of blisters and necrosis, the mucosa being damaged severely; in eyes they cause conjunctivitis, sometimes followed by blindness. Taken up orally, they cause vomiting, severe diarrhea, and in rare cases mydriasis, delirium and respiratory distress. Chemically, they are diterpene esters of the tigliane, daphnane, and ingenane type. Many of them possess also cytotoxic or co-carcinogenic activity. Envenomations in humans are limited to improper application in traditional medicine. Poisoning of animals occurs accidentally only, because of the burning taste. Animals usually avoid these plants, but grazing animals sometimes cannot avoid the uptake of such plants. In sheep, cattle and horses poisonings have been described with the signs of hemorrhagic gastroenteritis, liver damage, and central nervous disturbances. The most potent substance is TRA, a diester of phorbol. Croton tigliurriy Croton. Croton is endemic to the tropical parts of Asia; it has been imported to Mauritius and to East Africa. Croton oil was a well-known purgative in human as well as in veterinary medicine (57, 58). The active compounds are esters of phorbol, a tetracyclic, tiglian-type diterpen. Croton oil possesses a burning taste and causes vomiting and diarrhea; applied locally it produces skin irritation followed by formation of blisters and edema. Closely related to phorbol is huratoxin (daphnan-type frame) from Hura crepitans^ a tree endemic to the tropical part of America (59). 3.12 Family: Ranunculaceae. Aconitum napelluSy Monkshood, Wolfsbane. Delphinium species, Larkspur. Both plants are toxic. Poisoning in humans has been reported from the roots as well as from the pollen (honey) (60, 61). Among animals, horses are especially sensitive (51), but due to the bad taste the plant usually is avoided.
20 /0CH3
H3CO
^CH3
Aconitin
Ri = C O - C Q H S ; R2 = CO-CH3 ; R3 = C2H5
Aconin
R-| = R2 = H
;
R3 = C2H5
J)CH3
Cf^CH2-f-|
Lycoctonin
Taxicin I
21
\^
H 11
P
QH
OHO 1
li
R-C-NH-ChKH^O-C
Taxol A: R = CgHs Taxol B: R = CH2CH=CHCH3
Daphnetoxin: R-j = CgHs ; R2 = H Mezerein: R-j = CgHs ; R2 = OCO-(CH=CH)2-C6H5 Simplexin: R-j = C9H-19 ; R2 = H
22
The symptoms are more or less identical with those already reported, namely salivation, vomiting, diarrhea, and in severe cases death due to respiratory paralysis. The compounds responsible are aconitin, aconin, lycoctonin and related compounds. 3.13 Family: Taxaceac Taxus species. Common yew, English yew. Taxus species are far distributed in Europe, North Africa, West Asia, and North America (Ground Hemlock). More than 30 compounds have been isolated and investigated chemically, and pharmacologically (62). Poisoning in humans is rare (63, 64) but some fatalities have been reported. Taxus branches, however, are dangerous in animals, especially in horses and pigs. Envenomations in wild living animals are rare. Death occurs very fast from respiratory paralysis (51, 65). Typical compounds are taxicin and the taxols which have been falsely attributed as alkaloids. The taxols have gained interest because of their potential antitumor activity. 3.14 Family: Thymelaceae. Daphne mezereum, Mezereon. Mezereon is endemic to Europe, West Asia, and Canada. Poisoning is due to Daphnetoxin, Mezerein, and Simplexin (66); their toxicity has been determined (51). The structures of the compounds are closely related to huratoxin. The symptoms are identical, but more severe; the kidneys are concerned especially with nephritis, albuminuria, hematuria, and stranguria. Envenomations are observed mainly with children, eating the red berries. In animals poisoning has been reported, but is a rare event because of the burning taste of the plant. This is probably also the reason why poisoning in wild animals is unknown so far. 4.
CONCLUSION. This review does not cover all terpenes isolated from plants nor all plants with terpenes as secondary metabolites. This is , however,a selection in respect to their activities. As may be seen, the toxicity is varying,, but what is common in all, is a bad taste, bitter, burning, and causing irritation of mouth lips and tongue. This clearly shows that these compounds have been developed during the evolution as protection, thus establishing an equilibrium between herbivors and plants. Such protection has been especially important in the "early" times of the angiosperms. So it is worth looking for their biological relevance, and it is also interesting that wild animals obviously have learned to deal with the dangers of their feed. And this may explain, too, why many herbivors became extinct some 60 million years ago, while carnivors did not show this break in evolution.
23
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24
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
J.PJ. Joubert, J. S.Afr. Vet. Assoc. 54 (1983) 255-258. F. Bohlmann, Naturwissenschaften 67 (1980) 588-594. J.C. Mitchell and G. Dupuis, Brit. J. Derm., 84 (1971) 139-150. B.M. Hausen, Planta med., 50 (1984) 229-234. R. Gmelin, Arzneimittelforschung 13 (1963) 771. G. Rosenberger, Krankheiten des Rindes, Parey, Berlin, Hamburg, 1978. T. Takemoto and N. Nakajima, J. Pharm. Soc. Japan 77 (1957) 1157. P.R. Enslin, F.J. Joubert and S. Rehm, J. Sci. Food Agr., 7 (1956) 646. D. Lavie, D. Willner and Z. Merenlender, Phytochemistry, 3 (1964) 51. M.R. Cooper and A.W. Johnson: Poisonous plants in Britain and their effects on animals and men: Ministry of Agriculture Fisheries and Food, London, 1984. A.A. Forsyth: British Poisonous Plants: Ministry of Agriculture Fisheries and Food. Bulletin 161, pp.73-74, London, 1979. Hagers Handbuch d. pharm. Praxis (P.H. List and L. Horhammer, Eds), Springer, Berlin, Heidelberg, New York, 1980. K. Hiller and G. Bickerich: Giftpflanzen, Enke, Stuttgart, 1988. F. Zymalowski, P. Pachaly and S. Auf dem Keller, Planta med., 17 (1969) 8-13. H. Schindler, Planta med., 10 (1962) 232-237. H.J. Hapke: Toxikologie fiir Veterinarmediziner, Enke, Stuttgart, 1988. E.G.C. Clarke, D.J. Humphreys and T. King: Nachweis von Andromedotoxin, in E.Kaiser (Ed): Tier- und Pflanzengifte, Goldmann, Miinchen, 1973. A.M. Chambers, Scott. Med. J. 29 (1984) 107-108. R.J. Higgins, D.A.R. Hannam, D.J. Humphreys and J.B.J. Stodulsky, Vet. Rec. 116(1985)294-295. L.A.M.G. van Leengoeld and J.J. van Amerongen, Tijdschr. Diergeneesk., 108 (1983) 41-42. D.H.E. Tattje and R. Bos, Planta med., 41 (1981) 303-307. E. Hecker and R. Schmidt: Phorbolesters, the irritants and cocarcinogenes of Croton tiglium L. in: Fortschr. Chem. Org. Naturst. 31 (1974) 377-467. E. Hecker, Dtsch. Apoth. Ztg. 126 (1986) 2599. G. Fiirstenberg and E. Hecker, Planta med., 22 (1972) 241-266. H.H. Kalbfleisch, Arch. Toxicol.,13 (1943/44) 17-20. Y. Saito, A. Midura, K. Sasaki, M. Satake and M. Uchiyama, Bull. Natl. Inst. Hyg. Sci., 9 (1980) 32-35. R.W. Miller, J. Nat. Prod. 43 (1980) 425-437. S. Ritter, Dtsch. Apoth. Ztg. 125 (1985) 1834-1836. S. Ritter and E.G. Krienke: Vergiftungen im Kindesalter. Enke, Stuttgart, 1986. B. von Dach and R. A. Streuli, Schweiz. med. Wschr. 118 (1988) 1113-1116. H. Schildknecht and R. Maurer, Chemiker Ztg. 94 (1970) 849. H. Schildknecht, Angew. Chem. 93 (1981) 164-183.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
25
Crinitol, an Acyclic Diterpene Diol from Marine Algae I. Kubo and L.R. Smith Crinitol, (1), is a diterpene diol whose properties can in some ways be taken as representative of the current challenges in studying and finding uses for secondary plant metabolites. It is readily availability from a natural source and has a relatively uncomplicated structure; despite this, its function in the plant is not fully clarified. As will be discussed below, crinitol shows a modest level of antimicrobial and other biological activity. We can already control many troublesome microorganisms that contaminate food and cosmetics with natural products, including plant secondary metabolites, that are currently available. Nevertheless, discovery H. OH I
I
I
I ^^Q..M
x'^V^'^v--^^^ 9R and 9S Crinitol (1 and 2) of new effective antimicrobial agents continues to be important since microorganisms have developed resistance to many commonly used antimicrobial agents. There is no doubt that plants are still a good source for new antimicrobial agents. However, a typical difficulty with antimicrobial phytochemicals is that they are not usually potent enough to be considered for practical applications. Hence, studies to enhance the total biological activity are needed. In order to proceed with this, understanding the effects on microbial systems on a molecular basis is essential. The moderate activity of crinitol (1) isolated from several marine brown algae is an example of this level of potency, and its chemistry and bioactivity can be taken as a case study of the sort of questions that can arise in current studies of natural products. ISOLATION AND BIOGENESIS Crinitol (1) was first isolated from one of the most studied marine brown algae, Cystoseira crinita Bory (Cystoseiraceae), and its structure was established as 3,7,11,15tetramethylhexadeca-2,6,10,14-tetraene-l,9-diol (1); it was later found in the closely related species Cystoseira baleahca (2). The absolute configuration at C-9 was not determined in the Cystoseira-dQTivQd samples, but was determined as R in crinitol isolated from another brown
26 alga, Sargassum tortile C. Agaedh (Sargassaceae) (3). The Sargassutn identification had been pursued on the basis of assays of insect growth inhibition against the pink bollworm, Pectinophora gossypiella, by an artificial diet feeding assay (4). The larger biosynthetic context of crinitol has been extensively studied in brown algal species, which have yielded a wide variety of related diterpenes (5,6). It has been suggested that in the uncommon cases where plants accumulate geranylgeraniol (3) metabolically, oxidation products of geranylgeraniol "are possibly nothing more than the expression of its catabolism" (6). Such catabolic oxidation products reported have included a number of diols isomeric with crinitol, including 12-(5)-hydroxygeranylgeraniol (12-(5)-3,7,11,15-tetramethylhexadeca-2,6,10,14tetraene-l,12-diol) (4) (7,8); 13-(S)-hydroxygeranylgeraniol (eleganediol, or 13-(5)-3,7,ll,15tetramethylhexadeca-2,6,10,14-tetraene-l,13-diol) (5) (8-12); and bifurcanol (3,7,11,15tetramethylhexadeca-2,6,10,13-tetraene-1,15-diol) (6) (8). In view of the suggested
OH HO H
12-(S)-Hydroxygeranylgeraniol (4)
Geranylgeraniol (3)
OH
Eleganediol (5)
Bifurcanol (6)
^^
16-Hydroxygeranylgeraniol (7)
origin, it perhaps ought to be expected that similar compounds would be present in other plant sources, not just in algae; 16-hydroxygeranylgeraniol (7) has been reported to occur in a Japanese fungus, Boletinus cavipes (13). ABSOLUTE CONFIGURATION AND CONFORMATIONAL CONSIDERATIONS At the time that crinitol was isolated from 5. tortile (3), the absolute configuration at C-9 was not known. To define the absolute configuration of the stereocenter, the circular exciton chirality method (3,14) was applied. Four derivatives (8-11) were made and their CD
27
Ax'0::>^C-^^
xiv'v.^'^^^
(8)
(9) Br
(10)
(11)
spectra were recorded. Among them, only 10 and 11, whose C-9 hydroxy! group was pbromobenzoylated, showed the predicted benzoate Cotton effects. The absolute value (Ae) of both of these benzoate Cotton effects were almost identical; 10, -5.3 (247 nm) and 11 -5.4 (244 nm). These data clearly indicate that the C-1 benzoate chromophore does not interact with the C-9 benzoate chromophore, unlike dibenzoates of cyclic compounds (15). The observed negative Cotton effect is due to only to the interaction between the benzoate ^La transition and the double bond TT-T* transition. If all possible rotomers were equally present the sum of the Cotton effect should cancel. However, it has been shown for a large number of secondary allylic benzoates the rotomer having the carbinyl hydrogen and double bond eclipsed is favored. The presence of a large /yjc of 9.0 Hz between the olefinic and C-9 carbinyl protons in 10 and 11 is consistent with such a preferred conformer, as the size of the coupling is dependent on the dihedral angle. Thus, the absolute configuration of crinitol was determined to be 9R This was the first actual application of the allylic benzoate method (14) to a natural acyclic allylic alcohol. The configurational assignment did not quite end with this first determination, however. In the course of preparing a sample of the "unnatural" 9S crinitol isomer (2) by racemization, derivatization and recycle HPLC (discussed below), crinitol isolated from S. tortile was later found to contain a small amount (13%) of the 95 isomer (16). Mosher's NMR method (16-18) verified the original assignment of configuration for the dominant natural enantiomer. Given the significant presence of both enantiomers in S. tortile, one might conclude that the biosynthesis of crinitol is not highly stereospecific, and crinitol's optical purity will probably vary from source to source.
Before leaving the topic of absolute
configuration determination, we should mention a third method applicable to this type of compound, that of Horeau (19), which was used to determine the configuration of eleganediol (12). Horeau's method is based on the differential reactivity of racemic 2-phenylbutyric anhydride with chiral alcohols.
28 Given the size and flexibility of the crinitol molecule, statements about preferred conformations must be regarded as tentative. However, a clue may lie in an interesting concentration effect, found in the 300 MHz ^H NMR spectrum of natural 9R crinitol (15) as shown in Figure 1. The C-9 hydroxy results in a clearly separated C-9 proton (H9) at 6 4.4, whose coupling provide clues to conformational preferences near the center of the molecule. In particular, the observed splitting goes from a quartet (7=7.4 Hz) at ca. 15% in CHCI3, to a triplet (7=8.4 Hz) in ca. 1% solution. Coupling to the C-10 olefinic proton (HJQ, a doublet at 6 5.15) remains almost constant, but a clear differentiation appears in coupling to the diastereotopic methylene protons on C-8. A predominantly anti H9/H10 orientation throughout would be consistent with recent studies reported by Gung et al. (20). The spectra seem to indicate that there is no particular C-8/C-9 conformational preference at high concentration. On dilution, however, the different couplings that emerge would be more consistent with a predominantly extended C-8/C-9 conformation in which H9 is anti to the pro-5 proton (Hg^) on C-8, and therefore gauche to the pro-i? proton (Hg;^; H85 and Hg;^ appear as a partially visible multiplet around 6 3.2. A "hairpin" conformation around C-8/C-9 might also rationalize the observed couplings, but would appear sterically unreasonable and at variance with reported studies on similar analogs (20). Other concentration-related differences appear, for example in the C-1 methylene proton pattern at ca. 6 4.1, but interpretation is not so straightforward. Crinitol may exert its bioactivity by interaction with membranes, and the C-9 proton could perhaps provide an NMR "probe" of the environment in which the center of the crinitol molecule finds itself in such interactions; one might speculate similarly about the related diols 4-7. ANTIBACTERIAL AND OTHER BIOLOGICAL ACTIVITY Crinitol was isolated from 5. tortile by following a bioassay for growth inhibition of P. gossypiella{3). The only active principle isolated in the methanol extract, a viscous colorless liquid, ([«]£) -5°, cO.43, CH3OH) was identified as crinitol (1, [a]D -3°), previously isolated from C crinita, by comparison of spectral data (1). The effective dose (ED50) for 50% growth inhibition of P. gossypiella was 500 ppm. In our continuing search for new antimicrobial agents from botanical sources, crinitol was later found to exhibit moderate antibacterial activity against several Gram-positive bacteria (21-23) as shown in Table 1. For comparison. Table 1 also includes results from assays of geranylgeraniol (3), famesol (12) and geraniol (13). Crinitol exhibited weak to moderate activity against all the Gram-positive bacteria tested but not the Gram-negative bacteria up to 800 /xg/ml. It did not show any activity against fungi, except T. mentagrophytes. This dermatologically pathogenic fungus was
29
Figure 1. Downfield Portion of the 300 MHz Proton NMR Spectrum of Crinitol, in Chloroform-d.
30
susceptible to crinitol with the minimum inhibitory concentration (MIC) of 25 figlml (23). Interestingly, T. mentagrophytes was relatively susceptible to many long-chain alcohols (23). Among the Gram-positive bacteria tested, P. acnes was the most sensitive bacterium, with an MIC of 25 jwg/ml. By contrast, S. aureus was the least sensitive, with an MIC of 400 jitg/ml. In addition, crinitol was found to be bactericidal against P. acnes. This was determined as follows: 30 III taken from the medium that was incubated for 2 days with the MIC of crinitol was added into 3 ml of the crinitol-free fresh medium and incubated for 2 days. No recovery of this follicular bacterium was observed.
MIC (fig/ml) 1
3
12
13
Bacillus subtilis Brevibacterium ananoniagenes
50 100
>800
12.5 12.5
400 400
Streptococcus mutans
50
>800
12.5
400
Staphylococcus aureus
400
>800
25
800
Propionibacterium acnes
25
>800
>800
>800
6.25 >800
400
Escherichia coli
>800
800
Pseudomonus aeruginosa
>800
>800
>800
>800
Enterobacter aerogenes
>800
>800
>800
Saccharomyces cerevisiae Candida utilis Pityrosporum ovale Penicillium crustsum
>800 >800 >800
>800 >800 >800
>800 >800 >800 >800
400 400 200
>800
—
Trichophyton mentagrophytes
25
—
>800 12.5
200 200
, The rate of activity varied slightly with the seed culture mediaL, the physiological age of the culture, and the type of culture medium. — , Not tested. In addition to crinitol, two structurally related minor acyclic diterpene alcohols, geranylgeraniol (3) and phytol, were also isolated from the active fraction in S. tortile, although neither of them exhibited any activity up to 800 fig/ml as listed in Table 1. In addition, several derivatives of crinitol as well as its two common natural congeners, famesol
31
(12) and geraniol (13) were also assayed. The hydroxy group at C-9 in crinitol seemed to be essential for the antibacterial activity. In fact, the oxidized compound, 9-oxocrinitol, and mono- and diacetyl derivatives, did not exhibit any activity up to 8(X) /ig/ml. Among the three naturally occurring alcohols tested (3,12,13), a sesquiterpene alcohol, famesol (12), exhibited the most potent activity against several Gram-positive bacteria and T. mentagrophytes. In contrast, a diterpene alcohol, geranylgeraniol (3), did not show any activity up to 800 ixglml. Alcohols are among the most versatile of all natural products. Free and esterified alcohols occur widely in nature, e.g. in fruit. The antimicrobial effects of alcohols have been reported. However, studies have been limited to short-chain ( < Cg) alcohols, almost exclusively ethanol. Antimicrobial activity of long-chain ( > C^) alcohols, on the other hand, has been demonstrated only with restricted microorganisms because of their limited solubility in water. To place our results with crinitol in the more general context of antimicrobial activities of alcohols, we investigated a series of simple aliphatic alcohols as a model (23,24). The study was focused towards understanding the role of the hydrophobic portion since all the molecules studied possess the same hydrophilic hydroxyl group. The mechanisms of antibacterial activity of long-chain alcohols are due to a balance between the polar hydrophilic (head) and nonpolar hydrophobic (tail) portions of the molecule. The fluidity of a cell membrane can be disturbed maximally by particular hydrophobic moieties attached to hydroxyl groups. They could enter the molecular structure of the membrane with the polar hydroxyl group oriented into the aqueous phase by hydrogen bonding and nonpolar carbon chain aligned into the lipid phase by dispersion forces. It may not be illogical to suppose that a hydrophobic chain length greater than that for the maximum activity disperses into the lipid layer, resulting in the breaking of the hydrogen bond. Thus, when the balance between hydrophilic and hydrophobic portions of the alcohol was destroyed by the dispersion forces, on the other words when the hydrophobic character, log P, exceeded the maximum value, log PQ, the activity disappeared. The maximum activity depends on the hydrophobic chain length from the hydrophilic hydroxyl group, and also the microorganisms being tested (23,24). This general rule may apply to many other membrane-active antimicrobial agents analogous to crinitol. C-9 CONFIGURATION AND BIOACTIVITY: RACEMIZATION AND RESOLUTION VIA MTPA ESTERS As part of our studies of the antimicrobial activity of crinitol and related compounds (23), it was of interest to test whether the two enantiomers of crinitol exhibit the same or different activities, as a clue to whether the antimicrobial action might be due to some specific interaction beyond the above hypothesized general lipophilic action on cell membranes.
32
However, a source of the non-natural 9S isomer (2) was first required. The ready availability of natural crinitol made a racemization/resolution route, as illustrated in Scheme 1, attractive. Racemization was accomplished by Collins oxidation (16,25) to the dicarbonyl compound (14), followed by lithium aluminum hydride (LAH) reduction to give the racemic mixture (1 -I- 2). Resolution via diastereomeric derivatives seemed plausible. Esterification with enantiomerically pure a-methoxy-a-(trifluoromethyl) phenylacetic acid (MTPA) (17), followed by separation of diastereomers by recycle-HPLC (R-HPLC), had earlier been used to purify enantiomers of ipsenol and ipsdienol (26). A model system, the resolution of ^3-nonen-2-ol, a secondary ally lie alcohol naturally occurring in Rooibos tea (16,27), also worked satisfactorily. Therefore, the route using the te-(MTPA) esters was selected for crinitol. With crinitol, use of MTPA chloride/dimethylaminopyridine (16,28) gave the desired mixed diester (15 + 16) cleanly and in high yield. The mixed diesters were then separated by recycle-HPLC (16). It is of interest that recycle-HPLC of the te-MTPA ester of natural crinitol showed the presence of a small amount (13%) of the 95isomer, as mentioned above. Absolute configurations were checked in the crinitol bis-MTPA esters 15 and 16 by their iH-NMR spectra, using the Mosher method, as extended by Ohtani et al. (16-18). This method presumes a Sj^-periplanar arrangement of the C-H bond at the chiral center, the ester carbonyl bond, and the a-C-trifluoromethyl bond of the ester moiety. In this geometry, the ester's phenyl and methoxy groups, respectively, shield and deshield nearby groups in the alcohol moiety. While the C-9 configurations of the crinitol bis-MTPA esters were already reasonably assignable by comparison with material prepared from the known 9i? natural diol (3), it was nonetheless reassuring that the relative shifts of the clearly identifiable C-8 methylene hydrogens (6 2.37 and 2.17 in 15, vs. 2.42 and 2.21 in 16) were consistent with predictions by the Mosher/Ohtani NMR method. Diastereomeric excesses of the separated MTPA esters 15 and 16 were determined to be >96%, based on NMR measurements. Higher diastereomeric excesses could have been obtained by further chromatography, but the values obtained sufficed for our needs. The "unnatural" 95crinitol was assayed for antimicrobial activity simultaneously with a sample of natural crinitol, against the same battery of Gram-positive microorganisms originally used with the natural material alone (16). Against B. subtilis, B. ammoniagenes, 5. aureus, 5. mutans and P. acnes, the natural and unnatural material showed identical activity, which also matched the results of earlier tests (21) on natural material alone. This indicated that, for these test species at least, the configuration at C-9 is irrelevant to antimicrobial activity. This seems to indicate that antimicrobial activity is not due to interaction with a specific receptor, but rather to the above mentioned more general lipophilic interaction affecting cell membranes (24).
33
Scheme 1
x^>x-v^x^t^^
Collins Oxidation
OH
14 LiAIH/
A > - 0 : v ' 0 ^ > ^ - x ^ ^
+ xW^-vJ^V^^^
MTPA Chloride/DMAP ^ OMTPA
.A>-^\..A>X.^^
OMTPA
15
OMTPA
+
H
OMTPA 16 1. Recycle HPLC 2. NaOH
Separated
1 + 2
34
ENHANCED ANTIMICROBIAL ACTIVITY BY SYNERGISTIC COMBINATION Although crinitol may play an important role in the defense of living plants, and can easily be obtained in relatively large quantities from S. tortile or other sources (3), its antibacterial activity may not be potent enough to be considered for practical use as a single active ingredient. For this reason, the enhancement of the antibacterial activity of crinitol by combining it with other substances was explored (29), although the rationale for selecting "other substances" remains in a preliminary stage. The initial selection of adjuvants to crinitol was based largely on the above mentioned structure-activity relationship study. Thus, the hydroxy group at C-9 in crinitol seems to be essential to its antibacterial activity. The ally lie C-9 hydroxy group is easily oxidized, a possibility which might detoxify crinitol in bacterial systems. Because crinitol possesses two ally lie alcohol groups in its molecule (at C-1 and C-9), we combined it with several antioxidants which have been used as food and cosmetic additives. Thus two natural antioxidants, vitamins C and E, and two synthetic antioxidants, BHA and BHT, were chosen for the experiment. While the synthetic antioxidants exhibited antimicrobial activity against all the test bacteria (30), natural antioxidants did not show any activity up to 800 jug/ml. In combination with crinitol, neither of the natural antioxidants showed any enhancing activity against the bacteria tested. Neither synthetic antioxidant exhibited any synergistic effect against 5. aureus at all. Table 2 shows the MIC of crinitol in combination with the two synthetic antioxidants at the concentration of each 1/2MIC. The activity of crinitol against 5. mutans was significantly increased by both BHA and BHT. The antibacterial activity of crinitol against this cariogenic bacterium was increased 64-fold by BHT; the MIC was lowered from 50 to 0.78 fjLg/ml. Similarly, BHA also synergized crinitol against S. mutans, but not as much as BHT; the MIC was reduced only from 50 to 12.5 /xg/ml. Interestingly, crinitol also enhanced the antibacterial activity of BHT and BHA against S. mutans 16- and 8-fold, respectively. The data were obtained by the broth checkerboard method (31). By way of comparison, we might consider the famesol/anacardic acid synergism against P. acnes. Its antibacterial activity against this follicular bacterium was significantly increased in combination with ViMlC of anacardic acid isolated from the cashew Anacardium occidentale (Anacardiaceae) apple, nut and nut shell oil (32). More specifically, the MIC of famesol was lowered from 6.25 to 0.78 ^g/ml, in combination with 0.39 figlml of anacardic acid. Interestingly, this synergism was found to be vice versa; the MIC of anacardic acid was reduced from 0.78 to 0.2 /xg/ml when it was combined with 3.13 /xg/ml of famesol (33). The mode of action of the combination, however, remains unclear.
35 Table 2. MICs of crinitol alone and in combination with V2MTC of BHA and BHT MIC (Mg/ml) Microorganisms tested 1 + BHA 1 1 + BHT B. subtilis
50
50
12.5
B. ammoniagenes
100
100
50
S. mutans
50
12.5
0.78
S. aureus
400
400
400
P. acnes
25
6.25
6.25
ECOLOGICAL ASPECTS Crinitol was originally reported from C. crinita, "a brown seaweed rather common along the Sicilian coasts" (1); C. baleahca, another species from which crinitol has been reported, also was collected from Sicily for study (2). A wide range of Mediterranean algae have been studied and characterized (5,6); although crinitol itself seems to have been reported from only two species in the region, many related compounds have been described, and there seems to be significant variability in seaweed populations in the region (5,6,8,11,12,34,35). Much less has been reported on congeners of the marine brown alga S. tortile, which is known as "yoremoku" in Japan. This seaweed is commonly found on rocks from below the low-tide level to depths of several meters or more. In order to investigate the biological activity of crinitol, more S. tortile was collected from different geographic locations along coasts of the Pacific Ocean and the Sea of Japan during different seasons. The isolation of crinitol was monitored by an antibacterial activity assay against B. subtilis. As a result of this study, the quantity of crinitol in S. tortile was also analyzed in terms of its geographical and seasonal variation. More specifically, S. tortile was collected from two different geographic location in Japan, one at Kata, Wakayama, which is along the coast of the Pacific Ocean, and the other near Maizuru, Kyoto, which is along the coast of the Sea of Japan. The antibacterial crinitol was isolated as the only active principle based on bioassay-guided fractionation of the CH2CI2 extracts of 5". tortile collected during both spring and fall at Kata. It should be noted that crinitol was not only the antibacterial principle but also it was one of the major compounds (yielding about 1 % of the fresh weight) from 5. tortile collected at Kata. In contrast, it could not be isolated from those S. tortile collected near Maizuru, regardless of the season. Although seasonal variation of crinitol in S. tortile was not observed, the geographical
36 variation was remarkable. This variation could be caused by genotypic and/or by phenotypic factors. These results seem to be interesting from a chemotaxonomic point of view, since species of Sargassum are often difficult to identify. It should, however, be noted that geographical and seasonal variation among marine natural products seems to be common. Morphologically, it is noticeable that S. tortile grows better around Kata (the Pacific Ocean variety) than Maizuru (the Sea of Japan variety); the water temperature around the latter is cooler than the former. Thus, the algae collected from around Kata were larger than those of Maizuru. The observed geographical variation of crinitol in S. tortile may be caused by an environmental difference, if crinitol is hypothesized to be a defense substance. The water temperature around Kata is warmer than Maizuru, and bacteria at the former location may be more active than at the latter. If this hypothesis is true, then the algae growing around Kata may need more antibacterial agent to combat them. Clearly, further study is needed to confirm this. Based on the above findings, we also collected several other species of Sargassum algae along the coast of the Western United States. For example, S, muticum was collected near Trinidad, California. Although its /i-hexane extract exhibited weak antibacterial activity against B. subtilis, its CH2CI2 extract (from which crinitol was isolated from S. tortile) showed no activity. More specifically, crinitol was not identified in S. muticum at all. CONCLUSION Crinitol's natural role in seaweeds remains unclear, but it can be classified as a nonionic surfactant judging from its chemical structure. Its biological activity suggests that it may possibly exert a chemically protective function. The basis of crinitol* s antibacterial activity apparently derives from its properties as a nonionic surfactant that may disrupt cell membranes. The binding of nonionic surfactants can only involve relatively weak head-group interactions, such as hydrogen bonding, so that the predominant interactions will be hydrophobic (36). However, the specific binding site of crinitol still remains to be established. A wider range of bioassays may be appropriate forjudging possibly useful bioactivity; related substances, for example, have shown cytotoxicity in sea urchin egg development (8) and physiological effects on intestinal contractions (37). If crinitol is to find use as an antibacterial agent, this will probably require use of adjuvants to enhance its activity. Combining two or more substances to enhance the total biological activity may not only be a most efficient way to use renewable natural products, but also the microorganisms may take longer to develop their resistance to two or more toxins
37
whose mode of action is diverse. Factors such as potential human toxicity or irritancy (for which there is no current evidence) would also need to be checked. Conceivable uses of crinitol, as with many other natural products, may simply be defined by availability, physical properties, and the relative ease with which possible options might be explored. Acknowledgement This work was supported in part by NOAA, National Sea Grant College Program, Department of Commerce, under grant number NA85AA-D-SG140, project number R/MP-32, through the California Sea Grant Program, and the California State Resources Agency for financial support. The work has involved a number of scientists. We are greatly indebted to the colleagues cited in the references, especially Dr. T. Matsumoto, Dr. N. Ichikawa and Dr. M. Himejima. S. tortile was collected by Dr. N. Ichikawa and identified by Dr. H. Nakahara. We also thank Gretchen Peterson for assisting in the literature search. REFERENCES 1
2 3
4 5 6 7 8
9
E. Fattorusso, S. Magno, L. Mayol, C. Santacroce, D. Sica, V. Amico, G. Oriente, M. Piattelli, and C. Tringali, Oxocrinol and crinitol, novel linear terpenoids from the brown alga Cystoseira crinita, Tetrahedron Lett., (1976) 937-940. V. Amico, P. Neri, M. Piattelli and G. Ruberto, Linear diterpenoids from Cystoseira baleahca, Phytochemistry, 26 (1987), 2637-2639. L Kubo, T. Matsumoto and N. Ichikawa, Absolute configuration of crinitol, an acyclic diterpene insect growth inhibitor from the brown algae Sargassum tortile, Chem Lett., (1985) 249-252. I. Kubo, Screening techniques for plant-insect interactions, in Methods in Plant Biochemistry (K. Hostettmann, ed.), Academic Press, London, 1991, Vol. 6, pp 179193. E. Fattorusso, Recent results in the chemistry of mediterranean algae, in Marine Natural Products Chemistry (D. J. Faulkner and W. H. Fenical, eds.), Nato Conference Series, Plenum Press, New York and London, 1977, pp. 165-178. M. Piattelli, Chemistry and taxonomy of Sicilian Cystoseira species. New. J, Chem., 14 (1990) 777-782. R. Vails, B. Banaigs, C. Francisco, L. Codomier and A. Cave, An acyclic diterpene from the brown alga Bihrcaria bifurcata, Phytochemistry, 25 (1986), 751-752. R. Vails, B. Banaigs, L. Piovetti, A. Archavlis and J. Artaud, Linear diterpene with antimitotic activity from the brown alga Biiurcaria biAircata, Phytochemistry, 34 (1993), 1585-1588. J. F. Biard, J. F. Verbist, R. Floch and Y. Letoumeux, Epoxyeleganolone et eleganediol, deux nouveaux diterpenes de BiAircaria biAircata Ross (cystoseiracees). Tetrahedron Lett., 21 (1980), 1849-1852.
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10 11 12
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25 26
L. Hougaard, U. Anthoni, C. Christophersen and P. H. Nielsen, Eleganolone derived diterpenes from Bifiircaria blAircata, Phytochemistry, 30 (1991), 3049-3051. R. Vails, V. Mesguiche, M. Pellegrini, L. Pellegrini and B. Banaigs, Variation de la composition en diterpenes de Bifurcaria bifurcata sur les cotes atlantiques frangaises, ActaBot. Gallica, 142 (1995) 119-124. R. Vails, L. Piovetti, B. Banaigs, A. Archavlis and M. Pellegrini, (5)-13Hydroxygeranyl-geraniol-derived furanoditerpenes from Bifurcaria bifurcata, Phytochemistry, 39 (1995), 145-149. T. Wada, K. Kobata, Y. Hayashi and H. Shibata, Two chemotypes of Boletinus cavipes, Biosci. Biotech. Biochejrn., 59 (1995), 1036-1039. N. C. Gonnella, K. Nakanishi, V. S. Martin and K. B. Sharpless, General method for determining absolute configurations of acyclic allylic alcohols, J. Amer. Chem. Soc., 104 (1982) 3775-3776. H. Liu and K. Nakanishi, Additivity relation found in the amplitudes of excitonsplit circular dichroism curves of pyranose benzoates^ J. Amer. Chem. Soc., 103 (1981) 5591-5593. L. R. Smith and I. Kubo, The racemization of crinitol. Resolution of crinitol and of 3nonen-2-ol enantiomers by recycle-HPLC, via MTPA esters, J. Nat. Prod., 58 (1995) 1608-1613. J. A. Dale, D. L. Dull and H. S. Mosher, a-Methoxy-a-trifluoromethylphenylacetic acid, a versatile reagent for the determination of enantiomeric composition of alcohols and amines, J. Org. Chem., 34 (1969) 2543-2549. I. Ohtani, T. Kusumi, Y. Kashman and H. Kakisawa, High-field FT NMR application of Mosher's method. The absolute configurations of marine terpenoids, J. Amer. Chem. Soc., 113 (1991) 4092-4096. A. Horeau and A. Nouaille, Extension et simplification de la methode de determination de la configuration des alcool secondaires par "dedoublement partiel" VIII. Application a la genipine, Tetrahedron Lett., 12 (1971), 1939-1942. B. J. Gung, M. A. Wolf, K. Ohm and A. J. Peat, Conformational preferences of Cloxygenated acyclic chiral alkenes: The effect of vinyl and allyl substituents, Tetrahedron Lett., 34 (1993) 1417-1420. I. Kubo, M. Himejima, K. Tsujimoto, H. Muroi and N. Ichikawa, Antibacterial activity of crinitol and its potentiation, J. Nat. Prod., 55 (1992) 780-785. I. Kubo, H. Muroi and A. Kubo, Antibacterial activity of long-chain alcohols against Streptococcusmutans, J. Agric. Food Chem., 41 (1993), 2447-2450. I. Kubo, H. Muroi and A. Kubo, Structural functions of antimicrobial long-chain alcohols and phenols, Bioorg. Med. Chem., 3 (1995) 873-880. I. Kubo, H. Muroi, M. Himejima and A. Kubo, Antibacterial activity of long-chain alcohols: the role of hydrophobic alkyl groups, Bioorg, Med. Chem. Lett., 3 (1993) 1305-1308. L. F. Fieser and K. L. Williamson, Organic Experiments, D. C. Heath, Lexington, 1979, 4th ed., p. 148. I. Kubo, S. Komatsu, T. Iwagawa and D. L. Wood, Analytical and preparative separation of bark beetle pheromones by high-performance liquid chromatography, J. Chromatogr., 363 (1986) 309-314.
39 27 28 29 30 31 32 33 34 35 36 37
M. Kawakami, A. Kobayashi and K. Katoh, Volatile constituents of Rooibos tea {Aspalathus linearis) as affected by extraction process, J. Agric. Food Chem., 41 (1993) 633-636. H. S. Byun and R. Bittman, Stereospecific synthesis of thioether glycerophospholipids. Catalytic asymmetric epoxidation followed by in situ borohydride-mediated opening of glycidol with hexadecylmercaptan, J. Org. Chem., 59 (1994) 668-671. I. Kubo and M. Taniguchi, Polygodial, an antifungal potentiator, J. Nat. Prod., 51, (1988) 22-29. P. M. Davidson, Phenolic compounds, in Antimicrobials in Foods (A. L. Branen and P. M. Davidson, eds.). Marcel Dekker, New York, 1983, pp. 37-74. C. W. Norden, H. Wenzel and E. Keleti, Comparison of techniques for measurement of in vitro antibiotic synergism, J. Infect. Dis., 140 (1979) 441-443. M. Himejima and I. Kubo, Antimicrobial agents from the Anacardium occidentale (Anacardiaceae) nut shell oil, J. Agric. Food Chem., 39 (1991) 418-421. I. Kubo, H. Muroi and A. Kubo, Naturally occurring antiacne agents, J. Nat. Prod., 57 (1994) 9-17. G. Combaut and L. Piovetti, A novel acyclic diterpene from the brown alga Bifurcaria bifurcata, Phytochemistry, 22 (1983), 1787-1789. L. Semmak, A. Zerzouf, R. Vails, B. Banaigs, G. Jeanty and C. Francisco, Acyclic diterpenes from Bifurcaria biiurcata, Phytochemistry, 27 (1988), 2347-2349. M. N. Jones and D. Chapman, Micelles, Monolayers, and Biomembranes, Wiley-Liss, New York, 1995. F. Delia Pieta, M. C. Breschi, R. Scatizzi and F. Cinelli, Relaxing activity of two linear diterpenes from Cystoseira brachycarpa var. balearica on the contractions of intestinal preparations, Planta Med., 61 (1995), 493-496.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
41
Stereoselective Synthesis of Methylcyclopentanoid Monoterpenes A. Bianco
1. Introduction. 2. Structure and biogenesis of methylcyclopentanoid monoterpenes. 3. Synthesis of methylcyclopentanoid monoterpenes. 3.1. 2-Hydroxy-3-oxobicyclo[4.3.0]non-4-ene-type skeleton. 3.1.1. Synthesis via bicycloeptene derivatives. 3.1.2. Synthesis via bicyclooctene derivatives. 3.1.3. Synthesis via bicyclononadiene derivatives. 3.1.4. Diels-Alder reactions. 3.1.4.1. Intramolecular Diels-Alder reactions. 3.1.4.2. Intermolecular Diels-Alder reactions. 3.1.5. Photochemical cycloadditions. 3.1.6. Nitrile oxide cycloaddition. 3.2. 3-Oxobicyclo[4.3.0]nonan-2-one- or 4-one-type skeleton. 3.2.1. 3.2.2. 3.2.3. 3.2.4.
Tricyclo[3.3.0.0]octanone degradation. Favorskii rearrangement. Homer-Wadsworth-Emmons reaction. Norrish I type reaction.
1. Introduction
The higher plants routinely, rapidly and efficiently carry out syntheses that offer challenging synthetic targets to the greatest organic chemists. The biogenetic processes, starting from the simplest building bricks, create the asymmetric molecules
42
in a relatively simple manner, being the consequence of specific batteries of enzymes. We have the problem to invent something suitable to take the place of enzymes if we wish to prepare these natural products. Methylcyclopentanoid monoterpenes are a group of isoprenoids characterised by the presence in their skeleton of a cyclopentanoid residue. Both hydrophilic and hydrophobic compounds are known ( ref s 1-3 ), which may be both volatile and non volatile. Products of alkaloidal nature (ref 4 ) are also included in this family.
OOCH3
OOCH3
OOCH3
O 0-glc Loganin
CH3 Iridomyrmecin
Nepetalacton
Fig.l The principal groups of natural products belonging to this family of monoterpene derivatives are: glycosidic and non-glycosidic iridoids ( see fig 1, ref s 5-7 ), together with related compounds secoiridoids and bisiridoids, and monoterpene alkaloids ( see fig 2).
OOCH3
Gentianine
Fig. 2
43
Methylcyclopentanoid monoterpenes represent key intermediates of monoterpene metabolism of this type; in fact, starting from this skeleton, the typical biotransformation of the cyclopentanoid residue begins, as an oxidative cleavage. In the case of iridoids, they are ring opened to secoiridoids and are incorporated as monoterpenoid part of important groups of alkaloids: indole, ipecacuanha, cinchona, pyrroloquinoline alkaloids and related compounds (ref 8 ). Another aspect of methylcyclopentanoid monoterpenes is their biological role for the organisms which biosynthesise them. They are in fact often involved in the plant/insect and in the plant/plant biochemical interactions; the biochemical role of these compounds has been extensively studied in recent years and the role of methylcyclopentanoid monoterpenes in plant-animal and plant-plant interactions has been demonstrated (ref 9 ). Methylcyclopentanoid monoterpenes and their metabolites also show several biological activities in man and animals (ref 10 ). Therefore there were many reasons which attracted natural products chemists to take up on the total stereoselective synthesis of this class of isoprenoids In this article the stereoselective syntheses of methylcyclopentanoid monoterpenes are reviewed. The chemical procedures performed for the synthesis of glycosidic and non glycosidic iridoids will be presented.
2. Structure and biogenesis of methylcyclopentanoid monoterpenes
Methylcyclopentanoid monoterpenes are structurally characterised by a (1R,6S) 3oxobicyclo[4.3.0]nonane moiety. Two carbons, belonging to the isoprenoid unit, are generally linked to the 9 and 5 positions. So the basic skeleton is that depicted in fig.3.
44
Fig. 3 The biogenesis of this family of compounds constitutes a separate chapter in the metaboHsm of monoterpenes, and does not involve the carbocationic Ruzika's scheme but consists perhaps of an aldolic condensation-type process on an oxidated derivative of geraniol. The biogenesis of methylcyclopentanoid monoterpenes has been carefully studied allowing the key steps of the process to be depicted (ref s 11-13 ). The methylcyclopentanoid
monoterpenes
arise from
geranyl-PP via an
intramolecular stereocontroUed Mannich type condensation of a dialdehydic or a trialdehydic intermediate, as shown in fig.4. The resulting cyclopentanaldehydic compound has been named iridodial or iridotrial, depending on the nature of the biosynthesized compound and of its biogenetic pathway.
.CHO
OPP
^
k
_
R^^CHO R=CH3 or CHO
Fig.4
— •
1
V—CHO /^CHO R
R=CH3 or CHO
45
Iridodial and iridotrial intermediates can arise by different biogenetic pathways, giving rise to different groups of methylcyclopentanoid monoterpenes. Intramolecular hemiacetalization between an aldehyde group and the enolic form of a second aldehyde, affords the 2-hydroxy-3oxobicyclo[4.3.0]non-4-ene type structure depicted ( fig.5 ).
OR [^\—CHO
R R=CH3 or CHO
R=CH3 or CHO
R=CH3 or CHO R'=H or sugar
Fig. 5 The hemiacetalic C-2 function can be blocked by a sugar residue, affording the methylcyclopentanoid monoterpenes known as glycosidic iridoids. Otherwise the same function can be esterified giving rise to the non-glycosidic iridoids of plumeria or valeriana-type (fig.6).
CH2OAC
oX^-K^o
Isovaleroyl-O'
Valtrate
Fig. 6
Piumericin
46
Intramolecular redox reaction of iridodial or iridotrial intermediates afford two different skeleton of non-glycosidic iridoids: the iridomirmecin-type and the nepetalactone-type (fig.7).
Fig, 7 Reduction of one or both aldehydic functions of iridodial or iridotrial, responsible for the closure of the pyranoid ring, gives rise to the non-glycosidic iridoids, known as iridodiols, generally having only the methylcyclopentanoid moiety (fig.8 ).
OH OH
Fig.8 The iridodial intermediate can also suffer a well known transamination reaction, followed by intramolecular Schiff base formation, giving rise to the formation of 3azabicyclo[4.3.0]nonane-type
skeleton,
typical
of
monoterpene
alkaloids.
47
Heterocyclic ring formation can then lead to a piperidine or a pyridine-type ring system, giving rise to tiie two principal groups of monoterpene alkaloids (fig.9 ).
Fig, 9 In the end, it is necessary to cite, in this biogenetic discourse, the secoiridoid group which is derived from methylcyclopentanoid monoterpenes, even if it does not maintain the characteristic methylcyclopentanoid moiety. Secoiridoids arise, in fact, from the oxidative opening of the cyclopentane ring of both glycosidic or nonglycosidic iridoids by cleavage of the C-8/C-9 bond. hifig.10 is depicted the biogenetic transformation of loganin to secologanin.
COOCH3
COOCH3
COOCH3
OHC •^sC
o-gic
Loganin
Fig. 10
Secologanin
48
Secologanin is probably one of the best known secoiridoids, being the monoterpenic key intermediate in the biogenesis of indole and related alkaloids.
3. Synthesis of methylcyclopentanoid monoterpenes.
Numerous syntheses of methylcyclopentanoid monoterpenes have been reported, but we will restrict our discussion here to a selected number of the more general and stereoselective syntheses of the basic skeletons, giving particular attention to the stereoselective syntheses of optically active forms of these terpenes. hi view of the very different approaches, we prefer to divide the chapter into two topics. hi the first one ( section 3.1. ) we will consider the syntheses of methylcyclopentanoid monoterpenes having a hydroxyl group at the C-2 carbon and a double bond between the C-4 and C-5 carbons ( essentially glycosidic iridoids and related compounds, having both C-2 and C-4 carbons at the same oxidation level). hi the second one ( section 3.2. ) we will present the syntheses of methylcyclopentanoid monoterpenes having the C-2 and C-4 carbons at different oxidation levels (iridomyrmecin and nepetalactone-type compounds).
3.1.2-Hydroxy-3-oxobicyclo[4.3.0]non-4-ene-type skeleton. This skeleton is typical of iridoid glycosides, but several non-glycosidic iridoids, e.g. plumeria-type and valeriana-type compounds, are also characterised by the same skeleton.
49
It can be synthesised via several methods. Some of these employ bicyclic systems as starting material ( see sections 3.1.1. - 3.1.3. ), while others involve the construction of bicyclic iridoidic skeleton via cycloadditions ( see sections 3.1.4. 3.1.6. ). All the procedures lead only to the aglyconic moiety, the insertion of glucose being accomplished always in the final step.
3.1.1. Synthesis via bicycloeptene derivatives. The strategy, pioneered by Vandewalle, involved a completely stereospecific scheme based on the stereoselective fragmentation of precursors having a norbomane-type skeleton ( ref 14 ). The stereoselective synthesis of the suitable norl)omane-type precursors is shown in scheme 1. The norbomene 1, which is the starting materials for the synthesis of loganin depicted in scheme 2, has been obtained from cyclopentadiene and methyl Ecrotonate in classical Diels-Alder conditions, with a high endo-exo stereoselectivity.
^ COOCH3
, COOCH3
O
Scheme 1
2
50
After reduction with lithium aluminium hydride and upon treatment with mcMoroperbenzoic acid, exclusively the endo-dXcohoX cyclized to 2, with yields higher than 80%. Swem oxidation followed by reductive cleavage of the ether bond, afforded the desired norbomanone 3 in a stereoselective manner. Fragmentation of norbomanone-precursors like 3 can be performed by two different strategies, depending on the skeleton desired to be obtained. Fragmentation via Norrish I type reaction afforded
iridomyrmecin and
nepetalactone-type compounds and will therefore be discussed in section 3.2.4. Fragmentation via Baeyer-Villinger oxidation ( ref 15 ) allows access to the 2hydroxy-3-oxobicyclo[4.3.0]non-4-ene-type skeleton and the synthesis of loganin by this approach is reported in scheme 2.
kO^
u COOCH3
COOCH3
COOCH3 MEMO"
-
XHO
HO'"
Scheme 2 Norbomanone 3 has been first transformed into the chetal 4 by Swem oxidation and selective transacetalization with 2-methyl-2-ethyl-dioxolane. Oxidation with m-
51
chloro-perbenzoic acid gave the lactone 5 which has been opened to the methylcyclopentanoid derivative 6. The missing carbon has been introduced on the compound 6, after MEM protection of the hydroxyl fiinction, upon metallation with with a 1:1 mixture of tBuOK-LilCA ( compound 7 ). Simple acid work-up afforded the p-methyl acetal of loganin aglucone 8 which is a known intermediate in the synthesis of loganin.
3.1.2. Synthesis via bicyclooctene derivatives This approach has been first used by Sakan and Abe ( ref 16 ) for the total synthesis of the verbenalin aglycon 19 which confirmed the structure and configuration of the corresponding glucoside, verbenalin. The key bicyclooctene derivative 14 has been prepared in stereoselective manner as depicted in the scheme 3.
AcO
H3C
O
Is, 14
Scheme 3
COOH
O13
H3C
11
COOH
52
The dienone 9 has been stereoselectively methylated, from the less hindered endo face of the molecule, to compound 10 having the methyl group in an a configuration. Hydride reduction afforded to the formation of the alcohol in which the hydroxyl function was disposed in anti configuration with respect to the methyl group, again in consequence of the hydride attack from the less hindered face of the molecule. The secondary hydroxyl group has been then protected by acetylation ( compound 11). Permanganate/periodate oxidation of 11 afforded the diacid 12, having the bicyclooctane structure with the correct relative configuration of the chiral centers. The intermediate 12 has been transformed in acid medium to the lactone 13 which on lead tetraacetate oxidation gave the key bicyclooctene derivative 14.
-^
14
O
COOCH3
0
COOCH3 CHO "CHO
18
17
Scheme 3 contd Compound 14 has been oxidised with osmium tetroxide to the glycol 15 which has been protected as the acetonide 16. Oxidation in basic medium with ruthenium oxide afforded a ketoacid which has been transformed into the corresponding methyl ester 17.
53
The acetonide protecting group has been then removed in acid conditions and subsequent lead tetraacetate oxidation opened the cyclopentane ring because of the vicinal diolic function, furnishing the hemiacetalic structure 18. The compound 18 gave rise to the more stable hemiacetalic structure of verbenalol 19, corresponding to the methylcyclopentanoid monoterpenic structure of verbenalin. A similar strategy has been employed by Vandewalle et al in the total synthesis of verbenalin aglycone ( ref 17 ), but a different approach has been used for the stereoselective synthesis of the bicyclooctene key synthon.
COOCH3
V^^o 20 R=OH 21R=CI
22 R=0 23R=N2
^^
Scheme 4 Starting from 2-cyclopentene-l-acetic acid 20, the intermediate 24 has been constructed through a four step sequence as described in scheme 4. Acid 20 has been transformed to the acid chloride 21 which has been condensed with Meldrums acid to give, after solvolysis with metfianol, the p-keto ester 22. Subsequent treatment with/?-tosylazide gave the diazo compound 23 which has been stereoselectively cyclized to 2-carbomethoxy-tricyclo[3.3.0.0]octan-3-one 24 with copper acetylacetonate as catalyst. Tricycloderivative 24 is the starting product for the synthesis of verbenalol 19 reported in Scheme 5. Solvolysis of 24 in acetic acid and subsequent cyanoborohydride reduction has been performed in one pot to afford the alcohol 25 which, after dehydration, gave a,P-unsaturated ester 26 and successively the key compound 27, after methanolysis and Jones oxidation.
54
Selenenylation in the a-position to the carbonyl of 27 with subsequent direct oxidation, gave the enone 28. Double bond migration of the unsaturated ester function of 28 has been performed via allyUc bromination and subsequent reduction with zinc, yielding, in a satisfactory stereocontrolled way, the compound 29. Subsequent conjugate addition with dimethylcopperlithium yielded the desired isomer 30 with good stereoselectivity.
COOCH3
AcO
COOCH3
COOCH3
AcO
O 25
24
O
COOCH3
q
29
26
C00CH3
28
27
\ O
COOCH3
COOCH3
30
Scheme 5 The double bond of 30 has been cleaved with ozone and successive acid work-up of the reaction mixture afforded in one step the verbenalin aglycone 19.
55
3.1.3. Synthesis via bicyclononadiene derivatives This procedure has been used essentially for the total synthesis of genipin which has been described by Buchi et al ( ref 18 ) and which allowed the confirmation of the structure of this compound as well as the stereochemical assignments. The procedure is depicted in scheme 6 and starts from the bicyclononadiene derivative 31, prepared by thermal decomposition of the ethyldiazoacetate adduct of cyclooctatetraene ( ref 19 ), which has been reduced with lithium in ammonia to the bicyclononadiene derivative whose methylester 32 is the starting product.
COOH
COOCH3
31
32
OOOCH3
r
COOCH3
r
HO'
C00CH3 N
HO^.^"'^^^-^
^N.^
CHO
CHO
OH
OH
36
33
35
34
Scheme 6 Osmium tetroxide oxidation of 32 gave a mixture of the isomeric tetraols 33 whose vicinal diolic function on the cyclopentane ring has been regioselectively cleaved with one mole of lead tetraacetate to give the dihaldehydic derivatives 34 which have been isolated as lactols 35. Periodate cleavage of the isomeric lactols 35 afforded the dihaldehyde 36.
56 COOCH3
COOCH3
COOCH3
OHC
Scheme 6 contd Regioselective intramolecular aldolic condensation of 36 with piperidinium acetate gave only the compound 37 with the desired 2-hydroxy-3-oxobicyclo[4.3.0]non-4ene-type skeleton. The regioselectivity of this reaction can be explained on the basis of a less hindered
transition
state
which
corresponds
to the
formation
of
the
methylcyclopentanoid derivative 37. (+)-genipin 38 has then been prepared by a regioselective reduction of the free aldehyde function of the intermediate 37 with lithium tri-/-bythoxyaluminium hydride.
3.1.4. Diels-Alder reactions The Diels-Alder reaction has been used for the synthesis of the iridoidic skeleton, starting from both acyclic (described in section 3.1.4.1.) and cyclic (descibed in section 3.1.4.2.) intermediates.. 3.1.4.1. Intramolecular Diels-Alder reactions. This approach has been proposed by Tietze et al which constructed a suitable acycKc intermediate shown in scheme 7. By means of an intramolecular Diels-Alder
57
reaction ( ref. 20 ) the direct preparation of the iridoidic-type skeleton has been described as depicted in scheme 7.
OCH3
Scheme 7 3.1.4.2. Intermolecular Diels-Alder reactions An intermolecular Diels-Alder reaction has been also employed starting from a suitable cyclic diene and the iridoidic skeleton has been prepared in a stereoselective manner (ref 21) as shown in scheme 8.
40
cCf41
39
H I
4r° 42
R=H or COOCH3 R', R"= Alkyl
H I
OR"
Hr° OR" 43
Enantiomeric mixture
Scheme 8 The cyclopentene-carbaldehyde 39 reacts with the enol-ether 40 to form the adduct 41, having the skeleton of 3-oxobicyclo[4.3.0]non-l-ene. An alcohol has been then regioselectively and stereoselectively added (compound 42 ) at the double bond from
58
the less hindered p side of the molecule. Finally a regioselective acid catalysed elimination of an alcohol afforded the 2-alkoxy-3-oxobicyclo[4.3.0]non-4-ene moiety ( compound 43), characteristic of methylcyclopentanoid monoterpenes. The reaction can be performed using the enol-ether 40 with both hydrogen or carbon
substituents; depending
on the
employed
enol, the 2-alkoxy-3-
oxobicyclo[4.3.0]non-4-ene-type skeleton may be prepared in substituted or unsubstituted forms at 5 position.
3.1.5. Photochemical cycloadditions The
photochemical
addition
of
fimctionalized
cyclopentenes
and
tricarbonylderivatives has been also used for the preparation of iridoidic skeleton, via a bicylo[3.2.0]heptane intermediate, as shown in the scheme 9.
o
H
HsCOOC^
OH
COOCH3
cti
COOCH3
COOCH3
H I
H
44
Scheme 9 This procedure is an adaptation of a photoannulation reaction, originally developed by de Mayo ( ref 22 ) and leads initially to the stereoselective formation of a cyclobutanol ring (intermediate 44 ) which undergoes a spontaneous ring opening to the typical iridoidic dialdehyde ( intermediate 45 ) which is theoretically in equilibrium with the lactol iridoidic moiety ( intermediate 46 ), as depicted in the scheme 9. The first two syntheses of loganin aglycone 58 have been performed in a similar manners by Buchi, Tietze et al ( see scheme 10-13, ref 23 ) and by Partridge et ah.
59
( see scheme 14, ref. 24 ). They differ in the structure of the starting cyclopentene derivative. Buchi started from 3-cyclopentenol 47 which is photochemically added to the enol double bond of 2-formyhnalonaldehyde 48 in a stereoselective way (scheme 10 ).
H3COOC
THPO-^
1
V ^
COOCH3
>=0 THP
OH
47
48
COOCH3 THPi
50 and 50a
Scheme 10 The hemiacetalic iridoidic intermediate 49 obtained has been successively transformed into the two corresponding enantiomers methylacetals 50 and 50a and the secondary alcoholic function on the cyclopentane ring oxidised to a carbonyl group (enantiomeric mixture 51 and 51a obtained). The missing one carbon unit is introduced at this stage utilising the reactive aposition to the carbonyl and different procedures have been employed, generally in relation to the desired iridoid. In the case of the first synthesis of loganin ( see scheme 11 ) the ketonic intermediate 51 ( as an enantiomeric mixture ) has been formylated in high regioselective way to the aldehyde 52 as the major product, owing to the steric
60 hindrance constituted by the carbomethoxyl group which is more important than the hindrance of methoxyl group in consequence of the structural constitution of the molecule. Aldehyde 52 has been successively derivatized with w-buthyhnercaptan to produce the thiomethylene derivative 53.
COOCH3
COOCH3
COOCH3
o=rtS
OHC^H, 51
._„ n-BuS"'
. . . / ^^^ ^H3 53
52 Major products
Enantiomeric mixture
COOCH3
COOCH3 COOCH3
H3C
OCH3 55R=H 56 R=Ts
^^sC
54
0CH3
0CH3 54 and 54a
COOCH3
COOCH3
COOCH3
H T
H T
AcO'
AcO' H3C
0CH3 57
>- AcO--<
I
^sC
0-glcAc4 59
Scheme 11 Desulphurization and reduction of 53 produced a mixture of epimeric ketones 54 and 54a which have been transformed into the thermodynamically more stable ketone 54 via sodium methoxide treatment.
61 As a general rule the substituents on the cyclopentane ring in 3 configuration in this type of molecules suffer a lower steric hindrance with the consequent major thermodynamic stability for compounds having substituents in this absolute configuration. The introduction of a secondary hydroxyl function on the cyclopentane ring in the desired syn configuration with respect to the methyl, has been performed in three steps, firstly by reducing the ketone function with sodium borohydride. The more stable anti hydroxyl ( compound 55 ) has been obtained and, to invert its configuration, it has been esterified with p-toluensulfonate ( compound 56 ), and substituted, in bimolecular nucleofilic substitution conditions, with acetate (compound 57). The classical glucosylation with 2,3,4,6-tetraacetylglucose, afforded the acetate of loganin 59, via the aglycone 58. A simple modification of this procedure has been performed by Tietze (ref 25-28) as shown in scheme 12. COOCH3
COOCH3
COOCH3
H I
HO-
')nr. HOOC
H i.
OCH3
HOOC
60 and 60a
0CH3
61 and 61a /
COOCH3
AcO (I
I
HOH2C
OCH3
63 Scheme 12
COOCH3
62
The procedure has been utiUsed to synthesise the hydroxyloganin, the key ring intermediate in the metabolism of iridoids to secoiridoids and subsequently to indole and related alkaloids having this monoterpenic part. The difference in the procedure consists essentially in the different introduction of the missing one carbon unit, at the same stage after the photochemical addition, utilising methyhnagnesium carbonate. The ketone 51, prepared as an enantiomeric mixture as described in scheme 9, is regioselectively carboxylated in the a position to give the p-ketoacid 60 which, without any manipulation, is immediately stereoselectively reduced to the Phydroxyacid 61, with sodium borohydride. Tietze proposed a thermodynamic control in the high regioselectivity of carboxylation of 51 and a steric control, as consequence of convex geometry of molecule, in the stereoselectivity of reduction of 60. Resolution of the enantiomeric mixture can be performed at this step, after acetylation of the secondary hydroxyl, by separation of diastereoisomeric salts 62 and 62a with 1-phenylethylamine. The carboxyl group of the desired enantiomer of 62 is then reduced to a hydroxymethyl group in the intermediate 63 which can afford hydroxyloganin 72 ( ref 25-28 ) or 7-epihydroxyloganin 68 ( ref 25, 28 ) following two different procedures as depicted in scheme 13. Diacetate 64 is transformed into the hemiacetal 66 by a mixture of acetic and perchloric acid in water. Glucosylation of 66 with 2,3,4,6-tetra-O-acetyl-P-D-glucose in the presence of boron trifluoride afforded the hexaacetate of 7-epihydroxyloganin 67 and, after alkaline hydrolysis, the corresponding iridoid 68.
63
A\
COOCH3
COOCH3
RO"'
/
ROH2C
AcO"
HT
ACOH2C
0CH3
64 R=Ac 65 R=Ms
COOCH3
RO' "
^^
.„rtS
n ROH2C
AcO—<
I
I
OH^r ACOH2C
H i _ . OCH3 69
—
COOCH3 AcO-
w.r ACOH2C
0-glcR4
67 R=Ac 68R=H
66
COOCH3
HT
COOCH3
RO—<
Hi.. 70
ROH2C
I 0-glcR4 71 R=Ac 72R=H
Scheme 13 For the synthesis of hydroxyloganin 72, the same starting intermediate, as dimesylate 65, is treated with tetraethylammonium acetate to invert the configuration at C-7. Hydrolysis of acetal as before allowed to prepare the hemiacetal 70, and successive glucosylation afforded hydroxyloganin hexaacetate 71 and thereafter hydroxyloganin 72. An improvement in the glucosylation procedure has been performed by Tietze ( ref 29 ) which utilized P-trimethylsilyl-2,3,4,6-tetraacetylglucose which reacted with the acetylated hemiacetalic fimction in the presence of catalytic amounts of trimethylsilyl trifluoromethanesulfonate in acetonitrile or liquid sulphur dioxide at -30 or -50 T respectively.
64
Loganin acetate 59 has been rapidly obtained by Partridge et al.X scheme 14, ref. 24 ) starting from enatiomerically pure (-)-(lS,2R)-2-methyl-3-cyclopentenyl acetate 76.
H3COOC AcO'
H3C-<^
H3C 73
COOCH3
AcO-Y
I
^sC
I 0-glcAc4
59
48
76
74R=H 75 R=Ms
COOCH3
AcO—<
I
H3C
OH 58
Scheme 14 The optically pure starting material has been prepared from the prochiral 5methylcyclopentadiene 73 by hydroboration with (+)-bis(cis-pinan-trans-3-yl)borane. (-)-(lR,2R)-2-methyl-3-cyclopentenol
74 obtained has been mesylated (
compound 75 ) and the desired (-)-(lS,2R)-2-methyl-3-cyclopentenyl acetate 76 has been prepared by displacement with acetate on heating with tetraethylammonium acetate. This olefin undergoes photoannelation in regioselective and stereoselective manner with methyl-diformyl acetate 48 to produce directly the loganin aglycone derivative 58 in about 20% yield. Classical treatment with 2,3,4,6-tetra-O-acetyl-D-glucose in anhydrous conditions, in the presence of boron trifluoride etherate, afforded loganin pentaacetate 59.
65
A further utilisation of the Patemo-Buchi enone photoannulation reaction has been performed by Chaudhuri et al ( ref 30 ) who, by employing 5-(trimethylsilyl)-l,3cyclopentadiene 77 in acetonitrile at -50 °C ( see scheme 15 ), obtained in a regioselective way only the photoadduct 78, by reaction with methyl-diformyl acetate 48.
H3COOC
MeaSi
-O
H ?°°^"'
MeaSi
48
77
78
COOCH3
R
MeaSi
u 9^^^"3
H 0CH3 L.
80 R=CH3C0
79
Scheme 15 The intermediate 78 is stabilized by acetalization with methanol ( compound 79 ) and the introduction of missing carbon can be easily performed in stereoselective way by allylic displacement of the silyl group with acetyl chloride or other reagents (compound 80).
66
3.1.6. Nitrile oxide cycloaddition The Claisen rearrangement - nitrile oxide cycloaddition sequence has been adapted to the synthesis of methylcyclopentanoid monoterpenes by Curran et al (ref. 31-32 ). This reaction sequence is in fact a useful, stereocontroUed annulation procedure in a carbocyclic ring system which the authors demonstrated to be applicable to the synthesis of heterocyclic systems.
o ^ CHsOOC^ . k . ^O R
OEt 82
83
OMs
N
O
OEt 86
85
^ 87
Scheme 16
88
84
67
The starting glycal 81 has been prepared by Ferrier rearrangement of D-xylal; the acetalic P-anomer has been employed in the synthetic procedure. As shown in scheme 16, the nature of substituted acetic acid which esterifies the secondary hydroxyl depends on the target structure. freland-Claisen rearrangement of 81, followed by methylation, afforded the intermediate 82 as a single stereoisomer. DIBAL reduction to the aldehyde and successive WoUemberg homologation afforded the key nitro-olefin 83. The nitrile oxide cycloaddition has been performed on 83 under Mukaiyama conditions giving the isoxazoline 84 in about 50% yields with high stereocontrol of the annulation reaction. The
2-hydroxy-3-oxobicyclo[4.3.0]nonane-type
skeleton,
typical
of
methylcyclopentanoid monoterpenes, has been then constructed in a stereospecific and regiospecific manner. Successive steps can be designed according to the target molecular moiety. In the case depicted in scheme 16, nickel-Raney reduction of 84 provided the Phydroxyketone 85 which can be easily transformed to the desired skeleton by mesylation (compound 86) and successive treatment with DBU. The 2-hydroxy-3-oxobicyclo[4.3.0]non-5-ene-type structure ( compound 87 ) has been obtained which in turn has been equilibrated in the same conditions to the thermodynamically more stable 2-hydroxy-3-oxobicyclo[4.3.0]non-4-ene-type one ( compound 88). The reported model scheme had shown that Claisen rearrangement-nitrile oxide cycloannulation represented an excellent procedure for the stereoselective synthesis of 2-hydroxy-3-oxobicyclo[4.3.0]non-4-ene-type skeleton differently substituted on the cyclopentane ring.
68
3.2.3-Oxobicyclo[4.3.0]nonan-2-one- or 4-one-type skeleton.
Different approaches have been proposed for the stereoselective synthesis of methylcyclopentanoid
monoterpenes
oxobicyclo[4.3.0]nonan-2-one-type)
with and
nepetalactone-type iridomirmecin-type
skeleton
(3-
skeleton
(3-
oxobicyclo[4.3.0]nonan-4-one-type). We will divide the discussion on the basis of the key reactions used in the synthetic strategy.
3.2.1. Tricyclo[3.3.0.0]octanone degradation. 2-Carbomethoxy-tricyclo[3.3.0.0]octan-3-one 24, obtained as described in section 3.1.2., has been utilized as a chiral starting material for the preparation of isoiridomirmecin 92 (ref 17, scheme 17).
H3C
COOCH3
COOCH3
O 89
24
H3C OSi{CH3)3 CH3 91
Scheme 17
90
69
As shown in scheme 17, compound 24 has been methylated in a stereocontroUed way by reaction with methyl iodide in the presence of lithium diisipropylamide, affording the derivative 88. Reaction of 88 with dimethylcopperiithium afforded, again in a stereocontroUed way, the ketoester 89. The hydrolysis of ester function of ketoester 89 led to the decarboxylation, affording a single ketone 90. The kinetic enolate derived from ketone 90 has been trapped as the trimethylsislyl derivative 91. Ozonolysis of 91, followed by reductive work-up with sodium borohydride, afforded directly the isoiridomyrmecin 92.
3.2.2. Favorskii rearrangement. The starting idea of this approach arises from the knowledge that fiinctionalized cyclopentanecarboxylates can be obtained from the Favorskii rearrangement of cyclohexanone derivatives (ref 33 ). (+)-carvone 93 has been chosen as the starting cyclohexanone derivative and it has been transformed into the chlorohydrin derivative 95 via the epoxide intermediate 94, as described in scheme 18.
THPO.
^
o 93
Scheme 18
94
95
70
The monoepoxide 94 has been obtained by oxidation of (+)-carvone 93 with hydrogen peroxide in alkaline medium (NaOH/MeOH) for 1 h at room temperature. Treatment of 94 with chlorotrimethylsilane in acetonitrile/dimethylsulphoxide opened the epoxide affording, in high regioselective yields, the corresponding chlorohydrin which has been transformed, without isolation, to its tetrahydropyranyl ether 95. When 95 has been treated with sodium methoxide in methanol at room temperature, the Favorskii rearrangement occurred in a stereoselective manner and the cyclopentanecarboxylate 96 has been obtained in high yields as described in scheme 19.
THPO. THPO'
CI "tY
'COOCH3
o
95
96
Scheme 19 In schemes 20 and 21 are depicted the strategies for the stereoselective transformations
of the key intermediate 96 in the methylcyclopentanoid
monoterpenes with iridomyrmecin- and nepetalactone-type skeleton. For the synthesis of dihydronepetalactone 99, the intermediate 96 has been manipulated as depicted in scheme 20. The key step is represented by the stereoselective hydroboration of 96 with disiamylborane and by the successive oxidation, which led to the formation of the free primary alcoholic function of the intermediate 97. The stereoselectivity of hydroboration is due to the attack of the bulk borane from the less hindered (3-side of
71
the molecule and allowed to have the methyl group in the intermediate 97 in the required a-configuration.
THPO'
THPOCOOCH3
96
97
H \
HO'
99
n? 98
Scheme 20 The hydrolysis of 97 in alkaline conditions, followed by acid treatment, allowed the intramolecular lactonization, affording the hydroxylactone 98. Barton-type deoxygenation of 98 yielded the expected (+)-dihydronepetalactone 99. Alternatively ( see scheme 21 ), the same key intermediate 96 can be transformed into the iridomyrmecin 102. The compound 96 has been transformed into the acetate 100 by reduction with lithium aluminium hydride, followed by acetylation of the free primary hydroxyl and successive deprotection of the secondary hydroxyl. Barton-type deoxygenation of 100, with the same procedure previously described in scheme 20, gave the acetate 101 which is a known intermediate in the synthesis of iridomyrmecin 102.
72
THPO' COOCH3
96
H f
OAc 102
101
Scheme 21
3.2.3. Homer-Wadsworth-Emmons reaction. The Homer-Wadsworth-Emmons reaction is an important methodology used in the synthesis of natural products, but side reactions in many cases do not allow the application of this reaction in stereoselective synthesis. However, Nangia et al ( ref 34 ) recently described, by using the HomerWadsworth-Emmons reaction in a controlled way, the stereoselective synthesis of boschnialactone as depicted in scheme 22. R-pulegone 103 is known to give rise to a rearrangement product 104 as a mixture ofsyn and anti isomers in which (approximately 6:4) the syn one prevails.
73
C00CH3
105
104
103
/ OEt
107
106
Scheme 22 Lithium aluminium hydride reduction of the mixture 104, followed by a chromatographic purification, allowed the preparation of the alcohol 105 as pure stereoisomer. Ozonolysis in buffered conditions, using dichloromethane and sodium hydrogen carbonate, afforded the p-hydroxy alcohol 106 which has been esterified with diethylphosphonoacetic acid in the presence of DCC to the key cyclopentanoid derivative 107. The ester 107 has been submitted to the Homer-Wadsworth-Emmons reaction, using diisopropyl ethyl amine as the base in the presence of lithium chloride. Under these conditions, after 16 h the lactone 108 has been obtained in high yields in a regioselective way, without (3-elimination and a-epimerization.
74
II .OEt ^OEt
^ 0 107
108
109
Scheme 22 contd Highly stereoselective hydrogenation of 108 from the less hindered exo face of the molecule, afforded boschnialactone 109. The authors also showed the possibility, by employing suitable starting products, such as S-pulegone, to obtain iridomyrmecin, isoiridomyrmecin and teucriumlactone with the same Homer-Wadsworth-Emmons reaction.
3.2.4. Norrish I type reaction. Norbomanone-type compounds can be fragmented by a Norrish reaction giving with high stereoselectivity iridomyrmecin-like compounds. Vandewalle et al ( ref 15 ) have demonstrated an alternative route which starts from the norbomanone 3, whose synthesis has been described in scheme 1, and from its epimer 110 which has been prepared in a similar manner as reported in scheme 23. The well known endo adduct 111 of cyclopentadiene and maleic anhydride is the starting material for the preparation of nomomanone 110. The compound 111 has been reduced to the diol 112. Treatment with mchloroperbenzoic acid, in the same conditions previously described in scheme 1 for the preparation of 3, afforded the diol 113.
75
As in the case of the preparation of 3, a cyclization occurred between the two syn primary and secondary alcohoUc fiinctions, with formation of an additional heterocyUc ring.
Hcr-^
111
-f
'
-
^
HO
no
113
Scheme 23 Regioselective tosylation of the primary free alcohoHc function of 113, followed by reductive removal of the tosylate, Swem oxidation of secondary free alcoholic function and reductive cleavage of the ether bond of the tetrahydrofurane ring, gave the required norbomanone 110. Norrish I type reaction has been carried on the norbomanone-type compound 3 and on its epimer 110 (see scheme 24 ). The described procedure allowed to obtain, with satisfactory yields, the lactols 114 and 115, in which the required 4-hydroxy-3-oxobicyclo[4.3.0]nonanane-type skeleton has been prepared in a single step and in a satisfactory stereocontrolled manner. Depending on the required configuration of the methyl group on the cyclopentane ring, the lactols 114 and 115 have been manipulated as depicted in scheme 24.
76
OH
H3C
3 p-methyl llOa-methyl
9^
H3C
116
114 p-methyl 115a-methyl
ccr
H3C
109
Scheme 24 The lactol 115, after oxidation of hemiacetalic fiinction and saturation of the double bond, gave boschnialactone 109. The lactol epimer 114, on introduction of the a-methylene group by means of the Grieco's method, has been transformed into teucriumlactone 116.
References 1. J.M.Bobbitt and K.P.Segebarth, The iridoid glycosides and similar substances in, W.I.Taylor and A.R.Battersby (Ed), Cyclopentanoid terpene derivatives. Marcel Dekker hic. New York, 1969. 2. A.Bianco, The Chemistry oflridoids in, Atta-ur-Rahman (Ed), Studies in Natural Products Chemistry, Elsevier, 1990, vol. 7.
77
3. G.W.K.Cavill Insect terpenoids and nepetalactone, in, W.LTaylor and A.R.Battersby (Ed), Cyclopentanoid terpene derivatives. Marcel Dekker Inc., New York, 1969. 4. G.A.Cordell, Monoterpene alkaloids, in, R.H.Manske (Ed), The alkaloids. Academic Press, New York, 1977, vol. 16. 5. L.J.El Naggar and J.L.Beal, J. Nat Prod., 43, 649 (1980). 6. C.A.Boros and F.R.Stermitz, J. Nat. Prod., 53, 1055 (1990). 7. C.A.Boros and F.R.Stermitz, J. Nat. Prod., 54, 1055 (1991). 8. M.Luckner, Secondary metabolism in microorganism, plants, and animals, Springer-Verlag, Berlin 1990. 9. J.B.Harbome, Introduction to ecological Biochemistry, Academic Press, London, 1989. 10. H.Inouye, Iridoids, in Methods in Plant Biochemistry, Academic Press, London 1991, vol. 7. 11. S.Damtoft, Phytochemistry, 11, 1929 (1983). 12. F.Bellesia, R.grandi, U.M.Pagnoni, A.Pinetti and R.Trave, Phytochemistry, 23, 83 (1984). 13. H.Inouye and S.Uesato, Prog. Chem. Org. Nat. Prod., 50, 169 (1986). 14. P.Callant, H.De Ville and M.Vandewalle, Tetrahedron, 37, 2079 (1981). 15. P.Callant, P.Storme, E.Van der Eycken and M.Vandewalle, Tetrahedron Letters, 24, 5797 (1983). 16. T.Sakan and R.Abe, Tetrahedron Letters, 2471 (1968). 17. P.Callant, R.Ongena and M.Vandewalle, Tetrahedron, 37, 2085 (1981). 18. G.Buchi, B.Gubler, R.S.Schneider and J.Wild, J. Am. Chem. Soc, 89, 2776 (1967). 19. K.F.Bangert and V.Boekelheide, J. Am. Chem. Soc, 86, 905 (1964). 20. L.F.Tietze and T.Eicher, Reaktionen und Synthesen im organisch-chemischen Praktikum, Thieme, Stuttgart 1981, p.364. 21. L.F.Tietze, Chem. Ber., 107, 2491 (1974). 22. P.de Mayo, Ace. Chem. Res., 4, 41 (1971).
78
23. G.Buchi, J.A.Carlson, J.E.Powell and L.F.Tietze, J, Am, Chem. Soc, 92, 2165 (1970). 24. J.J.Partridge, N.K.Chadha and M.R.Uskokovic, J, Am. Chem, Soc, 95, 532 (1973). 25. L.F.TiQtzQ.Angew. Chem. Int. Ed Engl., 22, 828 (1983). 26. L.¥.TiQtzQ,Angew. Chem. Int. Ed. Engl., 12, 757 (1973). 27. L.F.Tietze, Chem. Ber., 107, 2499 (1974). 28. A.R.Battersby, N.D.Westcott, K.H.Glusenkamp and L.F.Tietze, Chem. Ber., 114, 3439(1981). 29. L.F.Tietze, R.Fischer and G.Remberg, LiebigsAnn. Chem., 971 (1987). 30. R.K.Chaudhuri, T.Ikeda and C.R.Hutchinson, J. Am. Chem. Soc, 106, 6004 (1984). 31. D.P.Curran, P.B.Jacobs, R.L.Elliot and B.H.Kim, J. Am. Chem. Soc, 109, 5280 (1987). 32. B.H.Kim, P.B.Jacobs, R.L.Elliot D and. P.Curran, Tetrahedron, 44, 3079 (1988). 33. E.Lee and C.H.Yoon, J. Chem. Soc. Chem. Commun., 479 (1994). 34. A.Nangia, G.Prasuma and P.B.Rao, Tetrahedron Letters, 35, 3755 (1994).
Current Address Professor Armandodoriano Bianco Dipartimento di Chimica - Universita La Sapienza Piazzale Aldo More 5,1-00185 Roma (Italy) Fax 39 6 490631 E-mail
[email protected]
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
79
Chemical Constituents of Taxus Species V.S. Parmar and A. Jha 1. INTRODUCTION The genus Taxus belongs to the family Taxaceae and consists of much branched trees or shrubs. Taxaceae is a family of five genera: Amentotaxus Pilger, Austrotaxus Compton, Pseudotaxus Cheng, Taxus Linn, and Torreya Am (1). Various species of this genus are also trivially termed as yew and are specified by the region of their occurrence, e.g. Himalayan yew, European yew, Canadian yew, etc. They are evergreen shrubs or trees. The wood of Taxus is one of the heaviest of soft woods. It is also mechanically strong and durable. Its timber is oily and irregular growth rings in the wood provide decorative value. Different Taxus species have proved to be of special significance among phytochemists in recent years because they have been found to possess a number of biological activities. Taxus baccata is a poisonous plant (all parts except the fleshy aril) and has been recorded as a fish poison. Its leaves possess tremendous medicinal properties in Unani system of medicine: they are antispasmodic and emmenagogue and are used for epilepsy and as lithontriptic (2). A tincture made from young shoots has been in use for diarrhoea and severe biliousness. Vohora and Kumar in 1971 reported that the water extract of the leaves oiTaxus baccata possesses good tranquilizing activity (3). The chemical constituents of different Taxus species have been studied for over a hundred years. Taxane alkaloids, diterpenoids with taxane skeleton, lignans, biflavonoids, steroids, sugar derivatives and diterpenes possessing tropone skeleton are commonly found to occur in this genus. The phytochemistry of the genus Taxus has evoked a great deal of interest because of the occurrence of antitumour and antileukemic taxanes, molting hormones, alkaloids, antineoplastic diterpenes and other physiologically important compounds in its various species, in particular because of the importance of taxol/paclitaxel (Taxol is a registered trademark of Bristol-Myers Squibb Co., hence the generic name paclitaxel should be used instead), the world known new antitumour drug. This has made Taxus as one of the most intensely investigated genus of all the plant genera. A few reviews have appeared in the literature (4-7) on this subject while the one that covered maximum information on taxoids, the typical secondary metabolite of the yew tree was published in 1993. Phytochemistry of Taxus species, being a hot topic, a number of reports on the isolation of newer metabolites poured in thereafter. The latest review (5), to our knowledge, covers the literature on the subject from 1987 to mid 1995 and at least fifty new compounds have been reported from the species Taxus after that. There is no updated comprehensive review that enlists all the isolates from various Taxus species. Our present review covers the literature upto June 1996 listing all chemical constituents isolated from different species of Taxus giving due emphasis to the biological activities possessed by them.
80
2. TAXANE DERIVATIVES Taxanes are typical cyclic diterpenoids. A normal taxane (I) contains an eight membered ring sandwiched between two six membered rings, but there are numerous compounds which have rearranged taxane skeleta. 3,11-Cyclotaxanes (11) are tetracyclic systems with an additional bond between C-3 and C-11. Taxanes having ring system III are called 11(15—> 1) aZ?eotaxane. They have linearly fused five, seven and six membered rings. A six/ten/six membered ring system (FV) is known as 2(3—>20) abeoiaxants. An 11(15—>1), 11 (10—>9)bisabeoi^x?ine having skeleton V is also known. Compounds with skeleton VI are referred to as pretaxanes as the bond between C-3 and C-8 is not yet formed.
x < \
IV
VI
2.1 Normal Taxanes The naturally occurring diterpenoids with the taxane skeleton are the typical constituents of Taxus. The constituents of the yew became the active subject of phytochemical and synthetic work since the discovery of terpenes belonging to the taxane group (8-10). Roberto and Jelmoni (11) reported a case of poisoning following the ingestion of berries of T. baccata. The alkaloid taxine is the toxic constituent that occurs in the leaves and berries, while the aril is free of alkaloids. Taxine is in fact a mixture of at least 11 compounds, only five of which [taxine A (229) and B (70), 13-deoxo-13a-acetyloxytaxine B (30), 13deoxo-13a-acetyloxy-l-deoxytaxine B (31) and 13-deoxo-13a -acetyloxy-1-deoxy-nor-taxine B (35)] have been structually characterized so far. The action of the alkaloid was only apparent after ingestion of the berries when the latter were chewed. The leaves of T. cuspidata
81
and T. floridana also tested positive for alkaloids (12). The symptoms of taxine poisoning are mainly associated with cardiac and respiratory disturbances and death results from heart failure, accompanied by respiratory paralysis (13). The unusual diterpene, taxol (116) (14) was reported in 1971 by Wani etal. (15) as the major cytotoxic, antineoplastic (16) and antileukemic constituent occurring in yew (1719). It was found to possess confirmed activity in the L-1210, P-388 and P-1534 leukemia, the B-16 melanoma and the Walker 256 carcino-sarcoma m vivo assays. It is speculated that to some extent the activity of taxol may be due to the easily cleaved allylic a -hydroxy ester function at C-13 which may be capable of acting as a leaving group under physiological conditions. Taxol is an interesting drug in that it appears to act by a mechanism that is different from other known anticancer drugs; it has been recognized as a strong antimitotic agent (20). This important biological activity has been related to the in vitro inhibition by taxol and some of its derivatives of the depolymerization process of tubulin. It acts by promoting microtubule formation (21-23) by decreasing the lag time for microtubule assembly and shifting the equilibrium in favour of the microtubule (24). Taxol blocks the processes, essential for prostate tumour cell (PC-3ML) invasion and metastasis (25). The taxol content in various parts of several Taxus species have been determined (26-28). The activity of taxagifine (82) is lesser than that of taxol (ID^^ 5 x lO^M, cf. 10^ for taxol) (29). The taxane esters 10-deacetyltaxol (114), 7-e/7i-10-deacetyltaxol (120), 10deacetylcephalomannine (135) and 7-epr-lO-deacetylcephalomannine (138) have KB activities (ED^Q, g/ml) 2.7X10-2, 3.4x10% 3.1 X 10- and 4.6X10-2, respectively (30). 19Hydroxybaccatin III (104) was inactive in vivo in the PS mouse lymphocytic leukemia system at the doses tested (0.26-4.3 mg/kg). The greater water solubilities of these more polar taxane derivatives {Le. 114 and 135) may help to circumvent in vivo toxicities of the parent compounds and could show some clinical advantages over taxol and cephalomannine (30). The KB and P388 activities of cephalomannine (134) are roughly comparable to those of taxol (116). Cephalomannine gave typical T/C values in the range 152-180 at dose levels of 1-3.3 mg/kg against PS, while taxol showed T/C values of 148-152 in the dosage range 1.4-2.2 mg/kg. Both these compounds showed cytotoxicity (ED^^) against KB cell cultures at 10'^ g/ml, whereas baccatin III (102) had an ED^^ of 2.0 g/ml and 16-hydroxybaccatin I (92) had an ED^^ of 2.9 g/ml (31). Some of the taxoids isolated recently have been demonstrated to be equally or in some cases more potent anti-tumour agents than taxol. Taxol C (129), isolated from the bark of T.media possesses antitumour activity (e.g. IL^^ against L1210 murine leukemia cells of 3.6±0.3 nM) higher than that of taxol (e.g. 57.0±3.0 nM) and is also active against adriamycin resistant cells (32). Taxol C possesses selective cytotoxicity in the NCI human cell line screen (33). 10Deacetyltaxol C, A^-methyltaxol C (130) and taxol D (133) also showed activity close to that of taxol in the tubulin assembly assay (32,33). Table 1 lists the different taxanes isolated thus far from different Taxus species.
82
TABLE 1 Normal taxane derivatives isolated from different Taxus species. Compound
Species
References
Taxa-4(20),ll-diene-5a,9a,10p,13a-tetraol (1) T. haccata
(34,35)
16,2a,5a,9a,106,13a-Hexaliydroxytaxa4(20),ll-diene (2)
T. chinensis
(36,37)
Taxusin (3)
T. baccata T, hrevifolia T. cuspidata T. floridana T. mairei T. yunnanensis
(38,39,40) (39) (41,42) (39) (43-46) (47)
Taxa-4-(20),ll-diene-5a,76,9a,106,13apentaol pentaacetate (4)
T. haccata T. mairei
(38) (43,48,49)
Taxa-4-(20),ll-diene-2a,5a,9a,106,13apentaol pentaacetate (5)
T. baccata T. mairei
(38) (48)
Taxa-4-(20),ll-diene-2a,5a,76,9a,106,13ahexaol hexaacetate (6)
T. baccata
(38)
2-Deacetyl-5-decinnamoyltaxinine J (7)
T. canadensis T. wallichiana T. yunnanensis
(50,51) (52) (53)
Taxa-4(20),ll-diene-5a-hydroxy16,76,9a,106-tetraacetate (8)
T. baccata
(40)
5-Decinnamoyltaxinine J (9)
T. brevifolia T. yunnanensis
(54) (53)
2-Deacetoxydecinnamoyltaxinine J (10)
T. baccata T. wallichiana
(55) (56)
Decinnamoyl-l-hydroxytaxinine J (11)
T. baccata
(55)
2-Deacetyldecinnamoyltaxinine E (12)
T. baccata
(55)
Taxa-4(20),ll-diene-5a,9a,106,13a-tetraol9a,106-diacetate (13)
T. baccata T. mairei
(38) (43,48,49)
83
2a,5a,9a-Trihydroxy-10p,13a-diacetoxytaxa-4(20),ll-diene (14)
T. chinensis
(57)
16,5a-Dihydroxy-9a, 106,13ot-triacetate2a-benzoate taxa-4(20),ll-diene (15)
T. chinensis
(36,37)
16,5a, 13a-Trihydroxy-9a, 106-diacetate-2abenzoate taxa-4(20),ll-diene (16)
T. chinensis
(36,37)
Taxa-4(20),ll-diene-2a,5a,76,9a,106pentaol 7a,9a, 106-triacetate-2a-amethylbutyrate (17)
T. haccata
(38)
Taxa-4-(20),ll-diene-2a,5a,76,106-tetraolT. baccata 5a,7p,10p-triacetate-2a-a-methylbutyrate(18) T. mairei
(38) (48,49)
Taxa-4(20),ll-diene-lp,2a,5a,19-tetraol9a,10p,13a-triacetate (19)
T. chinensis
(58)
Taxinine E (20)
T. cuspidata T. mairei T. yunanensis
(59) (48) (60)
Taxinine J (21)
T. cuspidata T. mairei T. yunanensis
(59) (61,62) (47,60)
2a-Deacetoxytaxinine J (22)
T. baccata T. cuspidata T. mairei T. wallichiana T. yunnanensis
(40) (63) (61) (52,56,64) (47)
2a,13a-Dihydroxy-9a, 106-diacetoxy5a-cinnamate taxa-4(20),ll-diene (23)
T. chinensis
(58,65)
106-Hydroxy-2a,9a,13a- triacetoxy5a- ciimamate taxa-4(20),ll-diene (24)
T. chinensis
(58,65)
9a,106,13a-Triacetoxy-5a-cinnamoyltaxa-4(20),ll-diene (25)
T. chinensis T. cuspidata T. mairei
(65) (63) (66)
l-Hydroxy-2-deacetyltaxinine J (26)
T. wallichiana
(52)
2a,9a-Dihydroxy-106,13a-diacetoxy5a-cinnamate taxa-4(20),ll-diene (27)
T. media
(67)
84
2a, lOfi-Dihy droxy-9a, 13a-diacetoxy5a-cinnamate taxa-4(20),ll-diene (28)
T. media
(67)
16,13a-Dihydroxy-9a, 106-diacetate2a-benzoate-5a-cinnamatetaxa-4(20), 11-diene (29)
T. chinensis
(36,37)
13-Deoxo-13a-acetyloxytaxine B (30)
T. haccata
(68)
13-Deoxo-13a-acetyloxy-l-deoxytaxine B (31)
T. baccata
(68)
2'-Desacetoxyaustrospicatin (32)
T. baccata T. cuspidata T. wallichiana
(70) (63,71) (72)
7,2'-Didesacetoxyaustrospicatin (33)
T. cuspidata T. wallichiana
(32) (72,69)
Taxuyunnanine D (34)
T. yunnanensis
(47)
13-Deoxo-13a-acetyloxy-l-deoxy-nortaxine B (35)
T. baccata
(68)
2a-Acetoxy-2'-deacetyl-l-hydroxyaustrospicatine (36)
T. baccata
(55)
Taxuspine P (37)
T. cuspidata
(73)
Taxuyunnanine C (38)
T. chinensis T. yunnanensis
(77) (47)
Taxuyunnanine G (39)
T. yunnanensis
(74,76)
Taxuyunnanine H (40)
T.yunnanensis
(76)
Taxuyunnanine I (41)
T. yunnanensis
(76)
Taxuyunnanine J (42)
T. yunnanensis
(76)
2a,5a, lOfi-Triacetoxy- 146-propanoy 1oxy-4(20), 11-taxadiene (43)
T. chinensis
(77)
2a,5a,106-Triacetoxy-146-(2'-methyl)propanoyloxy-4(20),ll-taxadiene (44)
T. chinensis
(77)
Taiwanxan (45)
T. mairei
(78,79)
85
Taxuyunnanine B (46)
T. yunnanensis
(47)
2a,5a,106-Triacetoxy-146-(2'-methyl)butanoyloxy-4(20), 11-taxadiene (47)
T. chinensis T. cuspidata
(77) (42)
Taxa-4(20),ll-cliene-106-methoxy2a,5a-diacetoxy- 146-a-methyIbutyrate (48)
T. haccata
(40)
Yunnanxane (49)
T. chinensis T. yunanensis
(77) (60,80)
Taxinine A (50)
T. chinensis T. cuspidata
(81) (63,82,83)
76-Acetoxytaxinine A (51)
T. mairei
(48)
Taxinine H (52)
T. cuspidata
(84)
2,10-Di-O-acetyl-5-decinnamoyltaxicin I (53)
T. baccata
(55)
Triacetyl-5-decinnamoyltaxicin I (54)
T. baccata
(55)
Taxuspine F (55)
T. cuspidata
(32)
Taxuspine G (56)
T. cuspidata
(32)
9a,10p-Diacetyltaxicin I (57)
T. cuspidata
(85)
5-Cinnamoyltaxincin I triacetate (58)
T. cuspidata
(63)
10-Deacetyltaxinine B (59)
T. cuspidata
(83)
2-0-Acetyl-5-0-cinnamoyltaxicin I (60)
T. baccata
(86)
Taxinine (61)
T. T. T. T.
(87,88) (81) (63,71,75,83,84,89-91) (43)
Taxinine B (62)
T. cuspidata
(59,63,71,75,83,89,91)
5-Cinnamoyl-9,10-diacetyltaxicin I (63)
T. baccata
(92)
5-Cinnamoyl-9-acetyltaxicin-I (64)
T. baccata
(93)
5-Cinnamoyl- 10-acetyltaxicin-I (65)
T. baccata
(93)
5-Cinnamoyl-9-acetyltaxicin-II (66)
T. baccata
(93)
canadensis chinensis cuspidata mairei
86
5-Cinnamoyl-10-acetyltaxicin-II(67)
T. haccata
(93)
l,9a-Dihydroxy-2a,106-diacetoxyT. media 5a-cinnamoyltaxa-4(20),ll-diene-13-one(68)
(67)
l,106-Dihydroxy-2a,9a-diacetoxyT. media 5a-cinnamoyltaxa-4(20),ll-diene-13-one(69)
(67)
Taxine B (70)
T. haccata
(94,95)
Taxinell (71)
T. haccata T. cuspidata
(96) (63,89)
Taxine I (72)
T. haccata
(95,97,98)
2,9-Dideacetyltaxine II (73)
T. haccata
(99)
9-Deacetyltaxine I (74)
T. haccata
(99)
2,10-Dideacetyltaxine II (75)
T. haccata
(99)
10-Deacetyltaxine I (76)
T. haccata
(99)
5-Decinnamoyltaxagifine (77)
T. canadensis T. chinensis
(50,51) (100)
5-Decinnamoyl-5-acetyltaxagifine(78)
T. chinensis
(100)
Taxinine M (79)
T. hrevifolia T. cuspidata
(101) (83)
2-Deacetyl-5-decinnamoyltaxagifine (80)
T. canadensis T. chinensis
(50) (65)
19-Debenzoyl-19-acetyltaxinine M (81)
T. wallichiana
(102)
Taxagifine (82)
T. haccata T. cuspidata
(29,35) (83,103)
Taxacin (83)
T. cupsidata
(83,103)
5-Decinnamoyl-ll-acetyl-19-hydroxytaxigifine (84)
T. yunnanensis
(53)
Taxuspine L (85)
71 cuspidata
(104)
TaxchinA (86)
T. chienensis
(105,106)
Taxchin B (87)
T. chienensis
(105,106)
87
5a,76,9a,106,13a-Pentaacetoxy-2abenzoyloxy-4a,20-dihydroxytax-l 1-ene (88)
T. mairei
(107)
7B,9a,106,13a,20-Pentaacetoxy-2abenzoyloxy-4a,5a-dihydroxytax-ll-ene (89)
T. mairei
(107)
Taxumairol A (90)
T. mairei
(108)
Baccatin I (91)
T. baccata
(34,109)
16-Hydroxybaccatin I (92)
T. T. T. T.
(109) (110) (48) (31,64)
16-Hydroxy-7-deacetylbaccatin I (93)
T. baccata T. brevifolia T. media
(111) (112,113) (114)
ip-Hydroxy-7,9-dideacetylbaccatin I (94)
T. canadensis T. chinensis T. mairei
(51) (115) (108)
5-Deacetylbaccatin I (95)
T. baccata T. yunanensis
(109) (116)
16-Acetoxy-5,7,10-deacetylbaccatin I (96)
T. canadensis
(50,117)
16-Hydroxy-2,5,7,10-tetradeacetylbaccatin I (97)
T. chinensis
(58)
l-Acetoxy-5-deacetylbaccatin I (98)
T. mairei T. yunanensis
(108,118) (60)
l-Hydroxy-2,7,9-trideacetylbaccatin I (99)
T. yunnanensis
(53)
16,9a-Dihydroxy-46,20-epoxy-2a,5a,76,106, 13a-pentacetoxy-tax-ll-ene (100)
T. brevifolia
(112)
76-Acetoxy-9-acetylspicataxine(101)
T. media
(119)
Baccatin III (102)
T. baccata T. brevifolia T. cuspidata T. yunanensis
(34,43,111,120-123) (124,125) (19) (60,126)
Baccatin V (103)
T. baccata
(127,128)
baccata chinensis mairei wallichiana
19-Hydroxybaccatin III (104)
71 haccata T. chinensis T. wallichiana T. yunanensis
(35) (36) (30) (116)
10-Deacetylbaccatin III (105)
T. haccata T. brevifolia T. canadensis T. cuspidata T. wallichiana T. yunanensis
(122,129-131) (54,111,125,132,133) (50,51) (83) (52,134) (116,135)
9-Hydroxy-lO-deacetylbaccatin III (106)
T. haccata
(136)
1-Acetyl-lO-deacetylbaccatin III (107)
T. canadensis
(50)
l-Dehydroxybaccatin III (108)
T. yunanensis
(116)
7-epi-lO-Deacetylbaccatin III (109)
T. canadensis
(51)
7-Xylosyl-lO-deacetoxybaccatin III (110)
T. yunnanensis
(53)
19-Hydroxy-7-e/7i-baccatin III (111)
T. chinensis
(137)
13-e;?i-10-Deacetylbaccatin III (112)
T. haccata
(123)
2-Debenzoyl-2-tigloyl-10-deacetylbaccatin III (113)
T. haccata
(123)
10-Deacetyltaxol (114)
T. haccata T. hrevifolia T. canadensis T. cuspidata T. wallichiana T. yunnanensis
(35) (124,125) (51) (83) (30) (135,138)
106-Hydroxybutyrate-lO-deacetyltaxol A (115^) T. haccata
(111)
Paclitaxel [Taxol (116)]
(15,17,26,35,111, 122,130,139,140) (124,125,132,141,142) (50,51) (143,144) (15,17,19,26,63,83,91) (17,26) (31,52,134) (60,126,138,145,146)
T. haccata T. hrevifolia T. canadensis T. chinensis T. cuspidata T. media T. wallichiana T, yunnanensis
89 7-Xylosyl-lO-deacetyltaxol A (117)
T. baccata T. yunanensis
(111) (60)
7"Xylosyltaxol A (118)
T. baccata
(111)
7-epi-Taxo\ (119)
T. brevifolia T. canadensis
(147) (51)
7-ep/-10-Deacetyltaxol (120)
T. yunnanensis
(135)
lO-Deacetyltaxol-7-xyloside (121)
T. baccata T. brevifolia T. wallichiana T. yunnanensis
(70) (125,132) (52) (60)
7-Xylosyltaxol (122)
T. brevifolia T. yunnanensis T. wallichiana
(132) (53) (52)
10-Deacetyltaxol-2',7-diacetate(123)
T. brevifolia
(54)
Taxol-2',7- diacetate (124)
T. brevifolia
(54)
Yunnanxol (125)
T. yunnanensis
(145)
7-Epitaxuyannanine A (126)
T. yunnanensis
(138)
7-Epi-lO-deacetyltaxuyannanine A (127)
T. yunnanensis
(138)
10-Deacetyltaxuyannanine A (128)
T. yunnanenis
(138)
Taxol C (129)
T. media T. yunnanensis
(148) (149)
A^-Methyltaxol C (130)
T. media
(148)
7-Xylosyltaxol C (131)
T. baccata
(111)
7-Xylosyl-lO-deacetyltaxol C (132)
T. baccata T. wallichiana
(70,111) (52)
A^-Debenzoyl-A^-butanoyltaxol [Taxol D (133)] T. media
(119)
Cephalomannine (134)
(35,120,122,150) (124,125,132,151,152) (50,51) (19,83)
T. T. T. T.
baccata brevifolia canadensis cuspidata
90 T. wallichiana T. yunnanensis
(31,52,134) (60,126,138,145)
10-Deacetylcephalomannine (135)
T. haccata T. brevifolia T. cuspidata T. wallichiana T. yunnanensis
(35) (124) (83) (30) (135,138)
lO-ft-Hydroxybutyrate-lO-deacetyltaxol B (136)
T. haccata
(111)
7-ep/-Cephalomanine (137)
T. media
(153)
7-e/7/- 10-Deacetylcephalomannine (138)
T, yunnanensis
(135)
7-Xylosyl-lO-deacetyltaxol B (139)
T. haccata
(HI)
7-Xylosyltaxol B (140)
T.haccata
(111)
lO-Deacetylcephalomannine-7-xyloside (141)
T. hrevifolia
(132)
146-Hydroxy-lO-deacetylbaccatin III (142)
T. wallichiana
(154)
2-Debenzoyl-146-benzoyloxy10-deacetylbaccatin III (143)
T. wallichiana
(155)
10-Deacetyl-lO-oxo-baccatin V(144)
T. chinensis
(137)
lO-Deacetyl-lO-dehydro-7-acetyltaxol A (145) T. media
(114)
10-Deacetyl-10-oxo-7-e;?f-taxol(146)
T. hrevifolia
(147)
7 -Xylosyl-10-deacatyltaxol D (147)
T. haccata
(70)
10-Deacetyl- 10-oxo-7-e/7/taxuyannanine A (148)
T. yunnanensis
(138)
Baccatin IV (149)
T. T. T. T.
(34,121) (113) (61) (64)
Baccatin VI (150)
T. haccata
(111,121)
l-Dehydroxybaccatin IV (151)
T. haccata T. mairei
(121) (44,49,61)
haccata hrevifolia mairei wallichiana
91
1-Acetylbaccatin IV (152)
T. yunanensis
(116)
4-Deacetylbaccatin IV (153)
T. media
(119)
l-Dehydroxy-4-deacetylbaccatin IV (154)
T. mairei
(118)
7,9-Deacetylbaccatin IV (155)
T. brevifolia T. canadensis
(113) (51)
1-Dehydroxybaccatin VI (156)
T. mairei
(62)
9-Dihydro-13-acetylbaccatin III (157)
T. brevifolia T. canadensis T. chinensis
(113) (50,51,156) (115)
lO-Hydroxyacetylbaccatin VI (158)
T. canadensis
(51)
7,9,10-Trideacetylbaccatin VI (159)
T. canadensis
(51)
7-e'/7/-9,10-Trideacetylbaccatin VI (160)
T. canadensis
(51)
Taxuspine E (161)
T. cuspidata
(32)
9p-//-9-Dihydro-19-acetoxy-10-deacetylbaccatin III (162)
T. baccata
(86)
9,10-Dideacetyl-9,10-dibenzoylbaccatin VI (163)
T. brevifolia
(157)
10-Deacetyl-lO-benzoylbaccatin VI (164)
T. brevifolia
(157)
7-Deacetyl-7-benzoylbaccatin VI (165)
T. brevifolia
(157)
Baccatin VII (166)
T. baccata
(121)
Taxuspine N (167)
T. cuspidata
(73)
Yunnanxamine (168)
T. yunnanensis
(145)
Taxagifine III (169)
T. chinensis
(65)
4-Deacetyltaxagifine-III (170)
T. chinensis
(65)
Taxuspine K (171)
T. cuspidata
(104)
92
1
R-|=R3=R4=R5~OH, R2=R6=R7=H
2
R-|=R3=R4=R5=R6=R7=OH, R2=H
3
Ri=R3=R4=R5=OAc, R2=R6=R7=H
4
Ri=R2=R3=R4=R5=OAc, R6=R7=H
5
R-|=R3=R4=R5=R7=OAc, R2=R6=H
6
Ri=R2=R3=R4=R5=R7=OAc, R6=H
7
Ri=R7=0H, R2=R3=R4=R5=OAc, R6=H
8
Ri=OH, R2=R3=R4=R6=OAc, R5=R7=H
9
Ri=OH, R2=R3=R4=R5=R7=OAc, R6=H
10 Ri=OH, R2=R3=R4=R5=OAc, R6=R7=H 11 R-,=R6=0H, R2=R3=R4=R5=R7=OAc 12 Ri=R7=0H, R2=R6=H, R3=R4=R5=OAc 13 Ri=R5=0H, R2=R6=R7=H, R3=R4=OAc 14 R-,=R3=R7=OH, R2=R6=H, R4=R5=OAc 15 Ri=R6=0H, R2=H, R3=R4=R5=OAc, R7=0Bz 16 Ri=R5=R6=OH, R2=H, R3=:R4=OAc. R7=0Bz 17 Ri=OH, R2=R3=R4=OAc, R5=R6=H, R7=OCOCH(CH3)C2H5 18 Ri=R2=R4=OAc, R3=R5=R6=H, R7=OCOCH(CH3)C2H5
21
Ri =:R5=H, R2=R3=R4=R6=0AC Ri =:R2=R3=R4=R6=OAc, R5=H
22
Ri =:R2=R3=R4=0AC, R5=R6=H
23
Ri =:R5=H, R2=R3=OAc, R4=R6=OH
20
24 Ry- :R5=H, R2=R4=R6=OAc, R3=0H 25
Ri =:R5=R6=H, R2=R3=R4=OAC
Ry. :R2=R3=R4=OAc, R5=R6=OH 27 Ry. :R5=H, R2=R6=OH, R3=R4=OAc 28 Ry ;R5=H, R2=R4=OAc, R3=R6=OH 29 Ry :H, R2=R3=OAc, R4=R5=OH, R6=0Bz 26
93 AcO
AcO
.OH
R3
R2
Ri
AcO\^^^* R5 Re 30 Ri=H, R2=R5=R6=OH. R3=R4=OAc 31 Ri=R5=H, R2=R6=OH, R3=R4=OAc 32 Ri=R2=R3=R4=OAc, R5=R6=H 33 Ri=R5=R6=H, R2=R3=R4=OAc
19
DAc
AcO\^^^^
AcO.
OH
34
Aca 35 AcO AcO
oAc O
^ H ^
AcO
AcO
OAc OAc
AcO OAc 37
94 R3
R2
38 Ri :R3P=R4: R5=0Ac, R2=H 39 Ri :R5=0AC, R2=H, R3ft=R4=OH 40 Ri =R3p=0H R2=H, R4=OCOC2H5,R5=OAc 41 Ri =R3p=0H R2=H, R4=OCOCH(CH3)2. R5=0Ac 42 Ri =OAc, R2 R5=H, R3p=R4=OH 43 Ri =R3p=R5=|OAc, R2=H, R4=OCOC2H5 44 Ri =R3p=R5='OAc, R2=H, R4=OCOCH(CH3)2 45 Ri OH, R2:R3p=R5=OAc, R4=OCOCH(CH3)C2H5 46 Ri R2=R3p= R5=0Ac. R4=OCOCH(CH3)C2H5 47 Ri R3p=R5=OAc, R2=H, R4=OCOCH(CH3)G2H5 48 Ri R5=0AC , R2=H, R3p=OCH3. R4=OCOCH(CH3)C2H5 49 Ri :R3P=R! '5=OAc, R2=H, R4=OCOCH(CH3)CH(OH)CH3 R4
R5
50 51 52 53 54 55 56 57
R3
R2
^6
Ri=OH, R2=R5=H, R3=R4=R6=OAc Ri=OH, R2=R3=R4=R6=OAc, R5=H Ri=R3=R4=R6=OAc, R2=R5=H Ri=R3=R5=OH, R2=H, R4=R6=OAc Ri=R5=0H, R2=H, R3=R4=R6=OAc Ri=OH. R2=R3=R4=R6=OAc, R5=H Ri=R6=0H, R2=R5=H, R3=R4=OAc Ri=R5=R6=OH, R2=H, R3=R4=OAc
95 R3
R2
58 59 60 61 62 63 64 65 66 67 68
Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri
69
Ri = H, R2=R5=OAc, R3=R4=OH
R2
70 71 72 73 74 75 76
H, R2=R3=R5=OAc, R4=0H R2=R5=OAc, R3=0H, R4=H H, R2=R3=R4=OH, R5=0Ac R4=H. R2=R3=R5=OAC R2=R3=R5=OAc, R4=H H, R2=R3=OAc, R4=R5=OH H, R2=0Ac, R3=R4=R5=OH H. R2=R4=R5=OH, R3=0Ac R4=H, R2=0Ac, R3=R5=OH R4=H, R2=R5=OH, R3=0Ac H, R2=R4=OH. R3=R5=OAc
Ri
Ri=R2=R3=OH, R4=0Ac Ri=R2=R4=OAc, R3=H Ri=R2=R4=OAc, R3=0H Ri=R4=0H. R2=0Ac, R3=H Ri=R3=0H. R2=R4=OAc Ri=OAc, R2=R4=OH, R3=H Ri=R4=0Ac, R2=R3=OH
77 R i = R 4 = 0 H , R2=H, R3=0Ac 78 Ri=R3=0Ac, R2=H. R4=0H 79 Ri=R4=0H, R2=0Bz, R3=0Ac 80 Ri=R3=R4=OH, R2=H 81 Ri=R4=0H, R2=R3=OAc 82 Ri=OCOCH:CHPh, R2=H, R3=0Ac, R4=0H 83 Ri=OCOCH:CHPh, R2=0Bz, R3=0Ac, R4=0H 84 Ri=R2=0H, R3=R4=OAc
96 85 Ri :R4=0Ac, R2=H, R3=0H 86 Ri O H , R2=H, R3=R4=OAc 8 7 R i =:R4=0Ac. R2=H, R3=0C0CH:CHPh 88 Ri :OBz, R2=R3=OH, R4=0Ac 89 Ri :OBz, R2=R4=OH, R3=0Ac 90 Ri :R2=0H, R3=0Bz, R4=0Ac
91 92 93 94 95 96 97 98 99
OAc OAc
AcQ
AcO^^^^"
=R2=R3=R4=R5=R7=OAc, R6=H =R2=R3=R4=R5=R7=OAC, R6=0H =R3=R4=R5=R7=0AC, R2=R6=OH =R4=R5=R7=OAC, R2=R3=R6=OH =0H,
R2=R3=R4=R5=Fl7=OAc, R6=H
=R2=R4=OH, R3=R5=R6=R7=OAC =R2=R4=R6=R7=OH, R3=R5=OAc =0H,
Rs^^^"
R2=R3=R4=R5=R6=R7=OAc
=R4=R5=0AC, R2=R3=R6=R7=OH
100 R =R2=R4=R5=R7=OAc, R3=R6=OH 101 R =OCOCH2CH(Ph)NMe2. R2=R3=R4=R5=R7=OAc. R6=H
102 R
=R4^=R5=OH, R2=H, R3=0AC
103 R
=R4a=R5=OH. R2=H, R3=0Ac
104 R
=R2=R4a=R5=OH, R3=0AC
105 R
=R3=R4^^=R5=OH, R2=H
106 R
=R2=R3=R4a=R5=OH
107 R
=R3=R4a=OH, R2=H, R5=0AC
108 R
=R4„=0H, R2=R5=H, R3=0Ac
109 R
=R3=R4ot=R5=OH, R2=H
110 R
=axylosyl, R2=R3=H, R4„=R5=OH
111 R
= R2=R4a=R5=OH, R3=0AC
112
=R3=R4p=R5=OH, R2=H
R
R.
HO^^^
\
OH t)
113
OAc
t)Bz OAc
97
114Rip=R2=OH, R3=0Bz 115 Rip=OH. R2=OCOCH2CH(OH)CH3. R3=0Ac 116 Rip=OH, R2=0Ac, R3=0Bz 117 Rip=axylosyl, R2=0H, R3=0Ac 118 Rip=0-xylosyl, R2=R3=0Ac 119 Ri«=OH, R2=0Ac, R3=0Bz 120 Ri„=R2=0H. R3=0Bz 121 Rip=axylosyl, R2=0H, R3=0Bz 122 Rip=axylosyl, R2=0Ac, R3=0Bz R
AcO
P
OAc
OH t)Bz OAc
123 R=OH 124R=OAc
OH t)Bz OAc
125
126 Ria=OH, R2=0Ac, R3=H 127 Ri^=R2=0H, R3=H 128 Rip=R2=0H, R3=H
C51
129 Rip=OH, R2=0Ac, R3=H 130 Rip=OH, R2=0Ac, R3=CH3
Ph-
131 Rip=0-xylosyl, R2=OAc, R3=H 132 Rip=0-xylosyl, R2=OH, R3=H
OH t)Bz OAc
98 Acq
o
OH R3
OAc
134 Rip=OH. R2=0Ac, R3=0Bz 135 Rip=R2=0H, R3=0Bz 136 Rip=OH, R2=OCOCH2CH(OH)CH3, R3=0Ac 137 Ria=OH, R2=0Ac, R3=0Bz 138 Ri„=R2=0H. R3=0Bz 139 Rip=0-xylosyl, R2=OH, R3=OAc 140 Rip=0-xylosyl, R2=R3=0Ac 141 Rip=0-xylosyl. R2=OH, R3=OBz
142 Rip=R3=R4=R5=R6=OH. R2=H, R7=0Bz 143 Rip=R3=R4=R6=R7=OH. R2=H, R5=0Bz 144 Rip=R4=R6=OH, R2+R3=0, R5=H 145 Rip=OAc, R2+R3=0, R4=0C0CH(0H)CH(Ph)NHBz, R5=H, R6=0H, R7=0Bz 146 Ria=OH. R2+R3= =0, R4=0C0CH(0H)CH(Ph)NHBz, R5=R6=H, R7=0Bz 147 Rip=0-xylosyl. R2=R5=H, R3=R6=OH. R4=0C0CH(0H)CH(Ph)NHC0C3H7, R7=0Bz 148 Ria=OH. R5=R6=H, R2+R3= =0. R4=OCOCH(OH)CH(Ph)NHCOC5Hii, R7=0Bz
99 R4
149 150 151 152 153 154 155 156
Ra
=R2p=R3=R4=R5=R7=OAc, R6=0H, R8=H =R2p=R3=R4=R5=OAc, R6=0H, R7=0Bz. R8=H =R2p=R3=R4=R5=R7=OAC, R6=R8=H =R2p=R3=R4=R5=R6=R7=OAC, R8=H =R6=0H, R2p=R3=R4=R5=R7=OAc, R8=H = 0 H , R2p=R3=R4=R5=R7=OAc, R6=R8=H =R4=R5=R7=0AC, R2p=R3=R6=OH, R8=H =R2p=R3=R4=R5=OAc, R6=R8=H, R7=0Bz
157
R =R4=R5=OAc, R2p=R3=R6=OH, R7=0Bz, R8=H
158 159 160 161 162 163 164 165 166 167 168
R =R2p=R3=R5=OAc, R4=R6=OH, R7=0Bz, R8=H =R5=0Ac, R2a=R3=R4=R6=OH, R7=0Bz, R8=H =R5=0Ac, R2p=R3=R4=R6=OH, R7=0Bz, R8=H =R2p=0Ac, R3=R4=R5=R6=OH, R7=0Bz, R8=H =R8=0Ac, R2p=R3=R4=R5=R6=OH. R7=0Bz =R2p=R5=OAc, R3=R4=R7=OBz. R6=0H, R8=H
R R R R R
=R2p=R3=R5=OAc, R3=R7=OBz, R6=0H, R8=H =R3=R4=R5=0AC, R2p=R7=OBz, R6=0H, R8=H =R6=0H, R2p=R3=R4=R5=OAc, R7=OCOC5Hii, R8=H =R2p=R3=R7=OAc. R4=0Bz, R5=OCOCH2CH(Ph)NMe2, R6=0H, R8=H =R4=0Ac, R2p=R3=R6=OH, R5=0C0CH(0H)CH(Ph)NHBz, R7=0Bz, R8=H
Acq
g>Ac
AcO^^^'
HO^^^"
OH
169 R=OAc 170 R=OH
O
171
oAc
100
2.2 3,11 'Cyclotaxanes 3,11-Cyclotaxanes (Table 2) can be formed upon irradiation of corresponding 13-oxotaxa-11-enes (63,86,158). However, such naturally occurring compounds have^"- geometry in the cinnamoyl side chain, while the irradiated products are a mixture of E- and Zdiastereoisomers. Thus, it is difficult to infer that these compounds are artefacts. TABLE 2 3,11-Cyclotaxanes isolated from different Taxus species. Compound
Species
Taxinine K (172)
T. cuspidata
(82)
Taxinine L (173)
T. cuspidata
(82)
5-Cinnamoyl-lO-acetylphototaxicin I (174)
T. baccata
(158,159)
Taxuspine C (175)
T. cuspidata
(63)
References
9-0-Acetyl-5-0-Cinnamoylphototaxicin I (176) T. baccata
(86)
Taxuspine H (177)
(32)
172 173 174 175 176 177
T. cuspidata
Ri=OH, R2=R3=R5=OAc, R4=H Ri=R2=R3=R5=OAc, R4=H Ri=OCOCH:CHPh, R2=R4=R5=OH, R3=0Ac Ri=OCOCH:CHPh, R2=R3=R5=OAc, R4=H Ri=OCOCH:CHPh, R2=0Ac, R3=R4=R5=OH Ri=OCOCH2CH(NMe2)Ph, R2=R3=R5=OAc, R4= :H
2.3 11(15—>l)-Abeotaxanes A number of taxoids isolated from different Taxus species, on structural reinvestigation were found to contain a rearranged nortaxoid skeleton or 11(15—>iyabeo taxoids, as they are more correctly described (133,160,161) and not a taxane skeleton as originally proposed. Brevifoliol (181) was the first natural taxoid with 11(15—>iyabeoiaxant skeleton which was initially assigned a normal taxane skeleton (162) but was later corrected to the
101
11(15—>l)-a6eotaxane skeleton (57,133,163). In a similar way a number of taxoids whose basic structure, based upon their spectroscopic data were previously reported as derivatives of originally proposed structure of brevifoliol with the normal taxane skeleton or by considering them as baccatin VI derivatives have now been proposed to be rearranged 11(15—>l)-abeotaxanes (mainly by HMBC spectrum and X-ray studies). Taxchinin A (189) was the first naturally occurring taxoid to be correctly assigned the 11(15—>l)-flbeotaxane skeleton (164) and that with an oxetane ring was taxchinin B (220) (137). However, the fact that the first example of a taxoid with 11(15—>l)-afee(9taxane skeleton was a rearrangement product of taxol (19) raises the question as to whether all these compounds are simply artefacts. Although this possibility cannot be completely ruled out, the fact that the rearranged taxoids are being obtained from different species by different research groups, coupled with the fact that rearrangement of taxol requires rather vigorous conditions, suggests that some, if not all of the isolated nortaxoids are genuine natural products. Table 3 contains 11(15—>l)-a6eotaxanes isolated so far from various species of yew. TABLE 3 11(15—>l)-A/?eotaxanes isolated from different Taxus species. Compound
Species
References
Taxchinin D (178)
T. chienensis
(105,106)
Taxchinin E (179)
T. chienensis
(106,165)
Taxchinin G (180)
T. chienensis
(105,106)
Brevifoliol (181)
T. hrevifolia T. wallichiana
(125,133,162) (134)
13-Acetylbrevifoliol (182)
T. hrevifolia T. wallichiana
(133) (64)
9-Deacetyl-9-benzoyl-10-debenzoylbrevifoliol (183)
T. hrevifolia
(160)
10-Debenzoyl-2a-acetoxybrevifoliol (184)
T. wallichiana
(102)
7-Deacetoxy-lO-debenzoylbrevifoliol (185)
T. wallichiana
(161,166)
10-Debenzoylbrevifoliol (186)
T. wallichiana
(161,166)
2a-Acetoxy-7-deacetoxy-10-debenzoylbrevifoliol (187)
T. haccata
(86,161)
9-Deacetyl-9-benzoyl-10-debenzoyltaxichinA(188)
T. haccata
(131)
102 Taxchinin A (189)
T. baccata T. chinensis T. wallichiana
(98,161,167) (137,164) (64,134)
106-Benzoyloxy-16-hydroxy-5a-(3'-dimethyl- T. brevifolia amino-3'-phenylpropanoyloxy-76,9a,13atriacetoxy-ll(15—>l)-fl^eotaxa-4(20),lldiene (190)
(133)
106-Benzoyloxy-16-hydroxy-5a-(3'-methylamino-3'-phenyl)-propanoyloxy-76,9a,13atriacetoxy-ll(15—>l)-aZ?eotaxa-4(20),lldiene (191)
T. brevifolia
(133)
Taxuspine A (192)
T. brevifolia T. cuspidata
(133) (63)
Taxchinin H (193)
T. chienensis T. wallichiana
(106,165) (102)
Taxuspine J (194)
T. cuspidata
(32)
Taxuspine M (195)
T. cuspidata
(104)
Taxacustone (196)
T. cuspidata
(85)
Taxuspine 0 (197)
T. cuspidata
(73)
5 -0-(6-£)-Glucopyranosyl)106-benzoyltaxacustone (198)
T. cuspidata
(85)
Taxayuntin G (199)
T. yunnanensis
(168)
Yunantaxusin A (200)
T. yunnanensis
(169)
TaxuchinA(201)
T. chinensis
(110)
Taxacustin (202)
T. cuspidata T. wallichiana
(83) (52)
7,9,10-TrideacetylflZ7eobaccatin VI (203)
T. baccata
(92)
Taxayuntin (204)
T. baccata T. cuspidata T. wallichiana T. yunnanensis
(170) (83) (52) (145,171,172)
103
7,13-Dideacetyl-9,10-didebenzoyltaxchinin C (205)
T. brevifolia
(160)
4-Deacetyl-9,10-didebenzoyl-9,10diacetyltaxchinin C (206)
T. media
(72)
10-Debenzoyltaxchinin C (207)
T. media
(72)
10-Debenzoyl-lO-acetyltaxchinin C (208)
T. media
(72)
Taxchinin K (209)
T. chienensis
(106,165)
Taxchinin I (210)
T. chienensis
(106,165)
Taxuyunnanine F (211)
T. yunnanensis
(172,173)
10,13-Deacetyl a^eobaccatin IV (212)
T. wallichiana
(174)
Taxayuntin E (213)
T. yunnanensis
(175)
Taxayuntin F (214)
T. yunnanensis
(175)
Taxayuntin A (215)
T. yunnanensis
(176)
13-Acetyl-13-decinnamoyltaxchinin B (216)
T. baccata
(177)
7, 9-Dideacetyltaxayuntin (217)
T. yunnanensis
(178)
10-Benzoyloxy-2,4-diacetoxy-5,20-epoxyl,7,9,13-tetrahydroxy-ll(15—>1)abeoiaxsine (218)
T.brevifolia
(113)
Taxchinin C (219)
T. chinensis
(137)
Taxchinin B (220)
T. chinensis T. cuspidata
(137) (16)
Taxchinin J (221)
T. chienensis
(106,165)
13-Deacetoxy-13,15-epoxy-ll(15—>1)abeo-13-epi-b^cc2iim VI (222)
T. media
(72)
10,15-Epoxy-ll(15—>l)-fl^^o10-deacetylbaccatin III (223)
T. wallichiana
(155)
Taxuyunnanine E (224)
T. yunnanensis
(172,173)
104
178 179 180 181 182 183 184 185 186 187 188 189 190 191
Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri Ri
O H , R2=R3=R5=R6=OAc, R4=0Bz : 0 H . R2=R3=R6=OAc. R4=0Bz, R5=0C0CH:CHPh :R4=0H, R2=R3=R5=R6=OAc :R5=0H, R2=R3=OAc. R4=0Bz, Re^H OH, R2=R3=R5=OAc, R4=0B2, R6=H :R4=R5=OH, R2=0Ac. R3=0Bz, R6=H :R4=R5=OH, R2=R3=R6=OAC :R4=R5=OH, R2=R6=H, R3=0AC :R4=R5=OH, R2=R3=OAc, R Q ^ H
R.^^^^"'
:R4=R5=OH, R 2 = H . R 3 = R 6 = 0 A C :R4=R5=OH, R 2 = R 6 = O A C , R 3 = 0 B Z :R5=0H, R 2 = R 3 = R 6 = O A C , R 4 = 0 B Z
OCOCH2CH(Ph)NMe2, R2=R3=R5=OAc, R4=0Bz, R6=H Ri=
192
Ri=R2=R4=OAc, R3=0Bz, R5=H
193
Ri=R2=0Ac, R3=0Bz. R4=0H. R5=H
194
Ri=R2=R3=R4=OAc. R5=H
195
Ri=R2=R4=OAc, R3=0H. R5=H
OAc
PAC R3
196 R i = R 3 = 0 H , R2=H 197 R i = R 2 = 0 H , R3=0Ac
199
198 Ri=0-D-glucosyl, R2=H, R3=OBz
200 Ri=R5=0H. R2=R3=OAc, R4=0Bz
R i = H , R2=R4=OH, R3=R5=OAc
105
AcQ
QAc
OAc AcQ
pAc
OAc
t>Ac
Aca 201
222
9
OH
223 202 R =R2=R3=R6=OAc, R4=R5=OH 203 R =R5=0Ac, R2=R3=R4=OH, R6=0Bz 204 R =R2=R3=R6=OAc, R4=0Bz, R5=0H 205
=OAc, R2=R3=R4=R5=OH, R6=0Bz
206
= 0 H , R2=R3=R4=R5=OAc, R6=0Bz
207
=R2=R5=OAc, R3=R6=OBz, R4=0H
208
=R2=R4=R5=OAc, R3=R6=OBz
209
=R2=R5=R6=OAc, R3=R4=OBz
210
=R2=R5=OAc, R3=R6=OBz, R4=0H
211
=R2=R3=R5=R6=OAc, R4=0H
212
=R2=R3=R6=OAc, R4=R5=OH
213
=R2=R3=OAc, R4=R5=OH, R6=0Bz
214
=R2=R6=OAc, R3=0Bz, R4=R5=OH
215
=OAc, R2=R6=OBz, R3=R4=R5=OH
216
=R2=R3=R5=R6=OAc, R4=0Bz
217 218 219 220 221
R R R R R
PBz
AcO^^^"'
=R6=0Ac, R2=R3=R5=OH, R4=0Bz =R6=0Ac, R2=R3=R5=OH, R4=0Bz =R2=R5=OAc, R3=R4=R6=OBz =R2=R3=R6=OAc, R4=0Bz, R5=0C0CH:CHPh =R2=R6=OAc, R3=0Bz, R4=0H, R5=0C0CH:CHPh
224
OAc
106
2.4 2(3—>20)-Aheotaxanes These compounds have a distinct biogenetic origin than the other taxoids, resulting from a transannular cyclisation, which is different from that involved in the formation of normal taxanes (92). They are tricyclic systems with a ten membered ring sandwiched between two six membered rings. Isolates of this class from Taxus species are listed in Table 4. TABLE 4 2(3—>20) A^eotaxanes isolated from different Taxus species. Compound
Species
References
Taxin B (225)
71 yunnanensis
(179)
2-Deacetyltaxin B (226)
T. yunnanensis
(179)
Deaminoacyltaxine A (227)
T. baccata
(92)
Taxuspine B (228)
T. cuspidata
(63)
Taxine A (229)
T. cuspidata
(180,181)
7-0-Acetyltaxine A (230)
T. baccata
(55)
2-Deacetyltaxine A (231)
T. baccata
(55,182)
AcOl'"
225 226 227 228 229 230 231
Ri=OH, R2=R3=R4=OAc Ri=R4=0H, R2=R3=OAc Ri=R2=R3=OH, R4=0Ac Ri=OCOCH:CHPh. R2=R4=OAc, R3=0H Ri=0C0CH(0H)CH(NMe2)Ph, R2=R3=OH, R4=0Ac Ri=0C0CH(0H)CH(NMe2)Ph, R2=R4=OAc, R3=0H Ri=0C0CH(0H)CH(NMe2)Ph, R2=R3=R4=OH
107
2.5 11(15—>1), 11(10—>9) Bisabeotaxane The only known taxoid with 11(15—>1), 11(10—>9)bisabeoi?iX?Lnt skeleton is wallifoliol (232) from T. wallichiana (134). A benzil-benzilic acid type rearrangement of 10-dehydro10-deacetyl-11(15—>l)-flfeeobaccatin III (A) might be the biosynthetic pathway of this compound. OH
HO""
AcO
232
2.6 Pretaxoids Paclitaxel, because of its unique mode of action and enormous medicinal potential is under an intensive research effort. The core structure of taxol consists of four rings (six membered A and C rings, eight membered B ring and four membered D ring) and a very little is known about the biosynthesis of this taxane core. Verticillol, a natural product isolated from Scidopitys verticillata, with rings B, C and D not yet formed has been much in the focus of speculation about the biosynthesis of the taxane skeleton (6,183). Verticillol, however differs in the stereochemistry at the C-1 and lacks oxygenation at C-2, C-5, C-7, C-9, C-10 and C-13 and numerous attempts of its cyclisation have failed (184). Very recently a novel pretaxoid, canadensene (233) in which rings B,C and D are not yet formed has been isolated from T. canadensis (185). The isolation of canadensene in the genus Taxus and its structural similarity with the core structure of taxol and related taxanes has considerable importance for biosynthetic studies. Two more such compounds were isolated from T. chinensis (Table 5).
Verticillol
TABLE 5 Pretaxoids isolated from different Taxus species. Compound
Species
References
Canadensene (233)
T. canadensis
(185)
Taxachitriene A (234)
T. chinensis
(186)
Taxachitriene B (235)
T. chinensis
(186)
108 OAc
OAc
233 Ri=OAc, R2=R3=OH 234 Ri=R2=0Ac, R3=0H 235 Ri=R2=0H, R3=0Ac
AcO^^^^"'
3. LIGNANS Lignans are found in several species of Taxus. Secoisolariciresinol (236), isotaxiresinol (249), isolariciresinol (250) and isotaxiresinol-6-methyl ether (251) are found in several species of Taxus (39,187). Secoisolariciresinol (236), isotaxiresinol (249) and taxiresinol (255) were isolated by Majumdar et a/.(188) from T. haccata, whereas a-conidendrin (252) was isolated from T. marei (43,45). Table 6 lists various lignans isolated from the genus Taxus. TABLE 6 Lignans isolated from different Taxus species. Compound
Species
References
Secoisolariciresinol (236)
T T. T. T T
(-)-4-0-Methyl-3'-0-demethylsecoisolariciresinol (237)
T baccata
(187)
(-)-3-Dimethylsecoisolariciresinol (238)
T. baccata
(192)
2-(3,4-Dimethoxybenzyl)-3(3,4-methylenedioxybenzylidene)butane-1, 4-diol (239)
T baccata
(193)
(-)-2-(3, 4-Methylenedioxybenzyl)-3(3,4-dimethoxybenzylidene)butane-l,4-diol (240)
T. baccata
(194)
baccata brevifolia cuspidata floridana mairei
(39,187,188,190) (39) (39) (39) (43,44,49,191)
109
(-)-2-(3,4-Methylenedioxybenzyl)-3(3,4-methy lenedioxybenzy lidene)butane-1, 4-diol (241) Hydroxymatairesinol (242)
T. media T. wallichiana
(119) (189)
(-)-Matairesinol (243)
T. haccata
(194)
(-)-2-Epinortrachelogenin (244)
T. media
(119)
Isoliovil (245)
T. wallichiana
(189)
4'-0-Demethylsuchilactone (246)
T. haccata
(193)
(-)-Isohibalactone (247)
T. haccata
(194)
(-)-Hibalactone (248)
T. haccata
(194)
Isotaxiresinol (249)
T. haccata T. hrevifolia T. cuspidata T. floridana T. mairei
(39,187,188,190) (39) (39,195) (39) (43,44,49,60,191)
Isolariciresinol (250)
T. haccata T. hrevifolia T. cuspidata T.floridana
(39,187) (39) (39,195) (39)
Isotaxiresinol-6-methyl ether (251)
T. haccata T. hrevifolia T. cuspidata T. floridana
(39) (39) (39) (39)
a-Conidendrin (252)
T. haccata T. mairei T. wallichiana
(194) (43,45) (189)
6-Conidendrin (253)
T. wallichiana
(189)
Lariciresinol (254)
T. cuspidata
(39)
110
Taxiresinol (255)
T. baccata
(188)
Z>-Sesamin (256)
T. mairei
(44)
4-Hydroxysesamin (257)
T. mairei
(44)
RiO.
OH 236 Ri=R3=Me, R2=H 237 Ri=R2=Me, R3=H 238 R-|=R2=H, R3=CH3
239 Ri+R2=CH2, R3=R4=CH3 240 Ri=R2=CH3, R3+R4=CH2 241 Ri+R2=R3+R4=CH2
H3CO,
OCH3
242 R=OH 243 R=H H3CO.
246 Ri=OH, R2=0Me 247 Ri+R2=OCH20
Ill H3CO,
CH2OH
RpO'
CH2OH
249 Ri=R2=H 250 Ri=CH3; R2=H 251 Ri=H; R2=CH3
248
H3CO.
H3CO.
H3CO.
254 R=Me 255 R=H
256 R=H 257 R=OH
112
4. FLAVONOIDS Taxus species contain the biflavonoids mainly of the amentoflavone series. A1% emulsion of flavonoidal mixture obtained from T. haccata (196) with tween-80, suitably diluted with normal saline or physiological solution was used by Vohora et al (197). for all studies on pharmacological properties with reference to nervous system. Their observations showed CNS effects by flavonoidal mixture as evidenced by potentiation of pentobarbitone narcosis, hypothermia and delayed reaction of fasted rats in Hebb-William maze. Flavonoidal mixture also showed antipyretic and analgesic effects. Table 7 lists all the flavonoids isolated from the genus Taxus. TABLE 7 Flavonoids isolated from different Taxus species. Compound
Species
References
Ginkgetin (258)
T haccata T canadensis T cuspidata T. wallichiana
(196) (88) (91,198,199) (200,201)
Sciadopitysin (259)
T haccata T. hrevifolia T canadensis T. cuspidata T. wallichiana
(196,202) (75) (88) (91,198,199) (200,201)
Sequoiaflavone (260)
T haccata T wallichiana
(196) (200,201)
Sotetsulflavone (261)
T haccata T. cuspidata
(203,204) (198)
Kayaflavone (262)
T cuspidata
(198)
Amentoflavone (263)
T. haccata T wallichiana
(205) (201,205)
4',7,7"-Tri-0-methylamentoflavone (264)
T. haccata
(206)
4',4",7,7"-Tetra-0-methylamentoflavone (265)
T haccata
(205,206)
113
4',7"-Di-0-methylamentoflavone(266)
T. haccata
(205)
(+)-Taxifolin (267)
T. mairei
(49)
Kaempferol (268)
T. hrevifolia
(75)
Quercetin (269)
T. brevifolia T. cuspidate
(75) (91)
Isorhamnetin (270)
T. brevifolia T. cuspidata
(75) (91)
RiO,
258 R-i=R2=CH3', R3=R4=H 259 Ri=R2=R4=CH3; R3=H 260 Ri=CH3; R2=R3=R4=H 261 R-j=R2=R4=Hi R3=CH3 262 R-|=R3=R4=CH3; R2=H 263 R-|=R2=R3=R4=H 264 Ri=R2=R3=CH3, R4=H 265 R-|=R2=R3=R4=CH3 266 R-|=R4=H, R2=R3=CH3
268 R=H 269 R=OH 267
270 R=0CH3
114
5. STEROIDS Phytoecdysteroids, the well-known molting hormones are another important class of naturally occurring organic compounds isolated from Taxus species. Insect molt hormone activity associated with extracts of yew leaves has been reported by Takemoto et a/. (207). Table 8 lists phytoecdysteroids alongwith other common steroids isolated from the yew. These consist of a number of closely related active compounds (insect metamorphosis hormones) possessing insect molting activity. The most common of these phytoecdysteroids is ecdysterone (271). TABLE 8 Steroids isolated from different Taxus species. Compound
Species
References
Ecdysterone (271)
T. baccata T. chinensis T. cuspidata
(208,209) (210) (211,212)
Taxisterone (272)
T. cuspidata
(15,213)
Ponasterone A (273)
T. brevifolia T. cuspidata
(113) (211)
Makisterone A (274)
T. cuspidata
(214)
Campesterol (275)
T. cuspidata
(89)
6-Sitosterol (276)
T. baccata T. brevifolia T. canadensis T. cuspidata T. floridana T. mairei T. wallichiana
(39) (39) (88) (39) (39) (39,43,44,49) (52)
Daucosterol (277)
T. wallichiana
(52)
Stigmasterol (278)
T. cuspidata
(89)
p-Sitostenone (279)
T. mairei
(191)
115
271 Ri=OH 272 Ri=H
273
276 R=H 277 R=glucosyl
278
279
116
6. SUGAR DERIVATIVES A novel glycoside, taxicatin (280) was isolated from the leaves of T.haccata (215-218). The glycosides of aliphatic and aromatic alcohols, as well as of monoterpene alcohols and eugenol have been detected in Taxaceae (219). Recently we have isolated betuloside (283) from T. haccata, reported its absolute stereochemistry and have found it to possess hepatoprotective activity (220). Table 9 lists various glycosides and other sugar derivatives isolated from the genus Taxus. TABLE 9 Glycosides isolated from different Taxus species. Compound
Species
References
Taxicatin (280)
T. baccata T. canadensis
(215-218) (88)
(7?)-Taxiphylline (281)
T. haccata T. cuspidata
(221) (222)
(5)-Dhurrin (282)
T. baccata
(221)
Betuloside [Rhododendrin (283)]
T. haccata T. canadensis
(130,196,220) (88)
L-4-/7-Coumaroyl-m>'o-inositol (284)
T. haccata
(223,224)
D-1-O-Methyl-mMco-inositol (285)
T. haccata
(225)
Taxuside (286)
T. haccata
(187,192)
Triglochinine (287)
T. haccata
(221)
Isotriglochinine (288)
T. haccata
(221)
Raffinose (289)
T. haccata
(217,226)
Z)-Pinitol (290)
T. haccata
(225)
Sequoitol (291)
T. haccata
(225)
117
CN
\
O
>=< I OH
281 R=a-0-glucosyl 282 R=p-0-glucosyl
OCHa
280
-OH
/
O HO
287 288
CH2OH OH
H
H
290
OH
H
OH
291
118
7. MISCELLANEOUS Kojic acid (294), a y-pyrone, isolated from T. mairei is a typical microbial metabolite and fungal contamination of the plant extract might be a reason of its formation (227). The diterpenestaxamairinsA(297)and B (298) possessing tropone skeleton displayed inhibitory activity [IC^^ 30.21 g/ml for taxamairin A and 26.78 g/ml for taxamairin B] against hepatoma cells in vitro (228). (+)-Abscissic acid (317) was extracted from the seeds of T. haccata (229-231). The needles of T. haccata contained 26 carboxylic acids, 80% of which were shikimic (335) and quinic acids (336) (232). Rhodoxanthin (300) has been found to be present in all the species of Taxaceae (87,233). Escholtzxanthone (301) was isolated from T. haccata leaves (234). Various compounds isolated from genus Taxus which are not otherwise classified in the text are listed in Table 10. TABLE 10 Novel compounds of different types isolated from Taxus species. Compound
Species
References
Coniferaldehyde (292)
T. mairei
(43,235)
Vanillin (293)
T. mairei
(43,191)
Kojic acid (294)
T. mairei
(227)
10-Nonacosanol (295)
T. mairei
(227)
Brevitaxin (296)
T. brevifolia
(236)
Taxamairin A (297)
T. mairei
(228,237)
Taxamairin B (298)
T. mairei
(228,237)
Taxamairin C (299)
T. mairei
(236)
Rhodoxanthin (300)
all species
(87,233)
Escholtzxanthone (301)
T. haccata
(234)
3-Phenylpropanol (302)
T. haccata
(187)
3-(3',4'-Dihydroxyphenyl)propanol(303)
T. haccata
(187)
3-(4'-Hydroxy-3'-methoxyphenyl)propanol (304)
T. haccata
(187)
119 3-(4-Hydroxyphenyl)propyl tetracosanoate (305)
T. canadensis
(88)
3-(4-Hydroxyphenyl)propyl pentacosanoate (306)
T. canadensis
(88)
3-(4-Hydroxyphenyl)propyl hexacosanoate (307)
T.canadensis
(88)
3-(4-Hydroxyphenyl)propyl dotriacontanoate (308)
T. canadensis
(88)
3-(4-Hydroxyphenyl)propyl tetratriacontanoate(309)
T. canadensis
(88)
4-(4'-Hydroxyphenyl)-2/?-butanol [(-)-Rhododend^ol^etuligenol (310)]
T. brevifolia T. baccata
(133) (187,196,238)
4-(4'-Hydroxyphenyl)-25-butanol (311)
T. baccata
(239)
4-(3',4'-Dihydroxyphenyl)-2/?-butanol (312) T. baccata
(187,238)
4-(3'-Methoxy-4'-hydroxyphenyl)2/?-butanol (313)
T. baccata
(187,238)
Deglycosylicariside B^ (314)
T. wallichiana
(240)
Dehydrovomifoliol (315)
T. wallichiana
(240)
Vomifoliol (316)
T. wallichiana
(240)
Abscissic acid (317)
T. baccata
(229-231)
d5-5,cis-9-Octdecadienoic acid (318)
T. baccata T. cuspidata
(241) (242)
Vanillic acid (319)
T. baccata
(243)
Pyrocatechuic acid (320)
T. baccata
(243)
Protocatechuic acid (321)
T. baccata
(243)
p-Resorcylic acid (322)
T. baccata
(243)
120
Gentisic acid (323)
T. baccata
(243)
Y-Resorcylic acid (324)
T. baccata
(243)
Pholoroglucinic acid (325)
T. baccata
(243)
Gallic acid (326)
T. baccata
(243)
Syringic acid (327)
T. baccata
(243)
o-Hydroxyphenylacetic acid (328)
T. baccata
(243)
/7-Hydroxyphenylacetic acid (329)
T. baccata
(243)
Homoprotocatechuic acid (330)
T. baccata
(243)
Cinnamic acid (331)
T. baccata
(243)
/7-Coumaric acid (332)
T. baccata
(243)
Ferulic acid (333)
T. baccata
(243)
Caffeic acid (334)
T. baccata
(243)
Shikimic acid (335)
T. baccata
(232,243)
Quinic acid (336)
T. baccata
(232,243)
Benzoic acid
T, baccata
(243)
2-Hydroxy-, 3-hydroxy- and 4-hydroxybenzoic acids
T. baccata
(243)
CHO
CHO
OCH3 OH
292
293
294
295
121
297 R=OH 298 R=0CH3
296 0CH3
301
OH
HO'
302 R=H 303 R=OH 304 R=OMe
305 306 307 308 309
n= 22 n= 23 n= 24 n= 30 n= 32
122
>^N)H
HO
312 R=H 313 R=Me
310 R 311 S
315
314
316
COOH 317
COOH 318
COOH 319 Ri=H, R2=OCH3, R3=0H 320 Ri=R2=0H, R3=H 321 Ri=H, R2=R3=OH 322 Ri=R3=0H, R2=H
COOH 324 Ri=OH, R2=H 325 Ri=R2=0H
COOH
3 2 8 R i = O H , R2=R3=H 329 Ri=R2=H, R3=0H 330 Ri=H, R2=R3=OH
123 COOH
R2
HO//,,^ ^ C O O H
COOH
Ri
331 332 333 334
Ri=R2=H Ri=H, R2=0H Ri=0CH3, R2=0H Ri=R2=0H
OH
335
336
8. CONCLUSIONS This review of literature covering phytochemical investigations on ten Taxus species have resulted in the isolation of nearly three hundred and fifty compounds belonging to the classes of taxanes, lignans, flavonoids, steroids, sugar derivatives, aromatic acids, etc. Last few years have witnessed a revolution in the phytochemistry of the yew tree; it is evident from the figures that out of the 246 references cited in this review, 151 range in the period 1990-96. Pharmaceutical industries are concentrating too much on this genus to obtain paclitaxel (taxol) and equivalent compounds of paclitaxel. As paclitaxel has become an approved agent to cure cancer, issues regarding its supply have now come to the forefront. The way the yew trees are being cut world-wide to obtain this drug, it is estimated that the source of supply would last for only 5-10 years (244) and it cannot be regarded as an environmentally acceptable source. Of late, four possibilities of supply are being considered: (i) find a better source for the supply of paclitaxel such as a different species or a cultivar of Taxus, or a different plant part or cultivation condition, (ii) semi-synthesis of paclitaxel from a more abundant precursor, (iii) total synthesis of paclitaxel, and (iv) tissue culture production of paclitaxel or its close precursor (245). Thus, we clearly visualise that this area demands still more intensive research to make it fruitful, and this updated and comprehensive review will help fellow scientists in locating information in one source. A cknowledgement We thank the Danish International Development Agency (DANIDA, Denmark) for financial assistance to carry out phytochemical investigations on a few Taxus species.
124
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
135
Withanolides, Biologically Active Natural Steroidal Lactones : A Review A.S.R. Anjaneyulu, D. S. Rao, P.W. Lequesne
INTRODUCTION Withanolides mainly Solanaceae
in the SolsLnaceous
occur
is a large family comprising over 2000 species which
have great economic and medicinal importance. potato (Solanum tuberosum) 3
2
brinjal 4
melongena esculentum
) , chili ( Capsicum frutescens ) , ) and tobacco {Nicotiana tobaccum
includes
several
medicinally
{ Atropa
nightshade
These are generally Solanaceous
The important cash crops among
annual shrubs. are
plants.
belladona
important 7
) ,
the
plants
(ambergine)
( S.
tomato ( Lycopersium ) . This family
plants
like
thorn
apple
the deadly ( Batura
8
9
stramonium ) , Solanum species
xanthocarpum
12
constituents.
.
the poisonous henbane especially s. dulcamara
{Hyoscyamus niger , S. indicum
)^ and and S .
These herbs are well known for their alkaloidal In the indigenous
tubers and the leaves great importance.
Withania
Ayurvedic medicinal system the 13 14 and Physalis species have
Occurrence of Withanolides During the last two and a half decades many withanolides and their related compounds have been isolated from Withania other Solanceous
species
physiological
activity.
and
some
of
Withanolides
from the following species :~
them
possess
have been
and
remarkable
isolated
so far
136 Withania 1. 2. 3.
species
W, somnifera I/, coagulins
15
W, arboritus
Physalls
(frutescens)
20 alkekengi •. . 21a,b,29 P. minima P.
22
angulata 23 viscosa
2^a,b
P. pubescens C.
Datura species
D.
D. stromoniufli
2.
D,
1.
£.
G.
5.
W.
6
P. ixocarpa
7.
P
peruviana
8.
P
iancifolia
9.
P.
10.
P.
obtusifolia
19
species
Nicandra
25 26a ,b 27
phyladelphica 28 curasavica
28
31
4,
D. ferox
5.
D.
4.
J.
31,31a
tatura
29a
: physaloides
34
Lycium species ; 1.
F.
30
quercifolia 32,33 D. metel
Nicandra
aristata
;
I.
3.
W.
species
P.
P.
17
18
4.
Lycium
Jaborosa
chinense 35
species
: 36
leucotricha
1.
J. integrifolia
2.
J. Jbergii^^
5.
J. odonelliana
3.
J. magellanica
6.
J. sativa
3.
A. australis
4.
A.
39,39a, 40
Acnistus species : 1. 2.
41 A. Jbreveflorus GresiJb 42 A. ramifluoruiD ffeirs
H.
Trechonaetes laciniata
I.
lochroma 1.
Griseb 44 arborescens
43
45
species
lochroma cocconeum
46
lochroma
fuchsioides
46a
137 J.
Witharingia
K.
Tubocapsicum
L.
Dunalis 1.
W.
coccoloides 48 anonaium
species
;
australis
JD.
47
49
D.
2.
D.
tuJbuiosa
49a
Deprea species ; D. orinocensis 50
N.
2.
procumbens
51
origan!folia 52
Salpichroa
Withanolides : Chemical Classification The
withanolides
are
a
group
of
naturally
occurring
steroids built on an ergostane skeleton, in which C-22 and C-26 are appropriately oxidised in order to form a "S-iactone ring*
The name withanolide was originally proposed by Lavie et al 53 for a group
of
steroidal
lactones
of
the
ergostane
six-membered lactone in the nine carbon side chain-
series
with
a
The names in
current use for structure (1) are either withanolide, or according to the Chemical Abstracts system, 22-hydroxyergostan-26-oic 26,22-olide. are
acid-
In the CAS Chemical Substances index the compounds
catalogued
as
ergost
derivatives.
The
name
ergost-26,22-olide based on lUPAC rules, does not seem to be used. 54 The generic 'witha steroids' which has been proposed does not seem to have gained much popularity. carboxylic
For compounds with a modified
skeleton and or side chain, only
CA names
used, along with the trivial names.
(1)
Withaphysalin B ( 2 )
are being
138 Withaferin known
A
(12) the
Aswagandha)
was
Subsequently, withaferin
a
A
first
isolated
number
of
(12)
withanolides.
active
were
constituent
Withania
Indian medicinal plant,
somnifera
by
Lavie
compounds
isolated
The interesting
of
Dun
et
al.
in
structurally
and
these
well 1965.
related
to
known
as
were
biological activities
the
(Sanskrit :
elicited
by
withaferin A (12) and its congeners augmented the search for this type of compound, and the number of withanolides from different plants
has
already
surpassed
two
hundred.
Withanolides
are
generally defined as C-28 steroidal lactones built on a skeleton of ergostane intact or rearranged. There
exist
several
this group of compounds.
interesting
review
articles
on
To make a comprehensive account of these
compounds and to depict an overall picture of withanolides, this review was highlighted with some recent developments in the field till the end of August 1996. Withanolides
generally
contain
28
carbon
atoms
with
polyoxygenated functionalities.
These compounds in general possess
the a, 0-unsaturated-l-oxo-2-ene
in ring A and a 5-lactone between
C-22
and
saturated A
C-26
in
the
side
chain.
A
few, however,
contain
a
6-lactone system. common
feature
of
all
known
withanolides,
including
the related modified ergostane derivatives, is oxidation at C-1, C-22 and C-26.
Over 90% of withanolides
contain
a
1-oxo-group.
C-26 is oxidised in most instances.to the carboxylic acid level, thus allowing the formation of a 26,22 lactone. In contrast to the lactone, which in most instances is a ,3 -unsaturated, the precursor a ,0 -unsaturated
lactol
is
never
found
as
such,
but
found
as
epoxylactols or the corresponding epoxylactones. In addition to the oxidative processes at the above three centres, one of the characteristic features of the plants producing withanolides
is
their
extraordinary
ability
to
introduce
oxygen
functions at almost every position of the carbocyclic skeleton and side chain.
Till now, there are only one secondary (C-11) and two
tertiary (C-8 and C-9) carbon atoms which have not been found to be oxidised. Withanolides are classified into two major groups
.
139 A - Withanolides with an unmodified skeleton a) with a regular.: 6-oriented side chain b)
with an unusual «-oriented side chain.
B - Withanolides with modified carbocyclic skeletons or side chains These withanolides were classified initially on the basis of chemotypes of Withania
species
depending on the region of the
plant collected, to the compounds obtained as most of these have been
initially
several
isolated
withanolides
Solanaceae species,
the Withania
from
have
been
family, eg. Physalis Acnistus
species.
isolated
species.
species.. from
other
Later
on
species
of
Datura species,,
Jaborosa
Chemically, these may be classified as
ergostane derivatives-
From their structural pattern, these can be
broadly divided into seven groups. 1.
5B,63-epoxides
2.
6a,7a-epoxides
3.
5-enes
4.
Intermediate
5.
5a,6 a-epoxides
6.
63,73-epoxides
7.
Phenolic withanolides
compounds
Among these the 53 ,63-epoxides are most common. the compounds possess a 43-hydroxy1 group. which possess an a ,3 -unsaturated-Y-lactone 25 lactone A (61), the rest have ^-lactones. Withanolides 5a-hydroxyl
eg.
originate from number,
having
lycium
5 3,63 -epoxides.
eg. withanolide
B
generally
(62) and
are
Lavie
et
contain
believed
The 5-enes also form a
(107).
G
system, eg. ixocarpa-
6a ,7a -epoxides
substance
Most of
Except for four members
to
sizable
al. suggested
that
60,70-epoxides are originated from 5-enes by migration of A -ene to .6-ene with simultaneous hydroxylation at C-5 followed by epoxidation on A . inspection of the intermediates suggests that most of them originate from 53,63-epoxides either by trans-diaxial 181 or diequatorial opening. Sarma et al. suggested that 134 chlorowithanolides are formed by the reaction of chloride ion present in the plant with the 53,63-epoxide system (Tables la-ln) . Hydroxylation Hydroxylation
is
a
common
feature
in
withanolides.
Hydroxyl at C-4 is the most common feature in 53 ,63-epoxides, eg.
TABLE
la
Compound
Hydroxyls Epoxide Ene
140
5 3,6 8-EPOXIDES UTE 17&
SIDE CEAIN
Other groups
m.p.
("C)
[ UjD
Source
Ref.
Without hydroxyls :
Withaphysalin B (2)
2, 24
18-20 lactol 161-62 (EtOAc) (epimers)
-
P. minima
64
Monohydroxy Compounds :
27-deoxywithaferin A ( 3 )
48
5%,6%
2,24
--
268-9 (EtOAc)
+
101.5
W. somnifera ( S . African variety)
65
48-Hydroxy-5B ,6%-epoxy1-0~0-22R-wltha-2,14,24trienolide (4)
4%
5%,6%
2,14,24
--
239-40(~to~c)
+
122
W. a r i s t a t a Pauq
66
4~-hydrory-l-ox0-58,6%epoxy-wltha (24S,25K)dihydro-2-enolide ( 5 )
48
5%.68
2
--
252 (CHC13-EtOkc)
+
32.5
W. somnifera
65
Jaboroaalacrone A ( 6 )
27
5%,613
2,24
--
215-20
+
95
58,68
2,24
4-0x0
273-4 (EtOAc)
+
113.5
~ U E - H Y ~ ~ O X ~ - ~ , ~ - ~ ~ O X 20 O -8
58 ,6%-epoxy-ZOR,22R-
( s . "African variety) Jaborosa i n t e g r e f o l i a
67
W. somnifera
68
LiUll
(s. African Vs.
Chemotype 111)
w i tha-2,24-dienolide (7)
2WE-iLydroxy-l,4-dioxo5 8, bbepoxy-20R,22Rw i :ha-2-enoli de ( 8 )
20 B
58,bB
2
&ox0
260-63
+
2,3-Di.hydro lactone A ( 9 )
27
5%,68
24
--
180-82 (EtOAc)
_-
49.2
-do-
68
-do-
69
Compound
Hydroxyls Epoxide Ene
[ QID
Other groups m.p. ("C)
2:3, 24:25-Tetrahydro-27deoxywithaferin A (10)
209-210
+ 17.4
2,3-dihydro-27-deoxywithaferin A (11)
236-37 (EtOAc)
- 3.0
Source Acnis tus brevifluorus
-do-
Ref. 70
70
Dthydroq Compounds
Withaferin A (12)
4e, 27 56,6e
2,24
--
243-45
+
114
2,3-dihydrowithaferin A
46,17a 5 6,6%
24
--
229-30
+
8.0
27-Deoxy-14C-OHwfthaferin A (14)
4e,lk
2,24
--
265-67
+ 67
17 *Hydroxy-27-deoxywithaferin A -4acetate (15)
4~,17a 56,66
2,24
--
195-96 (EtOH)
+
175
U. somnifera Dun
66
Withacnistin i16)
46,18
56,66
2,24
--
130-35 (amorphous solid)
+
123
A. aroborescecs
44
4 6,27-Dihydroxy-58,66epoxy-1-0x0-22R-witha2,14,24-trieno1l.de (17)
40,27
50,66
2,14,24 --
256-59
+
98
U. frutescens Pauq
17
Jaborosalactone L
17427 56,66
--
214-15
+ 49
( 13)
(18)
5 ,6
2,24
W.somnifera Dun
71
-do-
71
-do-
72
(lndian Cfiemotype)
141
56,@-EPOXIDES
Compound
Hydroxyls Epoxide Ene
142
TABLE la
( Ccntd. )
WITH 176- SIDE CHAIN
Other groups m.p.
[a ID
("C)
Source
Withanolide D (19)
48,20 8 55,@
2,24
253-55
+
80
W. somnifera
24,25-Dihydro-withanolide D ( 2 0 )
46,20 8 5 8,68
2
275 (EtOAc)
+
14
Y. somnifera ( 5 . African variety)
7 a,17a-Dihydroxy-55,65epoxy-1-oxo-22R-witha2,24-dienolide (21)
7a,17a
55,68
2,24
252-54 (MeOH)
V i scosalactone A' ( 2 2 )
45,27
55,65
24
4 8,2Gcl-Dihydroxy-l-oxo5 8,68 -epoxy-ZOR,22R-
45,20 5 58,68
24
A withanolide isomeric to withaferin A (24)
4~,27
24 q25a-epoxywi thanelide D ( 2 5 ) Jabarosalactone 0 ( 2 6 )
witha-24-enolide (23)
Irrnrcrnclide(Z7) Physagulin A ( 2 8 )
-* -28.0
2,24
*
48,208 58,65
2
*
*
17a,19 5 4 6 8
24
*
*
5 8,65
73
(Chsmorype 111)
W. somnifera
65 74
(Seeds)
* 277-80 (Acetone )
Ref.
*
P. Viscosa
23
W. s o n n i f e r a
68
(5. African Vs. Ctmotype 111) W. o b t u s i f o l i a
(Sudan)
4 EJ16cr-OAc5 8,66
2,24
146-54 (EtOAc)
14a,l%-OAc 5 3,65
2,24
A whi.te powder
--
+
21
19
P . angulata
75
Jabarosa l e u c o t r i c h a Leaves Iochroma coccinium
76
P . angulata
77
46
TABLE la ( Ccntd. ) :B.;6E-EPOKu)JS WITH 178- SIDE C M I N
Compound
Hydroxyls Epoxide Ene
[a ID
Other groups m.p. ("C)
)
17a ,19
5 46B
24
--
287-90
--
Jaborosalactone W (26b)
193,28
5 8,6E
24
--
220-22
---
27-Hydroxywithanolide D (29)
4 E,208,27
5 E,6E
2,24
--
202 (4,27dtacetate)
+ 17
1 4-Hydroxywithanoltde D (30)
@,14 5208 5 B,6B
2,24
--
211-13 (EtOAc) (4-acetate)
-
17a-Hydroxywithanoltde D (31)
48,17a,20
5E,6E
2,24
--
196-97 ( Et0Ac (4-acetate)
+ 2
7~-Hydroxywithanolide D (32)
48,78,208
5E,6E
2,24
--
260 (Acetone:Hex)
+
78- k e toxywithanolide D (33)
48,78-OAc 20
5 8,6 8
2,24
--
173-75
17-Isowithanolide E(34)
14a,17a,208 58,68
2,24
--
262 (EtOAc)
Physapubescin (35) (26-eptmers)
4!3,15a-OAc, 5 E,6 E 26
2
Jaborosalactone V @&
Source
Ref.
Jaborosa leucotricha 39a -do-
39a
R-Lhydroxy Compounds :
22,26-lactol
W. somifera Dun (Chemotype 111)
18
118.5
A.
78
-do-
78
-do-
78
australis
43
Griseb
-+
Noncrystalline
21.3
--
A.
arborescens
79
Y. somnifera (Chemotype LII)
80
P. pubescens L
81 143
144
TABLE la ( Ccntd. ) 56,68-EPOXU)ES kT’M 17% SIDE CHAIN Compound
Viscosalactone B
(36)
Physapubenolide
(37)
Hydroxyls
38,48,27
Epoxide
Ene
Other groups
[ a ID
m.p. ( “ C )
*
58,68
144-45
48,148 , 1 5 - 58,68
* +
76
OAc Substance A
(38)
Withangulatin A
(39)
Ref.
P . viscosa
23
P . pvbescens (Waltair variety)
24b
W . somnifera (Egyptai.n variety)
82
83
14a,17a,208 55,M
2,24
--
*
48,14a,
5@,68
2,24
--
152-53
+
23.9
P . angulata
58,68
2,24
--
148-53
+
71.9
Iochroma f u s c h s i o i d e s 46a
5B,68
2,24
--
b
58,68
--
38,48,206 2 4 a , 258
55,63
15cl-OAc 1.8-Acetoxywithanolide D
*
Source
3 , 18-OAc
(39a) Tetrahydroxy Compounds Substance C (@-OHsubstance A) (40) Withajardin B
(41)
45,14a 17a,208
17a ,25
21,24-cy
*
*
*
*
W . somnifera (Egyptfan variety)
82
*
Deprea o r l c e n s i s
50
*
P . angulata Frli.age
84
methyl
Pentahydroxy Compounds: Physangultde
(42)
145 Fig.la.5p,6p-Epoxides with 17p-side chain
3 R, =OH,R2 = R3=H 4. R, = OH,R2 = R3=H 5. R, =OH,R2=R3=H, 24,25-di-H 6. Ri = R2=H,R3=OH 7 R, = = 0 , R2 = OH, R3 = H 8. R, = = 0 , R2 = OH,R3 = H, 24,25-di H 9. R, = R2 = H,R3 = OH, 2,3-diH 10. R, = OH,R2 = R3 = OH 2,3,24,25-tetra H l l . R , = OH,R2 = R3 = H, 2,3-diH 12. R, = R3 = OH,R2 = H 13. R, = OH,R2=R3=H, 17a-OH,2,3-di H
14. Ri =OH,R2=R3=H, 14a-0H 15 R, = OAc,R2=R3=H, 17a-0H 16 R, = OH, R2 = R3 = H,18-OH 17 R, = OH, R2 = H, R, = OH, 18. R, = R2 = H,R3 = OH, 17a-0H 19. R, = R2 = OH,R3 = H 20. R, = R2 = OH,R3 = H 24,25-di H 21 R, = R2 = R3 = H, 7a-0H, 17a-0H 22. R, = R3 = O H , R 2 = H 2p,3p - epoxy 23. Ri = R2 = OH, R3 =H, 2,3 - di H 24. R, = R3 = OH, R2 - H
146 Fig. l a . 5p,6p- Epoxides with 17p- side chain
25 26
R, = Rz = OH, R3 = H, 24,25 - epoxy Ri — R2 ~ R3 ~ H, 17a-0H, 1 9 a - O H , 2 , 3 - d i H
26a. R, =R2 = R3 = H, 17a-0H, 1 9 a - O H , 2 , 3 - d i H 26b. R,=R2 = R 3 = H , 19-OH, 2 8 - O H , 2 , 3 - d i H 27 R, =OH,R2=R3=H, 16a-0Ac 28 Ri = R2 = R3 = H, 14a-0H,15a-0AC 29 . R, = R2 = R3 = OH 30 R, = R2 = OH, R3 - H 14a-0H 31 R, = R2 = OH, R3 = H, 17a-0H 32 Ri = R2 = OH, R3 = H, 33 R, = R2 = OH,R3 = H, 7P-0AC
34 R, = R3 = H, R2 = OH 14a-0H 35 .R, = OH, 1 5 a - 0 A C , R2 = R3 = H, H 26,
TABLE lb 56,6B-EYOXIiIES WITH 17- SIDE CHAIN Compound
Hydroxyls Epoxide Ene
Other groups m.p. ("C)
[a],
Source
Ref.
Monohydroxy Compounds :
*
*
J. magellanica
38
--
--
--
J. bergii haves
37
2
--
--
_-
17
58,68
--
12-OX0
(44) 148,178
58,68, 24a,25a 58,68 24a,25a
--
Jaboromagellsne (43) Dthydroxy Compounds :
Jaborosalactone M
2,3-Dehydrojabcrcsalactone M (45).
148,178
-do-
37
Rihydroxy Compounds :
Withanolide E (46)
14a,178, 20 8
58,68
2,24
--
Withaperuvin E (47)
14a,175, 20 6
56,68
24
4-ox0
48,178,208 56,68
24
--
14a,178,
56,66
24
--
Jaborosalactol M (50)
148,176, 26a
58,68 i 4 ~25u ,
--
2,3-dehydrojabcrcsalactol M (51)
148,178, 26a
56,68 24a,25"
2
Withaperuvin G
(48)
2,3-dihydrowithanolide E (49)
20
167-68 (Acetone)
+
103.5
P. peruviana
85a,b
248-50 (Acetone)
-
72.5
P. peruviana roots
34
163 (CHC13)
_-
273-75 (Acetone)
--
P. peruviana
22,26-lactol
234-36 (EtOAc)
--
J. bergii
22,26-laccol
--
_-
-do-
leaves -do-
26b 26a
37 37
147
148
TABLE
l b ( Contd.
% , 6 6-EPOXIDES W I l l I 17a - S D E CHAIN
bID
Compound
Hydroxyls
Epoxi.de
Ene
Other g r o u p s
Physagulin C (52)
48,14a, 15a-OAc
56,68 168,176
2,24
--
242.5-243
+
105.8
P. a n g u l a t a ( L e a v e s & Stems)
86
48,14a 178,208
56,166
2,24
--
197-98 (EtOAc)
+
95.8
P . peruviana
26a 87
Visconolide (54)
48,14a, 178,208, 28
58,66
2,24
--
P. v i s c o s a
88
Physalactone (54a)
36-OMe, 46 -OH, 14a-OH, 178 -OH, 206-OH
58,68
24
--
P . alkekengi
58
Tetrahydro-
m.p.
("C)
Source
Ref.
Compounds:
4 -Hydroxywl.thanoll-de E (53)
Pentahydroxy compounds:
*
Not a v a i l a b l e
149 Fig. l b . 5 p ,6 p - Epoxides with 17a - side chain
43. R, = R3 = H,R2-CH, 12.0XO,2,3-diH
49 R, = R3 = H, Rz - OH Ma^OH ,2,3-di H
44. R , - R3-H,R2=OH, 14p-OH,2,3-diH
50. Ri = R3 = H,R2^0H, 14p -OH, 22-26, lactd, 24a,25a-epoxy, 2,3-di H
45 R, -R3-H,R2-OIi 24a, 25a - epoxy
51 R, =R3 = H,R2-OH, 14P-0H, 22-26 lactol, 24a,25a-epoxy
46. R, =H,R2 - R3 - OH, 14a-0H
52 R, =0H,R3 = H, 15a-OAc,R2=16p, 17p-q)oxy 53. R, = R2 - R3 = OH, 14a - OH
47. Ri =OXO,R2=R3= OH, 14a-OH,2,3-diH 48. R, = R2 = R3 = OH, 2P, 3p - epoxy
54. R,-R2=R3=OH, 14a - OH, 28-OH 54a. R, - R2 = R3 = OH, 14a - OH, 3p - OMe, 2,3-diH
150
TABLE l c 5B,6 6-EPOXIDES WITH MODIFIED SIDE CHALN ~
Compound
Hydroxyls Epoxide Ene
PI,
Other groups m.p. ("C)
Source
Ref.
Monohydroxy Compounds :
NS 1
Dihydroxy Compounds
vi th
Y-lactone side chain : (+)
Jabnrnsalactnne M (55)128,178
58,66
2,24
Trechonolfde B (56)
178,228
Trechonnlfde B (57)
176,22,2
56,65 2,24 12,22hemiacetal 58,66 2,24
Acnistin A ( 5 8 )
175,25
58,66
Jabcrosalactone U (55a) 128.176 Trihydroxy Compounds with Y-lactone in the side
12,22-~xidc 22,2641 de 22,26-olide 12 -Me0
---
* 264 (CH2C12:hex)
--
--
-
J. mageiianica
a9
Trechonates l a c i n i a t a
45
-do-
45
A . ramifluorum
Meirs
Leaves
56 66 2 248 ,258
42 215
J. sativa
chain :
Ixocarpalactnne B (59)
4 6,200,
56,65
2
Acnistin E
4 6,178
56 ,66
2
(60)
22
~
25
P . ixocarpa Brnt
20,24-cyclo methylene
--
--
A. ramifluorum
Meirs
25 42
Tetrahydroxy Compounds in the side chain:
idth I -lactone
Ixocarpalactnne A (61)
46,166, 205,22a
56 ,613
2
Withajardin E
36,48,
58 ,66
-
(61a)
1 7 ,24a ~
-21,25-cycln
294-95 (Et0Ac:MeOH)
+
a4
P . ixocarpa
Brot
Depreaorinocensis
25 25a
151 Fig, k .
3p,6P- Epoxides with Modified side chain O
OH
55 .
55a . 24P,25P - epoxy
5 6 . R = H , R, = OH 5 7 . R = OIVIe,Ri=OH
OH OH
Acnistin A (58) Acnistin £ (60)4P - OH OH
HO Ixocarpolactone B (59) HO O
OHE
OH 6 1 . Ixocarpolactone
OH
61 ( a )
Id
152
TABLE
6a ,7=-EPOXIDES W I T H 178- SIDE CHAIN ~
~
Compound
Other groups m.p.
Hydroxyls Epoxide Ene
[a ID
("C)
~~
Source
Ref.
Monohydroxy Cmpounds :
Lycium substance B (62)
f3,7a
2,24
--
260-65
+ 107
Lycium chinenseleaves 35
Withanicandrin (63)
6a,7Q
2,24
12-0x0
267-69
+ 105
Nicandra physaloides
90
Daturalactone A (64)
6w ,7a 24a,25a
2
12-OX0
303-05
+
Datura guerolfolia 8 D stromoniurn
91 31
Daturalactcne D (65)
6a ,7a
2
--
Nicalbin B (66)
6a ,7a
2
5-Dehydroxywithanolide R (67)
6a ,7a
2,24
--
Withancne (68)
w,ia
2,24
--
5-27-dihydrcxy-1-0x06a,7a-epoxy-22R-witha2,24-dienclide (693
@,7a
2,24
--
Withanolide R (70)
6a ,7a
2,24
-_
6a,7a
2
'
16,26-rxidc
--
282 275-76 (EtOAc)
63
+
--
24.2
--
.
D . queroifolia
92
Nicandra physaloides var. albiflora
93
W. somnifera (Pak variety)
94
Gi. somnifera Dun
66
Dihydroxy Compounds :
Nic-3 (71)
24 a,25a
275-76 (CHC13:EtOAc) 230-32 (EtOAc) (27-acetate)
f
+
81 73
- 64 182-83 (23-Acetate) -22,26-lactcl 248
(Indian chemctype) -do-
66
W. somnifera 95 (Chemctype I V s . 111)
Nicandra pysaloides
96a,b
TABLE
Id ( Ccntd.
6a,7a-EWXIDES WITH 178- SIDE CHAIN Compound
Hydroxyls Epoxide Ene
Other groups m.p. ("C)
Nic-7 ( 7 2 )
5a,26a
12-cxc; 22, 26-lactcl
5 a, 20aF-dihydrcxy6 a,7 wepoxy-l-cxc-witha2,24-dienclide (73)
5a,206
Daturalactone B (74)
5a,12a
Daturalactcne C (75)
5a,12B
Nicandrin B (76)*
5a,12a
m,7a 24a,25a W,7a
@
,7a
@,7~ 24a,2Sa 6a,7a
2
263 (Benz :CHC13) 282-4
I a ID _-
+
Source Nicandra physaloides
96a,b
w.
coagulans
16a
91
34
2,24
--
2
--
260-61
0
2
--
285-86
-I-73
N. Physaloides 8 D. quercifolia D. quercifolia
2,24
--
246-48
+
N. physaloides var.
88
110.7
Ref.
97
albiflora
Withaferoxolide (76)*
5a,12a
6a,7a
2,24
--
Vamcnolide (77)
5a,14a
6 ,7
2
--
*
*
P. angulata leaves
98
Ixccarpanclide (78)*
5a,206
6a,7a
2
--
*
*
P. ixocarpa aerial parts
99
W,20 6
6 ,7
2
-
281-83
-123.59
W. somnifera r m t s
217
256-58 (EtOAc) (3-Acetate)
+
71.5
W. somnifera (Indian chemctype)
66
257
+
60.5
W. somnifera (Chemotype I11 Vs. Ind. I)
100
Withasomnifercl B (29
>
278 (mci3)
116.4
D. ferox aerial parts 31
Trihydroxy Compounds :
1 a,3 6 , 5 &trihydrcxy6 a, 7 wepcxy-1-cxc-22Rwitha-24-enclide (80)
1~,36,5a ~ , 7 a 24
Withanolide T (81)
5",14", 20%
6",7a
2,24
-
--
153
154
TABLE Id ( Ccntd. 6a,7oEPOXIDES WITH 178- SIDE CHAIN ~~
~
~~~~
Compound
~
Hydroxyls Epoxlde Ene
Vitastramonolide (82)
5 SlP, 27
Nicalbin A (83)
5";1@, 26
i elD
Other groups m . p . ("C) 269-70 ( MeOH
6a,7'3 2 24a,25a
+ 37
W ,7a
2,24.
--
250 (CHC13:MeOH)
i29.8
140 -hydroxywithancne (85) 5 a, 148, 17
W,7"
2,24
--
280 (EtOAc)
+
148 -hydrexyixccarpanelide 5 a, 146, (86) 20 a
6a,7a
2
5 a, 120,
Withascmniferol A (86b)
5 a,208, 27
(86a)
N . physaloides
albifl ora
14"-hydrcxywithancne (84) 5 a, 14a, i7a
158 -hydroxynicandrin B
*
do-
36.7
*
*
Nct available
$'*
--
+
271-73
: May be identical
+
74.8
var.
93
s o m n i f e r a (Ind. VS. 102 Chemotype i11)
P. a n g u l a t a D. ferox
2,24
101
w.
150
6a,7a
Ref.
D . strarnonium
22,26-lactol 241-43 (EtOAc)
24,25-dihydr@
Source
leaves
W. somnifera rcots
Identity nct established
102 103
318 21T
155 Fig.Id. 6 a,7 a - Epoxides with 17 P - side chain
OH 62. Lycium substance B
72. 1 2 - 0 X 0 , 26 < , 24a,25a-epoxy OH
63. 1 2 - 0 X 0 , 64. 12 - 0X0 , 24a,25a - epoxy, 65. 24,25-diH
73. 20P-OH 74. 12a -OH , 24a,25a - epoxy
66. 16,26-oxido,24,25-diH
75. 12P-OH, 24a,25a-epoxy
67. 5 - deoxy, 23 a - OH ,
76. 12a-OH
68. 17a -OH
77. 14a - OH , 24,25 - di H
69. 27 - OH
* 78. 20p - OH , 24,25 -di H
70. 23a-OH
• 79. 20p OH, 24,25-diH
71. 24a , 25 a - epoxy H 26 < OH * identity not established , appear different by physical constants.
156 iMg. Id. 6a,7a- Epoxides with 17 (]-side chain
80. l a - 0 H , 3 p - 0 H , 2,3 - di H 81. 14a - OH, 20p - OH 82. I2a-OH,27-OH
83. 16a-OH, 26 < 24a,25a - epoxy OH 84. 14a - OH, 17a - OH 85. 14p-OH,17a.OH
86 . 14a-OH,20p-OH, 24,25a - di H 86a. 12a-OH,15p-OH 86b . 20p - OH, 27 - OH
Fig. le. 6 a,7a-Epoxides with 17 a-side chain
87. Withasomniferin A, 4en , 17p - OH H 88. 5a - OH , 17p - OH , 26 < ,24a ,25 a - epoxy, 2 en OH OH 89. 5a - OH, 25 a - OH, 26 < , 17p , 24 p - Oxido , 24,25 - di H H
TABLE
le
6a,7crEPOXIDES WITH 17
Ccmpound
Hydroxyls Epoxtde Ene
-
SIDE CHAIN
Other grcups m.p. <"C)
[a],
Source
Ref.
Mcnohydrcxy Compounds
Withascmniferin
ZL
--
W. scmnifera (Pak. variety)
224-45
--
N. physaloidesleaves 104
277
_-
(87)
94
Dihydrcxy Compounds
Nil Trihydrcxy Compounds :
5a,17B,
Nic-2 ( 8 8 )
6 a , 7a 24a,25a 2
--
26a
5a,25a, 26 B
Nic-11 (89)
6a,7o
2
17i.245 cxidc
m L E
-do-
96b
If
6a,7a-EPOXIDES WITH AROMATIC R I N G D (ME0 COHPOUNDS)
Compound
Hydrcxyls Epoxide Ene
Nic-1 ( 9 0 )
5a,26a
Nic-1-lactcne (91)
50
5 a,26a 24a,25a 5u, 6 a 24a , 2 P
Other groups m.p. I ' C )
2
--
138
2
--
230-32
[%ID
--
+
Source
N. physaloides
Ref
105
34.6
-do-
106
-dc-
107
5 a, 22
6Q
la
2
24-rxo
86
Nic-12 (93)
5a
6a
la
2,22
24-cxo
174-75
_-
-do-
107
Nic-10 ( 9 4 )
5a
6a
la
2
20-ow
107 (EtOH)
_-
-do-
1 U7
157
Nic-17 ( 9 2 )
--
158 Fig. If. 6 a,7 a - Epoxides with aromatic ring D
Nic - 10 ( 94 ),
Nic-17(92),
R=
Nic- 1 - lactone ( 9 1 ) , R =
Nic-1 (90),
R=
OH H
TABLE lg 5-ENES WITH 1 7 a SIDE C"
Compound
Hydroxyls Epox-Lde Ene
Other groups m.p. ( " C )
Source
[a],
Ref.
Mcnchydrcxy Compunds:
-
*
57
i7.5
*
57
2,5,8(14) 24
tic name ( 9 6 )
Withaphysalin A (97)
2,5,14, -184-85 24 -* 5,24 2,5,24 18-20 lactcne 222-23
Withaphysalin D (98)
3,5,24 18-20 lactcne
Seccwithametelin ( 9 9 )
2,5,24 27-Me0
190
i28.53
Jaturametelin C (100)
5,24
27-Me0
199-202 (CHC13:MeOH)
i68.7
-do-
110
Daturametelin E (101)
5,24
3 -OS03H
An amcrphcus pcwder
i25.8
-dc-
110
Ccagulin (101a)
3,5,24 14 ,20 -cxidc
coagdens
126a
Withascmifercl
14,155-epoxywithanclide I 20 (101b)
14,155
2,5,24
--
210-11
i56.5
No najle ( 9 5 )
*
* i43.6
*
W. somnifera
5 7.3
P. minima
64
(Indian variety) P . minima var. I n d i c a
108
D . metel
109
w.
Withania c o a g u l e o s
16c
159
lg ( Ccntd.
160
TABLE
5 - W S WITH 178- SIDE CHALN
Compound
Hydroxyls Epoxide
Ene
Other groups m.p.
("C)
[a
ID
Source
R2f.
Dihydrcxy Ccmpcunds :
17Q, 27-dihydr~xy-1-cxc 22a-witha-2,5,24trienclide (102)
17a,27
7,27-dihydrcxy-l-cxc witha-2,5,24-trienclide
78,27
--
2,5,24
--
205
+
74
(27-acetate) (acetcne:hex)
--
66
2,5,24
--
208-09 (7,27diacetate)
-
119
-do-
66
2,5,8(14), 24
--
224-25
+
48
*
57
Withanelide 0 (105)
2,5,8(14), 24
--
201-02 (CHC13:EtOAc)
+
112.5
Withanclide N (106)
2,5,8(14) 24
--
Amcrphous + 31 (27-acetate)
Withanclide G (107)
2,5,24
--
194-95 (EtOAc)
+
52.5
Withanclide J (108)
2,5,24
--
215-16 ( CHCl :EtOAc
+
32.5
-do-
111
Withanclide L (109)
2,5,14,24
--
213 (EtOAc)
+
9.6
-do-
111
(103)
Nc name
(104)
Withanclide M (110)
17a,205
14 ,15
2,5,24
--
240 (EtOAc)
+ 44.6
Withanclide I (111)
17a,20E
--
3,5,24
--
184 (EtOAc)
+
118
W. somnifera (Chemctype I) -dc-
W. somnifera (Chemctype 111)
w. somnifera (Chemctype 111) -do-
78 78 111
111 111
TABLE
lg ( Ccntd.
5-ENES WITH 176- SIDE CHAIN
Compound
Hydroxyls Epoxi.de
Ene
Other groups m.p.
[ QID
("C)
Scurce
*
Ref.
4 6,208
--
2,5,8(14) 24
--
230 (4-acetate)
+
110.6
la -hydrcxywithanclide (113)
la,14a
--
5,24
--
152-55
+
48
W. aristata Pauq (aerial parts)
112
Withaphysalin C (114)
14 8
+
33.4
P. minima (Indian chemctype)
113
114
J6-Withanclide
13,14 2,5,24 epoxy (13,14-secc)
8-20 lactcl 202-03
14a,208
--
2,5,16,24
--
212-13 (EtOAc)
+
44
W. sornnifera (Chemctype 111)
5,6-desoxywithaferin A (116)
4 8,27
-
2,5,24
--
218-20 (MeOH)
+
42.5
A.
2,3-dihydrc-5,6-desrxywithaferin A (117) (20S,20R)-la-acetcxy-3 hydroxyitha-5,24dienclide
46,27
--
5,24
27.4
-
5,24
212-13 (EtOAc) Amcrphcus
+
la-OAc,36
---
--
2,5,24
--
--
--
2,5,24
--
200-02 (EtOAc)
(115)
18-acetoxy-4-decxy208,18-OAc 5,6-decxy-5-withanclide D (11W
+ 58.8
--
breviflorus Gresib -dr-
57
70 70
Dunalia australis
115
Iochroma fuchsioides
46a
W. somnifera (Indian chemvtype)
95
rrcts
Trihydroxy Compeunds :
Withanclide Q
(119)
17a,236,27
-
6.6
161
lg ( Ccntd. 1
162
T&LE
5-ENES WIITH 17 - SIDE CHAIN ~
~~
Compound
Hydroxyls Epcxide Ene
L QID
Other groups Q . P . ("C)
Uithanolide H (120)
140,20~, 27
2,5,24
141-42 (EtOAc) (27-acetate)
Withanelide K (121)
140,170, 20
2,5,24
218-19 (CHC13:EtOAc)
17bhydrexywithanclide K (121a)
140,178,
2,5,24
45,76,20cctrihydrexy-1rxc-witha-2,5,24trienolide (122)
46,7B ,208
2,5,24
lB-acetoxy-5,6-deoxy5-withenclide D (118b)
45,208,18CH20Ac
2,5,24
Withanclide U (123)
45,140,
2,5,24
140,206,27
3,5,24
3 8,2C 0 -trihydrexyF 20R,22R-witha-5,24dienrlide (125)
10,35 ,208
5,24
Pubescenolide (126)
lQ,36,27
5,24
Physalclactcne B (127)
la-OAc, 38,206
5.24
(
124)
i0
~
+
35.5
9.2
Ref.
Y. somnifera
111
-do-
111
W. coagulens
16c
205
206
27-hydroxywithanrlide I
+
Source
234
+ 9.8
Dunalia australis (Griseb) Sleum
116
Iochroma fuchsioides 46a 230 (4-acetate)
+
* 273 (EtOAc)
110.6
* +
19.8
W. somifera Chemotype 111 Vs. Ind. I)
* W. somnifera
100
58 117
(Chemctype 111)
* *
* *
P. pubescens
118
P. peruviana
119
TaBLe lg
5-ms WIm Compound
Hydrexyls Epoxide Ene
( Ccntd.
1
178- SIDE CHAIN
Other groups m.p. ("C)
[.I,
Ref.
14a-20a,27-trihydrexy20R-22R-1-cxc-witha3,5,24-trienclide (128)
14a,20J, 27
--
3,5,24
--
Scminene (129)
la,36,27
-
5,24
-
Withajardin (130)
46,14Q, 17Q,25
--
2,5
3a-hydrcxy-2,3-dihydrcwithanelide H (131)
36,14a, 20aF,27
--
5,24
--
190-92 (EtOAc:MeOH)
i
64.7
W. coagulaos fruits
121
(ZOR,22R)-3 Eacetcxy-1a, lZB,ZO-trihydrcxy-l-cxewiiha-2,5,24-trienclide (132)
h ,36-OAC, 126,205
--
5,24
--
135-37
-
16.7
Dunalia a u s t r a t i s
115
184 (EtOAc)
i
98.7
W. somnifera (Chemctype 111)
117
*
*
W . somnifera
120
*
*
Deprea procumbens
51
Tetrahydrcry Ccmpouuds :
*
Nct
21,24-cycle
leaves
available
163
164
Fig.lg . 5 Eiics with 17 p- side chain
95 96 97 98 99
. . . . .
14a-OH, A^A«^''^ 20P - OH, A^ , A^^
14a - OH, A^ 18,20-lactone 14a-OH, A\18,20-lactone 21-OH,A^27-OMe 100 . 21-OH,27-OMe 101 . 21-OH,3p-0-S03H
101a. 101b. 101c. 102 . 103 . 104 . 105 . 106 . 107 . 108 . 109 .
27-OH,A\l4a,20a-oxido 20P-OH, 14,15 p-epoxy,A^ 3p - OH, 14a, 15a - epoxy 17a-OH, 27-OH, A^ 7p - OH, 27 - OH, ^ 17a-OH,27-OH,A^A^^''*^ 4P-0H, 17a-0H,A',A''^'^^ 7a - OH,27 -OH, A^A'^'^^
4a - OH,20p - OH, A' 17a- OH,20p-OH,A^ 17a -OH, 20p - 0 H , A ' ^
110 . 17a - OH,20P-OH, 14a ,15a - epoxy, A^ 111 . 17a- OH;20p-OH,A' 112 . 4p - OH,20p - OH, A^A'8(14) H 113 . 1< , 14a-OH OH
114 . 14P-OH, 13,14, seco - 13,14 - epoxy, A^ 115 . 14a-OH,20p-OH,A%A'^ 116 . 4p - OH, 27 - OH, A' 117. 4p-OH,27-OH, H 118 . 1< , 3P-0H OAc 118a. 20P-OH, 18-OAc, A^ 118b. 4p - OH, 20p - 0H,18 - OAc, A^ 119 . 17a - OH,20p - OH,27 - OH, A^ 120 . 14a - OH, 20p - OH,27 - 0H,A' 121 . 14a - OH, 17a - OH, 20p - OH, A' 122 . 20p-OH,A^ 123 . 4P-0H, 14a-OH,20p-H,A' 124 . 14a - OH, 20p - OH, 27 - OH,A^ 125 . la - OH, 3P - OH,20p - OH 126 . la - OH, 3P - OH, 27 - OH H 127 . 1>< , 3p - OH, 20p - OH OAc 128 . 14a - OH, 20a - OH,27-OH, A^ 129 . la - 0H,3p - OH, 27 - OH 130 . 4P-0H, 14a-OH,17a-OH,25-OH, A-,21 - 24 - cyclo, 24,25 - di H 131 . 3P-0H, 14a-OH,27-OH,2,3-diH, 20p - OH H 132. 1>< ,3p-OAc,12p-OH,20p-OH OH
TABLE
lh
5-ms WITH 17aCompound
Hydroxyls Epoxi.de Ene
__
Withanclide withnchydrcxyl k%ydroxy Compounds : Neb? nithznclide (13%) 17 B
SIDE CHAIN
Other groups m.p. ("C)
[ 0ID
Source
Ref.
- -.
3,5,24
14-20 Oxy
W . somnifera
85b
--
2,5,24
14-20 Oxy
W. somnifera
05c
__
2,5,24
--
216-17 (EtOAc)
W. somnifera
85b
2,5,24
-dc-
85c
_-
2,5,24
--
192-93
--
W . somnifera
85a
*
*
P. alkekengi (Turkmenisrhan)
122
*
*
P . viscosa
123
Dihydroxj Compounds :
Withanclide P (133b) New withanclide
140,176 173,200
+
51
Trihydroxy Compounds :
Withanclide F
(134)
1 4 ,176, 205
5.24
Physanclide (135)
4-CXO
(Chernctype 111)
Tetrahydroxy Compounds :
Nil Pentahydroxy Compounds :
28-hydrcxywithaphysanclide (136)
Nct available
--
5,24
--
165
*
46,14a, 1)B ,206,28
166
Compound
Hydroxyls Epoxlde Ene
--
Daturametelin D (137)
21,24epoxy
2,5
Other groups m.p.
("C)
[al,
Source
Ref.
199.5-201.5
- 90.6
D . metel aerial p a r t s
110
--
210
-
64.4
D . metel leaves
12 6
--
280
-
77.7
Amorphous pcwder
i5.0
27-Me0
CYCl3-MeOH
Wlthametelin (138)
--
21,24
2,5, 25(27)
Isowlthametelin (139)
--
21,24
335, 25(27)
Daturametelin F (140)
--
21,24 epoxy
5, 25(27)
126,176,
--
2,5,24 12,23-cyclo; 23,26-olide
--
2,5
26,28-olide
217-18 (EtOAc)
i56.8
P. peruviana leaves (Varanasi variety)
127
--
5
26,28-olIde
239-40 (MeOH) 244-45 (EtOH)
-
P. peruviana (Varanasf. variety)
128
Jaborosalactone P (a splranic withanolide) (141)
22
Perulactone B ( 1 4 2 )
14a,176, 206,22a
Perulactone (143) (176-oriented side chaln)
la-OAc, 36,206, 22a
3 -OS03H
*
*
3.2
-doD. metel aerial parts J . odenelliana
126 110 40
167 Fig. Ih , § - Enes with 17a - side chain
I33b R =H,A^ 133c. R = OH, 14-deoxy,A^ 134 . R=OH 135 . R = 0 H , 4 - 0 X 0 136 . R = OH,28-OH,4p-OH
3,4-diH,A^
Fig.li. 5 Enes with Modifled side chain
137 . A%27-CH20Me 138 . A''"<">
139 .A''25^27)
140 . A ' " ^ ' ' \ 3 p - 0 - S 0 3 H
168
OH
141 Jaborosalactone P.
142 Perulactone B OH 0H =
143 Perulactone
(145)
OH 144 With aphysaJian £
TABLE lj INTEXMDIATE COMPOUNDS WITH 178- SIDE CHAIN
Compound
Hydroxyls Epoxide Ene
Ocher gxups m.p. ("C)
[a],
Source
Ref.
Hcnchydrcxy Ccmpcunds : 68
Withascmidiencne (145)
27
W. s o m n i f e r a
126a
Jaborosalactone B (146)
68,27
Jaborosa i n t e g r e f o l i a Lam
67
Jabcrcsalactone C (147)
58,27
--
2,24
6 -Cl
218-20 (CHC13:EtOH)
-
-dc-
130
Jabcrcsalactcne E (148)
6 8 , 27
--
2,24
5 -C1
225-28
--
-dc-
130
Withacoagulin (149)
5u 208
*
*
Withania coagiilans
131 66
-
2,4,24 18-20 lactcne 311-12
+
Withaphysalin E (144)
61.4
var.
P . minima Indica
129
Dihydrcxy Ccmpcunds :
I
2,6,24
-
2,6,24
-
238-39 deccmpcses ( CHC13 :EtOAc )
+
75.5
W. SOmnifera Dunal
5a,17a-dihydrcxy-l-cxc22R-witha-2,6,24trienolide (150)
5a,17a
-
2,4,6-trien-l-cne (151)
14u,206
-
2,4,6,24
-
220-22 (EtOAc)
+
12.2
W. s o m n i f e r a L (Chemctype I1 Vs. Ind. I)
132
Pubescencl (152)
4u.7a
24 ,25
-
--
180-81
+
11.4
P . pubescens
24a
Scminclide (153)
48,27
14 ,15
-
*
*
W. s o m n i f e r a
120
2,24
(Indian chemctype)
(Panistan variety)
169
170
TABLE lj INTEWJDUl"E
( Ccntd. )
COIPOUNDS WITH 176- SIDE CHAIN ~
Compound
Hydrcxyls Epaxi.de Ene
Other graups m.p. ("C)
Source
[QID
Ref.
5a-methcxy-4,5-dihydrcJabcrcsalactcne B (154)
6 427
--
2,24
5 a-Me0
200-01
--
5*ethoxy-4,5-dihydrcJabcrcsalactcne B (155)
68,27
--
2,24
5a-ethcxy
203-04
--
GO-chlcrc, 5 Ehydrcxywithaferin A (156)
4Bt5B,27
-
2,24
6Q-Cl
281-84
+
68
4B,5 B,20 F -trihydrcxy-
48,5B,16Q
-
2,24
6a-Cl
247-49
+
76
Dunal i a tubul osa
5a
Acnistcferin**(158)
5Q,6B ,27
285-88
+
99.6
Acnistus b r e v i f l o r u s
41
Jaborosa i n t e g r e f o l i a
130
Acnistus b r e v i f l o r u s
-do-
69 69
Trihydrcxy Ccmpeunds :
Q
Withania f r u t e s c e n s
133
Pauq
6-chlcrc-1-cxc-witha2,24-dienclide (157)
227-29 (EtOH)
Jabcrcsalactcne D** (159) 5a,68,27
-
Withametelin C (160)
5a,68,21
--
24
Physagulin B
6B114Q 15a-OAc
--
2,16,24 5Q-Cl
(161)
**
* A white powder
May be identical
--
* 159.5
Lam D. metel
135
P. angulata L
77
Compound
Hydroxyls
Epoxide
Ene
Other groups
m.p. ( " C )
294-95
--
2,7,24
-_
Jaborosalactone F (163) 5a,65,12a, 27
-
2,24
--
Withamf.nCmin (164)
5a,66,14a. 15a-OAc
--
2,16,24
--
48, 5a,66,
19,27
WCthasomnCferol C (162) 5a,14@,205
[a],
+
67.78
Source
U. somnifera
Ref.
217
Tetrahydroxy Compounds:
268-70 (Acetone: Pet. ether)
-
208 (Amorphous powder)
-.-
Jaborosa i n t e g r e f o l i a 136 Lam P . minima
21b
Jaborosa l e u c o t r i c h a
3~~
Pentahydroxy Compounds: Jaborosalactone X (165)
24
204-05
171
172 Fig. IJ , Intermediate Compounds with 17(3- side chain
R,A^'<=
146 . A \ 6p . OH, R, = H, R2 = OH 157. 4P - OH, 5p - OH, R, = OH, R2 =H,6a-Cl 147. 5p - OH, 6a - CI, R, = H, R2 =0H 158. 4p-OH,5p-OH,16a-OH,6a-Cl, 148. 5a - CI, 6p - OH, R, = H, R2 =0H R, = R2 = H 149. 5a - OH, R, = OH, R2 = OH, A' 159. 5a - 0H,6p - OH,R, = H,R2 = OH 150. 5a - OH, 17a - OH, R, = R2 = H,A^ 160. 5a - 0H,6p - 0H,21 - OH,R, = R2 =H, 151. 14a - OH, R, = 0H,R2 = H , A \ A^ 2,3 -di H 152. 4a - 0H,7a - OH,24a ,25a - epoxy, 161. 5a-CI,6p-OH,14a-OH,15a-oAc, R, = R2 =H R, = R2 = H 153. 4p - OH, R2 = OH, R, = H, 162. 5a - OH, 14a - OH, R, = R2 =H 14a, 15a -epoxy 163. 5a - 0H,6p - OH, 12a - OH,R, =H,R2=0H 154. 5a - 0Me,6p-0H,R2 =OH,R, = H 164. 5a.OH,6P-OH,15a-OAc,R, = R2=H 155. 5a - OEt, 6p - OH, R2 = OH,R, = H 165. 4p - OH, 5a - 0H,6P - OH, 19 -OH, 156. 4p - OH, 5p - 0H,R2 = OH, 6a - CI, R2 = 0H,2 ,3 - di H R,=H
lk
TABLE
INTERMEDIATE COwpouNDS WITH 17a SIDE CHAIN ~~~
~~
Compound
Hydroxyls Epoxide Ene
~ ~ _ _ _ _ _ _ ~
~
Other groups m.p. ("C)
I QID
Source
Ref.
Monohydroxy Compounds : Ni1
Dihydroxy Compounds N o Name (166)
:
56.178
12.22oxi.do
-Jaborosalactone T (166a) 58,178 Trihydroxy Compounds : Jaborosalactone R (166b) 66,128,178 -Tetrahydroxy Compounds :
--
Wi thaperuvi.n C (167)
66,14a 178,206
Wtthaperuvin D (168)
48,56, 14a, 2a,6a178 ,206 oxido
Physalolactone C (169) 46,58, 176,20 8
--
Withaperuvin F (170)
46,56, 176,206
3a,6aoxido
4-Deoxyphysalolactone (171)
56,14a, 176,208
--
Jaborosalactol N (172) 68,148, 178,26a Withanolide C (173)
--
2
6a-C1: 12 -Me0 * 23,26101Cde Q-C1; 12-0Me 234-35
2,4
--
2,4,24
--
*
24
.-
209-11
2,14,24 6a-Cl 2,14,24 2,24 2,4
2,24
-6a-Cl
-5 -C1
*
J. magelianica
138
*
J.
sativa
219
sativa
219
*
263-64
+
*
*
P . peruviana
139
12
-do-
140
*
P . peruviana
141
174-75 (CHC13)
+
207-208 (Et0Ac)
i103
183-85
--
J.
39.70
-doP . peruviana
(Varanasi variety)
--
Jaborosa bergii
--
Y. somnifera
26b 142 37 137 173
68,14a, 178,206
24a,25a
2.24
174
TABLE lk (Contd.) IMZRWDIAlZ COMPOUNDS WITH 17'-
SIDE CHAIN ~~~
Compound
Hydroxyls EpoxCde Ene
Other groups m.p. ("C) 5a-Cl
*
--
265-66
68,14a,178, 206 Jaborosalactone S (166~)5a ,613,128,178 Pentahydroxy Compounds:
---
2,24
WCthanoltde S (174)
50,66,14a, 7 6,206
--
2,24
--
Physalolactone (175)
48,56,14a, 176,206
--
2,24
6a-c1
Wtthaperuvtn B (176)
48,58,6a, 176,206
--
2,14,24
Wtthaperuvtn H (177)
48,58,14a, 178,208
-
2,24
28-Hydroxywithaperuvtn C (178)
66,14a,i76, 208,28
--
2,4,24
--
*
--
2,24
--
*
--
2,14,24 6GCl.
Wtthanolide C (17%)
Wtthaphysanoltde (179) 46,5",14a, 178,208
2
--
[a],
Ref.
Source
* *
3. sativa
219
137
W. somnifera
272
+
97.5
P. peruviana
143
227-28
-k
29.4
-do-
144
*
*
4,6-mercapto 213-14 acetaldehyde adduct
+
84.3
P. peruviana
roots 139
-do-
P.peruviana -do-
145
leaves
88
146
Hexahydroxy Compounds:
23-Hydroxy physalolactone C (180)
48,58,17B, 20 8,23a
202-04
-k
60
P. peruviana
leaves 147
lk (Contd.) UITB 170- SIDE CIIBLN
TAE4IZ
INTERMEDIATE Epoxtde
Wtthaperuvh (181)
4 8,5 8,6 Q, 14~,178,208
--
2,24
--
28-Hydroxywithaphysanoltde (182)
48,5~,14~, 178,206,28
--
2,24
-
*
Physagultn F (183)
58,68,148,
2,24
--
A white powder
*
15Q-OAc
16?,178
Other groups
m.p. ("C)
Hydroxyls
Compound
Ene
COMPOUNDS
225-27
(MeOH)
Ref.
Source
+
156.8
*
+
70.7
P. peruviana (Varanast vartety)
142
P. viscosa leaves
123
P. angulata
148
L
Not available
175
176
lAoA^
OH
(166)
OH
OH ( 177) With aperuvinU
166a . 5P - OH , 6a - CI, R = OMe 1 6 6 b . 6 P - O H , R = OH,A4 166c . 5a - OH , 6p - OH, R = OH , 24,25 - di H
177 Fig. 1 k. Intermediate Compounds with 17a - side chain
167 . 6p - OH, 14a - OH, Ri = R2 =0H, A^ 168 . 4p - 0H,5P - OH, 14a - OH, R, = R2 = OH,2,3 - di H,3a -,6a -Oxido 169 . 4p - OH, 5p - OH, Ri = R2 =0H, A^\ 6a - CI 170. 4p - OH, 5p - OH, Ri = Rz = OH, A'\ 2 ,3 - di H,3a , 6a - Oxido 171. 5P - OH, 14a - 0H,6a - C1,R, = Rz =0H 172. 6P - OH, 14P - OH,R, = 0H,R2 =H, H 26 -C < , 24a ,25a - epoxy OH 173. 6p - OH, 14a - OH, R, = R2 ^ OH, 5a - CI.
174. 5a-OH,6p-OH,14a-OH,R, = R2=OH 175 4p-OH,5p-OH,14a-OH,R, = R2 = OH, 6a-CI 176. 4p - OH, 5p - OH, 6a - OH,R, = R2 =0H, 178. 6p - 0H,14a - OH,R, = R2 =OH,28-OH, A^ 179. 4p - 0H,5a -OH, I4a -OH,Ri =R2 K)H 180. 4p - 0H,5P - OH,R, =R2 =0H 23a - 0H,6a - CI 181. 4p -0H,5p-0H, 6a - 0H,14a-0H, R, = R2 = OH 182. 4p - 0H,5a - 0H,14a - 0H,R,=R2 =0H, 28-OH 183. 5p-OH,6p-OH,14p-OH,15a-OAc, 16p,17p-epoxy
Compound
Hydroxyls Epoxide Ene
Wi thanolide Y (184)
7Q17Q,205
5ah
2,24
Salpl.chrolide A (Ring D-arornatlc) (185)
26
5a,6a
2,13,15,17 22-26 (i3-18) lactol abeo
Somniferin (184a)
58 ,27
5a,6a
24
178
TABLE 1L 5 a,6 -Epoxu)ES
Other groups m.p. (“C)
--
4 -0Me
[a1 ,
270-73
--
179-80
--
--
--
Source Y. somuifera (Chemotype 111 vs. Ind. I)
Ref. 149
S. o r i g i n i f o l l a
52
W. somoifera
57a
QI,
Source
Ref.
-47.6
W. somnifera
132
I “I”
Source
Ref.
J. magelienica
150a,d
J. leucotricha
151
TABU l m 6 478-EpoXDES
Compound
Hydroxyls
66,7B-Epoxywithanone ( 186)
5a,14% 17a
Epoxide Ene
Other Groups m.p. (“C)
--
6 6,75 2 9 24
TABU
242-44 (CHC13)
[
In
pI1woLIc UI-LUIES Compound Jaborol (187) Jaborosalactone Q (188)
Hydroxyls
-1~,65,27
Epcxide Ene
---
Other groups m.p. (“C)
-1,3,5(10) 24
--
135 (MeOH)
--
176-77
---
179 Fig. 1.1. 5a,6a - Epoxides
(186)
^"'H
Fig, l.n. Phenolic Withanoiides O
(187)
OH
(188 )
180 71 withaferin A place
73 (12), withanolide D
at primary, secondary
(19). Hydroxylation
as well as tertiary
takes
positions.
A
primary alcoholic group is generally observed at C-27 in these 44 compounds while that at C-18 as in withacnistin (16) and lochroma withanolides ^ and at C-28 as in visconolide (54) is rare. Even hydroxylation at C-19 has been recently noticed ' Hydroxylation at C-14, C-17, C-20 is very common in withanolides eq. wi thanolide E "''"'^ (46). A close observation of the withanolides containing 6 a, 7 of-epoxides suggests the absence of C-4 hydroxyl in them unlike in 5 3,6 3-epoxides. Hydroxylation or an oxof unction at C-12 are 97 noticed in some of the compounds eg. daturalactone C (75). 14 3-hydroxy compounds have been isolated from Physalis pubescens 24b eg. physapubenolide (37) and from Withania somnifera eg. 102 (85) and even recently from Jaborosa 143-hydroxywithanone bergii Hydroxylation at C-15 was observed only in five compounds either
free
or
in
the
form
of
Physapubescein (35) and physapubenolide 95 95 Q (119) and R (70) possess 23-hydroxyl. noticed in nicalbin A93(83).
acetoxy
groups
eg.
(37). Withanolides C-16 hydroxylation is
Hydroxylation at C-1 and C-3 is mostly noticed in 5-enes. Epoxidation at the C-5 and C-6 positions with Epoxidation 5 0 , 6 3-conf iguration and at 6,7 position with G «, 7 «-conf i gura l.icjn is most common in withanolides. The isolation of withanolide Y149 (184), a 5a ,6a-epoxide 132 and 6 3,73-epoxywithanone (186) with 63,7B-configuration provides rare examples. Similarly epoxidation at C--2 and C-3 as in viscosa23 (22) (2 3,3 3-epoxy compound) and at C-14 and C-15, as in lactone A withanolide
M
(110) are also
rare.
In the side
chain,
epoxidation takes place prominently at C-24 and C-25 positions as , m . „. ^ . . . 104-107,96b , pubescenol , ,24a, datura, ^ ^ in Nicandra NiCi found steroids c' ,91,97 lactones^ 86 (52) has been ^^ig^ ~^i 7^ " Very recently physagulin C epoxide) isolated by Shingu et al. from Physalis angulata L. The
oxirane
bridge
is noticed
between
C-12 and C-22
181 positions, eg. Jaborosalactone M (44), trechonolide A (56), datumetelin"^^^ (137) and daturilin"*"^^ (138). The 3o',6a-oxiao (171), the 163,23-oxido bridge in bridge in withaperuvin F 25 ixocarpalactone B (59) and the 16,26-oxirane bridge in nicalbin B 93 (66) are a few other rare examples, Two new withnolides of Got, 7 a-epoxide series94 have been recently isolated, one without 5ot-hydroxyl 4 A -ene having 2,3-dihydro system.
and
the
other
with
Unsaturation Unsaturation at C-2 and C-24 positions is common in withanolides, while it is present at C-5 in twenty-five compounds. Unsaturation at C-14 and C-16 is rare. The /r compound ' was supposed to be the intermediate in the formation of compounds with 25(27) 17°^-oriented side chain. Unsaturation at ^ has been noticed 125 7 in daturilin (138). Withasomniferol (61) with A unsaturation has been reported very recently along with two more new withanolides, withasomniferols A & B from the roots of W. .^ 217 somnifera Dihydro systems A number of 2,3-dihydro derivatives were isolated with saturation of the 2,3-double bond eg. 2,3-dihydrowithaferin A 71 (13). The side chain may also possess saturated lactonic system as in 24,25-dihydro compounds. Incidentally, some tetrahydro derivatives have also been isolated as found in 2,3,24,25-tetra hydro-27-deoxywithaferin A (10). A dihydrosystem at C-5 and C-6 was also noticed in pubescenol (152) which was isolated by Row 4- al. 1 24a et Aromatisation Aromatisation in expanded ring D is common in steroids 07,96b^ ^^^ nicandrenone (90). Even
Nicandra phenolic
withanolides with B-secosystem were also isolated, eg. (+)-jaborol ' (187). It is observed that aromatisation takes place in ring D in 6 a, 7 o-epoxides and in 5 a, 6 a-epoxides. Nicandra
Steroids Nicandra
steroids possess a 5^-hydroxy-6 «, 7 o-epoxy pattern.
182 Additionally, thoy can bo classified into two types, (a) ring-D aromatic compounds and (b) So^-0H-6a , ?« ; 24 a, 25 a-diepoxy lactols. Furthermore, the side chain has been extensively modified to yield such complex structures as in Nic-11 (89) and even with cleaved side chains as in Nic-10''"^^ (94) or Nic-l?-^^^ (92). Several Nicandra steroids are closely related to withanolides (Chart I ) . Among them withanicandrin 90 (63) is the simplest. Their structures were established by X-ray crystallography and by chemical and spectroscopic data. The most important structural modification is the aromatisation of the ring-D in Nic-1 , 10, 12, and 17 . The side chain is also variously modified. The biogenetic relation 107 among these compounds was suggested by Begley et al. (Chart II). The aromatisation of ring-D involves the participation of 18-Me and especially 17 and 14 hydroxyls.
Crombie
presumed that
the aromatisation involves the 16-hydroxyl which is not commonly found in this group of steroids. A simple and convenient scheme (Chart III) with commonly occurring 17,14-dihydroxy compounds was suggested by L.R. Row for these compounds . The removal of 17-OH gives rise to C-17 carbocation which sets forth a series of rearrangements involving the intermediate 17-18 cyclosteroids giving rise to expansion of ring-D into a benzenoid ring. It is, of course, possible to visualise other schemes, but the scheme suggested has the merit and involves common reactions well known to occur biogenetically. Withanolides - relation with physalins Physalin A^° (89), B"'-^^ (190) and C"^^^ (191) were first reported by Matsuura et al. from Physalis alkekengi in 1969 and they estblished their structures beyond any ambiguity by X-ray analysis and also by chemical and spectroscopic study. Chemically, they are also related to ergostanes and bear resemblance to withanolides. The physalins are 13,14-secowithanolides with a bicyclolactone system in the side chain. (Table 1.0) T • ^ al. - 154-156, 22 have ^ - 1 4 . ^ severaln new physalins u Row et isolated from P' angulata and P- lancifolia . They are physalins D 154 (192), E 1 5 5 (193), F 1 5 6 (194), G^^ (195), H^^^ ^^^^^ ^ ^22 ^^^^^^ 156
(3^93) ^nd
K^^
(199).
The
structures
of
the
above
were
183 CilART - i BIOGENETIC RELATION OF NICANDRA STEROIDS TO WITHANOLIDES
6H
OH
^
Nic -3 (71)
H Nic-17(92)
{
O Nic -10 (94)
Nic -1 (90)
O Nic -12 (93)
184 Chart -11 Biogenetic Scheme ( Begley ct al) 1^7 ( for aromatisation in ring - D involving C - 18 methyl ) R
185 Chart - HI Biogenetic Scheme (L.R. Row ) 1^1 ( Ring - D aromatisation )
(O)
186
TABLE la PHYSALINS AND THEIR PHYSICAL CONSTANTS
Compound
m.p.
[a
1,
P h y s a l i n A (189)
266OC ( A c e t o n e ) 262°C (MeOH)
-
126
P h y s a l i n B (190)
25OOC ( A c e t o n e ) 271°C (MeOH)
-
150
Source
P.alkekeng1
var
.
franchelli
152
-do-
153
191)
274-277'C
EtOAc)
-160
Physalin D
192)
262-263OC
EtOAc)
-
68
P. minima
Physalin E
193)
305-307OC
MeOH )
-
83
P. angulata a n d P. (Leaves & Stems)
Physalin F
194)
295-296OC
-
20
Physalin G
19s 1
295-296°C
+
17
238-240°C
P h y s a l i n I (197)
305-306
(MeOH)
(MeOH)
+
12
20
-do-
Physalin C
P h y s a l i n H (196)
Ref
154
-doP.
lancifolia
P.
angula(Stems1 -do-
anc fo
I
155 156 22 155
22
TABLE lo ( Contd.)
PHYSALINS AND THEIR PHYSICAL CONSTANTS ~
Compound
[“I,
m.p.
-
Physalin J (198)
268-270°C
Physalin K (199)
280-282°C
Physalin L ( 2 0 0 )
248-249OC
- 118
Physalin N (201)
252-54’C
-
Physalin 0 ( 2 0 2 )
272-73°C
-
Physalin P (203
* *
Epidihydrophysalin C ( 2 0 4 ) Physalin (205)
270-274°C
60
Source
P.
--
angulata 8 P. l a o c i f o l i a
P . a n g u l a t a leaves
Re€
156 22
P. alkekengivar. franchelli
157
124
-do-
158
115
-do-
158
*
-do-
159
*
Wit h a r i n g i a c o c c u l o l i d e s
--
P . minima
47 160
187
188 O Phvsalins
O^ /P
Physalin A ( 1 8 9 ) ; R = OH PhysaliiiC(191);R = H EpidihYdrophYsalin C ( 2 0 4 ) ;
-24,25-diVdro
PhysaliiiO(202)R = OH, 25 pn - dihydro Physaiin L (200 ) AJ. 2,3-dihydro
Physalin B ( 190 ) , K> PhvsalinD(192); 5 a - O H , 6 p - O H Phvsalin E (193 ) ; 5 a - O H , 7 a - O H PhysaUn F ( 1 9 4 ) ; 5 p , 6 (5 - cpoxy Physalin G ( 1 9 5 ) ; A*. 6 p - OH PhysalinH( 1 9 6 ) ; 7 P - O H Physalin I (197 ) ; 5 a - OMe , 6 P- OH Physalin J (198 ) ; 5 a , 6 a - epox> Physalin K ( 199 ) ; 4 a» 5 a - epoxy - 6 a - OH PhysaUn (205 ) ; A4,6-OH
CH2 Physalin IS ( 2 0 1 )
physalin P ( 2 0 3 )
189 established
on the basis of chemical and spectroscopic evidence.
These are all derivatives of physalin B (190). 47 C (204) from Witharingia cocculolides
Epidihydrophysalin 4 , and a A ,
1fi0
(205) from P- minima
6-hydroxyphysalin
have also been reported
later. (200) was isolated from P. alkekengi
Physalin L
by Kawai
et al. and was shown to be 25-epi~4-dehydro-2,3^25,27-tetrahydro1 co
physalin A. Physalin O (202) (25S) 25,27-dihydrophysalin A and Physalin N (201a) were isolated from P. alkekengi var. franchetti. 159 (203) has been recently isolated from the same Physalin P species. There are several characteristic features of the physalins, A, B and C, in particular. a)
The lactone between C-18 and C-2 0.
b)
The oxido bridge between C-14 and C-17 with a 14 6-hydroxyl and oxido bridge between C-14 and C-27 giving seven membered oxygen heterocyclic system across the ring-C, D to the Y-lactone in physalin B.
c)
13,14-Seco ring system (C,D rings) with a 13a-hydroxyl.
d)
A ketone at C-15. The
biogenesis
of this
complex
and highly
oxygenated
steroids (8 rings) containing a minimum of nine oxygen atoms calls for
an elaborate
system
mainly formed in Physalis plants.
on the part of the plant. species
These
, not in the other
were
Solanaceous
A hypothetical biogenetic scheme, (Chart IV) was proposed
by Crombie et al. An insight into the biogenetic pathway can be gained from a study
of the structures
Physalis
species.
suggesting
that
of several
withanolides
cooccurring in
They were appropriately named as withaphysalins they
share
the general
characteristics of both 64 64 physalins and withanolides especially withaphysalins A (97), B (114) from P. minima ; 24b pubescens^ physapubenolide (37) from P. {2),
C
physapubescin
(35),
The withaphysallins A^"^ (97), B^^ (2) and C"^^"^ (114) share in common with physalins, the lactone (or lactol) between C-18 and C-20
and withaphysalin
C is a 13-14 secosteroid
with
an oxide
bridge between C-13 and C-14. It is noteworthy that withaphysalin A 64 (97) carries a 14 oe-hydroxyl while withaphysalin C (114) has
190 Chart - IV
OH OH Withaferin A (12 )
Withanicandrin ( 6 3 )
OH "OH Physlain A ( 189)
191 Q1
143-hydroxyl. Physapubescin (35) has the 24b function whorcas physapubenolide possesses along
with
14P-hydroxyl.
It
has
already
rare 15a-acetoxy 15^-acotoxy group
been
remarked
that
hydroxyl function is frequently noticed at C-17 and C-20 in several c o
withanolides.
A
corresponding
new
withanolide,
to withanolide
Turkhmenistan.
(54a)
physalactone in P. alkekengi
E occurs
found
in
This particular withanolide was shown to possess a
double bond between C-8 and C-14 which might be in fact an artifact from the parent 14a-hydroxywithanolide
(later A
structure was
corrected to 14a-hydroxy compound). CO
Thus
(54a)
physalactone
precursor
withanolide
essential
prerequisite
with
formation
of physalins.
to
can
be
regarded
as
14a,17a,20B~trihydroxy
start
the
biogenetic
the
ideal
system,
process
an
for
the
to
the
CO
Thus
physalcatone
is
the
key
biogenetic process, which is now represented in Chart V. step
is the oxidative
cleavage
of
C-13
and
C-14
bond
The key with
the
formation of 13-methylene, 14-ketone which sets the process to the course as shown in the Chart V Hypothetic biogenetic scheme to withaphysalin C physalin
(191)
C
by
17 o^OH-15-Oxophysalin scheme.
Odidative
transient
A
Glotter
14 a,i7 a-hemiketal
is
may
be
the
cleavage
of
13,14
13a,17a-dihydroxy-14-one
carbocyclic activates
E.
formation
frame work
from
16-methylene
which
precursor bond
(d)
(e).
in
(114) to
Chart
according
should
which
Thus
(d) due by
shown
is
to
result
the in
stabilised
rearrangement
to the presence
Michael-type
VI.
in of
addition
a by
the
15-one
at
24-25
olefinic bond affords the final product. Withanolide Glycosides Twenty withanolide glycosides have been isolated so far. Adam et al.49 isolated dunawithanine A (206) and dunawithanine B (207), m.p. 213° from the leaves of Dunalis
australis.
identified
evidence
on
the
basis
chemical degradation. (208)
was
isolated
(Varanasi variety). al.
from P- pubescens
of
spectroscopic
They were as
well
as
Physalolactone B-3-0-3- (D)-glucopyranoside by
A.B.
Ray
et
al.
from P-
peruviana
Pubescenin (209) was isolated by M. Sahai et 24b (Waltair variety) and it was shown as
la -acetoxy-2 0a -.hydroxy-24 a,25 a-epoxywitha-5-ene-22,26olide-33-0-glucopyranoside.
192 Chart - V Hypothetic biogenetic path of physalins ( L.R. Row ) ^^^
HO^ 5 .R
OH
OAc
OH
O HOL?^R
OH
OAc
H^
OAc
193 Chart -VI Biogenetic Scheme to 114 & 191 ( E . Glotter^3
Withaphysalin (114)
Physalin C ( 191)
194 Four new withanolide glycosides were isolated australis
by
Adam
et
al.
very
recently.
Dunalia
from
Among
the
four
glycosides, three contain physalolactone B as aglycone. Two
glycosides
were
isolated
Datura
from
tatura
L
29a
they were named as daturataturin A (210) and daturataturin B Very
recently
28-0-withanolide
glycosides
and G (223) have been isolated from Physalis Withanolide
glycosides
isolated
(211).
eg. physagulins E 1 48
angulata
so
far
and
(222)
L
are
listed
with
their physical constants (Table - Ip) Spectroscopic Studies of Withanolides U.V. Spectra Withanolides containing
5B,63-epoxides
/Nw^ 210-218 nm whereas 5 a-OH-6 a,7 a-epoxides /N^219-228 nm.
show absorption at
exhibit absorption at
Withanolides containing 5-enes also absorb similar
to 5a-OH-6ot,7a-epoxides in their U.V. spectra (See Table - 2 ) . I.R. Spectra Withaferin A^ (12), 2,3-dihydrowithaferin A^ (13) and 73 -1 -1 withanolide D (19) exhibit peaks at v 1692 cm , 1695 cm , -1 max 1700 cm respectively due to overlapping of the peaks of two chromophores.
Generally
«:3-unsaturated six membered ketone in -1 25 ring A is observed at/^'1685 cm . Ixocarpalactones A (61) and oc
19R
1?7
B (59), Perulactone (143), Perulactone B (142) contain a saturated-Y-lactone in the side chain which is recorded generally -1 24a at^^-'1770 cm
whereas pubescenol
epoxy- <s-lactone at 1728 cm
(152) exhibits its saturated
and its saturated six membered ketone
at 1710 cm""'". (See Table - 3 ) . "*"H NMR Spectra The
H NMR
spectra
(Tables
4a-d) of
the
polyoxygenated
steroids have been diagnostic to derive some basic functionalities present in these molecules.
For example an a:3-unsaturated ketone
is commonly present in ring A, the others being an a:3-unsaturated<5- lactone comprising the side chain, 5 3,6 3-epoxide; 6a ,7a-epoxide etc.
The chemical shifts of the 18 & 19-methyls give some specific
information regarding the prsence of 63,73-epoxide or 143-hydroxyl. The C-2
and C-3 protons of the a : 3-unsaturated
carbonyl
system
TABLE l p WITHANOLIDE GLYCOSIDES
Compound
r a1,
m.p.
Source
Ref.
Dunawithanine A (206)
208-213'C (Decomposed)
-+ o
Dunawithanine B (207)
192-195°C (Decomposed)
+
12.5
Physalolactone B -3 B - 0 D-c,lucopyranoside (208)
Amorphous solid
+
3.7
P . peruviana
Pubescenin (209)
188-189OC
-
38.6
P . pubescens
24b
Duturataturin A (210)
Amorphous
-
38.1
Datura t a t u r a L
29 a
Daturataturin B (211)
Amorphous
-
17.7
Dunawithanine C (212)
Amorphous
+
15.2
Dunawithanine D (213)
Amorphous
+
5.7
-do-
163
Dunawithanine E (214)
Amorphous
+ 12.3
-do-
163
Dunawithanine F (215)
Amorphous
-
-do-
163
18
Dunalia a u s t r a l i s
49
(dried sprouts) -do-
49
leaves
-doDunalia a u s t r a l i s
162
29 a 163
195
TABLE
196
I p ( Contd.
WITHANOLIDE GLYCOSIDES
r Ol0
Compound
S.ource
Ref.
Datura metelin A (216)
Amorphous
-
22.4
Datura metelin B (217)
Amorphous
+
1.4
-do-
110
Datura metelin G-AC ( 2 1 8 )
Amorphous
-
48.4
-do-
llOb
Sito indoside IX (219 ) Sito indoside X (220) Physagulin D
221)
*
*
*
*
A white powder
+
21.0
Physagulin E
222)
A white powder
+
56.7
Physagulin G
223)
A white powder
+
31.3
TA
-
*
Not available
TA
a (224) b (225)
.(
*
*
*
L
Datura m e t e l
Y. s o m n i f e r a
164
-do&
164
77
P. a o g u l a t a
leaves
llOa
stems
P. a n g u l a t a
berries
-doT . ano.valum
berries
-do-
148 148 48 48
197 Fig. Ip. Withanolide glycosides
OGIc
•OH Dunawithanine A ( 206 ) ; Ri=Glc-Glc, R2 = OAc
I
Glc Ri = OH , R4 = H Dunawithanine B ( 2 0 7 ) R, = Glc - Glc , R2 = OAc, R3 = 0H, R4 = H Physalolactone - 3p - 0 Glucopyranoside (208 ) R, = Glc , R2 = OAc, R3 = OH , R4 = H Pubescinin { 20P) R, = Glc , Rz - OH , R3 = R4 = H ,
24a , 25a - epoxy Duturataturin A ( 2 1 1 ) R, = Glc , R2 - OH , R3 = R4 = H , 7a - OH
Daturaturin A (210)
198 Fig. Ip. withanoiide glycosides
R'O Dunawithanine C ( 212)
XYI
o-
HO O
XYIO OH Dunawithanine D (213 )
HO GIcO O
XYIO OH Dunawithanine £ ( 214 )
GIcO
HO
Glc OH Dunawithanine F (215 )
GIcO
HO O
GIcO OH 12-OH
199 Fig. Ip. Withanolide glycosides
OH
Daturametelin A ( 216 )
Daturamctclin B ( 217 ) r-0-6'
DaturameteUn G - Ac ( 218 ) Physaguiin D ( 221)
OHoH Physaguiin E(222) Physaguiin G ( 223 ) 16P,17p-epoxy
Sitoindoside IX (219)
Sitoindoside X (220)
200 TABLE
2
U.V. SPECTRAL VALUES OF SOME WITHANOLIDES Compound
Bands observed at (x
)
Ref.
max Withaferin A (12)
214 nm (e, 17,500) over-lapping of tv70 chromophores
71
Withanolide D (19)
217 n^ (e, 19,500) over-lapping of two chromophores (» :^ unsaturated ketone in ring A, a :3 -unsaturated- o-lactone in side chain)
73
2,3-Dihydrowithaferin A (13)
210 nm (e, 10,000)
27-Deoxywithaferin A (3)
215 nm (^, 17,500)
2,3-Dihydro v/ithanolide D (224) acetate
225 nm (e, 9,600)
27-Deoxy-14-ot-OHwithaferin A (14)
216 nm (e, 16,000) over-lapping of two chromophores
72
24,25-dihydrowithanolide D (20)
214 nm (e, 9,500)
65
43~Hydroxy-l-oxo-56,63 epoxywitha-2-enolide
214 nm ( e ,
65
9,500)
71 65 73
(5) 25
A (61)
218 nm (^ , 8,200)
Ixocarpalactone B (59) Pubescenol (152)
218 nm (e , 8,300) 212 nm
24a
Lycium substa,nce B ( 62)
224 nm (log ^4.19)
35
5a,20a^(R)-dihydroxy-
223 nm {^,
16a
Ixocarpalactone
r
14,000)
25
l-oxowitha-2,24dienolide (73) 219 nm (log e ,
4.28)
101
27-Acetoxy-5 a-hydroxy221.5 nm (^ , 16,400) l-oxo-6a,7a-epoxy-22Rwitha-2,24-dienolide (69)
66
Withastramonolide (82)
Nicalbin A (83)
225 nm {^,
9,900)
93
201 TABLE
2 ( Contd. )
U.V. SPECTRAL VALUES OF SOME WITHANOLIDES
Compound
Bands observed at (x
)
Ref.
max Nicabin B (66)
225 nm (e,9,750)
Withanolide G (107)
223 nm (e,19,400)
111
Withanolide H (120)
220 nm (G,17,200)
111
Withanolide T (81)
224 nm (e:/18,000)
100
233, 227 Sh (£.13,000)
126
Withametelin (138)
222 nm (e,7,000)
126
Perulactone B (142)
225 nm (e,8,500)
127
Nicandrin B (76)
226 nm
Daturilin (138)
218
Jabarol (Unusual Phenolic withanolide (187)
(MeOH) 224, 281, 291 nm (log €A,29, 3.51, 3.34)
Isowithametelin
(139)
nm
93
34 (G/16,318)
(MeOH + OH ) 224, 282, 301 nm (log E' 4.3, 3.51, 3.41)
125 150a,b
202 TABLE
3
I.R. SPECTRAL VALUES OF SOME WITHANOLIDES
Compound
Peaks observed at (cm
Withaferin A (12)
1692
71
Withanolide D (19)
1695
73
2,a-Dihydrowithaferin A (13)
1700
71
27-Deoxywithaferin A (3)
1690
(a:B-unsaturated six membered ketone)
65
2,3-Dihydrowithaferin D (224)-4-acetate
1738
(a:3-unsaturated-6-lactone)
73
24,25--Dihydrowithanolide D (20)
1730
(Saturated-6-lactone)
65
1681
(a:3-unsaturated six membered ketone)
65
1703
(Saturated-6-lactone)
65
^.0 -Hydroxy--1-OXO-5 B, 63 epoxywitha -2-enolide (5)
Ixocarpalactone
A (61)
Ixocarpalactone B
Perulactone (143)
Perulactone B (142)
(59)
)
Ref.
1681
(a:B-unsaturated six membered ketone)
65
1770
(Saturated-Y~lactone)
25
1675
(a:3-unsaturated ketone)
25
1685
25
1735
(acetate)
1775
(Saturated-Y-lactone)
1762
(Saturated-Y-lactone)
1732
(acetate)
1770
(Saturated-Y-lactone)
1680
(fit:3-unsaturated six membered ketone)
Nicalbin A (83)
1685
(a:3-unsaturated six membered ketone
Nicalbin B (66)
1685
-do-
128
127
93 93
203 TABLE
3 ( Contd. )
I.R. SPECTRAL VALUES OF SOME WITHANOLIDES Compound
Peaks observed at (cm
)
2,3-dihYdro-27-deoxy withaferin A (11)
3400, 1715, 1697
65
2,3,24,25-Tetrahydro-27deoxywithaferin A (10)
3420, 1730, 1705
70
Lycium substanceB (62)
3450
35
Withaphysalin C (114)
1712, 1685
Acnistin E (60)
1675
(°' :P-unsaturated ketone)
1730
(lactone carbonyl)
(OH), 1705 (unsaturated-6lactone, 1690 (0:3-unsaturated six membered ketone)
Ref,
113 42
Daturalactone A (64)
3590-3500 (-0H), 1730 (Saturated-«S -lactone); 1690 (a:3unsaturated six membered ketone)
91
Acnistin A (58)
1655
(a :3-unsaturated ketone)
42
1730
(lactone carbonyl)
3460
(-0H); 1745 (Y-lactone)
1690
(ot : 3-unsaturated-Y-lactone)
1655
(a :3-unsaturated ketone)
Withaphysalin E (144)
129
Daturilin (138)
1650, 1680, 1720, 1125
125
(+) Jaborol (Phenolic withanolide) (187)
3400
(-0H); 1700 (12-ketone, six membered saturated ketone)
150a,b
1690
(Lactonic carbonyl)
1655
(2,5-di-en-l-one)
1718
(Carbonyl)
Withametelin (138)
126
204 with a 1-keto group are characteristically
noticed
at/xJ6 6.18
as
doublet and at r-^ 6 6.79 as d/d in compounds having 4-hydroxyl as in withaferin
A
(12) .
In a compound
with
proton appears a little upfield at around withanolide E
no 4-hydroxyl
the
C-3
6.6-6.9 as d/d/d as in
(46).
Of the 5,6-epoxy derivatives, except for two, the rest are 5B,63-epoxides.
The
6a-H
generally
appears
as
a broad
peak
at
around 6 3,20 and the 63-H slightly at lower field at/^'6 3.3 as a doublet. epoxy
The chemical shift difference of this proton in isomeric
compounds
is
too
small
stereochemistry of 5,6-epoxide.
to
distinguish
the
exact
However, using high resolution 500
MHz NMR spectra to observe the coupling constants of 6H ( a or 3 ) with
the
methylene
coupling
protons
constants
stereochemistry,
at
C-7
calculated
of
and
from
5,6-epoxide
was
comparing Altona
them
with
equation
decided.
A
larger
,
the the
coupling
constant 4.5 Hz (calcd. 4.9 Hz) has been noticed for 5a,6a-epoxide for 6 3-H and 7 3-H while a smaller coupling constant 2.7 Hz has been noticed for 5 3,6 3-epoxides for C-6a-H^ and C-7o-H^ coupling. In
the
case
of
6,7-epoxides,
except
example, all the rest are 6a, 7o'-epoxides. have two protons the 6-H
for
an
isolated
The epoxides in general
and 7-H, around/N/ <5 3.0, the former as a
doublet and the latter as d/d.
Of these the 7-H, appears at lower
field. The
chemical
shift
of
the
influence of 5,6- or 6,7-epoxides
19-methyl
is
subject
to
the
as well as the presence of the
4 3-hydroxyl and the presence of a:3-unsaturated system or saturated -1-keto
system
in
ring
A.
In
the
case
of
6ot , 7a-epoxides
the
19-methyl generally comes around 6 1.20 while the same comes around 6 1.30 in 53,63-epoxides because of the deshielding influences of the 63-epoxy oxygen on the 19-methyl in the latter. with
this
In agreement
19-methyl moved downfield to 132 63,73-epoxywithanone (186) with 6 3,7 3-epoxy system. The
the
configurational
assignments
of
6 1.32
in
53,63-epoxides
and
5a,6a-epoxides are decided by solvent induced chemical shifts C-6H.
Addition
of
causes a negative
benzene
to
CDC1-. solution
shift on the 19-methyl
(-5
of
of
5 3,6 3-epoxides
Hz) and a positive
shift (+11 Hz) on the same in the case of isomeric 5a,6a-epoxides. The shift of 19-methyl is more pronounced in pyridine where it
moves
from
6 1.32
to
1.69
with
53 ,6 3-epoxide
as
in
205 o -|
physapubescin pyridine for from
(35). Similar strong deshielding was noticed in 132 6 3/70-epoxywithanone where the 19-methyl moved
1.32 to 1.69 but in its 6a,7a-epoxy isomer the shift is just
marginal 0.07 ppm. in
Thus the solvent induced shifts, in particular,
pyridine form diagnostic evidence for the stereochemistry
of
the 5,6 and 6,7-epoxy systems. The chemical shift of 18-methyl is also affected to some extent by the presence or absence of hydroxy groups at C-17 and C-14.
In
the
absence
of
both
it
appears
at
6 0.75;
with
17a-hydroxyl it comesr^'^O .85 and with 17- and 14 a-dihydroxy system at
6 0.95.
The presence
of
a 143-hydroxyl
lowers
its chemical 1 no
shift very much to <5 1.16 as in 14B-hydroxyl withanone (85). The pyridine induced shift noticed on 18-methyl is much more (-2.4) with 14«-hydroxy system. The
21-methyl
usually
comes
around
1.0
without
much
deviation even with a hydroxyl at 14 or 17 but the same is very much subjected to solvent induced shifts. The
pyridine
induced
shifts
in
14a-0H-withanone
102
(84)
(14a,17a) indicate a significant deshielding effect of 17 a-OH on the signal of 21-methyl but not on that of 18-methyl. been
found
18-methyl
to
be
with
in
respect 102
143-hydroxywithanone
agreement to
with
17a-0H
(85)
the
relative
(dihedral
angle
(143 ,17a-hydroxyls)
the
This has
position
of
150°).
In
geometry
of
the molecule is changed to a cis C/D junction and the effect of the hydroxy groups is clearly felt on both the 18 & 21-methyls with a downfield shift of 0.3 6 ppm and 0.37 ppm respectively in pyridine. The
appearance
singlets between groups of the
of
two
methyls
on
double
bond
as
two
6 1.85 and 2.0 account for the 27 and 28-methyl
a : 3-unsaturated-<5-lactone
system.
In the presence
of an epoxide (24,25) these methyl groups move upfield and appear around <5i,45.
These two methyls, however, appear upfield
around
^ 1.2 in the example of having saturated five membered-'^-lactone or a spiro system 25 . In a fair number of examples either of these methyls may be missing to be found as hydroxymethyl which can be diagnosed easily. Another
characteristic
proton
generally as d/t is that of C-22 H.
that
appears
around
6 4.60
It varies from 6 3.9 to 4.6,
In the case of 5,6-enes, the 6-11 can easily be located at 6 5.6 as a multiplet.
2-n
Cn-puru
3-ii
4-H
6-ii
3.20~ 4.40dt (Wlw4)
22-.2
1u-ri3
19-r13
206
TABLE 4a H' NMR SPEC'IRBI. DATA OF 5,6-EPOXIDES
21-H3
26-113
27-33
Other chsr,ps
58,66-Cpcxides
i;itrefcrin A
6.16~ 6.97c.d (10) (1O.i)
3.75d (6)
Withanclide
tj.21d (1U)
6.97dd (lU,6)
6.206 (10)
7.08dd (10,6)
--
0.68s
1.38s
0.97d (7)
4.35Lrs 2.03 (!.lethjrlene)
3.77u 3.25 4.25dd (6; (;Jiy4)
0.85s
1.41s
1.27s
3.76d (6)
0.68s
1.39s 0.98d (6)
0.70s
1.32s 1.01d 2.09 (6.8)
4.41
(27-C€&
1.16s 0.97d (6)
4.37
(27-CF12 OH)
2
27-&c:;ywitha-
ieric A (3165
3-23 4.37dt (Wj-94) (12,3.5)
1.91 Trrc
aethjrls
--
1.91
Twc methyls
2,3-dihydrc71 withaferin A (13)
--
--
2,3-dihydrc-Jabcrcsalactcne A (9169
--
--
--
Jab? csalactcl M (50157
--
--
--
3.22d 0.04d (3) (12,4)
1.10s
1.21s 0.97d
--
3.20
4.88~1
1.10s
1.25s 1.42s
1.93s
1. 88s
--
3.20m 4.56dd (Wr6) (12,4)
0.98s
1.25s 1.29s
1.92s
1.88s
-_
Withanclide E (46Iasa
6.03dq 6.87dq
3.55t 3.18br 4.40dt (3.5)
4.42dt 0.67s (J22,23'3Hz, (J22,20=12Hz)
2.03s
1.40
3.56d(11),26-OH; 4.99d(11),26-2
(10)
E 6.03dd 6.86dq -(10,2.7) (10,6, 2.5)
*
--
OH)
Ccupling ccnstants in parenthesis.
TdBLE
4a ( Ccntd. 1
H' NMR SPECTRAL DATA OF 5,6EPOXLDES
Ccmpcund
2-H
3-H
4-H
6-H
22-H
18-H3 19-H3 21-H3 27-H3
28-H3
Physagulin C (in Bgridine)
6.4d (9.8)
7.26dd (9.8,6.2)
3.96d
--
4.43m
1.26s
1.78s
0.99d (7)
1.68s
1.87s
2.09~,15-CHCO 5.21s,15-15~ 3.72~,16-E
6.19d
6.92dd
6.73d 3.24
3.96s
1.15s
1.43s
1.38s
1.23s
1.17s
4.50,16-H; 4.47d,23=&
Ixcc palactcne B (59)
6.28
7.05
4.66
4.14
1.06
1.41
1.47
1.18d
1.21d
4.48,16-&
Phys3gibenclide (37)
6.18d (10)
6.95dd (10,5.5)
3.79d 3.36m 4.32dt (5.5) (W; 4)
1.11s
1.40s
1.02s
1.87s
1.94s
4.97d(4.4), 1% -g; 1.99s ,CH,CO
6.95dt (10,6)
3.75d (6)
4.18dd (12,4)
0.88s
1.40s 1.27
1.92
2.12
2.10 (acetate) 4.82dd( 9,2), 7a -g
4.51dd (13.5,3.5)
0.83s
1.33s
1.27s
1.87s
1.94s
3.9lddd( 11,5,5), 7-g 1.98ddd (12,12,5), 8-E axial
3.84ddd (11.3,5.6, 2.6)
--
1.38s
1.24d (7)
1.37s
1.35s
6.99d(8),15a -E; 7.12dd(8,1),16-2; 6.90d(1),17-3; 4.99d(9.5),26-Z; 3.40d(9.5),26-%
(6.2)
(52)
Ixcc2fpalactcne A (61)
If
6.20d 78 - Acetoxy- 45, 20B-dihydrcxy-56, (10) 63 -epcxy-1-cxcwitha2,24-dienclicie (33)79
3.23
3.33d (2)
Other charges
5 a, 6a-Epcxides
Wi thanrlide Y (184)
Salpichrclide A (19515'
5.98dd 6.72ddd 1.93dd 3.36d (10,3) (10,5,2.5) (20,5) (5) (4.&H), 3 103t (?0,2.5) ( 4 8-g) 6.0ddd 6.77ddd 3.14ddd 3.24d (10.1, (10.1.5, (19.4, (4.9) 2.8,l) 2.5) 2.5,Z.B) (46-g)
207
Compound
2-H
7-H
22-H
Lyciun substance B 5.86dd 6.60ddd 3.05d (10.1,2.2) (10.1,5.0, (3.7) (62)35 2.2)
3.32brs
4.39dt 0.77s (13.2,3.4)
1.19s (6.7)
1.03d
5a,2CilF(R)5.86d d (10,3,1) Dihyarcxv-Fa,70epoxy-i-o::rvi tha2,24-dienclide(73)16a
6.61dd 3.05d (10,4.5,3)
3.33dd (4,l)
4.26dd (1234)
1.18s
1.31s
Withastramonclide (82)lo1
5.84dd (10,Z.l)
6.61ddd (10,4.9, 2.1)
3.04d (4)
3.32dd 4.44dt 0.78s (3.7,1.8) (13.4,3.3)
1.18s
1.10d (6.1)
12-Oxowithanclide
5.85d
6.6Ad !-d,4.5, 5.2.2.4)
3.08d (4)
3.42d (4.1)
4.551~
1.1s
1.26
0.9d (7)
3.05d (4)
3.28dd (4,l)
4.21dd (12,5)
0.95s
1.20
1.31
(10)
2,3-Dihydrc-5Q, 20aF(R)-dihydrcxy 6 a,7o-epcxy-l-cxcritha-2,24-dienclide (234)16a
3-H
6-B
18-H3 19-H3 21-H3
0.95s
208
TABLE 4b H' NMR SPECTRAL DATA OF 5u-HYDRoxY-6,7-EwxU)Es 27-H3 28-H3 Other g r w p s 1.95s
1.89s
1.93
---
Two methyls
-1.52s
2.05s 4.04brs(WtN6), 12-H; 4.38d(5.6),27-CH2 1.58s
1.91 Two methyls
---
Withancne (68)66
5.81dd (10,2.3)
3.06d 6.60ddd (10,5,2.3) (4.1)
3.34dd 4.64ddd 0.86 (3.8,1.6) (12.3,4.6, 4.0)
1.18
1.03d (6.3)
1.88
1.93
2.69brd(18.7),4-xaxial; 2.53dd(18.7, 5.3), 4-Heq; 2.41111, 20-g
14a-hydrcxy- 102 withancne (84)
5.85dd (10,2.3)
2.98d 6.64ddd (10,5,2,3) (4.1)
3.32dd 4.70ddd 0.97 (3.8,1.6) (10,6.8, 2.6)
1.20
1.08d (6.3)
1.88
1.94
2.68brd(18.7),4-Haxial; Z.Xdd(18.7, 5.3),4-Heq;2.4n,ZO-H
TABLE 4b ( Ccntd. ) H' NMR SPECTRAL DATA OF 50-HYDROXY-6,7-EF'OXU)ES Ccmpcund
2-H
3-H
6-H
7-H
22-H
18-H3
146-hydrcxywithancne ( 85)lo2
5.86dd (10.6, 2.6)
6.61ddd (10.5,5.2 (2.4)
3.04d (3.8)
3.78dd 4.71ddd 1.16 (3.8,1.6) (12.9,4.4)
19-H3 21-H3 1.18
27-H3 28-H3 Other grcups
1.05d (6.3)
1.89
1.95
2.67brd(19.0).4-Haxial; 2.59dd' (19.2, 5), 4-11dd; 2.67~1, 20-11
1.09d (6)
1.94s
1.88s 4.02~,12-02
Nicandrin B (76)34 5.83dd (10,2.5)
6.61ddd 3.04d (10,5,2.3) (4)
3.37dd (4,1.7)
4.40~
Daturalactcne-3 (75197
5.86dd (10.1,Z)
3.06d (3.7)
3.33d (3.7)
1.18d (6.7)
1.95s
1.88s 3.55dd (10.4, 4.61, 12-
(5.82dd (10,2.5)
3.04d (3.5)
3.26dd (3.5,1)
4.49ddd 0.77s (13.1,3.4, 4.6) 3.97dt 0.76s
1.18s
Nicalbin A (83)93
6.61ddd (10.1,4.8, 2;4) 6.62ddd (10,5,2.5)
1.18s
0.93d (7)
1.42s
1.40s 4.18t, 16-11; 5.08s, 26-H 26-11
Nicalbin B (66lg3 5.83dd (10,2.5)
3.03d 6.59ddd (10,5,2.5) (3.5)
3.27dd (3.5,l)
3.57t (6.5)
0.82s
1.17s 1.03d (6.5)
1.43s
1.36s 3.98dd-(7.5, 4.5), 16-H; 5.02~,26-g; 1.81dd (10, 7.51, 17-11; 1.59~,20-g
Withanclide R 95 5.85dq (as acetate) ( 7 0 ) (10,5,1)
3.05d 6.56dd (10,4.5,3) (4)
4.73dd (3.5,2)
0.80s
1.17s 0.93d (7)
5.52 cctet 2.97d (4)
4.41dt
0.75s
0.85s
I a, 36,,50-trihydrcxy-6a ,7aepcxy-witha-24enclide (as acetate) (80)66
--
66,76-epcxy 132 5.83d withanone (186) (9.4)
6.61m
3.728
3.7910
(4)
4.64m
0.747s 1.17s
0.86
1.32
1.01d (7)
1.07d (6.3)
1.97
Twr methyls Twr
CH-CO-2.15;5.538 (Zj, 23-11
1.92 3.68,1--2;2.05, methyls acetate
1.86
1.96
209
3.19brd(19.5), 4-11 6-axial; 2.15dd (19.2, 5.0),4-gdq)
210
Ccmpcund
2-H
3-H
4-H
6.76dd -Withanelide G (107)11'5.83dq (10,391) (10,5,2.5)
6-H
18-H3
19-H3
21-H3
1.05s
1.25s
1.30s
4.65brs 0.71s (Wyd 8.8)
1.22s
3.95d(12.7)6.76brs 1.44s (12.7) 6.02brs 3.73dd (12.7,3.0) ( 2 l-CZ2)
5.86
4.48dt 0.73 (12, 3.5)
1.26s
1.05d (7)
1.41s
1.30s
5.60 4.21dd (Wtw 8) (10,6.5)
Wlthametelin (138)lZ6 5.88d 6.77dd 3.30dt 5.58t (9.8,2.5) (9.8,5.1, (20,2.5) (5.8) 2.5) 4Pi; 2.80dd (20,5.1), 40-1!
-
22-H
27-H3
28-H3
1.91
Two methyls
4.90s 2.07 (27-Cl&,)
Other grcups
---
2.07 (acetate)
7a,27-dihydrcxy-l5.99dq cxc-witha-2,5,24trienclide (as acetate) (103)66
6.83dq
Withanelide'U (as acetate) (123)
6.75dd (10,4.7)
5.78d (4.7)
6.15 (Wp4)
4.25dd (10,6.5)
1.08s
20K, 22R-4 B, 76, 5.92d 20-Trihydrexy-1-cxc- (10) 24-trienclide (122)"6
6.76 (10,5)
4.62d (5)
5.78d (1.5)
4.22dd (12.5,4)
0.91
1.44
1.28
1.87
1.93
3.85d(6),
48,17,27-trihydrcxy- 6.15 l-cxr-22R-witha-2,5, (10) 24-trienclide112
6.70 (10,6)
5.79 (6)
6.10t (2.0)
4.65
0.86
1.47
1.07
--
2.07
4.39, 27-CZ2;
6.05d (10)
1.91
Twc methyls
2.10 (acetate) 7-3
TABLE 4c ( Ccntd. ) ‘H N!lR SPECIRBI, DATA OF 5-=
Ccrnpcund
2-H
3-H
78 Withanclide N (as acetate) (106)
5.90dq
6.83dq
Withanclied I
--
(111)11’ Withanclide K (121)l” Iscwithametelin (139)lZ6
--
4-H
--
6-H
22-H
18-H3
19-H3
21-H3
27-H3
28-H3
Other grcups
5.62t
4.66dt
1.03
1.25
1.05d
--
5.71t
4.25dd
1.07s
1.40s
1.30s
1.87
1.94
--
1.88
1.95
--
2.07 4.93s, 27-CX2; 2.07, acetate; 5.25, 15-11
5.62dt
6.08dt
(10)
(10)
5.64dt (10)
6.08dt (10)
5.70
4.66dd (10,5)
1.07s
1.3%
1.32s
6.02dd (lo,,?.5)
5.62dd 4.63brs (5,2.5) (Wt@lO)
0.68s
1.33s
3.7dd 5.99s (13,2),1-H; 27-H; 3.89d, (13T, 6.73s. 1-I! 27-&
1.42s
--
0.89
1.29
1.25
1.89
1.95
-
0.87
1.03
1.25
1.8;
1.96 3.84 (Wd7.2), f 1-26
2.72dd 5.58dt (20,5), 2-H(10,5) 3.28d ( 2012-€J
(11.5,4.5)
--
3.87 (Wfrv19.5)
--
5.62
10,36,23trihydrcxy-l-cxrwitha-2,24-dienclide (125)”7
--
3.99 br.cctet
--
5.58 4.23dd (WfN9.9 ( 13,3.6
4.22
Twc methyls
Twc methyls
211
36 ,2G0 -dihydrcxyI-cxcwitha-2,24117 dienrlide
Ccnpcund
2-H
3-H
4-H
6-H
22-H
212
TABLE 4d H' NMR SPEC'IRAZ. DATA OF 1NTEKi.WJATE
DERIVATIVES
18-H3 19-H3
21-H3 27-H3
28-H3 ~
2,4,6-trien-l-cne (151)~~~
5.97d (9.3)
6.99dd (9.4,6.5)
5.97d 5.86dd 4.22dd 1.15s (5.3) (9.4,1.8) (13.2,3.8)
Withaphysalin E ( 144)lZ9
5.96d (9.7)
6.9dd (9.7,6)
6.14d 4.54t (6) (3)
Jabcrcsalactcl
4.60dd (13.4,3.6)
--
~
Other grcups ~~
1.30s
1.20s
1.89
1.95
6.30dd,(10,2.9),7-g 2.74brd(12), 8-g
1.48s
1.52s
1.88s
1.96
--
1.49s
0.96d (7)
5.0d(11),26-H; 3.56d(11),26-g
6.Oldd 6.92dd (9.5,l) (9.5,6)
6.14dd 6.42brs 4.07~ (6,l)
1.18s
Withaminimin (164)
5.87dd 6.58ddd (10,2.5)(10,5,2)
3.19dt 3.60brt 4.34ddd (20,2.5) ( 2 ) (12,7,4) 48-2
1.16s
1.21s (7)
1.12d
1.87brs
Physalclactcne C (169)141
5.9dd
5.00t 4.391~ 4.76dd (2.4) (12.6,4)
1.06s
1.22s
1.38
1.85s
1-97s
--
(10.2,
4.67d (8.5)
1.04s
1.22s
1.35s 1.85s
i.97~
4.34d (8.5), 230g;
5.72~1 4.63dt
0.85s
1.21s
1.04s (7)
Twc methyls
0.68s
0.91s
0.84d (6)
1.45s Twc methyls
S
6.55dd (10.2,2.5)
2.1) 23-Hydroxy-physalc- 5.90dd 6.55dd lactcne C (180)'47 (10.2,2)(10.2,2.5)
5a,17a-Dihydrcxy- 5.91 l-rxcwitha-2,6,24trienclide (151)66 Pubescencl (152)24a
--
6.66
--
4.99dt 4.3810 (10.3,
1.40s Twc methyls
1.98brs 5.33d(2.5),15-H; 5.60brd(2.5),16-g; 2.53m,20-g; 2.31dd ( 17,4), 23%; 2.01s, 15-O&
2.5)
4.2801
--
4.38m
1.90
--
3.64m,7-11;5.33d, 4a-OH; 4.31111,7a-OH
TABLE 4d
( Ccntd.
1
H ' NMR SPECTRAL DATA OF DEEiWEDIA'lT
DWIVATIVES
~
4-H
6-H
6.OOdd 6.47dd 200 -trihydrrxy-1- (10, (10,2.5) F cxr-22R-witha-2,24- 1.9) 134 dienclide ( 157)
5.08m
4.44dd (12.3, 5.3)
4.18dd (13.2,3.5)
0.81s
1.22s
1.25s
1.87s
1.94s
Withaperuvin (in pyridine) ( 181)14'
6.28dd 6.89dd
5.77t
4.53dd
5.16dd
1.31s
1.60s
1.78s
1.98s
1.82s
Withaperuvin D ( 168)140
2.37dd 4.46m (18,3*5), 1-11; 2.82d (181, 1-1!
5.2id (6.5)
4.27d (7.5)
4.48dd (10,4)
1.07s
1.14s
1.40s
1.87s
1.94s
Ccmpcund
2-H
3-H
6a-Chlcrc-4 0,5 6;
22-H
18-H3
19-H3 21-H3
27-H3
28-H3
Other grcups
--
Ccupling ccnstants in parenthesis
213
214 1 f. f.
The
solvent
induced
saturated-6-lactone
CDC 1
shifts
A
systems were used
3
on
25~methyl
to decide
the
in
conformations
of lactone system. A fairly chain.
large number
occur with
17a-side
In all these the presence of 14a-hydroxyl, 17B-hydroxyl and
20-hydroxyl
has
withnolide E groups
been
^'
held
noticed. on
the a -face
bonding,
thus
decreasing
and
17 3-hydroxyl
between
20-hydroxyl
reflected (strong
in
the
the
and
solvent
shifts
of
of
structure
21-methyl
and
22-H
structural
changes
are
'
(46)
to
1.37,
Similar
been
and
distance
E
5 1.10
-
also
hydrogen
the
withanolide
have
by
173 -hydroxy1
increasing
18-methyl
of
14a,20hydroxy
molecule
between
These
spectrum
the
and 22-H; 4.88 to 5 . 1 6 ) .
- 61.42 to 1.76
18-methyl,
the
22-H^ while
18-methyl.
to
analysis the
of
distances
and
pyridine
negative
21-methyl
According
(46) derived by X-ray
together
18-methyl
on
of withanolides
shifts
noticed
with
40-hydroxy withanolide E ^ ^ ^ ' ^^ (53) (See Tables 4a to 4 d ) .
"*-^C NMR Spectra A
detailed
study
of
withanolides
(Table
5) was
followed
Kirson
and
by
13
made
C
NMR
by
Gottlieb-*-^^
find useful applications
spectral
Pelletier in
data of various 1 fi8 al. in 1979
et
1981.
The
•^'^C NMR
in structural and conformational
data
analysis
of withanolides. The downfield unambigously and
the
6132.0 and The
the
lactone
two
singlets around
6202.0
carbonyl
groups of
at
respectively.
C-26
and 6167.0
«:3-unsaturated The
two
correspond
to
the
«: ^-unsaturated
shift
singlets
double
lactone
bond
system
around
carbons as
in
6149.0 C-24
(13).
The presence of a hydroxymethyl at C-25 the
C-24
withaferin A Next
carbon
shifts
to
at
lower
respectively. and
and
6 121.0
C-25
of
instead of a
field
to
A
the lfi8
simple
6 153.5
as
in
(12). in
carbons
connected
The
and
C-7
order, to
an
carbons
the
carbons
epoxy of
easily
oxygen,
a
recognisable
hydroxy1
5oi-hydroxy-6,7-epoxy
or
are
an
660.0 ppm for eg. lycium substance B
compounds (62)
the
acetate. can
easily recognised where both come as doublets with a chemical lower than
C-1
around
2,3-dihydrowithaferin
methyl
C-6
ketone doublets
6 142.0 ppm are of the C-2 and C-3 carbons chemical
recognise
be
shift
(56.2
and
215 1 ^ ( C NMR
57.3)
data
for
the
C-6
and
C-7
carbons
of
only
one
isolated example of 66,73-epoxy derivative (186) is not available). Similarly,
the
more
common
53,66-epoxy
recognised by the chemical shifts
system
can
also
be
664.0 as a singlet for C-5 and
662.0 as a doublet for C6 carbon as in withaferin A
(12).
The presence of 5a,6a-epoxide has marked shielding effect 52 on C-6 carbon<\^ 4 ppm as noticed in salpichrolide (185). In the case of Nic-2 lactone
(91)
v/ith an epoxide at
C-24/ C-25 in the side chain-6--lactone, the two carbons 24 and 25 apper at 659.1 and 63.1 as singlets. In the case of viscosalactone A
29a
(22) (having 2B,3 3-epoxy
system) the carbons appear at 6 54.9 and 55.8 as doublets. The C. carbon with 43-hydroxyl as in withaferin A
(12)
appears at <5 69.8 as a doublet which shifts to lower field to 672.I in its acetate.
The 2 7-carbon with a primary hydroxyl appears at
^ 57.0 as a triplet which deshields to "Sss.O on acetylation. C-10
and
C-13
carbons
come
as
singlets
at
648.O
and
The 6 42.5
respectively, of which the former is more affected by the presence of a functional group in ring A and B. Of the chemical shifts of methyl carbons C-18, C-19, C-21, C-27 and C-28 the chemical shifts of C-18, C-21 remain more or less constant ( 6 11.5 and 13.5). methyl
carbons
in
The chemical shifts of the remaining
particular
that
of
C-19
is
affected
by
the
functionalities as well as their stereochemistries present in rings A and B. The chemical shift of C-2 2 carbon with the 6-lactone oxygen is around 6 78.0 in all the compounds. The presence of 43-hydroxyl produces a deshielding effect on C-3 ir^l
ppm) and shielding effect on C-2 (^^'2-3 ppm) as well as
a shielding effect on C-19 hyf^2
ppm by 1,3-dixial
interaction
1 fift
The presence of 5 3,6 3-epoxy ring, saturation of 2,3 double bond of ring A
ketone
(f^3 ppm), and
has
a deshielding
C-4
by (r03
effect
ppm ). The
on C-5
presence
(r^ 3 ppm) of
Y -effect not only on C-10 but also on C-S^
produces
C-10
53 ,63-epoxide A similar
Y-effect has been noticed on C-9 by 6a,7a-epoxy systems. In the tives, C-5
commonly
appears
trans and 5-hydroxyl
occurring
around is
5a-hydroxy-6oi,7a-epoxy
73.0 where the ring
a-axial.
deriva-
junction A/B
With 53-hydroxyl
with A/B
is cis
5
216
TABLE
13C NMR DATA OF W I m O L I D E S Carbrn Nr.
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
2,3-dihydrr Witha- Witha17-1sc Withawithaferin A nclide D ~ ~ A nclide ~ E nclide ~ (12)168
(19)168
202.3 132.3 142.5 69.8 63.9 61.7 29.8 31.1 44.0 47.8 21.8 27.2 42.5 56.0 24.2 39.2 51.8 11.6 17.0 38.7 13.3 78.7 29.8 153.5 125.6 167.0 57.0 20.0
202.2 132.5 142.2 69.9 64.0 62.1 31.1 29.1 44.1 47.7 21.7 39.6 42.7 56.3 23.8 21.9 54.6 13.5 17.1 75.1 20.7 80.9 31.4 149.1 121.9 166.2 12.4 20.5
(11)168 211.5 31.6 26.2 72.8 66.7 58.5 29.6 31.4 43.1 50.4 21.3 27.2 42.6 56.2 24.2 39.0 51.9 11.5 15.2 38.8 13.4 78.3 29.4 149.1 121.9 167.0 12.4 20.5
(46)16’
(34)16’
203.2 129.8 143.9 32.9 62.2 64.2 26.2 34.1 36.9 48.6 22.9 34.3 54.5 82.3 30.1 37.7 87.8 20.6 14.6 80.0 19.5 80.3 32.5 151.1 121.4 166.6 12.3 20.6
129.7 144.0 33.0 62.1 64.1 25.8 33.3 37.1 48.6 21.9 26.5 50.7 85.3 33.3 32.7 90.9 19.2 14.8 76.2 21.6 81.4 31.6 150.9 120.9 164.8 12.3 20.7
-
Ixccarpa ~ lactcne A? (61)25 202.0 132.6 143.2 73.0 64.1 61.2 31.2 29.0 44.2 48.1 21.3 40.0 43.3 54.5 37.2 70.0 57.6 14.1 16.8 79.5 22.1 73.2 78.9 41.9 40.3 181.6 14.5 13.0
2,3,24,25Ixrcarpa Physapube- Physapube- Viscrsa Tetrahydrclactrne ~ ~ nnrlide lactcne A 27-derxy (acetate) withaferin A (59)25 (35I8l (37)24b (22)23 201.2 202.5 134.0 132.2 143.2 139.9 72.2 69.7 61.1 63.6 60.1 61.2 31.0 30.4 29.2 28.9 44.3 43.7 48.3 47.6 21.4 20.7 39.4 39.5 42.8 42.9 54.4 58.8 33.7 76.2 37.0 75.2 62.5 5C.2 12.7 14.9 17.0 15.6 74.9 38.4 12.6 27.0 71.9 65.1 107.6 29.5 64.1 49.3 42.4 63.6 177.6 91.7 13.9 16.6 12.2 18.8 AcO 20.7 AcO 21.4 170.1 171.1
202.6 131.1 143.5 69.6 63.4 62.7 26.1 40.3 36.1 47.9 21.8 41.4 46.1 84.1 80.8 33.6 52.3 15.6 17.2 37.5 17.2 78.5 31.2 149.5 121.9 166.8 12.4 20.5 AcO 21.5 169.8
206.9 54.9 55.8 74.6 64.0 59.6 29.6 31.0 42.7 48.6 20.0 27.3 42.7 56.1 24.3 40.7 51.9 11.5 14.5 38.9 13.3 78.7 29.8 153.8 125.8 167.4 57.2 20.1
211.6 31.6 26.2 72.8 66.7 58.5 29.4 31.4 43.0 50.4 21.2 27.5 42.6 56.1 24.2 38.9 51.9 11.5 15.2 38.2 12.7 80.0 29.2 27.2 38.9 176.2 12.0 18.1
TABLE 5
( Ccntd.
1
13C NMR DATA OF WITHANOLIDES Carbrn Nr.
Lycium Withanone substan B ( 6 ~ ) ~ ~(68)lo2
1
2 3 4 5 6 7 8 9 10 11 12 13. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
203.0 129.1 139.5 36.8 73.3 56.2 57.3 35.8 35.6 51.5 21.9 39.9 43.6 52.0 23.6 27.3 51.0 12. i* 14.7 39.2 13.3* 78.3 29.7 149.0 122.1 167.0 12.5 20.4
203.4 128.1 140.9 36.9 72.9 55.1a 55.Za 35.7 34.5 50.4 21.1 32.4 48.2 45.1 22.5 36.3 83.1 14.2 14.7 42.9 9.4 78.5 32.0 150.R 120.1 166.0 12.0 20.1
14wOH 14b-OH Witha- Witha- DaturaNical- Nical- 5a-OH withancne witha- nican- fercxc- lactcne C bin A bin B -6-ene ncne drin lide
(84)"' 203.5 127.8 141.1
-
72.9 52.7a 56. - 6a 25.3 49.8 20.lb 33.5 50.6 84.4 28.1 86.0 18.8 13.8 43.4 9.7 78.7b 33.1 151.3 119.3 166.1 12.4 .19.8
(85)102 (63l3I (76)31
(75I3l
203.1 202.1 128.8 128.9 139.6 139.8 36.7 36.8 73.2 73.3 56.2 56.2 56.8 57.0 35.7 36.1 37.7 28.7 51.5 50.6 38.4 30.1 212.0 72.5 57.7 47.1 52.9 43.7 23.6 23.1 27.7 26.6 42.8 42.9 11.5* 12.C' 14.7 14.7 39.5 39.1 13.1* 12.4" 76.3 79.0 29.1 29.7 149.3 149.2 121.8 122.0 166.8 167.0 12.5 12.5 20.5 20.5
202.8 129.0 139.9 36.7 73.2 56.3 57.0 38.6 35.1 50.9 31.3 77.6 48.5 53.4 22.8 26.9 49.6 7.6 14.6 37.7 14.9 79.0 32.7 149.1 122.0 166.9 12.5 20.5
203.6 127.8 141.2 72.6 53.1a 53.7a 30.8 50.6 21.5 34.3 52.4 83.5 31.6 83.1 16.8 13.8 41.6 10.0 78.4 32.9 151.9 119.9 166.1 12.1 20.1
*
tnterchangeable
202.5 202.9 129.0 128.9 140.0 139.8 36.8 36.7 73.3 73.3 56.2 56.0 57.0 56.9 35.3 35.4 35.3 35.4 51.0 51.0 21.7 21.1 40.2 38.9 45.1 44.8 48.6 49.5 35.8 35.2 75.2 81.8 64.0 59.4 13.6 13.2 14.7 14.7 38.3 43.2 14.3 19.2 67.2 75.9 32.0b 32.6 65.0 60.3' 63.5b 59.1' 91.9 95.8 16.5 14.4 18.8 21.8
204.8 129.1 141.0 36.5 74.9 132.8 129.5 38.1 40.8 51.4 22.2 32.7 48.9 47.8 23.3 36.5 84.9 15.1 14.6 42.9 9.5 78.9 32.7 150.9 121.3 167.5 12.4 20.6
(
202.8 128.7 140.1 36.9 72.8 56.9 55.7 38.6 31.7 51.7 24.4 29.3 137.0 135.2 124.1 125.4 141.5 128.9 14.1 43.1 17.3 67.4 33.7b 64.7b 63.3 91.7 16.6a 18.7a 217
a,b &
(83Ig3 (66)93 (159)16'
Nicandrencne (Nic-1)
218
Carbcn WithaNc. nclide G (107)"~ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
204.3 128.0 145.3 33.5 135.2 125.0 25.3 35.2a 36.3a 50.8 22.2 32.5 47.5 85.0 32.1 20.6 49.4 17.4 18.9 75.3 21.2 18.4 31.7 149.2 121.9 166.7 12.4 20.5
WithaChlcrchydrin Chlcrchydrin 3S-hydrrxy2,3dihydrcwithacf withanclide cf withanrlide nclide-H acetate nclide D ( 1 2 0 ) ~ ~ (~ 1 2 1 ) ~ ~(~1 ~ ) ' ~ ~( 1 7 4 ) ~(~1~5 7 1 ~ ~ ~ (131)121
Withanclide H
204.2 128.0 145.5 33.5 135.2 125.0 25.3 35.2a 36.3' 50.9 22.2 32.5 47.4 85.1 31.9 20.7 49.4 17.5 19.0 75.3 21.1 81.7 31.9 153.7 125.6 166.4 57.1 20.1
Withanclide K
Perulactrne B
-
205.2 127.8 146.1 33.6 135.4 125.2 25.5
39.8 127.4 129.4 140.7 121.6 25.2 34.Za 34.ga 52.6 20.7 26.5 51.4 85.9 33.0 33.0 89.2 18.9 20.4b 76.gb 20.2 81.0 32.4 151.6 120.9
-
12.2 20.7
35.A
37.2 50.9 23.2 32.3 54.4 83.4 31.7 37.2 88.5 20.5 19.0 78.8 18.8 73.1 30.3 38.4 38.0 181.8 10.5 72.6
'-
204.3 128.6 141.4 35.0 76.3 76.0 25.9 33.5 34.6 52.0 22.8 34.6 54.9 83.5 30.9 37.3 88.2 21.5 15.4 79.0 19.6 81.1 32.6 151.4 121.1 167.2 12.3 20.6 OdC 21.5 170.3
a,b interchangeable
201.1 127.7 143.1 66.6 78.3 66.0 39.1
206.8 127.0 144.5 34.5 77.6 66.5 31.6
1~,38,20-trihydrcxy l-cxrwitha-5,24dienclide ( 125)117
212.2 52.9 68.7 41.0 134.9 126.0 25.6
72.9 38.3 66.3 41.4 137.6 125.2 31.7 3 1. .?
31.5
39.1
35.9
45.7 55.7 22.5 39.2 43.1 57.2 23.5 21.9 54.4 13.7 9.9 74.9 20.6 80.7 31.3 149.3 121.8 165.6 12.5 20.5
37.3 55.7 22.9 34.5 54.9 82.2 30.2 37.6 87.4 20.6 8.6 78.8 19.0 81.0 32.3 152.0 121.3 167.8 12.2 20.6
34.1 51.3 20.9 32.1 47.5 84.9 31.9 20.8 49.4 17.4 19.1 75.2 21.1 82.0 31.9 154.7 125.5 166.9 56.6 20.1
.
41.5 41.7 20.2 39.8 43.0 56.8 23.9 22.0 54.7 13.6 19.4 75.2 20.8 80.1 31.5 149.1 122.0 166.2 12.5 20.5
TABLE 5 ( Contd. ) 1 3 C NMR DATA OF WITHANOLIDES
Carbrn Nr
.
Daturilin (138)~’~
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
23-hydrcxy Physalc- WithaWithaphysalclactrne peruvin peruvin D lactcne (164)’~~ (18~1)’~~ (175)’~~ ( 1 ~ 1 )( 168)140 ~ ~ ~
Withaminimin
204.06 204.15 128.0 128.71 145.11 141.33 33.524* 36.03 136.06 77.22 74.26 124.60 33.334* 26.47 33.297 35.43 39.963 35.43 50.544 52.20* 23.678 23.18 39.749 38.82 52.23* 42.662 56.051 82.28 24.124 83.36 26.556 120.37 47.699 161.30 12.751 16.80 18.976 15.10 42.939 36.11 60.574 17.21 75.629 78.47 30.795 32.35 150.25 69.355 139.2 121.44 165.26 167.44 129.74 12.37 25.627 20.64 -CH 3 CO 21.36 C H - C O 170.63
203.6 126.8 147.5 65.8 80.0 66.0 35.8 40.0 39.9 58.3 23.7 31.0 55.9 83.7 32.8 37.1 88.2 20.9 9.8 80.4 19.3 85.7 67.5 153.8 122.5 166.9 12.7 16.2
203.8 126.9 147.6 66.0 80.0 66.0 35.7 40.1 39.9 58.3 23.8 35.5 55.8 83.5 31.2 37.8 88.3 21.1 3.7 79.7 19.3 82.5 34.0 153.3 121.8 168.9 12.2 20.5
202.4 127.2 145.2 67.0 79.4 74.5 32.0 37.4 37.8 55.6 22.7 34.5 54.0 82.8 30.7 37.4 87.4 20.7 9.7 78.8 19.1 80.9 32.2 151.1 121.2 166.7 12.2 20.7
214.0 42.3 77.6 74.5 79.8 77.3 26.4 35.8 34.9 55.8 21.6 35.4 55.6 83.9 31.1 37.4 88.7 20.6 15.3 78.3 19.4 83.0 32.8 154.2 121.7 165.7 12.3 20.8
Withaphysalin E
150a,b
(+) Jabarrl (187)
(144)~” 206.1 117.5 140.4 126.0 160.0 72.8 37.1 37.8 44.7 53.8 22.7 36.3 60.0 83.1 34.8 25.7 57.7 177.3 20.0 83.9 26.3 78.4 31.9 148.7 121.9 165.4 12.5 20.7
a,b,c,d : : Interchangeable
219
-3-
149.76 114.04; 126.55 116.56a 140.62 76.14‘ 39.57, 43.50, 73.23 121.04b 58.90 212.81 45.5gd 46.96 24.52 34.44 83.35 16.09 11.01 40.85 13.04, 78.29 33.72 153.97 121.67 166.10 12.28 20.52
220 ring junction and the hydroxyl being equatorial as noticed in for 134 (171), the C-5 carbon appears
eg. chlorohydrin of withanolide E deshielded at
677.6.
The stereochemical effect of C/D ring junctions can also be noticed, though to a lesser extent, on crbons - 13, 14 and 18 for 102 eg. in 14o-hydroxywithanone (84) with C/D trans stereochemistry, the C-14 appears at 6 84.4 while it appears at 6 83.5 in 143-hydroxy 102 isomer with C/D cis stereochemistry. The 146-hydroxyl has a deshielding effect on C-13
(1.8 ppm) and a shielding effect (2.0
ppm) on C-18 in the isomers. Effect of 14a-0H The presence of 14 0'-axial hydroxyl compared to those with no hydroxyl produces a gauche shielding effect of 5-8 ppm on C-7, C-9 and C-12 in rings B and C.
A similar Y-interaction might have
also been expected on carbons-16 and 17 in the pentacyclic D ring but only C-17 has been found
shielded.
This has been explained
that in a five-membered ring no more than one carbon experiences a Y-effect.
The
17 B-side
chain
forces
position to receive the Y -effect.
the
17a-H
into
an
axial
However, if a 17^-hydroxyl is
present no change is observed in the shifts of either C-16 or C-17 since C-17 carbon is non-protonated. Effect of 17a-OH The oxygen atom at C,- shields C-12 and C-14 (but not C-15, also in the pentacyclic, vide supra).
In the side chain, C-21 is
shielded but not C-22; the former carbon is thus gauche 17-OH
but
not
the
latter.
results are explained by a 3 The J(HH) between H-20 and H-22
conformation such as structure I. was measured
from the 270 MHz
(12) acetate and withanone
to the
These
H NMR spectra of withaferin A (68) and found to be 3 and 3.5 Hz,
respectively, in agreement with I.
The gauche relationship between
the lactone ring oxygen and C-2 0, in this conformer explains the quite high field position of the resonance of this methyl group. The effects of 17 °'-hydroxylation
in the presence of 2 0-OH 169 to or 14 o'-20-(OH)^ functionalities respectively are very similar the case without the additional OH groups, for C-14 in the latter case, where this carbon is nonprotonated and, therefore, does not feel the
Y-effect, indicating
conformations
similar to 1 in the
221 vicinity of C - 1 7 . Effect of 20-OH The
presence
r^ 5 ppm on C-16 relationship
of
possess
group
is
causes
a
strong
group
17a-oriented
side-chains
which
a
causes
(6.1 ppm) (Structure I I I ) .
expected
is
Y-alcohol axial
containing
17^-hydroxyl
effect on C-21 carbon
20-hydroxyl
Y-effect
the carbon and the oxygen are in
gauche
(Structure II) whereas C-23 is unaffected.
Withanolides to
a
indicating
to
almost
shield
C-12.
unchanged.
replaces
the
oxy-substituent
one
may
from
a
C-20.
caused
where a hydrogen bond is possible
known
deshielding
The presence of a 14a-0H
Instead,
Probably
have
strong
are
the
Steric the
shift
Y-effect
of
from
hindrance
rotation
this
the
14
from
around
the
C,^-Cppj
(Structure I V ) .
The introduction of 2 0-hydroxyl
does not cause shielding
on
C-16 because of formation of a hydrogen bond between the 14 and
20
alcohols
by
(Structure
V).
The
deshielding
of
C-21
caused
20-hydroxylation is larger than usual. The C-17 epimeric compounds with conformations interesting. where
as
I and V
in
V
the
same
Y-effect
is
noticed
from
C-20.
interactions are absent in the case of compounds with an side chain such as withanolide E The presence
of
a
double 3.5
allyl bond
alcohol
2 7-hydroxyl
causing
causes
(ring
polarisation
shielding
These
«-oriented
E)
on
C-25
and
in
the
side
chain
Acetylation of
of
the
C-24
deshielding
and
on
this C-25
C-24
by
ppm. The axial 12o-OH in withaferoxolide
axial
hydrogens
at
C-9, C-14
through shielding by 6.9 in daturalactone C-17
C-16
(46).
causes deshielding on C-24 and C-25 by 4 ppm. primary
are
In I the 17^-011 experiences Y-effect on C-12 and
and
C
produces
and
to 8.3
C-17
and
(76) is gauche to the experiences
a
Y-effect
ppm, whereas the equatorial
123
OH
(75) produces minor shielding effects on C-9, a characteristic
high
field
C-18
methyl
signal
( 'S 7.6 ppm) . In
withanolides
containing
5-enes
eg.
withanolide
(107) the C-5 and C-6 carbons appear at<*^<5 135.2 and The C-22
{f^'5
presence
of
23-hydroxyl
ppm) and produces
3 and
produces
an
G
125.0 ppm.
upfield
shift
Y-effects on C-24 and C-25
on
as in
222
OH
HO
H
c^"P OH
20 ^ 2 1
«H
y
1 ,/
^
. ^ ^
1 OH
\
«\
/23 )^22
\y ^0
"^•-HO
H
^21
ri vi
223 23-hydroxyphysalolactone
(180).
Mass Spectra Mass
spectrometry
is
a
useful
characteristics of a withanolide.
tool
in
diagnosing
the
The molecular ion in electron
impact is either weakly present or totally absent in some cases. 65 Cleavage of the side chain at C-20 and C-22 in (3) and 31 91 (62) ' produces m/z 125 whereas in a saturated- <5-lactone it produces m/z 127 by same type of fission.
A fragment m/z 125 may
also be produced by a system containing 5 a--0H-6 a,7a-epoxide as in 31 91 ' (74) or 5,6-glycolic system as in Jaborosa-
daturalactone B
F""""^^ (163)
lactone
resonance.
(Chart
Vila).
This
will be stabilised by The presence of an epoxy- lactone 24a in the side chain,
revealed by a fragment m/z 141, further disintegrates to m/z 125 by the
loss
of
oxygen
1 fi7 .
The
same
27-hydroxymethyl disintegrates
ion m/z
further
141
which
by acquiring
comes
from
a hydrogen
a
and
losing water molecule to give m/z 124 (Chart V i l a ) .
Carbonyl group at C-12 in withanicandrin
90
(63) triggers a
fragmentation involving the cleavage of C Q - C, . bond on one side and a McLafferty rearrangement with the participation of 2 0-proton and the 12-ketone leading to a peak m/z 275 (Chart Vila). In 24,25-dihydrowithanolide D
(20) fragmentation between
C-20 and C-22 produces m/z 127 to confirm the dihydrosystem in the 127 side chain (Chart Vllb). In perulactone B (142) and 125 ixocarpalactone A (61) scission between C-20 and C-22 produces the ion m/z 143.
If the side chain contains a hydroxyl at C-20 or
glycol system at C-20 and C-17 scission occurs at C-17 and bond
producing
m/z
169,
as
in
eg.
4
C-20
-hydroxywithanolide
E^^^'^"^(Chart Vllb). In the compounds with 5a-0H-6a ,7 a-epoxide
in
ring
a 12 a- or 3-OH
A/B
the
scission
system at
C-17
containing and
C-20
produces m/z 299 (M-side chain-H^O)
as in withaferoxolide (77). 142 In chlorowithanolides, eg. 4-deoxyphysalolactone the molecular + + ion M could not be located. Under electron impact (M -HCl) is the peak with highest intensity (m/z 486). In B
the
spectra
of
5a-methoxy-4,5-dihydrojaborosalactone
(154) and 5a-ethoxy-4,5-dihydrojabarosalactone
B
(155);
the
224 Chart - Vila
m/zHl
m/zl42
m/zl24
m/z275
225 Chart - VII b
Perulactonc B ( 142 )
Ixocarpalactonc A ( 6 1 )
/
\ OH
O m / z 143
OH e
V o
o^ o m/z 113
m / z 143
226
m / z 185
227
CHART-Vlld MASS SPECTRAL FRAGMENTATION OF METHANOL ADDUCTOF ISOWITHAMETELIN126 (139)
(139) m /z 468
in/z436
m/zl34 -28
CH2
-«r ^^'^
OMe -OMe
m / z 155
in/zl24
-CO "O^ ^O
+
iii/zl23
in/zl06
' Q^ m/z95
228 TABLE 6a C D . VALUES OF SOME WITHANOLIDES
Compound
CD values observed at
Ref.
43-Hydroxy withanolide E (53)
400(0) ;340 (+1.42) cis ring junction of A/B; 304(0), 294 (-0.32), 282(0), 22R, 217.5(4-13.91), 252(+4.25), strongly negative at shorter wave-lengths.
26a
Withaperuvin (181)
370(-0.25), 354(-0.53), 322 (-0.60) due to strong compression. Due to the interaction of substituent and carbons 4,5 and 6.
142
Withanolide S (174)
336(-1.32)
143
Withaperuvin E (47)
345(-0.71), 245(+7.24)
Physalolactone (175)
363(+0.7), 353(+0.1), 348(+0.14), 338(+0.11) cis fusion of A/R, 306(-0.33); 239(+12.35), (22R), 205(-3.53) .
144
Withaperuvin D (168)
288(-4.55) (cis A / B ) , 251(+5.31)
140
4-DeoxyF>hysalolactone (171)
342(+0.89), 334(+0.85)
142
Withanolide O (105)
336(-0.29), 302 (-0.09), 256(+3.R6)
78
Withanolide U (123)
337(-0.28), 302 (-0.09), 256(+3.86)
100
Withanolide T (81)
339(-2.17), 253(+5.10)
100
Nicandrin B (76)
338(-1.95), 250(+3.83), (22R)
34
Withanone (68)
338.5(-2.16), 252.3(+5.02), 195(+29.5)
66
Withanicandrin (63)
336.5(-1.96), 287 (+0.8), 252(+5.01) 195(+31.0)
90
2,3-Dihydroderiv., of 5 a,2 Oa (R)-dihydroxy-
302 inflection (+0.35), 253(+4.32), 198(+16)
16a
r
6a,7a-epoxy-l-oxowitha-2,24-dienolide (234) Nic-1 (90) (Nicandrenone)
338(-1.7), 224(+6.22)
34
105
229 TABLE
6a ( Contd. )
C D . VALUES OF SOME WITHANOLIDES Compound
CD values observed at
Ref.
Withanolide Q (acaetate)(119)
340(-3.14), 260.5(+3.85)
95
Withaphysalin C (114)
346(-3.64), 251(+5.80)
113
Oxidative product at C-20 of perulactone (234)
286.8(+3.5), indicative of 17aoriented side chain
128
18-20 Lactone of withaphysalin B (2)
345(+1.30) cis A/B, 256(+3.8) (22R)
64
Oxidation product of physapubescin (233)
348,294, 254.5. A positive band 294 nm (e+2.76) is characteristic of 14aH-15-one.
81
Physapubescin & Nic-2-lactone (232)
230 nm (Ae_2.9) 22R, 24S, 258, configurations
81
230 TABLE 6b 2 111 COTTON EFFECTS OF A -EN~l-ONE AND 1-ONE CHROMOPHORES
Compund
X
max
nm
Ae
226
345
+ 0.96
227
335
- 0.76
228
294
- 3.32
229
291
+ 0.67 (shoulder)
TABLE
6c
111 COTTON EFFECTS OF 20-ONE CHROMOPHORE
CD
Compound X
max 36-Hydroxy pregn-5-ene-20-one
(230)
Compound (230a) 3B,17a-Dihydroxy-pregn-5-en-2-one Compound ( 2 3 1 a )
(231)
287.5
+ 3.00
294.0
+ 3.59
285.8
+4.03
298.2 303.0
+2.83 + 2.29
296.6
+3.89
231 Table 6a. A positive Cotton effect at r^ 250 nm suggests 22R configuration in ring E ( a: e-unsaturated-6~lactone) (See Table 6a). Even the stereochemistry of 17~0H ( a or B ) can be decided. A comparison of the CD spectrum of 36 -hydroxypregn-5-ene-20~one (230) with that of 36,17a-dihydroxypregn-5-ene-20-one (231) is shown in Table 6b. 1 oo
An oxidation product of perulactone was confirmed to possess 17a-oriented side chain Ae, 286.8 (+ 3.50). A negatice Cotton effect atro 230 nm ( Ae, - 2.9) in lactone 81 of physapubescin similarly indicates 22R, 24S, 25S configuration as in Nic-2-lactone-'-^^ (232). A positive Cotton effect at 287 (+ 0.80) is indicative of 90 the presence of 12-ketone in withanicandrin (63) as in 5«-pregnane-12-one and other ketosteroids. A positive band at 294 nm ( AG, -f 2.76) is characteristic of 81 14a-Ii-15-one as in the derivative of physapubescin (233). A very weak triplet of bands at 275.5, 268 and 263 nm are observed for the aromatic ring absorption in Nic-1 (90) due to the dissymmetric environment and 224 nm for side chain epoxy lactone. Even the stereochemistry of ring E (27- and 28-methyls) 1fifi were confirmed from the CD data and from molecular rotation differences (See Table 7 ) . Microbial Transformations and Cell Free Cultures There are several investigations on microbiological transformations of withanolides. Withaferin A (12) is hydroxylated by CuDiiinghamella elegans to give two metabolites , one of them originally identified as 14a-hydroxywithaferin A (236). In a 171 subsequent paper , it was shown that the previous assignment was wrong and that the structure of the above compound is 15 3 -hydroxywithaferin A (237), whereas that of the second compound is 12B-hydroxywithaferin A (238). However, in a patent 172 of 1986, it was claimed that fermenttion of withaferin A with the same microorganism afforded 14a-hydroxywithaferin A (236). 33-Methoxy2,3-dihydrowithaferin A (239) was also transformed by C. elegans
232 TABLE - 7 MOLECULAR ROTATION DIFFERENCES ( A M D ) IN CERTAIN WITHANOLIDES65 Saturated Lactones
Unsaturated Lactones
AMD
- 425.3
155.3
R MD -412.5
t OH
Ci ^oK
R MD - 546.0
-342.8
-do-
OH,
I R
H
-351.6 1
^ o \
r]^
O
-^S^
- 195.3
^
AcO
^
233 into
its 12 3-hydroxy
derivative
173
(240).
Biotransformations of 1 74 simplex . Of the
withaferin A were also induced by Anthrobacter several
derivatives
thus
obtained,
only
5 a,63-epoxy-2 7-hydroxy-
was identified. Anthrobacter l ,4-dioxowitha--2,24-dienolide citreus 175 deacetylated withaferin A (12) 4,27-diacetate t o the free withaferin A"^^^ ( 1 2 ) . From
tissue
of W, somnifera
cultures
several
sterols such as 24-methylene and 24-ethylidenechlesterol as well
a s sitosterol 177
(242),
stigmasterol
(244) were isolated
. No withanolides
extract
tissue
of
investigation
these
isolated
24-methylenecholesterol.
in the
Concomitantly, in an 1 7fi plants , 24-methylcholesterol,
24-methylcholesta-5,24-dien-33-ol, were
(243) and campesterol
could be detected
cultures.
of W. somnifera
28-isofucosterol
simple
(247, 241)
sitosterol, along
with
stigmasterol and small
amounts
of
This was the starting point for a study
on the biosynthesis of withanolides, to be discussed. By using labeled physalin B (190) and physalin F (194) in 179 that both were
cell free extracts of P. minima , it was observed rapidly
converted
(192)).
However, callus
sysnthesized callus
into
only
derived
the corresponding 5a,6 0-diol (physalin D 180 derived from diploid P. minima plants
small
from
amounts
triploid
of physalin P. minima
D, in contrast to which
synthesized
physalins B (190), D (192) and F (194). Biosynthesis Goodwin and co-workers
178
isolated radioactive
withaferin 3 A(12) and withanolide D (19), by administering [28-^ H]-24-methylene cholesterol to young leaves of Withania somnifera 3 Similar experiments with [28- H]-24(R,S)-24-methylcholesterol gave negative results, whereas [28H]-24-methylcholesta-5,24-dien-3 -ol gave ambiguous results. Later 184 Whiting et al. found that it was incorporated in Nic-1 (90). r. 4-u w ,24(28) . 24 . ^^ ^ From the above A and A isomers are the precursors of withanolides. 1ftR — 1ft7
Investigations of Gros and co-workers by 14 administration of [2- C]-mevalonolactone to seeds and seedlings of 1 QC
A, breviflorus resulted in the isolation of withaferin A (12) and jaborosalactone A (6). The results of the incorporation 185 confirmed the suggestion that jaborosalactone A (6) must be the
234
( 226 ) 5P , 6P. ( 227 ) 5a ,6a (228) 2,3.dihydro-5p,6p. (229) 2,3-dihydro-5a,6a-
OH
(230a)
MeO (232)
(233)
(90)
235
OH (236); (237): (238); (239); (240); (248);
( 241 ) R = CHCH3 (242) R = CH2CH3 (243) R = CH2CH3,A22 I4a -OH (244) R = C H 3 , A 7 , 5,6-diH ISp -OH ( 244a) R = C2H3 , A25. 24 4oH 12p -OH (247) R= =CH2 3p - McO, 2,3 - di H 3P-lVIeO, I2p - OH, 2,3. diH 3 - (1 - aziridinyl) ,2,3 - di H
OH HO HO
HO
a - ecdysone ( 245 ) ; 22a - OH P - ecdysone ( 246 ) ; 22p - OH
Physalindicanol A,
:x^
OH
(246a) and ( 246b) OH
Phsalindicanol B, R = (247a)
236
(249)
(250)
HO H O Ajugalactone ( 252 )
237 plausible precursor of withaferin a (12) and biosynthesized prior to withaferin A. Whiting
and
co-workers
18 4 18 8 '
published
two
papers
concerning with the biosynthesis of Nic~l (90) in N.physaloides; 18 8 the first , with the origin of the aromatic D-ring and the 184 second with the elaboration of the epoxylatol side chain (Chart VIII).
From the biosynthetic experiments performed by Mulchandani 189 14 et al. with Physalis minima radioactive acetate (1- C and 14 14 2-
C)
and
mevalonic
acid
(2-
C ) , were
incorporate into physalin D (192). A
found
to
efficiently
Similarly tritiated withanolide
(12) was found to incorporate into physalin D (192). From the
above
the authors
concluded
that
withanolides
are
the
possible
precursors of physalins. Ergosta-5,25-diene-3 3 ,24 e-diol (24Aci) isolated from the fruits of W. coagulans and two isomers (246a,b) named 1 90 physalindicanols isolated from P. minima var. indica (whole plant) along with other withanolides, were proposed as potential biogenic precursors. Physalindicanol whereas
(246a,b)
A
physalindicanol
(247a)
B
is
the
is
24-epimer
of
(244a),
25-hydroxy-24-methylene-
cholesterol. Biogenesis of Withanolides (247) is the biogenetic precursor
24-Methylenecholesterol
of withanolides. The formation of 24-methylenecholesterol from 1 78 cholesterol was explained by Lockley et al. . They synthesised it and also isolated from Withania somnifera cultures. Hydroxylation followed
by
at
oxidation
C-1
from
produces
the
1-oxo
rear
side
compound.
i.e.,
« -side
Dehydration
of
3-hydroxyl produces a: &-unsaturation in ring A. Among withanolides the 4P-hydroxyl is generally noticed in 5-enes and
53 ,6 3-epoxides.
4 3-Hydroxylation
is easy
as C-4
is
allylic to A^'^ system. It is presumed that formation of 4-hydroxyl takes place first and epoxidation at C-5 and C-6 follows later. This explains the formation of more 53,63-epoxides with 43-hydroxywithanolides 7-hydroxyl (Chart IX).
and
might
then be
with
formed
4d-hydroxyl by
the
containing
allylic
5-enes.
oxidation
of
The
5-enes
238 CHART - Vlll Possible pathways for Ring D > Aromatisation O
II
1,7
{Xn^{
R
HQ
(O)
SAM = S - adenosyl methionine
itf^-^ixy R
M
SAM
T
Possible pathways to the biosynthesis of the epoxvlactol side chain
OH
(O)
(0)
= H
239 CHART-IX BIOGENETIC SCHEME IN RINGS A&B OH
OH
OH
OH
0H£
a
a OH
240 The hydroxylcction at the tertiary positions i.e., 14, 17, 20
is
common
in
both
the
173-
and
17a-side
chain
derivatives
although this system is more dominant in the 17a-oriented system. Lavie et al. isolated a A
-withanolide
it is the precursor of 17a-oriented
(115) and suggested that side chain.
It might
have
originated due to the dehydration of 17a-hydroxyl and the highly crowded side chain will rearrange to the a-side. Hydroxylation at C-27 is frequent whereas at C-28 is rare. ^-lactone are produced by allylic 95 95 oxidation eg. withanolides Q (119) and R (70) whereas ^-lactone 128 (145). ring is noticed in a few compounds eg. perulactone 25 Ixocarpalactones form C-23 and C-26-olides with 22-free 23-hydroxy
compounds
with
hydroxy1 as in ecdysones
(245,246).
'
r ,-a.u u 1- 64,108,129,133 are obtained u^ • ^ w 4- • *. • Withaphysalms by i lactonisation of 18-methyl and 20-hydroxvl. Hydroxylation at C-18, C-19, C-21 is rare. C-21-hydroxyl
and their related derivatives
C-19-hydroxyl
'
and C-18-hydroxyl have been recently reported.
Aromatisation of ring D produces Nicandra Formation
of
saturated
dominent in w. somnifera
lactones,
formation
of
A
steroids
24,25-dihydrolactones
South African variety.
hydrolactones were isolated from The
Compounds with , compounds with
breveflorous
5a-hydroxy-6a
,7a -epoxides
explained, from 53 ,6 3-epoxides as given in the scheme. explained
from the following
less in number.
is
2,3,24,25-Tetra-
reason.
can
be
This was
4-Deoxy-53,63-epoxides
are
Transdiaxial opening of epoxide forms glycol at
C-5 and C-6 position.
Dehydration at C-6 followed by epoxidation
from the rear side produces 5a-hydroxy-6a,7a-epoxides. 5a,63 and 53,6a-dihydroxy systems arise from the scission of
53,6 3-epoxides
opening
by
the trans
respectively.
diequatorial
opening
Formation of
diaxial of
and trans
diequatorial
chlorowithanolide
by
the
53,63-epoxide
bulky nature of chloro group.
ring might be due to the 181 Sarma et al. proved this point by
performing the reaction of NaCl on 53,63-epoxides indicating the presence of NaCl in the plant itself. Hydroxylation at 12, 15, 16 positions is rare. Hydroxyla91 97 tion at C-12 was noticed in daturalactones B (74) and C (75)
241 (164) whereas a 12-oxo group was found
and Jaborosalactone F
in
Qfl 91 31 withanicandrin^^ (63) and daturalactone A ' (64). Even 12-methoxy group is noticed in (23R)-6a-chloro-5B,17a-dihydroxy-12B 138 ~methoxy-l-oxo-12,22--epoxy-ergosta-2,24-diene-23,26-olide (167). 81 15a-Acetoxy group is noticed in physapubescin (35) and in 24b 152-4 physapubenolide (37) whereas physalins contain 15-oxo group in common. But 15-hydroxy compound as such has been recently , . -,31a isolated q •] 93 25 Nicalbins A'* (83) and B (66), ixocarpalactones A (61) 25 and B (59) possess 16-hydroxyl. Formation of 14B-hydroxyl in 102 146-hydroxywithanone (85) was explained through the formation of carbocation, which produces either 14 3~OH or 14a-OH by
hydration
(Chart X I ) . Dehydration of 14-hydroxyl gives A by
subsequent
epoxidation
withanolide M (110).
producing
14
a
-withanolide followed
14,15-epoxide
as
in
These compounds are naturally occurring.
The
frequency of epoxidation is found to be higher at C-5 and C~6 than at C-14 and C-15. ^
-withanolide
formation
of
Similarly, elimination of 17-hydroxyl produces (115) which
17a-oriented
is
side
the
key
chain
intermediate
compounds,
for
the
followed
by
oc
epoxidation gives C-16 and C-17 epoxides, eg. physagulin C
(52).
Hydroxylation at C-22 and C-27 takes place in 24-methylene cholesterol.
The oxidation is carried up to the aldehyde stage to
form a 6-lactol, which yields a 6-lactone by oxidation. will
give
nicalbins
epoxy ,
pubescenol
lactol
as
physapubescin (153),
Nicandra
in .
Epoxylactone
nic-1-lactone
The above
steroids
(91).
is The
'
,
noticed
in
formation
of
lactonic ring is found to be stereospecific. Modifications of
side chain during
withanolide
formation
are shown in Chart X. The formation of the novel side chain of withametelin is considered acnistins
to
follow
a mechanism
similar
to
that
proposed
for
(Chart X I ) .
Pharmocological Activity Withanolides
are
polyoxysteroids
and
several
of
them
possess significant pharmacological acitivity, eg. Withaferin A(12)
242 Chart-X MODIFICATIONS IN THE SIDE CHAIN
,
OH H -:
^ CH20H !
24 - Methylene cholesterol
243
Chart-XI
\ /
r^ OH
OH 0 O''^—OH
OH R=*-OH X ^ leaving group
Acnistins
Withametelins
244 exhibits
tumor inhibitory
activity
in
activity, bactereostatic
experimental
tumors
immunosuppressive properties. activity
mainly microorganisms 57
flavus,
against and
Epidermophyton
in
mice
and as
cytotoxic well
as
Withaferin A possesses antibacterial
acid-fast
antifungal
floccosum
bacilli
activity
and
grampositive jergj Aspergillus
against
.191
and Cladosporium
herbarum
and invivo growth inhibitory and radiosensitizing effect on mouse 2 14 ascites carcinoma ' . It showed significant activity in
Ehrlich
vitro against cells derived from human carcinoma of the nasopharynx (KB) and in vivo against sarcoma 180 (SA) in mice. shown to produce tumour
significant
systems in mice.
growth
retardation
Withaferin A
(lymphocytes)
strongly
versus
inhibited.
It
host
seems
reaction that
It was also a number
(12) suppressed
lesions in adjuvant induced arthritis in rats. graft
in
of
secondary
The locally induced in
chicks
besides
was
also
antiproliferative
activity, withaferin A (12) has a complex influence on inflammation 57 and immune response . Withaferin A (12) and its microbial transformation products, 123-hydroxy and 153-hydroxy derivatives, were found to inhibit the growth and biochemical functions of in 171 . The adduct of vitro grown P-388 lymphocytic leukemia cells aziridine
to
2,3
double
bond
i.e.,
2,3-dihydro-3(l-aziridinyl)
A (248) possesses antitumor activity against P-388 192 lymphocytic luekemia 27-Deoxywithaferin A is found to be more 193 active than other compounds
withaferin
Withanolide P-388
luekemia
E
(46)
exhibits
cytotoxic
activity
against
system, activity against B-16 mouse 57 melanoma and Lewis lung carcinoma , antifeedant property against Shodoptera
and
L-1210
littorolides
,
suppressive properties.
antineoplastic
activity
4 3-Hydroxywithanolide
and
immune
E (53) possesses a o n
considerable lifespan increasing activity against L-1210 leukemia and showed less antifeedant property against larvae of 195 Spodoptera littaralis . Even the model compound having resemblance in ring A/B, i.e., (249) , prepared
53,63-epoxy-l-oxocholest-2-en-43-ol
synthetically
has inhibitory
activity
against
the growth of sarcoma 180 ascites tumor. Withanolide
S
(174)
and
53,63-epoxy-la,14a,173,20-tetra-
hydroxy-witha-24-enolide (250) possess antifeedant activity against 195 the larvae of 5. littoralis 5 a,20a (R)-DihYdroxy-6 a, 7 a-epoxy-l-oxo-5 oi-witha-2 ,24-dieno-
245 lide (73) exhibits immuno modulating properties
197
33-Hydroxy-2,3-dihydrowithanolide F (251) has a protective effect in the cases of CCl. induced hepatoxicity rats.
in adult albino
It also produces a moderate fall of blood pressure in dogs
and has a significant anti-inflammatory . 198 inflammations produced by formalin
effect
in
subacute
24,25-Et>oxywithanolide D (25) and physangulide (42) have 199 effects when administered mtra-peritioneally
antiinflammatory
to mice and rats (10 mg/kg) in exudative and proliferative types of experimentally
induced inflammation.
The activity was comparable
to that of hydrocortisone. Topoisomerase
Withangulatin A (39) acts in vitro on
II
to
induce topo isomerase II mediated DNA damage Several daturalactones show, to some extent, antifertility 201 . Daturalactone B (74) is more
effects on albino female rats active
than daturalactone A
23~hydroxyphysalolactone
(64) and 2,3-dihydrodaturalactone
(180) exhibited
cytotoxic activity
B.
(ICcp
value 40 ug/ml) against L-5178 Y cell in vitro"''^^. The epoxide and quinoid like moiety in AB ring as well as the unsaturated lactone in the side chain have been shown to have tumor-inhibitory
activity
as
found
in
several
classes
of
•J 202 terpenoids
Withaferin A (12) and withacinstin
(16) showed cytotoxity
invitro, against KB cell cultures derived human carcinoma of the nasopharynx and invitro against sarcoma 180 in mice and walker 25 6 , . 44,203 intramuscular carcinoma Physalolactone was
inactive
against
175
, a chlorohydrin
P-388
lymphycytic
of
4P-0H withanolide
leukemia,
indicates
E
the
5,6-epoxide is a requisite for the activity 17P-Hydroxy
group
along
with
5P,63-epoxy
system
is
essential for the antitumor activity, eg. withanolide E (46), 4P-0H withanolide E (53). Thus 17-isowithanolide (34) possessing 17°'-0H is found to be inactive
.
17 3-Hydroxywithanolide K was found to
be active against a number of potentially pathogenic fungii 16c
Dunal
204
Even the crude extracts of the leaves of Withania
somnifera
have been reported in Ayurveda and Unani Medicine (India)
used for the treatment of tumor and tubercular glands and besides
246 being sedative and hypnotic. W. somnifera
The glycowithanolides from effective
on
Swiss
mice
in
alleviating
were shown to be
the
adverse effects of 215 . The same were
morphine and attendant immunosuppression action also found
to act on an animal model of Alzheimers disease and
perturbed central cholinergic markers of cognition in rats. findings
were
"medharasayan"
taken
to
support
(Promoter
of
the
of ^'
effect
learning
and
These
somnifera
memory)
as
^s
used
in
Ayurvedic medicines A. arborescens and tubercular glands Physalins
is also used
44
which
resemble
for the treatment of tumors
closely
in
their
withanolides occur mainly in the plants of Physalis
structure species.
to The
plants as such as well as the isolated physalins have been reported to exhibit varied pharmacological activity. P. alkegengi
was
diuretic, anthelmintic
reported
in
Chinese
medicine
and anticancer activities
P.angulata^^^
andP. peruviana^^^
P. angulata
and P. peruviana
are
diuretic.
are
.P.
The
anthelmintic
and
to
have
minima
leaves P-
of
pubescens
(Puerto Rico) has been used in indigestion. Physalin B (190) and epidihydrophysalin from
Witbaringia
cocculolides
were
shown
to
C
(204) isolated
possess
promising
antitumor activity Physalin B (190) was found to be moderately active in PS. The
favourable
intensification
influence of
of
antitumor
a
5a,6ot-epoxy
activity
was
grouping
discolsed
in
the
from
the
observation that 5a,6a-epoxyphysalin B is more cytotoxic in 9 KB than physalin B
'
Antitumor activities of physalins A, B, E, F and L were determined
by
using
He
La
cells
moderate in vitro cytotoxicity Physalin
B
cytotoxic
(190) and
physalin
against mouse
tumor
physalin
(IDCQ F
A
(18)
exhibited
yg/ml) against He La cells.
(194), which were known
cells
show
higher
activity
to be (IDc^
0.32 -0.35 yg/ml) than physalin A, while physalins E (193) and L (200) were found to be inactive. Physalin X, a compound of unknown modifying physalins of Pbysalis ^ ^.^ . ^54,211 abortifacient
minima
structure, obtained by
has been reported to be an
247 Nicandra physalolides
104
a South American plant is noticed
for its fly repellent activity. Ecdysones which are yet another closely related group of polyoxy steroids and Nicandra
steroids are knwon for their powerful
antifeedant properties. These are regarded as insect moulting 212 213 hormones , eg. « - and 3 -ecdysones (245,246), ajugalactone (252). Synthesis The methods
efforts
on synthesis
for the synthesis
substitution
patterns
of withanolides
of model
of rings
A
compounds and B
comprise (a)
with
the required
(b) methods
synthesis of the unsaturated 6-lactone side chain
for
starting
the from
androstane skeleton and (c) attempts involving rings A and B and the side chain finally leading to several natural withanolides, as withaferin A, withanolide D etc. reviewed by E. Glotter
These details have been recently
and hence no description has been included
in this article on the synthetic aspects. ACKNOWLEDGEMENTS One of the authors
(A.S.R. Anjaneyulu) wishes to express
his gratitude to the authorities of Andhra University for granting sabbatical leave from January to June, 1996 and the authorities of Northeastern visiting
University,
Professor during
update and finalise
Boston
for extending
him facilities as
this period which made it possible to
this review.
The assistance
rendered
inthe
preparation of manuscript by Messers D.V. Jagannadha Rao (typing), V.V.S. Prasada
Rao (drawings) and M.V.R.
reading) is gratefully acknowledged.
Krishna
Murthy
(proof
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
263
Natural Products by Oxidative Phenolic Coupling Phytochemistry, Biosynthesis and Synthesis G.M. Keseru and M. Nogradi
1.
INTRODUCTION
2.
NATURAL PRODUCTS BIOSYNTHESIS OF WHICH MAY INVOLVE OXIDATIVE PHENOLIC COUPLING*
2.2 Curare alkaloids 2.3 Amino acids 2.4 Compounds formed from precursors containing one aromatic ring 2.4.1 Allylphenols, isoprenylphenols and cinnamic acids 2.4.2 Aromatic carboxylic acids 2.4.3 Benzoquinones 2.4.4 Phenols 2.4.5 Lignans 2.4.6 Ellagic acid derivatives (ellagitannins) 2.5 Compounds formed from precursors containing two aromatic rings 2.6 Compounds formed from precursors containing condensed aromatic rings 2.6.1 Naphthols, naphthoquinones 2.6.2 Anthraquinones, anthracenes and reduced derivatives thereof 2.6.3 Phenanthrenes and dihydrophenanthrenes 2.7 Compounds formed from precursors containing four aromatic rings. Bis(bibenzyls) 2.8 Compounds formed by oxidative phenolic coupling of oxygen heterocycles 2.8.1 Benzofurans 2.8.2 Chromenes, chromones, dihydrochromones 2.8.3 Coumarins 2.8.4 Naphthols and naphthoquinones combined with oxygen 2.8.6 Xanthones 2.9 Terpenoids 2.10 Antibiotics 2.11 Marine metabolites
264 3. OXIDATIVE PHENOLIC COUPLING IN THE BIOSYNTHESIS OF SECONDARY METABOLITES. 4. SYNTHETIC METHODS 4.1 Synthetic methods for the formation of biaryls 4.1.1 The Ullmann biaryl synthesis 4.1.2 Nickel- or palladium-catalysed Grignard-type coupling 4.1.3 Pschorr-type diazo-couplings 4.1.4 Photochemical syntheses 4.1.5. Oxidative phenolic coupling 4.1.6 Non-phenolic oxidative coupling 4.1.6.1 Vanadium(V) compounds 4.1.6.2. Thallium(III) compounds 4.1.6.3 Other oxidants 4.2 Synthetic methods for the formation of diarylethers 4.2.1 Ullmann diarylether synthesis 4.2.2 Oxidative coupling 4.2.3 Coupling using arene-metal TT-complexes APPENDIX 1. INTRODUCTION "Oxidative coupling is an important reaction in the biosynthesis of
secondary
plant metabolites. E.g.
it was
estimated
that
the
biosynthesis of more than 10% of alkaloids involves one or more oxidative coupling steps [1]. In oxidative coupling more often than not a phenolic hydroxyl group plays an important role role
is dual,
radical
it
may
both act
active as
the
and
passive.
attacking
In
agent
form
of
proper,
[la]. This an or,
aryloxy as
an
electron donating group, activate the aromatic ring in ortho and para positions. Attack of an aryloxy radical leads to compounds of diarylether type, while activation by a phenolic hydroxyl not only facilitates such an attack but may promote the oxidative formation of aryl radicals leading to diaryl type compounds. Considering the vast number
of natural products the structures of which
suggest
that their biosynthesis may involve oxidative phenolic coupling, it is not surprising that rigorous proof of such pathways has only been presented for a few. In the first part of this review we present typical examples of compounds presumably involving oxidative phenolic coupling in their biosynthesis. The
compounds
are grouped
according
to
structural
265 features rather arbitrarily. A more or less complete list of such compounds compiled from the Dictionary of Natural Products [3] is given as an appendix. It is noteworthy that while with some classes of plant metabolites (e.g. terpenes) oxidative phenolic coupling is exceptional, for others (such as curare alkaloids and ellagic acid derivatives) this reaction produces an amazing variety of products (161 and 164 compounds respectively) listed. In the second part we describe the outline of their biosynthesis, while in the third part some general synthetic methodologies for the formation of aryl-aryl and aryl-0-aryl bonds, the characteristic structural feature of our compounds, are presented. 2. NATURAL PRODUCTS THE BIOSYNTHESIS OF WHICH MAY INVOLVE OXIDATIVE PHENOLIC COUPLING* 2.1 Alkaloids (except curare alkaloids) Although a wide range of alkaloids show structural characteristic
of
compounds
arising
from
oxidative
features phenolic
coupling, an overwhelming majority of such compounds are derived from
isoquinolines,
mainly
from
1-benzyl-l,2,3,4-
tetrahydroisoquinolines. Some of them are formed from two identical units through C-C bonds (e.g. bisacnadinine) or from two different isoquinolines
through
an
ether
bond
berberidine).
(e.g.
In
melantolide two identical units are joined both by a C-C and an ether bond, while in medellin two different units were attached to each
other
by
a
C-C
bond,
an
ether
bond
and
a
methylenedioxy
bridge. Isoquinolines may be linked to other structures as well, such as a naphthol (e.g. dioncophylline) or an aromatic ester (e.g. isonoyanine). Intramolecular bridge formation by oxidative coupling is
also
common,
just
to
mention
morphine
and
the
aporphine
alkaloids, or as a less well known structure, linaresin. Spermidine alkaloids, such as cadabicin, are essentially macrocyclic cinnamic amides involving a diarylether bond (Schemes 1-4).
*Compounds are mentioned by their trivial names and respective codes of the Dictionary of Natural Products [2] are given in parentheses. When no trivial names were coined, only the codes are given. The bonds which may have been formed by oxidative phenolic coupling are indicated by heavy lines.
266 MeQ
Scheme 1
OMe
MeO OH
6
Atalanine (A-2988)
Bisjatrorrhizine
iMe
Bismurrayafolin (B-584)
OH (B-562)
JOQ"^ MeO,
Berberidine
(B-287] OMe
HN^^O
MeO* MeO Me Cadabicin (C-19)
OH
Bisacnadinine
(B-491)
267
o 12,12'-Bis(11-hydroxycoron a r i d i n e ) .(B-539)
OMe
OMe Decaline
9CI
(D-176)
NMe
Dioncophylline (D-3353)
MeO
OH
Emestrin B (E-206)
MeO Disazecin (D-3758)
OMe Heimidine (H-146)
268
Scheme 3 MeN
0*
0
MeO MeO Isonoyaine
(1-572)
Jolantinine (J-116) MeO.
TTH^
MeO,
NMe
.^'^^^^ Jolantamine (J-113)
MeO Kreysgenine (K-317) OH MeO
MeO Linaresin (L-588)
Lithramine (L-974)
269 Scheme 4 MeO
MeO.
NMe
OMe Medellin
Melantolide
(M-368)
(M-420)
MeO
Me Me-'^'^'^Me
OMe
Pilocerein (P-1271)
Secolucidine
OH (S-457)
.OH MeO,
MeN
MeO'
MeO y-Morphine
{M-1697)
Speciosin
{S-967)
2.2 Curare alkaloids Curare
alkaloids
tetrahydroisoquinolines
are
dimers
of
l-benzyl-1,2,3,4-
linked by one, two, or even three
ether
bridges positioned in a most varied manner. Discussing them in a
270 separate section is justified by their impressive number and variety (more than 160 compounds are listed in ref. 3 ) . In some of them, as in baluchristanamine B, oxidative cleavage has severed one of the benzyl groups from the isoquinoline ring. Interestingly compounds derived by C-C coupling do not occur in this group (Scheme 5 ) . Scheme 5
NMe
Me-N
Berbamunine (B-284)
NMe
Me-
o OH
(
Me7^r>v.^J^
CHO istanamine (B-88) Baluchri
MeO
Cycleaneonine {C-2215) 2.3 Amino acids Amino acids rarely undergo oxidative coupling, the most important example being thyroxin. Two other examples are shown below (Scheme 6). Bractazonine, B-756, containing a nine membered ring is biosynthetically also related to amino acids. The prerequisite of oxidative phenolic coupling is an aromatic ring and therefore, for want of a more logical ordering principle.
271 nitrogen free compounds will be discussed according to the number of aromatic rings in the precursors.
Scheme 6
HoNOC
CO2H
Aldostatin HO
)
H-4
"NHCHO (A-716)
^
/
N=C D-1691
OMe
J.
HO I Bractazonine
(B-756)
P^°^^"
.CO2H
<
NHo
I
Thyroxine
{T-1457)
2.4 Compounds formed from precursors containing one aromatic ring 2.4.1 Allylphenols, isoprenylphenols and cinnamic acids All the examples in this group are products of C-C coupling. In the isoprenyl substituted members the isoprenyl chain often forms, like in tectol, a dihydropyran ring. Isodunnianol
is one of the
rare trimeric compounds in this group (Schemes 7 and 8 ) .
272 Scheme 7 Me
Me
Bisclausarin (B-503)
Dehydroisopeniclide (D-316) CO2H OH
MeO
>\ ^
OH
r
loy v^_/
MeO
/
r^ u ^ ^^ Dehydrodieugenol (D-296) C02Me
^ OH
Gr .
^)
r.'^ ^' Diferulic a c i d (D-1072)
V
I
/
Magnolol (M-lOO)
nCsHii Me
MeO
"^
OH
OH Epiphorellic acid {E-342)
^^' ^^ HO
OH
Mastogophorene D (M-317)
2.4.2 Aromatic carboxylic acids Both diary1 and diarylether type compounds occur in this group. Formally ellagic acid derivatives should also be dicussed here, but due to their large number and high complexity they are treated separately. Two gallic acid molecules are linked by an ether bond in chesnatin, while three such molecules with one ether linkage and one C-C bond occur in valoneanic acid. Canesolide, although formally a furan derivative, also belongs to this group.
273 Scheme 8
Eudesmagnolol (E-18 59)
Hermonionic acid (H-484) Me Me 0'
OH Isodunnialol (1-430)
Tectol (T-170)
2.4.3 Benzoquinones Biperezone, B-405, is the only benzoquinone known to exist in a dimeric form (Scheme 10). 2.4.4 Phenols Monocyclic phenols readily form dimers and oligomers involving both C-C and diary 1 ether bonds (Scheme 10) . In the dibenzodioxin M-677 probably two ether bonds are formed by oxidative coupling although it is unclear which ether oxygen is derived from which precursor. Oligomers can be exemplified by pentafuhalol, a homopentamer of pyrogallol (Scheme 11). 2.4.5 Lignans Lignans, themselves products of dimerization of cinnamyl alcohols, can undergo further dimerisation to yield compounds like cerberalignan A (Scheme 12).
274
Scheme 9 HO
O
OH
OH
OH -OGluc
95H11 C3H7
CO2H
OH MeO-
H02C'''^^
HO^
OH
Chestnatin Me
v\
-0 .
Isodidymic
(C-885)
acid
(1-417)
Me OH
CH3 (CH2) 6
CO2H
. (CH2) 6CH3 CO2H
MeO HO
HO ^ Mennegaziac acid (M-467)
MeO
OH Micaric acid (M-1480)
H-1296 CO2H HQ
HO
OH HO2C
pH HO2'
HO
OH
Valoneanic acid (V-46)
CI Canesolide (C-241)
275
HO
Me
Biperezone
(B-405)
M^v^ ^Me
HO-"^^;^-^
OH D-3060
Vi::-X/^^OH
Hematoxylol (H-13)
M e ^ ^Me
.OH
OMe
Heyderiol A {H-845)
Leprolomin (L-446)
MeO
Scheme 11
DH
^H
Pentafuhalol (P-413) Scheme 12 HOCH2.
OH
OMe Cerberalignan A (C-750)
276 2.4.6 Ellagic acid derivatives (ellagitannins) Oxidative dimerization of two molecules of gallic acid give rise to ellagic acid, which is, in the dicarboxylic acid form, the basic building block of a very large number of highly complex natural products containing at least one but usually two or more molecules of ellagic acid, linked to glucose or some of its transformation products.
Sometimes
additional
gallic
acid
units
and
other
polyphenols are incorporated into the molecule. These features are well illustrated by acutissimin C and agrimonic acid In the
latter
the glucose
unit
is
linked
as
an
(Scheme 13) .
open-chain
C-
glucoside both to a catechin and a gallic acid unit, the latter being attached to ellagic acid by a C-C bond. 2.5 Compounds formed from precursors containing two aromatic rings Often the two aromatic rings involved
in oxidative phenolic
coupling are already linked in some way, in other words coupling proceeds
in
an
diaryIheptanoids
intramolecular or
way.
For
dibenzocyclooctanes
some (e.g.
compounds,
as
the
acerogenin
A
and
binandkatsurin A resp.) this order of events is highly probable, while for others, as in several of the alkaloids discussed above (e.g. lithramine) or in protosappanin B, it is less evident (Scheme 14). 2.6 Compounds formed from precursors containing condensed aromatic rings 2.6.1 Naphthols, naphthoquinones With naphthols and naphthoquinones C-C coupling
seems to be
exclusive, although the ether bond in D-2250 may have been formed oxidatively too (Schemes 15 and 16). Both symmetrical
(e.g. 3,3"-
bisjuglone) and unsymmetrical homodimers (e.g. bisisodiospyrin) are common. The units can be linked by a double bond, as in diosindigo A, and by a second C-C bond, as in hypocrellin. Naphthoquinones may also
form
interesting
trimers
as
e.g.
combination
in galpinone. Ancistrocongine of
a
naphthalene
dihydroisoquinoline. Here the naphthalene moiety group.
ring
is an
with
a
is the acceptor
277
Scheme 13
HOCH
Ellagic acid (dilactone
UO^
form)
OH
Agrimonic acid (A-580) 2.6.2 Anthraquinones, anthracenes and reduced derivatives thereof This group comprises a large variety of types from simple anthraquinone dimers, as chrysotalunin, to combinations of an anthraquinone with an amino acid or a prenylated naphthol (e.g. laccaic acid C and dermocanarin I) , tetrahydroanthracenes and anthraquinones such as setomimycin and julichrome Ql, as well as to compounds with multiple cross-links as, for example, fagopyrin. Biogenetically the tetrahydro derivatives can be rather regarded as substituted naphthols. This is rather obvious e.g. with karviskione. Finally the anthraquinone moiety may combine with other oxygen heterocycles an in the case of euxanmodin A (Schemes 17 and 18).
278
Scheme 14 OH
MeO Acerogenin A (A-144) HO
Q
OMe Binandkatsurin A (B-399)
OH
OH HO Caurarinondiol {C-545)
(CH2)i4
Robustol (R-309)
MeO HO OH MeO Diacylkatsulignan C (D-146) Protosappanin B (P-1986)
279
Scheme 15 DMe
OH Ancistrocongine
6 O (A-1666Bisjuglone (B-563) Bisisodiospyrin ;)H
(B-561)
0
OMe
Me'
Diosindigo (D-3359)
Diospyrol 0
(D-3362)
OH
COMe OMe 0
6
OH
D-2250
OH
Hypocrellin
(H-323 3)
OMe
0 Galpinone
0 (G-88)
0 Toddacoumaquinone
(T-1524)
280
Scheme
17 MeO
MeO
OH
OH . 0 A s p e r i n i n e A (A-2902
^NH
OH
0
Dermocanarin I (D-532)
OH MeO
.NH
OH 0 OH Fagopyrin (F-18)
Karviskione (K-81)
2.6.3 Phenanthrenes and dihydrophenanthrenes The title compounds are much less common than the anthraquinones. An example for a heterodimer, probably formed from a homodimer, is blestrin (Scheme 19) . 2.7 Compounds formed rings. Bis(bibenzyls)
from precursors
containing
four aromatic
It is a reasonable assumption that macrocyclic bis(bibenzyls) such as the marchantins arise by oxidative C-C or ether bond formation from acyclic precursors already containing all four aromatic rings (e.g. perrotetin E). The ether linkage between the second and third rings is probably also established by phenol oxidation. Gnetifolin, though phytochemically unrelated, is structurally rather similar to bis(bibenzyls) (Scheme 20).
281 Scheme
Me^
18
OH
0
OH
0
Chrysotalunin
9
,iOAc
(C-1283)
0
OH
Julichrom
Ql
(J-141) CO2H
QH
-NH2 HO2C 9
OU
HO2C.
ir^T
Euxanitiodin
(E-2018)
OH
OH
0
0
OH
OH
_ OH HO'^ ^^^'^'-^ ^ ^ 1 ^ ^ V ^ ^ O H 0 OH Laccaic acid (L-157)
OH Setomimycin
(S-661)
TOT
0
Antibiotic
OH MA 1 4 4 A E 1
Scheme 19 MeOv
—-
.OH
Blestrin
D
(B-653)
0-200
(2201)
282 Scheme 20
OH Marchantin A (M-258)
Perrottetin E (P-845) HO^ . x ^ ^OH
HO
OMe Plagiochin D {P-845)
2.8 Compounds heterocycles
formed
by
Gnetifolin
oxidative
phenolic
(G-615)
coupling
of
oxygen
2.8.1 Benzofurans From the mulberry tree a large number of 2-arylbenzofurans (e.g. dimoracin), typical products of oxidative phenolic coupling, have been isolated. The sequence of events with dibenzofurans (C-C or OC coupling first) is less clear (Scheme 21). 2.8.2 Chromenes, chromones, dihydrochromones Simple symmetrical homodimers of chromenes with a C-C linkage such as ageratoriparin and non-symmetrical ones with an ether bond, like
pterochromene
13
are
both
known.
Chromones
can
form
homodimers, such as compound D-1580, or combine with a flavonoid as in the case of compound D-1419. Ergoxanthine is better considered as a xanthone precursor rather than a proper chromanone. 22) .
(Scheme
283
Scheme 21
Dimoracin
(D~3315)
MeO
OMe Candidusin
(C-229)
Penioflavion
(P-308)
2.8.3 Coumarins A trimer with both C-C and ether likages is triumbellatin. I-sophysodic acid is an example of an isocoumarin linked to an aromatic acid of very similar structure (Scheme 23). 2.8.4
Naphthols
and
naphthoquinones
combined
with
oxygen
heterocycles Naphthols
and
naphthoquinones
anellated
with
an
oxygen
heterocycle like a pyrone or isopyran ring usually form homodimers through C-C bonds (Scheme 24). 2.8.5 Flavonoids Almost diarylether dimeric found.
every
type
of
flavonoids
linkages, but dimeric such
as
dimers
flavones are more
isoflavones. In batramiaflavone Homodimers
form
two biaryl
biscyclolobin
and
via
C-C
common
or than
bonds can be
heterodimers
such
abiesin both exist. In the latter even the oxidation level of the monomers
may
differ.
Important
members
of
this
group
are
the
284 oligomeric catechins ranging from dimer to hexamer or probably even to higher polymers (Schemes 2 5 and 26).
Scheme 22 MeO
OMe
Me
0 AH-11
Pterochromene 13 (P-2106)
^^
^Ph
(D-1580)
OH
D-1419
C,H 5^11
Scheme 23 0" ^0
H02C
Triumbellatin
(T-2962) Isophysodic acid
(1-601)
285
Scheme
24
MeO'
MeO" Crisamicin A
(C-2000)
T a l a r o d e r x i n e A (T-36) 2.8.6
Xanthones
A few dimeric xanthones and xanthone precursors linked through a C~C bond are known (Scheme 27).
286
HO^
"^ 0 Biscyclolobin (B-50 4
0
OH
Batramiaflavone
2, 3-Dihydrohinokiflavone
(D-1213)
(B-109)
287 Scheme 2 6
Oligomeric catechins
HO' (P-528-532)
^^
0 ^Ph T-589
'Me Me02C E Eumitrin Al (E-1960) HO
Ploiarixanthone
(P-1512)
2.9 Terpenoids Terpenoids undergo oxidative coupling only exceptionally. The only examples recorded in ref 1 are shown below. Note that B-538 is not a phenol.
288
Scheme 28
^e-
.CO2H
Macrophyllic acid
Me^
Me
(M-530)
2.10 Antibiotics Some important groups of antibiotics and antineoplastic agent such as the vancomycins (e.g. vancomycinic acid) and the so called OF peptides (e.g. peptide 49491) are evidently products of oxidative phenolic coupling, while with the tetracylines this process seems to be marginal. In antibiotic A 4103 0 as many as three ether linkages and one C-C bond is formed by oxidative coupling. The dimeric tetracycline A-2201 is shown with the anthraquinones (Scheme 29). 2.11 Marine metabolites Oxidative phenolic coupling giving rise to ether bonds is common with marine metabolites (Scheme 30).
289
Scheme 29
'NHCO
_NHCO • " ^ NHCO
\
NHCO^''«NHCO
00
HO2C OH
•'•NHO
OH
A n t i b i o t i c A 41030
(A-1923)
^OMe
CI
JL H2N^
NH I if
^ y
1^^ HO^
HO.^ CO2H
^H
^C0NH2
HO2C
OF p e p t i d e 49491
(A-2245)
"NH2
HO2C"" ^NH2
Vancomycinic a c i d
Br Br 0^ I
Br
Br NH
JU
^ 1 ^ "Br OH
^OH Bastadin
4
{B-158)
Br HO D-727
HO2C
(V-63)
''NH2
290 3. OXIDATIVE PHENOLIC COUPLING
IN THE BIOSYNTHESIS
OF
SECONDARY
of
secondary
oxidative
phenolic
METABOLITES. Current metabolites
knowledge
concerning
the
involving
in
formation
their
biosynthesis
coupling is rather fragmentary. While a vast body of information has
accumulated
precursors
about
the
(alkaloids,
biosynthetic
flavonoids,
pathways
etc.)
leading
undergoing
to
the
oxidative
phenolic coupling, much less is known about the coupling process itself. It
was
Barton
and
Cohen
who
first
suggested
that
the
biotransformation of benzylisoquinolines to morphine alkaloids may involve phenolic radicals, which by radical pairing form new C-C and
C-0
bonds
either
intra-
or
intermolecularily
[4].
This
hypothesis was supported, among others, by the finding that in in vivo experiments (R)-reticuline was transformed to salutaridine. Among the enzyme systems performing redox reactions oxidations copper containing oxidases perform the irreversible
oxidation of
phenols. Two main types of phenol oxidases are known, the catechol oxidases and the laccases of which the latter is supposed to be involved
in oxidative phenolic coupling
[5, 6 ] . Laccases contain
+
2+
four atoms of copper, two Cu and two Cu mechanism of oxidation, generating phenoxide follows: enzyme-Cu "*" +
Ar-OH -> enzyme-Cu"*"
+
Ar-0*
ions. The basic radicals is as
+ H"*"
In a second step the enzyme is reoxidized by molecular oxygen: 2 enzyme-Cu"*" +
1/2 O2 +
2H
—>
enzyme-Cu"*"
+
H2O.
The phenoxy radicals are subsequently transformed by reactions which
are generally
not enzymatically
mediated
and
reasons for the usual absence of enantioselectivity
this
is the
in oxidative
coupling. For instance no optical activity has been recorded for the macrocyclic bis(bibenzyls) contaning a biaryl bond plagiochins),
although
we
have
shown
that
they
(e.g. the
possess
stable
chiral conformations at room temperature [7]. Formation of aryl-aryl bond can be explained by an electrophilic attack by the mesomeric form of the phenoxy radical:
have
Investigations been
impeded
involving
very
therefore
it
specific infancy. A
is
enzyme
the
general
by
not
transformed
Using
to
they
and
laccases,
of
Berberis
31).
intermolecular
A
similar
Note that no berbamunin
[8].
was
(R)-
in
the
and
of
use
culture
the
reaction
Their
involved
in
the
is
tissue
the
in
its
to shed new light results
in
of
the
may
have
Papaver
presence
(R)-reticuline
phenol the
of
fraction
means
in
from
process
plants
that
while
oxidases,
of
was
the
such as
above
transformation
the
stereospecific
microsomal
and
(S)-coclaurin
to
the
to
fraction
berbamunin.
was formed when only one of the enantiomers
Of course the above results
products.
least
the
intact
coupling
identification
that
This
system
32).
phenoloxidases
in
enzyme
is correct,
at
coclaurin
and
following
the
microsomal P-450
of
stereospecificity
was
of
by
alkaloids.
a
enzyme
coupling
for
demonstrated
role,
stolomifera
the
phenolic
Zenk and his coworkers
cytochrome
no
that
possible
salutaridine
play
of
intermediates
of Barton and Cohen
the
(Scheme
only
oxidative
difficulty
of morphine
capsules
of
responsible
w h i c h permitted
oxygen
details
surprising
was
relevance.
hypothesis
the
systems
biosynthesis
somniferum NADPH,
the
short-lived
breakthrough
techniques on
on
supplied
regioselectivity
of
system,
the
do not exclude
biosynthesis
of
other
enzyme the
proving [8a]
(Scheme
intervention
classes
of
the of
coupling
Scheme
Meo0
31
MeO~~~/~
M e O ~
~o~~~~_ ~ e ~o- ~ ~
M e O ~ OH
MeO .....
.....
MeO
H NMe
OH (R)-Reticuline HO
______~.
MeO~
~*/~~~_- NMe
~eO~Ji 0
i~Me
O (+)-Salutaridine
Scheme
32
MeO~/~~
MeO
HO"~.~.2,. _NMe v __-~H -
+
H O ~ (R)-Coclaurin MeO~~/~
H0~HNMe
NMe
HO HO MeO H
OH
Berbamunine
'IH
(S)-Coclaurin NMe
'IH
293 4. SYNTHETIC METHODS 4.1 Synthetic methods for the formation of biaryls General methods for the synthesis of biaryls will be reviewed in this section. Electrochemical processes are outside the scope of this work. Asymmetric syntheses have been excellently reviewed recently by Bringmann [9] and therefore will not be overviewed here. 4.1.1 The Ullmann biaryl synthesis The classical Ullmann reaction has been for long the exclusive method for the formation of aryl-aryl bonds [10]. In a typical procedure two equivalents of an aryl halide react in the presence of copper powder or other copper salts to give biaryls (Scheme 33). Scheme 33 Ar-X + Ar*-X
— A
• Ar-Ar + Ar-Ar* + Ar'-Ar'
By the same procedure differently substituted aryl halides can also be coupled to biaryls and heterobiaryls but the method is rather impractical because the desired unsymmetrical product is always accompanied by the symmetrical ones. Yields in the Ullmann reaction are generally low, aryl iodides and bromides are more reactive than the corresponding chlorides, while the fluorides are totally inactive. Introduction of nitro or ester groups in ortho position relative to the halogen atom of the aryl halide may dramatically increase yields. The presence of proton donor substituents such as amino, hydroxy1 and carboxyl groups are, in turn, unfavourable, because these functional groups may also react with the aryl halide [11]. Aldehyde, ester and lactone groups normally remain unchanged [12]. The Ullmann reaction is sensitive to steric effects, large substituents in ortho position may inhibit the reaction completely. The mechanism of the Ullmann reaction has not been fully clarified, however, it should proceed via aryl-copper intermediates [13]. This proposal was supported by an improvement of the method by Koten and Noltes, who reacted an aryl-copper compound with
294 copper(I)
trifluoromethane
sulphonate
and
obtained
symmetrical
biaryls in high yield [14] (Scheme 34). Scheme
34 Ar
V -cui
+
CF3SO3CU
V-c^ i
CUv
rOaSCFs
-
it CUO3SCF3
Ar—Ar
+
Cur'
^*CuO
This
approach,
products, biaryls
was
which also
[15].
The
Ar
^ Ar /
^
obviates
applied
to
aryl-copper
the the
IJ.CU :t03SCF3 "•Cu n formation synthesis
compound
of of
was
symmetrical unsymmetrical
prepared
from
corresponding aryl-lithium obtained by direct lithiation of an aryl halide and was coupled with the other aryl halide.
Owing to stabilisation by a nitrogen or sulphur containing ortho substituent stabilisation
relatively and
high
activation,
yields
were
effected
by
achieved. masking
an
Both ortho
positioned aldehyde function as an oxazoline ring, were achieved in an elegant synthesis of steganicin by Meyers et
al.
[16].
295
Scheme 36
(-)-Steganone 60% de This
so-called
"oxazoline"
synthesis
of
biphenyls
[18], and
several
method
crowded binaphthyls
was
also
biaryls,
applied
[17]
[19], as
for
the
tetrasubstituted
well
as
the
highly
functionalized cyclic biphenyl moiety of vancomycin [20].
Scheme 37 OTMS OTMS Mg, BrCHoCHoBr THF, 80%
o
•OMe
OMe MeO
OMe
OMe
MeO
OMe
Improved yields in the Ullmann reaction could be realized using activated iodide
copper powder
with
prepared
potassium
using
a
obtained
by
in situ
[21]. Unsymmetrical
salicylic
alcohol
reduction
of
Cu(I)
2,2•-biphenyls
were
template
combined
with
the
catalytic effect of activated copper [22]. Lithium-cuprate iodide applied
and in
alternative
the the
complexes
corresponding synthesis
strategy
obtained
by the reaction
aryllithium of
involves
were
unsymmetrical the
low
also
successfully
biaryls
temperature
coupling of higher order mixed diarylcuprates
of Cu(I) [23].
An
oxidative
[24]. The classical
Ullmann reaction can be accelerated by using, instead of copper
296
powder, copper(II) oxide, or by ultrasonic irradiation during the reaction [25]. In order to prevent high reaction temperatures a combination of copper(I) triflate and aqueous ammonia in acetone was recommended, which allowed the reaction to be run efficiently at room temperature [26]. The intramolecular version of the Ullmann reaction was useful for the preparation of condensed systems [27] (Scheme 39).
Scheme 39 MeO
OMe
•OMe
MeO Cu,210QC ^ 68% ^
C02Me
C02Me
The classical methodology of the Ullmann biaryl synthesis was significantly nickel using
improved
complexes
by
Semmelhack
[28]. Aryl
al.
using
iodides were coupled
bis(1,5-cyclooctadiene)nickel(0)
further
et as
improved by the application of
zero-valent
in high yield
catalyst.
Yields
were
tetrakis(triphenylphosph-
ine)nickel(O) [29] (Scheme 40). While the original methods require stoichiometric nickel
complexes, Zembayashi
et
al.
claimed,
however,
amount of that
the
reaction is also feasible with a catalytic amount of nickel(II) ions and Zn powder [30]. The method was also applied by Colon
297 Scheme 4 0 /^^"2)2X
Ni(0)[Ph3P]4 ^
76-83% et al. for the synthesis of unsymmetrical biaryls [31]. Interestingly aryl chlorides afforded a better ratio of coupling to reduction than the corresponding bromides and iodides. A stoichiometric amount of activated nickel powder was also found to be effective [32]. The mechanism of Ni(0)-catalysed coupling was investigated by Tsou and Kochi [33]. Readily available nucleophilic aryl Grignard compounds can be coupled with aryl halide electrophiles to give unsymmetrical biaryls in the presence of Ni, Co, V, Ti, Cu, Cr, Fe, Pd and Tl salts [34] (Scheme 41). Scheme 41 2 Ar-MgX + MX2 • [Ar2M] M = N i , Co, V, T i , Cu, C r ,
• Ar-Ar F e , U, Tl
Ni and Pd catalysts were found to be most useful for this purpose [35]. Palladium catalysed reactions show better chemoselectivity, but are restricted to activated aryl bromides while nickelcatalysed processes are more tolerant to steric hindrance of aromatic substituents [36]. These reactions are the best alternative to Ni(0) catalysed high temperature coupling of aryl halides [37]. With stericaly hindered aryl halides generation of a Grignard reagent is recommended. Preferably the component with an ortho substituents should be converted the Grignard compound[38]. Chiral ferrocenyl phosphine nickel complexes are useful for the preparation of optically active biaryls [39]. Ultrasonic irradiation is often applied to accelerate the reaction [40]. Aryllithium reagents are generally inactive, while arylzinc compounds can be efficiently coupled in the presence of nickel catalysts [41]. Compatibility with a wide range of functional groups and easy access to arylzinc compounds from the corresponding aryllithiums is the main advantage of their usage. Effectivity of this approach was demonstrated in the synthesis of steganon by Larson and Raphael [42] (Scheme 42).
298
1) Ni.THF^ 2)HC1 ^
Steganon
R = -CH=NC8Hi2, 85% MeO.
R = -COzMe, 0%
MeO MeO MeO The reaction was exploited very recently in a solid phase synthesis of biaryls. Aryl zinc bromides undergo palladium catalyzed coupling reactions with
aryl bromides bound
to a polystyrene
resin. The
product can be released from the resin by transesterification [44]. Ni(0)
catalysed
homocoupling
of arylzinc
reagents
could
also be
realised using aryl triflates [45], as well as aryl tosylates and mesylates [46]. As with nickel catalysts, Pd(0) complexes are also effective in the
coupling
demonstrated
of
aryl
iodides
with
arylzinc
in the synthesis of biphenomycin
compounds
by Schmidt
as
et
al,
[47]. Aryl triflates are also useful as electrophiles in the reaction of arylzinc compounds [48]. Pd(0) and Ni(0)catalysed cross-coupling and homocoupling reactions of aryl triflates were reviewed recently [49]. Aryl fluorosulfonates gave biaryls in this reaction too. The first application of arylstannanes was presented by Stille who
demonstrated
the
transferability
of
an
aryl
group
from
tetraphenyltin [50]. This approach was generalised by the improved preparation halides
of
arylstannanes
followed
by
their
from
Pd(0)
trialkylstannanes catalysed
coupling
and
aryl
with
aryl
halides [51]. Similarly to aryl halides, aryl triflates also react with
arylstannates
triflates
are
to
give
especially
biphenyls suitable
[52]. Electron-rich
coupling
reagents
aryl
in
the
synthesis of unsymmetrical biaryls [53]. Symmetrical biaryls can be easily obtained by the reaction of an aryl triflate with 0.5 eq. of hexamethyldistannane. Arylcoppers particularly couplings.
[54]
efficient
Symmetrical
and
mercurials
nucleophiles biaryls
[55] in
were
found
to
be
palladium-catalysed
can be efficiently
prepared
from
299 arylthalliuin catalyst of
bis(trifluoroacetate)
with
Li2PdCl4 as
[56]. It is interesting that due to the induction effect
fluorine
coupled
on treatment
atoms, arylfluorosilanes
with
aryl
halides
could
also
in the presence
of
be a
successfully
Pd(0)
catalyst
(Scheme 43) [57]. Scheme 4 3
[(n3-G3H3PdCl)2] DMF,KF,lOQOC, 85%
One of the best ways to prepare unsymmetrical biaryls is the Suzuki-coupling utilising aryl bromides and as the nucleophilic partner aryl boronic acids and esters (Scheme 44) [58]. Scheme 4 4 ^B(0H)2 Br
The
Suzuki
Pd(0)[PPh3]4
R»
+ reaction
was
1
Na2C03 or NaHC03
applied
to
the
synthesis
of
several
unsymmetrical biphenyls [59]. It is compatible with a wide range of functional successful
groups
in the aromatic
coupling
compounds
and
aryl
prepared
in
situ
has
been
chlorides. from
ring
[60], but up to now no
reported However,
NiCl2(dppf)
involving
using and
a 4
arylborono
Ni(0)
catalyst
equivalents
of
butyllithium, aryl boronic acids could be successfully coupled with aryl chlorides in the presence of K3PO4 [61]. Ni(0) catalysts were also
effective
in the
coupling
of
readily available arene sulfonates reactive
towards
organoboranes
aryl
boronic
acids
with
the
[62]. Aryl triflates are also
[63] and
boronates
such
as
aryl
boronic acids [64]. Boronates may be applied more efficiently, but triflates were found to be less reactive than the aryl
halides
towards
organoborono
species
corresponding
[65]. Note
that
aryl
boronates are more accessible than aryl boranes and can be prepared by transmetallation from the corresponding aryllithium compound and the
air-stable
boronates
require
only
weak
bases
for
cross-
coupling. Base-free couplings can be performed using an equimolar quantity of tetraarylborates
[66]. Coupling of aryl boronic acids
300 and aryl halides or triflates is also possible in high yield in the presence of readily available and less expensive heterogeneous palladium catalysts [67], 4.1.3 Pschorr-type diazo-couplings The Pschorr reaction is the intramolecular coupling of arenes involving aryl radicals generated by the reduction of aryl diazonium salts [68]. The reaction was extensively studied during the last 100 years [69]. This Cu(I)-catalysed reaction proceeds via an aryl diazenyl radical intermediate (Scheme 45) [70]. Non-protic solvents from which competitive hydrogen abstraction is impossible, are to be preferred [71]. Scheme 45 +R-H, -R;^^ Ar-Ar' Ar-N2-^.X-
Cu(I)X
^ Ar-N2* "-^2 » Ar.+ Ar'-H -H^""^^ Ar-H+Ar'-
One of the most important fields of application of the Pschorr reaction is the synthesis of isoquinoline alkaloids. The syntheses of_ thalicsimidine [72] (Scheme 46) and aporphine [73] are typical examples. Modifications of the classical method are mainly concerned with the decomposition of the diazonium salt. Thermal [74], photochemical [75], and electrochemical [76] processes were found to be effective for the generation of the radical intermediate. Ti(III)-catalyzed Pschorr reaction of aryl diazonium salts and phenols gave symmetrical biaryls in excellent yield [77]. Scheme 4 6 MeOv
^^^
^^^
MeO>. NMe
NMe
Thalicsimidine The Gomberg-Batchman-Hey reaction is closely related to the Pschorr synthesis and gives unsymmetrical biaryls by treating aryl
301 diazonium salts with bases [78]. Moderate yields may be improved using phase-transfer conditions and crown-ether catalysis [79]. 4.1.4. Photochemical synthesis Since the first photochemical synthesis of phenanthrene from (E)stilbene was realized [80] photochemical methods became a convenient way of aryl-aryl bond formation (Scheme 47). Scheme 47
Dehydrogenation of the dihydrophenanthrene intermediate formed by the
cyclization
substituted
of
(Z)stilbene
phenanthrenes
presents
[81]. Yields
can
a
general be
route
improved
addition of hydrogen abstractors such iodine or
by
to the
tetracyanoethene
[82]. The method was extended to the preparation of phenanthridones [83] and
led to application of the synthetically
aryl halides
[84] from which
aryl
radicals
can
more
effective
be more
easily
generated. Heterolytic cleavage of the carbon-halogen bond effected by irradiation results in the formation of aryl radicals which can then recombine to biaryls
[85]. The catalytic effect of dialkyl
anilines was also recognised. This strategy was first demonstrated by Kihara et al. synthesis
of
using an aryl iodide as radical precursor in the
dibenzazocine
[86]. Aporphines
and
other
types
of
isoquinoline alkaloids were prepared by this method [87]. The closure of macrocyclic rings represents a special field of intramolecular chain
aryl-aryl bond formation. Irradiation of an open-
dibromide
afforded
myricanone
(Scheme
48)
represented the first synthesis of a macrocyclic [89].
[88],
which
diaryIheptanoid
302 Scheme 4
hv MeO The
OMe
synthesis
accomplished
by
MeO of a macrocyclic an
lactam,
intramolecular
OMe
RO
azoninone, was
photocyclization
also
of
the
corresponding open-chain aryl halide precursor [90] (Scheme 49).
MeO ^ ^
IV-'ST
hv
-^
HO
OMe
MeO'
4.1.5. Oxidative phenolic coupling As mentioned in Section 3, oxidative phenolic coupling plays an important
role
in the
biosynthesis
of
a wide
range
of
natural
products. This stimulated the application of this reaction to the biomimetic
synthesis
of
natural
biaryls.
Unlike
the
in
vivo
processes, very probably following a radical dimerization pathway [91] yields of in vitro syntheses are generally moderate or low. Classical work in this field has been extensively reviewed [92], and
important
developments
after
the
70s
will
therefore
be
summarised here. Methodological development was focused on the exploration of various new oxidants in order to replace the traditional but still popular iron(III) chloride and potassium hexacyanoferrate [93]. The non-selective obstacle
nature
in their
of
these
oxidizing
agents
is
synthetic application. Note that
the
major
the products
themselves are usually phenols too, which can be further oxidized to
give
polymeric
hydroxylated
and
FeCl3/AlCl3 system
by-products.
methoxylated
The
biaryls
selective could
be
synthesis achieved
in
of a
[94]. Application of phase transfer conditions
might be useful in suppressing overoxidation
[95]. The amount of
303 by-products can be also reduced using silica gel-absorbed oxidizing agents [96]. Although silver(I) oxide is generally more reactive than FeCl3, there
are
synthesis
some of
examples biaryls
for
its
[97].
successful
Copper(II)
application
benzoate
in
[98],
the
tert-
butylperoxide [99], the more exotic iodosobenzenediacetate [100], as
well
as
traditional
dichromate
[101]
transition
metal
Manganese(III)
were
oxidizing
used
chelates
agents
in
oxidative
were
found
. acetoacetonate
as
couplings.
to
[102]
ethylenediimino cobalt complex
such be
and
potassium First
row
effective
too.
bis(salicylidene)-
[103] are typical examples. It is
interesting to note that molecular oxygen may be also used in the oxidative
coupling
of
phenols
[104]
and
alkaloids were synthesized
in this way
also
the
be
exemplified
isoquinoline alkaloid
by
several
isoquinoline
[105]. This approach can
synthesis
of
corytuberine,
an
[106]. Intramolecular oxidative coupling of
reticuline N-oxide effected by molecular oxygen in the presence of Cu(I)
chloride
afforded
the
natural
product
in
moderate
yield
(Scheme 50).
Scheme 50 MeOv HO^
©^ y J
HOv
MeO
MeO,
1 ^0" Me
l ) C u ( I ) CI, O2/pyridine 2)NaHS03
HO' HO.
•
& MeO"
4.1.6 Non-phenolic oxidative coupling Oxidative
radical
electrochemical
coupling
as well
substrates. Application
as
of
chemical
arenes
can
oxidation
of anodic oxidation
be of
performed electron
by rich
in the synthesis of
natural [107] and unnatural biaryls [108] are outside the scope of this
review,
and
as
previous
sections,
this
one
will
also
be
devoted only to chemical syntheses. Non-phenolic aryl-aryl coupling of electron rich aromatics is based
on
the
formation
of
a
radical
cation
effected
by
the
304 oxidizing agent via electron transfer [109]. Methods can be classified according to the oxidizing agent generating the radical intermediate. Vanadium, thallium and manganese salts were found to be the most effective reagents. 4.1.6.1 Vanadium(V) compounds The use of vanadium(V) salts in oxidative coupling reactions was prompted by the work of Funk et al. who recognized the ability of vanadium oxytrichloride to form phenoxyvanadium complexes with phenols [110]. As it was shown by Schwartz et al., such complexes can be isolated and used for oxidative couplings [111]. Vanadium oxytrifluoride, a superior reagent [112] was found to be effective not only with free phenols but also with phenol ethers revealing the non-phenolic mechanism of this oxidation [113]. The method was successfully adapted to the oxidative macrocyclisation of a vancomycin subunit [114]. The importance of 3,4-dioxy function was underlined by Halton et al. who observed that the reaction did not proceed in the absence of these substituents [115]. However, the presence of this function is only a necessary, but not a sufficient condition for the success o/ the coupling reaction. Elegant syntheses of lignans and aporphine alkaloids are typical examples demonstrating the synthetic value of this approach [116]. In contrast to benzylic hydroxyls, tertiary or acylated nitrogen atoms do not inhibit the reaction. The formation of macrocyclic rings can be also accomplished in this way [117] (Scheme 51).
4.1.6.2. Thallium(III) compounds The versatile applicability of thallium salts in organic synthesis pioneered by McKillop [118] prompted Schwartz to try them for
305 oxidative coupling [119]. Thalliuin(III) tristrifluoroacetate (TTFA) was
found
e.g.
to
be
useful
for
the
oxidative
intramolecular
coupling of phenols to cyclic quinones [120] (Scheme 52). Scheme 52
*
The use of air-sensitive TTFA was later replaced by its in situ preparation
from thallium(III) oxide and TEA
[121].
This method
represented a new and effective route to biaryls. Conversions were significantly
improved, the formation of overoxidized
by-products
could be decreased and in most cases yields were better than those in
classical
phenolic
couplings
or
in
other
previously
known
methods [122]. Phenolic ethers could also be transformed to biaryls in
this
way
occasionally
[123] used
(Scheme
53).
The
for the regioselective
TTFA/
TFA
formation
system of
was
aryl-aryl
bonds [124]. Scheme 53
MeO
•OMe
MeO
T1(02CCF3)3 zTT^ •
84%
MeO
•OMe
MeO
OMe
OMe
4.1.6.3 Other oxidants Manganese
chelates, such as manganese(III)
acetonylacetonate
were also tried as oxidants in the coupling of phenols and phenol ethers [125]. In one of the first examples binaphthol was prepared by the oxidative dimerization of 2-naphthol
[126]. Although with
this reagent overoxidation can be mostly avoided, low conversions make this method inferior to others. Oxidation
by
selenium
dioxide
[128],
tellurium(IV)
chloride
[129], and ruthenium(IV) tetrakis(trifluoroacetate) [130] have also been reported for a limited number of substrates. Notorious toxicity of heavy metal compounds can be avoided using hypervalent iodine(III) reagents [131]. Diaryliodonium salts yield biaryls
in
the
reaction
with
methylmagnesium
bromide
in
the
306 presence of NiCl2 [132]. Moderate conversions in the oxidative coupling of phenols or phenol ethers can be significantly improved using phenyliodine(III) bis(trifluoroacetate) [133]. 4.2 Synthetic methods for the formation of diarylethers 4.2.1 Ullmann diarylether synthesis As shown structural drawbacks,
in Section 2 diarylethers are also widely units up
to
in
natural
relatively
products. recently,
Despite
Ullmann•s
occurring
its
serious
procedure
was
almost exclusively the method of choice for preparing diarylethers. According to the original methodology, phenols and aryl halides can be coupled
to diarylethers
in the presence
of
activated
copper
powder and potassium carbonate [134] (Scheme 54). Scheme 54 HO
[CU]
R-
-R'
Despite long reaction times and high temperatures, conversions in this
nucleophilic
aromatic
substitution
are
generally
low.
As
expected, activated aryl halides, such as nitroaromatics however, react faster and give higher yields [135] (Scheme 55).Diarylethers synthesised in this way can be converted to corresponding amines, and the amino group can then be replaced in the usual way either by a hydrogen atom or a hydroxyl group via the Scheme 55 OH
NO2
OH HOv^^s^OH
COsMe
NO2 C02Me DMF,r.t.,l h
C02Me
\-^C02Me
+
307 diazonium salts. This approach, which avoids high temperatures, has served well for the preparation of several diarylethers [136]. An aromatic copper compound was postulated as an intermediate and this hypothesis led to the introduction of improved copper catalysts. Activated copper powder, Ullmann's original catalyst, was replaced by Cu(I) salts, such as CuCl [137], Cu(I)Br [138], and Cul [139]. Cu(I)Br was also found to be effective in intramolecular Ullmann macrocyclizations [140]. Cu(II) salts may also have a catalytic effect under similar conditions. CUSO4 [141] Cu(0Ac)2, [142] and CUCO3 [143] were occasionally used. Air sensitive copper(I) salts, such as CuBr, can be stabilised as the dimethyl sulphide complex [144] and we used this catalyst in our laboratory on the multigram scale. In addition to copper(I) halides, copper(II) oxide was found to be an effective catalyst in the Ullmann diarylether synthesis [59, 145, 146] and was used for the preparation of several diarylethers in our laboratory [146]. Cu(I) oxide was first applied by Tomita et al, in the reaction of bromobenzene with 4-hydroxyphenyl acetic acid and they obtained the corresponding diarylether in 60% yield [147]. Organocopper reagents, such as methylcopper prepared from MeLi were also used as catalyst recently [148]. Yields could be further improved by ultrasonic irradiation [149],.as well as under phase transfer conditions [150]. It is surprising that, in contrast to the Ullmann biaryl synthesis, activated aromatic fluoro compounds (e.g. fluoro-esters, -nitrils, -aldehydes and -acetophenones) can couple with phenols in good yield. The reaction does not require any catalysts, but only K2CO3 as a base, [146] (Scheme 56) .
Scheme 56 COoMe
1
MeO
x
C02Me K2C03,DMF
87%
^
MeO'
MeO
This method and its modified version utilising potassium fluorideon-alumina and 18-crown-6 represents an efficient alternative to the copper catalysed Ullmann synthesis [151]. A combination of the aryl fluoride approach with the activating effect of the aromatic nitro group was useful in intramolecular
308 coupling reactions as for the synthesis of macrocyclic diarylethers [152]. Best results were obtained using K2CO3 and 18-crown-6 in DMF [153] (Scheme 57). Scheme 57
K2CO3, 18-crown-6 DMF, 87% ^ ^OH
F
Solvent
effects
in Ullmann
diarylether
synthesis were
also
studied and pyridine was found to be the most suitable [154], but the necessary reaction temperature could also be maintained in DMF [155],
diglim
[159].
Solvents having a high dielectric constant
[156],
DMSO
[157],
dioxan
[158] and
power such as 1,3-dimethyl~2-imidazolidinone
nitrobenzene and
solvating
(DMI) might be useful
in chemoselective nucleophilic substitution. The phenolic component in
Scheme
58
is
an
ambident
nucleophile,
but
gave
only
the
corresponding diarylether with chlorobenzene in DMI [160]. Scheme 58 OH
-
-
^
^
^
CI
.
K2C03,Cu(I)Cl
HO'
Scheme 59 CHO
o
a o CHO
KI03,l2 H2S04,KBr
OMe
OMe
CHO
Br" OMe
CHO ,C02Me NaOMe 50^ OMe
^C02Me + HO-
309 A method of limited scope but involving mild conditions, introduced by Doskotch
[161] and used also by us successfully
[161] is the
reaction of a phenolate anion with a diary1 iodonium salt (Scheme 59). 4.2.2 Oxidative coupling In contrast to the synthesis of biaryls, oxidative coupling has found only limited application in the synthesis of diarylethers. Side-reactions due to the sensitivity of the products toward the oxidising
agent, concurrent reactions, such as the
formation of
biaryls, as well as the fact that several sites at the substrate may be reactive, are all responsible for the lack of interest in this method. Thallium (III) nitrate was used as an oxidant for the synthesis of various diarylethers [163] (Scheme 60) among them the isodityrosin moiety of a number of cyclic peptides [164]. Oxidative macrocyclization of the open-chain precursor of deoxybouvarin was also accomplished by thallium(III) nitrate [165]. VOF3
effected
the
oxidative
cyclization
of
an
open
chain
precursor of a vancomycin fragment [166]. 4.2.3 Coupling using arene-metal 7C-complexes The use of arene-metal complexes as the halide component is an effective modern alternative of the Ullmann method. Arylhalides can be activated via their metal TT-complexes, and give diarylethers in high yield as was first demonstrated by Pauson and Segal. Although the method was originally developed for the preparation of simple diarylethers, synthesis
of
it
could
[167].
groups
be
diarylethers A
extended substituted
by
Pearson
with
chloroarene-manganese
et
sensitive
al.to
the
functional
tricarbonyl
complex
substituted with a chiral side chain was reacted with the phenolate anion
to
give
diarylether
manganese
complex
from
which
the
diarylether could be liberated without racemization by irradiation in
acetonitrile
(Scheme
construction
of
diarylether
unit
highly of
61) .
Mild
functionalized glycopeptide
diarylether moieties [168].
conditions diarylethers antibiotics
allowed
the
such
the
as
incorporating
310
Scheme 60 OH
Scheme 61
Me02C'^^-'''''^^
^^V^^ If 11
^ v
NHAc Me02C
Mn fro) n
MeCN
serious
PF,
NHAc
hv
A
-Mn(C0)3
drawback
^
Me02C
of the method
is that
only
a
substituents are compatible with Tl-complex formation
few
types of
in the aryl
halide component. This problem is eliminated with the use of
Scheme 62 CI
•Cl
; Fe+Cp.PFg
^
. Me02C^
H [O] ^ ^ ° 2 C - \HCO2CH2Ph
NHC02CH2Ph
[VU\ r ' e ^ c p . PFi
IV J\ H M e 0 2 C ^ ^NHC02CH2Ph
311 Schollkopf^s chiral glycine enolate equivalents, as in the elegant synthesis of the central diarylether unit of ristocetin [169]. It is remarkable that these limitations were partly lifted by using, instead of the manganese tricarbonyl complex the corresponding iron or ruthenium cyclopentadienyl complexes [170] (Scheme 62). 7l-Complex
activation
was
the
method
of
choice
for
the
construction of a cyclic peptide model related to the antibiotic teicoplanin, where ring closure of the ruthenium
complex
of the
corresponding open-chain hydroxy-chloroarene was effected by sodium 2,6-di-t-butylphenoxide [171] (Scheme 63). Scheme 63 OMe
.Ru+Cp.PFg
OMe
2,6-Di-tBuphenoxide
[(
Ru+Cp.PFg'
.0
^ )
H NHBOC
MeOoC
NHCO
NHBOC
312 APPENDIX Codes in "Dictionary of Natural Products' for Natural
Products
Involving in Their Biosynthesis Oxidative Phenolic Coupling* Footnote. *Classification
in this section may deviate from that followed in
Section 1. Alkaloids (aporphins mostly omitted): A-574, 1660-1667, 1669, 2988, 2995, B-490, 491, 497, 539, 562, 584, 586, 587, C-10, 19, 313, 314, 797, 798, 1607, 1727, D-176, 253, 256, D-585, 3352, 3758, E-184, 205, 206, H-146, 490, 1-572, J-113116, K-317, L-200, 201, 347, 585, 588, 974-978, M-368, 420, 701, 1697, N-99, 138, 185, 276, 836, P-47, 48, 271, 323, 1271, 1993, S-457, 967, T-194, 1168, 1169, 1237, 1289, 2963, U-99, 106 , V-215, 218, 228. Curare alkaloids: A-457, 2513, 2537, 2804, 3004, B-88, 89, 282-284, C-106, 107, 481, 741, 882, 900, 1222, 1600, 1602-1604, 2160, 2173, 2174, 2189, 22162218, D-67, 79, 274, 275, 340, 413, 1408, 1429, 3318, 3711, E-57, 351, 1605, F-25, 149, G-357, 762, 925, H-139, 488, 968, 1067, I178, 344, 408, 826, 832, 834, 890, 927, 951, J-103, K-216, 262, L342, 610, M-97, 139, 303, 468, 899, 939, 1341, N-151, 0-1, 5, 83, 546, 589, 661, 683, 825, 826, 836, P-8, 11, 123, 141, 324, 899, 1406, 1651, 1958, 2045, 2057, 2192, 2222, 2223, R-116, 117, 158, 159, 316, S-60, 322, 325, 382, 388, 471, 772, 1214, 1279, 1284, T42,
196, 1149, 1230, 1233, 1236, 1238, 1241, 1250, 1252, 1255,
1258, 1259-1262, 1270, 1272, 1276-1278, 1283, 1292-1297, 1472-1478, 1758, 1914, 2749, 2752, 3029, U-173, V-66, 93, 260, W-11, Y-10, 11. Aminoacids: A-716, B-756, D-2924,D-1691, F-391. Allyl- and
isoprenylphenols
and naphthols:D-296,297,
2063, 2252,
3430, E-1859), 1925, 1926, 1-430, M-lOO, 317,1673, 0-18, P-1885, R42, T-170 Z-30. Anthracenes: H-1819, K-81, 0-46, P-1050, S-661, 776, 777, T-443, 1562. Antibiotics: A-342, 358, 1925, 1923, 1946, 1953, 1952, 2198, 2223, 2240, 2243, 2244, 2245, 2243, 2728, 3148, C-1038, 1053, 1684, R-72, 299, 0-498, P-205, T-177, V-62, 63. Anthraquinones: 533, 795, E-2018, 2019, F-18, 274, 681, 1-234, 806, J-139, 140-150,
313 A-1754, 1777, 2902, 2926, 3108, B-609, C-518, 1283, 1417, D-532, 533, 795, E-2018, 2019, F-18, 274, 681, 1-234, 806, J-139, 140-150, 155, K-251, L-155, R-204, 205, 372, S-718, 832, 833, T-656, 668, 669, 1613. Benzoic acids, gallic acids and other bis-acids: C-885, 886, D-298, 1072, E-342, F-542, H-1298, 2023, 1-339, M-467, 1480, 0-545,P-304, 1985, U-97, V-46, 246, 352. Benzofurans: D-3315, M-1746, 1748. Benzopyrans: E-1490, P-2106. Benzoquinones: B-405. Bis-bibenzyls: 1-538, 776, K-217, M-268, 271, 272, N-230, 231, P-845, 846, 1425, R-263, 264, 265, 266. Catehins: P-526-552. Chromenes: A-355, Coumarins: A-574, D-557, 1566, 2080, N-510, 0-489, T-1524, 2962. Diarylethers (oligomeric): B-526, 946, 947. DiaryIheptanoids: A-144-146, 1078, 1087, 2849, C-545, M-1920, P-1677, T-1861. Dibenzodioxins: M-677. Dibenzofurans: P-308. Dibenzocyclooctans: B-399, D-146-148, 513, G-634-642, 1-470, 791, K-8-11, N-212, 215, R-458, S-291-295, 1217, 1218, W-102. Dihydrophenanthrenes: B-647-650, 652, 653. Dityrosines: A-2125, 2245, B-406, 737. Ellagic acid derivatives: A-580-582, 734, 735, 1084, 1086, 2510, B-354, C-108, 183, 184, 536538, 1959,
544, 911, 1777, 1824-1829,
D-138,
1978, 1979, 1984, 2016, 2054-2056,
139, E-114,
1773, 1774,
F-277, G-194-197,
243,
244, 700, 701, 711, 880, 881, 884, H-170, 171, 511-514, 694-701, 900, 909, 910, 1-530, 779, 780, 790, 827, J-118, L-194-199, 631, M-
314
14-18, 872,
130-133,
0-265,
143-148,
154,
P-272,
1045,
266,
467-471,
1046,
373,
1650-1655,
1117-1120,
118-120,
377,
63,
207, 211, 212, 1491, 1503, V-45, 232,
206,
378,
156,
S-119-125,
285,
N-517-521,
1706,
286,
R-94-96,
1258-1261,
871, 105, T-57,
W-88-93.
Flavonoids: A-27,
549,
1191; B-109,
370,
B-504,
619,
984,
D-82-84,
840,
1213,
1419, 1580, M-616, 1147, 0-48, 198, 199, P-534, 1540, 1554, 1667, 2050, R-240, 305, S-523, 524, 1468, 1522, T-589, 1309, 1311, 1564. Lignans: C-750, 751, 752, 756, 757, 759, 760, 761, 762. Marine metabolites: B-156, 157, 158, D-714, 728, 751. Naphthoquinones, naphthols: B-16, 561, 563, D-2083, 2250, 3059, 3359, 3360, 3361, 3362, 3363, E-4, 177, 1794, 1795, G-88, 1248, H-3233, 1-422, 562, M-299, N-186, 272(, P-1055, 1950, 1951, S-1493, T-590. Phenols, polyphenols: D-3060, 1901, 1902, 2931, 2932, 2933, H-845, P-413, 486, T-1161, 2393, 2542. Prenil-allyl-phenols: H-484. Pyranocouinar i n : B-503. Pyranonaphthols: A-2067-2069,
2309, 2310, 3058-3060, C-721, 2000, 1-292, K-81, N-
411, T-36, U-179. Stilbenes: G-615, 616. Terpenes: B-538, C-242, 243, M-53 Xanthones: C-916, E-1960, 1-928, P-1512, S-1579, 1583.
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E. Fujita, A. Sumi, Y. Yoshimura, Chem. Pharm. Bull. 20
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Ann. Chem. 1989, 1141; Nogradi, B. Vermes, M. Kajtar-Peredy und V. P. Novikov, Liebigs Ann. Chem. 1990, 299; N. T. Thu Ha, M. Nogradi, J. Brlik, M. Kajtar-Peredy und A. Wolfner, J. Chem. R e s . 1991 (M) 1240 ( S ) , 137; B. Vermes, Gy. M. Keseru, G. Mezey-Vandor, M. Nogradi and G. Toth, Tetrahedron 49 (1993), 4893; Gy. M. KeserG, M. Nogradi, and M. Kajtar-Peredy, Liebigs Ann. Chem. 1994, 361. 146
Gy. M. Keseru, Z. Dienes, M. Nogradi and M. Kajtar-Peredy,.J. Org. Chem. 58 (1993) 6725;
147
M. Tomita, K. Fujitani, Y. Aoyagi, Chem. Pharm. Bull. 13
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(1992)
1319; D. L. Boger, Y. Nomoto, B. R. Teegarden, ibid. 58
(1993)
(1965) 1341.
1425. 149
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150
G. J. Soula J. Org. Chem. 50 (1985) 3717.
151
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
323
Narcissus Alkaloids
Jaume Bastida, F. Viladomat and C. Codina
1.
INTRODUCTION Since the isolation of the first Amaryllidaceae alkaloid lycorlne (1) from Narcissus
pseudonarcissus by Gerrad in 1877, around 200 species belonging to this plant family have been examined for alkaloids. Although this group of alkaloids is of minor pharmaceutical importance, it has aroused increasing interest over the last years. The several reviews in this field are a valuable source of information (1-7) and, likewise, this topic Is regularly reviewed by the journal Natural Products Reports of The Royal Society of Chemistry (8-16). Out of the different genera which make up the Amaryllidaceae family, the genus Narcissus is one of the best known from the horticultural point of view. Due to their phytochemical interest, a chapter about Narcissus alkaloids has been prepared, following the usual general lines. The literature up to August 1996 has been covered. 1.1 Geographical distribution, botanical and taxonomical aspects The Amaryllidaceae are widely distributed. They are richly represented in the tropics and have pronounced centres in South-Africa and in Andean South America. Several genera have their centre in the Mediterranean, e.g. Narcissus. The majority of Narcissus species are found in the Iberian peninsula and North Africa, which substantiates the hypotheses that landmasses of Europe and Africa were once joined taking into consideration the shared distribution of many genera and species. Narcissus can adapt to the different habitats found in this area as well as the different soils, preferring slightly acidic conditions. The genus Narcissus, belonging to the tribe Narcisseae, consists of small to medium-sized herbs with linear leaves and a solid scape bearing an inflorescence of one to several (rarely numerous) flowers. Its spathal bracts are basally fused into a tube. The flowers are actinomorphic and have six equal tepals. Inside this, a corona is
324 generally present. The colours of the tepals usually vary from white to bright yellow and the corona fronn white or light yellow to orange or orange-red. Most are spring-flowering, but a few flower in autumn. The ovary is trilocular and the fruit is a capsule with globose to angular, dry and black seeds. The taxonomy and nomenclature of the genus Narcissus is complex because of its very varied wild populations, its suitability for hybridation and also for historical reasons because many descriptions of taxons have been based on garden specimens, several of which were probably of hybrid origen. The polymorphism of this genus has meant that its taxonomical classification has been constantly changing over the years. 1.2 The Amaryllidaceae family and their alkaloids There has been considerable taxonomical controversy over which genera belong to the Amaryllidaceae family. The revisions of Dahlgren's group (17, 18) have contributed to clarify this aspect. In another direction, one of the best tools for classification of several genera and species of this family has been the type of alkaloids that are exclusively synthezised by Amaryllidaceae species. Thus, e.g. the genus Behria in spite of having been classified as Amaryllidaceae (19), does not have Amaryllidaceae type alkaloids (20) and should therefore be included in another family. Furthermore, it is unusual to find other types of alkaloids in Amaryllidaceae species, but if present, they are always accompanied by true Amaryllidaceae alkaloids. Up to now, only three alkaloids isolated from this family do not belong to this specific type but to the mesembrane {=Sceletium) type -generally found in the family Aizoaceae-, which In spite of having the same aminoacids as precursors has a quite different biosynthesis -for more information about Sceletium alkaloids see Ref. (21, 22)-. These three compounds are amisine (83), mesembrenol (84), and mesembrenone (82) isolated from Hymenocallis arenicola (23), Crinum oliganthum (24) and Narcissus pallidulus (25), respectively (Figure 1). For this reason the Amaryllidaceae alkaloids have a high chemotaxonomical value. The general characteristics of the Amaryllidaceae alkaloids could be summarized by the following points: - a fundamental ring system composed of a Ce-Ci and a N-Ca-Ce building block, derived from L-Phenilalanine
(L-Phe) and L-Tyroslne (L-Tyr),
respectively. - they are moderately weak bases (pKa 6-9) - each alkaloid contains only one nitrogen atom which is secondary, tertiary or even quaternary, and the carbon content varies from 16 to 20 atoms.
325 NMep
OMe OMe
" ^ - ^
•N'
i
Me 83
Ri
82 R,+ R,= = 0 84 R,= OH, R2 = H
Fig. 1. Sceletium alkaloids isolated from Amaryllidaceae species Most of the Amaryllidaceae alkaloids may be classified Into nine principal skeletally homogeneous subgroups although there are several other alkaloids with structures derived from these main molecular frameworks. Representative alkaloids from each of these classes include: norbelladine (85), lycorine (1), homolycorine (23), crinine (86), haemanthamine (49), narciclasine (64), tazettine (59), montanine (87) and galanthamine (70). With the aim of unifying the numbering system of the different types, Ghosal's proposal will be used in this chapter (6) (Figure 2).
2.
NARCISSUS
ALKALOIDS AND THEIR OCCURRENCE
The alkaloids isolated from Narcissus species, classified in relation to the different ring types, are provided in Table 1, and the distribution of the different alkaloids is listed in Table 2.
326
OH
85
0
1
23 OH
OH 12
8
6
N' 7
86
6
OH
49
0 64
OMe
4
2
Me0 NMe 7
6
59
7
7
6
87
Fig. 2. Amaryllidaceae alkaloid types
6
70
327
TABLE 1a Narcissus alkaloid structures. Lycorine type.
Alkaloid name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
lycorine poetaminine pseudolycorine 1 -0-acetylpseudolycorine 2-0-acetylpseudolycorine 9-0-methylpseudolycorine galanthine goleptine jonquilline caranine pluviine norpluviine 9-0-demethylpluviine 1 -O-acetyl-9-O-demethylpluviine 1,9-0-diacetyl-9-0-demethylpluviine
Ri
H Ac H Ac H H H H Ac H H H H Ac Ac
Structure R, R3 OH GH2 OH GH2 OH H OH H OAc H OH Me OMe Me OMe H 0 GH2 H CH2 H Me H Me H H H H H Ac
OAc 1
OMe I
HO..
X
..OH
AcO.
X
MeO,
MeO
16 narcissidine
17 nartazine
_3i Me Me Me Me Me Me
Me H Me Me Me
328
TABLE l a (continued) Narcissus alkaloid structures. Lycorine type.
r^"^ MeO,
MeO,
MeO
MeO
18 assoanine 19 oxoassoanine
R^z=R2=H Ri + R2 = 0
20 vasconine 21 tortuosine
MeO.
MeO
22 roserine
R^ = H R^ = OMe
329
TABLE 1b Narcissus alkaloid structures. Homolycorine type.
Structure 23 24 25 26 27 28 29 30 31 32 33 34 35
Alkaloid name homolycorine 8-0-demethylhomolycorine 8-0-demethyl-8-0-acetylhomolycorine 9-O-demethylhomolycorine masonine normasonine 9-0-demethyl-2a-hydroxyhomolycorine hippeastrine lycorenine O-methyllycorenine oduline 6-0-methyloduline 2a-hydroxy-6-0-methyloduline
Ri
Me Me Me H
R2
Me H Ac Me
R3
CH2 CH2
Me
H CH2
Me Me CH2 CH2 CH2
Me Me
OH OMe OH OMe OMe
Me-CHOH-CHg-COO, OAc MeO
36
dubiusine
0 0 0 0 0 0 0 0
A_
H H H H H
R5
H H H H H H OH OH H H H H OH
Re Me Me Me Me Me H Me Me Me Me Me Me Me
330
TABLE 1b (continued) Narcissus alkaloid structures. Homolycorine type.
MeO. OCOEt
37
8-0-demethylhomolycorine-A/-oxide
38
poetinatine
TABLE 1c Narcissus alkaloid structures. Haemanthamine type.
- R 7
Structure R, R4 ~W^ ~^3 H H CH2 H Me Me H H Me H H H H Me H H Me Me H H Me Me H H Me Me OH H Me Me OMe OMe Me Me H OMe Me Me OH H H CH2 H GH2 H H H CH2 H OH CH2 H OMe GH2 H H H Me 1
Alkaloid name 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
vittatine maritidine 8-O-demethylmaritldine 9-O-demethylmaritidine 0-methylmaritidine papyramine 6-epipapyramine O-methyl-6-epipapyramine 6a-hydroxy-3-0-methylepimaritidine 6p-hydroxy-3-0-methylepimaritidine haemanthamine 11-O-acetylhaemanthamine haemanthidine 6-epihaemanthidine crinamlne narcidine
Ri
OH OH OH OH OMe OMe OMe OMe H H OMe OMe OMe OMe H OMe
Re
H H H H H OH H H OH H H H OH H H H
R7
H H H H H H H H H H OH OAc OH OH OH OH
331 TABLE 1c (continued) Narcissus alkaloid structures. Haemanthamine and Crinine types. .OMe --R4 — R.
55
cantabricine R^ = H, Rg = Me, R3 = H, R4 = OAc, R5 = H
57 6a-hydroxybuphanisine 58 6p-hydroxybuphanisine
56
narcimarkine R^ + Rg = CHg, R3 = OMe, R4 = H, R^= 0CH2CH(0H)Et
R^ = H, Rg = OH R^ = OH, Rg = H
TABLE 1d Narcissus alkaloid structures. Tazettine type. OMe
NMe
59 60
tazettine criwelline
R^ = OMe, Rg = H R^ = H, Rg = OMe
NMe
OH 61
pretazettine
63
obesine
OMe
NMe
62
3-epimacronine
332
TABLE 1e Narcissus alkaloid structures. Narciclasine and Montanine types. OH
r i ^ ^ ^
O
OH
64
OH
narciclasine
65
r^===^
66 trisphaeridine
O
narciprinnlne
r^^^=^
67
bicolorine
OH
r ^ ^
<
^^Ij^^^L
NHMe OH
68
ismine
69
pancracine
333
TABLE I f Narcissus alkaloid structures. Galanthamine type.
MeO
70 71 72 73 74 75 76 77
Alkaloid name galanthamine epigalanthamine 0-acetylgalanthamine norgalanthamine epinorgalanthamine A/-formylnorgalanthamine narcisine narwedine
Structure R, H OH H H OH H H
R, OH H OAc OH H OH OH 0
R3
Me Me Me H H OHO Ac Me
MeO.
Structure Alkaloid name 78 lycoramine 79 norlycoramine 80 epinorlycoramine
Ri
OH OH H
Rg
R3
H H OH
Me H H
334
TABLE 1g Narcissus alkaloid structures. Miscellaneous.
OMe
OMe
81
pallidiflorine (heterodimer)
OMe OMe
k^ 1
Me
82 mesembrenone (Sceletium type)
335 TABLE 2 Occurrence of Narcissus alkaloids. Alkaloid
Wild species / cultivars
Section
References
18 assoanine
A^. assoanus Leon-Duf. A^. jacetanus Fdez. Casas N. pseudonarcissus L. cv. King Alfred
JQ PN
(26) (27) (28)
19 oxoassoanine
A^. assoanus L6on-Duf. A^. jacetanus Fdez. Casas
JQ PN
(26) (27)
67 bicolorine
A^. bicolor L. A^. obesus Salisb.
PN BC
(29) (30)
57 6a-hydroxybuphanisine
A^. cantabricus DC.
BC
(31)
58 6(3-hy(lroxybuphanisine
A^. cantabricus DC.
BC
(31)
55 cantabricine
A'; cantabricus DC.
BC
(31)
10 caranine
N. cv. Livia
53 crinamine
A^. bujei (Fdez. Casas) Fdez.Casas N. cantabricus DC. A^. cv. Salome
PN BC
(33) (31) (34)
60 criwelline
A^. tazetta L.
TZ
(35)
36 dubiusine
N. dubius Gouan A'; tortifolius Fdez. Casas
DB DB
(36) (37)
70 galanthamine
N. confusus Pugsley A^. eugeniae Fdez. Casas N. X gracilis Sabine A^. X incomparabilis Mill. cv. Daisy Schaffer A^. X incomparabilis Mill, cv Flower Record N. X incomparabilis Mill, cv Fortune Helios A^. X incomparabilis Mill, cv John Evelyn N. X incomparabilis Mill, cv A^. X incomparabilis Mill, cv Marion Cran A^. X incomparabilis Mill, cv Nova scotia Pluvius A^. X incomparabilis Mill, cv A^. X incomparabilis Mill, cv Suda N. jonquilla L. A^. jonquilla L. cv. Golden Sceptre A^. jonquilla L. cv. Trevithian A^. kristalli N. lobularis Hort. A^. nivalis Graells A^. obesus Salisb. A^. X odorus L. var. rugulosus N. papyraceus Ker. Gawl. A'^. poeticus L. A^. poeticus L. var. ornatus Hort. A^. poeticus L. cv. Actaea A^. poeticus L. cv. Daphne
PN PN JC
(38) (39) (40) (32) (32) (32,41) (32) (32) (32) (32) (32, 42) (32) (43) (40,44, 45) (40) (46) (32) (47) (30) (40) (48) (49, 50) (51,52) (49) (32)
(32)
JQ
PN BC BC JN TZ NC NC
336 TABLE 2 (continued) Section
Alkaloid
Wild species / cultivars
70 galanthamine (cont.)
A^. poeticus L. cv. Sarchedon A^. pseudonarcissus L. cv. Carlton N. pseudonarcissus L. cv. Covent Garden A^. pseudonarcissus L. cv. Early Glory A^. pseudonarcissus L. cv. Grand Maitre A^. pseudonarcissus L. cv. Imperator N. pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Mrs. Ernst H. Krelage A^. pseudonarcissus L. cv. Oliver Cromwell A^. pseudonarcissus L. cv. Rembrandt A^. pseudonarcissus L. cv. Unsurpassable A^. pseudonarcissus L. cv. Van Sion N. pseudonarcissus L. cv. Wrestler N. tazetta L. TZ A^. tazetta L. var. chinensis Roem. TZ A'^ tazetta L. cv. Geranium A^. tortifolius Fdez. Casas DB A': triandrus L. cv. Tresamble A^. cv. Inglescombe A^. cv. Texas A^. cv. Twink
(49) (53, 54) (32, 42) (32) (32) (32) (28, 32, 42, 55) (32, 42) (32) (32) (32) (32) (32, 42) (32) (35, 49 56) (57) (49) (37) (40) (32) (32) (32, 42)
72 O-acetylgalanthamine
A^. pseudonarcissus L. cv. Carlton
(58)
71 epigalanthamine
A^. tazetta L. var. chinensis Roem.
TZ
(57, 59)
73 norgalanthamine
A^. leonensis Pugsley A^. nivalis Graells A^. pseudonarcissus L. cv. Carlton A^. pseudonarcissus L. cv. Covent Garden N. pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Rembrandt A^. pseudonarcissus L. cv. Rockery Beauty A^. pseudonarcissus L. cv. Spring Glory A^. pseudonarcissus L. cv. Van Sion A'', pseudonarcissus L. cv. Victoria A^. cv. Salome
PN BC
(60) (47) (53, 54) (32, 42) (28, 32, 42) (32, 42) (32) (32) (32) (32, 42) (32) (34)
74 epinorgalanthamine
A^. leonensis Pugsley
PN
(60)
75 A^-formylnorgalanthamine
A^' , confusus Pugsley
PN
(38)
7 galanthine
A^. cyclamineus DC. cv. Beryl N. cyclamineus DC. cv. Cairhays N. X incomparabilis Mill. cv. Daisy Schaffer A^. X incomparabilis Mill. cv. Deanna Durbin A^. X incomparabilis Mill. cv. Flower Record. A^. X incomparabilis Mill. cv. Helios A^. X incomparabilis Mill. cv. John Evelyn A^. X incomparabilis Mill. cv. Marion Cran A'^. X incomparabilis Mill. cv. Nova scotia A^. X incomparabilis Mill. cv. Pluvius A^. X incomparabilis Mill. cv. Sempre Avanti 1A^' , X incomparabilis Mill. cv. Suda
References
(40) (40) (32) (32,42) (32) (32) (32) (32) (32) (32, 42) (32) (32)
337
TABLE 2 (continued) Alkaloid 7 galanthine (cont.)
8 goleptine 49 haemanthamine
Wild species / cultivars A^. X incomparabilis Mill. cv. Toronto A^. jonquilla L. cv. Golden Sceptre A^. panizzianus Pari. N. poeticus L. N. poeticus L. var. ornatus Hort. A^. poeticus L. cv. Actaea A^. poeticus L. cv. Sarchedon A^. pseudonarcissus L. cv. Covent Garden A^. pseudonarcissus L. cv. Early Glory A^. pseudonarcissus L. cv. Grand Maitre A'^. pseudonarcissus L. cv. Imperator A'^, pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Magnet A^. pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Mrs. Ernst H. Krelage N. pseudonarcissus L. cv. Music Hall A'^, pseudonarcissus L. cv. Oliver Cromwell A^. pseudonarcissus L. cv. Queen of Bicolors N. pseudonarcissus L. cv. Rockery Beauty A^. pseudonarcissus L. cv. Romaine A'^, pseudonarcissus L. cv. Spring Glory A^. pseudonarcissus L. cv. Van Sion A^. pseudonarcissus L. cv. Victoria A^. pseudonarcissus L. cv. Wrestler A^. tazetta L. A^. tazetta L. cv. Scarlet Gem N. cv. Insulinde A'^. cv. Livia A^. cv. Twink
Section
TZ NC NC
TZ
(32) (45) (61) (49, 50, 62) (51,52) (49) (49) (32, 42) (32) (32) (32) (28, 32,42, 62) (32) (42) (32) (32) (32) (32) (32) (32) (32) (32, 42) (32) (32) (49) (49) (32) (32) (32, 42) (63, 64)
A^. jonquilla L. cv.Golden Sceptre A^. asturiensis (Jordan) Pugsley A^. bujei (Fdez. Casas) Fdez. Casas A'^ canaliculatus Guss A^. confusus Pugsley A^. X incomparabilis Mill. cv. Deanna Durbin A^. X incomparabilis Mill. cv. Flower Record A^. X incomparabilis Mill. cv. Fortune A^. X incomparabilis Mill. cv. Helios A^. X incomparabilis Mill. cv. John Evelyn A^. X incomparabilis Mill. cv. Marion Cran A^. X incomparabilis Mill. cv. Nova scotia A^. X incomparabilis Mill. cv. Pluvius A'^, X incomparabilis Mill. cv. Sempre Avanti A^. X incomparabilis Mill. cv. Suda A^. X incomparabilis Mill. cv. Toronto A^. jonquilla L. cv. Golden Sceptre A^. lobularis Hort. A^. obesus Salisb. A^. pallidiflorus Pugsley A^. poeticus L. var. ornatus Hort. A^. primigenius (Lainz) Fdez. Casas & Lainz N. pseudonarcissus L. cv. Carlton A^. pseudonarcissus L. cv. Covent Garden A', pseudonarcissus L. cv. Early Glory
References
PN PN TZ PN
PN BC PN NC PN
(65) (33) (49) (38) (32, 42) (32) (32) (32) (32) (32) (32) (32, 42) (32) (32) (32) (40, 44, 45) (32) (30) (66) (49,51) (67) (53, 54) (32, 42) (32)
338 TABLE 2 (continued) Alkaloid
Wild species / cultivars
49 haemanthamine (cont.)
A^. pseudonarcissus L. cv. Grand Maitre A^. pseudonarcissus L. cv. Imperator A^. pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Magnet A^. pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Mrs. Ernst H. Krelage A'^ pseudonarcissus L. cv. Music Hall A^. pseudonarcissus L. cv. Oliver Cromwell A^. pseudonarcissus L. cv. Queen of Bicolors A^. pseudonarcissus L. cv. Rembrandt A^. pseudonarcissus L. cv. Rockery Beauty N. pseudonarcissus L. cv. Romaine A^. pseudonarcissus L. cv. Spring Glory A^. pseudonarcissus L. cv. Unsurpassable A^. pseudonarcissus L. cv. Van Sion N. pseudonarcissus L. cv. Victoria A^. pseudonarcissus L. cv. Wrestler A'^, tazetta L. TZ A^. tazetta L. cv. Cragford A'^. tazetta L. cv. Early Perfection A^. tazetta L. cv. Geranium A^. tazetta L. cv. La Fiancee N. tazetta L. cv. Laurens Koster A^. tazetta L. cv. L'innocence N. tazetta L. cv. Scarlet Gem A^. tazetta L. cv. St. Agnes N. triandrus L. cv. Silver Chames A'^. triandrus L. cv. Thalia A^. triandrus L. cv. Tresamble A^. cv. Inglescombe A^. cv. Insulinde N. cv. Livia A^. cv. Texas A^. cv. Twink
50 11-O-acetylhaemanthamine
A^. bujei (Fdez. Casas) Fdez. Casas
PN
(33)
51 haemanthidine
A^. A^. A^. A^.
asturiensis (Jordan) Pugsley cyclamineus DC. cv. Cairhays tazetta L. tazetta L. var. chinensis Roem.
PN
(65) (40) (46) (57)
A^. A^. A^. A^.
asturiensis (Jordan) Pugsley cyclamineus DC. cv. Cairhays tazetta L. tazetta L. var. chinensis Roem.
PN
52 6-epihaemanthidine
30 hippeastrine
A'^ X incomparabilis Mill. cv. Fortune A^. jonquilla L. cv. Golden Sceptre N. X odorus L. var. rugulosus N. pseudonarcissus L. cv. Carlton A^. pseudonarcissus L. cv. King Alfred A^. tazetta L. A^. tazetta L. cv. Early Perfection 1N. cv. Salome
Section
TZ TZ
TZ TZ
JN TZ
References (32) (32) (28, 32, 42, 55) (32) (32, 42) (32) (32) (32) (32) (32) (32) (32) (32) (32) (32, 42) (32) (32) (35, 49, 56) (49) (49) (49) (49) (49) (49) (49) (49) (40) (40) (40) (32) (32) (32) (32, 68) (32,42)
(65) (40) (46) (57) (32) (40, 44) (40) (53) (55) (35) (49) (34)
339 TABLE 2 (continued) Alkaloid
Wild species / cultivars
Section
References
23 homolycorine
N. bujei (Fdez. Casas) Fdez. Casas N. confusus Pugsley N. cyclamineus DC. cv. February Gold N. eugeniae Fdez. Casas N. X incomparabilis Mill. cv. Helios A^. jonquilla L. cv. Golden Sceptre N. munozii-garmendiae Fdez. Casas N. X odorus L. var. rugulosus N. pallidiflorus Pugsley N. panizzianus Pari. N. papyraceus Ker. Gawl. N. poeticus L. N. poeticus L. var. ornatus Hort. N. poeticus L. cv. Daphne N. primigenius (Lainz) Fdez. Casas & Lainz N. pseudonarcissus L. N. pseudonarcissus L. cv. Carlton N. pseudonarcissus L. cv. Grand Maitre N. pseudonarcissus L. cv. Imperator N. pseudonarcissus L. cv. King Alfred N. pseudonarcissus L. cv. Van Sion A^. radinganorum Fdez. Casas A'^ tazetta L. N. tazetta L. var. chinensis Roem. N. tazetta L. cv. Cragford N. tazetta L. cv. Early Perfection N. tazetta L. cv. Geranium A^. tazetta L. cv. La Fiancee A': tazetta L. cv. Laurens Koster A^. tazetta L. cv. L'innocence A^. tazetta L. cv. Scarlet Gem N. tazetta L. cv. St. Agnes A^. tortifolius Fdez. Casas A^. triandrus L. cv. Thalia A^. vasconicus Fdez. Casas N. cv. Inglescombe
PN PN
(33) (69) (40) (39) (32) (44) (70) (40) (66) (61) (71,72) (49) (49,51,73) (32) (67) (74) (53) (32) (32) (28, 55, 62) (32, 42) (75) (76-78) (57) (49) (49) (49) (49) (49) (49) (49) (49) (37) (40) (79) (32)
PN PN JN PN TZ TZ NC NC PN PN
PN TZ TZ
DB PN
A^. bujei (Fdez. Casas) Fdez. Casas N. pallidiflorus Pugsley A^. papyraceus Ker. Gawl. A^. primigenius (Lainz) Fdez. Casas & Lainz N. pseudonarcissus L. cv. King Alfred A^. radinganorum Fdez. Casas A^. tazetta L. A^. tortifolius Fdez. Casas
PN PN TZ PN PN TZ DB
(33) (66) (71,72) (67) (55) (75) (77, 80) (37)
37 8-O-demethylhomolycorine-A^-oxide
A^. papyraceus Ker. Gawl.
TZ
(71,72)
25 8-0-demethyl-8-0-acetylhomolycorine
N. vasconicus Fdez. Casas
PN
(79)
26 9-O-demethylhomolycorine
A^. bicolor L. A^. confusus Pugsley
PN PN
(29) (69)
24 8-O-demethylhomolycorine
340
TABLE 2 (continued) Alkaloid 29
Wild s p e c i e s / cultivars
9-0-demethyl-2a-hydroxy- A^. dubius Gouan A^. tortifolius Fdez. Casas homolycorine
68 ismine
9 jonquilline 78 lycoramine
A^. asturiensis (Jordan) Pugsley N. bicolor L. N. obesus Salisb. A^. pallidiflorus Pugsley
Section
References
DB DB
(81) (37)
PN PN BC PN
(65) (29) (30) (66) (44)
A^. jonquilla L. cv. Golden Sceptre A^. cyclamineus DC. cv. February Gold A^. jonquilla L. A^. papyraceus Ker. Gawl. A^. pseudonarcissus L. cv. Carlton N. tazetta L. var. chinensis Roem.
JQ TZ TZ
(40) (43) (48) (53, 54) (57) (32, 42) (32, 42) (32, 42) (32) (32) (32) (32,42) (32)
79 norlycoramine
A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus A^. pseudonarcissus
80 epinorlycoramine
A^. leonensis Pugsley A^. pseudonarcissus L. cv. Carlton
PN
(60) (54)
31 lycorenine
A^. bujei (Fdez. Casas) Fdez. Casas A^. cyclamineus DC. cv. February Gold A^. cyclamineus DC. cv. Peeping Tom A^. eugeniae Fdez. Casas A'^, X incomparabilis Mill. cv. Helios A^. X incomparabilis Mill. cv. Suda A^. jonquilla L. cv. Golden Sceptre A^. jonquilla L. cv. Trevithian A'^ munozii-garmendiae Fdez, Casas A^. poeticus L. A^. poeticus L. var. ornatus Hort. A^. poeticus L. cv. Actaea A^. poeticus L. cv. Daphne A^. poeticus L. cv. Sarchedon A^. pseudonarcissus L. cv. Carlton A^. pseudonarcissus L. cv. Covent Garden A^. pseudonarcissus L. cv. Imperator A^. pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Romaine A^. pseudonarcissus L. cv. Unsurpassable N. pseudonarcissus L. cv. Van Sion A^. tazetta L. var. chinensis Roem. A'^, triandrus L. cv. Thalia A^. triandrus L. cv. Tresamble A^. cv. Inglescombe
PN
(33) (40) (40) (39, 82) (32) (32) (44) (40) (70) (62) (49) (49) (32) (49) (53, 54) (32, 42) (32) (28, 32, 42, 55, 62) (32) (32) (32, 42) (57) (40) (40) (32)
32
O-methyllycorenine
L. cv. Covent Garden L. cv. King Alfred L. cv. Magnificience L. cv. Rembrandt L. cv. Rockery Beauty L. cv. Spring Glory L. cv. Van Sion L. cv. Victoria
A^. bujei (Fdez. Casas) Fdez. Casas A^. munozii-garmendiae Fdez. Casas
PN
PN NC NC
TZ
PN PN
(33) (70)
341 TABLE 2 (continued) Alkaloid
Wild species / cultivars
Section
32 0-methyllycorenine (cont.) N. pseudonarcissus L. cv. Carlton 1 lycorine
A^. cyclamineus DC. cv. Beryl N.folli N. X gracilis Sabine A^. X incomparabilis Mill. cv. Deanna Durbin N. X incomparabilis Mill. cv. Flower Record N. X incomparabilis Mill. cv. John Evelyn N. X incomparabilis Mill. cv. Marion Cran N. X incomparabilis Mill. cv. Nova scotia A^. X incomparabilis Mill. cv. Pluvius A^. X incomparabilis Mill. cv. Sempre Avanti A^. X incomparabilis Mill. cv. Toronto A^. jacetanus Fdez. Casas A^. jonquilla L. cv. Trevithian A^. kristalli N. leonensis Pugsley A^. X odor us L. var. rugulosus N. papyraceus Ker. Gawl. A^. posticus L. A^. poeticus L. var. ornatus Hort. A^. poeticus L. cv. Actaea A^. poeticus L. cv. Daphne A^. poeticus L. cv. Sarchedon N. pseudonarcissus L. A'^ pseudonarcissus L. cv. Covent Garden N. pseudonarcissus L. cv. Early Glory A^. pseudonarcissus L. cv. Grand Maitre A^. pseudonarcissus L. cv. Imperator A^. pseudonarcissus L. cv. King Alfred A'^, pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Mrs. Ernst H. Krelage A^. pseudonarcissus L. cv. Music Hall A^. pseudonarcissus L. cv. Queen of Bicolors A^. pseudonarcissus L. cv. Rembrandt A^. pseudonarcissus L. cv. Spring Glory A^. pseudonarcissus L. cv. Van Sion A^. pseudonarcissus L. cv. Victoria A^. tazetta L. A^. tazetta L. var. chinensis Roem. A^. tazetta L. cv. Cragford A'^. tazetta L. cv. Early Perfection N. tazetta L. cv. Geranium A^. tazetta L. cv. La Fiancee A^. tazetta L. cv. Laurens Koster N. tazetta L. cv. L'innocence A^. tazetta L. cv. Scarlet Gem A^. tazetta L. cv. St. Agnes A^. tortuosus Haworth A^. triandrus L. cv. Silver Chames A^. triandrus L. cv. Thalia A^. vasconicus Fdez. Casas A^. cv. Inglescombe A^. cv. Insulinde A^. cv. Irene Copeland
References (53, 54)
JC
PN PN JN TZ NC NC
PN
TZ TZ
PN PN
(40) (46) (40) (32, 42) (32) (32) (32) (32) (32, 42) (32) (32) (27) (40) (46) (60) (40) (48,71,72) (49, 50, 83) (49,51,52) (49) (32) (49) (84) (32, 42) (32) (32) (32) (32, 42) (32, 42) (32) (32) (32) (32) (32) (32,42) (32) (35, 46, 49, 56, 7678) (57) (49) (49) (49) (49) (49) (49) (49) (49) (85) (40) (40) (79) (32) (32) (32)
342
TABLE 2 (continued) Alkaloid 1 lycorine (cont.)
Wild species / cultivars
Section
References (32) (32) (32, 42)
A^. cv. Livia N. cv. Texas A^. cv. Twink
62 3-epimacronine
A^. asturiensis (Jordan) Pugsley A^. bicolor L. A^. obesus Salisb.
PN FN BC
(65) (29) (30)
40 maritidine
N. papyraceus Ker. Gawl. A^. tazetta L. var. chinensis Roem.
TZ TZ
(48) (57)
41 8-O-demethylmaritidine
A^' , primigenius (Lainz) Fdez. Casas 8L Lainz
PN
(67)
42 9-0-demethylmaritidine
A'^. radinganorum Fdez. Casas
PN
(75)
43 O-methylmaritidine
A^. papyraceus Ker. Gawl. A^. tazetta L. A^. tazetta L. var. chinensis Roem.
TZ TZ TZ
(71,72) (77) (57)
47 6a-hydroxy3 - 0-methy lepimaritidine
A^. tazetta L. var. chinensis Roem.
TZ
(57)
48 6p-hydroxy3 - O-methy lepimaritidine
A^. tazetta L. var. chinensis Roem.
TZ
(57)
27 masonine
A'^ bujei (Fdez. Casas) Fdez. Casas A^. jonquilla L. cv. Golden Sceptre A'^, pseudonarcissus L. A^. pseudonarcissus L. cv. Carlton A^. tazetta L.
PN
(33) (44) (74) (53, 54) (35)
PN TZ
(53, 54)
28 normasonine
A^. pseudonarcissus L. cv. Carlton
82 mesembrenone
A^. pallidulus Graells
CM
(25)
64 narciclasine
A^. canaliculatus Guss var. tipica N. cyclamineus DC. var. tipica N. X incomparabilis Mill. cv. Carabinieri A^. X incomparabilis Mill. cv. Helios A*". X incomparabilis Mill. cv. Mercato A^. X incomparabilis Mill. cv. Orange Bruid A'^. X incomparabilis Mill. cv. Mrs. R. O. Backhouse A^. X incomparabilis Mill. cv. Scarlet Elegance A^. X incomparabilis Mill. cv. Sempre Avanti A^. X incomparabilis Mill. cv. Tunis A^. X incomparabilis Mill. cv. Walt Disney A^. jonquilla L. A^. jonquilla L. cv. Trevithian A^. X odorus L. var. rugulosus N. poeticus L. cv. Actaea A^. poeticus L. cv. Cheerfulness A^. pseudonarcissus L. cv. Carlton A^. pseudonarcissus L. cv. Flower Carpet A^. pseudonarcissus L. cv. Golden Harvest IA^. pseudonarcissus L. cv. King Alfred
TZ CM
(86) (86) (86) (86, 87) (86) (86) (86)
JQ JN
(86) (86, 87) (86) (86) (43) (86) (86) (86) (86) (86) (86) (86) (62, 86, 87)
343
TABLE 2 (continued) Alkaloid
Wild species / cultivars
64 narciclasine (cont.)
A^. pseudonarcissus L. cv. Mount Hood A^. pseudonarcissus L. cv. President Lebrum A^. pseudonarcissus L. cv. Rembrandt N. serotinus Lofl. ex L. var. tipica N. tazetta L. var. tipica N. tazetta L. cv. Geranium A^. triandrus L. cv. Thalia A^. triandrus L. cv. Tresamble A^. cv. Celebrity A^. cv. Clamor A^. cv. Texas A^. cv. Totus Albus N. cv. Verger
54 narcidine
A^. pseudonarcissus L. cv. King Alfred
56 narcimarkine
A^. poeticus L.
65 narciprimine
Narcissus sp
76 narcisine
N. tazetta L.
16 narcissidine
N. cyclamineus DC. cv. Beryl A^. X incomparabilis Mill. cv. Deanna Durbin A^. X incomparabilis Mill. cv. Pluvius A^. X incomparabilis Mill. cv. Sempre Avanti A^. poeticus L. N. poeticus L. cv. Actaea A^. poeticus L. cv. Sarchedon A^. pseudonarcissus L. cv. King Alfred N. pseudonarcissus L. cv. Rockery Beauty A^. tazetta L. A^. tazetta L. cv. Geranium A^. tazetta L. cv. L'innocence
Section
ST TZ
References (86) (86) (86) (86) (86-88) (88) (86, 87) (86, 87) (86) (86) (86) (86) (86) (55)
NC
(50, 89) (87)
TZ
NC
TZ
(56) (40) (32, 42) (32, 42) (32) (50, 62, 83) (49) (49) (55) (32) (49) (49) (49)
17 nartazine
N. poeticus L. N. tazetta L.
NC TZ
(49) (35, 49)
77 narwedine
A^. cyclamineus DC. N. kristalli N. pseudonarcissus L. cv. Carlton N. tazetta L. N. cv. Irene Copeland A^. cv. Texas
CM
(90) (46) (53, 54) (35) (32) (32, 91)
63 obesine
A^. obesus Salisb.
BC
33 oduline
A^. X incomparabilis Mill. cv. Fortune A^. jonquilla L. cv. Golden Sceptre A^. X odorus L. var. rugulosus N. pseudonarcissus L. cv. Carlton
34 6-(9-methyloduline
A^. bujei (Fdez. Casas) Fdez. Casas N. pseudonarcissus L. cv. Carlton
TZ
JN FN
(30) (32) (40, 44) (40) (53, 54) (33) (53, 54)
344
TABLE 2 (continued) Alkaloid
Wild species / cultivars
Section
References
35 2a-hydroxy6-0-methyloduline
A^. cv. Salome
81 pallidiflorine
N. pallidiflorus Pugsley
PN
(66)
69 pancracine
A'', poeticus L.
NC
(92)
44 papyramine
A^. panizzianus Pari. A^. papyraceus Ker. Gawl. A^. tazetta L. var. chinensis Roem.
TZ TZ TZ
(61) (48,71,72) (57)
45 6-epipapyramine
A^. panizzianus Pari. A^. papyraceus Ker. Gawl. A^. tazetta L. var. chinensis Roem.
TZ TZ TZ
(61) (48,71,72) (57)
46 0-methyl6-epipapyramine
A^. papyraceus Ker. Gawl.
TZ
(71,72)
11 pluviine
A^. cyclamineus DC. cv. Cairhays A^. cyclamineus DC. cv. Peeping Tom A^. X incomparabilis Mill. cv. Daisy Schaffer A^. X incomparabilis Mill. cv. Deamia Durbin A^. X incomparabilis Mill. cv. Flower Record A^. X incomparabilis Mill. cv. Helios A^. X incomparabilis Mill. cv. John Evelyn A^' , X incomparabilis Mill. cv. Pluvius A^. X incomparabilis Mill. cv. Sempre Avanti A^. X incomparabilis Mill. cv. Suda A^. poeticus L. NC A^. pseudonarcissus L. cv. Covent Garden A^. pseudonarcissus L. cv. Imperator A^. pseudonarcissus L. cv. King Alfred A^. pseudonarcissus L. cv. Magnet A^. pseudonarcissus L. cv. Magnificience A^. pseudonarcissus L. cv. Mrs. Emst H. Krelage A^. pseudonarcissus L. cv. Oliver Cromwell A^. pseudonarcissus L. cv. Queen of Bicolors A^. pseudonarcissus L. cv. Romaine A^. pseudonarcissus L. cv. Spring Glory A^. pseudonarcissus L. cv. Unsurpassable A^. pseudonarcissus L. cv. Van Sion A^. pseudonarcissus L. cv. Victoria A^. pseudonarcissus L. cv. Wrestler A^. tazetta L. cv. Early Perfection A^. tazetta L. var. chinensis Roem. TZ A^. cv. Inglescombe A^. cv. Insulinde A^. cv. Livia A^. cv. Texas A^. cv. Twink
(40) (40) (32) (32, 42) (32) (32) (32) (32) (32) (32) (62) (32, 42) (32) (42, 62) (32) (32, 42) (32) (32) (32) (32) (32) (32) (32, 42) (32) (32) (49) (57) (32) (32) (32) (32) (32, 42)
13 9-O-demethylpluviine
A^. pseudonarcissus L. cv. Carlton
(54, 58)
14 1-O-acetyl9-O-demethylpluviine
A^. pseudonarcissus L. cv. Carlton
(54, 58)
(34)
345
TABLE 2 (continued) Alkaloid
Wild species / cultivars
15
N. pseudonarcissus L. cv. Carlton
(58)
A'^ cv. Texas
(93)
l,9-(9-diacetyl9-O-demethylpluviine
12 norpluviine
Section
References
A^. poeticus L. var. ornatus. Hort.
NC
(51)
38 poetinatine
A^. poeticus L. var. ornatus. Hort.
NC
(52)
61 pretazettine
A^. bicolor L. A^. confusus Pugsley A^. obesus Salisb. A'^. pallidiflorus Pugsley A^. panizzianus Pari. A^. tazetta L. A^. tazetta L. var. chinensis Roem.
PN PN BC PN TZ TZ TZ
(29) (38) (30) (66) (61) (56, 76, 77) (57)
3 pseudolycorine
A^. assoanus Leon-Duf. A^. dubius Gouan A^. jacetanus Fdez. Casas A^. papyraceus Ker. Gawl. A^. tazetta L. A^. tazetta L. var. chinensis Roem. A^. cv. Salome
JQ DB PN TZ TZ TZ
(94) (81) (27) (48,71,72) (56, 76, 77) (57, 95) (34)
4
1-O-acetylpseudolycorine
A^. assoanus Leon-Duf.
JQ
(94)
5 2-0-acetylpseudolycorine
A^. assoanus Leon-Duf.
JQ
(94)
6
A^. nivalis Graells A^. poeticus L. A^. pseudonarcissus L. cv. King Alfred
BC NC
(47) (62) (28, 62)
22 roserine
A^. pallidulus Graells
CM
(96)
59 tazettine
A^. asturiensis (Jordan) Pugsley A^. bujei (Fdez. Casas) Fdez. Casas A^. canaliculatus Guss A^. cantabricus DC. A'^. cyclamineus DC. cv. Peeping Tom N.folli N. X gracilis Sabine A^. jonquilla L. cv. Golden Sceptre N. jonquilla L. cv. Trevithian A^. kristalli N. X odorus L. var. rugulosus N. papyraceus Ker. Gawl. A^. poeticus L. var. ornatus Hort. A^. pseudonarcissus L cv. Grand Maitre N. pseudonarcissus L. cv. Van Sion A^. tazetta L.
PN PN TZ BC
A^. tazetta L. var. chinensis Roem. A^. tazetta L. cv. Cragford A^. tazetta L. cv. Early Perfection
TZ
(65) (33) (49) (31) (40) (46) (40) (40, 44, 45) (40) (46) (40) (48) (51) (32) (32) (35, 46, 56, 77, 78, 97, 98) (57, 95) (49) (49)
2 poetaminine
9-O-methylpseudolycorine
JC
JN TZ NC
TZ
346
TABLE 2 (continued) Alkaloid
Wild species / cultlvars
59 tazettine (cont.)
N. tazetta L. cv. Geranium A^. tazetta L. cv. La Fiancee A^. tazetta L. cv. Laurens Koster N. tazetta L. cv. L'innocence N. tazetta L. cv. Scarlet Gem N. tazetta L. cv. St. Agnes N. triandrus L. cv. Silver Chames
21 tortuosine
A^. tortuosus Haworth A': cv. Salome
PN
(85) (34)
66 trisphaeridine
A'^ asturiensis (Jordan) Pugsley
FN
(65)
20 vasconine
A^. bicolor L, A^. vasconicus Fdez. Casas A^. cv. Salome
PN PN
(29) (79) (34)
39 vittatine
A^. cantabricus DC. A^. pseudonarcissus L. cv. Carlton
BC
(31) (53, 54)
Section
References (49) (49) (49) (49) (49) (49) (40)
The taxonomical aspects are based on the works of J. Fernandez Casas (99-105) and other authors (106-110) for wild Narcissus species and hybrids, and those of Jefferson-Brown (111) and Boit and Piozzi (32, 40, 42, 49, 86) for Narcissus cultivars (cv.). Key to sections: BC = Bulbocodii DC; CM = Cyclaminei DC; DB = Dubii Fdez. Casas; JC = x Joncissus Fdez. Casas ( Jonquillae DC x Narcissus L.); JN = x Jonissi Fdez. Casas {Jonquillae DC x Pseudonarcissi DC); JQ = Jonquillae DC; NC = Narcissus L.; PN = Pseudonarcissii DC; ST = Serotini Pari.; and TZ = Tazettae DC
3.
BIOSYNTHETIC
PATHWAYS
The work on the biosynthesis of Amaryllidaceae alkaloids reached a peak in the period 1960-1976 with a great number of studies related with this subject. However, since then, little new work has been produced apart from the isolation of compounds predicted as biosynthetic intermediaries of a certain pathway or, more recently, a new biosynthetic proposal to obtain galanthamine (70), which differs from the initial one. Although L-Phe and L-Tyr are closely related in chemical structure, they are not interchangeable in plants. In the Amaryllidaceae alkaloids, L-Phe serves as a primary precursor of the Ce-C^ fragment, corresponding to ring A and the benzylic position (C-6), and L-Tyr is the precursor of ring C, the two-carbon side chain (C-11 and C-12) and nitrogen, C6-C2-N. The conversion of L-Phe to the Ce-Ci unit requires the loss of two carbon atoms from the side chain as well as the introduction of at least two oxygenated
347
substituents into the aromatic ring, wiiich is performed via cinnamic acids. Tiie presence of the enzyme PAL has been demonstrated in Amaryllidaceae plants (112) and the elimination of ammonia mediated by this enzyme is known to occur in an antiperiplanar manner to give frans-cinnamic acid, with loss of the B-pro-S hydrogen (113). Thus, it may be expected that L-Phe would be incorporated into Amaryllidaceae alkaloids with retention of the B-pro-R hydrogen. However, feeding experiments in Narcissus pseudonarcissus cv. King Alfred showed that tritium originally present at C-3 of L-Phe, whatever the configuration, was lost in the formation of several haemanthamine and homolycorine type alkaloids, which led to the conclusion that fragmentation of the cinnamic acids involves oxidation of C-B to ketone or acid level, the final product being protocatechuic aldehyde or its derivatives (Fig. 3). On the other hand, L-Tyr is degraded no further than tyramine before incorporation into the Amaryllidaceae alkaloids. Thus, tyramine and protocatechuic aldehyde -or derivatives of the latter- are logical components for the biosynthesis of the precursor norbelladine (85). This reaction occupies a pivotal position since it represents the entry of primary metabolites into a secundary metabolic pathway. The junction of the amine and the aldehyde results in a Schiff's base, two of which have been isolated up to now: craugsodine (114) and isocraugsodine (115). The existence of Schiff's bases in nature as well as their easy conversion into the different ring-systems of the Amaryllidaceae alkaloids allow the presumption that the initial postulate about this biosynthetic pathway was correct. Barton and Cohen (116) proposed that norbelladine (85) or related compounds could undergo oxidative coupling of phenols in Amaryllidaceae plants, once ring A had been suitably protected by methylation, resulting in the different skeletons of the Amaryllidaceae alkaloids (Fig. 4). a.- Lycorine and IHomolycorine types. The pyrrolo[de]phenanthridine alkaloids (lycorine type) and the 2-benzopirano[3,4-g]indole alkaloids (homolycorine type) both originate from an orttio-para' phenoloxidative coupling (Fig. 5). The biological conversion of cinnamic acid via hydroxylated cinnamic acids into the Ce-Ci unit of norpluviine (12) has been used in a study of hydroxylation mechanisms In higher plants (117). When [3-3H, P-^^Q] cinnamic acid was fed to Narcissus pseudonarcissus cv. Texas a tritium retention in norpluviine (12) of 28% was observed. This is very near a predicted value of 25%, resulting from para-hydroxylation with
348
L-Phe PAL
COOH
Tyr-decarboxylase
frans-cinnamic add HO
COOH
para-coumaric acid: R= H caffeic acid: R= OH
HO^
HO"
^
^CH
protocatechuic aldehyde HO
Sciiiff's base (isomeric structures in solution) OH
HO
HO
rL3 85
Fig. 3. Biosynthetic pathway to norbelladine (85).
M
HO
e
HO O
a
/
q
NH
88
s
M
HO
e
O
n
Q
OH
/
NH
88 ortho-para’
c
OH
M e O & q
/
NR
88/93 para-para‘
tvPes
tVDeS
lycorine (1) homolycorine (23)
crinine (86) haemanthamine (49) tazettine (59) narciclasine (64) montanine (87)
c
para-otfho’
galanthamine (70)
349
Fig. 4. Oxidative phenyl-phenyl coupling in Amaryllidaceae alkaloids.
I
M e O & q
HO
NH
/
MeO&
/
I t MeO&
Me0
Me0
88
/
11
MeO&
Me0
/
/
Me0 OH 31
1
MeO&
/
Me0 6
I )MeO&
-
OMe
0 23
Fig. 5. Alkaloids proceeding from an ortho-para' coupling.
7
350
OH
351 hydrogen migration and retention, where half the tritium would be lost In the first hydroxylation and half the remainder in the second. In the conversion of 0-methylnorbelladine (88) into lycorine (1), the labelling position [3-3H] on the aromatic ring of L-Tyr afterwards appears at C-2 of norpluviine (12), which is formed as an intermediate, the configuration of the tritium apparently being p (93). This tritium is retained in subsequently formed lycorine (1), which means that hydroxylation at C-2 proceeds with an inversion of configuration (118) by a mechanism involving an epoxide, with ring opening followed by allylic rearrangement of the resulting alcohol (Fig. 6). Supporting evidence comes from the incorporation of [2p-^H]caranine (10) into lycorine (1) in Zephyranthes Candida (119). However, an hydroxylation of caranine (10) in Clivia miniata occuring with retention of configuration was also observed (120). Further, [2a-^H; ll-^'^Clcaranine (10) was incorporated into lycorine (1) with high retention of tritium at C-2, indicating that no 2-oxo-compound can be implicated as an intermediate. The conversion of the 0-methoxyphenol to the methylenedloxy group may occur late in the biosynthetic pathway. Tritiated norpluviine (12) is converted to tritiated lycorine (1) by Narcissus x incomparabilis cv. Deanna Durbin, which not only demonstrates the previously mentioned conversion but also indicates that the C-2 hydroxyl group of lycorine (1) is derived by allylic oxidation of either norpluviine (12) or caranine (10) (121). Regarding the conversion of [23-^H, S-OMe-^'^Clpluviine (11) into galanthine (7), in Narcissus pseudonarcissus cv. King Alfred, the retention of 79% of the tritium label confirms that hydroxylation of C-2 may occur with inversion of configuration (62). It was considered (122) that another analogous epoxide (89) could give narcissidine (16) in the way shown by loss of the pro-S hydrogen from C-11, galanthine (7) being a suitable substrate for epoxidation. Labelled [a-^'^C, p-^H]-0-methylnorbelladine (88), when fed to Narcissus x incomparabilis cv. Sempre Avanti afforded galanthine (7) (98% of tritium retention) and narcissidine (16) (46% tritium retention). Loss of hydrogen from C-11 of galanthine (7) was therefore stereospecific.
Recently,
Kihara et al. (123) have isolated a new alkaloid, incartine (89), from flowers of Lycoris incarnata, which could be considered to be the biosynthetic intermediate of this pathway (Fig. 7). The biological conversion of protocatechuic aldehyde into lycorenine (31), which proceeds via 0-methylnorbelladine (88) and norpluviine (12), first involves a reduction of
352
1
12
Fig. 6 . Biosynthesis of lycorine (1) with inversion of the configuration.
Me0
Me0
Me0
Me0 7
HS
\
/
Me0
Me0 89
16
Fig. 7. Conversion of galanthine (7) to narcissidine (16) via epoxide (89).
353
the aldehyde carbonyl, and afterwards, in the generation of lycorenine (31), oxidation of this same carbon atom. The absolute stereochemistry of these processes has been elucidated In subsequent experiments (124), and the results show that hydrogen addition and removal take place on the re-face of the molecules concerned (125), the hydrogen initially introduced being the one later removed (126). It is noteworthy that norpluvilne (12), unlike pluviine (11), is converted in Narcissus pseudonarcissus cv. King Alfred primarily to alkaloids of the homolycorine type. Benzylic oxidation of position 6 would afford (90), followed by ring opening to form an amino aldehyde and then a hemiacetal formation and methylation could provide lycorenine (31) (62),
and, on subsequent
oxidation, could give homolycorine (23) as can be seen in Fig. 8. b. Crinine, Haemanthamine, Tazettine, Narciclasine and l\/lontanine types The alkaloids derived from 5,10b-ethanophenanthridine (crinine and haemanthamine types), 2-benzopyrano[3,4c]indole (tazettine type), phenanthridine
(narciclasine
type) and 5,11b-methanomorphanthridine (montanine type) originate from a para-para' oxidative phenolic coupling (Fig. 9). Results of experiments with labelled crinine (86), and less conclusively with oxovittatine, indicate that the two naturally occurring enantiomeric series, represented in Figure 9 by crinine (86) and vittatine (39), are not interconvertible in Nerine bowdenii (127). Incorporation of 0-methylnorbelladine (88), labelled in the methoxy carbon and also in positions [3,5-^H], into the alkaloid haemanthamine (49) was without loss of tritium, half of which was sited at C-2 of (49). Consideration of the possible mechanisms involved in relation to tritium retention led to the suggestion that the tritium which is expected at C-4 of (49) might not be stereospecific (128). The conversion of Omethylnorbelladine (88) into haemanthamine (49) involves loss of the pro-R hydrogen from the C-3 of the tyramine moiety, as well as a further entry of a hydroxyl group at this site (129). The subsequent benzylic oxidation results in an epimeric mixture (51/52) which even HPLC can not separate. The epimeric forms were proposed to be interconvertible
through
(91a).
The
biosynthetic
conversion
of
the
5,10b-
ethanophenanthridine alkaloids to the 2-benzopyrano[3,4-c]indole was demonstrated by feeding tritium-labeled alkaloids to Sprekeiia formosissima. It was shown that this plant converts haemanthamine (49) to haemanthidlne/ epihaemanthamine subsequently to tazettine (59) in an essentially irreversible manner
(51/52)
and
(130). This
transformation was considered to proceed through (91a) or the related alkoxide anion.
354
[ol
MeO&
HO
/
-
MeO&
HO 12
/
MeO&
HO
/
CH
II
0
OH 90
1
-
Me0
Me0
-
methylations
n Me0& HO
Me0
0
OH
23
31
rotation
Fig. 8 . Conversion of norpluviine (12) to homolycorine type alkaloids.
CH
II 0
(&--OH 0
0
OH 61
OH 51
355
Fig. 9. Alkaloids proceeding from a para-para’ coupling.
356 although this intermediate (91a) and Its rotational equivalent (91b) have never been detected by spectral methods (131) (Fig. 10). It has also been proved that the alkaloid narciclasine (64) proceeds from the pathway of the biosynthesis of crinine and haemanthamine type alkaloids and not through norpluviine (12) and lycorine (1) derivatives. In fact, in view of its structural affinity to both haemanthamine (49) and lycorine (1), narciclasine (64) could be derived by either pathway. When 0-methylnorbelladine (88) labelled in the methoxy carbon and in both protons of position 3 and 5 of the tyramine aromatic ring, was administered to Narcissus plants, all four alkaloids incorporated activity. The isotopic ratio [^H:^'^C] for norpluviine (12) and lycorine (1) was, as expected, 50% that of the precursor, because of its ortho-para' coupling. On the contrary, in haemanthamine (49) the ratio was unchanged. These results prove that the methoxy group of (88) is completely retained in the alkaloids mentioned, providing a satisfactory internal standard and also, the degree of tritium retention is a reliable guide to the direction of phenol coupling. Narciclasine (64) showed an isotopic ratio (75%) higher than that of lycorine or norpluviine (12) though lower than that of haemanthamine (49). However, the fact that more than 50% of tritium is retained suggests that 0-methylnorbelladine (88) is incorporated into narciclasine (64) via para-para' phenol oxidative coupling. O-methylnorbelladine (88) and vittatine (39) are implicated as intermediates in the biosynthesis of narciclasine (64) (132-134), and the loss of the ethane bridge from the latter could occur by a retro-Prins reaction on 11-hydroxyvittatine (92). Strong support for this pathway was obtained by labelling studies. 11-Hydroxyvittatine (92) has also been proposed as an intermediate in the biosynthesis of haemanthamine (49) and montanine (87)
(a 5,11b-methanomorphanthridine
alkaloid)
following the observed
specific
incorporation of vittatine (39) into the two alkaloids in Riiodophiala bifida (127) (Fig. 11). Fuganti and Mazza, (133, 134) concluded that in the late stages of narciclasine (64) biosynthesis, the two-carbon bridge is lost from the oxocrinine skeleton, passing through intermediates bearing a pseudoaxial hydroxy-group at C-3 position and further hydrogen removal from this position does not occur. Noroxomaritidine was also implicated in the biosynthesis of narciclasine (64) and further experiments (135) showed that it is also a precursor for ismine (68). The alkaloid ismine (68) has also been shown (136) to be a transformation product of the crinine-haemanthamine series. The precursor, oxocrinine labelled with tritium in the positions 2 and 4, was administered to Sprel<elia formosissima plants and the radiactive ismine (68) isolated was shown to be specifically labelled at the expected positions.
=( 0
CH
H’
0 51
I OH 52
91a
III OMe
OMe
1
NH
methylation
1 -
0
CH
II 0
91b
61
Fig. 10. Biosynthesis of pretazettine (61). 357
358
39
92
87
Fig. 11. Proposed biosynthetic pathways to haemanthamine (49) and montanine (87).
359 c. Galanthamine type. The alkaloids with a dibenzofuran nucleus (galanthamine type) are obtained from a para-ortho' phenyl oxidative coupling. Although norbelladine (85) was shown not to be a precursor of galanthamine (70) in Narcissus pseudonarcissus cv. King Alfred, an incorporation of this labelled compound has been obtained in Leucojum aestivum (128). The initial studies of this pathway suggested that the phenyl oxidative coupling does not proceed from 0-methylnorbelladine (88) but that the order of methylation of the precursors should be norbelladine (85) --> A/-methylnorbelladine -->A/,0-dimethylnorbelladine (93) to finally give galanthamine (70) (137), the conversion of (94) to narwedine (77) either being not reversible or, if so, enzymically controlled (128). The precursor N,0dimethylnorbelladine (93) was first Isolated in 1988 from Pancratium maritimum (138) a species that also contains galanthamine (70) (Fig. 12a). Chlidanthine (95), by analogy with the known conversion of codeine to morphine, might be expected to arise from galanthamine (70) by 0-demethylation. This was shown to be true when both galanthamine (70) and narwedine (77), with tritium labels, were incorporated into chlidanthine (95) (139). However, the most recent studies related to the biosynthesis of galanthamine (70) seem to contradict the evidence set forth here. Experiments carried out in cell suspension cultures of Leucojum vernum have shown that the methylation of the nitrogen atom occurs after the phenolic coupling and is brought about through an acetyl intermediate (140) (Fig. 12b).
360
93
77
94
70
95
Fig. 12a. Biosynthesis of galanthamine (70) proposed by Barton et al., 1963. 0
Me0
00
HO
HO
Me0
Me0
Me0
-
&NH
Me0
HO
/
Me0
&NH
I I )
/ 73
Me0
&NMe
/ 70
Fig. 12b. Biosynthesis of galanthamine (70) proposed by Eichhorn and Zenk, 1995.
NH
361 4.
SPECTROSCOPY Only ^H NMR, ^^C NMR, and mass spectrometry, the three most important
spectroscopic methods for the Amaryllidaceae alkaloids, will be treated here. A list of names of the different Narcissus alkaloids, their physical and spectroscopic properties, as well as literature is given in Table 3. With respect to the spectroscopic results, the most recent data about the mentioned alkaloids are provided, even when isolation was made from species of genera other than Narcissus.
TABLE 3 Narcissus alkaloids data
Alkaloid
melting Molecular MW point °C Formula (Ref)
[a]D° (Ref.)
Additional data (Ref.)
18 assoanine
CnHnNO^
267
175-176° (141)
UV(26),IR(26),MS(26), ^H NMR (26), *^C NMR (26)
19 oxoassoanine
CnHisNOs
281
247-250° (26)
'H
261-263° (29)
'H
67 bicolorine
Ci5H,2N02
238
UV(26),IR(26),MS(26), NMR (26), ^'C NMR (26) IR (29), MS (29), NMR (29), '^C NMR (29)
57 6a-hydroxybuphanisine
C17H19NO4
301
[ah'' +37 128-130° MeOH; c 0.25 (31) (31)
UV(142),IR(142),MS(142), 'H NMR (142), '^C NMR (143)
58 6P-hydroxybuphanisine
C,7Hi9N04
301
128-130° [OC]D'" +37 (31) MeOH; c 0.25 (31)
UV (142), IR (142), MS (142), 'H NMR (142), ^^C NMR (143)
55 cantabricine
C,8H23N04
317
75-76° (31)
M D ' " -7.14 MeOH; c 0.52 (31)
[alo''-196.6 CHCI3; c 2.0 (145)
IR(31),MS(31), ^ H N M R ( 3 1 ) , *^CNMR(31)
UV (144), IR (144), MS (144), *H NMR (144), CD (146)
10 caranine
Ci6HnN03
271
176-178° (144)
53 crinamine
CnH,9N04
301
190-192° [a]D''+180 CHCI3; c 0.55 (147) (147)
UV (148), IR (147), MS (147), ^H NMR (148), ^^C NMR (148), CD (147), X-Ray (149)
60 criwelline
C18H21NO3
331
211-212° (150)
[a]D''+278 CHCl3;c0.21 (150)
UV (150), MS (151), ^H NMR (152), '^C NMR (152), CD (153)
36 dubiusine
C23H27NO8
445
226-228° (36)
UV(36),IR(36),MS(36), 'HNMR(36),''CNMR(36)
362 TABLE 3 (continued) Alkaloid
melting Molecular MW point °C Formula (Ref.)
[a]D° (Ref.)
Additional data (Ref.) UV(38),IR(38),MS(38), 'H N M R (38), ''C NMR (154), CD(153),X-Ray(155)
70 galanthamine
C17H21NO3
287
124-126° (38)
[aC-115 EtOH;c0.5 (154)
72 0-acetylgalanthamine
C19H23NO4
329
129° (58)
MD' -61 MeOH;cl.3 (58)
71 epigalanthamine
C17H21NO3
287
199° (156)
[ah'' -327A EtOH;c0.27 (155)
UV(156),IR(155),MS(156), 'H NMR (155), CD (153)
73 norgalanthamine
Cl6H,9N03
273
156-158° [a]D''-74 (47) CHCI3; c 0.28 (157)
UV(158),IR(47),MS(47), *H NMR (47), '^C NMR (47), CD(159),X-Ray(160, 161)
74 epinorgalanthamine
C16H19N03
273
150-152° [a]D''-62 (60) CHCI3; c 0.73 (60)
IR (60), MS (60), ^H NMR (60),^^C NMR (60)
75 JV-formylnorgalanthamine
CnH,9N04
301
190-192° (38)
UV(38),IR(38),MS(38), 'H NMR (38),'^C NMR (38)
7 galanthine
C,8H23N04
317
160-162° [a]D''-94.7 CHCI3; c 0.69 (61) (162)
UV (162), MS (61), 'H NMR (61),'^C NMR (61)
8 goleptine
C,7H2,N04
303
49 haemanthamine
CnHi9N04
301
195-198° [a]D''+38.3 CHCI3; c 0.45 (38) (163)
50 11-O-acetylhaemanthamine
C19H21NO5
343
amorph. (33)
51 haemanthidine
C,7H,9N05
317
[a]o''-14 176-179° CHCI3; c 0.28 (167) (167)
UV(168),IR(169),MS(169), 'H NMR (164), '^C NMR (65), CD (153)
52 6-epihaemanthidine
C,7H.9N05
317
176-179° CHCI3; c 0.28 (167) (167)
UV(168),IR(169),MS(169), 'H NMR (164), ^^C NMR (65), CD (153)
30 hippeastrine
CnH^NOs
315
[a]D+138 212-213° CHCI3; c 0.47 (138) (138)
' H N M R ( 3 4 ) , *'CNMR(34),
23 homolycorine
C,8H2iN04
315
141° (64)
169-171° (69)
'H
IR(64)
[aL^^-99 CHCI3; c 0.2 (64)
[a]D''-9.1 MeOH; c 0.55 (33)
[a]D''+93.5 CHCl3;cl.2 (28)
UV (58), MS (58), NMR (58), •^C NMR (58)
UV(38),IR(38),MS(38), ^H NMR (164), *^C NMR (143), CD(165),X-Ray(166) 'H
IR (33), MS (33), NMR (33), ^^C NMR (33), CD (33)
UV(168),IR(34),MS(34), CD (170) UV(69),IR(69),MS(69), ^H NMR (69), *^C NMR (170), CD (170)
363 TABLE 3 (continued) Alkaloid
melting Molecular MW point °C (Ref.) Formula
[a]D°
Additional data (Ref.)
(Ref.)
24 8-0-demethylhomolycorine
C17H19NO4
301
138-140° (170)
[a]D''+89.6 CHCl3;c0.41 (171)
UV(172),IR(172),MS(75), ^H NMR (172), ^^C NMR (172), CD(172),X-Ray(173)
37 8-O-demethylhomolycorineA^-oxide
CnHi^NOs
317
153-154° (72)
M D ' ' +19 MeOH;c0.1 (72)
UV (72), IR (72), MS (72), *H NMR (72), *^C NMR (72)
25 8-O-demethyl8-O-acetylhomolycorine
C19H21NO5
343
186-188° (79)
[aJo'' +70.4 EtOH; c 0.54 (79)
IR (79), MS (79), ' H NMR (79),^^C NMR (79)
26 9-0-demethylhomolycorine
CnHi9N04
301
270-272° (69)
29 9-O-demethyl2a-hydroxyhomolycorine
CnHigNOs
317
272-275° (37)
68 ismine
CisHisNOs
257
98° (174)
CigHnNOs
327
188-190° (44)
9 jonquilline
UV(69),IR(69),MS(69), ^H NMR (69), *^C NMR (69)
[aJo +98.8 EtOH; c 0.52 (37)
IR (37), MS (37), ^HNMR(37),*^CNMR(37)
UV(174),IR(174),MS(174), *H NMR (174), " C NMR (65), X-Ray (65)
[aC-325
UV(44),IR(44)
CHCI3; c 0.5 (44)
78 lycoramine
C17H23NO3
289
120-121° (159)
[aJo'' -67.4 CHCI3; c 0.33 (159)
IR (159), MS (159), *H NMR (159),^^C NMR (159)
79 norlycoramine
C16H21NO3
275
123-124° (175)
[a]D''-39.1 CHCI3; c 0.95 (175)
IR (175), MS (175), ^H NMR (175),
80 epinorlycoramine
C,6H2,N03
275
[a]D''+79.1 CHCl3;c0.51 (60)
IR (60), MS (60), ' H NMR (60),"C NMR (60)
31 lycorenine
C18H23NO4
317
199-203° (176)
[alo''+172.4 CHCI3; c 0.7 (176)
UV (177), MS (178), *H NMR (82), *^C NMR (82), X-Ray (179)
32
C19H25NO4
331
125° (70)
[alo'"+164.1 CHCI3; c 0.53 (70)
IR (70), MS (70), ' H NMR (70),^^C NMR (70)
C,6Hi7N04
287
250° (148)
[ah'' -62 EtOH; c 0.1 (148)
UV(148),IR(148),MS(148), ^H NMR (148),'^C NMR (148), CD (153), X-Ray (180, 181)
C18H19NO5
329
126-128° (29)
[a]D^' +225.6 CHCl3;c0.15 (175)
IR (29), MS (29), ' H NMR (175),^^C NMR (29)
0-methyllycorenine
1 lycorine
62 3-epimacronine
364 TABLE 3 (continued) Alkaloid 40 maritidine
melting Molecular MW point °C (Ref.) Formula C,7H2,N03
287
273
[a]D° (Ref.)
250-252° [ah'' +26.8 (163) MeOH; c 0.78 (163) 139-141° (175)
[ah'' +28 EtOH;c0.53 (67)
Additional data (Ref.) UV(48),IR(182),MS(163), 'HNMR(163),CD(183),
X-Ray(184)
41 8-(9-demethylmaritidine
C,6H,9N03
42 9-O-demethylmaritidine
C,6H,9N03
273
238-239° (75)
43 O-methylmaritidine
C,8H23N03
301
240-242° (72)
47 6a-hydroxy3-(9-methylepimaritidine
C18H23N04
317
[a]D''-10.2 CHCI3; c 0.45 (57)
UV(57),IR(57),MS(57), ' H NMR (57), CD (57)
48 6p-hydroxy3-0-methylepimaritidine
C18H23N04
317
[a]D''-10.2 CHCI3; c 0.45 (57)
UV (57), IR (57), MS (57), ' H NMR (57), CD (57)
27 masonine
C,7HnN04
299
180° (185)
[alo'' +55.6 CHCI3; c 0.5 (53)
UV(53),IR(53),MS(53), ^H NMR (53),^^C NMR (53), CD (170)
28 normasonine
C16H15N04
285
229° (picrate) (53)
[a]D''+9.1 CHCI3; c 0.4 (53)
'HNMR(53),^^CNMR(53)
82 mesembrenone
C,7H2iN03
287
177-180° (picrate) (25)
64 narciclasine
CuHnNOv
307
231-233° (88)
54 narcidine
C,7H2,N04
303
IR (175), MS (175), ' H N M R (164),^^C N M R
(67)
IR (75), MS (75), ' H NMR (75) W D ' ' +16 MeOH; c 0.1 (72) j
UV(72),IR(72),MS(72), ^H NMR (72),'^C NMR (72), CD (57)
UV(53),IR(53),MS(53),
IR (25), MS (25), ^H NMR (25), ^^C NMR (186) [aJo'' +140 EtOH;c0.1 (88) M D +16 MeOH; c 0.11 (55)
UV (87), IR (87), MS (88), 'HNMR(88),^^CNMR(88),
X-Ray(187) UV(55),IR(55),MS(55), *HNMR(55)
56 narcimarkine
C2,H27N05
373
110° (50)
IR (50), MS (50),
65 narciprimine
C14H9N05
271
315-320° (188)
UV(87),IR(87),MS(188), ' H NMR (87)
76 narcisine
Ci8H2,N04
315
158-160° (56)
[aC-18 CHCl3;c0.5 (56)
UV(56),IR(56),MS(56), 'HNMR(56),'^CNMR(56)
365 TABLE 3 (continued) Molecular Formula
melting MW point °C (Ref.)
16 narcissidine
C18H23NO5
333
17 nartazine
C20H23NO6
77 narwedine
[a]D° (Ref.)
Additional data (Ref.)
203-205° (189)
[a]D''-28.3 CHCI3; c 0.32 (189)
UV (190), IR (191), MS (192), *H NMR (193), X-Ray (191)
373
185-186° (49)
[a]D''-120 CHCI3; c 0.25 (49)
IR(49)
CnHi9N03
285
189-190° (159)
MD''+112.2 CHCl3;c0.13 (159)
UV(156),IR(159),MS(159), *H NMR (159)
63 obesine
C,6HnN04
287
[ah'' -5.6 MeOH; c 0.69 (30)
MS (30),'H NMR (30), '^C NMR (30)
33 oduline
Ci7H,9N04
301
Alkaloid
165° (53)
[alo''+149.4 MeOH; c 3.0 (53)
UV(53),IR(53),MS(53), 'HNMR(53),*^CNMR(53)
34 6-0-methyloduline
C,8H2,N04
315
164° (picrate) (53)
[a]D''+148 MeOH; c 0.13 (53)
UV (53), IR (53), MS (53), *HNMR(53),'^CNMR(53)
35 2a-hydroxy6-O-methy loduline
Ci8H2,N05
331
245-248° (34)
[a]o''+91.1 CHCI3; c 0.34 (34)
IR (34), MS (34), ^H NMR (34),^^C NMR (34)
81 pallidiflorine
C34H40N2O7
588
69 pancracine
C,6Hi7N04
287
44 papyramine
C18H23N04
317
IR (66), MS (66), 'H NMR (66),'^C NMR (66) 272° (194)
MD^+74
MeOH; c 0.6 (194)
110-112° MeOH; c 0.25 (61) (72)
45 6-epipapyramine
C18H23N04
317
110-112° MeOH; c 0.25 (61) (72)
46 O-methyl6-epipapyramine
C19H25N04
331
amorph. (72)
[a]D''-31 MeOH; c 0.1 (72)
11 pluviine
C17H21N03
287
220-222° (93)
[a]D-165 CHCI3; c 0.35 (93)
13 9-O-demethylpluviine
C16H19N03
273
148° (58)
[a]D''+61 MeOH; c 2.4 I (58)
UV (194), MS (194),*H NMR (194), ^'C NMR (194), CD (194) UV(72),IR(61),MS(61), 'HNMR(61),^^CNMR(61)
UV(72),IR(61),MS(61), ^HNMR(61),''CNMR(61) UV(72),IR(72),MS(72), NMR (72), ''C NMR (72)
'H
UV (93)
UV (58), MS (58), ^H NMR (58),'^C NMR (58)
366
TABLE 3 (continued) Alkaloid
melting Molecular MW point °C (Ref.) Formula
[a]D° (Ref.)
Additional data (Ref.)
UV (58), MS (58), ^HNMR(58),'^CNMR(58)
14 1-O-acetyl9-O-demethylpluviine
C.8H21NO4
315
203° (58)
[ot]D''+38 MeOH;cl.O (58)
15 1,9-0-diacetyl9-O-demethylpluviine
C20H23NO5
357
147° (58)
[a]D''+8.3 MeOH;cl.l (58)
12 norpluviine
C,6H,9N03
273
[a]D-232 239-241° (93) MeOH; c 0.06 (93)
CisHipNOs
329
221-223° (196)
[a]D^VlOO EtOH; c 0.2 (196)
UV(196),IR(196)
38 poetinatine
C20H23NO6
373
212-213° (52)
[a]D''+50 CHCl3;c0.15 (52)
IR (52), MS (52), 'H NMR (52)
61 pretazettine
C,8H2,N05
331
227-229° (175)
[aJo^'+189.9 CHCI3; c 0.64 (197)
UV(198),IR(198),MS(198), 'H NMR (198), CD (199)
3 pseudolycorine
C,6H,9N04
289
[a]D''-60 237-240° (94) MeOH; c 0.04 (72)
UV (94), IR (94), MS (94), 'H NMR (94),'^C NMR (94)
4 1-O-acetylpseudolycorine
C,8H2iN05
331
248-250° (94)
5 2-O-acetylpseudolycorine
C,8H2,N05
331
168-170° (94)
6 9-O-methylpseudolycorine
C,7H2lN04
303
22 roserine
C,8H22N03
300
59 tazettine
C,8H2lN05
331
2 poetaminine
222-226° (200)
UV (58), MS (58), (58), '^C NMR (58)
'H NMR
UV(93),IR(195)
UV(94),IR(94),MS(94), 'H NMR (94) UV(94),IR(94),MS(94), NMR (94), '^C NMR (94)
'H
[a]D''-43 EtOH; c 0.4 (200)
UV(200),IR(28),MS(200), 'H NMR (200) MS (96),'H NMR (96), '^C NMR (96)
202-203° (167)
[(X]D''+138
CHCI3; c 0.23 (167)
UV(201),IR(202),MS(198), 'H NMR (198), *^C NMR (203), CD (153)
21 tortuosine
Ci8H,8N03
296
242-243° (85)
IR (85), MS (85), *H NMR (85),'^C NMR (85)
66 trisphaeridine
C,4H9N02
223
132-134° (174)
UV(174),IR(174),MS(174), 'H NMR (174), '^C NMR (65)
20 vasconine
CnH,6N02
266
233-235° (79)
'HNMR(34),'^CNMR(34)
IR (79), MS (79),
367
TABLE 3 (continued) melting Molecular MW point °C Formula (Ref.)
Alkaloid 39 vittatine
C,6HnN03
271
206-208° (31)
[a]D°
Additional data (Ref.)
[a]D''+26 MeOH; c 0.25 (31)
UV (138), IR (138), MS (138), ' H N M R (164), ''C NMR (143), CD (204)
(Ref.)
The pairs: 6a-hydroxybuphanisine/6p-hydroxybuphanisine (57/58), haemanthidine/6-epihaemanthidme (51/52), 6ahydroxy-3-0-methylepimaritidine/6p-hydroxy-3-0-methylepimaritidine (47/48), and papyramine/6-epipapyramine (44/45), exist in solution as a mixture of C-6 epimers, and their data were obtained from these mixtures. In the case of narwedine (77), a rapid racemisation occurs in the presence of base (89). Narciprimine (65) is considered an artifact produced from narciclasine (64) during acid extraction (89), and tazettine (59) has been shown to be an artifact produced by base-catalysed rearrangement of pretazettine (61) (199, 205). Narcissamine (32, 42) is a quasi-racemic mixture consisting of equimolar amounts of norgalanthamine (73) and norlycoramine (79)
4.1 Proton Nuclear Magnetic Resonance ^H NMR spectroscopy gives important information about the different types of Amaryllidaceae alkaloids. Several early contributions about homolycorine and crinanehaemanthamlne type alkaloids were made by Hawksworth et al. (206) and Haugwitz et al. (207). In the last decade, the routine use of 2D NMR tecniques (COSY, NOESY, ROESY, etc.) has facilitated the structural assignments and the settling of their stereochemistry. A compilation of the different ^H NMR spectra, arranged according to the different skeleton types is shown in Table 4. a." Lycorine type This group has been subject to several ^H NMR studies and lycorine (1) as well as Its main derivatives have been completely assigned. The general characteristics of the ^H NMR spectra are: i
Two singlets for the para-oriented aromatic protons in the range 5 6.5-7.2 ppm.
ii
A unique olefinic proton around 5.5 ppm.
iii
Two doublets as an AB system corresponding to the benzylic protons of C-6.
Iv
The deshielding observed in the p protons of positions 6 and 12 In relation to their a-homologues is due to the effect of the c/s-lone pair of the nitrogen atom.
368
V
Like almost all other lycorine type examples, the alkaloids isolated from Narcissus genus show a trans B/C ring junction, the coupling constant being ^4a.10b~11HZ.
In the plant, the alkaloid lycorine (1) is particularly vulnerable to the oxidation processes giving several ring-C aromatized products. b.- Homolycorine type This group includes lactone, hemiacetal or cyclic ether (unusual) alkaloids. The general traits for this type of compounds could be summarized as follows: i
Two singlets for the para-oriented aromatic protons. In lactone alkaloids, differentiation of the H-7 and H-10 signals is readily made by virtue of the deshielding of H-7 by effect of the per/-carbonyl group,
ii
The hemiacetal alkaloids always show the substituent at C-6 in a-disposition, and the benzylic proton H-6p appears as a singlet between 5-6 ppm, depending on the substituent at C-6.
iii
The majority of compounds belong to a single enantiomeric series containing a cis B/C ring junction, which is made clear by the small size of the coupling constant J^ ^ob- '" *he Narcissus genus no exception to this rule has been observed,
iv
The large coupling constant between H-4a and H-10b (J^g^^Q^^~^OHz), is only consistent with a frans-diaxial relationship.
V
In general, the C ring presents a vinylic proton around 5.5 ppm.
vi
The singlet corresponding to the A/-methyl group is in the range 6 2.0-2.2 ppm, its non existence being very unusual,
vii If position 2 is substituted by an OH, OMe OAc group, it always displays an adisposition. viii The H-12a is more deshielded than H-12P as a consequence of the c/s-lone pair of the nitrogen atom. An interesting study of homolycorine type alkaloids with a saturated ring C has been made by Jeff and coworkers (208). They describe empirical correlations of A/-methyl chemical shifts with stereochemical assignments of the B/C and C/D ring junction.
369 c- Crinine - Haemanthamine types The absolute configuration of these alkaloids, which allows
both series to be
differentiated, is determined through the circular dichroism spectrum. The alkaloids of the Narcissus genus usually belong to the haemanthamine type, while in genera such as Brunsvigia, Boophane etc., the crinine type alkaloids are predominant. It is also noteworthy that the alkaloids isolated from the Narcissus genus do not show additional substitutions in the aromatic ring apart from those of C-8 and C-9. On the contrary, in the genera where crinine type alkaloids predominate, the presence of compounds with a methoxy substituent at C-7 is quite common. Thus, taking into account the previous considerations, haemanthamine type alkaloids show the following characteristics: i
Two singlets for the para-oriented aromatic protons In the range 5 6.4-7.0 ppm.
ii
Using CDCI3 as the solvent, the magnitude of the coupling constants between each olefinic proton (H-1 and H-2) and H-3 gives information about the configuration of the C-3 substituent. Thus, in those alkaloids in which the twocarbon bridge (C-11 and C-12) was cis to the substituent at C-3, H-1 shows an allylic coupling with H-3 (J^ 3~1-2 Hz) and H-2 a smaller coupling with H-3 (^2,3-0" 1.5 Hz), as occurs in crinamine (53). On the contrary, in the corresponding C-3 epimeric series, e.g. haemanthamine (49), a larger coupling between H-2 and H3 (Ja.sS Hz) is shown, the coupling between H-1 and H-3 not being detectable,
iii
It is frequently possible to observe an additional coupling of H-2 with the equatorial H-4p, in a W-mechanism.
iv
The proton H-4a shows a large coupling with H-4a (J4a4a~13 Hz) due to their frans-dlaxial position, characteristic of the haemanthamine series.
V
Two doublets for an AB system corresponding to the benzylic protons of position C-6.
vi
The pairs of alkaloids with a hydroxy substituent at C-6, like papyramlne/6epipapyramine
(44/45),
haemanthidine/6-epihaemanthldine
(51/52)
etc.,
appear as a mixture of epimers not separable even by HPLC. vii Also in relation with position C-6, It is interesting to note that ismine (68), a catabolic product from the haemanthamine series, shows a restricted rotation around the biarylic bond, which makes the methylenic protons at the benzylic position magnetically non-equivalent.
370 d.- Tazettine type Although tazettine (59) is one of the most widely distributed alkaloids
in the
Amaryllidaceae family, it was found to be an extraction artifact of pretazettine (61) (209). The presence of an A/-methyl group (2.4-2.5 ppm) in tazettine type alkaloids inmediately distinguishes
them from the haemanthamine
type, from which
they
proceed
biosynthetically. Moreover the ^H NMR spectrum always shows the signal corresponding to the methylenedioxy group. We have also included the alkaloid obesine (63) in this group, although it exhibits some structural differences with the skeleton type. e.- Galanthamine type Among the Amaryllidaceae alkaloids, only the galanthamine type shows an orthocoupling constant between both aromatic protons of ring A. The general characteristics of their ^H NMR spectra are: i Two doublets for the two OAt/7o-oriented aromatic protons with a coupling constant of J^g-SHz. ii The assignment of the substituent stereochemistry at C-3 is made In relation with the coupling constants of the olefinic protons H-4 and H-4a. When coupling constant J^^ is about 5 Hz, the substituent is pseudoaxial while if It is ~0 Hz this indicates that the substituent at C-3 is pseudoequatorlal. ill Two doublets as an AB system corresponding to the benzylic protons of C-6. iv The existence of the furan ring results in a deshielding effect in H-1. V This type of alkaloids often show an A/-methyl group but occasionally A/-formyl or A/-acetyl derivatives have been reported.
TABLE 4a 'H NMR data of Lycorine type alkaloids. Alkaloid H-1 H-2a
1 4.27 br s 3.97 br s
3 4.85 br s 4.50 t
4
5
5.58 m 4.15 dd
4.43 t 5.29 dd
7
10
13
14
4.53 dd 4.18 rn
4.55 s 3.72 rn
4.70 m 2.59 m
4.26 dd 2.3-2.6 m
5.54 dd 2.3-2.5 m
5.51 rn 2.36 rn
I _
---I-_
6
15
16 4.66 rn 3.4-4.2 rn
2.59 m
2.3-2.4 m
2.3-2.5 m
2.36 rn
H-3
5.37 br s
5.60 t
5.69 dd
5.45 t
5.55 rn
5.55 br s
5.41 rn
5.54 br d
5.39 br s
5.32 br t
4.66 m
H-4a
2.60 d
3.02 br d
2.90 br s
2.85 d
2.87 dd
2.65 s
2.78 dd
2.70 br d
2.3-2.5 m
2.2-2.3 m
3.4-4.2 rn
H-6a
3.32 d
3.68 br d
3.61 dd
3.54 d
3.55 dd
3.40 br d
3.52 dd
3.31 d
3.54 br d
3.5-3.6 br s
3.54 d
H-6P
4.02 d
4.16 d
4.17 d
4.14 d
4.16 d
4.05 d
4.13 d
3.93 d
3.59 br d
3.5-3.6 br s
4.09 d
H-7
6.68 s
6.71 s
6.70 s
6.61 s
6.75 s
6.52 s
6.58 s
6.59 s
6.64 s
6.71 s
6.68 s
H-10
6.81 s
6.89 s
6.74 s
6.83 s
6.96 s
6.78 s
6.82 s
6.82 s
7.08 s
7.14 br s
6.88 s
H-lob
2.50 m
2.74 br d
2.90 br s
2.69 d
2.76 ddd
2.65 s
2.41 ddd
2.98 dd
3.33 dd
3.27 dd
2.70 d
H-11 (2H)
2.44 m
2.6-2.7 m
2.66 t
2.63 rn
2.64 m
2.4-2.6 m
2.59 rn
2.5-2.6 rn
2.3-2.5 m
2.2-2.4 rn
5.56 br s (1H)
H-l2a
2.19 ddd
2.6-2.7 m
2.59 br t
2.42 dt
2.42 br q
2.25 dd
2.33 br q
2.3-2.4 rn
2.79 rn
2.6-2.8 m
3.4-4.2 m
H-12P
3.19 dd
3.37 dd
3.33 m
3.35 dt
3.35 ddd
3.25 ddd
3.32 ddd
3.32 m
2.79 m
2.6-2.8 rn
3.4-4.2 m
OCH,O
5.95 s
3.74 s
3.86 s
H-2P
OMe OMe OMe
____
3.84 s
____ ____ ---_
OAc
5.91 (2d) 3.84 s
1.92 s
3.82 s
3.85 s
3.78 s
3.80 s
3.74 s
I -
3.40 s
I -
3.89 s
3.86 s
3.82 s 3.44 s 1.98 s
2.06 s
1.92 s
OAc
solvent MHz
2.23 s
a
b
b
b
C
d
d
d
d
d
d
300
200
200
200
270
200
270
400
400
400
400
Ref.
371
Solvent a: DMSO-d,, b: CDCI,-CD,OD, c: CD,OD, d: CDCI,.
372 TABLE 4a (continued) ^H NMR data of Lycorine type alkaloids. Alkaloid
20
21
H-1
7.31 dd
7.85 br d
8.32 d
8.13 d
H-2
6.75 t
7.27 t
7.84 t
H-3
6.99 dt
7.33 br d
7.70 d
18
19
I
7.59 brs
22 2.85 ddd 3.13 dd
a
1.8-1.95 m 2.25-2.4 m
a
1.38 qd
a
2.25-2.4 m
p
3.4-3.5 m
H-4 H-6
4.09 s
10.42 s
9.55 s
9.50 s
H-7
6.64 s
7.57 s
8.09 s
7.92 s
6.95 s
H-10
7.17 s
7.81 s
7.86 s
8.34 s
H-11 (2H)
3.00 br t
3.45 brt
3.76 t
3.88 t
2.05 ddd
a P
5.35 t
2.70 dt 4.75-4.95 m
3.32 t
4.45 br t
OMe
3.93 s
4.09 s
4.19 s
4.33 s
4.15 s
OMe
3.87 s
4.03 s
4.08 s
4.20 s
4.05 s
4.17 s
1 3.90 s
H-12(2H)
5.42 t
OMe d
b
b
c
MHz
200
200
500
500
Ref.
(26)
(26)
(34)
(85)
solvent
Solvent a: DMSO-c/e, b: CDCI3-CD3OD, c: CD3OD, d: CDCI3.
1 ^ 1
250 (96)
TABLE 4b
'H NMR data of Homolycorine type compounds: lactone alkaloids. 27
Alkaloid
23
25
26
29
30
36
H-1 H-2 (2H)
4.81 ddd 2.49 rn
4.78 rn 2.59 rn
4.86 br d 2.5-2.7 rn
4.80 ddd 2.51 rn
4.75 rn 2.60 rn
4.85 s 2.62 rn
4.59 br d 4.28 br t (HP)
4.58 br s 4.38 br s (HP)
4.63 dd 5.46 dd (HP)
4.83 rn 2.64 rn
H-3
5.50 rn
5.50 d
5.63 br d
5.55 rn
5.50 br d
5.64 rn
5.68 br s
5.63 br s
5.62 rn
5.76 br d
H-4a
2.72 dd
2.73 dd
2.96 br d
2.71 br d
2.74 rn
5.50 br d ?
2.66 br d
2.62 d
2.74 br d
3.87 br d
H-7
7.57 s
7.60 s
7.47 s
7.54 s
7.49 s
7.52 s
7.47 s
7.45 s
7.52 s
7.44 s
24
28
37
H-10
6.99 s
6.98 s
7.13 s
6.91 s
6.96 s
7.11 s
6.95 s
6.92 s
6.97 s
7.24 s
H-lob
2.64 dd
2.63 dd
2.86 dd
2.60 dd
2.74 rn
2.66 d
2.83 dd
2.85 dd
2.79 br d
3.50 dd
H-11 (2H)
2.6-2.7 rn
2.50 rn
2.5-2.7 rn
2.5-2.6 rn
2.50 rn
2.52 rn
2.5-2.6 rn
2.48 rn
2.5-2.7 rn
3.57 rn
H-12a
3.14 ddd
3.14 ddd
3.31 ddd
3.15 ddd
3.18 ddd
3.15 ddd
3.17 ddd
3.13 ddd
3.19 ddd
2.88 rn
H-12P
2.24 ddd
2.25 dd
2.45 dd
2.30 ddd
2.27 dd
2.89 dd
2.31 dd
2.23 ddd
2.37 dd
2.72 rn
6.07 (2d)
6.05 d
6.04 (2d)
---
_--_
3.94 s
3.96 s
OCH,O OMe
3.96 s
OMe
3.95 s
NMe OAc
2.00 s
3.95 s 2.00 s
3.97 s 2.16 s
3.94 s 2.01 s
2.00 s
CHA
3.95 s 2.06 s I-__
2.08 s
-___
_ I -
__-____
_______
2.03 s
2.06 s 2.00 s
_-_
___I__
--__ 2.94 s
_______
2.53 dd 2.65 dd
CHB CHOH
5.24 rn
I _ _
----
Me
1.29 d
____I_
d
d
b
b
d
d
b
d
b
b
MHz
200
400
250
200
400
400
200
500
400
200
Ref.
(69)
(172)
(79)
(69)
(53)
(53)
(37)
(34)
(36)
(72)
solvent
, c: CD,OD, d: CDCI,.
373
Solvent a: DMSO-$, b: CDCI,-CD,OD
374 TABLE 4b (continued) ^H NMR data of Homolycorine type compounds: hemiacetal alkaloids. Alkaloid
31
32
33
34
35
4.35 br d 2.35 m
4.29 br d 2.35 m
4.35 d 2.31 dm
4.17 d 2.26 dd
H-2p
2.65 m
2.53 dm
4.21 brs
5.50 br s
5.46 br d 2.72 br d
5.39 br s 2.69 br d
5.69 br s
2.78 br d
2.65 m 5.50 br d 2.77 dd
2.62 dm
H-3 H-4a H-6p
5.93 s
5.54 s
5.99 s
5.39 br s
5.43 s
H-7
6.93 s
6.80 s
6.85 s
6.67 s
6.73 s
H-10
6.99 s
6.93 s
6.90 s
6.77 s
6.94 s
H-10b
2.44 dd
2.44 dd
2.4-2.5 m
2.35 dd
2.85 d
H-11 (2H)
2.4-2.6 m
2.4-2.6 m
2.4-2.5 m
2.3-2.4 m
2.54 m
H-12a
3.15 ddd
3.15 ddd
3.14 ddd
3.08 dd
3.33 m
H-12|3
2.27 dd
2.23 dd
2.25 dd
2.17 dd
2.38 dt
5.97 d
5.83 d
5.91 (2d)
3.43 s
3.51 s
2.22 s
H-1a H-2a
OCH2O OMe
3.88 s
3.89 s
OMe
3.87 s
3.88 s
OMe NMe
4.15 brs
2.87 d
3.55 s 2.08 s
2.10 s
2.11 s
2.05 s
solvent MHz
c
d
d
d
d
250
200
400
400
500
Ref.
(82)
(70)
(53)
(53)
(34)
Solvent a: DMSO-dg, b: CDCI3-CD3OD, c: CD3OD, d: CDCI3.
TABLE 4c 'H NMR data of Haemanthamine type alkaloids. Alkaloid
39
41
42
43
44
45
49
50
51
52
53
54
6.33 d 6.27 dd
6.33 d 6.27 dd
6.22 brs 6.22 br s
6.48 d 6.35 dd
H-1 H-2
6.44 d 6.06 dd
6.65 d 5.99 dd
6.58 d 5.91 ddd
6.49 d 6.08 dd
6.65 d 6.00 dd
6.66 d 6.00 dd
6.36 d 6.25 dd
6.40 d 6.21 dd
H-3
4.46 m
4.36 rn
4.31 td
3.85 rn
3.88 rn
3.85 m
3.82 rn
3.91 rn
3.85 rn
3.85 m
3.98 rn
3.86 rn
H-4a
1.85 ddd
1.75 ddd
1.73 td
1.73 ddd
1.77 ddd
1.60 ddd
2.11 ddd
2.07 ddd
2.36 ddd
2.21 ddd
2.08 m
2.11 ddd
H-4P
2.58 ddd
2.03 ddd
1.90 dddd 2.95 ddd
2.21 br d
2.09 m
1.96 ddd
1.98 ddd
2.12 ddd
2.00 ddd
2.08 m
2.04 ddd
H-4a
3.87 m
3.37 rn
3.43 dd
3.90 dd
3.59 dd
3.90 m
3.25 dd
3.41 rn
3.56 dd
3.20 m
3.22 dd
3.38 rn
H-6a
4.07 d
3.79 d
3.81 d
4.25 d
5.16 s
3.72 d
3.77 d
5.02 s
3.66 d
3.70 d
H-6P
4.71 d
4.39 d
4.41 d
4.80 d
5.86 s
4.25 d
4.40 d
5.69 s
4.28 d
4.31 d
H-7
6.53 s
6.56 s
6.53 s
6.57 s
7.03 s
6.89 s
6.41 s
6.52 s
6.94 s
6.79 s
6.47 s
6.53 s
H-10
6.87 s
6.82 s
6.87 s
6.81 s
6.80 s
6.83 s
6.74 s
6.95 s
6.70 s
6.73 s
6.79 s
6.78 s
H-11 endo
2.15 m
1.95 m
1.8-2.0 rn
2.22 ddd
2.00 rn
2.00 rn
3.96 dd
5.05 dd
3.92 m
3.92 rn
3.92 m
3.99 ddd
H-11 exo
2.32 m
2.15 m
2.19 ddd
2.37 ddd
2.00 rn
2.00 rn
H-12 endo
3.17 rn
2.95 m
2.91 ddd
3.27 ddd
3.02 ddd
2.84 ddd
3.30 dd
3.41 rn
4.20 dd
3.30 m
3.40 rn
3.39 m
H-12 exo
3.87 m
3.37 rn
3.31 ddd
4.08 ddd
3.73 ddd
3.39 rn
3.19 dd
3.41 m
2.96 dd
3.20 rn
3.40 m
3.26 dd
OCH,O
5.95 s
5.81 (2d)
5.95 s
5.83 (2d)
5.86 (2d)
5.86 s
3.89 s
OMe OMe OMe
_--_I_
3.86 s
3.88 s
3.88 s
3.82 s
3.88 s
3.88 s
3.34 s
3.37 s
3.31 s
3.36 s
---__--
3.41 s
3.32 s
3.28 s
d
d
-___I
3.40 s
3.35 s
2.03 s
OAc solvent
3.86 s
I_---
-_--
d
d
d
d
d
d
d
d
d
d
MHZ
360
360
200
200
200
200
360
500
360
360
300
360
Ref.
(164)
(164)
(75)
(72)
(61)
(61)
(164)
(33)
(164)
(164)
(148)
(55)
, c: CD,OD, d: CDCI,.
375
Solvent a: DMSO-$, b: CDCI,-CD,OD
TABLE 4e
'H NMR data of Tazettine type alkaloids
'H NMR data of Narciclasine type alkaloids.
Alkaloid
I
59
63
62
60
Alkaloid
I
376
TABLE 4d
68
66
67
4.23 ddd
8.36 dddd 7.61 ddd
8.73 dd 8.02 td
6.98 dd 6.81 ddd
64
I 6.17 ddd
H-1 H-2
5.60 ddd 6.15 ddd
5.78 d 6.20 dd
5.44 ddd 5.97 d
5.86 d 6.13 d
H-1 H-2
H-3
4.13 rn
3.89 ddd
4.12 rn
4.3-4.4 rn
H-3
3.92 ddd
7.67 ddd
8.09 td
7.28 ddd
H-4a
2.20 rn
1.93 ddd
2.39 br d
H-4
3.90 dd
8.11 ddd
8.31 dd
6.73 dd
H-4P
1.60 rn
2.09 ddd
1.68 ddd
H-4a
4.35 ddd
3.15 br s
H-6
4.02 d
H-6
-_I--_-
4.38 d
H-7
H-4a
2.83 rn
2.95 t
H-6 H-6'
4.65 d 4.95 dd
4.68 d 4.94 d
3.10 rn
H-7
6.50 br s
6.55 s
7.52 s
6.66 s
H-10
6.85 s
6.52 s
6.73 s
6.70 s
H-1Ob
__-_I__
H-I 1
2.81 br d
9.06 s
10.42 s
--I---
7.32 s
7.97 s
H-10
6.75 s
7.89 s
8.16 s
6.67 s
OCH,O
6.01 (2d)
6.15 s
6.41 s
5.99 s
4.73 s
2.73 s
4.20 d
NMe solvent
4.43 dd
4.26 d 7.00 s
C
d
b
d
2.65 d
2.83 d
2.76 dd
3.01 d
MHz
500
200
200
200
H-12'
3.30 d
3.30 d
3.16 dd
3.10 d
Ref.
(88)
(174)
(29)
(174)
OCH,O OMe
5.90 s 3.45 s
5.92 s 3.45 s
6.01 s 3.40 s
5.99 s
NMe
2.50 s
H-12
2.40 s
2.38 s
solvent
d
d
d
C
MHz
90
300
100
250
(1981
(152)
f 175)
(30)
Ref.
I
Solvent a: DMSO-de, b: CDCI&D,OD,
C:
CD,OD, d: CDCIa
TABLE 4f 'H NMR data of Galanthamine type alkaloids. Alkaloid
70
71
72
73
74
H-1 H-2a
4.73 rn 2.12 ddd
4.58 m 1.69 ddd
4.58 m 2.10 ddd
4.67 m 2.24 ddd
4.62 br s 2.04 ddd
75
77
78
80
4.50 m 1.95 ddd
4.72 rn 2.73 dd
4.28 m 1.4-1.9 m
4.37 t 1.88 m
3.14 m
2.40 br d
2.50 ddd
3.98 m
4.09 m 1.5-1.7 rn (2H)
H-2P
2.80 ddt
2.77 dddd
2.69 ddd
2.60 m
2.68 dd
2.61 ddt
H-3
4.25 m
4.61 dddd
5.34 dd
4.28 m
4.15 m
4.07 m
H-4
6.10 ddd
6.05 dt
5.92 d
6.06 ddd
5.98 d
6.00 ddd
6.02 d
1.4-1.9 m (2H)
H-4a
6.21 dd
5.79 dt
6.28 d
6.23 dt
6.05 d
5.88 dd
6.92 d
1.4-1.9 m (2H)
1.7-1.9 m (2H)
H-6
3.79 dd
3.61 d
3.77 d
4.21 d
3.93 d
3.94 dd
3.76 d
3.54 d
3.98 s
H-6
4.21 d
4.06 d
4.22 d
4.44 d
4.04 d
4.80dE15.10dZ
4.12 d
3.92 d
3.98 s
H-7
6.74 d
6.55 d
6.61 d
6.84 d
6.62 d
6.60 d
6.64 d
6.51 d
6.65 d
H-8
6.70 d
6.61 d
6.68 d
6.90 d
6.68 d
6.76 d
6.68 d
6.57 d
6.62 d
H-lla
2.20 ddd
2.16 dt
2.18 dd
2.08 m
1.88 dt
1.71 ddd
2.26 dt
1.4-1.9 rn
1.7-1.9 m
H-llP
1.69 ddd
1.63 ddd
1.65 dd
2.08 m
1.80 td
1.81 ddd
1.87 d
1.4-1.9 m
1.7-1.9 m
H-12a
3.16 dt
3.04 br d
3.14 br d
2.42 rn
3.22 m
3.82 ddd E 13.15 ddd Z
3.14 m
2.96 t
3.19 dt
H-12P
3.39 ddd
3.25 dt
3.40 br t
3.57 rn
3.38 dt
3.61 ddd
3.27 t
3.12 t
3.43 m
OMe
3.95 s
3.82 s
3.85 s
3.91 s
3.84 s
3.84 s
3.82 s
3.76 s
3.85 s
NMe
2.52 s
2.56 s
2.45 s
_-____-__
2.45 s
2.29 s
0.02 s 18.07 s
NCHO 2.04 s
OAc solvent MHz
______-__ _______-_
d
d
d
C
d
d
d
d
d
200
400
400
200
200
200
400
400
200
Ref.
c: CD,OD, d: CDCI,.
377
Solvent a: DMSO-$, b: CDCI,-CD,OD,
378
4.2 Carbon^^ Nuclear Magnetic Resonance ^^C NMR spectroscopy has been extensively used for determining the carbon framework of Amaryllidaceae alkaloids, and the major contributions are due to Grain et al (203), Zetta and Gatti (210), and Frahm et al. (143). The assignments are made on the basis of chemical shifts and multiplicities of the signals (by DEPT experiment). The use of 2D NMR tecniques such as HMQG and HMBC allow the assignments to be corroborated. Table 5 shows a compilation of the different ^^G NMR spectra classified according to the different types. The ^^G NMR spectra of Narcissus alkaloids can be divided in two regions. The low-field region (>90 ppm) contains signals of the carbonyl group, the olefinic and aromatic carbons as well as that of the methylenedioxy group. The other signals, corresponding to the saturated carbon resonances are found in the high-field region, the A/-methyl being the only characteristic group, easily recognizable by a quartet signal between 40-46 ppm. The effect of the substituent (OH, OMe, OAc) on the carbon resonances is of considerable importance in localizing the position of the functional groups. The analysis of the spectra allows conclusions to be drawn about the following aspects: - The number of methine olefinic carbons. - The presence and nature of the nitrogen substituent. - The existence of a lactonic carbonyl group. - The presence of a quaternary carbon signal assignable to G-lOb in the chemical shift range of 42-50 ppm.
Table 5a NMR data of Lycorine type alkaloids. Alkaloid c-1 c-2 c-3 c-4 C-4a C-6 C-6a c-7 C-8 c-9 c-10 C-lOa C-1Ob c-11 c-12 OCH,O OMe OMe OMe OEMe OOCk OEMe
1 70.2 71.7 118.5 141.7 60.8 56.7 129.7 107.0 145.2 145.6 105.1 129.6 40.2 28.1 53.3 100.6
7
3 70.7 71.8 118.6 141.7 61.4 56.4 127.3 110.5 146.2 145.1 111.3 126.5 39.4 28.3 53.9
68.5 73.9 114.0 144.6 60.9 56.7 127.9 110.4 146.0 146.0 111.3 127.2 41.1 28.8 53.9
68.3 81 .o 115.1 143.9 60.9 56.6 129.3 110.8 147.8 147.6 108.0 126.6 41.5 28.5 53.8
56.1
56.0
57.3 56.0 55.9
5
~~
13 69.8 33.5 116.2 139.6 59.5 55.3 126.0 112.4 146.0 144.4 110.2 128.8 41.6 27.3 52.1
70.3 28.8 115.0 139.2 60.4 51.9 125.0 113.4 145.4 144.0 110.2 129.2 38.0 28.4 52.4
15 69.9 28.8 114.6 139.4 60.2 51.8 129.2 111.5 149.4 137.5 121.2 132.5 38.2 28.6 52.3
55.9
56.0
55.9
a
19 119.4 123.9 123.9 124.3 131.3 157.5 129.0 108.6 149.8 153.3 103.3 120.3 117.0 27.5 46.8
_--
170.9
170.6
170.6
21.3
21.4
20.6 169.2 21.3
__---
b
b
d
d
d
d
56.3 56.2
22 31.7 21.8 23.0 40.0 162.4 138.5 119.3 96.8 150.5 146.8 142.0 127.1 135.5 26.8 57.4
20 120.0 131.2 125.6 123.2 136.0 144.7 121.3 110.5 151.7 157.9 102.4 130.6 136.0 27.4 55.4
21 102.2 164.5 118.0 139.7 133.0 142.6 122.9 111.0 153.1 159.1 104.6 131.4 126.1 27.8 57.0
--
---
56.7 56.4
57.8 57.4 57.0
62.5 62.4 56.8
I -
oocm solvent MHz
14
-_-_ ____
---
-----I -
b
b
C
d
Ref. c: CD,OD, d: CDCI,
379
Solvent a: DMSO-d,, b: CDCI,-CD,OD,
380
Table 5b I3C N M R data of Homolycorine type alkaloids. Alkaloid
c-I c-2 c-3 c-4 C-4a C-6 C-6a c-7 C-8 c-9 c-10 C-lOa C-lob c-11 c-12 OCH,O OMe OMe OMe NMe OEMe OOCk OsCH,CHOHMe OOCaCHOHMe OOCCH,CHOHMe OOCCH,CHOHh solvent MHz Ref
23
24
25
26
27
28
77.7 31.3 115.2 140.9 66.6 165.9 116.9 111.9 148.9 153.1 110.8 137.8 43.8 28.1 56.6
79.2 32.1 117.1 141.3 68.0 167.9 117.9 116.9 147.9 153.8 112.3 137.6 43.7 28.6 57.3
77.5 30.9 116.5 138.7 66.8 166.3 116.5 115.9 146.3 152.2 110.9 135.7 42.2 27.4 56.0
77.9 31.3 116.1 140.0 66.9 167.0 115.6 112.7 148.1 152.0 115.0 138.0 43.0 27.9 56.5
77.3 31.1 115.6 140.2 66.8 165.4 118.5 109.7 151.8 147.8 108.6 139.7 43.8 27.9 56.3 102.0
75.6 31.2 115.1 139.4 59.1 165.1 118.0 109.9 152.2 147.8 107.4 138.7 43.0 29.6 44.1 102.0
56.4 56.2
56.7
----___
I_____
44.2
44.1
------_
56.0
42.9 174.9 21 .o
I_____
56.2
_______
-------------------------
_______
-------
43.5
-__-___
_______
82.5 66.2 118.5 143.3 66.4 165.7 118.5 112.0 147.6 151.8 114.7 137.2 38.2 27.0 55.8 55.2
_--____
-______ 43.3
29
-------
42.2
30 82.2 66.2 119.4 142.4 67.1 165.0 118.0 109.8 148.0 151.9 108.5 138.8 38.4 27.3 55.9 102.1
_______
---____
_______
42.9
_______
31
32
33
34
67.6 32.6 117.2 140.8 68.5 92.3 131.3 111.9 149.6 149.6 114.0 128.6 44.7 28.7 57.6
66.6 31.3 115.7 140.7 67.3 98.2 130.5 109.7 148.3 148.3 112.4 125.6 44.0 27.8 56.6
66.7 31.7 115.7 140.6 67.5 91.8 132.0 107.4 147.0 147.0 109.8 128.2 44.2 28.1 56.7 101.0
56.5 56.5
55.8 55.5 55.1
_______
66.4 31.3 115.6 140.3 67.5 98.2 131.8 107.3 146.8 146.7 109.5 126.9 43.9 27.9 56.5 100.8 55.2
43.8
44.3
44.3
_______ _______
_______
35 72.8 68.3 119.4 144.0 68.3 98.5 126.9 107.6 147.1 147.1 109.8 131.2 39.2 27.7 56.3 101.1 55.5
_______
_______ __--___ _______ _______
37
79.7 69.2 115.1 147.4 67.5 165.4 115.4 115.0 148.2 152.0 112.7 136.7 39.9 28.0 56.3
78.8 31.4 121.0 135.6 79.3 167.1 117.6 117.1 147.9 153.4 I12.6 134.9 37.4 26.2 70.7
56.2
-------____--
_______
44.0
43.8
-------
____-__
___I__
_______
36
--
_I____
--------
. . . . . . .
_______
--
43.3 169.4 21 .I 170.8 40.8 66.7 19.9 ..
56.2
d
C
b
b
d
d
b
d
C
d
d
d
d
63 (170)
50 (172)
62 (79)
50 (69)
100 (53)
100 (53)
50 (37)
50 (34)
62 (82)
50
100
100
50
b 50
b 50
(70)
(53)
(53)
(34)
(36)
(72)
Solvent a: DMSO-$, b: CDCI,-CD,OD,
c: CD,OD, d: CDCI,
Table 5c
13C NMR data of Haemanthamine type alkaloids. Alkaloid
c-1 c-2 c-3 c-4 C-4a C-6 C-6a c-7 C-8 c-9 c-10 C-lOa C-lob c-11 c-12 OCH,O OMe OMe OMe OMe OEMe OOCk solvent
39 132.1 127.4 64.0 32.7 62.3 62.8 126.3 106.9 145.6 146.0 102.7 138.3 44.2 44.2 53.5 100.6
41 130.3 127.6 63.3 31.6 62.8 60.9 121.1 109.8 144.8 146.0 109.2 136.5 44.0 42.8 52.8
43 128.2 127.3 70.7 25.6 64.8 57.1 116.5 110.0 148.9 149.0 106.2 138.0 44.9 40.6 52.3
44 132.4 125.6 72.4 28.7 62.2 87.0 127.4 111.0 147.8 148.5 105.4 136.4 44.7 42.3 42.0
____
55.8
_______
56.1 56.1 56.1
56.6 56.6 56.1
56.6 56.6 56.0
56.8 56.0 55.8 55.7
d
45 132.0 125.9 72.4 28.1 56.3 88.9 126.5 112.5 147.8 148.7 105.4 137.4 44.1 40.9 47.9
46 132.4 125.2 72.4 28.5 57.1 96.4 125.5 112.4 147.3 148.2 105.0 138.1 43.7 41.1 48.4
_______
49 128.0 127.2 73.0 29.5 62.7 63.3 126.9 106.9 146.5 147.0 103.3 135.0 50.0 80.0 61.5 101.0 56.0
_ _ I _
50 127.5 129.5 72.4 28.2 62.8 61.0 126.2 106.6 146.5 146.5 103.4 134.2 49.2 80.2 60.4 100.8 56.5
51 127.7 130.4 72.7 27.5 62.2 85.8 127.5 108.4 146.8 148.0 103.1 135.2 50.1 78.9 51.4 101.4 56.9
52 127.8 130.5 73.1 27.5 56.5 88.3 127.3 109.6 146.6 148.1 103.1 136.6 50.0 78.2 58.3 101.4 56.8
53 123.6 136.0 76.0 30.1 66.1 63.4 126.5 106.9 146.2 146.5 103.2 135.3 50.3 80.0 61.2 100.9 55.8
55 25.4 26.4 69.9 30.3 66.0 59.1 118.4 109.0 146.3 145.4 109.6 137.1 42.4 35.5 50.8 55.8
---_
MHz
d 20
b 50
d 50
d 50
50
d 50
d 20
d 50
d 50
d 50
d 75
d 50
Ref.
(143)
(67)
(72)
(61)
(61)
(72)
(143)
(33)
(65)
(65)
(148)
(31)
c: CD,OD, d: CDCI,.
381
Solvent a: DMSO-d, b: CDCI,-CD,OD,
382
Table 5d ^^C NMR data of Tazettine type alkaloids. Alkaloid C-1 C-2 C-3 C-4 C-4a C-6 C-6a C-7 C-8 C-9 C-10 C-10a C-1 Ob C-11 C-12 OCH2O OMe NMe solvent MHz Ref.
59 130.5 128.4 72.4 26.6 69.9 65.0 127.8 103.7 146.3 146.3 109.1 125.4 50.1 101.7 61.7 100.6 55.6 41.9 d 16 (203)
60 130.1 128.9 72.1 25.4 68.2 62.6 126.2 108.5 146.6 146.2 104.2 130.9 50.0 102.6 64.5 100.9 56.7 40.6 d 75 (152)
62 131.3 126.0 72.7 29.8 63.3 168.5 118.6 103.8 147.1 152.3 111.0 142.2 46.2 80.1 53.5 102.1 56.2 42.8 d 50 (29)
63 132.4 136.5 63.6 34.4 68.5 62.2 131.0 107.3 148.4 147.3 111.0 125.0 50.3 82.7 55.7 101.9
c 62 (30)
Solvent a: DMSO-ofg, b: CDCI3-CD3OD, c: CD3OD, d: CDCI3.
Table 5e ^^C NMR data of Narciclasine type alkaloids. Alkaloid C-1 C-2 C-3 C-4 C-4a C-6 C-6a C-7 C-8 C-9 C-10 C-lOa C-1 Ob OCH2O NMe solvent MHz Ref.
64 124.7 69.1 72.3 68.8 52.8 172.1 129.2 168.9 152.3 144.8 95.7 132.1 133.3 102.0
66 122.0 126.7 128.1 129.9 143.8 151.8 123.1 105.5 148.1 148.2 99.9 130.3 124.3 101.9
a 68 (88)
d 50 (65)
67 120.2 125.9 131.0 133.0 136.5 152.8 126.6 108.3 159.2 152.0 102.2 122.0 135.3 105.7 45.9 c 50 (29)
68 129.9 118.0 129.1 110.7 146.7 63.5 134.0 109.7 147.5 147.4 110.2 131.2 127.2 101.3 30.8 d 50 (65)
383 Table 5f ^^C NMR data of Galanthamine type alkaloids. Alkaloid C-1 C-2 C-3 C-4 C-4a C-6 C-6a C-7 C-8 C-9 C-10 C-10a C-1 Ob C-11 C-12 OMe NMe NCHO OCMe OCMe solvent MHz Ref.
70 88.1 30.0 62.2 126.8 126.0 60.5 129.5 121.6 110.5 145.5 144.0 132.7 48.2 34.0 54.3 55.5 42.2
72 86.2 27.7 63.2 123.3 121.8 59.9 127.2 130.2 111.7 146.7 144.4 131.9 47.8 33.7 53.4 56.0 40.9
d
170.9 21.4 d 100 (58)
73 88.2 30.3 61.7 129.2 125.9 51.6 123.6 122.8 112.2 146.9 145.3 133.1 48.2 35.6 45.9 56.2
74 88.4 29.9 61.9 127.6 127.1 53.5 132.1 120.9 111.3 146.2 144.1 133.1 48.5 39.7 46.8 55.9
75 87.9/88.7 29.8 61.4 128.0/128.2 125.8/126.2 41.0/52.8 127.4 119.9/121.6 111.4 146.2/146.5 144.3/144.5 131.8/131.9 48.0/48.1 46.6/46.7 35.7/39.1 55.8
76 88.7 29.2 61.4 128.2 125.1 58.4 127.1 121.2 111.3 146.8 144.5 131.2 48.1 35.1 35.2 55.6
78 89.8 31.5 65.2 27.6 31.7 60.4 129.1 121.6 111.3 146.2 144.0 136.3 46.7 23.9 54.1 55.9 41.9
80 90.0 31.7 65.5 27.8 37.5 53.8 127.2 120.8 110.9 146.6 144.2 136.6 47.4 24.2 47.4 56.1
d 25 (159)
d 50 (60)
162.1/162.7
(154)
b 50 (47)
d 50 (60)
b 50 (38)
Solvent a: DMSO-ofe, b: CDCI3-CD3OD, c: CD3OD, d: CDCI3.
161.2 21.4 d 50 (56)
384
4.3 Mass Spectrometry Extensive studies on the mass spectrometry of Amaryllidaceae alkaloids by electron impact were reported in the sixties and seventies (92, 151, 178, 192, 211-218). The fragmentation patterns in the EIMS of various skeletal types are fairly well documented and have considerable diagnostic value. a.- Lycorine type The molecular ion appears as a quite intense peak, and generally suffers the loss of water, as well as C-1 and C-2 and their substituents by a retro Diels-Alder fragmentation (Fig. 13). The loss of water is not present in the spectra of acetyl derivatives. The ease of the loss of water from the molecular ion was found to be greatly dependent on the stereochemistry of the C-2 hydroxyl group. Thus, in the mass spectrum of lycorine (1) the relative intensity is low, while in 2-epilycorine it is the base peak (192). b.- Homolycorine type In this structure type the cleavage of the labile bonds in ring C by a retro Diels-Alder reaction is dominant, generating two fragments: one, the most characteristic, represents the pyrrolidine ring (plus substituents in position 2), and the other (a less abundant fragment) encompasses the aromatic lactone or hemilactone moiety (Fig. 14). A further general and noteworthy feature is the low abundance of the molecular ion in all compounds with a double bond A^'"* (211).
c- Crinine-Haemanthamine types Several general considerations should be taken into account for these types of alkaloids: The stability of the molecular ion, which is almost always the base peak. The important role played by the aromatic ring in the stabilization of the Ions, which is retained in all fragments of high mass while the nitrogen atom is often lost. The relatively large number of nitrogen-free ions. The fragmentation mechanisms are initiated by the rupture of a bond p to the nitrogen atom which implies the opening of the C-11/C-12 bridge (215, 216). c.1. Compounds with a saturated ring C and no bridge substituent. The configuration of the substituent on ring C plays a minor role in the fragmentation process.
385 C.2. Compounds with a double bond (A^'^) in ring C and no bridge substituent. The fragnnentation pattern involves ruptures of C-4a/C-10b and C-3/C-4 bonds. A characteristic feature is the loss of a nitrogen-containing nnoiety, C3H5N [M-55]. C.3. Compounds with a double bond (A^'^) in ring C and a hydroxyl substituent at C-11. The presence of a hydroxyl group on C-11 is responsible for dramatic changes in the fragmentation pattern (Fig. 15), and it is profoundly influenced by the stereochemistry. There are three fundamental patterns of fragmentation: Loss of CH3OH: it is more favorable when the two-carbon bridge and the substituent on C-3 are on the same side of the molecule. Loss of C2H6N: the relative significance of the loss of this neutral nitrogen moiety is governed by the ease with which the methanol is eliminated. Loss of CHO: A peak at m/z [M-29] due to the loss of an aldehyde radical is present In all compounds of this type. d.- Tazettine type Minor changes in stereochemistry are sufficient to cause appreciable differences in the stereoisomers in these kind of structures. Thus, in the MS of tazettine (59), with a p configuration of the methoxyl group at C-3, the dominant ion occurs at m/z [M-84], following a C-ring fragmentation by a retro Diels-Alder process. In contrast, the mass spectrum of criwelline (60), which differs only in the configuration of the mentioned methoxyl group, contains a peak of low abundance at A7yz[M-84] (Fig. 16). Ions occur in both steroisomers due to the successive loss of a methyl radical and water from the molecular ion (151). e.- Galanthamine type In this type of structures, the intense molecular ion as well as [M-1] peak, the breaking of ring C (losing a C4H6O fragment) and the elimination of elements of ring B (including the nitrogen atom) are characteristic (Fig. 17). This behavior is similar for the dihydro derivatives (213).
386
OH
OH
m/z 227
m/z 287 LYCORINE (1)
m/z 269
m/z 268 Fig. 13. Mass fragmentation pattern of lycorine (1).
m/z 226
n Me0 MeO$
0
m/z 315 HOMOLYCORINE (23)
c
m/z 109
rn/z 108
-
+.
-cot
JJ - )OeM Me0
Meo 1+ -
Me0
0
m/z 206
m/z 178
Fig. 14. Mass fragmentation pattern of homolycorine (23).
387
388
d z 269
m/z 240
m/z211
m/z 181 OMe
m/z 257
OMe
m/z 225
m/z 30 1 HAEMANTHAMINE (49) m/z 258
-
m/z 227
OMe
'0-H 0
\ m/z 258 Fig. 15. Mass fragmentation pattern of haemanthamine (49).
OMe
m/z 331 TAZETTINE (59)
m/z 247
- CH,O 1 1 1
0
m/z 30 1
dZ331
m/z 247
CRIWELLINE (60) Fig. 16. Mass fragmentation pattern of tazettine (59) and criwelline (60) 389
390
m/z 287
m/z 21 7
5
GALANTHAMINE (70)
m/z 1 74
Fig. 17.Mass fragmentation pattern of galanthamine (70).
-H'
m/z 21 6
391 5.
BIOLOGICAL AND PHARMACOLOGICAL ACTIVITIES Plants of the genus Narcissus L. have been used throughout history to treat a va-
riety of human medical problems (219). N. poeticus L., for example, was described in the Bible as a well-established treatment for symptomatologies now defined as cancer (220). In the fourth century BO, the Greek physician Hippocrates of Cos (the "Father of Medicine") recommended a pessary prepared from narcissus oil (probably N. poeticus L.) for the management of uterine tumours (221). In the first century AD, Pliny the Elder recorded the topical use of that extract and another derived from N. pseudonarcissus L. for this purpose (N. poeticus L. is now known to contain 0.12 g of the antineoplastic agent narciclasine (64) per kg of fresh bulb) (86). Arabian, North African, Central American and Chinese medical practitioners of the middle ages continued using Narcissus oil in cancer treatment (222). For example, bulbs of N. tazetta L. var. chiinensis Roem., cultivated in China as a decorative plant, were used for treatment of tumours in folk medicine. In this case, pretazettine (61) was proved to be one of the antltumour active compounds (57). It has been known for a long time that daffodil ingestions are very dangerous, resulting in toxic symptoms. After ingestion of N. tazetta L. (43) and N. jonquilla L. (223), the first visible symptoms are nausea, vomiting and diarrhea, followed by neurological (trembling, convulsions, etc.) and cardiac sequelae, and sometimes resulting in death if eaten in quantity. N. papyraceus Ker-Gawler is believed to be toxic for herbivorous mammalians; in this case, the toxicity might be related to the alkaloid content which is five times higher in the aerial part than in the bulbs (72). Moreover, N. pseudonarcissus L. showed irritant and allergenic properties on contact with animals and men (74, 224, 225). Apart from these harmful effects of daffodils, several Narcissus extracts have shown antiviral (226-231), antimicrobial (232, 233), cytotoxic (234), antitumour (228-230, 235) and allelopathic (236) activities. Table 6 shows the different pharmacological and/or biological properties exhibited by the alkaloids from the genus Narcissus L., but only some of the activities of a reduced number of Narcissus alkaloids are known. The most extensively studied effect is that of non-specific inhibition, e.g. antitumour, antimitotic, suppression of development of splenomegaly and increase in number of nucleated blood cells. The relationship of chemical structure and biological activity of these alkaloids is largely unknown (236), and further
392
studies of several alkaloids of this plant family for therapeutic purposes remain still to be done. TABLE 6 Biological and pharmacological activities of Narcissus alkaloids Alkaloid
Activity
References
57 6a-hydroxybuphanisine
• Moderate cytotoxic against human tumoral Molt 4 cells
(237)
10 caranine
• • • •
Weak analgesic Convulsant and hypotensive Acetylcholinesterase inhibitor Active against the murine P-388 lymphocytic leukaemia (as acetylcaranine)
(89) (89) (89) (238)
53 crinamine
• • • • • •
Powerful transient hypotensive in dogs Respiratory depressant Moderate antimalarial Antimicrobial Strong cytotoxic Cytotoxic against Molt 4 lymphoid and LMTK fibroblastic cell lines • Weak cytotoxic against HepG2 hepatoma
(89) (89) (148) (239) (148) (240)
36 dubiusine
• Cytotoxic against non-tumoral LMTK cells • Moderately active against Molt 4 lymphoma
(240) (240)
70 galanthamine
• • • • •
(89,241,242) (243) (244) (245) (246, 247)
• • • •
•
Powerful analgesic Anticonvulsive Hypotensive Inductor ofhypothermia in rat Cholinesterase inhibitor with peripheral and central pharmacological effects Centrally-acting competitive acethylcholinesterase inhibitor under investigation as therapeutic agent in treating Alzheimer's disease Reverses cognitive deficits induced by nBM (nucleus basalts magnocellularis) lesions in mice Anticurare and antimorphine as well as memory-influencing Combines both physostigmine and neostigmine properties: * like physostigmine, reverses opioid-induced respiratory depression, but not the concomitant analgesia * like neostigmine, antagonises muscle paralysis induced by cZ-tubocurarine, also antagonises the ganglionic blockade and increases the contraction response As hydrobromide has several central and peripheral effects: * has central effects such as antagonism of the respiratory depressant effect of morphine-like compounds * is capable of penetrating the blood-brain barrier and it is used in the treatment of the central effects of scopolamine (hyoscine) intoxication. It has certain advantages over physotigmine for this purpose. * is able to reverse the central anticholinergic syndrome
(240)
(89, 248-254) (230,249,251) (255) (256) (255, 257) (244)
(257- 259) (247, 260)
256,
(256, 258)
257,
393 TABLE 6 (continued) Alkaloid
Activity
70 galanthamine (cont.)
• • • •
* is used for its anticholinesterase activity in the treatment of disturbances in the peripheral sympathetic synaptic transmission. * reverses the neuromuscular blocking effect of curare-type muscle relaxants and has been used safely in anaesthesia and in the treatment of various neurologic disorders (pareses, paralysis of different origins, myasthenia gravis, progressive muscular dystrophy, etc.). Energix®, a preparation composed of galanthamine, increases endurance during exercise and delays the onset of fatigue Used in a pharmaceutical composition for treatment of alcoholism Cytotoxic against non-tumoral LMTK cells Produces poisoning of digestive, respiratory, neuromuscular and central nervous systems
References (259) (242, 246, 250, 255-258,261-263)
(264-266) (267) (240) (43)
71 epigalanthamine
• More hypotensive and less toxic than galanthamine • Anticholinesterase activity lower than galanthamine
(268) (268, 269)
73 norgalanthamine
• Cytotoxic against Molt 4 lymphoid and LMTK fibroblastic cell lines
(240)
75 7V-formylnorgalanthamine
• Moderate cytotoxic against Molt 4 lymphoma and HepG2 hepatoma • Cytotoxic against non-tumoral LMTK cells
(240) (240)
• Hypotensive • Weak cytotoxic against Molt 4 lymphoid cells
(89) (240)
• • • • • • •
Hypertensive Cytotoxic against a variety of cultured cells (in vitro) Cytotoxic against fibroblastic LMTK cells Moderately active against Molt 4 lymphoid cells Moderately active against Rauscher leukaemia Inhibitor of HeLa cell growth Inhibitor of protein synthesis, blocking the peptide bond formation step on the peptidyl transferase centre of the 60S ribosomal subunit • Slightly reduces DNA synthesis, whereas RNA synthesis is practically unaffected
(270) (271) (240) (240) (234) (272) (272, 273)
51 haemanthidine
• Active against A-431, KB, Lul, Mel2 and ZR-75-1 cell lines • Significantly active against LNCaP and HT cell lines
(167) (167)
30 hippeastrine
• • • • •
(274) (7) (167) (240) (240)
23 homolycorine
• Inductor of delayed hypersensitivity in animals • Cytotoxic against fibroblastic LMTK cells
(74) (240)
24 8-O-demethylhomolycorine
• Cytotoxic against fibroblastic LMTK cells • Weak cytotoxic against Molt 4 lymphoid cells
(240) (240)
7 galanthine 49 haemanthamine
Antiviral. Active against Herpes simplex type 1 (HS-1) Weak insect antifeedant Significantly active against the LNCaP and HT cell lines Cytotoxic against non-cancerous LMTK cells Weak cytotoxic against Molt 4 lymphoid cells
(272)
394 TABLE 6 (continued) A kaloid
Activity
References
29 9-O-demethyl2a-hydroxyhomolycorine
• Cytotoxic against fibroblastic LMTK cells • Moderately active against Molt 4 lymphoid cells
(240) (240)
68 ismine
• Cytotoxic against Molt 4 lymphoid and LMTK fibroblastic cell lines
(240)
78 lycoramine
• Central anticholinesterase activity stronger than galanthamine • Like neostigmine, antagonises muscle paralysis induced by dtubocurarine, also antagonises the ganglionic blockade, and increases the contraction response • Produces acute poisoning of digestive, respiratory, cardiovascular. neuromuscular and central nervous systems
(244) (244)
31 lycorenine
• Weak hypotensive • Cytotoxic against murine LMTK and human HepG2 cell lines
(270) (240)
32 O-methyllycorenine
• Cytotoxic against fibroblastic LMTK cells • Weak cytotoxic against HepG2 hepatoma
(240) (240)
• Respiratory stimulant • Relaxant of isolated epinephrine-precontracted pulmonary artery • Increases contractility and rate of isolated perfused heart. These effects are mediated by stimulation of p-adrenergic receptors • Specific inhibitor of ascorbic acid (AA) biosynthesis. Seems to act as a non-competitive inhibitor on galactano-gamma-lactone oxidase • Most of the effects of lycorine on physiological processes have been ascribed to its ability to inhibit ascorbic acid biosynthesis in
(89, 275) (275) (275)
1 lycorine
VIVO.
• The ability to inhibit the ascorbic acid biosynthesis has made this substance a valuable tool for studying the AA-dependent reactions • Inhibitor of cyanide-resistant respiration. AA is needed for the in vivo synthesis of hydroxyproline-containing proteins, specifically utilised for the development of KCN-resistant respiration. • Inhibitor of peroxidase enhancement, which seems related with the synthesis of hydroxyproline-containing proteins • Inhibitor of cell division in rat fibroblasts. Ascorbic acid is required for cell division • Inhibitor of protein blocking peptide bond formation • Inhibitor of DNA synthesis. • Moderate antitumoural • Cytotoxic against a variety of cultured cell lines • Inhibitor of HeLa cells growth • Decreases the growth of several viruses through its inhibitory action on viral protein synthesis • Active against several RNA and DNA viruses. Inhibits the growth of Herpes simplex type I, poliomyelitis, coxsackie, Semliki Forest and measles viruses • Active against RNA-containing flaviviruses and bunyaviruses • Does not inhibit the activity of reverse transcriptase • Weak protozoicide • Plant growth inhibitor by inhibition of protein synthesis • Inhibitor of growth and cell division in plants, inhibiting the cell cycle during interphase. Ascorbic acid is required for cell division
(43, 244)
(275-279) (144) (279-282) (144, 281)
277,
279,
(144,281) (240, 282) (272, 273, 283) (272, 283) (89, 274, 275) (148,271,240) (272) (284-287) (89, 274, 275, 286288) (289) (228) (89) (89) (144, 275, 277, 279, 282)
395 TABLE 6 (continued) Activity
References
• Inhibitor of germination of seeds and growth of roots. Lycorine-1O-P-D-glucose has the reverse effect, and may also produce mitogenic activity in animal cells • Ungeremine, a natural metabolite of lycorine, is responsible, at least in part, for the growth-inhibitory and cytotoxic effects of lycorine • Certain bacteries transform lycorine into pancrassidine, which is less cytotoxic than ungeremine. • The changes observed in response to stress suggest its role in protective and repair mechanisms of producer plants • Inhibits the feeding of the desert locust Schistocerca gregaria
(283, 290)
(294)
62 3-epimacronine
• Weak cytotoxic against human Molt 4 and murine LMTK cell Imes
(240)
27 masonine
• Inductor of delayed hypersensitivity in animals
(74)
82 mesembrenone
• Cytotoxic against Molt 4 lymphoid cells • Weak cytotoxic agamst fibroblastic LMTK cells
(240) (240)
64 narciclasine
• Antimitotic • Strong tumour inhibitor. One of the most important antineoplastic components of Amaryllidaceae species • Active against larynx and cervix carcinomas • Inhibitor of HeLa cells growth • Inhibitor of growth of Ehrlich tumour cells • Inhibitor of protein synthesis in eukariotic ribosomes, blocking the peptide bond formation on the 60S ribosomal subunit. • Resistance to narciclasine in a mutant strain of Saccharomyces cerevisiae is due to an alteration on the peptidyl transferase centre • Reduces DNA synthesis, whereas RNA synthesis is practically unaffected • Inhibitor of ascorbic acid biosynthesis in vivo • Strong antibiotic. Active against Corynebacterium fascians • Active against RNA-containingflavivirusesand bunyaviruses • Inhibitor of cell division in tobacco tissue culture • Inhibitor of Avena coleoptile sections and rice seedling test • Inhibitor of seed germination and root growth at low concentrations. The O-glucoside of narciclasine has the reverse effect • Narciclasine-4-O-P-D-glucopyranoside shows cytotoxic and antitumour activity very similar to narciclasine • The peculiar activity of narciclasine seems to arise from the ftmctional groups and conformationalfreedomof its C-ring
(88, 89, 295) (89, 222,296, 297)
• Increases the amplitude andfrequencyof respiratory movements • Increases the amplitude and decreases the frequency of cardiac contractions • Decreases the soporific effects of ethanol and barbiturics • Increases the analgesic effect of morphine • Protective against thiopental poisoning • Enhances the effects of caffeine, corazole, arecoline, and nicotine • Probably acts primarily on m-cholinoreactive structures of the brain
(304) (304)
Alkaloid 1 lycorine (cont.)
11 narwedine
(291) (292) (293)
(298) (272) (221,299,300) (89, 273, 299-301) (299) (272) (88, 144) (88) (289) (297) (297) (302) (303) (88)
(304) (304) (304) (90) (90)
44 papyramine
(240) • Cytotoxic against fibroblastic LMTK cells • Weak cytotoxic against human tumoral cell lines Molt 4 and HepG2 (240)
61 pretazettine
• Active against murine Rauscher viral leukaemia
(89, 205, 230, 234, 288)
396 TABLE 6 (continued) Alkaloid 61 pretazettine (cont.)
j Activity
References
• Active against spontaneous AKR T cells in mice • Active against intraperitoneally and subcutaneously implanted LLC (Lewis lung carcinomas) • Effective against Ehrlich ascites tumour, a non viral transplantable tumour • Cytotoxic against fibroblastic LMTK and Molt 4 lymphoid cell lines • Inhibitor ofHeLa cell growth • Inhibitor of protein synthesis in eukariotic ribosomes, blocking the peptide bond formation on the 60S ribosomal subunit. • Slightly reduces DNA synthesis; has no effect on RNA synthesis • Active against Herpes simplex type 1 (HS-1) • Active against neurotropic RNA viruses • Potent inhibitor of viral reverse transcriptase of RNA tumour viruses, binding to the polymerase enzyme • Demonstrates synergistic effect in combination with standard cytotoxic drugs • In combination with ryllistine inhibits nucleic acid synthesis • Can be administrated over a long period of time without apparent toxicity
(199, 305-308) (307)
• Effective against murine Rauscher leukaemia in mice, suppressing the splenomegaly and the increase of nucleated blood cells, and decreasing the virus level in plasma without apparent toxicity • Inhibitor of protein synthesis in tumour cells at the step of peptide bond formation. It has a different binding site than lycorine on the peptidyl transferase centre of the 60S ribosomal subunit • Reduces DNA synthesis. RNA synthesis is practically unaffected • Cytotoxic against human Molt 4 and murine LMTK cells • Moderately active against HepG2 hepatoma • Moderately active against Ehrlich ascites tumour cells • Inhibitor ofHeLa cell growth • Antiviral. Active against Herpes simplex type 1 (HS-1) • Active against neurotropic RNA viruses • Does not inhibit the activity of reverse transcriptase
(230, 312)
• Cytotoxic against Molt 4 lymphoid and LMTK fibroblastic cell lines • Weak cytotoxic against HepG2 hepatoma
(240)
59 tazettine
• • • •
(89, 270)) (167) (240) (205)
39 vittatine
• Weak analgesic in mice • Tachycardic in dogs
3 pseudolycorine
5 2-O-acetylpseudolycorine
Weak hypotensive Active against the Co 12 cell line Weak cytotoxic against fibroblastic LMTK cell lines The stereochemical rearrangement from pretazettine to tazettine inactivates the biological activity of pretazettine
(307-309) (240) (272) (205, 272, 307310) (272, 308) (274) (289,310) (167, 229, 230, 273, 288) (230, 307, 308) (311) (306) 235,
310,
(272,273,310) (272) (240) (240) (231) (272) (274) (289,310,312) (228, 230)
(240)
(89) (89)
397
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
407
Total Synthesis of Naphthylisoquinoline Alkaloids Mark A. Rizzacasa 1. INTRODUCTION The plant families Ancistrocladaceae and Dioncophyllaceae are the only identified sources of the unusual naphthylisoquinoline alkaloids (ref. 1). The family Ancistrocladaceae contains one genus, Ancistrocladus, which consists of approximately 20 species that are distributed in the Indian archipelago, tropical Asia and tropical West Africa. These isoquinoline alkaloids are structurally unique in that they appear to originate, biosynthetically, form the acetate-polymalonate pathway and not from amino acids (ref. 1). Another interesting structural feature of these compounds is that they exist as thermally stable atropisomers because of restricted rotation about the biaryl linkage.
OMeOMe
MeO" MeO'
OMeMe
(+)-Ancistrocladisine (4)
R = H (-)-Ancistrocladine (1) R = Me (-)-O-Methylancistrocladine (2) OH
OMe
OMeMe OH Me (-)-Ancistrocladidine (5)
MichellamineA(6)
MeO' {-)-Dioncophylline A (8)
408
Naphthyl-isoquinoline alkaloids can be grouped according to the location of the biaryl linkage with the largest group being 5-1' linked. Ancistrocladus alkaloids are exemplified by ancistrocladine (1) (ref. 2), the first of this family to be isolated, 0-methylancistrocladine (2) and ancistrocline (3) (ref. 3, 4), which possesses some tumour-inhibiting activity. Other examples include the 7-1' linked ancistrocladisine (4) (ref. 5, 6), ancistrocladidine (5) (ref. 7), which possesses an uncommon 7-2' linkage, and the recently isolated dimeric 5-8' linked alkaloid, michellamine A (6) (ref. 8, 9,10), which has been shown to inhibit the cytopathic effects of HIV-1 (AIDS virus) in vitro. Dioncophyllaceae naphthylisoquinoline alkaloids do not possess oxygenation at C-6 with some examples being dioncophylline A (7) (ref. 11) and dioncophylline C (8) (ref. 12), the only 5-1' Unked alkaloid isolated from this plant family thus far. 2. BIOMIMETIC SYNTHESIS As mentioned, naphthylisoquinoline alkaloids are structurally unusual on account of the methyl group at the 3-position and oxygenation at the 8- and/or 6-position of the isoquinoline ring which points to a polyketide origin. This postulate has been supported by extensive studies conducted by Bringmann on the in vitro biomimetic synthesis of the naphthalene and isoquinoline portions of these compounds (ref. 13, 14) and this work is worth briefly mentioning in a discussion on the total synthesis of these compounds.
OMe Me SCHEME 1: Proposed biosynthesis of naphthylisoquinoline alkaloids.
409
It was proposed that both the naphthalene and isoquinohne moieties of these compounds could arise from a common polyketide precursor 9 (Scheme I). Aldol condensation of 9 could provide diketone 10 or a reduction/aldol sequence could provide the C-6 deoxygenated analogue 11. Diketone 11 serves as a precursor to the naphthalene 12 or, by condensation with a biological ammonia equivalent (ie. pyridoxamine), isoquinoline 13 and various hydrogenated derivatives. Biaryl coupling between 13 and naphthalene 12 gives the C-6 deoxygenated alkaloids found in Dioncophyllaceae. Amination of diketone 10 then gives the 6,8-dioxygenated isoquinoline 14 and biaryl coupling with naphthalene 12 provides the alkaloids that occur in the Ancistrocladus genus.
2. NH4OH, MeOH
HO OMeMe 14
SCHEME 2 A source of the polyketide 9 was the protected equivalent 16 (ref. 13) which can be obtained by ozonolysis of the diene 15 (Scheme 2). Exposure of 16 to silica gel affords phenol 17 which serves as a precursor to a 6,8-dioxygenated isoquinoline. Methylation followed by condensation with ammonia gives isoquinoline 18 from which the isoquinoline 14 can be secured by selective cleavage of the hydroxyethyl group.
^^QQ-OMe
MeO,
SiOo
19 OMeOMe 1. KOH, MeOH
^-
2. KOH, Me2S04
21
1.KOH, MGOSO.
— ^
2. NH4OH, MeOH
„
I
SCHEME 3 Ozonolysis of diene 19 provides the reduced polyketide 20 which is cyclised to phenol 21 (ref. 13) (Scheme 3). Methylation and a further aldol condensation gives the naphthalene segment 12 while methylation followed by condensation with ammonia yields the C-6 deoxygenated
410 isoquinoline fragment 13. Not only did this short biomimetic route rapidly supply each of the segments from simple precursors it also served to demonstrate the possible biogenetic formation of an isoquinoline alkaloid from acetate units. For the biomimetic synthesis of 1,3-dimethyltetrahydroisoquinolines, Bringmann studied the condensation of diketones with pyridoxamine phosphate equivalents (ref. 15). It v^as found, however, that the desired reductive amination did not occur. Condensation of pyridoxamine 23 with dione 22 gave only the isoquinoline 24 which could be reduced by NaBH4 to the corresponding tetrahydroisoquinolines 25 (Scheme 4). Hydrogenolysis of the A^-benzyl group then gave the cw-isoquinoline 26 as well as the corresponding rran^-isomer 27. Since pyridoxamine did not display the biomimetic properties desired it could be replaced by the simpler benzylamine which gave analogous results. MeOH
' . A ^ N ® CI© OMeMe
H3C
NaBH4 88%
N
,Me NH OMeMe 27
SCHEME 4: Biomimetic synthesis of 1,3-dimethyltetrahydroisoquinolines 3. SYNTHETIC APPROACHES TO UNSYMMETMCAL CHIRAL BIARYLS Since the naphthylisoquinoline alkaloids occur as stable atropisomers, a key aspect of synthesis is the stereocontroUed construction of the biaryl linkage (ref. 16). A number of methods have been developed for the synthesis of unsymmetrical chiral biaryls and can be divided into inter- and /nframolecular approaches. Some intermolecular approaches are depicted in Figure 1 and majority utilise nucleophilic aromatic substitution on the aromatic ring (S^Ai reaction) activated by a functional groups such as oxazolines, esters, imines. Most of these approaches involve the use of a chiral auxiliary with the chiral oxazoline method developed by Meyers (ref. 17, 18, 19) finding a wide range of applications to date. The stereoselectivities observed in this coupling reaction can be explained considering both the steric and electronic effects of the ortho substituents on the Grignard reagent (ref. 20, 21). As depicted in Scheme 5, Grignard addition may lead to azaenolate 29a and 29b. The initial nucleophilic attack should occur mostly from the P-face (28P) as a-face (28a) attack is hindered by the substituent on the oxazoline moiety. The atropisomeric selectivity observed could be due to free rotation about the sigma bond
411 FIGURE 1: Intermolecular Approaches 1. Chiral 2-Oxazolines (Ref. 17)
^^^^ ....-
V
,J\^Ox Y - P 11 •^^
OX=K°T"
^^=^
R ^ H; R' = APr R = H;R' = f-Bu R = Ph; R' = CHaOMe
R = OMe or F 2. Chiral Ethers and Esters (Ref. 22,23)
^
-ROMgBr
+
l ^
*
^
X X )<
R = H-Menthyl M = MgBrorLi
MgBr
_^Q^^^
"^
*
„ , , .. ^^ , R = (-)-Menthyl
3. Chiral Sulfoxides (Ref. 24) -X MgBr
^^^^
•\^,0
*
^X.CO,R
^CT 4. Planar Chiral Tricarbonyl(arene)chromium Complexes (ref. 25)
B(OH),
+
pd(0)
[ ^
*
T
MeO MeO. X
^R^ •Cr(C0)3
"
J>^m k>Cr(CO)3
0^/^,
[I
--
MeO.
412
of the complex 29 before magnesium methoxy bromide elimination occurs. When the chelating ability of the R substituent is poor, the preferred rotomer is 29b where the magnesium atom preferentially chelates to the methoxy substituent as shown. Rotomer 29b then collapses down to give the (P)-isomer 30 as the major product. Increasing the steric bulk of the R substituent increases the selectivity for atropisomer 30 but this effect is minor (ref. 20, 21).
V=C ^^N^'^ , -"^=^ S^ "=^^^ |^VC ^ n O MeO-Mg vjPr J, MeO.
^ = : ^
-MeOMgBr J
MeO , „ MeO. J ^ D
'Pr
Me^Mg Br 29a
Br 29b
R = CH3d.e. = 80% R = CH2OTBS, d.e. = 86%
SCHEME 5 An application of this methodology can be found in a total synthesis of the lignan (-)schizandrin in which a coupling between the oxazoline 31 and the Grignard reagent derived from bromide 32 gave the atropisomer 33 as the major product with good selectivity (ref. 26). Cram has also reported a biaryl coupling procedure which involves the use of an oxazoline in the presence of a chiral nucleofuge (ref. 22). OMe OMe
OMe
MeO MeO^\^ OMe 31
OMe (-)-Schizandrin
413 An alternative to the Meyers or Cram oxazoline methods is the use of 2-menthoxybenzoates for the synthesis of axially chiral biaryls. Treatment of the ester 34 with Grignard reagent 35 provides the (Af)-biphenyl 36 in 94% e.e (ref. 23). However, poor enantioselectivities were observed in cases where an ortho chelating substituent on the Grignard reagent was absent. OMe OMe
RT, 24h
MeQ
36 34
The cross coupling reactions of aryl halides or aryl triflates with arylmetals catalysed by palladium(0) has found extensive use in the synthesis of unsymmetrical biaryls (ref. 27). A recent application of this coupling approach in the presence of chiral planar chromium complexes (ref. 25) has proved fruitful in the synthesis of chiral biaryls. Furthermore, Cr(CO)3-complexed biaryls can be isomerised under thermodynamic control, providing access to both atropisomers. Heating a solution of the complexed biaryl 37 in xylene under reflux resulted in a 1:99 ratio favouring the isomerised biaryl chromium complex 38 (ref. 28)
xylene i
'\ OMe Cr(C0)3
reflux 2h 98%
37
38
Ph
M
Ph
MeO 40 OMe toluene, -45°C then HgO"'
OHO
^-
90% ee
414
Recently, the use of a chiral catalysts in the asymmetric biaryl synthesis has received some attention. Unsymmetrical chiral binaphthyls can be obtained in high enantiomeric excess by SnAr reactions of naphthylimines such as 39 with lithium bromide free 1-naphthyllithium catalysed by a chiral ether Ugand 40 (ref. 29). Another example is the Ni(0) catalysed cross coupling of naphthyl Grignard reagents and naphthyl bromides in the presence of a chiral ferrocene ligand (ref. 30). FIGURE 2: Intramolecular Approaches 1. Oxidative Coupling (Ref. 31)
d-(OR)n [O]
2. Palladium Catalysed Coupling (Ether bridge)
Reduction
Pd(ll)
Internal v-^^ asymmetric '" induction'
ll
3. Palladium Catalysed Coupling (Ester bridge)
Pd(ll)
X Q
Axially Prostereogenic or Helical Lactones Often Stereochemically Unstable
Ring-opening with chiral or achiral nucleophiles Intemalor external asymmetric induction'
R' = CH2OH or COXR X = O, 0, N or S
Intramolecular approaches involve utilising a bridge between the two aryl fragments which is either present or not present in the target molecule (Figure 2). Oxidative phenolic and phenolic ether coupling methods have been utilised for the synthesis of lignans and alkaloids where the bridge is part of the target molecule (ref. 31). A recent example of a reagent that can effect aryl dimerisation of electron rich compounds without free phenolic groups is iron(III) perchlorate in the
415 presence of trifluoroacetic acid. The intramolecular oxidative coupling of butyrolactone 41 using this Fe(ni) reagent system gave the biaryl 42 in excellent yield as one atropisomer (ref. 32). OMe
MeO
OMe
MeO Fe(CI04)3»6H20 •
MeO,
CF3CO2H-CH2CI2 O (1:10) RTIh
MeO
MeO
In another example reported by Evans and coworkers, a VOF3 mediated coupling of the linear tripeptide 43 provided the macrocycle 44 as one only atropisomer (ref. 33). Of note was the fact that substrates related to 43, where the 5-arylglycine ring contained a single para oxygen substituent, failed to undergo any intramolecular oxidative coupling under a variety of conditions.
^^.NCOCFg MeNH
^ODOB
11 MeO'
y ^'^^^^OMe
" " ^ OMe 43
VOF3, BF3*OEt2 TFA, TFAA, CH2CI2
0°CthenZn
"^^NH
58% MeO
OMe 'OMe 44
For the stereoselective construction of the biaryl linkage of naphthylisoquinoline alkaloids via an intramolecular strategy, a temporary bridge must be installed and then removed after aryl coupling. Bringmann has studied this type of approach extensively and has applied it to the synthesis of naphthylisoquinoline alkaloids (ref. 16) detailed in Section 4 below. The types of bridge that have been utilised are ester and ether linkages with stereocontroUed formation of the biaryl linkage arising from internal or external asymmetric induction (Figure 2). Ether linkages cause the possible helical isomers to slowly interconvert at room temperature thus allowing for atropisomer separation (ref. 34) while, in some cases, ester linkages can allow for rapid helical interconversion and these compounds do not exist as stable atropisomers. Coined cucially prostereogenie (ref. 35), these lactones can be converted into stable atropisomers by ring opening with chiral H, O or N nucleophiles (external asymmetric induction) or, if the lactone possesses asymmetry elsewhere in the molecule, achiral nucleophiles (internal asymmetric induction).
416
The model benzonaphthopyranone system 46, obtained by Pd(II) catalysed intramolecular coupling of ester 45, has been utilised by Bringmann as a precursor for stereoselective biaryl synthesis by external asymmetric induction (ref. 36, 37). With bulky substituents (ie. R=isopropyl) the helical conformers of 46 are stable and exhibit chirality with the two methyl groups of the isopropyl residue ortho to the biaryl axis appearing as separate signals in the ^H NMR spectrum. The lactone bridge of compounds such as 46 can be opened atropisomerselectively with chiral Hnucleophiles to provide optically active diols (ref. 38, 39). Treatment of the lactone 47 with borane in the presence of the chiral oxazaborolidine 48 developed by Corey (ref. 40) gave an excellent atropisomeric ratio favouring the (M)-isomer 49 (ref. 38). The mechanistic course of this atropisomer selective reduction has been investigated by semiempirical AMI calculations and it was concluded that the stereochemically important step was the second hydride attack on the aldehyde intermediates generated by initial lactone reduction (ref. 41)
rf4
H Ph Ph
48
Me
OH
BH3*THF Me 49 M:Pratio = 98.5:1.5
Rapidly isomerising lactones can also be ring-opened to with chiral O- and A^-nucleophiles with high atropisomer-selectivity. Treatment of the axially prostereogenie lactone 50 with the potassium salt 51, derived from (+)-menthol, provided the (P)-biaryl 52 as the major atropisomer in a modest d.e. of 48% (ref. 42). Ring-opening of lactone 50 with the chiral potassium amide 53 provided the (Af)-biaryl 54 with greater selectivity (d.e. 86%). The use of the corresponding sodium or lithium salts of either 51 or 53 gave lower diastereoselectivities in both cases. Higher selectivities using chiral O- and A^-nucleophiles have been obtained for axially prostereogenic lactones that contain asymmetry elsewhere in the molecule (internal asymmetric induction) (ref. 43).
417 OMe
MeO" ^ f ^ ^C HO^ J N ^ Me
MeO--V-CO HO. Me 53
4. TOTAL SYNTHESIS OF NAPHTHYLISOQUINOLINE ALKALOffiS 4.1 Racemic Synthesis The first total synthesis of a naphthylisoquinoline alkaloid, the 7-1' linked Omethyltetradehydrotriphyophylline (55) isolated from the Dioncophyllaceae family (ref. 44), was reported in 1984 by Bringmann and coworkers (ref. 45). The requisite naphthalene 12 and isoquinoline 13 segments where available from biomimetic synthesis as previously detailed but attempted intermolecular coupling between the two moieties in their brominated forms under classical Ullmann type conditions (ref. 46) afforded only traces of the desired coupled product. Therefore, an intermolecular coupling approach utilising a temporary ether bridge was undertaken. OMeOMe
OMe OMe NBS, AIBN ,Me
PTC NaOH
1. H2, Pt02
MeO
58
2. BnBr, K2CO3 3. 48% HBr reflux
hv 254 nm "^ MeO' NEt3 MeO 15%
MeO
1.Li,(C6H5)2 •
Me
Bn 2. 10% Pd-C 3. Me2S04, NaOH 46%
(±)-59 SCHEME 6: Synthesis of 0-methyltetradehydrotriphyophylline (55)
418
The synthesis of the bridged biaryl precursor 58 began with bromination of naphthalene 12 to provide dibromide 56 (Scheme 6). Selective hydrogenation of 13 followed by A^-benzylation and demethylation provide the tetrahydroisoquinoline 57 which was coupled with 56 under phase transfer conditions to give ether 58. Intramolecular aryl-aryl coupling was then achieved in low yield by photolysis of 58 to yield biaryl 59 along with starting material and a byproduct resulting from hydrodehalogenation. Completion of the synthesis of 55 involved reduction of the ether bridge with lithium biphenyl followed by simultaneous debenzylation/dehydrogenation and methylation. Based on the above approach, a racemic total synthesis of the ^r^zn^-configured alkaloid dioncophylline A (8) (or triphyophylline) was achieved by the same research group (ref. 47) (Scheme 7). On this occasion, a lactone bridge was utilised to construct the biaryl linkage intramolecularly. Oxidation of dibromide 56 followed by acid chloride formation provided the naphthalene segment 60 (ref. 48). The isoquinoline fragment 61 was readily available by benzylation and demethylation of the trans configured isoquinoline 27 obtained by biomimetic synthesis (see Section 2). Esterification of 61 with 60 gave the ester 62 which smoothly underwent palladium(II) catalysed intramolecular coupling to give the planarised lactone 63. Reduction then gave the alcohol 64 and its respective atropisomer which were separated by chromatography. Deoxygenation by hydroxy/halogen exchange followed by further reduction and subsequent debenzylation then provided racemic dioncophylline A (8) and its atropisomer 65. OMeOMe
1. DMSO, HaHCOg
56
100°C 1^11-^
I
^
2. NaCI02 3. (C0CI)2, DMF,at 74% 1. BnBr, K2CO3 2. 48% HBr reflux
Br
o
60
\ D M A P
/ ,.Me
>
•
72%
MeO MeO
2. LiAIH4 3. H2, Pd-C
(±)-8
MeO^>^
(±)-65
SCHEME 7: Racemic synthesis of dioncophylline A (8)
419 OMeOMe CuCN g|, DMF, reflux OMe
78%
70 OMeOMe
CN
MeOH, H2O
OMe
84%
71 SCHEME 8 The first intermolecular synthetic approach to naphthylisoquinoline alkaloids was reported by Rizzacasa and Sargent and involved the use of a Meyers oxazoline coupling (Section 3) to introduce the biaryl linkage (ref. 49, 50). The target chosen to test this methodology was (±)dehydroancistrocladine (66) (ref. 5, 6), a derivative of the 7-1' Unked alkaloid ancistrocladisine (4). The naphthyloxazoline 67 was synthesised from 1,5-diacetoxynaphthalene 68 as shown in Scheme 8. Oxidation of 68 with NBS in acetic acid/water affords the bromojuglone 69 in high yield (ref. 51). This interesting reaction proceeds through a number of discernible intermediates and provides the desired highly functionalised ring system in one step from a readily available starting material. Reduction and tandem hydrolysis/methylation under phase transfer catalytic conditions gives the naphthalene 70 in good yield (ref. 52, 53). Cyanide displacement then provides nitrile 71 which is converted into the oxazoline 67 under standard conditions (ref. 49).
MeO
OMe
Mg, THF
1. Mel, MeNOg
then 67 reflux
2. NaOH 3. HgO"' 4. Mel, K2GO3 90%
^.
MeO
81%
MeO
MeO, MeOgC
CHO
CH3CH2NO2
MeO, MeOgC
NO2
^ •
KOAc, AcOH MeO' MeO'
80%
1.LiAiH4 2. AC2O, pyr.
MeO MeO
75% "^
75
SCHEME 9: Synthesis of dehydroancistrocladisine (66)
420
MeO, ROCH2
Me NHAc
TMSCI, Nal then Zn, HOAc
MeCN, reflux
97%
83%
naphthalene reflux 24%
MeO' MeO
(±)-4 + diastereoisomer
MeO' MeO
SCHEME 9 (cont): Synthesis of dehydroancistrocladisine (66) The completion of the synthesis of 66 is depicted in Scheme 9 and begins with the generation of the Grignard reagent from bromide 72. Addition of the oxazoline followed by heating provides the biaryl compound 73 in excellent yield which is then subjected to a series of deprotection steps involving quatemisation, base hydrolysis and acid hydrolysis of the acetal. Methylation then provides ester 74 and subsequent Henry reaction give the crystalline nitrostyrene 75. Reduction and acetylation yields the diacetate 76 which is O-deacetylated to give alcohol 77. A one pot hydroxy-iodine exchange/zinc reduction sequence gives acetamide 78 which smoothly undergoes Bischler-Napieralski cyclisation to provide racemic ancistrocladisine (4) and the corresponding diastereoisomer as an inseparable mixture. Dehydrogenation with Raney Ni then yields (±)dehydroancistrocladisine (66). 4.2 Asymmetric Synthesis: 5,1' Linked Naphthylisoquinoline Alkaloids The efficient synthesis of racemic triphyophilline (60) paved the way for the first asymmetric total synthesis of the 5,1-linked napthylisoquinoline alkaloid (-)-ancistrocladine (1) (ref. 47). The approach once again relied on the successful intramolecular strategy used previously and required the development of asymmetric synthesis of the tetrahydroisoquinoline portion of 1. The route to isoquinoline segment 79 began with the synthesis of homochiral amphetamine derivative 83 via a reductive methylation protocol (Scheme 10) (ref. 54, 55) Thus, condensation of ketone 80 with (5)1-phenylethylamine gave imine 81 and stereoselective hydrogenation over Raney nickel (92% d.e.) provided enantiomerically pure salt 82 in 85% yield after one recrystallization. Hydrogenolysis then afforded amine hydrochloride 83 which was acetylated to give acetamide 84. Ring closure of 84 to 85 was then achieved under standard Bischler-Napieralski conditions. Stereoselective reduction of dihydroisoquinoline 85 using a protocol developed for the synthesis of 2,6-transdisubstituted piperidines (ref. 56) proved highly effective providing ^on^-tetrahydroisoquinoline 86 in 92% d.e. The final steps to 79 involved benzylation followed by selective demethylation of
421 the C-6 ether with hot aqueous HBr (ref. 57).
Me
MeQ
H Ph
NH2
X 5 Me
K^
toluene -H2O
OMe
MeO. H2N+
Me
N
OMe 81
80 10% Pd-C NH4HCO2 MeOH, refluxc
1. Raney Ni 5 bar H2
^Me
MeO,
Me
2. HCIg
Ph
85%
I I J +NH3
AcCI, NEtg
T OMe
PI-
96%
83 MeO.
Me NHAc
MeO>,
P_Oa^MeCN^ 95%
OMe 84 MeO,
| T
,Me NH OMeMe
2. 47% HBr 70°G, 9h 65%
..Me
T T OMeMe 85
1.BnCI,K2C03
86
1
J:Z^ LiAIH4
-78-^0°C 92%
Me HO^ >r"'^V^^' NBn
OMeMe 79 SCHEME 10
The stereoselectivity observed in the reduction of 85 to 86 is not easily explained in the dihydroisoquinoline case. In the case of tetrahydropyridines, hydride approach of the n bond occurs antiperiplanar to the to the axial vicinal ac-H bond as in C on stereoelectronic grounds to give cis configured products (Figure 3). In the presence of a Lewis acid such as AlMes the complexed iminium ion adopts another conformation due to A^^'^) strain between the R and the AlMes groups. Hydride attack antiperiplanar to the to the axial vicinal CQ-K bond again (ie. T) occurs to give the trans isomer. Since the carbon adjacent to the imine carbon in the dihydroisoquinoline 85 is no longer sp^ hybridised, such a high selectivity may not be expected.
Me^
N H
R
FIGURES
422 OMeOMe Pd(PPh3)2Cl2
^-
Me
NaOAc MeaNCOCHa
LiAIH4 separate 95%
OMeOMe
OMeOMe 1. BrCHgCHaBr PhsP
^.
OMeMe
2. LiAIH4 3. H2, 10%Pd-C 91%
90 + OMeOMe
OMeOMe 1. BrCHgCHgBr PhsP 2. LiAIH4 3. H2, 10%Pd-C
OMeMe OMeMe 91 92 SCHEME 11: Asymmetric synthesis of ancistrocladine (1) and hamatine (92) With the optically active tetrahydroisoquinoline 79 in hand the total synthesis of (-)ancistrocladine (1), and its naturally occurring atropisomer (+)-hamatine (92), was completed as shown in Scheme 11. Esterification of compound 79 with acid chloride 60 gave the ester 87 which smoothly underwent Pd(n) catalysed intramolecular coupling to yield the 5-1' coupled lactones 88 and 89 as a 3:1 mixture respectively. None of the possible 7-1' linked regioisomeric lactone was detected. These lactones are helicene-like atropisomers (not axially prostereogenic) but they
423
interconvert quite rapidly making separation at this stage difficult. Therefore, lactones 88 and 89 were subjected to hydride reduction to yield the configurationally stable alcohols 90 and 91 in the same ratio, which were easily separated by chromatography. Deoxygenation and debenzylation converts into 90 ancistrocladine (1) and 91 into hamatine (92). An interesting point to note is the ratio of atropisomers observed in the biaryl coupling is similar to the ratio of the two atropisomeric alkaloids that occur in the plants. The low atropisomerselectivity in the above synthesis of ancistrocladine (1) prompted a study into a more efficient process (ref. 58).The helical lactones 88 and 89 interconvert quickly (Ti/2 < 1 min) and represent thermodynamic ratio favouring atropisomer 88. This dictates the ratio of atropisomers obtained in the reduction step to stable isomers 90 and 91. Since this ratio cannot be improved upon to give either atropisomer selectively and unstable lactones 88 and 89 are difficult to separate, a less planar ether bridge was utilised in the hope that epimerisation would occur more slowly. OMeOMe
OMeOMe 56
NaOH PTC
79
100%
Pd{PPh3)2Cl2 61%
ratb 1.5:1 separate
SCHEME 12: Atropisomer selective synthesis of 1 and 92 by thermodynamic isomerisation. The biaryl precursor 92 was obtained by coupling naphthalene 56 and isoquinoline 79 under conditions used previously (Scheme 12). Palladium(II) catalysed coupling then afforded the helical isomers (P)-93 and (M)-94 regioselectively. These ethers now isomerise more slowly (T1/2 ~ lOh) than their lactone counterparts and can be separated to provide atropisomerically pure 93 and 94 in a ratio of 1:1.5 favouring the ancistrocladine (1) type (M)-helical isomer 94. Evidence for the assignment of the major isomer initially arose from iR NMR analysis (ref. 57). The chemical shifts of the protons at C-4 and the methyl at C-3 are greatly influenced by the anisotropic ring current of the naphthalene substituent. In 94 Ha and the C-3 methyl group protons are shielded by the
424
naphthaleneringabove the plane while the He proton is deshielded when compared to the shifts for the same protons in compound 92 (Figure 4). Conversely, Ha and the C-3 methyl group in isomer 93 are now deshielded while He is shielded. 3.12
M N,
n
Bn
H OO 1.89
1.78
^ 0.73
n
M
o QQ
Bn
94
'
1.11
1.29
93
FIGURE 4: iH NMR chemical shift differences in compounds 92, 93 and 94. Compound 93 can be converted into hamatine (92) by reductive cleavage of the ether bridge with dilithio biphenyl followed by catalytic hydrogenolysis on the A'^-benzyl group. Re-epimerisation of 93 in solution at room temperature then gives the same thermodynamic ratio from which 94 is separated and can converted into 1. Thus, a chirally economical synthesis of each atropisomer is achieved. OMeOMe
93
Hg/Pd-C
Li/biphenyl
92 (+)-Hamatine
55% OMeMe OMe OMe
94
Hp/Pd-C
Li/biphenyl
(-)-Ancistrocladine (1)
OMeMe
Utilising a similar strategy, a total synthesis of the c/^-configured tetrahydroisoquinoline alkaloid (+)-ancistrocline (3) was then achieved by the same research group (ref. 59). The synthesis of the required isoquinoline 95 portion was first attempted from the known dihydroisoquinoline 85 (Scheme 13). C/^-selective reduction proceeded to give the tetrahydroisoquinoline 96 in high de (>95%) however attempted mono-demethylation gave a complex mixture due to the instability of the cis-configured tetrahydroisoquinoline 96 towards epimerisation at C-1 and oxidation (ref. 55). Therefore, a second route involving demethylation prior toring-closurewas utilised. Optically pure isoquinoline 84 was subjected to monoselective
425
ether cleavage to provide phenol 97. Bischler-Napieralski ring-closure gave dihydroisoquinolines 98 and 99 in a ratio of 33:67 respectively and stereoselective reduction (ref. 55) of isomer 99 followed by A^-methylation gives the required tetrahydroisoquinoline 95. MeO,
,Me
NaBH4
MeO.
OMeMe 85 MeO-
.Me NHAc OMe 84
MeO-
.Me
HO
OH Me 98 OMeOMe Pd(ll)
NEta, DMAP OMeOMe
^-
COGI 60
OMe Me 100
Br
OMeOMe
OMeOMe
LiAIH4
CH2OH Me
1. BrGH2CH2Br PhaP 2. LiAIH4
»>
,NMe OMeMe SCHEME 13: Synthesis of (+)-ancistrocline (3), The rest of the synthesis is described only briefly in the original communication (ref. 59) and no yields are quoted. Coupling of the isoquinoline with naphthoyl chloride 60 gives the ester 100 which undergoes intramolecular Pd(II) catalysed coupling to give the lactone 101. Reduction followed by deoxygenation and then provided (+)-ancistrocline (3).
426 OMeOMe
OM eOMe
ffSl
X^
^^'^^<^Me
1 MeO ^ J N ^ ^ ^ \
> ^
^^, M e
1 ^ ^ v ^ Me
M eCX
UvsJi^^NH OMeMe
rV
V
NHAc
OMe 102
2
OMeOMe
OAc
MgBrO
^
M e O y ^ ^ J ^ [f OMeN OMe 104
105 SCHEME 14
OAc
I OMe
The first asymmetric intermolecular synthesis of 0-methylancistrocladine (2) involved the use of a Meyers biaryl coupling, successfully utilised in the earlier synthesis of dehydroancistrocladine (66), to construct the biaryl linkage atropisomer-selectively (ref. 60, 61). It was envisaged that 2 could arise from the acetamide 102 which in turn could be derived from biaryl 103 (Scheme 14). A coupling between the Grignard reagent 104 and chiral oxazoline 105 could then provide 103 stereospecifically. This approach required the synthesis of chiral oxazoline 105 which is outlined in Scheme 15 and begins with the nitrite 71 available in three steps from 1,5diacetoxynaphthalene (see Scheme 8). OMeOMe
OMeOMe
OMeOMe f-BuOK CN
OMe
Et30BF4
^BuOH
CONH2
OEt
CICH2GH2CI
OMe NH2BF4
100%
71
107 OH
reflux
H2N\^p^
109 81% 105
I
OMe
© 0 OH
i-!!!El H 2 N v ^
Ph
"OH
^OMe
108
109
SCHEME 15 Partial hydrolysis of nitrite 71 gave the amide 106 which was treated with triethyloxonium tetrafluoroborate (Meerwein's reagent) to give the imino ether salt 107 (Scheme 15). This salt was
427
not isolated but simply allowed to react with the chiral amino alcohol 109 (ref. 62), derived from commercially available diol 108, in hot 1,2-dichloroethane to give the oxazoline 105. This one pot protocol was far superior to the acid chloride/ amide/ SOCI2 ring-closure route used previously. With the chiral oxazoline now in hand, the asymmetric biaryl coupUng reaction was investigated. Treatment of bromide 110 with 1,3-propanediol and acid gave the ketal 111 which was converted into the Grignard reagent 104 (Scheme 16). Addition of a solution of 105 in THF followed by heating provided the biaryls (AT)-103 and (P> 112 in a ratio of 45:20 respectively which were easily separated by chromatography. The debromo compound 113 and demethylated oxazoUne 114 were also isolated along with the desired biaryl products. It appears that magnesium bromide generated in the reaction facilitates demethylation of oxazoline 105 however the resultant naphthol 114 is easily recycled by remethylation. The stereochemistry of the major atropisomer 103 was later confirmed by its conversion 0-methylancistrocladine (2) (see below).
MeO^^CHO
p,30^^
Br O - ^ M e o y ^ ^ j
MgBrO^ UeO^J^J
Mg, THF trace I2, A
OMe 111 OMeOMe
105 THF reflux
^ MeQ
X)ro°^ OMe 103
M;P ratio 69:31 65% yield
112
OMeOMe NaH,THF 105 100% 114
OMe
SCHEME 16 Acidic hydrolysis of the acetal group in 103 was attempted but this also lead to slow ringopening of the oxazoline. Quatemisation of the oxazoUne 103 with methyl iodide followed by base hydrolysis was also problematic, giving only the amide 115 which resisted further hydrolysis under these conditions due to the sterically hindered carbonyl group. However, treatment of the quatemized oxazoline or amide 115 with KOH in wet DMSO provided the desired acid which was
428
then converted into the methyl ester 116. Reduction of with lithium aluminium hydride then provided the alcohol 117. OMeOMe
OMe OMe Me
103
OH
1. Mel, MeNOo 2. KOH. MeOH 99%
KOH, DMSO OMe
MeO.
2. Mel, K2CO3
115
MeO^
LiAIH4
c:
116 R=C02Me 17 R = CH20H 92%
The corresponding hydrolysis of the iodide salt derived from biaryl 112 also gave the atropisomeric ester 118 which was reduced to alcohol 119. Although each of the alcohols were optically active, the high temperature required in the base hydrolysis step may have caused some rotation about the biaryl axis. For example, the biaryl acid 120 undergoes racemisation at 25°C in chloroform with a half life of 220 min (ref. 63) while the binaphthyl 121 is thermally stable with no racemisation observed after 5 h heating at 140°C in a sealed tube in sodium hydroxide solution (ref. 64). The rates of racemisation of biaryl acids in aqueous sodium hydroxide solution are known to be greater than those in an organic solvent (ref. 65). Therefore, it was deemed necessary at this stage to verify the optical purity of alcohols 117 and 119. OMeOMe
112
OMeOMe
1. Mel, MeN02
^-
2. KOH, DMSO 3. Mel, K2CO3 96%
HO2C
120
121
The method chosen was that of Johnson and coworkers (ref. 66) which involves the use of a chiral oxazaphospholidine-2-thione as a chiral derivatising agent for ^ip NMR analysis of
429 enantiomeric excess. The chloride 122, derived from thiophosphoryl chloride and (-)-ephedrine hydrochloride (ref. 67), possesses an asymmetric phosphorous atom and when allowed to react with alkoxides, displacement occurs at phosphorous with inversion of configuration. The resulting derivatives can then be examined by ^H and ^^P NMR spectroscopy to determine diastereoisomeric purity. Treatment of the sodium salt derived from biaryl alcohol 117 with the chloride 122 gave the oxazaphospholidine derivative 123. Similarly, the biaryl alcohol 119 was converted into the derivative 124. The racemic biaryl alcohol 125 was also synthesised and derivatised by a similar route starting with achiral oxazoline 67 (Scheme 17). OMe OMe Q Me CI H2N-v^Ph HO-^Me
Me
Me S, N-Y^^^
PSCI3 NEta
^
0-a I
NaH. 117
-^v J w CI b - ^ M e
*
b-^Me
MeO
122
(-)-Ephedrine OMeOMe NaH 119
122
MeO
OMeOMe THF, reflux ^^. OMe N ^
1. Mel, MeCN
MgBrO'^ ' ^ ^ ^ V ' ^ ^ 0 ^
67 I OMe OMeOMe
87%
>< ' MeO,. ^ ^ Y - V 0 : : \ ^ ^ OMe 126
104
OMeOMe
NaH MeO,
LiAIH4
122
MeO.
'-^125R = GH20H 99%
SCHEME 17
2. KOH, 100X DMSO, HpO 3.Mel,K2C03 94%
430
Treatment of the oxazoline 67 with the Grignard reagent 104 gave the biaryl 126 in excellent yield (Scheme 17). Quaternisation followed by base hydrolysis in wet DMSO and subsequent methylation gave ester 127 which was reduced to the racemic biaryl alcohol 125. Treatment of 125 with sodium hydride followed by the chiral phosphorous reagent 122 then gave the derivative 128 as a mixture of diastereoisomers and the ^H and ^^P NMR spectra were then examined. The proton decoupled ^Ip NMR spectrum of 128 exhibited signals at 5 84.10 and 84.27 for the two diastereoisomers, whilst the 300 MHz iH NMR spectrum exhibited two doublets at 5 2.63 and 2.67 (7 = 12.4 Hz) assigned to the A^-methyl protons, and two singlets at 5 4.69 and 4.70, assigned to the acetal protons. The oxazaphospholidine-2-thione 123 derived from the alcohol 117 showed only the signals at 5 84.28 in the 3lp NMR spectrum and signals at 5 2.63 and 4.69 in the ^H NMR spectrum, while the derivative 124, which was obtained from the enantiomeric alcohol 119 showed only the other signals. This analysis firmly established the optical purity of the alcohols 117 and 119. OMeOMe
OMeOMe Ql_l MeO,
^ o2-\
LMsCI, pyr.
Me MeQ
2. LiAIH4 3. H3O+
EtNO,
CHO
81%
95%
OMeOMe
OMeOMe
Me
LiAIH4 MeO,
NO2
POCI3 Me
MeO,
98%
MeCN, reflux
NHR OMe AC2O
OMeOMe
Me
133 R = Ac 88%
RaneyNi Me
,,
OMeMe 134
132 R = H
OMeOMe
MeO
^,,
^-
AcOH, NaOAc
naphthalene reflux
MeO
27% from 133 OMeMe 129
SCHEME 17: Synthesis of (+)-0-methyldehydroancistrocladine.
431
To confirm the configuration about the biaryl linkage of the major atropisomer 103 obtained in the biaryl coupling, the derived alcohol 117 was first converted into (+)-0-methyldehydroancistrocladine (129), a degradation product of (-)-ancistrocladine (1) (ref. 2) (Scheme 17). Mesylation of 117 followed by hydride reduction and acid workup gave the aldehyde 130 which did not undergo any racemisation in boiling benzene for 5 h. Henry reaction yield the crystalline nitrostyrene 131 which was reduced to the amphetamine diastereoisomers 132 with lithium aluminium hydride in boiling THF. Acetylation then gave acetate 133 which smoothly underwent Bischler-Napieralski ring closure to the dihydroisoquinoline 134. Dehydrogenation finally furnished (+)-0-methyldehydroancistrocladine (129) which was identical in its properties, including sign and magnitude of optical rotation, with that obtained by degradation of (-)ancistrocladine (1). The amine mixture 132 was then converted into 0-methylancistrocline (2) as detailed in Scheme 18. Resolution of the diastereoisomeric mixture of amines was achieved using the 'directed resolution' method described by Helmchen and Nill (ref. 68). This involves initial conversion into (S, R) and (R, /?)-4-hydroxy-3-phenyl-A^-(l-phenylethyl)butanamides which can easily be separated by flash chromatography (ref. 69, 70). Amine 132 was allowed to react with (+)-(/?)-4,5-dihydro-4phenylfuran-2(3^-one 135 in refluxing toluene in the presence of 2-hydroxypyridine to give the butanamides 136 and 137 which were separated by chromatography. On the basis of the Helmchen analysis (ref. 67), the hydroxyamide 136 was expected to be eluted last and this proved to be the case since this diastereoisomer was converted into 2. Acid hydrolysis of 136 followed by acetylation provides amide 102. Ring-closure then gave dihydroisoquinoline 138 which was stereoselectively reduced to compound 2 under conditions described earlier. O
OMeOMe
OMeOMe 1. IMH2SO4
135 MeO.
MeO Toluene reflux separate
OMe
OMeOMe
OMeOMe
136R = H;R' = Me28% 137R = Me;R' = H27% OMeOMe
POOL MeO-
Me
NHAc
AIMea .Me
MeO
LiAIH4 -78°G
MeO.
^>K.^N OMe Me 138
SCHEME 18: Synthesis of (+)-0-methylancistrocladine (2)
432
OMeOMe
.Me S^NH
or
OMeMe 139 OMeOMe
137
RMe
MeO,
OMeMe 140 OMeOMe
1M H2SO4 MeO-
MeO
The alcohol 119 derived from the minor product of the biaryl coupling was then converted into (+)-0-methylhamatine (92) (ref. 71) as well as the enantiomer 140 of 0-methylancistrocladine (2). Hydrolysis of the butanamide 137 gives the amine 141 which was converted into (-)-Omethylhamatine. This successful intermolecular approach was next applied in first total synthesis of the 5,r-linked (-)-ancistrocladinine (143) (ref. 21, 72). It was envisaged that deprotection and Bischler-Napieralski ring closure of the amide 144 would yield the dihydroisoquinoline 143, and the amide 144 was available from a coupling between the oxazoline 105 and the Grignard reagent 145 (Scheme 19). OMeOMe OMeOMe
OMeOMe -Ph OMeN.^ 105 ''''—OMe
^xo +
MgBr
BnO
OMe 145
SCHEME 19
433
CHO
LBnBr, K2CO3
BnO.
Mg, THF
2. propane-1,3-diol pTsOH
OMe
Then 105 reflux
99%
146 OMeOMe
OMeOMe 1.Mel then KOH, DMSO
BnO.
BnQ
Tir^°'^
148
P:M ratio 23:77 64% yield
OMeOMe
2. Mel, K2CO3 3. LiAIH4 58%
^^
OMe 149 OMeOMe
1. ACgO, pyr. 7 steps
2. POCI3, 3. NaOH 71%
OMeOMe
OMeOMe
OMeOMe
SCHEME 20: Asymmetric synthesis of (-)-ancistrocladinine (143) The synthetic route began with the synthesis of the bromide 147 by benzylation and ketalisation of the known aldehyde 146 (Scheme 20). Generation of the Grignard reagent followed by addition of chiral oxazoline 105 afforded the biaryls 148 and 149 in 64% yield and a ratio of 77:23 respectively along with 22% of the demethylated oxazoline 114. Quaterisation, base hydrolysis and reduction then gave the alcohol 150 which was converted into the acetamide 144 by
434
an analogous sequence to that described in Schemes 17 and 18 above. Debenzylation provided phenol 151 which upon exposure to phosphorous oxychloride in acetonitrile gave only an intractable gum. However, acetylation followed by ring-closure smoothly gave a dihydroisoquinoline and treatment with base provided ancistrocladinine (143), which was further characterised by conversion to the known diacetate 152. Since ancistrocladinine (143) has been converted into ancistrocladine (1) by reduction, this also constituted a formal total synthesis of 1. OMeOMe OMeOMe
''-OMe OMe
MgBr
OMe
OMe
OMe 156
157
154R^ = Me R2
158
159
155R^=OBn R^
160
161
153R^=Me R2=
|—(
J
TABLE 1 Products
Yield (%) Yield (%) Ratio M:P of 114
d.e.
Reaction Time
156:157
77
16
4
65
20 15
42:58
103:112
69:31
38
5
158:159
72
19
70:30
40
6
149:148
64
22
77:23
54
20
160:161
69
28
88:12
76
20
The factors that govern the stereoselectivity in this type of coupling reaction were next investigated using the chiral oxazoline 105 and variety of 2,6-disubstituted aryl Grignard reagents (ref. 21, 72). The bromides 153,154 and 155 were converted into their corresponding Grignard reagents and allowed to react with the oxazoline 105 and the results, along with earlier findings, are detailed in Table 1. The stereochemistry of the products followed from their conversion to known intermediates and the demethylated oxazoline 144 was isolated from all the reactions
435
conducted. For the six-membered acetals, it was the (M)-atropisomer that predominated and the diastereomeric excesses are higher for the bulky dimethyldioxanes than for the unsubstituted ones.
O^p. ^Ph -MeOMgBr L^"-J •
MeQ
A*
157
Br
For the five-membered acetal, the (P)-atropisomer was the major product which indicated that the coupling is proceeding \ia transition state such as A* (ref. 20, 72) in which the fivemembered acetal competes more effectively with the methoxy group for chelation to magnesium atom. Meyers concurrently found that these reactions proceeded under chelation control (ref. 20, 26) and the selectivity of the couplings could be increased by changing one ortho substituent to a non-chelating group. OMeOMe Br MeO^ A .
^CHpOTBS 2 ^ 1 DO
l^g
BrCHgCHgBr OMe
OMeOMe
OMeOMe
M MeO
N-^'""LPr N ^pr
CHgOTBS
+
MeO
163 OMeN^ 162 '"'APr
yW.P ratio 92:8 76% yield
OMe 154
TBSOCH, MeO
-MeOMgBr
~\
/
/ -
SCHEME 21
^
164
OMe
436
OMe OMe
164
NHAc
1.TFA, H2O/THF
aq. NaOH » •
2. Ac^O. pyr.
^^Q
THF 55°C
80%
CH2OTBS
MeO,
98% OMe 166
OMeOMe
OMeOMe 1.TBAF 2. Dess-Martin reagent
1.TBSCI, NEt3 then DIBAL-H 2.MsCI.NEt3 MeO, 3. LiBEtgH
CH2OTBS
930/^
MeO-
63% OMe 167
OMe 130
SCHEME 22: Stereoselective formal synthesis of 0-methylancistrocladine (2). The fact that the outcome in these coupling reactions is dependant on chelation control was further demonstrated in a recent formal synthesis of 0-methylancistrocladine (2) (ref. 73) (Scheme 21 and 22). A coupling between the simpler chiral oxazoline 162 obtained from the amide 106 and (5)-valinol and the Grignard reagent derived from the bromide 163 gave a much improved diastereoselectivity favouring the (M)-atropisomer 164. This selectivity can be explained by considering the transition state B* in which the magnesium atom is chelated to the methoxy group rather than the CH2OTBS substituent (ref. 20, 26, 73). Acid induced rearrangement of the major atropisomer 164 followed by acetylation gave the ester 165 and hydrolysis afforded acid 166 (Scheme 22). One pot silylation followed by reduction and deoxygenation then afforded ether 167. Deprotection and oxidation of 167 then yielded the 0-methylancistrocladine (2) intermediate 130 which was identical to the material prepared by the earlier route (ref. 61). OBn OMe OBn OMe
OBn OMe
Mg BrCHpGHpBr
OBn
P:M ratio 91:9 70% yield
OBn
437
The stereocontrolled construction of the biaryl linkage required for the synthesis of dioncophylline C (7) was achieved by a coupling between the oxazoline 168 and the Grignard reagent derived from the bromide 169 (ref. 74). The desired (P)-atropisomer was obtained in high diastereoisomeric excess since there was only one substituent present on the aryl Grignard reagent available for chelation to magnesium. Proof of the stereochemical outcome of the coupling was obtained from a single crystal Xray structure obtained for the methiodide salt derived from the major isomer. The promising results obtained for the above couplings lead to the development of a more convergent intermolecular synthetic strategy to naphthylisoquinoline alkaloids. This proposal was successfully implemented in a total synthesis of O-methylancistrocline (170) (ref. 75) (Scheme 23). The key step involved an intermolecular coupling between the chiral oxazoline 162 and the Grignard reagent 171 derived from an optically pure tetrahydroisoquinoline. Under chelation control, this coupling should proceed through the intermediate C* to provide the desired atropisomer 172. The synthesis of the bromoisoquinoline 173 required was easily achieved as shown in Scheme 23 but there were initial problems in generating the Grignard reagent. These were overcome by the use of 1,2-dibromotetrafluoroethane as an entrainer and the coupling reaction proceeded smoothly to give the biaryls 172 and 174 in a 82:18 ratio as deduced by ^H NMR spectroscopy. The mixture of biaryls was then treated with trifluoroacetic acid and then acetylated to give the acetamides 175 and 176 which were separated by silica gel chromatography. Completion of the synthesis of 0-methylancistrocline (170) was achieved by LiAlH4 reduction of atropisomer 176 followed by deoxygenation via a halogen/oxygen exchange-Zn reduction protocol. The synthetic material was identical to a semi-synthetic sample prepared from the related alkaloid ancistrocladinine (143). Furthermore, the absolute configuration about the biaryl linkage was confirmed by comparison of the calculated and experimental CD spectra. The NMR spectrum obtained for 170 also compares well with that reported for the Ancistrocladus alkaloid ancistrocladonine (ref. 76), the stereochemistry of which has not be elucidated, however no extensive comparisons have been made as yet. OMeOMe
OMe OMe
MeQ MeO.
438
MeO,
LCICOgMe NaHCOa
Mg BrCF2CF2Br
MeO,
2. LiAIH4 3. Br2 55%
'APr
1.TFA, H2O/THF MeO,
MeO.
2. AC2O, pyr.
^.
OMeMe 172
OMeMe 174 OMeOMe
OMe OMe
NHAc MeO,
NHAc MeO,
P:M ratio 16:84 32% overall yield from 162
OMeOMe
1.LiAIH4 2. MegSiCI/Nal then Zn/HOAc
OMeMe ^ ^^ ^'^
50%
OMeOMe
MeO,
OMeMe 170
OMeMe 143
SCHEME 23: Synthesis of (+)-0-methylancistrocline (170) 4.2 Asymmetric Synthesis: 7,1' Linked Naphthylisoquinoline Alkaloids The first asymmetric synthesis of a 7,1' linked alkaloid was that of (-)-dioncophylline A (8) (triphyophylline) which resulted in the revision of the initially proposed stereostructure (ref. 77). The synthesis utilised the intramolecular approach that was successful for the synthesis of racemic 8 (see Scheme 7) and began with the production of optically pure tetrahydroisoquinoline 79
439 N-N CI HO,
,Me
PTC, NaOH
N.
•^sx
Bn
NH
2. H2, P d - C
OMeMe
89%
79
,Me
BnCI /•Pr2NEt
y
OMeMe
HBr
N.. Bn
Y
reflux
81%
OMeMe
177
60
..Me
(Ph3P)2PdCl2
DMAP
-N-Bn
Bn
NEt3
OH Me 178
»
DMA, NaOAc 130°C
Me
90%
75%
179 O
1 . C2Br2Cl4 PPh3 2 . LiAIH4
^-
3. H2, Pd-C 83%
1 . C2Br2Cl4 PPh3 * •
2 . LiAIH4
3. H2, Pd-C 82%
^.-^ .Me
(^
v^NH XJ^ OH Me
Me
184
MeO MeO
OH
OH
Me
Me
183
SCHEME 24: Asymmetric synthesis of (-)-dioncophylline A (8) (Scheme 24). Reductive deoxygenation of the derived tetrazole provided 177 which was Nbenzylated and demethylated with hot aqueous HBr to give tetrahydroisoquinoline 178. Esterification with acid chloride 60 gave ester 179 which smoothly underwent intramolecular coupling under Pd(II) catalysis to give the axially prostereogenic lactone 180. Reduction of 180
440
gave the stable atropisomeric alcohols 181 and 182 which were easily separated by silica gel chromatography. The arrangement of the NH and OH groups in atropisomer 182 apparently lowers the polarity of this isomer relative to isomer 181, allowing for easy separation. Subsequent deoxygenation of atropisomer 182 followed by debenzylation gave compound 183 which was found to be significantly different to the natural product. Similar treatment of atropisomer 181 provided (+)-dioncophylline A (184) which was identical to the natural material except for sign of optical rotation. The total synthesis of natural (-)-dioncophylline A (8) was ultimately achieved by using the enantiomer of 178.
Red-AI -60°to-40°C
MeO'
AIMea, THF rt2min
185
then Red-AI -60°to -40°C 2 min, then 25°C"
OH
187
Me 95% de
The low atropisomer selectivity obtained in the reduction of prostereogenic lactone 180 with LiAlH4 (59:41) prompted an investigation into atropdiastereoselective ring opening of the enantiomeric lactone 185 with achiral hydrogen nucleophiles (ref. 78) in the hope that internal asymmetric induction could provide higher selectivities. When lactone 185 was added to an excess of sodium bis(2-methoxyethoxy)aluminium hydride (Red-AI) alcohol 186 was obtained with an improved de of 87%. Conversely, reduction with the Lewis acidic reagent diisobutylaluminium hydride (DIBAL-H) gave the atropisomer 187, the precursor to (-)-dioncophylline A, in good de (71%) while pretreatment with trimethylaluminium followed by reduction with excess Red-AI gives atropisomer 187 in even higher de. The selectivity for the Lewis acidic hydrides can be explained by complexation of the aluminium reagent on the upper side of the lactone forcing attack of the hydride from the lower face as depicted in Figure 5. The intermediate lactolate then opens to an aldehyde that is rapidly reduced before the axial chirality is lost. lAA/NA/WWN/V*
•AAAAA/NAA/W
441 MeO,
Me Bn
CHoCHO
MeQ
60 DMAP
APrOH/HaO pH 4 45%
(Ph3P)2PdCl2 j
DMA, NaOAc 130°C 87% 1.NaOH 2. (COCI)2, NEt3 C02Me
MeOH 5mins 85%
OH Me COaMe
1.LiAIH4 2. Me2S04 MeO PTC/NaOH 3. C2Br2Cl4 MeO PPhg, LiAIH4 4. H2, Pd-C
MeO
OMe Me
194
68%
SCHEME 25: Synthesis of (+)-ancistrocladisine (4) The first total synthesis of the 7,1' linked (+)-ancistrocladisine (4) was also achieved using similar methodology to that described for dioncophylline A (ref. 35). To ensure that intramolecular coupling would occur regioselectively at the 7,1' position, the naphthalene moiety must be attached to the 8-oxygen position of the tetrahydroisoquinoline therefore compound 189 was required. The synthesis of this segment was accomplished by a regio- and stereoselective Pictet-Spengler condensation of the amphetamine 188 with acetaldehyde at pH 4 providing 189 as the major product (ref. 55). Esterification with naphthoyl chloride 60 then gave ester 190 which underwent intramolecular coupling to provide lactone 191 in high yield. Methanolysis of prostereogenic lactone 191 gave the stable atropisomers 192 and 193 in a 1:1 ratio. Atropisomer 193 was then subjected to reduction, deoxygenation and debenzylation to provide tetrahydroisoquinoline 194 which upon permanganate oxidation afforded (+)-ancistrocladisine (4) in low yield. Although the synthesis was not atropisomer selective at this stage, the undesired atropisomer 192 can be converted back to axially prostereogenic lactone 191 and ring opened again to provide the thermodynamically controlled mixture of isomers. A more efficient process was to simply expose isomer 192 to the NaOMe/MeOH conditions and this caused epimerisation to provide the
442
same equilibrium mixture. The selectivity of this ring-opening was finally greatly improved by kinetically controlled atropisomerselective ring-opening with various oxygen based nucleophiles (ref. 79). This study revealed that even small oxygen nucleophiles can open the lactone stereoselectively under kinetic control with /-propoxide being amongst the most selective (Table 2). Slightly higher selectivity was gained by using (+)-mentholate as the nucleophile however (-)mentholate gave only a modest reversal of atropisomerselectivity.
OH CO2R
Me
196
TABLE 2 RO-/ solvent
Reaction time
Ratio 195:196
NaOMe/MeOH NaOMe/MeOH
15 s 5min
15.3:1 1.3:1
NaHCOs/MeOH
60min
3.3:1
/-PrOK//-PrOH
20.9:1
/-PrOK//-PrOH
10 s 22 h
Li-(+)-mentholate/THF Li-(-)-mentholate/THF
lOmin lOmin
1.1:1 23.2:1 1:1.8
4.2 Asymmetric Synthesis: 5,8' LinkedNaphthylisoquinoline Alkaloids The dimeric naphthylisoquinoline alkaloids michellamines A (6) and B (197), isolated from the rare tropical liana Ancistrodadus korupensis (ref. 80, 81), have evoked great interest due to their cytopathic activity against human immunodeficiency viruses (HIV). These compounds are also interesting due to their high polarity, owing to the presence of 6 free phenolic groups and two secondary amino groups, and the rare 5,8' coupling between the naphthalene and isoquinoline rings. The corresponding monomers, the korupensamines A (198) and B (199), also isolated from Ancistrodadus korupensis (ref. 82), do not exhibit antiviral activity but do possess some antimalarial properties.
443 Me OH
Me OH
OH OMe
HO,
OH OMe
HO,
The first total synthesis of michellamine A (6) was achieved by the biomimetic oxidative dimerization of a derivative of korupensamine A (198) (ref. 83) which itself was synthesised by an intermolecular strategy involving a Stille-type coupling (ref. 84, 85). The total synthesis of both 198 and atropisomer 199 began with production of tetrahydroisoquinoline 201 obtained from optically pure 200 (Scheme 26). Bromination then provided the required tetrahydroisoquinoline segment 202. The synthesis of the naphthalene segment was achieved by the application of a Wittig surrogate to the Stobbe approach (ref. 86, 87). Aldehyde 203 was converted into half ester 204 which underwent ring closure in boiling acetic anhydride. Hydrolysis followed by methylation and reduction gave the naphthalene 205 which was deoxygenated to provide bromide 206. Metalhalogen exchange followed stannylation of the aryl lithium gave stannane 207 which was coupled with the isoquinoline segment under Pd(II) catalysis to provide atropisomers 208 and 209 in low yield. These were not separated at this stage but simply deprotected with boron trichloride followed by hydrogenolysis to provide korupensamines A (198) and B (199) which were separated as previously described (ref. 82). The synthesis of michellamine A (6) from korupensamine A (198) commenced with the A^formylation of 198 with pivalic formic anhydride (Scheme 27). Subsequent treatment with acetyl chloride provided diacetate 210 where the chelated C-5' hydroxy group was not protected. Dimerization was then achieved under conditions described by Laatsch for simple naphthol dimerisation (ref. 88) which involved treatment of with Ag20 in chloroform in the presence of
444
NEt3. The resulting binaphylidendione 211 was then carefully reduced with sodium borohydride and deprotection was effected in a single step with methanolic HCl to provide michellamine A (6).
Me
BnO,
I.BnBr, NaOH
NBn
Me
^ 2. /-PrSNa, DMF 3. BnBr, NaOH
OMeMe 200
r ^COgf-Bu
94%
OBn M e
OBn M e 202
201
/-Pro
APrO CO2H
(EtO)2PO'''^C02Et CHO
Bn
NBn
40%
APrO
Brp, DMF
rn
NaH then TFA
Ft
OMe
1. AC2O reflux 2. NaOEt 4. LiAIH4
57%
APrO 1. Br2C2Cl2, PhgP
f-BuLi
2. LiAIH4
BUgSnCI
^»
202 PdCl2(PPh3)2
89%
86%
OMe
15%
206 APrO
OMe
APrO
OMe
1. BCIo BnO,
Bna
2. H2 Pd-G 3. separation
1.4:1 OBn M e 208 OH
OMe
OBn M e 209 OH
^-
LiCI, CuBr DMF 135°C,40h
OMe
OH Me 199 SCHEME 26: Total synthesis of korupensamines A (198) and B (199).
445 OH
OMe
OH
OMe
Ag20 2. AcCI, EtgN
ACQ
NCHO
90%
0.2% EtaN 85%
OAc Me 210 Me
Me
OAc
OH
OHCN
1. NaBH4
^^
2. MeOH/HGI reflux 67% NCHO OAc Me 211
SCHEME 27: Biomimetic synthesis of michellamine A (6) from korupensamine A (198). Closely following the biomimetic synthesis of michellamine A (6), a report appeared describing the convergent synthesis of michellamine A (6) (ref. 89) (Scheme 28). Due to its higher activity, the synthesis of michellamine B (197) was also undertaken. The approach involved the use of binaphthyl 212 and an intramolecular Suzuki-type coupling (ref. 90), again mediated by palladium. The synthesis of binaphthyl 212 began with the Diels-Alder reaction between the quinone 213 and diene 214 which gave the naphthoquinone 215 in good yield. Copper-bronze mediated dimerization of 215 followed by reductive acetylation provided binaphthyl 216 which was selectively deacetylated and converted to the desired triflate 212. The required tetrahydroisoquinoline 217 portion was prepared from bromide 202 by lithiation and treatment with B(0Me)3 followed by aqueous work-up. Coupling of 212 with 217 in the presence of palladium(O) gave the quateraryl 218 as a mixture of atropisomers. Removal of all the protecting groups gave michellamine A (6), B (197) as a mixture which was separated as reported previously (ref 80, 81). Although a mixture of michellamine A (6) and B (197) can be equilibrated to produce a mixture of 6,197 and the corresponding M/M atropisomer named michellamine C (219), none of this material was detected in the coupling reaction mixture. The question as to why 219 does not appear to co-occur naturally and was not detected synthetically is most intriguing and is yet to be answered.
446
O
OTMS
O
1. Cu(bronze) DMF, Pd(PPh3)4
68%
214
213
OMe
OAc OAc OMe
1. DBU Me CH2Cl2/MeOH
^-
216
OAc
2. TfgO, GH2CI2 2,6-lutidine 79% Me
Pd(PPh3)4
OMe OAc OTf 212
OBn
\P/M'OAc OMe
OMe OAc
I
I
II
1H2,Pd-C ^
6
2.MeOH,HCI
^
o-'/o
197
OBn Me 217 74%
OBn Me 218
SCHEME 28: Convergent synthesis of michellamines A (6), B (197). Another synthesis of michellamines A (6) and B (197) as well as the unnatural michellamine C (229) was reported by Hoye and co-workers in 1994 (ref. 91). The approach also involved a Suzuki-type, intermolecular biaryl coupling followed by a biomimetic phenolic oxidative coupling effected by Ag20. The sequence commenced with the synthesis of the naphthalene portion 220 as shown in Scheme 29. Simultaneous generation of a benzyne and dienolate from dibromide 221 and amide 222 with lithium cyclohexylisopropylamide and subsequent cycloaddition provided the naphthalene 223. Although the yield was low, his annulation procedure compared well with longer alternative sequences. Methylation followed by halogen-lithium exchange and treatment with trimethylborate then gave boronic acid 220. The isoquinoline half 224 was synthesised from compound 201 by regiospecific iodination and this was coupled with naphthalene 220 to provide the atropisomers 225 and 226 after acid hydrolysis of the MOM ether. Silver induced dimerisation gave a mixture of the purple cross-ring quinones 227 which upon treatment with hydrogen over 10% Pd/C resulted in reduction of the quinones and simultaneous debenzylation to provide michellamines A (6), B (197) and C (219). HPLC separation of a portion of this mixture provided pure (6) as well as a 2:1 mixture of (197) and (219).
447
OMOM
OMOM
,Br
h
'I
LICA (2.2 eq)
MOMO
NEt2 LiO"
1.
^Me
20%
Me Me 222 MOMO
223
pMe BnO.
1. Me2S04
Me
BnO I2, Ag2S04
^-
NBn
2. n-BuLi 3. B(0Me)3 4. NH4GI, aq.HGI
66%
^
OBn Me
^(0^)2 220
201
OH
OMe
OH
OMe
1.220Pd(PPh3)4 2. HCIMeOH
OH
Ag20 g^Q
1^
CH2CI2
BnO
53%
-100% -4:3 ratio
Me
Me
OBn
O
OMe
Hp
OH
(6) + (197) +
Pd-C Me BnO^ A l
-100%
^ ^ ^ M e NBn
OBn Me 227
OH
Me
219
SCHEME 29: Total synthesis of michellamines A-C. Hoye and co-workers (ref. 92) later reported the total synthesis of ancistrobrevine B (228), isolated from Ancistrocladus abbreviatus (ref. 93) and korupensamine C (229), isolated from Ancistrocladus korupensis. The approach to these natural products also utilised a benzyne cycloaddition to construct the naphthalene moiety. Thus, treatment of the amide 222 and bromide 230 with lithium cyclohexylisopropylamide furnished naphthalene 231 in low yield (Scheme 30).
448
OMe
OMe LICA (2.2 eq)
OMeOH
NEt2
0' *"°\e
222
21-26%
Br
231 MOMO
OMe
precipitate from GH2CI2 with hexane^
1. Me2S04
^>
2. n-BuLi 3. B(0Me)3 4. NH4CI, aq.HCI
,
I B(0H)2
MOMO
OMe
Me
232
84%
Ar-^-O^^^r
MeO^
>Me i . H B r , A c O H NH OMeMe 86
^"^^
232 Pd(Ph3)4
*-
2. BnBr K2CO3 3.12, Ag2S04
OMeMe
27%
234
sat. NaHCOs PhOHs reflux
71% OMeOMe
OMeOMe
BnO,
HO,
Pd-C 100% HPLC separation
OMeMe 235
OMeMe 228 OMeOMe
OMeOMe
224 + 232
Pd(Ph3)4
^-
sat. NaHC03 PhCH3 reflux
H2 BnO.
Pd-C
76% OBn Me 236
100% HPLC separation
SCHEME 30: Total synthesis of ancistrobrevine B (228) and korupensamine C (229). Methylation and conversion to the boronic acid 232 followed by precipitation from dichloromethane with hexanes yielded the boronic anhydride 233. The isoquinoline portion 234 was synthesized from homochiral 86 by selective demethylation, benzylation and subsequent
449 iodination. Palladium mediated coupling of iodide 234 with either boronic acid 232 or anhydride 233 gave the biaryl 235 as a mixture of atropisomers. Deprotection and chromatographic separation provided ancistrobrevine B (228). Similarly, coupling of boronic acid 232 with iodide 224 gave biaryl 236 as an atropisomeric mixture and deprotection and separation furnished korupensamine C (229). ^if ^ V ^
Mg, CuBr.DMS OMe
82%
MeO
NHTs
Me
>
1. H2, Pd-C
2.Ac.O,NEt3
V
78%
HO,
Me
2. BBrg
OH Me
NOOsEt OBn Me 245
BnO^
1.LiAIH4 2.l2.Ag2S04 75%
1.TESCI, NEtg
*-
2. GIC02Et 3. TBAF 4. BnBr, KgCOg 63%
244 Me
''''''" OMe 242
;NH2Br
92%
BnO,
Me
MeO^
1.Na/NH3
OMe 241
100%
240
POCI3
K^
fX^Me TsN 239
7^
Me T I OBn Me
232 Pd(Ph3)4
^-
NMe
238
sat. NaHC03 PhCH3 reflux 73%
OH OMe
OH
OMe
1. HOI, MeOH
^-
HO
2. H2, Pd-C 92%
OBnMe 246
OH Me 247
OH Me 237
SCHEME 31: Synthesis of m^korupensamine D (237). The total synthesis of enf-korupensamine D (237) followed a similar methodology and began with a new sequence for the preparation of optically pure tetrahydroisoquinoline (238) (ref. 94) (Scheme 31). Copper catalysed ring-opening of (/?)-aziridine 239 with the Grignard reagent derived from chloride 240 provided the amphetamine derivative 241. Reductive removal of the A^tosyl group and acetylation gave acetamide 242 which was cyclised to dihydroisoquinoline 243. Stereoselective hydrogenation followed by demethylation then gave the salt 244. A selective A'protection and benzylation sequence yielded carbamate 245 which was converted into the tetrahydroisoquinoline fragment 238 by reduction and regioselective iodination. Palladium catalysed coupling of 238 with the boronic acid 232 gave a mixture of atropisomers 246 which
450
upon acid hydrolysis, MPLC separation and hydrogenolysis furnished the enantiomer of korupensamine D (237) and the corresponding atropisomer (247). MeOMeO
MeOMeO
OMe Me 1.LiAIH4 Me
Me
12 steps
MeO^Js^^cOgMe
2. PCC
OMeOMe
OMeOMe
1. (EtO)2POCH2C02Et NaH, benzene
LMsCI, NEtg
>. MeO-
2. DIBAL-H 3. (+)-DIPT, Ti(0APr)4 f-BuOOH 53% from 249
^-
OH
MeO
2. LiAIH4 91%
OMe 251
ratio-1:1 + atropisomer
OMeOMe 1.(PhO)2PON3 PPha, DEAD
^-
2. H2, Pd-G A2O, EtOAc 74%
OMeOMe
1. POCI3
MeO, NHAc
^.
MeO.
2. AJMea LiAiH4, -78°C 70%
OMeMe 248 SCHEME 32: Synthesis of 0,0,0-trimethylkorupensamine A (248).
The ether derivatives 0,0,0-trimethylkorupensamine A (248) and B have both been synthesised by a route which commenced with a lengthy sequence to the biaryl 249 from 3,5dimethylanisole (ref. 95) (Scheme 32). Reduction of 249 with LiAlH4 and oxidation gave aldehyde 250 which upon Wadsworth-Emmons-Horner extension, reduction and Sharpless asymmetric epoxidation provided epoxide 251 and the corresponding atropisomer in almost equal amounts which were separated by silica gel chromatography. The derived alcohol 252, obtained by mesylation of 251 and in situ reduction, was then converted into the acetamide 253 by displacement with azide under Mitsunobu conditions followed by reduction and acetylation. Ring closure followed by stereoselective reduction then yielded 0,0,0-trimethylkorupensamine A (248). The synthesis of 0,0,(9-trimethylkorupensamine B was accomplished in a similar manner using the atropisomer of 251 obtained in the epoxidation step.
451 OMe
OH LPhNMegBrg
OBn O M e
OMe
^-
2. BuLi, B(0/-Pr)3 aq. NH4CI
2. DBU, CH2CI2 3. Bu4NBr3
Me 255
68%
62%
256 OBn O M e
202 Pd(PPh3)4
OBn O M e
^.
OgHgNHBrg
Ba(0H)2. aq. DME 85°C 37h
BnO.
BnO,
82%
3:2 OBn M e 258
OBn OMe
W 11 Sr^^Me BnO^^
A^^^^vy^Me
OBn OMe BK
+
^^^jA^^NBn OBn Me
26%
n-BuLi — 259 R = Br 2-MeTHF -95°C B(0APr)3 '-*-261 R = B(OH)2 7 1 %
BnO^
TiVS ULJLM nr ^^ Me 1^
M
ksJi^^MBn OBn Me
^8%
1.Pd(PPh3)4 K3PO4, DMF 90°C 16h
^-
197
2. HCO2H MeOH, Pd-C then NH4OH 74%
260
SCHEME 33: Stereospecific synthesis of michellamine B (197) A stereospecific synthesis of the highly active michellamine B (197) has also been achieved by a palladium catalysed cross coupling between derivatives of korupensamines A (198) and B (199) (ref. 96). The route is based an adaptation of previous syntheses and commences with a new sequence to provide the naphthalene fragment 254 (Scheme 33). Bromination of tetralone 255 and subsequent dehydrobromination and /7ara-bromination provided naphthol 256. Benzylation and lithiation followed by boronation gave the boronic acid 254 which was coupled with optically pure bromide 202 to give the atropisomers 257 and 258. To assign the stereochemistry about the biaryl axis, each of these compounds was converted into korupensamines A (198) and B (199). Bromination of the mixture of 257 and 258 provided the 6'-bromonaphthalenes 259 and 260 in low yield and compound 259 was converted into the boronic acid derivative 261 by a slight modification of the standard condition which involved lithium-halogen exchange at low temperature (-95°C) followed by addition of B(0/-Pr)3 and aq. NH4CI. Palladium catalysed coupling of bromide 260 with boronic acid 261 under nonaqueous conditions and subsequent debenzylation gave michellamine B (197) in excellent yield.
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This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
457
DNA - Damaging Natural Products with Potential Anticancer Activity A.A. Leslie Gunatilaka and David G.I. Kingston Contents 1. General Introduction 2. DNA-Damaging Agents and Cancer Chemotherapy 3. Detection of DNA-Damaging Agents 3.1. Introduction 3.2. Mechanism-based Yeast Bioassays 4. General Methodology 4.1 Introduction 4.2. Extraction 4.3. Bioassay 4.4. Bioactivity-guided Fractionation 5. Natural Products with DNA-Damaging Activity 5.1. Introduction 5.2. Sesquiterpenes of Bryophytes 5.2.1. Porella cordeana 5.2.2. Frullania nisquellensis 5.2.3. Chiloscyphus rivularis 5.3. Diterpenoids 5.3.2. Rosane Diterpenoids of Vellozia Candida 5.3.2. Diterpene Dibenzoates of Caesalpinia pulcherrima 5.4. Steroids 5.4.1. Steroids of Pseudobersama mossambicensis 5.4.2. Structure-Activity Relationship Studies of 7-Hydroxy Steroids 5.5. Alkaloids 5.5.1. Dioxoaporphines of Artabotrys zeylanicus 5.5.2. Oxoaporphines of Xylopia aethiopica and Miliusa banacea 5.5.3. Azafluorenone Alkaloid from Cyathocalyx zeylanica 5.5.4. Piperidine Alkaloids from Cassia leptoptiylla 5.5.5. Guanidine Alkaloids from Pterogyne nitons 5.5.6. Steroidal Alkaloids from Solanum unrtbellifemm 5.6. Marine Diterpenoids from Eunicea toumeforti 5.7. Limonoids from Tricliilia emetica 5.8. Bloactive O-Heterocyclic Compounds 5.8.1. Furanonaphthoquinones from Crescentia cujete 5.8.2. Pterocarpans from EryttJiina burana 5.8.3. Coumarins from Sri Lankan Rutaceae 6. Summary and Conclusions 7. Acknowledgements 8. References
458
1.
General Introduction Cancer is a dreadful disease which has no geographic, national, religious or
other boundaries. It accounts for a vast number of deaths and suffering. Each year 6.5 million people are diagnosed with cancer worldwide [1]. In the United States alone, more than ten million people are living with a history of cancer and in excess of one million new cancer cases develop annually [2], and the death rate due to cancer has slightly increased over the past three decades. This may be attributed to the increasing longevity of the American population and the relationship between cancer and environmental factors, in particular cigarette smoking and Increased exposure to carcinogens. As with any other disease, solutions to the cancer problem involve both prevention and treatment, and advances on these two fronts require the continued development of novel and improved chemopreventive and chemotherapeutic agents. Natural products and their semisynthetic analogs have provided some of the most effective and widely used of the established chemotherapeutic agents for cancer. This is exemplified by such compounds as Taxol® (1), the vinca alkaloids vinblastine (2) and vincrinstine (3), the podophyllotoxin analogs etoposide (4) and teniposide (5), the camptothecin analogs topotecan (6) and CPT-11 (7) (Fig. 1), and the microbial products bleomycin (8), mitomycin C (9), and the anthracyclines adriamycin (10) and daunorubicin (11) (Fig. 2). It is noteworthy that these anticancer agents are all so complex that they would never have emerged from a synthetic program alone or from a combinatorial approach to drug discovery.
Thus the natural products approach to
discovery and development of new anticancer drugs is attractive in that it is complementary to synthetic and biosynthetic approaches. This review is concerned with our search for naturally occurring DNA damaging agents with potential anticancer activity as a part of a National Cooperative Drug Discovery Group funded by the U.S. National Cancer Institute and conducted in collaboration with the University of Virginia and SmithKline Beecham Pharmaceuticals. Many international collaborators from Brazil, Ethiopia, Guam, the Philippines, and Sri Lanka participated in this program directly or indirectly by providing extracts for bioassay and by working on the bioactive extracts. An essential step in an efficient natural product drug discovery program is the judicious selection of an initial screening strategy. The in vitro methods available for anticancer drug discovery can be grouped into two major categories. These are the
459 disease-oriented
approach and the mechanism-based
approach.
The disease-
oriented approach involves cellular assays in which one looks for general cytotoxic agents or agents which inhibit growth of cancer cells. Tumor cell lines utilized by the U.S. National Cancer Institute belong to this category.
Such bioassays are most
powerful when they are used, as in the N.C.I, program, to discover leads with unusual profiles of activity [3], but the implementation of a panel of multiple cell lines is laborintensive and thus costly. Compounds which show non-selective cytotoxicity do not turn out to have useful in vivo activity; thus recent compilations of "anticancer" natural products [4 - 6] contain many examples of compounds with no documented in vivo activity.
COzMe
(1) Taxol (2)
R = CHO (Vincristine)
(3)
R = CH3 (Vinblastine)
\OH (4) R = Me
(Etoposide)
(5) R = ^ ^ ^ - J
fTeniposide)
(6) Ri = OH ; R2 = CH2N(CH3)2 1 ?• (Topotecan) R3 = H (CPT-11) R2 = H ; R3 = CH3CH2
Fig. 1. Some clinically used natural product-based anticancer drugs of plant origin Thanks recombinant
to recent
developments
in genetics,
DNA technology, the last decade
has
molecular
witnessed
biology,
and
the evolution of
anticancer natural product drug discovery research from an empirical
search for
460
general cytotoxic agents to a more mechanism-based approach [7].
Unlike the
disease-oriented approaches involving mammalian cell-lines, the mechanism-based approaches utilize molecular assays involving DNA, oncogenic enzymes or their receptors [8,9].
0^55^NH2
(9) Mitomycin C o
(8) Bleomycin
NH2 (10) R = CH2OH Adriamycin (11) R = CH3 Daunorubicin
Fig. 2. Some clinically used natural product-based anticancer drugs of microbial origin 2.
DNA-Damaging Agents and Cancer Chemotherapy One of the paradoxes of the cancer problem concerns the reciprocal behavior
of the drugs used for its treatment; many of these drugs themselves are capable of producing cancer. Accumulated biochemical or pharmacological evidence suggests that DNA Is the principal cell target for many clinical antitumor agents.
Those
categories of antitumor agents which are known to interact with DNA include alkylating agents, antitumor antibiotics, some specific mitotic inhibitors, and miscellaneous drugs such as c/s-platinum complexes. Antimetabolites and steroidal hormones are two categories of antitumor drugs which exert their activity without Interacting directly with DNA. An ideal anticancer agent should exhibit high specificity and selectivity of action. There are various rational approaches to achieve these objectives of which the most promising has been the use of bifunctional or polyfunctional DNA-binding.
461 According to M. S. Waring "There are grounds for cautious optimism that future advances in our understanding of the molecular basis of drug-DNA interaction may lead to significant improvements in cancer treatment" [10]. However, until this promise is fulfilled there will still be a need for empirical approaches to drug discovery such as that described below.
3.
Detection of DNA-Damaging Agents 3.1. Introduction DNA, the biochemical repository of the genetic inheritance of all cells, is
subject to damage by a variety of chemicals and by radiation. Important DNA lesions include (a) alkylation of bases and/or phosphate residues, (b) inter- and intra-strand cross-linking, (c) single-strand breaks, (d) double-strand breaks, and (e) DNA-protein cross-links. lesion.
Anticancer agents are known to produce more than one type of DNA
Genetically engineered micro-organisms may be used as probes to detect
agents which interact with DNA.
The most
popular
genetically
engineered
microorganisms are yeasts and bacteria, and yeast is preferred in our work because of its closer genetic and biochemical resemblance to mammalian cells. What is the principle behind the use of genetically engineered yeasts to detect potential anticancer agents? An important and exploitable feature of many tumor cells is that they have defects in their ability to repair damage to DNA as compared with normal cells, suggesting that agents with selective toxicity towards DNA repair-deficient cells might be potential anticancer agents [11]. This rationale is strongly supported by the fact that DNA repair-deficient yeast mutants have been demonstrated to be hypersensitive to most known DNA-damaging agents [12], and yeast mutants can be developed that are hypersensitive to inhibitors of the enzyme topoisomerase I [11]. Presented in Fig. 3 are the principles underlying the use of DNA repair-deficient yeast mutants for the detection of DNA-damaging agents. 3.2 Mechanism-based Yeast Bioassays A recently developed mechanism-based approach for anticancer natural product drug discovery utilizes DNA repair- or recombination-deficient mutants of the
462 yeast Saccharomyces cerevisiae [12]. In our search for potential anticancer agents from natural sources we have employed this mechanism-based screen which depends on differential responses of DNA repair-deficient and repair-proficient yeast strains to the sample under investigation [13,14].
Normal Cells
DAMAGED DNA
Repair Y A Tumor Cells
^ ^
DNA
Agents with selective toxicity towards DNA repair-deficient cells
I DEFECTIVE IN DNA REPAIR DETECTION OF DNA-DAMAGING AGENTS
I -
Yeast Cells
.
Hypersensitive to most DNAdamaging agents
Fig. 3. Principles underlying the use of DNA repair-deficient yeast mutants In the detection of DNA-damaging agents Three major DNA repair pathways have been defined in yeast and these are known as the rad3, rad6 and rad52 pathways [15]. The rad3 pathway is associated with excision repair, the rad6 pathway is the error-prone repair pathway and rad52 is recombinational pathway associated with repair of double-strand breaks and melotic recombination. The principles underlying the production of these yeast mutants are depicted in Fig. 4 and are briefly described here. The cell wall of yeast is impermeable to many drugs.
Therefore, at first a mutation to make the cell wall of yeast more
permeable to drugs is effected.
Starting with this "wild-type" RAD+ (or RS 188N)
mutant, yeast mutants lacking all three known DNA repair pathways have been produced [12]. They are the rad3 mutant which lacks the excision repair pathway; the rad6 lacking the error-prone repair pathway and the rad52 lacking the DNA doublestrand break repair and meiotic recombination pathway. A fourth mutant is also known and it has been produced by the deletion of the topoisomerase I gene from the rad52 mutant and Is labeled rad52.top1. Hypersensitivity to topoisomerase II inhibitors likely occurs due to increase in topoisomerase II expression and/or activity to compensate for deletion of topoisomerase I. Thus, the differential sensitivity observed with rad52 and rad52.top1 can be used to detect the presence of both topoisomerase topoisomerase II Inhibitors.
I and
463
Repair-deficient mutation UV Radiation
Excision repairdeficient Rad3 mutant
^
Permeability mutation
Error-prone repair-deficient
^
\Rad6 mutant
>H
Rad+ mutant ("Wild-type" strain)
Yeast {Saccharomyces cerevisiae)
Repair- and recombinationdeficient mutation
|
Double-strand break repair- and meiotic recombinationdeficient Rad52 mutant
Double-strand break repair- and meiotic recombinationand topoisomerase Ideficient
in v
Deletion of topoisomerase I gene
Rad52.topl mutant Fig. 4. A diagrammatic representation of the production of yeast mutants capable of detecting DNA-damaging agents and topoisomerase inhibitors
General Methodology 4.1 Introduction During the past decade scientific methodologies for the discovery and study of bioactive natural products have changed dramatically for several reasons.
These
reasons have been enumerated by Cordell in a recent review [16] and include technological advances, novel molecules of substantial interest, changing ethical principles for organism collection and heightened awareness of the chemical and biological potential of the tropical rain forests. According to Cordell the areas in which strategies for the conduct of natural product chemistry have changed include plant selection and collection,
isolation
techniques,
structure
elucidation,
biological
evaluation, semlsynthesis, dereplication, and biosynthesis. The methodologies
involved in our search for DNA-damaging
natural
products with potential anticancer activity include extraction, bioassay, bloactivity-guided fractionation, dereplication, structure elucidation and structure-activity relationship (SAR) studies.
In this section the general methodologies used by us for extraction,
mechanism-based
yeast bioassay
and
bloactivity-guided fractionation
will
be
464 discussed.
Important aspects of structure elucidation and SAR studies will be
presented under each compound or class of compounds. 4.2. Extraction Extraction of shade-dried plant material is carried out with inert solvents under mild conditions to avoid any chemical transformations and/or decompositions that may occur otherwise. Ideally 50-100 g of plant material is allowed to soak three times in methyl ethyl ketone (MEK), followed by methanol. Extracts obtained with each solvent are separately combined and evaporated yielding two extracts. These are dried, weighed and subjected to our mechanism-based yeast bioassays. 4.3. Bioassay We have found that the use of the three yeast strains RAD+, rad52, and rad52.top1 provides the most reliable measure of bioactivity for topoisomerase inhibitors and other DNA-damaging agents. Fig. 5 depicts a cartoon representation of the basis of our mechanism-based yeast bioassay.
The assay is carried out by
introducing the test sample (crude extract, fraction or pure compound) dissolved in a 1:1 v/v mixture of methanol-dimethyl sulfoxide to a 100 fxL well in agar plates, separately impregnated with normal "wild-type" RAD+ yeast cells and with mutant yeast cells (rad52 and rad52.top1) and incubating the plates for 48 hrs at 30°C. If the test sample contains a DNA-damaging agent or a topisomerase inhibitor then it would inhibit the growth of one or more mutant strains producing a zone of inhibition i.e. a clear zone where growth of yeast cells had not occurred. The results are expressed as IC12 values which represent the concentration of the test sample in |ig/mL required to produce an inhibition zone of 12 mm diameter. An active lead should have an IC12 in the wild-type RAD+ strain equal to or greater than three times the IC12 in one of the mutant strains. For topoisomerase I inhibitors, the IC12 value in rad52.top1 should be at least threefold larger than that in rad52, while for topoisomerase II inhibitors the reverse should be true. For crude extracts doses ranging from 8000 ^ig/mL to 1000 M^g/mL are used whereas lower doses are required for fractions and for pure compounds, depending on the activity of the active compound and its concentration in the extract/fraction. Four or five different doses are used for each sample; knowing the dose and the zone of inhibition produced for each dose the IC12 values can be calculated by the application of regression analysis. The activity of yeast In each plate is tested by the use of a known
465 anticancer drug (positive control). For RAD+ and rad52 camptothecin (12) is used as the positive control and for rad6 streptonigrin (13) is recommended (Fig. 6). Table 1 shows the zones of inhibition and IC12 values for these standards in our commonly used yeast strains. Damaged DNA
Damaged DNA
DNA
^
, Repair pathways present I Normal yeast cells e.g. Rad+
Repair pathways absent I
Mutant yeast cells e.g. Rad6, Rad52,\ RadSl.topl Inhibition of growth
Agar plate + yeast cells
Normal growth
48h incubation
^
48 h incubation
100 \k\ well containing DNA damaging agent Fig. 5. Basis of mechanism-based yeast bioassay for the detection of DNA-damaging agents
HsCOy^ ^
CxX9^° (12)
Camptothecin
0 (13) Streptonigrin
Fig. 6. Positive controls used in yeast bioassay
466 TABLE 1. Response of yeast mutant strains to positive control drugs, camptothecin (12) and streptonigrin (13).
Drug
Conc./ICi2 (uff/mL)
Camptothecin (12)
Zone of inhibition RS167N RS321N RS322N RS322YK (rad6) (RS321) (rad52) (rad52Y) 21±3
5.0
21±3 16±3 110
200.0 8.7
ICl2 Streptonigrin (13)
4.0 8.0 ICi2
RS188N (RAD+)
0.6 20±4
20±3 2.4
0.4
4.4. Bioactivity-guided Fractionation Isolation of potential anticancer compounds from bioactive extracts involves bioactivity-guided fractionation. The DNA-damaging natural products encountered in our studies were extracted by MEK and/or methanol, and the general methodology which we have employed in our bioassay-directed fractionation of these extracts is schematically presented in Fig. 7.
These fractionations involved solvent-solvent
partition, Sephadex LH-20 gel filtration, normal phase and reversed-phase (RP) column, preparative thin-layer and high pressure liquid chromatography (HPLC). Silica gel chromatography was employed only if bioactive compounds were found to be stable under these mildly acidic conditions. During the bioactivity-guided fractionation of an MEK extract, it was first partitioned between hexane and 80% aqueous methanol. Water was then added to the 80% aqueous methanol fraction to give 60% aqueous methanol and this was extracted thoroughly with chloroform.
If the bioactive compounds were not extracted with
chloroform the aqueous layer was subsesquently extracted with ethyl acetate. If a methanol extract was found to be active it was first subjected to an n-butanol/water partition. The n-butanol layer, which usually contained the active compound(s), was then evaporated and treated in a manner similar to that used for an MEK extract described above.
467
ME K Extract
1
Butanol
1
MeOH Extract
1
1 1 1 Water
— _l_l
Hexane
80% ^\queous Methanol Add water to make it into 60%Aqueous ]Vlethanol and Extract with Chloroform
1
Chloroform
1
Sephadex LH-20 Gel Permeation - • Chromatography
Column Chromatography (RP) )|f Prep.TLC(RP) ^ ^
60% Aqueous Methanol
HPLC (RP)
Fig. 7. Fractionation scheme used for bioactive extracts
5.
Natural Products with DNA-Damaging Activity 5.1 introduction During our searcii for natural products with DNA-damaging activity utilizing
mechanism-based
yeast assays
we have investigated over 7,500 extracts of
bryophytes, higher plants and marine organisms collected mainly in Brazil, Ethiopia, Guam, Phillipines, Sri Lanka, mainland U.S.A. and the Virgin Islands.
Those
organisms which showed positive response to our DNA-damaging activity assays and from which we have isolated bioactive compound(s) are listed in Table 2 along with the families to which they belong and the class(es) of bioactive compounds encountered. Some important aspects of the structure elucidation of these bioactive compounds, along with their bioactivities and the structure-activity relationships (SAR) of some natural and synthetic analogs, will be discussed compound(s) encountered.
below under each class
of
468 TABLE 2.
Organisms exhibiting positive response to DNA-damaging yeast bioassay and the classes of compounds encountered in them. Family
JEecies
name
Class of compound(s)
Bryophytes Porellaceae Jubulaceae Geocalycaceae
Porella cordeana Frullania nisquallensis Chiloscyphus rivularis
Sesquiterpenes Sesquiterpenes Sesquiterpenes
Higher Plants Annonaceae
Artahotrys zeylanicus
Dioxoaporphine alkaloids Oxoaporphine alkaloids Azafluorenone alkaloid Oxoaporphine alkaloids Oxoaporphine alkaloids Furano- and Furofuranonaphthoquinones Pterocarpans Diterpene dibenzoates Piperidine alkaloids Guanidine alkaloids
Bignoniaceae Fabaceae Leguminasae Meliaceae Rutaceae Solanaceae Velloziaceae Marine Organism Gorgonian
Cyathocalyx zeylanica Miliusa banacea Xylopia aethiopica Crescentia cujete Erythrina burana Caesalpinia pulcherrima Cassia leptophylla Pterogyne nitens Pseudobersama mossambicensis Trichilia emetica Pleiospermium alatum Solatium umbelliferum Vellozia Candida Eunicea tourneforti
Steroids Limonoids Coumarins Steroidal alkaloids Diterpenoids Diterpenoids
5.2 Sesquiterpenes of Bryophytes Bryophytes, although less evolved than the higher plants, constitute one of the largest groups of land plants, accounting for about 20,000 species.
Taxonomically
bryophytes are placed between algae and pteridophytes and divided into 3 classes; Musci (mosses,
14,000 species), Hepaticae (liverworts, 6000 species),
and
Anthocerotae (hornworts, 300 species). The class Hepaticae is characterized by the presence of oil bodies which are easily extracted with organic solvents. Although a number of bryophytes find applications In traditional medicines of China, Europe, and North America, very little chemistry of these is known due to the difficulty of collecting
469 sufficient quantities of uncontaminated plant material. Recent studies have provided evidence that bryophytes elaborate a variety of secondary metabolites, some which are of biological and chemotaxonomic importance [17-19]. The first report of/>? vivo antitumor activity of a bryophyte appeared in 1986 in a review of results of screening of bryophytes in the National Cancer Institute's antitumor screening program by Spjut, Suffness, Cragg and Norris [20]. Subsequent studies, however, led to the conclusion that this bryophyte species,
Claopodium
crispifolium, was contaminated with a microorganism which contained maytansinoids, a well known class of antitumor macrolides [21]. Since the inception of our program in 1990, we have screened several hundred extracts of bryophytes for potential anticancer activity in our mechanism-based yeast bioassay. Out of about 200 bryophytes screened a total of 27 samples had IC12 values in the range of 4000-8000 [ig/mL, 22 had IC12 values in the 1000-4000 |ig/mL range, and only 3 yielded extracts with IC12 values less than 1000 jig/mL. Regrettably, many of the species which showed bioactivity in the preliminary screening failed to reconfimn activity on recollection. Problems associated with recollecting bryophytes for bioassay have been reviewed by Spjut, Kingston and Cassady [22]. Out of several hundred species of bryophytes screened only three species gave consistent results in our bioassay and the extracts derived from these were fractionated in order to isolate constituent(s)
responsible
for their
bioactivity.
Coincidentally the bioactive compounds encountered in all three species were found to be sesquiterpenes. 5.2.1. Porella cordeana Bioactivity-directed fractionations of the bioactive MEK extract of Porella cordeana (Hueb.) Moore (Porellaceae) collected in Oregon, USA, involving liquid-liquid partition, gel filtrations on Sephadex LH-20 and silica gel chromatography afforded five sesquiterpenoids, namely drimenin (14), 7-ketoisodrimenin (15), 7-ketoisodrimenin-5ene (16), aristolone (17) and norpinguisanolide
(18). The structures of all four
sesquiterpenoids were determined with the aid of spectroscopic data. This study also involved the complete assignment of ^^C-NMR spectra for drimenin (14) and aristolone (17) for the first time. Of these only drimenin (14) and aristolone (17) were found to be
470 toxic towards DNA repair-deficient mutants of Saccharomyces cesevisiae, with IC12 values of 200 and 390 ^ig/mL for the racl52 (RS322YK) strain [23].
Both
sesquiterpenoids were shown to be inactive towards rad6 (RS167N) and RAD+ (RS188N) strains.
Drimenin (14) was also tested in P-388 murine leukemia and
Chinese Hamster Ovary cytotoxicity (CHOC) assays and were found to be inactive. Bioactivity data of P. cordeana extract and its constituent sesquiterpenoids presented in Table 3.
(16)
(15)
TABLE 3. Bioactivity data of Porella cordeana extract and sesquiterpenoids. Extract/ Compound RS322YK (rad52Y) MEK Extract Drimerin (14) 7-Ketodrimenin (15) 7-Ketodrimenin-5-ene (16) Aristolone (17) Norpinguisanolide (18)
^Results expressed as IC12 (^ig/mL)
1641 200 >1000 >1000 390 >1000
Yeast Strain^ RS167N (rad6) 2438 >1000 >1000 >1000 >1000 >1000
RS188N (KAD+) 2475 >1000 >1000 >1000 >1000 >1000
are
471 5.2.2. Frullania nisquellensis The liverwort genus Frullania of the bryophyte family Jubulaceae has about 1000 described species [24] which have been subjected to extensive chemotaxonomic investigations [24-27] and are reported to be rich sources of sesquiterpene lactones some of which are known to cause allergic contact dermatitis [19]. An MEK extract of Frullania nisquallensis Sull. which showed activity in our yeast bioassay afforded the DNA-damaging sesquiterpene costunolide (19) on bioassay-guided fractionation. Costunolide showed moderate but selective activity with IC12 values of 50, 150, and 330 |ng/mL in the mutant yeast strains RS321N, RS322YK (rad52Y), and RS167N (rad6), respectively; it was inactive (IC12 > 1000 |ig/mL) in the "wild-type" strain RS188N (RAD+) [28]. Costunolide (19), when tested in a cytotoxicity assay employing the A-549 human lung carcinoma cell line, showed an IC50 of 12 ^xg/mL.
It is noteworthy that the
costunolide derivative 8a-angeloyloxy-costunolide (20) has been reported to possess potent cytotoxic activity in a number of human cancer cell lines [29].
(19) R = H
I
(20) R= ^""yS
OMe MeO
(22) During the fractionation of the bioactive MEK extract of F. nisquallensis two inactive compounds, the sesquiterpene (-)-frullanolide (21) and the tridepside tenuiorin (22) were also encountered. (-)-Frullanolide has previously been isolated from the
472
same plant species as the major allergic compound causing contact dermatitis [30]. Although tenuiorin (22) has been isolated from a lichen [31], it has not been reported previously from a Frullania species. Structures of all compounds have been confirmed by means of ^H-, ^^C-NMR, DEPT, HETCOR and HMBC spectra.
^^C-NMR spectral
assignments of tenuiorin (22) have been made for the first time. The bioactivity of costunolide (19) in both the DNA-damaging yeast bioassay and the cytotoxicity assay has led to the suggestion that it acted by interaction with DNA in some manner.
The lack of DNA-damaging activity of the structurally related
(-)-frullanolide (21) has led to the conclusion that the focus of activity in 19 is not simply the a,p-unsaturated lactone moiety as suspected in many other cytotoxicity studies with a-methylene-y-lactones [32]. 5.2.3. Chiloscyphus rivularis
(23) R = OH
(25) R = OH
(24) R = H
(26) R = H
(27)
pH
(30)
(28) The MEK extract of Chiloscyphus
rivularis
of
the
bryophyte
family
Geocalycaceae showed weak but reproducible activity in rad52 DNA repair-deficient yeast strain. Bioactivity-guided fractionation utilizing solvent-solvent partition resulted in concentration of bioactivity in the chloroform fraction.
Further fractionation of this
involving centrifugal partition chromatography (CPC), Silica gel column chromatography and preparative TLC afforded a moderately bioactive and new
sesquiterpene,
473
12-hydroxychiloscyphone (23) [33]. Several new and inactive sesquiterpenes were also isolated and characterized as 12-hydroxychloscyph-2,7-dione (25), chiloscyph-2,7dione (26), chiloscyph-2,7,9-trione (27), and rivulalactone (28).
The structure and
stereochemistry of rivulalactone (28), with the novel trinorchiloscyphane carbon skeleton, was confirmed by spectroscopic methods as well as semi-synthesis starting from chiloscypone (24) (see Scheme 1) [34]. It is possible that the biosynthesis of rivulalactone follows a similar sequence of reactions. Three known sesquiterpenes were
also
encountered
and were
identified
as
chiloscyphone
(24)
and
4-
hydroxyoppositan-7-one (29), and isointermediol (30).
(a)
^-
1
1
/
(b)
(28)
(24)
Reagents: (a) Os04/A/-methylmorpholine-A/-oxlde/acetone:H20 (8:1)/-10to -20°C/30 min; (b) m-CPBA/CH2Cl2/25oC/70 min.
Scheme 1. Semisynthesis of rivulalactone (28) from chiloscyphone (24). 12-Hydroxychiloscyphone
(23)
showed
moderate
but
selective
DNA-
damaging activity in the yeast-based assay with IC12 of 75 in 88 jug/mL in the rad52Y (RS 322YK) and rad52 (RS 321) assays and >1,000 jiig/mL in the RAD+ (RS 188N) assay. It also exhibited cytotoxicity to human lung carcinoma cell-line A-549 with an IC50 of 1.95 M-g/mL These bioactivity data are summarized in Table 4.
TABLE 4. Bioactivity data for 12-hydroxychiloscyphone (23) from Chiloscyphus
Yeast strain^ rad52
RS321
rivularis.
Cell line'^ jRAD+
88 75 >1,000 ^Results expressed as IC12 (M>g/mL) values ^Hiiman carcinoma ceU line; result expressed as IC50 (|Lig/mL) value
A-549 1.95
474
5.3. Diterpenoids 5.3.1. Rosane Diterpenoids of Vellozia Candida Plants belonging to the family Vellozlaceae occur predominantly in South America and Africa. They are interesting as they grow in rocky regions on dry soil, and in spite of their apparent fragility they show a surprising longevity. In our search for natural product-based DNA-damaging agents we have screened 20 extracts derived from 15 Brazilian species of this plant family. Eight of these extracts showed weak to moderate activity in our mechanism-based bioassay.
Vellozia Candida Mikan which
showed reproducible activity was selected for further study. Bioassay-guided fractionation of the bioactive alcoholic extracts of V. Candida involving solvent-solvent partition, Sephadex LH-20 gel filtration. Si gel column chromatography and reversed-phase HPLC as appropriate afforded a novel moderately bioactive 6,7-seco-rosane diterpenoid, candidalactone (31) [35]. The gross structure of candidalactone (31), Including relative stereochemistry, was determined by the application of ^H-,^^C-NMR, DEFT, COSY-45, HETCOR and NOESY spectroscopy. Another new but inactive diterpenoid, candidenoidiol, was also isolated and its structure was elucidated as 10S,11R-dihydroxy-5(6),15-rosanedien-7-one (32) with the help of ^H-,^^C-NMR, DEPT, COSY-45, HETCOR. HMBC and ORD spectroscopic techniques. HOaa.
(32) Candidalactone
(31) exhibited moderate
but selective
activity in our
mechanism-based yeast bioassay for DNA-damaging agents with the following IC12 values: RS 322YK (racf52;, 87 |Lig/mL; RS 188N (RAD*) >200 ^ig/mL. Considering the moderate bioactivity shown by the crude extracts it has been concluded that candidalactone represented the major bioactive compound in the crude extracts of V. Candida [35].
475
5.3.2. Diterpene Dibenzoates of Caesalpinia pulchem'ma In their search for DNA-damaging natural products with potential anticancer activity, the SmithKline Beecham group has recently reported the isolation of four novel diterpene dibenzoates, pulcherrimins A - D (33 - 36) from Caesalpinia pulcherrima (L) Swartz. [36]. Roots of C. pulcherrima, a tropical tree of the Fabaceae family collected in Mexico were sequentially extracted with ethyl acetate and methanol. The latter extract, which demonstrated selective activity in a DNA repair-deficient yeast mutant, was fractionated by RP chromatography followed by preparative TLC and HPLC to afford pulcherrimins A - D (35 - 38) of which only pulcherrimins A - C were selectively active in racl52 and rad52.top1 yeast mutant strains.
It was suggested that the pattern of
bioactivity exhibited is similar to that seen with inhibitors of topoisomerase II such as amscarine, ellipticine or etoposide. Pulcherrimins A and C were tested for cytotoxicity in Vero cells in culture and were found to have weak activity.
The bioactivity data for
pulcherrimins A - D are given in Table 5.
[ OH CO2H
""CH3
(33) R = OH (35) R = H
'CHs
(36) R = OAc
COC6H5
^OCOCeHs COCeHs
(34)
C02H
COCeHs
TABLE 5. Bioactivity data for pulcherrimins A - D (33 - 36). Compound
Pulcherrimin Pulcherrimin Pulcherrimin Pulcherrimin
A B C D
(33) (34) (35) (36)
radSl
Yeast strain^ rad52.topl
RAD+
Cell lineb Vero cells
>100 66 >100 >100
16 0.64 4.4 >100
88 >100 >100 >100
66 NTc 59 NTc
^Results expressed as IC12 (jig/mL) values Wero monkey cell line; results expressed as IC50 (|Lig/mL) values CNot tested
476
5.4. Steroids 5.4.1. Bioactive Steroids from Pseudobersama mossambicensis
Pseudobersama
mossambicensis
(Sim) Verde, a plant of the family
Meliaceae collected in Kenya, was subjected to our conventional extraction. The MEK extract was found to exhibit significant activity in the yeast-based bioassay employing the rad52 strain. Bioactivity-guided fractionation resulted in the isolation of three ergost5-ene-3p,7a-diol derivatives which were identified as ergost-5,24(28)-diene-3p,7a-diol (37), 24,28-epoxyergost-5-ene-3p,7a-diol (38), and ergost-5-ene-3p,7a,24,28-tetraol (39), with the aid of spectroscopic data [14]. Ergosta-5,24(28)-diene-3p,7a-diol (37) has been isolated simultaneously from another meliaceous plant [37] and comparison (TLC, RP-HPLC, ^H-NMR) of our sterol with an authentic sample confirmed its identity. The occurrence of ergost-5-ene-3p,7a,24,28-tetraol (39) as a diasteroisomeric mixture suggested that it may be an artefact arising from 24,8-epoxyergost-5-ene-3p,7a-diol (38) during the isolation process, most probably on silica gel chromatography.
(37) R =
(38)
(39)
..-r^ R ="-f^
The bioactivity data for Pseudobersama sterols are given in Table 6. All three sterols exhibited selective activity on the rad52 strain compared with the wild-type RAD+ strain.
Ergost-5,24(28)-diene-3p,7a-diol (37) was also tested in P-388 murine
lymphocytic leukemia assay and was found to be moderately cytotoxic.
477
TABLE 6. Bioactivity data for Pseudobersama sterols 37 - 39. Compound
Cell linet'
Yeast strain^ rad52 (RS322YK)
radS (RS167N)
RAD+ (RS188N)
P-388
8.0 0.4 1.0 0.6 0.4
12.0 6.0
> 1,500 >50
NIC
NTc 8.7 2.4
-
Ergost-5,24(28)-diene-3p,7a-diol(37) 24,28-Epoxyergost-5-ene-3P,7a-diol(38) Ergost-5-ene-3P,7oc,24,28-tetraol (39) Camptothecin (12) Streptonigrin (13)
4.3 NTc
^Results expressed as IC12 (^ig/mL) values bWild-type P-388 murine leukemia cell line; result expressed as IC50 (M^M) value CNot tested 5.4.2. Stmcture-Activity Relationship (SAR) Studies of 7-Hydroxy Sterols The DNA-damaging activity of steroidal 5-ene-3p,7a-diol derivatives noted above, their limited supply and the report that cholest-5-ene-3p,7a-diol derivatives are inactive in some cytotoxicity assays whereas their 7p counterparts are active [38] prompted us to undertake SAR studies of some of their analogs.
In addition to the
cytotoxic sterols, ergosta-5,24(28)-diene-3p,7a-diol (37) and 24,28-epoxyergost-5-ene3p,7a-diol (38) encountered in Pseudobersama mossambicensis, the following five 7hydroxy sterol analogs were synthesized starting from the readily available steroid stigmasterol (40); cholest-5-ene-24-one-3p,7a-diol (43), cholest-5-ene-24-one-3p,7pdiol (44), ergosta-5,24(28)-diene-3p,7p-diol (45), and 24,28-epoxyergost-5-ene-3p,7pdiol (46).
In order to investigate the role played by the 7-hydroxy function,
ergosta-5,24(28)-diene-3p-ol (48) and 24(28)-epoxyergost-5-ene-3p-ol (49) were also synthesized and their bioactivity evaluated [39]. Our synthetic strategy Involved the conversion of stigmasterol (40) into the key Intermediate 42 through 41 with the correct side-chain structure and absolute stereochemistry by methodology developed by Djerassi etal. [40]. The key intermediate was then converted to the requisite sterols by employing reactions outlined in Scheme 2.
478
v^OL-
V^i^ Hd-^CU
(T
v-^A-^
(40)
>r^
(41)
0
xtV^ - r f ? P ^ . X ^ (42)
(47)
\
*^'l
HO''^-^'^
\(0,(9)
1 0
riC^
d6c
(43)
/ (^
(37)
(44) 1
\ \(^
(38)
^^iXXy
r^x^
\
^^^^ \(e)
(49)
^^siXJO
HO^ (46)
(45)
Reagents: (a) See. ref. 40; (b) H2/Pd(C)/EtOAc/2h ; (c) Zn(OAc)2/ g. HOAc/120oC/2h; (d) CuBr/PhC020BuVH0Ac/Ar/120OC/0.5h; (e) Ph3P=CH2/DMSO/60°C/1 h; (f) Me2S=CH2/DMSO/0° to 250C/1 h; (g) 5% methanolic KOH/25°C/2h
Scheme 2. Synthetic routes to analogs of Pseudobersama sterols for SAR studies. All of the synthetic sterols were assayed in rad52 DNA-damaging and RAD* bioassays and some selected analogs have also been tested for cytotoxicity to Vero cells; the results are presented in Table 7. All synthetic 7a-hydroxy analogs showed activity in our DNA-damaging bioassay with 37 and 38 having activity comparable to natural sterols isolated from Pseudobersama mossambicensis. 7a-Hydroxy sterols were also active in Vero cell cytotoxicity assay. However, an inverse correlation between activity in the yeast assay and the cytotoxicity assay was noted. Thus, the 7a-hydroxy sterol which was the most active in the DNA-damaging assay was the epoxide 38, with
479 an IC12 value of 0.2 M,g/mL, but was inactive in the Vero cell assay, with an IC50 of >100 [AM. The second most active 7a-hydroxy sterol in the yeast assay was the olefin 37, which showed an IC12 of 7 p.g/mL and this was weakly active in the vero cell assay, with an ICsoOf 58 |iM. The 24-keto analog 44, the least active of the three 7a-hydroxy sterols in the DNA-damaging assay with an IC12 of 14 ^ig/mL, was the most active in the Vero cell culture with an IC50 of 9.9 inM. TABLE 7. Biological activity data for synthetic sterols 37, 38 and 4 3 - 4 9 .
Sterol
Yeast strain^ rad52
Cell line^ Vero cells
7.0 58.0 Ergost-5,24(28)-diene-3p,7a-diol(37) 0.2 >100 24,28-Epoxyergost-5-ene-3p,7cx-diol(38) 14.0 9.9 Cholest-5-ene-24-one-3p,7a-diol (43) >500 21.0 Cholest-5-ene-24-one-3p,7p-diol(44) > 8,000 31.0 Ergosta-5,24(28)-diene-3p,7Miol(45) > 8,000 16.0 24,28-Epoxyergost-5-ene-3P,7P-diol(46) > 8,000 NTc Cholest-5-en-24-one-3p-ol (47) > 8,000 NTc Ergost-5,24(28)-diene-3P-ol (48) > 8,000 NTc 24,28-Epoxyergost-5-ene-3p-ol (49) ^All sterols were inactive against "wild-type" strain KAD+; results expressed as IC12 (M,g/mL) values Wero monkey cell line; results expressed as IC50 (fiM) values CNot tested
In contrast to 7a-hydroxy sterols the 7p-hydroxy analogs 45 and 46 were completely inactive in the yeast assay, but showed moderate cytotoxicity in the Vero cell assay. These results suggested that the bioactivity of 7p-hydroxy sterols may involve a different mechanism of action than their a-counterparts. The sp-hydroxy sterols 47, 48 and 49 were found to be inactive in the yeast assay regardless of their side-chain functionalities and were not tested in the Vero cell assay. The results also indicated the requirement of a hydroxy function at C-7 of the sterol nucleus for biological activity in this class of compounds.
480
5.5. Alkaloids 5.5.1. Dioxoaporphines of Artabotrys zeylanicus As a part of our continuing efforts to discover molecules of biological interest from Sri Lankan flora, we initiated a program of screening Sri Lankan plants of the family Annonaceae for potential anticancer compounds employing the brine shrimp toxicity assay [41]. In our extension of this program carried out in collaboration with Dr. E. M. K. Wijeratne of the Medical Research Institute, Sri Lanka, we replaced this simple bench-top assay [42] with our mechanism-based yeast assay. One such plant which exhibited bioactivity in the latter assay was Artabotrys zeylanicus Hook. f. & Thoms., a plant endemic to Sri Lanka.
Although there is no reported work on A.
zeylanicus, previous investigations of Artabotrys species have resulted in the isolation of aporphine, noraporphine, oxoaporphine, protoberberine, and berberine alkaloids, as well as sesquiterpenes and steroids. OMe
OMe
Chromatographic fractionation of the bioactive chloroform extract of the stem bark of A. zeylanicus guided by the yeast bioassay afforded two bioactive alkaloids [43]. One of these was identified as the oxoaporphine alkaloid, atherospermidine (50) The major bioactive alkaloid was found to be new and was named artabotrine; it was identified
as
the hitherto
unprecedented
A/-methoxylated
alkaloid,
A/-methoxy-
norcepharadione A (51). Structure elucidation of artabotrine involved the use of UV, ^HNMR, ^^C-NMR, HMBC and MS techniques. The ^H-NMR spectrum of artabotrine (51) in the aromatic region consisted of complex overlapping signals, coupling pattern and constants of which were determined by computer simulation experiments employing Varian VNMR software based on the FORTRAN program LAME (also known is LAOCOON) with magnetic equivalence added.
Although the spectroscopic data,
including HMBC NMR, supported the structure 51 proposed for artabotrine, the presence
of an unprecedented
A/-methoxy function could not be
established
481 unequivocally.
Single crystal x-ray analysis was thus undertaken and the three-
dimensional x-ray diffraction data collected at reduced temperature further supported the proposed structure. An ORTEP diagram of artabotrine (51) is shown in Fig. 8. The HREIMS of artabotrine showed significant peaks at miz 305 (C18H11NO4; loss of oxygen from the molecular ion) and at mIz 291 C17H9NO4; loss of formaldehyde from the molecular ion). Scheme 3 depicts the proposed fragmentation pathways to explain the occurrence of these fragment ions in the mass spectrum of artabotrine.
Fig. 8. X-ray structure of artabotrine (51).
M+-30;m/z291
Scheme 3. Proposed mass spectral fragmentation of artabotrine (51). The interesting biological activity exhibited by artabotrine (51) prompted us to acquire as many natural 4,5-dioxoaporphines as possible in order to submit them to our mechanism-based yeast bioassay. Those tested included norcepharadione A (52) [44], norcepharadione B (53) [45], cepharadione A (54) [46], cepharadione B (55) [46], ouregidione (56) [47], compounds 57 [46], 58 [47], and 59 [48]. The bioactivity profile for these natural 4,5-dioxoaporphines along with data for atherospermldine (50) and artabotrine (51) are given in Table 8.
482 R2
R4
R3
(52)
H
H
0--CH2-0
(53) (54)
H Me
H H
OMe OMe O--CH2--O
(55) (56)
Me H
H
OMe
OMe
OMe
OMe
OMe
(57)
Me
H
OMe
(58)
H
H
OH
H
(59)
H
H
OH
OMe
H
TABLE 8. Bioactivity data for atherospermidine (50), and natural 4,5-dioxoaporphine alkaloids 51 - 59. Yeast strain^
Alkaloid
Atherospermidine (50) Artabotrine (51) Norcepharadione A (52) Norcepharadione B (53) Cepharadione A (54) Cepharadione B (55) Ouregidione (56) (57) (58) (59) Camptothecin (12)
Cell lineb
RS322YK (radSlY)
RS321N
RS188N {RAD+)
P-388 (wildtype)
P-388 (camptotheci n -resistant)
1.6 2.16 1.50 >100 7.60 >100 >100 9.30 >100 >100 0.60
27 1.20 0.10 20 4.70 >100 >100 3.50 >100 >100 -
>50 >500 >250 >250 >250 NTc >250 >250
NTc 1.59 NTc
NTC
1.12 NTC
NIC
NTC
NTc NTc
NTC NTC
NTC NTC
NTC NTC
NIC
NTC
NTC
NTc 100
NTC
NTC
0.012
>20
^Results expressed as IC12 (|Lig/mL) values t'Wild-type and camptothecin-resistant P-388 murine leukemia cell line; results expressed as IC50 (M^M) values CNot tested The above bioactivity data, although insufficient to make any firm conclusions, suggest some structural requirements of 4,5-dioxoaporphines for DNA-modifying activity. These are the presence of a methylenedioxy group at C-1 and C-2, and an unsubstituted ring C. Substitution on the amide N , whether it is H or Me, does not seem to have much effect on the bioactivity. It may be possible that the mechanism of action of these alkaloids involves intercalation into DNA.
483 5.5.2. Oxoaporphines of Xylopia aethiopica and Miliusa banacea Oxoaporphines represent another subclass of aporphine alkaloids peculiar to the Annonaceae and a few other alkaloid-bearing plant families.
Oxoaporphines
such as liriodenine (60) and oxoglaucine (64) have been reported to be cytotoxic [48,49]. Although no phytochemical investigations have been reported on Miliusa, a genus consisting of approximately 40 species indigenous to Australia and Indonesia [50], previous work on Xylopia aethiopica (Dunal) A Rich resulted in the isolation of kaurane and kaur-16-ene diterpenes from the fruits and kaur-16-ene, kolavane, and trachylobane-type diterpenes from the stem bark. Bioassay-guided
fractionation
involving liquid-liquid
partition, Sephadex
LH-20 gel filtration chromatography, and Silica gel chromatography of the MEK and methanolic extracts of X. aethiopica led to the isolation of five previously known oxoaphorphine alkaloids. The structures of these oxoaporphinoids were elucidated with the aid of spectroscopic data (UV, ^H-NMR, MS). Three of the alkaloids showed selective activity against rad52 and radd mutant yeast strains [51].
Of these,
oxophoebine (62) and liriodenine (60) showed significant activity whereas lysicamine (63) exhibited weak activity (Table 9). The remaining oxoaporphines, oxoglaucine (64) and 0-methylmoschatoline (65) were found to be inactive.
In addition to its activity
against rad52 and rad6 yeast mutants, oxophoebine (62) exhibited toxicity towards the yeast mutant lacking DNA topoisomerase I repair pathway, namely the. rad52.top.1 mutant.
Ri
(60)
H
O-GH2--O
H
H
(61)
H
O-CH2--O
OMe H
(62)
OMe OMe
OMe O-CHg-O
(63)
H
OMe
OMe H
(64)
H
OMe
OMe OMe OMe
(65) (66)
OMe OMe OMe H H 0~CH2--0 OH
H H H
484
TABLE 9. Bioactivities of Xylopia aethiopica and Miliusa Alkaloid Liriodenine (60) Lauterine (61) Oxophoebine (62) Lysicamine (63) Oxoglaucine (64) 0-Methylmoschatoline (65) lO-Hydroxyliriodenine (66) Camptothecin (12) Streptonigrin (13)
rad52 16.7 214 6.4d 300 >500 >500 295 0.6 0.4
cf. hanacea alkaloids 6 0 - 6 6 .
Yeast strain^ radSl.topl radS NTb 113 2.6e 390 >500 >500 72 >20 <0.4
15.0at500c NTb 4.7 >500 >500 >500 NTb S.7 2.4
RAD+ 500 NTb >500 NTb >500 NTb NTb NTb NTb
^Results expressed as IC12 ([ig/mL) values and are the result of a single determination unless specified otherwise ^Not tested ^Zone of inhibition in mm at the given cone. (^ig/mL) ^Average of 3 determinations ^Average of 2 determinations
The bioactive MEK extract of Miliusa cf. banacea when subjected to bioassayguided fractionation as for X
aethiopica above afforded
10-methoxyliriodenine
(lauterine) (61) and the novel oxoaporphinoid, 10-hydroxyliriodenine (66).
The
structures of 61 and 66 were determined by the usual spectroscopic data, and the structure of 66 was confirmed by conversion into 61 on treatment with diazomethane. Both alkaloids showed selective activity towards rad52 and rad52.top1 mutants. TABLE 10. Topoisomerase 11 unknotting activity of lauterine (61) and 10-hydroxyliriodenine (66).
Alkaloid
Topoisomerase II activity^
Lauterine (61) 25.0 10-Hydroxyliriodenine (66) 12.5 ^Inhibition of topoisomerase II unknotting activity in vitro; results expressed as IC50 (fig/mL)
485
cyathocaline (67) (see Scheme 4) as the only bioactive compound [52]. The structure of cyathocaline has been determined by the application of spectroscopic techniques for the parent alkaloid as well as its diacetyl derivative.
A probable pathway for the
biogenetic conversion of azaanthraquinones, which co-occur with azafluorenones in Annonaceous plants, into azafluorenones has also been proposed (Scheme 4).
Enz.
Azaanthraquinones
Azafluorenones (67) Ri = H ; R2 = OMe (Cyathocaline) Scheme 4. Proposed biogenetic origin of azafluorenones from azaanthraquinones. Cyathocaline exhibited moderate but selective activity in our yeast bioassay and also in a cytotoxicity assay against the A-549 human lung carcinoma cell line (Table 11). Three azafluorenone alkaloids have previously been tested for cytotoxic activity and were found to be inactive [53] but one has been reported to have anticandidal activity [54]. TABLE 11. Bioactivity of cyathocaline (67).
Yeast strain^ RS321N RS167N RS188N RS322YK (rad6) (RAD+) (rad52Y) Not active >400 87.0 90.0 ^Results expressed as IC12 (M^g/ii^L) values t'Human carcinoma cell line A-549; result expressed as IC50 (fiM) value
Cell lineb A-549 8.5±0.07
486 5.5.4. Piperidine Alkaloids from Cassia leptophylla In an effort to uncover anticancer active metabolites in Brazilian plants, in early 1993 we initiated active collaboration with several Brazilian natural products chemists. Out of the large number of extracts brought to our laboratory by Dr. Vanderlan da S. Bolzani and screened in our mechanism-based yeast bioassay, extracts derived from two leguminous plants were selected for further studies.
Based on preliminary
investigation the bioactive methanolic chloroform extract of the leaves of
Cassia
leptophylla which was shown to contain bioactive alkaloldal constituents was further processed for alkaloids.
The alkaloid fraction thus obtained was fractionated by
column chromatography on alumina, followed by Silica gel preparative TLC and reversed-phase TLC to afford seven piperidine alkaloids [55]; (-)-spectaline (68), (-)-spectalinine (69), canavaline (70), leptophyllin A (71), 3-acetylleptophyllin A (72), iso6-canavallne (73), and leptophyllin B (74). Of these, (-)-spectaline (68), (-)-spectalinlne (69), and canavaline (70) showed significant bioactivity in our mechanism-based bioassay utilizing the yeast strains RS 322YK (rad 52) and RS 321N. Leptophyllin B (74) exhibited moderate activity in these two strains whereas iso-6-canavaline (73) showed a positive response only to the RS 188N (RAD*) strain, suggesting it to be an antifungal agent. Cytotoxic activities of (-)-spectaline (68) and (-)-spectalinine (69) were evaluated in Vero monkey and Chinese hamster ovary cell lines and (-)-spectalinine (69) was found to be moderately active In both cytotoxicity assays. The biological activity data for the bioactive Cassia leptophylla alkaloids are summarized in Table 12.
Xi^, R.
H
(68) R = -(CH2)i2COCH3
(71) Ri = H; R2 = -(CH2)ioCH(OH)CH20H
(69) R = -(CH2)i2CH(OH)CH3
(72) R, = Ac; R2 = -(CH2)ioCH(OH)CH20H
(70) R = -(CH2)ioCH(OH)CH3
a,
487
HO.,,, H,C*
H
(73) R = -(CH2)ioCH(OH)CH3 (74) R = -(CH2)ioC02H
TABLE 12. Bioactivity data for piperidine alkaloids from Cassia leptophylla .
Alkaloid RS322YK (radSlY) (-)-Spectaline (68) (-)-Spectalinine (69) Canavaline (70) Iso-6-canavaline (73) Leptophyllin B (74) Camptothecin (12)
15.0 16.0 16.5 >500 >200 0.6
Yeast strain^ RS321N RS188N (RAD^) 17.5 27.0 26.0 >500 157 -
126 >50 >50 123 126 100
Cell lineb Vero CHO
>20 9.9 NTc NTc
15.0 9.7
NTC
NTC NTC NTC
0.019
-
^Results expressed as IC12 (|Lig/mL) values Wero monkey and Chinese hamster ovary cell lines; results expressed as IC50 (M^M) values CNot tested The structures of the piperidine alkaloids 68 - 74 were elucidated by application of FABMS. ^H-NMR, ^^C-NMR, DEPT, DQCOSY. HETCOR and HMBC techniques.
Out of the seven piperidine alkaloids encountered in C. leptophylla,
(-)-spectaline (68), leptophyllin A (71), 3-acetylleptophyllin A (72) and leptophyllin B (74) were found to be new [55]. Although piperidine alkaloids are abundant in nature and many of them are known to exhibit some biological activity [56], C. leptophylla alkaloids were the first to be reported to have DNA-damaging activity.
488
5.5.5. Guanidine Alkaloids from Pterogyne nitens
(75) RT = R3 = H ; R 2 = R 4 =
^^^'^^
(76) Ri = R3 = H ; R2 = R4 = NR1R2 HN==<^ 'NR3R4
(77) R i = R 2 = H;R3 = R4 = (78) Ri = R3 = Me ; R2 = R4 = (79) R i = R 2 = H;R3 = R4=
^ ^ ^ ^ ^
A methanolic chloroform extract of the leaves of the South American legume, Pterogyne nitens TuL, collected in Brazil exhibited a positive response to our yeast bioassay. Bioactivity-guided fractionation of this extract afforded the previously known pterogynidlne (75) and three new guanidine alkaloids named nitensldines A (76), B (77), and C (78) [57]. These alkaloids were tested in our yeast mutant strains RS 321, RS 167N (rad6), RS 322 YK (rad52) and RS 188N (RAD*) and were found to be active only in the strain RS 321, suggesting the potential of these alkaloids as DNA-modifying agents. The new alkaloids, nitensldines A-C (76 - 78) were also tested for cytotoxicity using the Chinese hamster ovary auxiliary B1 cell line, and all of them showed moderate cytotoxic activity. The bioassay data for these alkaloids are presented in Table 13. Although a number of unusual guanidine alkaloids displaying a broad spectrum of biological activities are known from marine organisms [58,59] and some plants of the families Euphorblaceae and Leguminosae [60-64], this constituted the first report of the DNA-modifying activity of guanidine alkaloids. TABLE 13. Bioactivity data for guanidine alkaloids 75 - 78.
Alkaloid RS321 Pterogynidlne (75) Nitensidine A (76) Nitensidine B (77) Nitensidine C (78) Camptothecin (12) Streptonigrin (13)
8,5 6.0 10.0 13.0 -
Yeast strain^ PIS167N RS322YK (rad6) (rad52Y) > 1000 > 1000 > 8000 > 1000 > 8000 > 500 > 8000 > 8000 8.7 0.6 2.4 0.4
RS188N (RADV >1000 >1000 >1000 >8000 -
Cell lineb CHOAux-Bl NTc 13.0 9.3 20.0 0.016 -
^Results expressed as IC12 (p-g/mL) values ^Chinese hamster ovary auxilliary Bl cell line; results expressed as IC50 (\ig/mL) values CNot tested
489 The structures of the guanidine alkaloids 75 - 78 encountered in this study were elucidated with the aid of spectroscopic data especially NMR techniques including DEPT, ''H-^^C H E T C O R and HMBC techniques.
Interestingly, pterogynine (79), which
has previously been isolated from Pterogyne nitens [60], was not encountered in this study. 5.5.6. Steroidal Alkaloids from Solanum umbelliferum In the course of our random screening of plants for potential anticancer activity using our yeast bioassay, a methanolic extract of Solanum umbelliferum Eschs. (family - Solanaceae) was found to exhibit promising activity.
Bioactivity-guided
fractionation afforded solasodine (80), 0-acetylsolasodine (81), and solasodine 3-0-pD-glucopyranoside (82) [65]. Of these, only solasodine (80) and 0-acetylsolasodine (81) were shown to have DNA-damagIng activity (Table 14). The inactivity of solasodine 3-0-p-D-glucopyranoside (82) may be attributed to its inability to permeate through the yeast cell wall. 92
(80) Ri^ = R2 = H
Nv
(81) Ri = Ac;: R2 = rOH1 R2 == H
(82) Ri <
^
<m
(83) Ri = H ; R2 = Ac (84) Ri = Ac ; R2 == Ac TABLE 14. Bioactivity data of steroidal alkaloids 80 - 82 and their derivatives 83 and 84. Steroidal alkaloid RS322YK (rad52Y) Solasodine (80) 0-Acetylsolasodine (81) Solasodine 3-0-P-D-glucopyranoside (82) N-Acetylsolasodine (83) N,0-Diacetylsolasodine (84) Camptothecin (12) ^Results expressed as IC12 (M^g/mL) values
5.0 21.0 >8000 >8000 >8000 0.9
Yeast strain^ RS321 RS167N (rad6)
RS188N (RAD+)
1.9 3.0 > 8000 > 8000 > 8000 -
6.1 15.7 >8000 >8000 >8000 100
> 10.0 30.0 > 8000 > 8000 > 8000
490
In an SAR study directed towards the determination of the functional moiety in solasodine (80) and its 3-0-acetate (81) responsible for their DNA-damaging activity, the two analogs, 83 and 84 were prepared and their bioactivity evaluated. On the basis of our previous studies on the biological activity of 3p-hydroxyergost-5-ene derivatives [14,39], it was hypothesized that the DNA-modifying activity of 80 and 81 may be due to the spiro-aminoacetal function present in the steroidal side chain of these compounds. This spiro-aminoacetal group may open up, producing an electrophilic iminium species
capable
of alkylating DNA (Scheme
5).
To test this
hypothesis,
A/-acetylsolasodine (83) and A/,0-diacetylsolasodine (84) were prepared, since the lone pair of electrons on nitrogen is unavailable to form the putative bioactive iminium ion intermediate in these compounds. The derivatives 83 and 84 were synthesized starting from commercially availabale solasodine (80) by the sequence of reactions outlined in Scheme 6. When subjected to our DNA-damaging bioassay both derivatives (83 and 84) were found to be inactive (Table 14), supporting the possible participation of the spiro-aminoacetal function in DNA-damaging activity of these steroidal alkaloids.
=
H
\xr^ — 1
^
^
B-(DNA)
Scheme 5. Proposed mechanism of DNA-damaging activity of steroidal alkaloids. It is noteworthy that preparations containing solasodine glycosides are currently being employed for the treatment of skin cancer [66]. The steroidal alkaloids verazine, veratroyi zygadenine, and germerin are reported to inhibit hepatoma and S 180 cells, probably by inhibiting DNA synthesis by affecting the DNA template [67]. Steroidal alkaloids and their glycosides occurring In numerous Solanum species are also known to possess a variety of other biological activities including antifungal, antiviral, molluscicidal, teratogenic and embryotoxic properties.
491
(a)
AcO'
(84)
(d) Et3Si
Reagents: (a)Ac20/pyridine/25°C/7h; (b)Et3SICI/DMF/imicla2ole/25°C/3h; (c) AcCI/CHgCla/EtsN/DMAP/O^C/lh; (d) HF-pyridine/rHF/250C/2.5h.
Scheme 6. Preparation of A/-acetylsolasodine (83) and A/,0-diacetylsolasodine (84) from solasodine (80).
5.6. Marine Diterpenoids from Eunicea toumeforti The gorgonian Eunicea tourneforti, collected in the waters off the U.S. Virgin Islands, was one of the select group of marine organisms that showed activity In our assay.
Bioactivity directed fractionation led to the Isolation of a number of new and
known diterpenoids, although regrettably these proved to be only weakly active against the RS321 yeast strain [68]. Typical of the diterpenoids isolated was 16-acetoxy-7,8epoxy-3,12-dolabelladlen-13-one (85).
(85)
492 5.7. Limonoids from Trichilia emetica Out of a large number of extracts derived from Ethiopian plants screened in our collaborative effort with Dr. Ermias Dagne of the University of Addis Ababa, Ethiopia, an MEK extract of Trichilia emetica Vahl. (family - Meliaceae) was found to exhibit selective activity against the rad52 yeast strain RS 322YK compared with the wild-type RAD* (RS 188N) strain. The usual fractionation guided by bioassay involving solventsolvent partition, normal and reversed-phase column chromatography, reversed-phase preparative TLC and reversed-phase HPLC furnished the limonoids nymania 1 (86), drageana 4 (87), trichllin A (88), rohltuka 3 (89), Trichilia substance Tr-B (90), and a novel seco-A-protolimonold identified as methyl-1S,23R-diacetoxy-7R,24^,25-trihydroxy20S-21,24-epoxy-3,4-seco-apotirrucall-4(28),14(15)-diene-3-oate (91) [69].
Of these,
only nymania 1 (86) and Tr-B (90) were found to be active in our mechanism-based yeast bioassay. Nymania 1 (86) was also tested in a Vero cell assay and was found to be moderately cytotoxic.
Bioactivity data for nymania 1 (86) and Tr-B (90) are
summarized in Table 15.
i V J / HO
(88)
OH O
(87)
'
493 The structure of the novel seco-A-protolimonoid (91) was elucidated with the aid of HREIMS, ^H-NMR, ^^C-NMR. DEPT, DQCOSY, HMQC and HMBC spectra.
In
addition this study involved the assignment of ^^C-NMR spectral data of drageana 4 (87), rohituka 3 (89) and Tr-B (90) for the first time and revision of ^^C-NMR assignments for trichilin A (88). Previous work on T. emetica have led to the isolation of a number of limonoids, some of which have been shown to have a wide range of biological activities including antifungal, bacteriocidal and antiviral activities [70], and insect antifeedant and growth regulating properties [70,71].
pAc
(90)
TABLE 15. Bioactivity data for the limonoids nymania 1 (86) and Tr-B (90) from Trichilia emetica. Limonoid
Nymania 1 (86) Tr-B (90) Camptothecin (12)
Yeast strain^ RS322YK RS188N (rad52Y) (RAD+) 0.7 16.7 0.6
25.0 > 100 110
Cell lineb Vero CHOAux-Bl 12.0 >20 0.054
^Results expressed as IC12 (M^g/i^L) values Wero monkey and Chinese hamster ovary auxilliary Bl cell lines; results expressed as IC50 (^ig/mL) values
494 5.8 Bioactive 0-Heterocyclic Compounds 5.8.1 Furanonaphthoquinones from Crescentia cujete Defatted wood chips of Crescentia cujete, a plant of the family Bignoniaceae, when extracted with MEK afforded a bioactive extract. Fractionation of this extract guided by bioactlvity and involving solvent-solvent partitioning and flash chromatography afforded two furofuranonaphthoquinones with a novel fused ring system and seven furanonaphthoquinones.
The two furofuranonaphthoquinones were identified as 3-
hydroxymethylfuro[3,2-b]naphtho[2,3-d]furan-5,10-dione (92) and 9-hydroxy-3-hydroxymethylfuro[3,2-b]naphtho[2,3-d]furan-5,10-dione (93), both of which were demonstrated to be active In our mechanism-based bioassay [72].
(97) R = H (98) R = OH
(94) Ri = R2 = OMe; R3 = OH (95) Ri = R2: OMe; R3 = H (96) Ri = R3: H; R3 = OMe
(92) R = H (93) R = OH
(99) Ri = R2 = R3 = H ; undefined stereochemistry (100) Ri = R3 = H ; R2 = OH ; undefined stereochemistry
The structures of the seven naphthoquinones were identified as {2S, 3S)-3hydroxy-5,6-dimethoxydehydroiso-a-lapachone (94), (2R)-5,6-dimethoxydehydroiso-alapachone
(95),
(2R)-5-methoxydehydroiso-a-lapachone
naphtho[2,3-b]furan-4.9-dione 4,9-dione
(98),
(97),
(96),
2-(1 -hydroxyethyl)-
5-hydroxy-2-(1-hydroxyethyl)naphtho{2,3-b}furan-
2-isopropenylnaphtho{2,3-b}furan-4,9-dione
(99),
and
5-hydroxy-
dehydroiso-a-lapachone (100), all of which were found to be bioactive [73]. Both
furofuranonaphthoquinones
92
and
93,
and
those
furano-
naphthoquinones showing the requisite differential in bioactlvity towards rad52
and
RAD"" yeast strains were subjected to cytotoxicity assay with Vero cells. The results of these
bioassays
are summarized
in Table
16.
It is
interesting
that
the
furofuranonaphthoquinone 92 exhibited selective activity whereas 93 was almost as active in the wild-type strain RAD* as in the DNA repair-deficient strain rad 52. Thus, 93
495 is presumably an antifungal or a general cytotoxic agent. All furanonaphthoquinones 9 4 - 1 0 0 showed moderate but selective activity against the DNA repair-deficient rad 52 yeast strain, indicating that they act as DNA-damaging agents.
The more active
compounds, 94 - 98, were also subjected to cytotoxicity assay against Vero cells; the highest potencies were shown by furanonaphthoquinones 97 and 98, both of which have an OH group in their side chain. The activity of 97 against Vero cells is about an order of magnitude greater than its previously reported cytotoxicity to KB cells [74].
TABLE 16. Biological activity data for furofuranonaphthoquinones 92,93 and furanonaphthoquinones 94 -100 from Crescentia cujete.
Compound
Yeast strains RS322YK RS188N (rad52Y) (RAD+)
Furofuranonaphthoquinone (92) Furofuranonaphthoquinone (93) Isolapachone (94) Isolapachone (95) Isolapachone (96) Furanonaphthoquinone (97) Furanonaphthoquinone (98) Furanonaphthoquinone (99) Isolapachone (100) Camptothecin (12)
10.0 2.0 47.0 33.0 48.0 14.0 3.0 60.0 80.0 0.6
30.0 3.0 >1000 120.0 280.0 180.0 10.0 130.0 140.0 110
Cell line^ Vero
2.3 2.9 3.7 4.7 4.3 0.41 0.21 NTc NTc 0.019
^Results expressed as IC12 (Mg/niL) values Wero monkey cell line; results expressed as IC50 (M^g/mL) values CNot tested The structures of all new compounds were determined by application of MS, ^H-NMR. ^^C-NMR, DEPT. ^H-^^C HETCOR. HMQC and HMBC techniques.
Structure
elucidation and stereochemical assignments including absolute stereochemistry of the furanonaphthoquinone 96 involved the use of the selective INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) NMR technique and comparison of the CD (Circular Dichroism) spectra with related compounds whose absolute stereochemistry has been determined by x-ray crystallography [75].
496 5.8.2. Pterocarpans from Erythrina burana Erythrina burana Chiov. (family - Fabaceae) is known to occur only in Ethiopia.
The genus
Erythrina is well known for elaborating
alkaloids
with
cardiovascular effects. Recent studies on the neutral and phenolic compounds of this genus have revealed the occurrence of flavanones and isoflavanones and antimicrobial pterocarpans [76]. A chloroform extract of the bark of E burana showing selective activity against the DNA repair-deficient racl52 yeast mutant when fractionated by flash chromatography over Silica gel and Sephadex LH-20 gel permeation chromatography, followed by Silica gel preparative TLC afforded two bioactive pterocarpans identified as phaseollidin (101) and cristacarpin (102)[77].
(101) Ri = R2 = H (102) R i = O H ; R2 = Me
TABLE 17. Bioactivity data for phaseollidin (101) and cristacarpin (102) from Erythrina burana. Compound RS322YK (rad52) Phaseollidin (101) Cristacarpin (102)
500 80
Yeast strain^ RS167N RS321 (rad6)
RS188N (RAD+)
P-388
Cell lineb CHCX: CHOC -PGO
> 1000 400 > 10.0 4.0 7.6 5500 > 10,0 > 20.0 4.0 330 540 108 ^Results expressed as IC12 (jig/mL) values bWild-type P-388 murine leukemia, Chinese hamster ovary and P-glycoprotein overproducing Chinese hamster ovary cell lines; results expressed as IC50 (fiM) values
497 The bioactivity data for phaseollidin (101) and cristacarpin (102) in our mechanism-based yeast bioassays are summarized in Table 17. Both pterocarpans showed selective activities in rad52 and RS 321 yeast assays compared with the wildtype RAD"" strain. Cytotoxic activities of phaseollidin (101) and cristacarpin (104) were determined in three cell lines, namely wild-type P-388 leukemia, wild-type Chinese hamster ovary (CHOC) and P-glycoprotein overproducing Chinese hamster ovary (CHOC-PGO) cells. Phaseollidin (101) was found to be moderately active in the wildtype CHOC cytotoxicity assay. Interestingly, cristacarpin (102) was inactive against wildtype CHOC but showed activity against a P-glycoprotein overproducing cell line; this reversal of activity is unusual. 5.8.3. Coumarins from Sri Lankan Rutaceae Our search for potential natural product-based anticancer agents employing a mechanism-based bioassay described in previous sections utilized the approach of random screening of extracts followed by bioassay-guided fractionation of those extracts exhibiting bioactivity. A second route to drug discovery is that of screening pure isolates obtained from other studies.
This approach was tested previously with 4,5-
dioxoaporphines aquired from various sources (see Section 5.5.1). In an extension of this approach we have evaluated twelve coumarins in our mechanism-based bioassay employing DNA repair-deficient (rad6 and rad52Y) and repair-proficient {RAD*) yeast strains [78]. Coumarins constitute a major class of 0-heterocyclic natural products with widespread distribution and broad pharmacological profile, including anticancer activity [79].
Coumarins occur commonly in plants belonging to the families Rutaceae,
Simaroubaceae, Meliaceae, and Burseraceae.
During our studies on Sri Lankan
Rutaceae we have encountered coumarins belonging to three structural types, namely simple coumarins {umbelliferone (103), suberosin (104), suberenol (105), osthol (106), and aurapten (107)}, furanocoumarins {bergapten (108), xanthotoxin (109), isopimpinellin (110), and marmesin (112)}, and pyranocoumarins {seselin (113), xanthyletin (114), and xanthoxyletin (115)}. Umbelliferone (103), subserenol (105), marmesin (112), seselin (113), xanthyletin (114), and xanthoxyletin (115) were isolated from Pleiospermium alatum [80,81], suberosin (104) from Luvunga angustifolia [81], and osthol
(106),
aurapten
(107),
bergapten
isopimpenellin (110) from Limonia acidissima [82].
(108),
xanthotoxin
(109),
and
498
(103)
(104) (105)
R1
R2
R3
H
H
H
^/V\
^/V^"
(106)
H
007)
H
Me
H
Me
H
Me
S/V'\
^/NA/^SA
(113)
H
(108) (109)
R^ =OMe R2 = H R2 = 0Me Ri = H
(110)
R^ = R2 = 0Me
(111)
R1=R2 = H
>--coo,
HO
(112)
(114) R = H (115) R = OMe
Coumarins 103 -110 and 112 - 115 were tested in our mechanism-based yeast bioassay employing the rad52 strain at a dose of 500 |ig/mL, and only seselin (113) and xanthyletin (114) showed detectable activity. Thus, these two were tested with other yeast strains (rad52Y, radd, and RAD*). Seselin (113) exhibited selective activity against the rad52 yeast strain as compared with the wild-type RAD" strain, indicating that it acts as a DNA-damaging agent. Seselin was also weakly active in a mammalian cytotoxicity assay against Vero monkey cells, with an IC50 of 12 |Lig/mL. It is more potent than the related coumarin, xanthyletin (114), perhaps due to the ability of seselin (113) to produce DNA damage as suggested by its selective toxicity to the DNA repair-deficient yeast mutant. Xanthyletin showed somewhat higher activity in the wildtype RAD* cell line, suggesting that its cytotoxicity is due to some other mechanism than DNA damage. Table 18.
The bloactivity data for these two coumarins are presented in
499 It is noteworthy that out of the three structural types of coumarins tested, only the pyranocumarins acted as moderate DNA-damaging agents, that the angular pyranocoumarin 113 was more active than its linear counterpart 114, and that the introduction of a methoxyl substituent at C-5 of the linear pyranocoumarin (e.g., 115) caused a total loss of its bioactivity.
It is interesting that the linear furocoumarins
included in this study {e.g., bergapten (108), xanthotoxin (109), and isopimplnellin (110)} were found to be inactive although the linear furocoumarin psoralen (111) is known to intercalate into DNA and to photobind with this macromolecule [83] yielding either monoadducts
or interstrand cross-links
[84] by one or two subsequent
photocycloaddition reactions with the pyrimidine bases, particularly thymine [85]. TABLE 18. Bioactivity data for the coumarins, seselin (113) and xanthyletin (114).
Compound radSl Seselin (113) Xanthyletin (114) Camptothecin (12)
33 52 0.6
Yeast strain^ RS167N RS322YK (rad6) (ra^52y) 480 87 318 87 8.7
RS188N
Cell lineb Vero
220 21 110
12 >20 0.02
^Results expressed as IC12 (^ig/mL) values Wero monkey cell line; results expressed as IC50 (|ig/mL) values
6.
Summary and Conclusions The work described in the preceding pages has demonstrated the utility of
the yeast bioassay for the detection and isolation of DNA-damaging agents in plant and marine extracts. The assay has many advantages. Thus it is relatively easy to set up and maintain, and It Is economical to run since no expensive reagents or radioisotopes are required. It is also appropriately selective, with only approximately 1 % of extracts tested showing significant activity in the assay. Finally, since it is a mechanism-based assay, those compounds isolated through its use have a mechanism of action that is at least partially understood, and thus can proceed more rapidly to a decision point on possible development as anticancer agents. The compounds isolated have come from a variety of biological sources, and have yielded a correspondingly diverse group of bioactive natural products. Except in a
500
few cases discussed, there appears to be no obvious correlation between structure and bioactivity, but it is noteworthy that several of these compounds, such as the furanonaphthoquinones, the aporphine alkaloids, and the azafluorenone alkaloids, are planar aromatic compounds which would be expected to intercalate into DNA. As noted earlier, the bioassay is capable of detecting topoisomerase I inhibition activity as well as topoisomerase II inhibition activity. It is thus interesting that we found no extracts that showed the topoisomerase I pattern of activity, indicating the scarcity of natural products with this activity and enhancing the uniqueness
of
camptothecin (12) and its derivatives. Of course, by the same token, the discovery of a new topoisomerase I inhibitor would be a noteworthy event. Finally, although none of the compounds described in this review are candidates for clinical development as anticancer drugs, some of them offer interesting target molecules for analog synthesis.
We are thus investigating some of the
compounds as pharmacophores for the development of improved analogs.
7.
Acknowledgements
The work described in this review depended first of all on an effective collaboration as part of a National Cooperative Drug Discovery Group (NCDDG) grant (1 U01 CA 50771) awarded to the University of Virginia (Prof. S.M. Hecht, Principal Investigator), and we are particularly grateful to the National Cancer Institute for this financial support. We are also grateful to Prof. Hecht for his leadership, advice and encouragement and the provision of bioactive extracts, and to Dr. Randall K Johnson (SmithKline Beecham Pharmaceuticals) for the provision of active extracts and of the yeast strains described in this review. The work carried out at Virginia Polytechnic Institute and State University was made possible thanks to the skillful efforts of our following coworkers: Dr. A Ahmad, Ms. N. J. Baj, Profa. V. da S. Bolzani, Ms. Claudia Brochini, Dr. Q.-M. Che, Prof. E. Dagne, Mr. T. E. Glass, Prof. M. Govindan, Ms. G. Govidan, Dr. Habib-ur-Rahman, Mr. K. Harich, Dr. G. G. Harrigan, Dr. C. E. Heltzel, Dr. Y C. Kim, Mr. W. Lennox, Prof. J.-Y. Liang, Dr. W. R. Phillips, Dr. G. Samaranayake, Prof. E. Silveira, Profa. L. M. M. Valente, and Mr. C. WU. We gratefully acknowledge their valuable contribution to this program. We also gratefully acknowledge the collaboration of Dr. G.W Chan, Dr. D.S. Eggleston, Mr. L. Faucette, Dr. K Haltiwanger, and Mr. G.A Hofmann from SmithKline Beecham Pharmaceuticals, and Prof. L.M.V. Tillekeratne and Dr. E. M. K. Wijeratne from Sri Lanka. We are grateful to Profs. H. Achenbach (Germany), A Cave (France) and A Banerji (India) for samples of 4,5-dioxoaporphlnes for SAR studies.
501 8.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
507
In Vitro Models of Human Disease States John M. Pezzuto, Cindy K. Angerhofer and Haider Mehdi Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612 1.
SUMMARY The treatment of most systemic diseases involves administration of drugs, and it is
apparent that drug-based therapeutic strategies will predominate into the 21st century. Unfortunately, efficacious agents are not available for the treatment of many conditions that debilitate humans. Examples are cancer, hepatitis and malaria. Drug discovery may be based on a variety of approaches, including random screening, combinatorial chemistry, "rational" design, or natural products, the latter of which comprises a main focus of our programs. In each case, a dominant feature of the programs deals with utilizing appropriate in vitro models of human disease states. The construct and design of these systems is of utmost importance for success. From the outset, it is typically appealing to consider the use of models employing laboratory animals. It can be demonstrated, however, that animal models are often precluded due to time, expense and ethical considerations. On the other hand, in vitro test systems are deficient in a number of respects, e.g., high rates of "false positives", little potential for metabolism, lack of ability to accurately reflect metabolic disposition, etc. Nonetheless, batteries or panels of test systems can be constructed to reflect selectivity or "in vitro therapeutic efficacy" by the generation of indices. We have illustrated these principals in the discovery of antimalarial, antihepatitis B, and cancer chemotherapeutic agents. In the area of cancer chemoprevention, the same notion of in vitro selectivity has been used, and active leads are then tested in a more physiologically-complicated mammary organ culture model. These approaches have been or can be applied to mimic other types of human disease states, e.g., AIDS. It is not a trivial matter to devise appropriate test panels, bearing in mind factors such as capacity, expense, safety and
*Some of the information described in this manuscript was presented at the American Association of Pharmaceutical Scientists Midwest Regional Meeting, Chicago, IL, May 20, 1996.
508
sensitivity, in addition to physiologic significance. However, if suitable models are not developed and employed, potentially useful drugs will be missed. 2.
INTRODUCTION Terrestrial plants are a well-established source of drugs useful for the treatment of human
ailments (1). Since conservative estimates suggest the existence of 250,000 species, various strategies have been devised for the selection of starting materials (2). Once starting materials are procured, however, it becomes of utmost importance to rapidly identify promising leads and to obtain the active chemical principles affiliated with these substances. The approach generally regarded as most practical for drug discovery is bioassay-directed fractionation. This entails evaluating plant extracts in a bioassay system, and those demonstrating a positive response are considered as active leads. After a number of active leads are identified, decisions are made to fractionate the most promising materials. Each fraction is monitored for potential to mediate a positive response in the bioassay test system, and this process continues until a pure active substance is obtained. The resulting substance is then subjected to procedures of structure elucidation. Once an active isolate is obtained, more thorough biological evaluation procedures are often performed and, based on the accumulated data, the material is considered as a candidate for more advanced testing and development. A question of paramount concern relates to the bioassay systems that are utilized for selection of active lead materials and for the activity-guided fractionation that leads to the procurement of active principles. We currently describe bioassay systems that are suitable for the discovery of antitumor, antimalarial and antihepatitis B agents, emphasizing aspects of selectivity that are designed to promote the discovery of agents capable of mediating physiological responses with therapeutic efficacy. 3.
CELL-BASED
MODELS
FOR
THE
DISCOVERY
OF
CANCER
CHEMOTHERAPEUTIC AGENTS 3.1 The Cancer Problem As summarized by the American Cancer Society (3), it is estimated that approximately 1.4 million new cancer cases will be diagnosed this year in the United States, and approximately 550,000 individuals will die of cancer. A steady rise in cancer mortality rate over the past 50 years suggests a lifetime risk for the development of cancer ranging from 1 in 3 (for women) to 1 in 2 (for men). It is generally agreed that cancer will be the leading cause of death in the U.S. by the year 2000, and it currently is the leading disease-based cause of death on a global level. Survival rates vary on the basis of cancer type and stage at the time of diagnosis, but
509 malignant, metastatic disease is rarely associated with a favorable prognosis. Agents currently in use, many of which are natural products, may lead to prolongation of survival time, but untoward side-effects and morbidity are common. These statistics and unpleasant observations all lead to the same conclusion: there is an urgent need for the provision and development of new and effective cancer chemotherapeutic agents. Drug discovery programs must continue in an unabated manner so that eventual management of disseminated disease may be accomplished. Historical precedent suggests natural products can mediate antitumor activity, and organisms spread throughout the biosphere continue to represent viable sources of starting material. 3.2 Evaluation of Cytotoxic Activity Utilizing Cultured Human Cancer Cells A large number of in vitro test systems and the issues that need to be considered when attempting to interrelate in vitro test results with in vivo efficacy studies have been described (cf. 4). In general, recent advances in molecular biology have led to a greater understanding of the molecular basis of human disease states. As a correlate, in vitro systems that monitor a response that is either closely related to or identical with the molecular event yielding the disease condition can be devised. In contrast to well-defmed in vitro test procedures, assays dealing with cell cultures have one major advantage: all potential mechanisms concerning cellular proliferation are simultaneously monitored. Obviously, it is recognized that cytotoxicity is neither necessary nor sufficient for antitumor activity. Therefore, it should be of no surprise that isolates derived from cytotoxicitybased assay procedures are often toxic toward mammalian species and do not demonstrate therapeutic efficacy. However, cytotoxicity is an activity that is consistent with antitumor activity and virtually every known naturally-occurring antitumor agent demonstrates a positive response in a cell culture system. Agents such as taxol and camptothecin were discovered on the basis of cytotoxicity. Thus, it is likely that a previously uncharacterized plant extract that contains a novel antitumor agent would also demonstrate a positive response, and many research programs dealing with the isolation and identification of potential antitumor compounds from plants have relied on cytotoxicity for bioassay-directed fractionation. In the past, cultured P388 or KB cells have been employed routinely. Briefly, the procedure involves treating the cells with various concentrations of the test substance and assessing cell growth after 48 or 72 hr of incubation with P388 and KB cells, respectively. The results are expressed as ED50 values (concentration required to inhibit cell growth by 50%). As established by the National Cancer Institute, ED50 values of <20 jug/ml for extracts and < 4 ji^g/ml for pure compounds are considered active. Although these test procedures are proven to be effective for the isolation of cytotoxic compounds that may be of novel structure, it is often the case that compounds identified as active toward cultured P388 cells are not active with P388-infected mice, and compounds active
510
with KB cells are often not active with in vivo tumor models. In many instances, the isolates are simply toxic. In an effort to discover agents with selective antitumor activity, the National Cancer Institute initiated a program wherein the cytotoxic potential of compounds or extracts are evaluated with a battery of human cancer cell types in culture. The objective is to uncover agents that demonstrate selective cytotoxic activity with a cell line derived from a single type of primary human tumor (5,6). The use of a battery of human cell lines derived from a variety of human tumor types offers great theoretical and practical advantage. For example, the cells can be carried as solid tumors in athymic mice. Thus, more advanced testing of active principles is obviously facilitated. Also, it can be speculated that certain test materials could demonstrate celltype specificity, and this would lead to isolation and identification of selectively cytotoxic agents. Based on structure and activity, agents capable of mediating selective cytotoxicity would certainly be candidates for more advanced testing. It is possible that an agent capable of demonstrating cell-type selectivity (or a derivative thereof) could be useful in the treatment of the specific type of cancer modeled by the cultured cells. Irrespective of the clinical outcome, however, the concept of selective cytotoxicity implicitly suggests the presence of a cell-specific receptor that differentiates the proliferative capacity of one tumor-type (or cell-type) from another. Such a discovery would be of monumental importance in terms of developing tumor-specific therapeutic strategies, and a cytotoxic agent specific for one cell type would greatly aid in identifying the appropriate subcellular target. Thus, agents that demonstrate the desired selectivity are anticipated to serve as important tools for probing tumor-specific receptor sites, and this is an entirely rational approach toward defining tumor-specific targets for chemotherapeutic agents. Based on these considerations, there is substantial merit associated with an approach that relies on selective cytotoxicity. Paradoxically, however, all of the well-known natural product antitumor agents that are currently utilized in clinical settings do not demonstrate selective cytotoxicity (2). Rather, it is typical to observe a general cytotoxic response with absolutely no cell-type specificity. Since it is not practical to assign a high priority to a plant extract based solely on non-specific cytotoxic activity, it is necessary to devise a broader-based bioassaydirected drug discovery program. At the present time we are engaged in a National Collaborative Natural Product Drug Discovery Group (7) involving the partnership of Research Triangle Institute and Bristol-Myers Squibb [during the first five years of the program (1990-1995), the industrial partner was Glaxo Group Research]. As with all cooperative agreements of this type, the National Cancer Institute is also a member of the consortium. Also, at the University of Illinois, cytotoxicity assays are
511 conducted (8) similar to those performed at the National Cancer Institute, although on a greatly reduced scale. Additional assays are performed to evaluate antimitotic potential with cultured ASK cells (9) and to assess ability to reverse the multidrug-resistant phenotype with KB-VI cells (10). At the other research facilities involved in the consortium, extracts derived from the same plants are evaluated in mechanism-based assays. The accumulated data are then evaluated in order to decide which materials should be subjected to fractionation procedures. In an ideal case, a plant extract would demonstrate activity in a mechanism-based assay and a limited number of cell lines. This type of activity profile leads to a high priority for fractionation. Alternatively, either type of activity (only mechanism-based or limited cytotoxicity) yields a high priority rating. General cytotoxicity alerts us to the fact that active principles are present in the plant extract which may or may not be of interest. In general, such an extract would be placed on hold due to lack of correlative activity, but these may be re-evaluated as additional knowledge is accumulated and mechanism-based assays are modified. Thus, cytotoxicity-based assays are useful for grouping plant materials into two broad categories (active or inactive); at some point in time, additional information may be accumulated to re-activate such leads. This is considered a more judicious procedure than ranking potentially useful materials as "false negatives" which is an unavoidable consequence of utilizing a highly discriminatory screen. 3.3 Summary of Isolates Obtained from Terrestrial Plants Reviews have been published summarizing lists of cytotoxic compounds that have been isolated from terrestrial plants using cell culture for bioassay-directed fractionation (11-13). In nearly every case, no obvious or meaningful cell-type specificity could be achieved.
One
notable exception is betulinic acid, originally noted in an extract derived from Ziziphus mauritiana, but readily available from other sources such as the bark of white birch trees (14). This compound mediates semi-selective cytotoxic responses with cultured melanoma cells, and inhibits the growth of human melanomas carried in athymic mice. The mode of action appears to involve induction of apoptosis (14).
This agent is currently undergoing preclinical
development. It is being considered for use in both chemotherapeutic and preventative settings. 4.
IN VITRO MODELS FOR THE DISCOVERY OF CANCER CHEMOPREVENTIVE AGENTS 4.1 Strategies of Cancer Prevention As described above, morbidity and mortality resulting from cancer is dismal, and it is
imperative that the search for new chemotherapeutic agents continue at an incessant pace. At the same time, prevention strategies must be developed and implemented on a grand scale. Some examples of cancer prevention are obvious. For example, 800,000 cases of skin cancer
512 are expected to be diagnosed this year, 90% of which could be prevented by limiting exposure to the sun. Smoking of cigarettes, a highly addictive habit that can be terminated with due diligence, probably leads to over 170,000 deaths per year in the United States alone (3). Thus, primary prevention, in conjunction with early diagnosis, could lead to massive reductions in cancer cases. Irrespective of the success of such programs, the number of cancer cases will remain significant. For example, certain individuals may be at a high risk of developing specific types of cancer, and this risk may not be negated by changes in diet or other elements of lifestyle. In these cases in particular, the concept of cancer chemoprevention is applicable.
Cancer
chemoprevention, as a discipline of pharmaceutical-based therapy, remains in its infancy. A number of intervention trials are presently ongoing (15,16), but no chemopreventive formulation is currently available or consumed by the general population, other then fruits, vegetables, and agents such as aspirin. Our focus has been the discovery of natural product cancer chemopreventive agents utilizing bioassay-directed fractionation as the primary approach (17-19). In vitro test systems have been devised to monitor alteration of the initiation, promotion and progression stages of carcinogenesis. Once active leads are identified, they are evaluated for potential to inhibit 7,12dimethylbenz(a)anthracene (DMBA)-induced preneoplastic lesions with a mouse mammary gland organ culture model. This secondary discrimination is used to select extracts for fractionation, utilizing the in vitro test system in which activity was originally observed as a monitor. A brief description of this approach is given below. 4.2 Test Systems Indicative of Inhibiting the Initiation of Carcinogenesis Antimutagenicity. A large number of plant materials are known to mediate antimutagenic effects (20). Clearly, however, most compounds isolated solely on the basis of an antimutagenic response observed with an in vitro test system, could not demonstrate chemopreventive activity. Since the process of antimutagenicity is much less complex than cancer chemoprevention, the basis for this lack of correlation is quite obvious. However, with reasonable certainty, we have shown that inhibition of DMBA-induced mutagenicity with Salmonella typhimurium strain TM677 correlates with inhibition of DMBA-induced preneoplastic lesion formation in mammary organ culture (21,22). This does not imply that a majority of antimutagens will demonstrate such activity, but it is reasonable to suggest that many compounds capable of inhibiting lesion formation will also demonstrate antimutagenicity in this test system. Therefore, it is reasonable to conduct bioassay-directed fractionation using antimutagenicity as a monitor with plant extracts that have been shown to be active in the mammary organ culture test system. Induction ofquinone reductase with cultured Hepa lclc7 cells. A second assay we utilize
513 that yields results indicative of cancer chemoprevention at the stage of initiation is the induction of quinone reductase in cultured Hepa IclcV cells. This system has been used by Talalay and coworkers for the isolation of sulforaphane from broccoli (23), and it was established as a useful system for evaluation of extracts (24). Like other in vitro assay systems used in our laboratory, induction of quinone reductase in cell culture correlates with in vivo activity that is consistent with cancer chemoprevention. While it is not generally clear that induction of phase I enzymes would result in chemoprevention since carcinogen activation is also possible, induction of phase II enzymes is more logically associated with protection. As an initial attempt to establish selectivity for induction of phase II enzymes rather than dual induction of both phase I and II enzymes, BP'cl and TAOclBP'cl cells are used in our laboratory. BP'cl cells have normal TCDD receptor but appear to be unable to translocate the bound receptor complex to the nucleus. On the other hand, TAOclBP^cl cells contain TCDD receptor that is either of reduced or altered affinity, but translocation to the nucleus is possible (25,26). Therefore, induction of quinone reductase in the Hepa lclc7 cell line, but not in the BP'cl or TAOclBP'cl cell lines, implies a receptor-mediated mechanism associated with induction of phase I and II enzymes. Conversely, activity in all three cell lines implies selectivity, and this is the major objective in our drug discovery program. A second component of selectivity concerns toxicity. Most test materials capable of enzyme induction can also induce a cytotoxic response. This leads to the concept of a chemopreventive index (CI), in this case defined as the concentration of compound that reduced cell growth by 50% (IC50), divided by the concentration of compound required to increase enzyme activity by 2-fold (CD). In the case of sulforaphane, evaluation with cultured Hepa lclc7 cells leads to a CI of 9.9 ^M/0.23 juM = 42 (27). The greater the CI value, the stronger the indication of selectivity. 4.3 Test Systems Indicative of Inhibiting the Promotion of Carcinogenesis Ornithine decarboxylase. One well-established consequence of tumor promoter action is the induction of ornithine decarboxylase activity. Interestingly, known chemopreventive agents such as retinoids are established as inhibitors of tumor promoter-induced ornithine decarboxylase activity, and this may relate to mechanism of action. For monitoring this activity, we have routinely employed cultured mouse 308 cells. Cells are treated with 12-0-tetradecanoylphorbol13-acetate (TPA), which induces ornithine decarboxylase. This is assessed by monitoring the generation of ^'^C02 from radio-labeled substrate, and the potential of test substances to inhibit enzyme induction is monitored. Again, to assess specificity, a chemopreventive index (CI) can be determined. Deguelin, for example, yields a favorable CI value of 0.64 ^g/ml (IC5o)/0.0003 /xg/ml (EC50) = 2133 (28).
514 Arachidonic acid metabolism. A good deal of evidence suggests that certain oxidants can play a critical role in the promotion of carcinogenesis (29). Included in this category of tumor promoters are UV light and chemicals such as hydrogen peroxide, peroxyacetic acid, chlorobenzoic acid, benzoylperoxide, decanoylperoxide, cumene-hydroperoxide, /7-nitroperbenzoic acid, and lO*'. One effect of these tumor promoters is the activation of phospholipase A2 which catalyzes the production of arachidonic acid. In turn, arachidonic acid serves as a substrate for cyclooxygenase which catalyzes the production of prostaglandins, thromboxane and prostacyclin. The precise role of these substances in tumor promotion is not clear, but a correlation of elevated prostaglandins in carcinogenesis has been established (30) and certain nonsteroidal anti-inflammatory agents (e.g., indomethacin and piroxicam) demonstrate chemopreventive activity (31). An assay for inhibition of cyclooxygenase is also sensible from a mechanistic viewpoint since inhibition of TPA-induced ODC activity by indomethacin is overcome by concurrent application of PGE2 (32). Accordingly, we have evaluated plant extracts for potential to inhibit cyclooxygenase (33). One of the most promising agents we have identified as an inhibitor of cyclooxygenase is resveratrol (34).
In addition to blocking the cyclooxygenase activity of the enzyme, the
hydroperoxidase activity is inhibited (34). The agent shows promising activities and is currently under investigation for development as a chemopreventive agent.
A related effect of
contemporary interest is the inhibition of cyclooxygenase-2, a form of the enzyme that is induced during the process of tumor promotion. Accordingly, one can search for specific inhibitors of cyclooxygenase-2, versus cyclooxygenase-1, and a more favorable profile of activity would be anticipated. 4.4 Test Systems Indicative of Inhibiting the Progression of Carcinogenesis Differentiation of HL-60 cells. Terminal differentiation can be induced in HL-60 cells by treatment with a variety of clinically interesting agents such as lof,25-dihydroxyvitamin D3 [la,25-(OH)2D3] (or analogs thereof), DMSO, actinomycin D, retinoic acid, etc. (35). Treatment of HL-60 cells with such agents results in the induction of granulocytes, monocytes, eosinophils, or macrophage-like cells, and in certain cases, this induction process is mediated without toxicity. One obvious clinical implication of agents found to be active in this test system is the treatment of leukemia since terminal monocytic differentiation suggests a mechanism of controlling the proliferation of progenitor cells. However, as judged by 1QJ,25-(OH)2D3, one of the most potent compounds known to mediate HL-60 cell differentiation, agents found to be active in this test system should be of interest for the control of other cancer types, in particular, cancer of the breast (36-38). We have established this assay and routinely evaluate extracts and compounds for potential
515 to induce nonspecific esterase (indicative of monocytes and macrophage-like cells), esterase (monocytes and other cell-types), and ability to produce superoxide when challenged with phorbol ester as judged by nitroblue tetrazolium (NBT) reduction (indicative of monocytes and granulocytes) (39). The potential to inhibit thymidine incorporation is also monitored to assess specificity and antiproliferative activity. In addition, similar to other assays in our panel, a chemopreventive index can be generated to assess specificity. This value is obtained by dividing the IC50 (concentration to reduce viable cell number by 50%) by the ED50 (concentration required to induce terminal differentiation by 50%). Using la,25-(OH)2D3 as a positive control, a CI value of > 1000 is obtained. Assays designed for the discovery of antihormones. Although many aspects of this overall program are directed toward chemoprevention of breast cancer, no component thus far described takes into account a strategy to exploit the hormone-dependence of this disease or certain other human cancers (e.g., cervical, ovarian). Thus, we have added components to focus on this class of chemopreventive agents. The first assay we employed simply detects displacement of estrogen binding to the estrogen receptor. One shortcoming of this procedure, as with all receptor binding assay methods, is that compounds capable of displacing estrogen may demonstrate either antagonistic (antiestrogenic) or agonistic (estrogenic) activity. Thus, more recently, we have utilized cultured Ishikawa cells for accessing estrogenic and anti-estrogenic effects. Treatment of this cell line with estrogen or estrogenic compounds has been shown to result in significant elevation of alkaline phosphatase that can be monitored easily with a chromogenic substrate using a 96-well plate format (40-42). Further, the response induced by adding known estrogens is effectively blocked by the addition of antiestrogens to the medium. Thus, the assay is suitable for large-scale assessment of either agonistic or antagonistic activity. In a typical assay, tamoxifen is used as a positive control, and this yields an antiestrogenic response with an IC50 of about 0.324 jitg/ml (575nM). In addition, as with other cell lines, cytotoxicity can be assessed to establish specificity. In general, follow-up work is performed in which competitive binding is assessed with the anti-estrogen (tamoxifen) binding site (43). 4.5 Correlative Approach Utilizing Mouse Mammary Organ Culture The assays described above are listed due to their capacity to suggest specificity.
The
chemopreventive index (CI) provides a good indication of having identified a promising starting material.
Some additional procedures are performed in our laboratory such as antioxidant
assays, or assays to identify inhibitors of aromatase. In these types of assays, it is difficult to assess specificity at an early stage. In either case, however, once a promising lead is identified with an in vitro test system, it is then evaluated in the mouse mammary organ culture model. This system is not suitable for bioassay-directed fractionation due to time and expense (17), but
516 resulting data tend to show a good correlation with full-term chemopreventive studies designed to monitor inhibition of mammary tumorigenesis (44). With our in vitro tests, approximately 6700 evaluations have been performed with plant extracts, and about 230 have proven active (i.e., 3.5%). Of these 230, about 160 have been assessed in the mouse mammary organ culture system, and approximately 35 of these leads have proven active. Accordingly, these 35 extracts represent our most promising leads, and activity-guided fractionation is being performed utilizing the in vitro test system as a monitor of activity. 4.6 Summary of Assay Results Following this experimental design, approximately 700 plant materials have been evaluated and this has resulted in over 6,500 bioassay results. A number of active principles have been obtained that are active with the in vitro test systems, and several of the isolates have retained activity by preventing formation of preneoplastic lesions in mammary organ culture. Thus far, three lead compounds have been shown to mediate considerable cancer chemopreventive activity in full-term tumorigenesis models (28,34,45,46) and are being studied in more advanced test systems. We remain hopeful that one or more discoveries resulting from this project will be deemed worthy of human intervention trials. 5.
MODEL SYSTEMS FOR THE DISCOVERY AND EVALUATION OF POTENTIAL ANTI-MALARIAL AGENTS 5.1 Overview of the Malaria Problem Malaria remains an enormous burden to global health. The 1995 World Health Report (47)
indicates that the disease is endemic in more than 90 countries which comprise 40% of the world's population. The incidence of malaria has been steadily increasing to a current annual estimate of 300-500 million clinical cases; 1.5-2.7 million of these result in death with the vast majority of lethalities due to infection with Plasmodium falciparum. The reasons for resurgence of malaria are numerous and complex, but a primary factor is the spread of resistance to currently available antimalarial drugs. The discovery and development of selective new agents for therapy and propylaxis is, therefore, of paramount importance. Malaria is caused by protozoan parasites of the genus Plasmodium, four species of which infect humans. (P. falciparum, P. vivax, P. malariae, and P. ovale) (48,49). The disease is initiated when infective sporozoites are injected into the human host during feeding by an infected mosquito. The sporozoites enter parenchymal cells of the liver within minutes where they multiply asexually into merozoites. After development for 6-16 days, depending on the species, merozoites are released from the hepatic cells into the peripheral blood where they quickly bind to and invade erythrocytes. This initiates the cycle of erythrocytic schizogony in
517 which the merozoites proliferate asexually, and upon reaching maturity after 36-72 hours (again a species-dependent process), the host red cell bursts and the liberated merozoites invade other erythrocytes to continue the cycle. Some merozoites of the intraerythrocytic stage differentiate into gametocytes which may be reingested by a mosquito wherein sexual reproduction and sporogony occur. New infective sporozoites are produced thus completing the life cycle. The hepatic stage of the infection is not generally associated with clinical symptoms; thus, the disease is recognized during the erythrocytic phase when the synchronized rupture of infected red blood cells and release of merozoites produces the characteristic paroxysms of chills and fever. This self-perpetuating phase of malaria can continue for weeks to months if unchecked by appropriate drug therapy, with attendant anemia and splenomegaly. Renal involvement or nephrotic syndrome can be severe, especially in infections caused by P. malariae. The most grave consequence of the disease is hypoxia, caused by the propensity of P. falciparum parasites to adhere to the endothelial lining of blood vessels and capillaries, limiting blood flow to many tissues and organs, most noticeably to the brain. 5.2 Current Status of Treatment Many different drugs are presently used for the treatment and prophylaxis of malaria around the world, but none is truly satisfactory in terms of safety and efficacy. Furthermore, resistance of particular strains of P. falciparum has been noted for most of these drugs and is a significant impediment to the practical use of many. It would be remiss if not impossible to discuss the status of antimalarial drug therapy without referring to natural products. Since the disease we call malaria has been recognized for millennia, a great number of plant species have been identified by various cultures as having antimalarial properties. While it is surely not possible to substantiate all of these claims in terms of modem therapeutic efficacy, it is certain that current antimalarial therapy consists substantially of natural products and related derivatives. Quinoline compounds and the plants that contain them have historically anchored the antimalarial arsenal, and they remain principal drugs today. Quinine and its diastereomer, quinidine, are quinoline alkaloids which were isolated in 1820 from the bark of the Cinchona tree, by virtue of the traditional South American use of this bark to treat intermittent fevers. Quinine is an effective schizonticide (i.e., it kills the form of the parasite in peripheral blood), but because it also affects mammalian lysosomes, the drug has been associated with significant adverse toxicity (48,50). The development of synthetic derivatives of quinine has resulted in improvement in potency and selectivity over the parent compound. Chloroquine and mefloquine are more potent and less toxic than quinine (49,51), thus, chloroquine had largely replaced quinine in clinical use; however, resistance of P. falciparum to chloroquine has been reported
518 from nearly every region in which the species is endemic (the exception being Central America), so that it is no longer a first-line drug in many regions of the world. Mefloquine is a secondgeneration quinoline derivative that is effective against chloroquine-resistant parasites, although mefloquine-resistance is rapidly increasing in regions of Southeast Asia (52,54). Despite their extensive record of traditional and modem usage, the potential mechanisms of action of the quinoline compounds remain obscure. These drugs have been established to accumulate in acidic compartments of mammalian cells and malarial parasites by virtue of their weak base properties (55). Furthermore, the enhanced selectivity of chloroquine and mefloquine has been attributed to the observation that they are actively concentrated in parasitic vesicles to levels 100 times higher than in mammalian lysosomes (56). It has been suggested that the schizonticidal effects of these drugs derive from the resulting alkalinization of acidic organelles such as lysosomes and food vacuoles. The compartmental increase in pH could conceivably inhibit enzymes with low pKa values (57), and could disrupt receptor-mediated endocytosis or other targets critical for the survival of the plasmodial trophozoite. However, alkalinization alone does not explain the mechanism of action of quinoline antimalarials (58). Another potential mechanism of action of quinoline-containing antimalarials involves ferroprotoporphyrin IX or 'haem' which is a byproduct of hemoglobin digestion by the developing parasite. Free haem is toxic to malarial parasites, thus most of the haem in an infected erythrocyte is found as haemozoin, a polymer in which ferroprotoporphyrin IX monomers are cross-linked via ferric iron-carboxylate bonds (59). Several groups investigating haemozoin formation first reported the process to be actively catalyzed by a parasite-specific enzyme known as haem polymerase (60,61), and reported that chloroquine and to some extent, other antimalarial drugs, could specifically inhibit this enzyme. More recent evidence, however, appears to indicate that haemozoin can form spontaneously from haem, and that this is the most likely explanation for its occurrence in Plasmodium-infQctod red blood cells (62,63). Intensive work in this area has revealed that the role of haem as an agent of plasmodial toxicity is a good deal more complex than initially suspected, and that inhibition of haemozoin formation may not alone constitute a viable antimalarial mechanism. The current status of haemozoin as a potential target for antimalarial agents has recently been reviewed (63). Additional attempts to define the mechanism of chloroquine have focused on specific interactions of the drug with binding proteins and DNA (64,65). Artemisinin (qinghaosu) provides a more recent example of a plant-derived antimalarial agent. Based on the reputed antimalarial use of Artemisia annua in the Chinese system of traditional medicine, artemisinin was isolated from the plant as the active compound, developed as a drug, and released for clinical use as a blood schizonticide (66-68). Artemisinin is a
519 sesquiterpene lactone possessing an interesting 1,2,4-trioxane ring system. The mode of action of this compound is under active investigation, and it has been demonstrated that the endoperoxide moiety is essential for antimalarial activity and that damage to parasitic membranes is a hallmark of the drug's action (69,70). The dependence of activity upon the presence of the endoperoxide suggested that free-radical generation played a role in the antimalarial mechanism of action. That this is indeed the case has been confirmed by a number of independent research groups through experimental evidence such as antagonism of activity by free-radical scavenging antioxidants (71). What is more unique to its mode of action is that, instead of exerting a toxic effect by initiating the chain reaction of oxidative damage through superoxide and hydroxyl radicals as is usually associated with free-radical generators, artemisinin itself becomes a carboncentered free-radical in a haem iron-dependent reaction (72). So activated, the artemisinin molecule has been found to alkylate a specific group of plasmodial proteins which may account for its lethality to the parasite. While this compound is effective against chloroquine-resistant P. falciparum in man, poor solubility has precluded its routine use, thus efforts have been directed at semi-synthesis of derivatives with improved solubility in lipid (artemether, arteether) or in water (artesunate, artelinate). Notwithstanding, there are still considerable problems with the routine, practical use of presently available artemisinin drugs. The short half-life of the drugs has resulted in a high clinical rate of recrudescence (i.e., reestablishment of infection by residual viable parasites which have managed to evade treatment) so that they are increasingly recommended to be used in combination with other agents having slower, more sustained antimalarial effects. In addition, evidence of significant neurotoxicity has recently been reported in cultured neural cell lines as well as in beagles and rats given high doses of the artemisinin derivatives, artemether and arteether (73,74). The synthesis of a wide varitey of secondgeneration endoperoxides which retain the endoperoxide function within a six-membered ring has demonstrated that the remainder of the artemisinin molecule is not essential for antimalarial activity (75). It is, however, likely that the chemical structure surrounding the peroxide will affect interaction of the compound with the specific cellular site(s) of action; thus, it may be possible to synthesize drugs with designed alterations of potency and selectivity that retain antimalarial activity but have less potential for toxicity or associated recrudescence. Halofantrine, a synthetic phenanthrene-methanol, is a recently introduced antimalarial drug which is a blood schizonticide. The drug is effective against chloroquine-resistant P. falciparum but has been associated with cardiotoxicity, so it must be used with caution (76). Another class of drugs that has proven clinically valuable against malaria are the antifolates. Sulfonamides such as pyridoxine and sulfones like dapsone inhibit the plasmodial enzyme dihydropteroate synthetase on the pathway of folic acid biosynthesis (48). The 2,4-
520 diaminopyrimidine, pyrimethamine, and the biguanide compound, proguanil, are selective inhibitors of plasmodial dihydrofolate reductase, a subsequent enzyme on the folate pathway. All of these compounds hinder the parasite's ability to synthesize purines and pyrimidines, thus they are slow-acting but effective schizonticides against P. falciparum. Pyrimethamine, and to a greater extent, proguanil, also possess activity against primary hepatic forms of malaria. Antifolate combinations are often used in the treatment and prophylaxis of chloroquine-resistant malaria, but adverse reactions may contraindicate their use (77,78). Furthermore, resistance of Plasmodium to many sulfa drugs is widespread (79). Tetracycline antibiotics such as doxycycline are also slow-acting schizonticides which have proven to be useful in prophylaxis of malaria and in treatment as one component of combination chemotherapy (80). A better understanding of the mechanism(s) of drug action represents an important element of the rational development of new antimalarial agents. Moreover, if a specific antiplasmodial mechanism of action can be established, the potential toxicity to host cells may be more readily predicted. However, as can be appreciated from the foregoing discussion, progress in this area of research has been relatively slow and the precise mechanisms of most of the potent antimalarials remain to be characterized. Since, clinical experience has demonstrated that the currently available drugs outlined above can be effective under the proper circumstances in given geographical regions, potential antimalarial mechanisms such as weak base-mediated disruption of parasite metabolism, oxidative stress, inhibition of haemozoin formation, antifolate activity and inhibition of protein synthesis must all be considered viable. Certainly such a short list can't begin to exhaust the possibilities for selective antiplasmodial targets, and it is likely that a number of distinct mechanisms could contribute to the antimalarial efficacy of a single drug. For this reason, our strategy for new drug discovery from natural products is grounded on a relatively broad initial requirement that test substances simply inhibit overall proliferation of intracellular parasites, and the search is subsequently refined through an additional experimental component to establish selectivity. A variety of factors have contributed to the resurgence of malaria, and continue to foster the disease (47). These include socioeconomic and political problems, as well as inadequacies in public health care. Two principal causes of malarial resurgence were: (i) the emergence of insecticide-resistant strains of the anopheline mosquitos which are the vectors for transmission of the disease, and (ii) drug-resistant strains of the parasite responsible for the pathology of the most lethal form of the disease, Plasmodium falciparum. Due to the latter, P. falciparum strains which are resistant to the antimalarial effect of chloroquine are spread throughout most of the areas where the disease is endemic, and resistance to more recently introduced antimalarial
521
agents is rapidly emerging. Since an effective human vaccine has been elusive despite intensive efforts in this area, new and more efficacious antimalarial agents are clearly needed as drug therapy will certainly remain a major component of comprehensive programs to limit the morbidity and mortality caused by malaria in all regions of the world. 5.3 Natural Products as a Resource for Antimalarial Agents A 1991 report of the Committee for the Study on Malaria Prevention and Control in the Institute of Medicine, National Academy of Sciences (81) recommended "increased emphasis on screening compounds to identify new classes of potential antimalarial drugs, identifying and characterizing vulnerable targets within the parasite, understanding the mechanisms of drug resistance, and identifying and developing agents that can restore the therapeutic efficacy of currently available drugs." Furthermore, the Committee endorsed "a systematic method for identifying plants of interest, screening them, and characterizing the structures or compounds responsible for their antimalarial activity" as a focus for botanical investigation in this field. Such strategies form the basis for our research program for the discovery of selective antiplasmodial agents from natural products. With over 250,000 species, the plant kingdom contains biodiversity which reflects known and novel compounds with potential therapeutic activity (2,82,83). Furthermore, the World Health Organization has estimated that 80% of people in developing countries are dependent upon plant-centered traditional medicines for their primary health care (84). The examples of quinine and artemisinin cited above illustrate the proven utility of compounds from plants in the treatment of malaria. Moreover, it is likely that additional plant-derived antimalarial drugs await discovery since the literature indicates that in vivo or in vitro antimalarial activities have been identified in a broad and varied spectrum of botanical families (85-92). Crude extracts of the root bark from Celastrus paniculatus Willd. (Celastraceae) demonstrated significant activity against cultured P. falciparum (93), and the active principle was shown to be pristimerin, a quinonoid triterpene. The family Simaroubaceae has also yielded a number of compounds with in vivo and in vitro antimalarial activity (94-96) including quassinoids. Bruceantin has been most extensively studied, but is very cytotoxic; however, other quassinoids are relatively less toxic to cultured KB cells while retaining potent antimalarial activity (88). Plants from the Meliaceae are commonly used as febrifuges in Africa, and several limonoids from this family, such as nimbolide and gedunin, have also been found to produce moderate inhibition of cultured P. falciparum (97,98). There are many additional examples of plant-derived antimalarial agents, most of which are alkaloids. Several acridine alkaloids obtained from members of the family Rutaceae exhibited strong activity against cultured P. yoelii (99), and protoberberine alkaloids (which are widely
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distributed across several plant families) have been shown to inhibit cultured P. falciparum (100). However, these compounds did not demonstrate in vivo activity against P. berghei. The quinazolone alkaloid, febrifugine, was found to be the antimalarial component of Dichroa febrijuga (Saxifragaceae), a plant used in traditional Chinese medicine (101), although its clinical use was precluded by hepatotoxicity. Recently, crude ethanol leaf extracts of Ficus pyrifolia (Moraceae) and of Rhus (= Baronia) taratana (Anacardiaceae), reputed antimalarial plants in Madagascar, have demonstrated substantial inhibition of cultured P. falciparum and of P. berghei in mice. Both extracts exhibited relatively low cytotoxicity towards cultured HeLa cells (89). In our laboratory, a number of potent and selective antimalarial bisbenzylisoquinolines have been isolated from many plants of the Menispermaceae and Berberidaceae, including Cyclea barbata (102), Stephania erecta and S. pierrei (103), Cyclea atjehensis and Hemandia peltata (104), and Nectandra salicifolia (105). Marine natural products are even more poorly characterized than terrestrial plants but are no less diverse. Chemical compounds with unique and significant biological activities are being discovered with increasing frequency as this immense resource is explored. Since many of these compounds are reported to possess antiprotozoal activities, (106-108) the marine environment may be reasonably expected to yield potential antimalarial agents. The range of taxonomic families which have demonstrated antimalarial effects in experimental systems suggests that many diverse natural products can inhibit the growth of the malarial parasite. The key to the discovery and development of clinically useful antimalarial drugs would appear to lie in our capacity to probe these resources and to identify the most potent and selective specimens. 5.4 Approaches to In Vitro Antimalarial Testing Faced with so much information concerning the potential occurrence of antimalarial principles in natural products, it is imperative that the drug discovery process accommodate high sample throughput while retaining the ability to distinguish the most promising active compounds. The test system must have the capacity to screen a diverse array of samples which act through any number of conceivable antimalarial mechanisms, and should then identify those which may exhibit specific inhibition of parasite growth without substantial toxicity toward host cells. This is achieved in practice by combining the use of a primary antimalarial assay with a secondary assay for general cytotoxicity (109). The primary assay is expected to expose any and all active test substances without regard to selectivity, so as to minimize the risk of omitting potentially effective agents. As a consequence, the *hits' in this assay will include false positives, i.e., substances which inhibit the growth of Plasmodium parasites and mammalian cells to the
523 same degree. In our experience, the major sources of false positives are general cytotoxins which inhibit the growth of many cell types non-selectively; these compounds are readily revealed as non-selective by the addition of an additional assay. This secondary assay should serve to detect additional bioactivities in those test substances which demonstrated antimalarial potential in the primary screen for the purpose of characterizing active compounds with respect to safety and antimalarial specificity. One strategy may be to test for a second activity that is thought to be necessary or complementary to that detected in the first assay, in which case samples are selected for further study only if they demonstrate activity in both assays. Conversely, the secondary activity may be an undesirable attribute of a putative antimalarial agent wherein a positive result would effectively exclude a test sample from further consideration. The antimalarial and cytotoxicity assays described below reflect the second strategy; however, either of these approaches could conceivably be applied to achieve the end result of enriching the test pool for the most promising samples. Primary assay. The primary assay is comprehensive with regard to its capacity to detect potential antimalarial activity, since there is no intent to exclude any antiplasmodial compounds at this stage, regardless of mechanism of action. This bioassay relies upon in vitro methodology for the culture of P. falciparum within human erythrocytes (110) and is widely used for investigation of potential antimalarial compounds. The method evaluates the ability of test substances to inhibit parasite proliferation by measuring the incorporation of a radiolabeled purine into plasmodial DNA (111). The protocol entails the addition of a cell suspension containing intraerythrocytic P. falciparum to a 96-well microtiter plate containing serial dilutions of the extract or compound(s) to be tested (103). The plates are then incubated at 37°C under an atmosphere of 5% O2, 5% CO2, and 90% Njfor 24 hours. Following the addition of 25JLI1 (0.5 ^lCi) pH]hypoxanthine to each well, the plates are incubated for an additional 18 hour period during which time parasites that survived the initial treatment will incorporate the radioisotope into nucleic acids. The assay is terminated by aspirating the contents of each well onto glass fiber filters and washing thoroughly, whereupon the incorporated radioactivity is determined in a scintillation counter. Finally, IC50 values (the concentration of test substance which inhibits [^H]hypoxanthine incorporation by 50%) are determined for each test substance using non-linear regression analysis. Secondary assays. All test samples which demonstrate activity in the primary assay described above are submitted to a secondary cytotoxicity assay. A standard protocol for the quantitation of cellular toxicity measures the ability of cultured cells to proliferate in the
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presence of a test extract or pure compound (112). In this case, KB cells (human oral epidermoid carcinoma) are utilized and are added to wells of a microtiter plate (10^ cells/well) containing various concentrations of the test compound(s). After a three day incubation period, the percentage of cells surviving the treatment is determined by measuring total protein with sulforhodamine B dye (113). The rationale for employing the [^H]-hypoxanthine incorporation assay as a primary screen is based on several lines of reasoning. First, it is possible to envision a multitude of mechanisms by which plasmodial growth could be inhibited; it's equally apparent that any or all of these mechanisms might be found in nature. Accordingly, the detection of a highly relevant but not highly specific biological activity (i.e., general inhibition of growth) is an inclusive approach to the discovery of novel and effective agents. Second, the requirement for development and proliferation of Plasmodium within human red blood cells reflects the conditions of a clinical malarial infection; moreover, this stage of infection coincides with the major pathology of the disease, and is also the stage most amenable to therapeutic intervention. Third, in vitro bioassay enables the use of cloned, human isolates oi P. falciparum, the species which is without question the most important agent of human malarial disease. Routine in vivo antimalarial testing has customarily relied upon the P. berghei-infectod mouse model, because stringent species specificity implies that only humans and a few simian species are viable hosts for infection with P. falciparum or the other human malarial parasites. Thus, an in vitro assay system circumvents this species restriction and also obviates the practical and ethical issues associated with largescale animal testing. The assessment of general cytotoxicity as a secondary assay is intended to facilitate the discrimination between general toxins and those samples which may exert more selective inhibition of Plasmodium. Practically, this has been accomplished through the experimental generation of a "selectivity index". Designating the IC50 value for cytotoxicity toward KB cells as one parameter, this selectivity index has been defined (109) as: IC 50 (KB) = Selectivity Index (SI) IC 50 (P. falciparum) This number is an estimation of selective inhibition of P. falciparum by a test compound or extract. The SI value affords a qualitative measure of activity which may be compared empirically to those of known antimalarial drugs. The parameter is used to prioritize active test samples and to guide bioassay-directed fractionation with the aim of isolating the most promising antiplasmodial compounds. Selectivity indices have also been employed in the analysis of structure-activity relationships among a group of closely related compounds in an attempt to
525 determine those characteristics which confer specific activity (104). 5.5 Results of Screening for Antiplasmodial Agents The two bioassays described above have been used in tandem to screen extracts, impure fractions and pure compounds, natural and synthetic, for antiplasmodial potential. This system has directed the isolation of many new compounds with potent and selective antiplasmodial activity, and has also enabled the demonstration of such activity for the first time in many other compounds whose structures were previously known. Two divergent groups of natural compounds will serve to illustrate the practical application of this screening system. Plants which contain bisbenzylisoquinoline alkaloids have been used traditionally in many different cultures for a variety of illnesses, including malaria. After detecting antiplasmodial activity in this class of compounds to substantiate the ethnomedical evidence, we have pursued the isolation and biological testing of several dozen of these compounds, employing the screening system described above (102-105,109,114-116). Despite closely related structural skeleta, it is interesting to note that these compounds differ widely in their biological activities. Structureactivity relationships have been difficult to assign based on functional groups and on number and location of bridges which join the benzylisoquinoline monomers. Consequently, application of the selectivity index has proven to be a valuable means of differentiating the generally-toxic compounds in the series from those with more selective action. In this case, isolates which exhibit selectivity indices of 100 or greater are considered to be better lead compounds than those with relatively lower preference for inhibition of Plasmodium. Another chemical class which has been studied extensively in this screening system is that of isonitrile-containing marine compounds, the antimalarial potential of this group was first reported in 1992 (117) after several sesquiterpene isonitriles were isolated from the marine sponge, Acanthella klethra, guided by conjunction of the antiplasmodial and cytotoxicity bioassays. Subsequent investigations have focussed on the isolation and structure elucidation of additional members of this class, and biological testing has confirmed the presence of potent and selective antiplasmodial activity in a number of new and known isonitrile-containing compounds (118-119). After preliminary disclosure of promising antimalarial leads, there is an ongoing requirement for the evaluation of selectivity in more advanced phases of the development process. By monitoring the effects of natural and chemical derivatization of lead compounds upon potency and selectivity in an in vitro bioassay system such as that described here, the safest and most effective candidates can be promoted for further study in in vivo models. Drug combinations are another conceivable means of influencing the potency and selectivity of antimalarial activity (48,120). Combinations which synergistically inhibit the growth of P,
526 falciparum may exhibit proportionally greater toxicity toward mammalian cells as well with no net increase in SI; however, this is not inherently the case. A prominent example is the combination of pyrimethamine and sulfadoxine in the drug, Fansidar. While not without mammalian toxicity, in concert, this pair is much more selective against Plasmodium since the parasite possesses both of these enzymes in the pathway of folate biosynthesis, while humans have only one. Many such interactions would be amenable to testing with the in vitro model, and bioassays have been described which evaluate the qualitative and quantitative effects of combining antimalarial drugs (121,122). 5.6 Practical Utility and Limitations of Selectivity Indices Under conditions of in vitro testing, compounds which inhibit the growth and survival of a variety of cell types can legitimately be called "antiplasmodial"; however, the meaning of the term is obscured due to the promiscuous nature of the toxicity. The inclusion of a selectivity parameter along with "antimalarial" data reported in the literature, is suggested here to facilitate a more qualitative evaluation of compounds with genuine potential as antimalarial lead compounds. In the SI parameter, we have attempted to establish a working model system to estimate the potential of a test compound for killing an intraerythrocytic malaria parasite without host toxicity. It is imperative that we draw a clear distinction between the concepts of "selectivity index" and "therapeutic index". The therapeutic index (TI) of a drug is defined as the median toxic dose/ median effective dose (LD50/ED50) in mammals, whereas we have defined the in vitro selectivity index as ED50 (i^ KB ceiis)/ED5o (j^ p. falciparum) (109). Since intact animals/patients are used in its determination, the TI value encompasses a much broader spectrum of toxic effects than would be detectable in our selectivity index; therefore, it is inappropriate for us to draw conclusions about clinical significance of the basis of SI values. Nonetheless, the SI values for several of the known antimalarial drugs have been determined (109), and based on the knowledge that these agents are clinically-relevant, the indices constitute a significant guideline for the prioritization of potential antimalarial agents. Intuitively, even the poorest selectivities of these drugs (i.e., SI >_ 300) appears to be quite good; however, it is important to point out that the range of SI values spans two orders of magnitude, and none of the drugs is without undesirable side effects. We have defined the antiplasmodial selectivity index of a test substance in terms of mammalian KB cells. These cells were selected by precedent (123) and also because, in our experience, this line exhibits an intermediate sensitivity to a large number of cytotoxic agents when compared to cell lines derived from a variety of other human tumors. At the present time, we are not certain if the selectivity index can be properly defined by utilizing cultured KB cells
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alone. It is possible that a specific cell line could be chosen to yield a more useful index, or that it is actually necessary to use a battery of cell lines and then evaluate the profile of activity. Evaluation of the antimalarial drug, artemisinin, provides a pertinent illustration of this. In late phase toxicity testing of artemisinin and several of its synthetic derivatives, significant neurotoxicity was noted when the drugs were administered to beagles. Cytotoxicity had not been revealed in vitro in KB cells, nor in any of a number of other mammalian cell lines which derived from a range of other tissues, including rat glial cells (124). However, when the compounds were tested with cultured neuronal cell lines, substantial cytotoxicity was observed, and these lines were further utilized to define some of the structural characteristics of the drugs which were associated with the specific neuronal toxicity (73). This example substantiates the potential of such in vitro models to contribute to the development of safer, more efficacious drugs. The SI value that is used as a 'cut-off parameter for prioritizing those samples that possess the most potent and selective antimalarial activity is somewhat subjective due to the nature of the work involved. In the preliminary stages of the work, a cutoff is established such that the volume of samples that are subjected to bioassay-directed fractionation and further analysis is limited to a practical number. Ideally, the SI cut-off should be as high as possible and in our laboratory it is presently around 100; however, we are limited by the activities of the samples at hand. It is also meaningful to consider that if the cut-off is set too high, many potentially important lead compounds may be discarded for the sake of pursuing the ideal drug. In addition, we have not intended that the selectivity index be the only criterion applied in the decision to subject pure compounds with significant antimalarial activity to further study. The discovery of compounds of novel structure possessing potent antimalarial activity but with relatively low (i.e., < 100) SI values, such may represent lead compounds which could be synthetically modified to yield drugs with higher selectivity, and may also provide valuable insight into potential mechanisms of antimalarial action or structure-activity relationships, and thus, such compounds may also be included in mechanistic studies to a limited extent. 6.
MODEL SYSTEMS FOR THE DISCOVERY AND EVALUATION OF POTENTIAL ANTI-HEPATITIS B AGENTS 6.0 The Hepatitis B Virus Problem Hepatitis B virus (HBV) (125) belongs to hepadnavirus family of DNA-containing viruses
that also include woodchuck hepatitis virus (126), ground squirrel hepatitis virus (127), duck hepatitis B virus (128), and heron hepatitis B virus (129). These viruses replicate primarily in the liver cells, and establish chronic infection in their respective hosts. In humans, primary
528 HBV infection and development of acute infection with transient liver dysfunction is characterized by hepatocellular injury and inflammation. Basically, there are two phases of infection: replicative and non-replicative or integrated stages. During the replicative stage, the patient actively produces HBV and hepatitis B surface antigen (HBsAg), HBV-DNA and HBVDNA polymerase is observed in serum; HBV-DNA and hepatitis B e antigen (HBeAg) are detectable in liver tissues (130). Patients typically have active hepatitis with elevated serum aminotransferases during this phase. Subsequently, the viral replicative markers (HBsAg, HBeAg, HBV-DNA and HBV-DNA polymerase) are not detectable, and anti-HBe appears in the serum, which is identified as the non-replicative or integrated stage that is frequently preceded by an increase in aminotransferase levels. During this stage, there is no viral replication, part of the hepatitis genome has become integrated into human DNA, and the patient remains HBsAg positive (131-134). Occasionally, a patient with chronic hepatitis B antigenemia may suffer a clinical relapse associated with symptomatology of acute viral hepatitis, including jaundice, tender hepatomegaly and elevations in serum aminotransferases. These features along with appearance of HBV-DNA, HBV-DNA polymerase and HBeAg, mark patients experiencing reactivation of HBV infection. Approximately 0.8% of chronic HBsAg carriers will relapse due to reactivation of HBV each year (135). The cause of spontaneous reactivation during the course of chronic HBV is unclear, although this has been reported in male homosexuals (136) and during withdrawal of cancer chemotherapy and immunosuppressive therapy (137). Much of the pathology of HBV infection arises from loss of immune response to the core antigen of the virus (HBcAg), which is expressed in infected liver cells. About 5-10% of individuals who are not able to resolve primary infection develop a persistent, usually life- long, chronic infection, with long-term liver disease which is clearly a major health problem. About 300 million individuals (nearly 5% of the earth's population) are chronically-infected with HBV, of which between 0.1-0.5 % (0.3-1.5 million) reside in the USA. It has been estimated that 25 % of these carriers will die of complications related to this disease, representing the ninth leading cause of death (138-140). People chronically-infected with HBV have a greatly increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) (141-155). 6.1 HBV Infection and Liver Cancer Hepatocellular carcinoma (HCC) is one of the major malignant diseases in the world. There is, however, striking geographical variations in its incidence throughout the world; it occurs commonly in sub-Saharan Africa, Southeast Asia, Japan, Greece and Italy (153-157) while in most western countries it is rare but alarming (155). There is a striking correlation between the incidence of HCC and the prevalence of the chronic HBV. Globally, HBV is probably the
529 etiologic agent that is responsible for 75-90% of these primary liver cancers (HCC) (153-155) which is responsible for over a million deaths annually (158). It is now well-accepted that chronic-infection with hepatitis B, C, or D is oncogenic and leads to the development of HCC (142, 153, 159-162). Early evidence for this claim was derived from a multitude of case control retrospective studies dealing with patients with HCC, and from compelling statistics derived from prospective studies of chronic HBV carriers. That infection of animals with their species-specific hepatitis B-like viruses is hepatocarcinogenic also supports this suggestion (163-166). Nonetheless, in spite of the abundance of evidence in support of the relationship between the HBV genome and tumor cell DNA, no clear mechanisms of transformation are available to explain the high incidence of HCC in patients or animals with chronic HBV. However, persistent infection, rather than acute infection, is a major risk factor. Thus, it is reasonable to anticipate that development of liver cancer may be prevented by treatments that cure the chronic infection. As a result, many investigators have attempted to define how HBV replicates itself and establishes a state of persistent virus production in most infected hepatocytes. A number of molecular mechanisms have been proposed, including: an acutely transforming viral oncogene; chromosomal aberrations due to HBV integration (i.e., deletions, translocations, duplication); activation of cellular proto-oncogenes; inactivation of cellular anti-oncogenes; and transactivation of cellular genes by HBV gene products (153). HCC usually develops only after 20-30 years of persistent HBV infection accompanied by hepatocyte necrosis, inflammation and regenerative hyperplasia. Since HBV is not directly cytopathic, liver injury must be immune-mediated. Factors that predispose HBV-infected individuals to develop HCC are chronicity, an immune response and liver injury rather than a direct genetic event. Hepatic injury and continuous hepatocyte regeneration may allow an accumulation of multiple mutational events sufficient for the emergence of HCC (167-169). 6.2 Prevention of HBV Infection The spectrum of diseases associated with HBV, including acute hepatitis, chronic hepatitis, cirrhosis and HCC, is preventable. Both active and passive immunization can protect against HBV infection. The HBV vaccine offers active immunization and is the recommended method of pre-exposure prophylaxis. Currently, plasma-derived and two recombinant yeast vaccines are available, and approximately 90% of healthy adults will develop anti-HBs after the three-dose regimen (170). Even if universal vaccination against HBV could be achieved (which would be very costly for developing countries), implementation would require several decades, and a large worldwide population of chronically HBV-infected individuals still would require medical care including an effective anti-HBV agent, which remains to be developed.
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6.3 Current Treatment for HBV Infections Alpha-interferon has emerged as the most effective agent for the treatment of chronic hepatitis B, but a positive response is obtained with only 30-40% of infected patients. However, recently developed inhibitors of HBV DNA synthesis, such as nucleoside analogs (171-174), have clearly demonstrated antiviral effects, with selective inhibition of active HBV replication. Unfortunately, the effect of treatment is usually transient, and significant toxicity has been encountered in longer treatment regimens (140, 175-180). Thus, interferon (181), when used alone or in combination with other strategies (ara-A or corticosteroid withdrawal), offers much promise, but the moderate to high doses required are associated with significant toxicity, the underlying mechanisms of antiviral action are not well-defined, the effects are often transient, and predictive markers of a beneficial response have not been established. During the past decade, a variety of additional anti-hepatitis B virus treatment regimens have been described (Table 1) (182), but efficacious therapy is still lacking.
TABLE 1: Proposed ideal anti-hepatitis B virus treatment regimen Property
Function
Anti-viral activity
Inhibition of viral genomic replication and all intermediates. Inhibition of viral supercoiled HBV DNA generation and processing.
Envelope antigen binding
Chelation and removal of excessive HBsAg load.
Immunomanipulation
Activation of cytotoxic T lymphocytes and NK cells (eradication of infected hepatocytes). Activation of B lymphocytes (prevention of horizontal transmission).
Hepatoprotective function
Free-radical scavenger. Inhibition of collagen deposition.
Additionally, it is recognized that HBV is not directly cytopathic, and immune mechanisms are important in both the persistence of, and recovery from, infection. Infection with HBV becomes chronic when a patient fails to clear the viremia within the first six months and the infected hepatocytes are not eradicated by cytotoxic T cells (141). Therefore, early treatment of chronic hepatitis B has been attempted as follows: (a) modulation of immune response (by suppression or stimulation), (b) inhibition of viral replication, (c) liver transplant with or without
531 combination of (a) and/or (b), and (d) inhibition of HBsAg processing/secretion. 6.3.1 Modulation of Immune Response Corticosteroids. Corticosteroids are immunosuppressant drugs which have had beneficial effects in autoimmune chronic active hepatitis. They were investigated for the treatment of chronic active hepatitis B (183-185), resulting in increased HBV replication, membrane expression of viral antigen, and delayed HBeAg seroconversion (132, 186). Stopping steroid treatment usually leads to a rebound in hepatic disease activity but may be followed by termination of viral replication within a few months (187). Thus, despite the decrease in transaminase activity, corticosteroids have little role in the treatment of chronic hepatitis B. However, corticosteroids may be useful for pretreatment of certain patients prior to interferon therapy (188) or for enhancing the efficacy of interferon or adenine arabinoside (ARA-A) treatment by prior steroid withdrawal in patients with mild inflammatory activity (189). Reticulose. Reticulose is a peptide-nucleic acid solution in which ribonucleic acid is combined with short-chain peptides. Recently, reticulose has been described as an immune system modulator, and early clinical studies have shown that reticulose has an ability to rapidly inhibit the course of a variety of viral diseases, particularly in the acute stage. However, it has also been reported to be effective in sub-acute and chronic phases of certain infections and has exhibited no reported side effects in over 20 years of use. Reticulose appears to act by alteration of the host cell response to viral replication. It seems to stimulate the immune system via increases in endogenous interferon and increases in lymphocyte production. In clinical trials with patients infected with hepatitis A or B viruses, treatment with reticulose demonstrated positive results in that serum bilirubin levels reached normal levels and white blood cell count showed significant increases, indicating stimulation of the immune system (190). Cytokines, An improved understanding of the control of the immune response by humoral factors, and the synthesis of these compounds by recombinant gene technology, may allow a more targeted enhancement of the immune response with cytokines such as interleukin-2, tumor necrosis factor (TNF), interferon-jS, or interferon-7 (interferon-a will be discussed separately) in patients with chronic hepatitis B. Interleukin-2 causes cytotoxic T cells to mature and increase in number and may thereby enhance elimination of infected hepatocytes. Tumor necrosis factor is an immunoregulatory cytokine with antiviral properties. Its effect in chronic HBV infection is complex and cannot be explained by its antiviral properties. A clinical study has shown that HBV replication is inhibited by low doses of tumor necrosis factor but increased by higher doses (191). An open study in 11 patients suggested that it may be beneficial for the treatment of chronic hepatitis B (192), although these results need confirmation by randomized, controlled studies. Interferon-7, which has low antiviral activity, enhanced the effects of interferon-jS in
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chronic hepatitis, probably as a result of its immunomodulatory properties (193). Interferon-j8 has exhibited antiviral activity used as a single agent as well as in combination with ribavirin (194). However, the most promising results have been achieved with interferon-a. Intetferon-oL. Interferon was first used for chronic HBV in 1976 by Greenberg's group (195). Interferon has a direct antiviral effect by inhibiting viral protein synthesis. Class 1 major histocompatibility complex (MHC) antigen displayed on hepatocytes is enhanced, and it is thought to aid recognition of infected hepatocytes by the immune system. Other potentially beneficial effects include increased natural killer (NK) cell activity (196). Various regimens of interferon-a have been attempted (197) and it has been found that longterm ( > 1 month) low-dose therapy was more effective and better tolerated than short-term highdose therapy (195, 198). Similar results were obtained with recombinant and lymphoblastoid preparations (199-201). The success rate was 30-40%, as judged by decreases in serum viral DNA, and patients responding to therapy have shown losses of HBeAg as well as decreased serum aminotransferase levels. Follow-up liver biopsies demonstrate reduced inflammation (198). It is hoped that this reduces the risk of subsequent cirrhosis and carcinoma which is a timedependent process. Several studies have been performed to evaluate restoration of hepatic markers during and after interferon-a treatment, simply or in combination with other therapies to treat chronic hepatitis B (see section 6.3.5 below). Thymosins. Thymosins are hormone-like polypeptides produced by thymus epithelial cells. They regulate the maturation of T-cells; moreover, they modulate interferon production and stimulate the expression of interleukin-2 and its receptor. A trial of thymosin fraction-5 in four homosexual patients with chronic hepatitis B demonstrated no effect after six months of treatment (202), while treatment of WHV-infected woodchucks with thymosin alpha-1 resulted in clearance of circulating WHV-DNA and a 50-300-fold decline in the levels of replicative intermediates in liver tissue (203). Subsequently, thymosin alpha-1 and thymosin fraction-5 were used to treat chronic hepatitis B in a human pilot study (204). Results were encouraging, and this was followed by several randomized, controlled multicenter trials of thymosin alpha-1 in the U.S. The primary action of thymosin is unknown, but it is probably related to its immuneenhancing effects. 6.3.2 Inhibition of Viral Replication A variety of antiviral drugs inhibit in vitro HBV-DNA production. These substances include nucleoside and non-nucleoside analogs. Nucleoside Analogues, Nucleoside analogues have shown great promise in inhibiting the replication of several pathogenic viruses. Among the 13 antiviral drugs currently approved by the U.S. Food and Drug Administration, 10 are nucleoside analogues. These agents include
533
acyclovir, gancyclovir, zidovudine, ribavirin, dideoxyinoside (ddl), dideoxycytosine (ddC), and vidabavirin, all of which have gained widespread use for the treatment of various viral diseases. These agents act by interfering with the replication of viral nucleic acid, either by inhibiting the relevant DNA or RNA polymerase or by affecting the function of other nucleic acid-modifying enzymes. Several nucleoside analogs have been examined for their ability to inhibit in vitro HBV replication (205-214). Although none has yet been shown to be clinically effective, several recent and ongoing studies suggest that these agents hold promise, either alone or in combination with other classes of antiviral agents such as the interferons (189, 215, 216), thymosins or other drugs with inhibitory activity on the expression/secretion of HBV/HBsAg particles. Treatment may be prior to or after liver transplantation, as discussed below. Non-nucleosides. Suramin has been reported as an inhibitor of duck hepatitis B virus DNA polymerase (DHBV DNAp) activity with an in vitro assay system (217). However, in vivo studies with three male patients with severe chronic active hepatitis showed no suppression of human hepatitis B virus DNA polymerase activity by suramin (218). This study also demonstrated that suramin treatment resulted in toxicity and side effects at the tested concentration. Therefore, higher concentrations were not tested, and the results suggest that suramin has no role in the treatment of chronic active hepatitis. 6.3.3 Liver Transplant Liver transplantation was considered as an ultimate choice for the patients with HCC, but this is problematic for patients with chronic viral hepatitis B, since the new liver is reinfected (selected reviews, 219-221). Therefore, liver transplantation should only be considered in a clinical setting wherein prevention of reinfection of the allograft is attempted (222). The use of hepatitis B immunoglobulin in high doses for prolonged periods delays rather than prevents recurrence (219). 6.3.4 Inhibition of HBsAg Processing/secretion Inhibition of a-glucosidase I. The imino sugar N-butyldeoxynojirimycin (NBDNJ), an inhibitor of the oligosaccharide-trimming enzyme a-glucosidase I, suppressed the secretion of HBV particles in HBV-producing HepG2.2.15 cells as well as HBV-infected HepG2 cells (223). This activity results because three hepatitis B surface antigens (HBs proteins) are singly or doubly N-glycosylated and are essential for the formation of infectious HBV particles. Currently, this substance is under investigation for in vivo studies. Other Inhibitors of HBsAg. Plant extracts have been used widely by traditional medical practitioners for the treatment of jaundice and liver diseases related to HBV (224); however, only a few systematic studies have been done to evaluate the anti-HBV activities of these plants under controlled in vitro or in vivo conditions. In these cases, lead plants as well as pure
534 compounds isolated from natural sources are reported to inhibit the secretion or processing of HBsAg (225-232). Further studies in this field might lead to the discovery of a clinically relevant anti-HBV drug. 6.3.5 Combination therapy Interferon-oL and steroids. As discussed above, the withdrawal of steroids from chronicallyinfected HBV patients has been associated with a surge of transaminase values and occasional clearance of virus, increased HBV DNA replication and membrane expression of viral antigen, while suppressing host cytotoxic T lymphocyte (CTL) activity (187, 233). Due to these effects, it was reasoned that withdrawal of steroids and consequent restoration of CTL activity might enhance the antiviral effect of interferon. The first large controlled trial to investigate combination therapy consisting of prednisone withdrawal and interferon administration was carried out by Perillo's group (189). This study confirmed the effectiveness of antiviral therapy, but it did not elucidate whether corticosteroids added to the benefit provided by interferon alone. Therefore, a subsequent study included comparison of interferon alone with the same regimen following prednisone withdrawal (200), and demonstrated that the prednisone withdrawal combination with interferon seemed no more successful than interferon alone. One patient with cirrhosis died of liver failure after prednisone and interferon therapy which emphasizes the potential risk of "immune lysis hepatitis" in such patients with severe liver disease. Therefore, interferon treatment is not recommended for patients with decompensated cirrhosis, in whom a high rate of adverse effects has been observed (234). Interferon also appears to be of no benefit for those patients with HBV who are East Asian, who have fulminant HBV, or who are chronically immunosuppressed (235, 236). At present, interferon remains the only accepted treatment for chronic hepatitis B with a success rate of approximately 30-40%, while the major problem with some other drugs include toxicity and lack of antiviral potency (138, 237, 238). Alpha-interferon represents a good compromise since it has both immunomodulatory and antiviral properties. Moreover, it is generally well-tolerated. Liver transplant in combination with other antiviral drugs. Nucleoside analogues. A clinical trial of famciclovir in combination with a short course of prostaglandin E in a patient with severe hepatitis B after liver transplantation has suggested complete recovery and normal functioning of the patient, who returned to work as a teacher (239). Immunomodulators. Since immunosuppression is associated with enhanced viral replication, Sheiner et ah, (240) have postulated that clinical recurrence of the disease may be associated with augmented immunosuppression consequent to anti-rejection therapy.
535 6.4 Need for Development of an Effective Treatment for HBV Infection As illustrated by the discussion above, it is important to develop an anti-HBV drug to control the disease. Systemic chemotherapy is the only practical approach. In searching for such agents, which may represent structurally complex or novel chemotypes, terrestrial plants comprise a viable starting point. The use of plants in traditional medicine has been recorded since 2100 BC. In some cultures in India and China, plant extracts have been used to treat jaundice associated with HBV infection (229, 230, 241, 242). Hudson (224) reports several genera of plants that have been evaluated for anti-HBV activity in humans or appropriate animal models, and at least one report exists which describes a plant extract that inhibits the HBV DNA polymerase (243). Thus, precedence exists among plants for anti-HBV activity. In this report we focus on a rapid, automated and selective evaluation of natural products and plant extracts for anti-HBV activity which could be utilized for the development of a drug. Such a drug could be used singly or in combination with other drugs (such as an inhibitor of HBV DNA and/or an inhibitor of HBsAg expression or secretion) to treat chronic HBV infection. 6.5 Development of an In Vitro Anti-HBV Assay System Most antiviral assays are performed utilizing classical virological methods involving the infection of host cells with virions at a certain multiplicity of infection, and subsequent viral quantification by procedures such as plaque formation and antigen expression. These protocols have the advantage of encompassing most, if not all, stages in the life-cycle of the virus. However, these protocols are often lengthy and their application as high-throughput assays is both tedious and cumbersome. Moreover, the analogous test system is not available for HBV. A major problem with test systems for evaluating potential anti-HBV compounds was that no suitable in vitro culture system was available that could accurately mimic acute HBV infection in humans. Prior to the development of a cell-based assay system, the effect of potential anti-HBV drugs was evaluated at the level of inhibiting the production of HBsAg by a HBV-carrying hepatocellular carcinoma cell line (PLC/PRF/5) (228, 244). This cell line was obtained from tumor tissue of a chronically-infected HBV patient (201, 245, 246). It contains multiple integrated copies of a partial HBV genome and produces HBsAg, but it does not produce extrachromosomal HBV DNA (247, 248). In addition, an in vivo woodchuck model was available to evaluate active "leads" obtained from in vitro studies. Woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV) are closely related to HBV. Therefore, these two in vivo models can be used to evaluate the active agents, but they are not very useful for the discovery process. These problems have been resolved by the development of HBV-producing cell lines which
536 have been developed by several investigators by stably transfecting hepatoblastoma cell lines, such as HepG2, with the molecularly-cloned HBV genome. These transfected cell lines produce replicative HBV DNA intermediates and release hepatitis B core (HBcAg, HBeAg) and surface (HBsAg) antigens, as well as mature infectious virions, into the culture medium (249-253). Due to these characteristics, HepG2.2.15 cells (stably transfected HepG2 cells) have been used as an in vitro model to screen for anti-HBV activity (205-213, 226, 254, 255). Recently, various methods have been reported for the screening of anti-HBV agents utilizing HepG2.2.15 cells. These procedures involve the analyses of intracellular extrachromosomal HBV DNA expression (205-207, 212, 226, 254, 255), extracellular HBV DNA secreted into the culture medium (207, 212, 254, 255), PCR amplification of extracellular HBV DNA captured on microtiter plates by an anti-HBsAg monoclonal antibody (209), or determination of HBsAg concentration in the culture medium (207, 226). We currently describe a procedure which was developed to evaluate a large number of plant extracts for anti-HBV activity, as well as cellular toxicity for hepatocyte-derived cells, using HepG2.2.15 cells (226). This assay is rapid, amenable to automation, and selective for the assessment of a large number of test materials for anti-HBV activity. Briefly, natural products and plant extracts are screened for anti-HBV activity, by assessing inhibition of expression/processing/secretion of HBsAg into the culture medium of HepG2.2.15 cells, as well as the inhibition of intracellular extrachromosomal HBV DNA. To eliminate variation, a single 96-well plate was used to evaluate both the cytotoxicity and HBsAg assessments, and a 24-well plate was used to assess intracellular extrachromosomal HBV DNA. 6.6 In Vitro Anti-HBV Assay System for Assessing Natural Products and Plant Extracts Cell Culture. The HBV-producing human blastoma cell line (HepG2.2.15) (253) was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (PCS), 2 mM glutamine, 100 units/ml penicillin, 100 ^g/ml streptomycin and 350 jug/ml gentamicin sulfate (G418) at 37°C under 5% CO2 in air. Similarly, HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% PCS, 2 mM glutamine, 100 units/ml penicillin and 100 ^g/ml streptomycin. Anti-HBV Assay. To evaluate test materials for anti-HBV activity, HepG2.2.15 cells were seeded on 96-well microtiter plates at a density of 3-4x10* cells per well in 190 /xl of medium (5% PCS, no G418). The test materials were then added in 10 /xl of 10% (v/v) aqueous DMSO to a final concentration of 0.5% (v/v) along with DMSO at the same concentration (0.5% v/v) and ddC (200 /xM) which were included as negative and positive controls, respectively. The treated cells were incubated at 3TC under 5% CO2 in air. The medium was collected every two days for six days, and replaced with fresh medium containing plant extracts, DMSO or ddC, to
537 the respective wells. At the end of the sixth day, the medium was removed and each plate was tested for cell survival using a sulforhodamine B staining procedure (see below). Culture medium collected throughout the course of the experiment was stored at -TO^C for subsequent analysis of HBsAg levels. The effect of the withdrawal of the plant extracts after six days of treatment was also studied. At the conclusion of the treatment period, cells were washed three times with phosphate buffered saline (PBS) and fed with fresh medium lacking test materials, DMSO or ddC. The cultures were allowed to recover for six days with the medium changed every two days. Culture medium collected was stored at -70°C and assayed for HBsAg levels as described below. At the end of 12th day, the plates were also examined for cell survival. A second set of experiments, representing a four-fold scale-up of the procedure described above, was carried out in 24-well plates, to determine the effect of test materials on the synthesis of intracellular extrachromosomal HBV DNA as outlined below. Assay for Hepatitis B Surface Antigen (HBsAg). The HBsAg released into the culture medium was assayed by using the Auzyme kit (Abbott Labs, Abbott Park, IL) which is based on capturing the HBsAg on monoclonal antibody-coated beads. Samples collected after six days of treatment of cultured HepG2.2.15 cells, were diluted 4-fold and assayed in triplicate wells. All samples from one experiment were analyzed using an antibody kit from the same lot in order to minimize variability among the experimental data points. The percent inhibition of HBsAg secretion after six days of treatment was calculated relative to the negative control (DMSO). Cytotoxicity Assay. Test materials were evaluated for cytotoxicity by measuring cell survival at the end of the each experiment using the sulforhodamine B staining method (8). DNA Extraction and Analysis. Intracellular extrachromosomal HBV DNA was extracted from cells by the method of Hirt (256) as adopted by Gripon et al. (257). Briefly, cells were lysed in buffer containing 0.5% (w/v) sodium dodecyl sulfate (SDS), 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10 mM EDTA and 200 ixglml proteinase K, and incubated overnight at 3TC. NaCl was added to the lysate to a final concentration of 1 M, and the mixture was stored overnight at 4*'C to allow cellular DNA to precipitate. Chromosomal DNA was pelleted at 29,0(X) X g for 1 hr at 4°C. The supernatant was extracted twice with an equal volume of phenol:chloroformlisoamyl alcohol (25:24:1, v/v/v) followed by two extractions with chloroform:isoamyl alcohol (24:1, v/v). Sodium acetate (pH 5.5) was added to the aqueous phase to a final concentration of 0.3 M, and nucleic acids were precipitated with 2 volumes of ethanol. Pellets recovered after centrifugation at 29,000 x g for 10 min were dissolved in 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA. DNA was analyzed by electrophoresis employing horizontal slab gels comprised of 0.8%
538
agarose, 40 mM Tris-acetate buffer (pH 7.4), and 1 mM EDTA. Gels were soaked in 0.25 N HCl for 15 min, and the DNA was denatured in 0.5 M NaOH containing 1.5 M NaCl for 1 hr at room temperature, followed by neutralization in 1 M Tris-HCl buffer (pH 8.0) containing 1.5 M NaCl for 15 min at room temperature and then transferred to Zeta Probe GT membranes (Bio Rad Laboratories, Hercules, CA) according to the manufacturer's recommended procedure based on that of Southern (258). After transfer, the membranes were rinsed twice with 2X SSC (IX SSC contains 150 mM NaCl and 15 mM sodium citrate, pH 7.0), air-dried and baked at 80°C for 2 hr under vacuum. The membrane was then blocked with pre-hybridization solution containing 7% (w/v) SDS and 0.25 M Na2HP04 (pH 7.2) at 65°C for 5 min, and hybridized at 53°C for at least 16 hr with a p^P] HBV probe. The probe was derived from the 3.2 Kb HBV DNA insert released from pSM2-HBV (gift from Dr. H. Schaller, ZMBH, University of Heidelberg, Germany) by EcoRl digestion, resolved by electrophoresis on 0.8% agarose gel, and recovered by electroelution. This insert was labeled with p^P]dCTP (Amersham Corporation, Arlington Heights, IL) using the "Prime a Gene" labelling system (Promega Corporation, Madison, WI). After hybridization, the membrane was washed twice with a solution containing 20 mM Na2HP04 (pH 7.2) and 5% (w/v) SDS at room temperature. After a 15 min wash at 50''C with the same solution, membranes were air-dried and exposed to film (X-OMAT-AR, Kodak, Rochester, NY). As molecular weight markers: non-radioactive lambda DNA size markers (New England Biolabs, Inc., Beverly, MA), cloned-HBV DNA (3.2 Kb), and PCR-amplified HBV DNA (1.32 Kb) were used. Intracellular extrachromosomal DNA extracted from HepG2 cells, the untransfected cell line, was used as a negative control in Southern blots. 6.7 Data Resulting from Analyses Performed with HEP62.2.15 Cells A number of plant extracts and pure compounds isolated from natural products were tested for anti-HBV activity utilizing HepG2.2.15 cells. Two parameters indicative of viral proliferation were evaluated: the inhibition of secretion of HBV/HBsAg into the medium of the cultured cells and the reduction of intracellular extrachromosomal HBV DNA. Since ddC is known to inhibit HBV replication (209, 213, 254, 255), it was evaluated along with the test materials (0.5% v/v) as a positive control. The results obtained with 84 plant extracts and 57 pure compounds are summarized in Table 2.
539 TABLE. 2.
Plant Extracts and Pure Compounds Evaluated for Anti-HBV Activity
Test Material*
Number Tested Number of "Active Leads" in Anti-HBV Assays HBsAg Secretion Intracellular Extrachromosomal (Inhibition)
Plant Extracts Pure Compounds
84 57
HBV DNA (Reduction)
2 1
Inhibition of HBsAg Secretion, Cultured HepG2.2.15 cells were treated with two-fold dilutions of the test materials ranging from 160-1.25 ^g/ml. The treatment was for a total of six days, with the change of medium containing freshly dissolved test materials every two days. At the end of the sixth day, HBsAg levels were measured in the medium and cytotoxicity was assessed from the same 96-well plates. These data were then used for the estimation of CC50 (50% cell survival) and ED50 (50% inhibition of HBsAg secretion) values, which in turn could be used for the estimation of an "zn vitro therapeutic index" or TI (CC50/ED50). In general, it is expected that this value should be > 1 to provide an indication of potential efficacy. However, for practical purposes, we considered a plant extract to be an "active" lead if the TI value is more than L5, while for pure compounds, the TI value should be more than 3.0. As summarized in Table 3, two plant extracts (Clerodendron inerme and Clematis sinensis) and one pure compound (7-fagarine) fall into this category, and are considered as "active" leads worthy of further study.
540 TABLE 3.
Examples of "active" leads capable of selectively inhibiting secretion of HBsAg with cultured HepG2.2.15 cells after six days of treatment.
Test* Materials
HBsAg Inhibition (ED50 fJLg/mlf
Cytotoxicity (CC50 fig/mlf
Clerodendron inerme (Aerial Part)
16
27
1.69
Clematis sinensis
29
45
1.55
11
66
6.0
TT^CC^JED^'
(Aerial Part) 7-Fagarine
Tlant extracts were dissolved in DMSO and evaluated for anti-HBV activity utilizing HepG2.2.15 cells. ''showed 50% inhibition of the secretion of HBsAg and 50% cell survival as the measure of cytotoxicity. T I is the in vitro therapeutic index. Results are expressed as the means of triplicate determinations relative to DMSO-treated controls. Reversibility of inhibition of HBsAg. Since HBV DNA is integrated into the genome of HepG2.2.15 cells, it was expected that when test materials are removed from the treatment wells after six days of incubation, the inhibitory effect on the secretion of HBV/HBsAg into the culture medium should be lost and the production/secretion of HBV/HBsAg should increase into the culture medium. To test this possibility, cultured HepG2.2.15 cells were treated with various concentrations of Clerodendron inerme, Clematis sinensis or 7-fagarine for six days and HBsAg levels were measured. The cells were then allowed to grow in fresh medium without test materials, for an additional six days, at which time HBsAg levels and cytotoxicity were assayed. During the entire course of the experiment, medium was replaced at two-day intervals, with or without plant extracts, as appropriate. The results are presented in Table 4. In this experiment, HBsAg was inhibited by 57 and 40%, respectively, as a result of treatment with 40 jLig/ml of Clerodendron inerme or Clematis sinensis extracts, for six days. Inhibition of HBsAg observed with 20 fjig/ml 7-fagarine was 82%. Six days after removal of the plant extracts from the medium, HBsAg secretion into the medium reached the level of the untreated control. As compared with the results observed in Table 3, a higher percentage of cell survival was observed in this experiment (Table 4), probably due to replacement of cells during this period. The inhibition of HBsAg at less cytotoxic concentrations suggests that the target for the inhibitory agent is a component or a process unique to the virus.
541 TABLE 4.
Reversal of the inhibition of the secretion of HBsAg into the medium of cultured HepG2.2.15 cells.
Materials Tested
Clerodendron inerme (Aerial Part) Clematis sinensis (Aerial Part)
7-Fagarine
Concentration Tested (jug/ml)
HBsAg
(% Inhibition)
Cytotoxicity (% Survival after 12 days)
After six days of treatment
Six days after removal
40 20 10 5
57 23 24 8
7 0 0 0
54 98 96 100
40 20 10 5
40 33 26 2
0 0 0 0
81 100 89 94
20 10
82 60
6 0
86 100
Results are expressed relative to solvent-treated controls and represent the mean of triplet determination. The above results are in accordance with previous reports concerning nucleoside analogs in similar situations (212). Therefore, we considered this protocol to be appropriate for the purpose of screening large numbers of plant extracts and pure compounds isolated from the natural products for the anti-HBV activity. Reduction of Intracellular Extrachromosomal HBV DNA In addition to assessing HBsAg levels, a known inhibitor of HBV DNA (ddC) and test materials were evaluated for potential to alter the intracellular extrachromosomal HBV DNA levels expressed by cultured HepG2.2.15 cells. Extrachromosomal DNA was extracted by the modified procedure of Hirt (256) as described by Gripon et al. (257) and analyzed by Southern (258) blot analysis with a HBV specific probe. As presented in Figure 1, ddC strongly inhibited intracellular extrachromosomal HBV DNA production at a concentration as low as 25 pM (Figure lA, Lanes 11 and 12, arrows), while solvent, DMSO [0.5% (v/v)] (Figure lA, Lanes 9 and 10) had no effect. In addition, nine plant extracts (Table 5; Fig lA, B, C, D, and E) significantly inhibited the intracellular extrachromosomal HBV DNA production in HepG2.2.15 cells. DNA extracted from HepG2 cells (not expressing HBV DNA) was used as a control for Southern blotting and, as expected, no band corresponding to HBV DNA was observed (Figure lA, Lane 6).
542 TABLE 5.
Plant Extracts Inhibiting the Intracellular Extrachromosomal HBV DNA Production in HepG2.2.15 Cells.
Test Material
Family Part*
Plant (Lanes)
Figure 1
Asparagus officinalis L.
Liliaceae
FU
25-28 (Fig lA)
Bidens pilosa L.
Asteraceae
LS
13-16 (Fig ID)
Caesalpinia bonducella (L.)
Caesalpiniaceae
LF
9, 10 (Fig IE)
Calotropis gigantea (L.) R,Br.
Asclepiadaceae
RT
33-36 (Fig ID)
Coleus X hybridus Hort.
Lamiaceae
PL
37-40 (Fig ID)
Gmelina arborea Roxb.
Verbenaceae
LP
25, 26 (Fig IC)
Picea glauca (Moench) Voss
Pinaceae
LS
37-40 (Fig IC)
Salix alba L. cv. tristis
Salicaceae
LS
29-32 (Fig ID)
Tinospora cordifolia Miers
Menispermaceae
ST
21-24 (Fig ID)
FIGURE 1. Southern blot analysis of intracellular HBV DNA following treatment with ddC or plant extracts for six days. For each lane, approximately 10 jug of intracellular DNA (also observed by ultraviolet-induced fluorescence emitted by intercalated ethidium bromide) was loaded. The experiment was performed in duplicate, and the intracellular DNA was analyzed on an 0.8% agarose gel along with lambda size and HBV-specific DNA markers. For all the Panels A, B, C, D, and E: Lane 1 contained non-radioactive lambda DNA size markers which are not visible in the Southern blot autoradiogram. Lane 3 (M) represents a mixture of electroeluted HBV DNA (3.2 Kb) from EcoRl-digested cloned HBV DNA sample and HBV DNA-specific PCR amplified product (1.32 Kb). Lane 6 represents intracellular DNA extracted from HepG2 cells which do not express HBV DNA, and was included as a negative control for Southern blot analysis. Lanes 2, 4, 5, 7, and 8 represent lanes without DNA which serve as an indication of background radioactivity. See Table 5 for additional designation of lanes. (Figure on following page)
543
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544
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FIGURE 2. Southern blot analysis of intracellular HBV DNA extracted from HepG2.2.15 cells following treatment with ddC, and after removal of ddC from the medium of the treated cells. Intracellular DNA ( - 1 0 fjLg) was loaded onto each lane. The experiment was performed in triplicate, and intracellular DNA was analyzed on a 0.8% agarose gel along with lambda size and HBV-specific DNA markers. Lane 1 represents lambda DNA size marker; Lane 3 represents cloned HBV DNA digested with EcoRl to release HBV cDNA (3.2 Kb) and plasmid DNA (3.6 Kb); Lane 5 represents a mixture of electroeluted HBV cDNA (3.2 Kb) from EcoRl digested cloned HBV DNA sample and HBV DNA-specific PCR-amplified product (1.32 Kb); Lane 8, which serves as a control for the probe specificity, contained intracellular DNA extracted from the parental cell HepG2 that lack the HBV DNA. Lanes 11-13 represent DNA extracted from HepG2.2.15 cells treated with DMSO; Lanes 14-22 represent DNA extracted from HepG2.2.15 cells treated with ddC at 100 juM (Lanes 14-16), 200 fxM (Lanes 17-19) and 300 [xM (Lanes 2022); Lanes 25-33 represent DNA extracted from HepG2.2.15 cells which were recovered for 6 days after a 6-day treatment with ddC at 100 fxM (Lanes 25-27), 200 (JLU (Lanes 28-30) and 300 fxM (Lanes 31-33). After removal of ddC from the medium, cells were cultured for six days post-treatment with changes of medium without ddC for every two days. Lanes 2,4,6,7,9,10,23 and 24 are lanes without DNA. The arrows indicate the HBV-specific DNA bands.
545
Reversibility of Reduction of Intracellular Extrachromosomal HBV DNA Additional experiments were then performed to assess the reversibility of ddC-inhibited expression of intracellular extrachromosomal HBV DNA. As shown by the Southern blot analyses presented in Figure 2, synthesis of intracellular extrachromosomal HBV DNA was completely inhibited after a six day treatment with ddC at all concentrations tested, including the lowest concentration, 100 /xM (Lanes 14-22). The expression of intracellular extrachromosomal HBV DNA, compared to DMSO-treated cells (Lanes 11-13, arrows), recovered after removal of ddC (Lanes 25-33, arrows). DNA obtained from HepG2 cells (Lane 8) presented no band corresponding to HBV DNA.
546 6.8 Summary of Anti-HBV Results and Procedures In summary, a simple cell culture assay system was developed for the evaluation of natural products for anti-HBV activity, the use of the stably-transfected HepG2.2.15 cell line not only simplifies the antiviral assay and makes it more amenable to automation, but also enables the evaluation of natural products as inhibitors of viral replication, expression, and maturation, in addition to the post-translational modification of viral protein(s), and virus release. Medicinal plants are used to treat viral jaundice in different parts of the world (227-231, 241), but few attempts have been made to confirm the anti-HBV activity of these plant extracts. These natural products remain a largely untapped source of anti-HBV compounds and we are focussing on the isolation of active components from medicinal plants with putative anti-HBV activity. Accordingly, the protocols described herein have been adopted for use with natural products, and they are suitable for bioassay-directed fractionation. Selectivity is an important criteria that was introduced at an early stage in the discovery process. All the test materials were evaluated for both cytotoxicity as well as anti-HBV activities, and a therapeutic index (TI) for the inhibition of secretion/processing of HBsAg was calculated.
This value is useful for the
selection of primary lead materials that can be subjected to more advanced testing.
7.
CONCLUDING REMARKS We currently describe drug discovery programs for three disease states that ostensibly have
little in common. In each case, however, it is clear that common strategies can be applied for the construct of biology-driven programs. The systems employed are obviously not designed for high-throughput or high-capacity screening; they are much too labor-intensive. Testing is limited to hundreds or perhaps thousands of starting materials on an annual basis, which leads to judicious decisions. Any available information, such as data from ethnomedical studies or scientific literature, is taken into account, and dereplication procedures are being used with increasing frequency. Moreover, the assay methods remain focussed on the identification of active leads with a relatively high probability of yielding therapeutically useful drugs. From a realistic viewpoint, the challenge of finding efficacious agents for the systemic treatment of life-threatening diseases such as cancer, AIDS, hepatitis, and malaria, is immense. The magnitude of past efforts that have led us to our current state of knowledge and technology is difficult to comprehend, in terms of human and economic resources and expenditures. This leads to the realization that a breakthrough discovery probably is not imminent, but the search must continue using the best tools and experimental designs that are currently available. The systems described herein, while labor-intensive and inappropriate for high-throughput programs, nonetheless yield leads in the range of 1-5%. This provides a reasonable workload for those
547
involved in isolation, structure elucidation, chemistry and other areas of development, and given the fact that even a single discovery capable of altering the course of a terminal disease would be of monumental importance, we are not concerned with limited capacity. Selectivity, as imposed by biological complexity, indices, and secondary discriminators, should lead to the discovery of agents capable of mediating pharmacological responses of practical utility, and this aspect remains of paramount importance. 8.
ACKNOWLEDGMENTS The authors are grateful to a number of generous collaborators who have supplied cell
lines and other materials described in this review. Experimental work in these areas is supported by the National Institute of Allergy and Infective Diseases, NIH (CKA), Shaman Pharmaceuticals (HM), and the National Cancer Institute, NIH (IMP). REFERENCES 1.
J.M. Pezzuto, Biochem. Pharmacol., 53 (1997) 121-133.
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G.A. Cordell, C.W.W. Beecher and J.M. Pezzuto, J. Ethnopharmacol., 32 (1991) 117133.
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561
Synthesis of Carotenoids
Hanspeter Pfander, Marc Lanz and Bruno Traber 1.
INTRODUCTION The brightly coloured carotenoids have aroused the curiosity of scientists since the
beginning of organic chemistry and the first publications to deal with the carotenoids date from the early part of the 19th century. They relate to well-known food materials and colours such as paprika, saffron, annatto, carrots and to autumn leaves. During the last century the emphasis was on isolation of the pigments and their characterization. The investigations during the first part of the 20th century were dominated by establishing structural formulae and developing synthetic methods. In 1930 Karrer established the correct constitution of lycopene (1) and p,p-carotene {= p-carotene, 2) (Figure 1) and in 1950 the total synthesis of p,p-carotene (2) was reported independently by Karrer,
Inhoffen and Milas. This labile molecule with its long conjugated polyene chain presented difficult practical problems that had not been encountered with other groups of natural products and required the development of new strategies and methods, especially for the construction of the polyene chain and linking together different parts of the molecule. The second half of this century was characterized by a continuous development of carotenoid research and current research encompasses a wide variety of fields and interests besides organic chemistry, such as plant physiology, food science, environmental science, taxonomy, industrial chemical synthesis, biotechnology and especially medical research. The continuous development of carotenoid research is reflected in various monographs and reviews published over the years and three generations of books under the title 'Carotenoids' have been published by Birkhauser. The first one, by Karrer and Jucker, published in 1948 [1] and the second, edited by Isler and published in 1971 [2], became classics which are still widely used as valuable sources of information. The book by Isler contains a major chapter of 250 pages on total synthesis and this comprehensive and authoritative review describes systematically the construction of many synthons and
562 the synthesis of many natural and unnatural carotenoids and related compounds. The list of naturally occurring carotenoids that was included in the Isler book, later appeared in an expanded form as a 'Key to Carotenoids' [3]. The rate of discovery and characterization of new carotenoids since then has been rapid, and a second edition of 'Key to Carotenoids', listing 563 carotenoids, was published by Birkhauser in 1987 [4]. Recently a new book series 'Carotenoids', again published by Birkhauser, was started. The main objective of this new series is for the older generation of carotenoid workers to pass on experience and expertise in a way that will be of value not only to established carotenoid researchers, but especially to workers coming new to carotenoids. The books in the new series are intended to be workbooks and this is especially true of the coverage of general methods pf carotenoid isolation and analysis which forms the subject of the two parts of Volume 1 [5,6]. Volume 2 of the series is the first book to be published that is devoted entirely to the total synthesis of carotenoids [7]. The main text consists of four chapters each dealing with a major topic and divided into a number of parts. The essential foundation for work in the field is a thorough appreciation of the principles and strategies of carotenoid synthesis and this is provided in Chapter 1. A major task in carotenoid synthesis is the construction of the polyene chain. The coupling reactions commonly used for carbon-carbon double bond formation are dealt with In Chapter 2. The various parts of Chapter 3 describe the preparation of polyene synthons and the total synthesis of different groups of carotenoids. Technical synthesis and the synthesis of isotopically labelled carotenoids are also covered. Eight 'Worked Examples' which describe in detail experimental procedures for key reactions in carotenoid synthesis are presented in Chapter 4. Finally two appendices provide tables of useful synthons and a list of natural carotenoids that have been prepared by total synthesis. In addition the published proceedings of the International Symposia on Carotenoids, held at 3-yearly intervals under the sponsorship of the International Union of Pure and Applied Chemistry, provide further important information on the synthesis of carotenoids [8-32]. Most of the carotenoids have a trivial name which is often derived from its natural origin and which gives no information on the structure. Therefore a semi-systematic nomenclature has been developed, which has been approved by the International Union of Pure and Applied Chemistry and the International Union of Biochemistry [33] and which is accepted by Chemical Abstracts for official documentation. According to the lUPAC-lUB rules 'Carotenoids are a class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls) consisting of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the centre of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship. All carotenoids may be derived formally from the acyclic C40H56 structure 1, having a long central chain of conjugated double bonds (Figure 2), by (i) hydrogenation,
563 (ii) dehydrogenation, (iii) cyclization, or (iv) oxidation, or any combination of these processes. This class also includes molecules which contain additional carbon atoms as
Figure 2
substituents to the normal C4o-skeleton and certain compounds that arise from rearrangements of the carbon skeleton 1, or by the (formal) removal of part(s) of this structure. All specific names are based on the stem name 'carotene', which corresponds to the structure and numbering shown in Figure 3. The broken lines at the two terminations represent two 'double-bond equivalents'.' As shown in Figure 3 Individual compounds may have acyclic or cyclic Cg-end groups.
9
11 10
Figure 3
12
14
564 The structure and nomenclature of the carotenoids according to the lUPAC-lUB rules were recently described with many examples by Weedon and Moss [34]. The main functional groups that occur in the xanthophylls, the oxygen-containing carotenoids, are the hydroxy, methoxy, carbonyl, carboxylic acid, ester and epoxy groups. Furthermore carotenoids with carbon-carbon triple bonds or an allenic groups are known. In addition carotenoids also occur as glycosides, glycosyl esters and carotenoproteins and a special case are the sulphates In which the hydroxy group of the carotenoid is esterified with sulphuric acid. The combination of the different end groups and functional groups leads to the great number of different structures of the carotenoids and up to date more than 600 carotenoids have been isolated from natural sources from which more than 400 bear one or more chiral centres [4,35]. Carotenoids are familiar to us through the orange-red colours of popular foods like oranges, tomatoes and carrots, and the yellow colours of many flowers. Carotenoids are essential to plants for photosynthesis, acting in light-harvesting and, especially, in protection against destructive photooxidation. Without carotenoids photosynthesis in an oxygenic atmosphere would be impossible. But carotenoids are not simply pigments of terrestrial plants. They also occur widely In archaea, bacteria, fungi and algae and the production of carotenoids in seaweed runs to hundreds of millions of tons per year. Some animals use carotenoids for coloration, especially birds e.g. flamingoes and canaries, and fish e.g. goldfish and salmon, and a wide variety of invertebrate animals, where complexation with protein may modify their colour to blue, green or purple. Above all carotenoids are important factors in human health. The essential role of p,p-carotene (2) and others as the main dietary source of vitamin A has been known for many years. More recently, protective effects of carotenoids against serious disorders such as cancer, heart disease and degenerative eye disease have been recognized, and have stimulated intensive research into the role of carotenoids as antioxidants and as regulators of the immune response system. Due to the ubiquitous occurrence and the various important biological functions of carotenoids, their synthesis is of high importance. On the other hand the great variety of structures and the relative instability of the carotenoids, i.e. their susceptibility to oxidation, addition of electrophiles including H"" and Lewis acids, and E/Z-isomerization caused by light, heat or chemicals make the synthesis demanding. In the following article the strategy and methodology for preparing carotenoids by total synthesis is discussed first; in the main part the syntheses of the most important carotenoids are outlined, followed by the discussion of the technical syntheses. Included is also an extended list of references which should allow the reader to obtain more and detailed information.
565 2.
STRATEGY AND METHODOLOGY Strategy In principle an almost countless number of strategies can be developed for the
preparation of a carotenoid, but in reality only a few strategies and methods have been worked out successfully and they have since been used repeatedly [36]. In the last step of the synthesis which leads to the entire carotenoid molecule, in principle either a carbon-carbon single bond or a carbon-carbon double bond has to be generated. Although the methodology for the formation of a carbon-carbon single bond between two double bonds is known, the alternative strategy, the formation of a carboncarbon double bond, is most often used in carotenoid synthesis. The planning of a synthesis for carotenoids consists mainly of two steps {Figure 4): I) formal dissection of the target carotenoid into a middle part and the end groups, ii) choice of the coupling reactions for the final step and definition of the appropriate functional groups (FG). end group
end group
middle part
middle part
end group
Figure 4
For the dissection one has to keep in mind that the molecule contains positions which permit a coupling in high yield [C(9)-C(10) and especially C(11)-C(12)] whereas others (especially C(7)-C(8) in cyclic carotenoids) give products only in low yields. Whereas the choice of the middle part, a conjugated polyene, and its synthesis have become a simple matter today, the design and the synthesis of the end groups is a challenging task. Experience has shown that the synthesis of end groups must be completed before they are attached to the middle part since chemical transformation of a complete carotenoid often results in a significant reduction of the overall yield. Most of the naturally occurring carotenoids are chiral and therefore targets for synthesis in enantiomerically pure form. This can sometimes be achieved by preparing the carotenoid
in
racemic form
and
then
resolving
the
optical
isomers
via
diastereoisomers or by chromatography on a chiral stationary phase. However, in most cases, the construction of the optically pure end group is the most efficient way and for
566 this task three different approaches have been applied. The first involves the preparation of a racemic intermediate from which the required enantiomer is resolved and used for the synthesis of the carotenoid in optically active form. In general this approach should only be selected if both optical isomers of the carotenoid are desired. More elegant are direct
asymmetric
syntheses,
as
e.g.
the
Katsuki-Sharpless
epoxidation
or
dihydroxylation, or the synthesis ex chiral pool in which a chiral natural product, such as a carbohydrate, amino acid or terpene, is partially or as a whole built into the target molecule. Coupling Reactions The most important coupling reaction for the synthesis of carotenoids is the Wittig reaction' or 'Wittig-carbonyl olefination' or 'Wittig condensation' in which a phosphorus ylide 3 reacts with a carbonyl compound 4 to give an olefin 5 and triphenylphosphine oxide (6) (Scheme 1). Ph
0=^
H
+ Ph
4
Ph
H
•
PhjP=( H
Ph3P = 0
Ph
H
3
+
}={ 6
6
Scheme 1
The reaction, which was discovered in 1953 by Wittig and Geissler, has developed into one of the most important methods for the synthesis of olefins and it has become indispensable for the synthesis of sensitive carotenoids and for the preparation of carotenoids on an industrial scale. The application of the Wittig reaction to the synthesis of carotenoids has recently been reviewed [37]. In comparison to other carbonyl olefination reactions, the Wittig reaction has a number of advantages. First of all the formation of the new carbon-carbon double bond proceeds regiospecifically, i.e. at the position of the original carbonyl function. Furthermore the starting materials, namely carbonyl compounds and phosphonium salts generally are readily accessible and the phosphorus ylides do not need to be isolated. As the Wittig reaction is carried out generally under mild conditions, i.e. alkaline or virtually neutral medium, it is an excellent method for the synthesis of the sensitive carotenoids. Furthermore many functional groups, such as acetals, epoxides, esters etc. are stable when suitable reaction conditions are selected. The principle disadvantage of the Wittig reaction is its sensitivity to steric hindrance. The yields of trisubstituted olefins are frequently low and the method fails in the synthesis of tetrasubstituted olefins. Sometimes it is also difficult to separate the olefin from the triphenylphosphine oxide which Is formed as a joint product. In addition the (E/Z)-selectivity of the Wittig reaction is often difficult to predict. The disadvantages of the Wittig reaction led to the development of modified methods and the most important modification is the olefination by means of phosphonate carbanions, as developed by Horner and by Wadsworth and Emmons. In the Horner-
567 Wadsworth-Emmons olefination, or in brief, the Homer-Emmons reaction, a phosphonate carbanion 7, produced by deprotonating a phosphonate 8, reacts with a carbonyl compound 9 to give an olefin 10 and a dialkyi phosphate anion 11 {Scheme 2).
?\ I'
-5
?\
base
"
(RIO^PV^/R' -B^se^ H H 8
5
(R^0)2P....R' H ^ 7
> R^^""
^
9
R' \
^—
/
ff
R'
) = / R H 10
+
C^
(RiO),P-oO 11
Scheme 2
Phosphonate carbanions are suitable starting materials for the synthesis of trisubstituted and tetrasubstltuted olefins. In general, the olefins are formed with high (E)selectivity. Another important advantage of the reaction is the fact that the by-product is a water-soluble phosphate which can easily be separated from the olefin. Since carotenoid synthesis began, the enol ether condensation has frequently been used for the formation of carbon-carbon double bonds and this reaction was also applied for large-scale industrial production of carotenoids. The reaction is an addition of an enol ether 12 to an acetal 13, promoted by a Lewis acid, especially BF3-etherate or ZnCl2, and involves a chain lengthening of two or more carbon atoms to yield an intermediary 3-alkoxyacetal 14 which subsequently is converted into an unsaturated aldehyde 15 (Scheme 3). 2
R^
_ OR 13
2
+
_ /
^^^ Lewis acid
r=r:i
R^
R
\
X-
A R
12
14
PR
< \
„2 OR^
R
R 15
Scheme 3
The reaction mechanism and the application of the enol ether condensation in the synthesis of carotenoids have recently been reviewed [38]. The main advantage of the enol ether condensation, compared with the aldol condensation, is that the enol ether reacts exclusively as the nucleophilic reaction partner and the acetal exclusively as the electrophilic one and this leads unequivocally to the desired, uniform, reaction product. As the alkoxy group of the starting acetal takes part in the formation of the new acetal grouping that results from the enol ether moiety, it is preferable for all the alkoxy groups of both reactant to be identical. Most of the examples published have been carried out with vinyl methyl ether (16) and vinyl ethyl ether (17), used to extend the carbon skeleton by two carbon atoms, or propenyl ethyl ether (18) and 1-methoxy-1-methylethene (19) for the extension by three carbon atoms (Figure 5).
568
O 16
17
19
18
Figure 5
A modification of the enol ether condensation is based on the observation that trimethylsilyl enol ethers 20 react readily with acetals 21 at very low temperatures in the presence of Lewis acids to give alkoxyaldehydes 22 with much higher reaction rates compared to the alkyl enol ethers (Scheme 4). OR 2
R^ H
0R2
21
\
^
R
0 - Si(Me)3 20
^ ^
0R2
R3 CHO
Ri
R^ 22
Scheme 4
The finding that 1-trimethylsllyioxybuta-1,3-dienes such as 23 react with acetals such as 24 not only in the expected 1,4-addition manner but also to give 5-alkoxy-a,punsaturated aldehydes in high yield and free from side products, established the silyloxydienes as important building blocks for carotenoid synthesis as shown in Scheme 5 for the synthesis of 25. OMe OMe
24
23
Scheme 5
The aldol condensation has been used in carotenoid synthesis In the formation of building blocks and in the construction of the entire carotenoid molecule. However the application of this reaction Is rather limited, especially because of side reactions which lead to low yields. The most important example for the application of the aldol condensation is the synthesis of carotenoids with K-end groups. The keto-end group 26 reacts with crocetindialdehyde (27) to give capsorubin (28) (Scheme 6).
27
Scheme 6 (continued....,)
OH
26
569
However, a major disadvantage of this synthesis is the fact that the keto building block, which is more difficult to synthesize, has to be used in a large excess. The most important organometallic reagents used for the carbon-carbon bond formation in carotenoid synthesis are the metal acetylides, metal alkenyls and the Grignard reagents. However the Refomnatsky reaction has also been applied and recently also transition metals such as Pd, Ni, Cu and especially low valent Ti have been used. The application of organometallic reactions for the synthesis of carotenoids and retinoids has recently been reviewed [39]. Because of their ready accessibility and their ease of reduction to give alkenes of predictable stereochemistry, metal acetylides have been widely applied for the synthesis of carotenoids. The reactions involve a nudeophilic addition of the metal acetylides RC^CM (M = Li, Na, K or MgHal) to aldehydes and ketones to give a-hydroxy alkynes. The metal acetylides are usually prepared by the deprotonation of terminal alkynes with strong bases, such as LiNHa, NaNH2, alkyllithium, potassium f-butoxide or with simple Grignard reagents. Examples of the application of these reactions for the synthesis of suitable building blocks and the construction of the carotenoid molecule are given in Scheme 22 and 23. A reaction with an acetylide was applied by Isler [40] for the first synthesis of vitamin A (29), which is still practised industrially. The C2o-skeleton is built up by addition of the Ci4-aldehyde 30 to an ethereal solution of the acetylide 31 to give the diol 32 which is afterwards transformed to vitamin A (29) (Scheme 7). Another prominent example is the synthesis of p,p-carotene (2), once practised on an industrial scale, by the reaction of the di-Grignard reagent of acetylene with a C19aldehyde according to the strategy C19 + C2 + C19 = C40 (see Scheme 13).
570
Scheme 7
Of utmost importance for the preparation of suitable building blocks in carotenoid synthesis is the preparation of vinyl alcohols by the reaction of aldehydes and ketones with vinyl magnesium halides, especially with the commercially available bromides. The most important application is the construction of a Ci5-building block by the reaction of a Ci3-synthon, most often a derivative of the ionones, with vinyl magnesium bromide or chloride (see Scheme 16). Alkenyl lithium reagents, which may be prepared by metal/halogen exchange, are much more reactive than the corresponding magnesium reagents and therefore mild reaction conditions can be applied. The reaction is exemplified by the synthesis of retinal (33) in which the alkenyllithium sllylenol ether 34 is condensed with p-cyclocitral (35) to give, after hydrolysis, the Ci5-aldehyde 25. A further condensation with 34 gives retinal (33) as a mixture of (E/Z)-isomers {Scheme 8).
34
34
OSiMe,
The sulphone coupling which was applied for the first time to the synthesis of vitamin A (29) by Julia [41] has frequently been used for the preparation of retinoids and carotenoids [42]. In this reaction a carbon-carbon double bond is produced by alkylation of the a-carbanion of a sulphone 36 by a halogen derivative 37 to give 38, followed by a base-promoted elimination to give the desired alkene 39 (Scheme 9).
571 SO,Ph
SO^Ph
,Je . Xr
->
37
36
p^"
38
39
Scheme 9
After the elimination, the water-soluble sulphinates can be readily recovered, in contrast to the Wittig reaction which suffers from the difficulty of recycling phosphine oxide. Modifications of the Julia olefination lead to more general routes to a variety of carotenoids. The success of this method is attributable to improvement of the reductive cleavage of the sulphonyl group by reduction with dithionites [29]. A prominent example of the application of the sulphone coupling is the synthesis of p,pcarotene (2) by the reaction of the Ci3-sulphone 40 with the dichloride 41 to give 15,15'didehydro-p,p-carotene, which was transformed to p,p-carotene (2) (Scheme 10).
Scheme 10
3.
SYNTHESIS OF POLYENE SYNTHONS
As outlined before, the construction principle that is most often used for the synthesis of carotenoids is to couple various end groups to central polyene synthons. As outlined in a recent review [43], most of the synthetic routes to C4o-carotenoids follow the strategy C15 + C10 + C15 = C40, confirming the importance of central Cio-compounds. A key intermediate for the synthesis of symmetrical central building blocks is the acetylenic did 42 which can easily be synthesized by a Grignard reaction of acetylene dimagnesium dibromide (43) with 2-methylprop-2-enal (44) (Scheme 11). OH -MgBr
BrMg44
43
44
Scheme 11
From compound 42, the most often used Cio-dial 45, the dibromide 46 and the diphosphonium sait 47, as well as the corresponding 15,15'-didehydro derivatives 48 - 50 have been synthesized (Figure 6). In addition, the synthesis of unsymmetrically
572 functionalized central Cio-building blocks, such as the C-io-phosphonium acetal 51 has been reported. O \ . - - ^ ^ ' ^ ^ .^'^^ / ^
Br. ^ ^ ^ ^-^^ ^.^-s. ^^^^
BrPh-P ^ / v > ^ / . c . ^ / v . \ / ^
47
46
BrPh^P, 49
48
50
MeO
Figure 6
Besides the Cio-dial 45, crocetindialdehyde (27), the central C2o-dialdehyde, is of utmost importance, especially for the synthesis of acyclic carotenoids. Partially protected dials, which are often necessary for the synthesis of unsymmetrical carotenoids, have also been prepared. The C2o-clial 27 is synthesized by the Wittig reaction of the Cio-dial 45 with the Cs-phosphonium salt 52 (Scheme 12). OEt
4.
SYNTHESIS OF THE END GROUPS AND THE CAROTENOIDS
4.1.
Carotenoids with the j^-end group P,p-Carotene p,p-Carotene (2) is the most common representative of the carotenoids and was
the first carotenoid to be synthesized. Three syntheses were reported nearly simultaneously by different authors [44-48] and, since then, many different approaches have been reported. p,p-Carotene (2) has been produced on a commercial basis since 1954 by F. Hoffmann-La Roche and since 1972 also by BASF, applying different processes. The synthesis of p,p-carotene (2) on a laboratory scale is described in
573
'Carotenoids' Volume 2 [49]. One of the most useful syntheses of p,p-carotene (2) starts from the Cig-unit 53 by application of the C19 + C2 + C19 = C40 strategy (Scheme 13) [46-48]. The C2-unit, acetylene dimagnesium dibromide (43), was condensed with the Cig-aldehyde 63, prepared from the Ci4-building block 30, to give the C4o-diol 54. Allylic rearrangement and simultaneous dehydration, partial hydrogenatlon over Lindlar catalyst and isomerization in high-boiling petroleum ether gave (all-E)-p,p-carotene (2).
Scheme 13 Zeaxanthin The 3-hydroxy-p end group is the most abundant chiral end group in carotenoids and is often called the zeaxanthin end group. Zeaxanthin (55) possesses two chiral centres at C(3) and C(3'), making possible three optical isomers, namely the (3R,3'R)isomer (most abundant in Nature) and the (3S,3'S)-isomer as well as the (3f?,3'S)-isomer which constitutes a meso-form. It is this optically inactive mixture of isomers which is usually obtained by synthesis of the so-called racemate [50]. The most important contributions to the synthesis of the optical isomers of zeaxanthin have been achieved by the research group of F. Hoffmann-La Roche. From 6oxoisophorone (56) as starting material, the chiral centre was introduced by an enantioselective fermentative
reduction of the double bond with baker's yeast.
Subsequent diastereoselective and regioselective reduction of the resulting diketone 57 gave a mixture of epimeric hydroxyketones, (3R)-58 and (3S)-58, which could readily be separated by Craig distribution (Scheme 14).
574
(3S)-58 Scheme 14
By reaction of the Cg-hydroxyketone (3R)-58 with isopropenyl methyl ether, the acetonide 59 was obtained. Subsequent reaction with but-3-yn-2-ol (60), acetylation and dehydration
gave
the
diacetate
61 which
was
reduced
with
sodium
bis-(2-
methoxyethoxy)aluminium hydride (SMEAH) to (3R)-3-hydroxy-p-ionol (62) {Scheme 15).
(3R)-58
OH 60 AcO
Scheme 15
For the synthesis of the C40 carbon skeleton the C13 + C14 +613 = 640 and the Ci5 + Cio + Ci5 = C4o strategies have been used, and the latter has been developed on a technical scale. The Ci5-phosphonium salt 63 was prepared by oxidation of 62 to (3R)-3hydroxy-p-ionone (64), followed by vinylation with 65 to the epimeric vinyl carbinols 66 and treatment with triphenylphosphine hydrochloride {Scheme 16).
Scheme 16
575
The reaction of 63 with the Cio-dialdehyde 45 gave, after isomerization, (allE,3R,3'R)-zeaxanthin (55) {Scheme 17).
Recently an efficient synthesis of the phosphonium salt 63, from the key building block (3R)-58 according to a Cg + C2 + C4 = C15 strategy, has been reported [51]. Violaxanthin The most abundant carotenoid with the 3-hydroxy-5,6-epoxy-5,6-dihydro-p end group is violaxanthin (67), which is formed in large quantities in higher plants. For the synthesis of this carotenoid in optically active form, the route Ci5 + Cio + Ci5 = C4o was used. As starting material for the synthesis of the Ci5-building block 68, the Cio-ester ketone 69 was selected, and this was protected, rean-anged and reduced to the allylic alcohol 70. Sharpless epoxidation of 70 gave, in high yield (93%) and high stereoselectivity (97% e.e.), the epoxide 71. The alcohol was oxidized by a Swern oxidation with oxalyl chloride and DMSO to the aldehyde 72. Chain elongation with the Cs-phosphonate 73 by a Horner-Emmons reaction resulted in the Ci5-ester 74 in an (E/Z)-r3i\o of 7:1. Deprotection of the ketal and simultaneous reduction of the ketone and ester groups resulted in a mixture of the diastereoisomeric diols (3/?)-75 and (3S)-75, from which the trans isomer (3S)-75 could be crystallized directly. A selective oxidation of (3S)-75 with I\/In02 gave the Ci5-aldehyde 68 which was reacted with the acetylenic C10diphosphonate 76. After hydrogenation and isomerization, (all-E)-violaxanthin (67) was obtained [52] {Scheme 18). CO,Et
o
OH
^ 69
70
Scheme 18 {continued...,)
576
jTo
(EtO)2Pv.AxrC02Me 73 w
CO,Me O ^
Canthaxanthin Canthaxanthin (77), containing two 4-oxo-p end groups, is produced on an industrial scale by partial synthesis from p,p-carotene (2). Many different approaches for the total synthesis of canthaxanthin (77) have been investigated and recently the application of the sulphone method has proved successful. As starting material, racemic a-ionone (rac-78) was transformed, with peroxyacetic acid, to the 4,5-epoxide, which was opened to give the 4-hydroxy-p compound, that led upon oxidation to the ketone 79. Successive Grignard reaction with vinylmagnesium chloride (80), halogenation, and reaction with a sodium sulphinate gave the sulphone 81, which was transformed to the corresponding ketal 82. Reaction with the Cio-dialdehyde 45, reduction with sodium dithionite/NHa, deprotection, and isomerization gave (all-E)-canthaxanthin (77) [29,53] (Scheme 19). ^
^^^ rac-78
Scheme 19 {continued...,)
MgCI 80
577
. ^ ^ ^ CH2S02-PhCI
Scheme 19
Astaxanthin The
most
important
carotenoid
bearing
3-hydroxy-4-oxo-p-end
astaxanthin (83). All three stereoisomers, Le. the (3S,3'S)-83 and
groups
is
(3R,3'R)-83
enantiomers, and the (3/?,3'S)-meso-form [(3R,3'S)-83], have been isolated from natural sources; the (3S,3'S)-isomer [(3S,3'S)-83] occurs most abundantly. A mixture of the three stereoisomers is produced on an industrial scale and is used mainly as a feed additive in aquaculture. The first synthesis of (3S,3'S)-astaxanthin [(3S,3'S)-83] starting from the Cghydroxyketone (3f?)-58 was reported by F. Hoffmann-La Roche [56]. A technical procedure for the synthesis of (3S,3'S)-83 and (3/?,3'R)-83 starting from 6-oxoisophorone (56) has recently been developed. For the synthesis of the (3S,3'S)-isomer [(3S,3'S)-83], 6-oxoisophorone (56) was transformed to the (3R)hydroxyketone (3R)-58 which was reacted with sulphuryl chloride to produce the diastereoisomeric
chloroketones
(3S,5R)-84
and
(3S,5S)-84.
Subsequent
dehydrochlorination with 5-ethyl-2-methylpyridine gave the chiral building block 85 in 92% yield referred to (3R)-58 [55,56] (Scheme 20).
CI (3S.5S)-84
Scheme 20
Different approaches have been investigated for the conversion of the building block 85 into the ketal 86, which constitutes a key intermediate in the synthesis of
578 (3S,3'S)-83. In the first approach, the functionalization was achieved by reaction of 85 with Br2 to give a mixture of the diastereoisomeric dibromoketones (4S)-87 and (4R)-87, from which, after repeated equilibration/crystallization, the desired c/s-compound (4R)-87 was isolated in 68% yield. Dehydrobromination of (4R)-87 in pyridine gave the monobromo compound 88 from which the dihydroxy compound 89 was obtained by mild hydrolysis with NaHCOa. By treatment of 89 with 2,2-dimethoxypropane, the desired synthon 86 was obtained in a yield of 4 1 % referred to 85 [Scheme 21).
Br {4S)-87
HO
Br
88
H
/:,,
Br
Br
(4/^)-87
OH 89
Scheme 21
According to previously described procedures [57], the chiral building block 86 was afterwards transformed to (3S,3'S)-astaxanthin [(3S,3'S)-83] in four steps in an overall yield of 48%. Alkylation of 86 with the Li derivative of the protected Ce-building block 90 gave the Ci5-dlhydroxyketone 91. Partial reduction of this with Zn/acetic acid followed by reaction with triphenylphosphine hydrobromide gave the phosphonium salt 92. According to the scheme Ci5 + Cio + Ci5 = C4o, the Wittig reaction of 92 with the dialdehyde 45, in the presence of NaOMe as base, gave (3S,3'S)-83 with an e.e. of 99.2% {Scheme 22).
Scheme 22 (continued...,)
579
(3S.3'S)-83 Scheme 22
Diatoxanthin Most of the natural acetylenic carotenoids have an acetylenic group in the 7and/or the 7'-position. An appropriate synthon to construct acetylenic carotenoids is (E)-3methylpent-2-en-4-yn-1-ol (93) which is a commercially available Ce-unit containing an acetylenic bond. Recently the synthesis of diatoxanthin (94) and its (9Z)-isomer have been reported [58,59]. A Grignard reaction of the acetylenic Ce-synthon 93 with EtMgBr and the protected 6-oxoisophorone 95 gave the Ci5-diol 96. Basic hydrolysis, recrystallization and subsequent peracetylation gave the diprotected triol 97 in optically pure form. Dehydration with POCI3 gave the diacetate 98. Deprotection and subsequent oxidation with Mn02 gave the desired end group 99 {Scheme 23).
580 For the synthesis of (aJl-E)-diatoxanthin [(all-E)-94] the strategy C15 + C10 + C15 = C40 was selected. The Cio-dial 45 was first converted into the monophosphonium salt 100 and then reacted with the acetylenic Ci5-end group 99 to give the acetylenic C25hydroxyaldehyde 101 which was reacted with the Ci5-hydroxyphosphonium salt 63 to (allE,3R,3'R)-diatoxanthin (94) {Scheme 24).
Mimulaxanthin A representative allenic carotenoid is mimulaxanthin (102), a symmetrical structure with allenic end groups. The allenic key building block 103 has been synthesized by several groups [60-66]. A rather short synthesis starts with the acetylenic Ci5-synthon 98, used for the synthesis of diatoxanthin (94). First, 98 was epoxidized with mchloroperbenzoic acid (m-CPBA) and, after separation of the diastereoisomeric (5R,6Sy 104 and (5S,6R)-104, the latter gave the allenic alcohol 105 in high yield upon reduction with DIBAH [23,67,68]. Mild oxidation to the aldehyde 103, followed by a Horner-Emmons reaction
with
the
acetylenic
Cio-bis-phosphonate
106,
led
to
the
15,15'-
didehydromimulaxanthin (107). Optically active mimulaxanthin (102) was prepared by partial hydrogenation of 107 followed by a thermal (E/Z)-isomerization [69] (Scheme 25).
581
AcO
Peridinin A unique structural feature of butenolide carotenoids is a 4-alkylidenebutenolide system displaying extended conjugation at the C(9)-position. A representative butenolide carotenoid, peridinin (108), an auxiliary light-harvesting pigment for photosynthesis in some algae, is an unusual Csy-dicyclic nor-carotenoid containing an additional 4alkylidenebutenolide ring structure, and carrying an allene function in the main polyene chain. In order to accomplish the total synthesis of 108, a new synthetic methodology (the sulphone method) has been developed for the construction of the conjugated alkylidenebutenolides [27,70 - 72]. The vinyl triflate 109 is prepared in high yield by the reaction of the TBS ether 110 with A/-phenyltrifluoromethanesulphonimide (Tf2NPh) in the presence of LDA. A coupling reaction of the triflate 109 with methyl acrylate, in the presence of a palladium-(ll)-catalyst,
582
affords the diene ester 111 which, on LiAIH4 reduction followed by acetylation, is converted into the allylic acetate 112. The allylic sulphone 113 Is obtained by the reaction with sodium sulphinate, catalysed by Pd(PPh3)4. Introduction of methoxycarbonyl and allyl groups into 113 and subsequent deprotection provides the compound 114. After deprotection, regioselective oxidation of 114 at the terminal vinyl group, followed by elimination of the sulphone group, affords a mixture of the (9Z)-formyl ester (9Z)-115 and the (9E)-isomer (9E)-115. Iodine-catalysed isomerization of the latter gives a mixture (ca. 3:4) of (9Z)-115 and (9E)-115. Epoxidation of the separated (9Z)-115 with m-CPBA gives a mixture of the syn-(P)-epoxide (5S,6R)-116 and the a/?f/-(a)-epoxide (5R,6S)-116 (Scheme 26),
TBSO
TBSO
(5R.6S)-116
(5S,6R)-116 Scheme 26
The synthon (117) is derived from the optically active C22-allenic apocarotenal 118 which is prepared via a Wittig condensation of the (3S)-Ci5-allenic aldehyde 103 with the Cy-phosphonium chloride 119. Reduction of the aldehyde group in 118 (a mixture of 11Z and 11E) with NaBH4, followed by acetylation, yields the acetate 120, which is converted into the sulphone 117 by heating under reflux with sodium sulphinate in propan-2-ol and water. Condensation between the (all-E)-allenic sulphone 117 and the formyl ester (5R,6S)-116 under the conditions of the sulphone method furnishes a mixture (ca. 1:1) of the optically active peridinin (108) and its (11'E)-isomer and these are cleanly separated by preparative HPLC in the dark (Scheme 27) [70,72].
583
HO
118R' = H. R2 = CH0 120 Ri = Ac. R= = CHpAc 117 Ri=Ac. R2=CH,S0,Ph OAc
108 Scheme 27
Fucoxanthin The synthesis of fucoxanthin (121), reported by Ito is clearly a highlight in the field of carotenoid synthesis [32-74]. The carotenoid was synthesized according to the strategy Ci5 + Cio + Ci5 = C40 with the Cio-dialdehyde 45 as central unit. As the allenic precursor 103 for the Ci5-end group 122 had previously been prepared for the synthesis of peridinin (108) the emphasis was on the preparation of the 3-hydroxy-8-oxo-p end group 123,
AcO
Scheme 28 {continued...,)
584
OAc
121 Scheme 28
The key step of the synthesis is the rearrangement of the a-acetylenic alcohol 97 into the unsaturated carbonyl compound 124. This rearrangement was carried out with tris(triphenylsilyl)vanadate, triphenylsilanol and benzoic acid to give a mixture of the isomers 124 and 125. The latter was converted by iodine catalysis into the desired isomer 124. This key intermediate was afterwards transformed into the Ci5-phosphonium salt 123 by standard procedures. The Wittig olefination of the Cio-dial 45 first with the fucoxanthin end group 123 and then with the peridinin end group 122 gave, in five steps, the C4o-carotenoid 126. Finally the epoxidation of this compound resulted in optically active fucoxanthin (121) and its (5S,6R)-isomer (Scheme 28). 4.2.
Carotenoids with the s end group a-Carotene In contrast to the unsubstituted p end group, the s end group possesses an
asymmetric carbon atom at position 6 and therefore, in the synthesis of compounds with this end group, the stereochemistry has to be considered. The most prominent carotene with the s end group is p,E-carotene (a-carotene) (127) which possesses the (6'/?)configuration. By analogy with the synthesis of p,p-carotene
(2), the strategy
Ci6 + Cg + Ci6 = C40 was used for the first synthesis of a-carotene (127) [75-77]. By use of the enantiomeric (S)-(-)- and (f?)-(+)-a-ionones [(S)- and {R)-78], the naturally occurring (6'R)-p,8-carotene (127) and its enantiomer have been synthesized. This constituted the first synthesis of an optically active carotenoid [78]. The enantiomers of a-ionone (78) were obtained by resolution of the racemate via the menthylhydrazones [79]. Recently an improved route to the enantlomerically pure a-ionones [(S)- and (R)78] on a preparative scale has been developed [80]. For the synthesis of the naturally occurring (6'/^)-p,c-carotene (127), (Rj-a-ionone [(R)-78] was reacted with vinylmagnesium chloride (80) to give the Ci5-alcohol 128 which was converted into the phosphonium salt 129. The subsequent Wittig reaction with 12'apo-p-caroten-12'-al (130) and NaOMe as base gave 127 in 45% yield referred to (R)-78 (Scheme 29).
585
Lutein The most important carotenoid with the 3-hydroxy-8 end group is lutein (131), the major carotenoid found in the leaves of higher plants. For the synthesis of (3R,3'R,6'R)lutein (131), the C15 + C10 + C15 = C40 strategy was applied [81], as used previously for the synthesis of the mixture of stereoisomers [12]. Since the Ci5-phosphonium salt 63 containing the 3-hydroxy-p end group has been synthesized before, the Ci5-phosphonium salt 132, containing the 3-hydroxy-s end group, was the key building block in this synthesis. As starting material, the optically active hydroxyketone 85, which had already been used for the synthesis of optically active astaxanthin [(3S,3'S)-83], was applied. Compound 85 was first converted into the corresponding mesylate and, by reaction with tetrabutylammonium
acetate,
the
corresponding
acetate
133
with
inverted
stereochemistry at C(3) was obtained. After hydrolysis, the hydroxy group was protected as the isopropenyl methylether to give 134. The key step in the synthesis was the diastereoselective epoxidation of 134 with dimethylsulphonium methylide (135) to give the epoxide 136. The selectivity in this reaction can be explained by the fact that, in 134, the conformation with the protecting group in an equatorial position is highly favoured, so the attack of the ylide on the C=0 double bond takes place preferentially from one side to give an axial C-C bond. The stereoselective opening of the epoxide in 136 with a Grignard reagent gave the aldehyde 137. After chain elongation by means of a Horner-Emmons reaction with 138, the resulting nitrile was reacted with MeLi (139) and, after hydrolysis, (3R,6R)-3-hydroxy-a-ionone
(140) was obtained. The reaction of 140 with vinyl
magnesium chloride (80) gave the Ci5-compound 141 which was transformed into the desired phosphonium salt 132 in a yield of 30% referred to 140 {Scheme 30).
586
O
CH^SMe, 136 •
IPMO
AcO 133
134 O
^^^ IPMO
(EtO)2P.^^CN 138
IPMO 137
136
140
PPhXI
141
132 Scheme 30
The Wittig reaction between 132 and (3/?)-3-hydroxy-12'-apo- -caroten-12'-al (142), with KOH as base, gave (all-E,3R,3'R,6'R)-lutein (131) in a yield of 25% referred to 142 (Scheme 31). ^,0H
.^> OH
Scheme 31
4.3.
Carotenoids with the K end group Capsorubin The 3-hydroxy-K end group is characteristic of capsorubin (28), and other
carotenoids isolated from paprika (Capsicum annuum). All syntheses are based on the 010 + 020 + 010 = 040 strategy, and use crocetindialdehyde (27) as central building block and the aldol condensation as the coupling reaction {Scheme 6). The synthesis of optically active capsorubin (28), reported in 1973, was the first synthesis of an enantiomerically pure xanthophyll [11]. For the optically active form of the key building block, the frans-Oio-hydroxyketone 26, two approaches have been reported. In the first reaction sequence, (+)-camphor (143) was converted into camphoric acid (144) by treatment with nitric acid. Camphoric acid (144) was then esterified with dimethyl sulphate
587 and the product selectively saponified with 1 equivalent of NaOH to give 145 which was decarboxylated to form the unsaturated ester 146. The crucial step in this synthesis was the regioselective and stereoselective introduction of the hydroxy group, whereby the second chirality centre was generated. The best results were obtained by hydroboration with (+)-diisopinocamphenylborane of the acetal 147, which was obtained from 146 by successive reduction, methylation and acetalization [11] (Scheme 32).
T-r^
/I > ^fH 0
I
w
CGjMe
(
H02C '*• • \ y
"•
\ /
k
HO2C
144
143
MeU 139 —•
V^146
\
C02Me
)
V "•
\ -V/ / 146
0
\
• " /
CO2H
\
\ "/
0
)^ \
0
', : ->
"
(
)
Y
•
OH 26
Scheme 32
4.4.
•w
Carotenoids with the y/end group Lycopene Some 50 carotenoids with an unsubstituted \\f end group have been found in
Nature, the most important one being lycopene (1). The first synthesis of lycopene (1) was reported by the school of Karrer [82] in 1950.
By
analogy
with
his
pioneering
synthesis
of
p,p-carotene
(2)
the
Ci6 + C8 + Ci6 = C4o route was used. The first synthesis of lycopene (1) via the Wittig reaction used the strategy Cio + C2o + Cio = C4o [83]. Linalool (148) was converted, with phosphorus tribromide and triphenylphosphine, into the phosphonium salt 149, The Wittig reaction with crocetindialdehyde (27) and BuLi as base gave lycopene (1) in an overall yield of 36% referred to 148 (Scheme 33). PPh^Br 149
148
Scheme 33
588 Phytoene (150), phytofluene (151), <;-carotene (152) and neurosporene (153), the biosynthetic precursors of lycopene (1), also contain the v|/-end group. They differ from lycopene (1) in the degree of saturation of the 7,8, 7',8', 11,12 and/or 11',12' double bond(s). Different building blocks as starting material have therefore been used for the synthesis of these compounds. (^-Carotene The synthesis of symmetrical (^-carotene (152), which has 7,8 and 7',8' single bonds, was carried out efficiently according to the C15 + Cio + C15 = C40 strategy by use of the Wittig reaction to couple the building blocks [84]. frans-Nerolidol (154) was converted, with phosphorus tribromide and triphenylphosphine, into the Ci5-phosphonium salt 155. The Wittig reaction with the Cio-dialdehyde 45 and BuLi gave ^-carotene (152) in an overall yield of 45% referred to 154 (Scheme 24).
Neurosporene The synthesis of carotenoids that are not symmetrical is more demanding and costly than that of symmetrical compounds, and different approaches have to be applied. This is illustrated by the two syntheses of neurosporene (153). By application of the C2o + C2o = C4o strategy the two different
C2o-building
blocks were
synthesized
Independently and coupled In one step. In this synthesis farnesyl bromide (156) was converted, with 2-nitropropane and KOH, into farnesal (157). Horner-Emmons reaction with the Cs-phosphonate 73 and reduction with LiAIH4 gave the alcohol 158 (32% yield referred to 156) which was transformed, with phosphorus tribromide and triphenylphosphine, to the C2o-phosphonium salt 152 (Scheme 35).
589
158
PPhaBr 159 Scheme 35
For the synthesis of the other C2o-building block, i.e. the lycopene half of the molecule, \|/-ionone (160) was used as starting material. The Grignard reaction of 160 with methoxyacetylene (161) and treatment with aqueous sulphuric acid gave the ester 162 which was reduced with LiAIH4 and reoxidlzed with Mn02 to the aldehyde 163. Subsequent condensation with the phosphonate 73, reduction and oxidation gave the C2o-aldehyde 164 in an overall yield of 12% referred to 160 (Scheme 36). MeO—ZZ:
H
163
164 Scheme 36
Wittig reaction of the C2o-phosphonium salt 159 with the aldehyde 164 and BuLi gave neurosporene (153) in a yield of 20% (Scheme 37). BrPh,P V
590 Phytoene and phytofluene For the synthesis of phytoene (150) and phytofluene (151) the C20 + C20 = C40 strategy has been applied [85,86]. For the preparation of phytoene (150), transgeranyllinalool (165) was treated with phosphorus tribromide to give geranylgeranyl bromide (166) which was reacted with triphenylphosphine to give the Cao-phosphonium salt 167. The C2o-aldehyde 168 was prepared by treatment of 166 with KOH and 2nitropropane. The Wittig reaction of 167 and 168 with PrLi gave a mixture of (E/Z)isomers of phytoene (150) in an overall yield of 6% referred to 165 (Scheme 38).
Scheme 38
(all-E)-Phytofluene (151) was isolated from a mixture of (^E^j-isomers synthesized by the Wittig reaction of aldehyde 168 with the Cao-phosphonium salt 159 and BuLi (Scheme 39). BrPh,P
169
168
Scheme 39
591 Spirilloxanthin Important carotenoids with the 1-methoxy-3,4-didehydro-1,2-dihydro-v|/ end group are spirilloxanthin (169), spheroidene and rhodovibrin. The starting material 6-methylhept-5-en-2-one (170) was converted, with methanol, into the methoxy compound 171 which was condensed with ethyl bromoacetate (172) in a Reformatsky reaction to give the ester 173. Reaction of 173 with NBS and then dehydrobromination led to the ester 174 which was reduced with LiAIH4 and transformed with triphenylphosphine hydrobromide to the Cio-phosphonium salt 175 and then coupled with crocetindialdehyde (27) in the presence of sodium methoxide as base in a Wittig reaction to give spirilloxanthin (169) in an overall yield of 2% referred to 170 [87] (Scheme 40). MeO
BrCKCOOEt 172
^ 171
170 MeO
MeO
COOEt 173
COOEt 174 crocetindialdehyde 27
MeO OMe
Scheme 40
This synthesis has been improved by using the condensation of 171 with ethyl diethylphosphonoacetate (176) and sodium hydride to produce the ester 173 [88] (Scheme 41). (EtO)2P ^
MeO
COOEt 176
MeO
^^ 171
COOEt
173 Scheme 41
Rhodopin The 1-hydroxy-1,2-dihydro-v|/ end group is present in several carotenoids such as rhodopin (177). For the synthesis of the unsymmetrical carotenoid rhodopin (177) the (Cio + C2o) + Cio = C4o strategy was used and the Wittig and the Horner-Emmons reactions were applied in the key steps [89]. The Horner-Emmons reaction of 6-hydroxy-
592
6-methylhept3n-2-one (178) with methyl diethylphosphonoacetate (179) and sodium methoxide followed by reduction with LiAIH4 resulted in the alcohol 180. Treatment of 180 with phosphorus tribromide and triphenylphosphine gave the Cio-phosphonium salt 181. The Wittig reaction with apo-8'-lycopenal (182) and sodium methoxide gave rhodopin (177) in an overall yield of 20% referred to 178 (Scheme 42). O
II (Et0)2p ^
HO
COOMe
179
180
178
apo-8'-lycopenal 182
Oscillol As starting material for the synthesis of oscillol (183) [90,91], the aglycone of oscillaxanthin containing two 1,2-dihydroxy-3,4-didehydro-1,2-dihydro-v|/-end groups, 3methylbut-2-en-1-ol (184) was chosen. Ester 185, obtained by the reaction of 184 with pnitrobenzoyl
chloride
(p-NBCI), was dihydroxylated
in a Sharpless
asymmetric
dihydroxylatlon [92] to give the diol 186 in a yield of 59% and an e.e. of 91%. The two vicinal hydroxy groups were protected with 2,2-dimethoxypropane and the ester group was saponified to give the alcohol 187. Swern oxidation and Wittig reaction with phosphorane 188 gave the ketone 189. Grignard reaction with vinylmagnesium bromide (65), followed by treatment with phosphorus tribromide and triphenylphosphine, resulted in the Cio-phosphonium salt 190 which was deprotected with aqueous acetic acid to produce the phosphonium salt 191. The Wittig reaction of 191 with crocetindialdehyde (27) and NaOH gave (all-E,2R,2'R)-oscillol (183) in an overall yield of 3.7% referred to 184(Sc/7eme43). HO "^^^^
'OH
OpNB
184
OpNB
185
OH 186
PhjP
0
.
OH 188 187
->
O 1
A" \
189
Scheme 43 (continued....)
<^
MgBr 65
593
crocetindialdehyde 27
pp
PPh^Br
191 OH
183
Scheme 43
1,2-Epoxylycopene Although several acyclic epoxycarotenoids occur naturally, only two, namely 1,2epoxylycopene (192) and 1,2-epoxy-^-carotene have been synthesized. Optically active fSj-1,2-epoxylycopene (192) has been synthesized by application of the Cio + C2o + Cio = C4o strategy [22,93,94]. The chiral centre was introduced by a stereoselective fermentative reduction of a ketone with baker's yeast. Racemic geraniol epoxide (193), obtained by the reaction of geraniol with AH-CPBA, was acetylated with acetic anhydride and the epoxide opened with acid ion-exchanger Dowex 50W\o give the racemic diol 194 which was oxidized with bipyridinium chlorochromate (BPCC) to the ahydroxyketone 195. Fermentative reduction with baker's yeast gave the optically active diol 196. The epoxide 197 was obtained by treatment of 196 with mesylchloride and NaOH. Subsequent treatment with phosphorus tribromide and triphenylphosphine led to the Cio-phosphonium salt 198. The Wittig reaction with apo-8'-lycopenal (182) and sodium ethoxide led to (all-E,S)-1,2-epoxylycopene (192) in an overall yield of 1 % referred to 193 (Scheme 44). HO OAc
OH OH
193
194
HO
HO ^^^^^^
'^-^
OAc
OH
195
OAc
196 , ^ ^ ^ •^^^:>^ PPh-Br
OAc 197
198
Scheme 44
apo-8'-lycopenal 182
•
594 2,2'-Diketospirilloxarithin The 1-methoxy-2-oxo-3,4-didehydro-1,2-dihydro-v|/
end group occurs in such
carotenoids as spheroidenone and 2,2'-diketospirilloxanthin (199), which possesses a conjugated system of 15 double bonds. 2,2'-Diketospirilloxanthin (199) was synthesized via a C5 + (Cio + Cio + Cio) + C5 = C4o strategy [95]. The central Cso-building block 200 was obtained by a Wittig reaction between two moles of phosphonium salt 201 [96,97] ^and 15,15'-didehydro-12,12'-diapo-carotene-12,12'-dial (48). Aldol condensation of 200 with two moles of 3-methoxy-3-methylbutan-2-one (202) and NaOH, and then partial hydrogenation in the presence of Lindlar catalyst led to 2,2'-diketospirilloxanthin (199) in an overall yield of 38% referred to 202 (Scheme 45).
OMe Scheme 45
4.5.
Higher Carotenoids The acyclic and the cyclic C45- and Cso-carotenoids are substituted at the C(2)-
and C(2')-positions with an additional isoprenoid unit. C.p. 450 A representative of the Cso-carotenoids with a substituted [3 end group is C.p. 450 (203) which has been isolated from Corynebacterium poinsettiae. For the synthesis of that carotenoid in optically active form, the C2o + Cio + C2o = C5o strategy and the Wittig reaction were chosen [98]. For the synthesis of the chiral C2o-end group 204 {Scheme 48), (~)-a-pinene (205) was selected, and was transformed with Pb(0Ac)4 to the ester 206. Rearrangement to the (-)-frans-verbenyl ester 207 and subsequent reduction with LiAIH4 gave {-)-transverbenol (208) which was pyrolysed to give the aldehyde 209 with an e.e. of 92%. Reduction of the aldehyde to the alcohol, tosylation, reaction with sodium cyanide, and subsequent reaction of the nitrile with DIBAH, gave the corresponding Cn-aldehyde, which was reacted with ethylene glycol to give 210. This key building block was also used for the synthesis of Cso-carotenoids containing the substituted s end group. Reaction of 210 with borane-dimethyl sulphide and H2O2 gave the secondary alcohol 211 which was oxidized and deprotected to give the keto aldehyde 212 (Scheme 46).
595
209
210
211
212 Scheme 46
Compound 212 was reacted in a Horner-Emmons reaction with ethyl-2(diethoxyphosphonyl)propionate (213) to give 214. Protection of the keto group, reduction of the ester, deprotection of the keto group and protection of the hydroxy group as an acetal gave compound 215. For the chain elongation, 215 was reacted with the protected but-3-yn-3-ol (216) and the tertiary alcohol was transformed, with CICOOEt, to the carbonate 217, which was reacted with p-TsOH to give the Ci8-building block 218 (Scheme 47). o (EtO)2P 213
COOEt EtOOC
212
1 1 JI OH 217
218 Scheme 47
The desired building block 204 was obtained by partial reduction of the triple bond in 218, followed by successive protection of the primary alcohol, oxidation of the secondary hydroxy group to the ketone, deprotection of the alcohol, Grignard reaction with vinylmagnesium bromide (65) and acetylation. The C2o-building block 204 was converted, with triphenylphosphine hydrobromide, into the phosphonium salt 219, reaction of which with the Cio-dialdehyde 45 gave C.p. 450 (203) (Scheme 48).
596
Decaprenoxanthin For the synthesis of decaprenoxanthin (220), the best known Cso-carotenoid with substituted e end groups, the acetal 210 was epoxidized to 221. This was opened with filtrol and the resulting alcohol was oxidized to the corresponding ketone 222. For the introduction of the second chiral centre, the same approach as for the synthesis of lutein (131) was selected. Reaction of 222 with dimethylsulphonium methylide (135), followed by ring opening with a Grignard reagent, gave the aldehyde 223 with an e.e. of 87%. The Horner-Emmons reaction of 223 with 138 gave the nitrile 224 which was converted, with MeLi (139), into the a,p-unsaturated ketone 225. After deprotection of the aldehyde function, chain elongation at C(2) was achieved with another Horner-Emmons reaction with 213 to give 226 which was reacted with vinyl magnesium bromide (65) to give the protected C2o-building block 227 [99] (Scheme 49).
210
221
o II (EtO^P^CN "^ ^ O
222
138
223 (EtO)2P ^COOEt I 213
224
225 Scheme 49 {continued...,)
597
MgBr 65
EtOOC
EtOOC 227
226 Scheme 4 9
The C2o-end group synthon 227 was afterwards transformed to the corresponding phosphonium salt 228 and the Wittig reaction with the Cio-dialdehyde 45 and epoxybutane as base gave the protected decaprenoxanthin 229, which was reduced to decaprenoxanthin (220) {Scheme 50). PPh,Br
EtOX
CO,Et
EtOX
220 Scheme 50
Sarcinaxanthin Sarcinaxanthin (230), a Cso-carotenoid containing two cyclic y end groups with an additional Cs-unit was first prepared as a racemate [100]. For the synthesis of the optically active compound the C20 + C10 + C20 = C50 strategy was chosen. For the synthesis of the C2o-phosphonium salt 231, the key building block of the synthesis, camphoric acid (144) was selected as starting material [101] (Scheme 51).
OTBDMS OH
/f^COOH
Y^COOH
Br
144
pph^cH.,Br233 ^
J \ /
232
Br, / = A
^^^ 235
s?<^'
Br
A,o^".
AcO"
236
S c h e m e 51
(continued...,)
598
AcO"
AcO"
238
PPh3 188
AcO"
240 (EtO),?.
.COOEt I 2
247
231
Reduction of 144 with NaBH4 and BFs-etherate and the subsequent selective protection with f-butyldimethylsilyl chloride (TBSCI) gave 232. The Swern oxidation and a Wittig reaction with MePPhsBr 233 gave the protected alkene 234, which was deprotected with tetrabutyjammonium fluoride and acetylated to 235. In the key step the acetate 235 underwent a rearrangement with 2,4,4,6-tetrabromocyclohexa-2,5-dienone to give the cydohexane derivative 236 that was epoxidized with /77-CPBA to 237. Subsequent dehydrohalogenation with 1,8-diazabicyclo[5,4,0]undec-7-ene yielded 238 containing the desired exocyclic double bond. Opening of the epoxide 238 with a Grignard reagent gave the aldehyde 239 that was immediately reacted with the phosphorane 188 to give the a,p-
599 unsaturated ketone 240. Reaction with ethylene glycol, basic hydrolysis and tosylation with TsCI afforded 241, which was reacted with NaCN 242 to give the nitrile 243. The nitrile 243 was reduced with DIBAH, and Horner-Emmons reaction with the phosphonate 213 gave the a,p-unsaturated ester 244. AftenA/ards careful reduction with DIBAH gave the alcohol 245. The two diastereoisomers of 245 were separated by column chromatography to give (2R,6S)-245 and (2R,6/?)-245. The latter was deprotected to give the ketone 246. The Grignard reaction with vinylmagnesium bromide (65) and selective protection gave the end group 247. Reaction with triphenylphosphine hydrobromide led to the desired phosphonium salt 231, which was coupled with the Cio-dial 45 in a two phase reaction with 5% NaOH in water/CH2Cl2 to give directly the deprotected sarcinaxanthin (230) (Scheme 51). Bacterioruberin The synthesis of racemic bacterioruberin (248), reported in 1979, was the first synthesis of a Cso-carotenoid [102]. The optically active end groups of bacterioruberin (248), bisanhydrobacterioruberin (249) and tetrahydrobisanhydrobacterioruberin (250) have been synthesized from the common building block 251 [103,104]. The chiral starting material fRJ-3-hydroxybutyric acid (252) was esterified with ethanol to produce the p-hydroxyester 253, which was alkylated with dimethylallylbromide (254) in the presence of LDA to give the Cg-ester 255. The benzyl ester 256 was obtained by reduction of 255 with LiAIH4 and protection with benzoyl chloride. Oxidation with PCC on AI2O3 and protection of the keto group with glycol formed the acetal 257. Saponification of this with KOH followed by oxidation with chromic anhydride and pyridine gave aldehyde 258 which was elongated with acetylmethylene-triphenylphosphorane (188) in a Wittig reaction to give the Ci2-keto acetal 259. Reduction of 259 with NaBH4 and acid-catalysed hydrolysis of the acetal gave the hydroxyketone 260. The Grignard reaction with methylmagnesium bromide (261) and oxidation with chromic anhydride in pyridine led to the building block 251 in an overall yield of 13% referred to 252 (Scheme 52). OH
OH
O
AJt.„„ 252
•
OH
O
A ^
JL ^
-^'^-^Br
253
Scheme 52 (continued...,)
O
--^^-'^^GEt
254 > •
Jl
255
600
A
PPh,
188
HO
^^
CHaMgBr 261
251
Scheme 52
For the synthesis of the end group of (2S,2'S)-bacterioruberin (248) [25,105], the hydroxyketone 251 was epoxidized with /77-CPBA to the epoxide 262. Treatment with LiAIH4 provided simultaneous ring opening of the epoxide and reduction of the carbony! group to give the triol 263 which was oxidized with chromic anhydride in pyridine to the dihydroxyketone 264. Chain elongation with vinylmagneslum bromide (65) in a Grignard reaction and treatment with triphenylphosphine hydrobromide gave the Ci5-phosphonium salt 265. The Wittig reaction with crocetindialdehyde (27) and sodium methoxide gave (2S,2'S)-bacterioruberin (248) in an overall yield of 9% referred to 253 {Scheme 53). HO
^^
X
261
OH
HO
HO
^<:^262
263 OH
OH
265 OH
crocetindialdehyde 27
Scheme 53
601
Bisanhydrobacterioruberin The end group of (2S,2'S)-bisanhydrobacterioruberin (249) [94,106] was obtained from 251 by treatment with vinylmagnesium bromide (65) followed by conversion into the phosphonium salt 266 with triphenylphosphine hydrobromide. Wittig reaction of 266 with crocetindialdehyde (27) and NaOH afforded (2S,2'S)-bisanhydrobacterioruberin (249) {Scheme 54). o crocetindialdehyde 27
Tetrahydrobisanhydrobacterioruberin The (2R,2'R)-3,4,3',4'-tetrahydrobisanhydrobacterioruberin end group 267 has been obtained by regioselective reduction of the a.p-unsaturated ketone 259 with copper(l)-chloride, sodium f-butoxide, triphenylphosphine and hydrogen. The resulting ketone 268 was then elongated with vinylmagnesium bromide (65), and deprotected to give ketone
269
which
was
reacted
with
methylmagnesium
bromide
(261)
and
triphenylphosphine hydrobromide to give the phosphonium salt 267. A Wittig reaction with crocetindialdehyde (27) gave (2R,2'R)-3,4,3',4'-tetrahydrobisanhydrobacterioruberin (250) in 5% yield referred to 259 [105,107] {Scheme 55).
Scheme 55 (continued...,)
602
4.6.
Diapocarotenoids Methylbixin The most important acyclic apocarotenoids
are the
C2o-cliapocarotenoids
crocetindialdehyde (27), crocetin, and bixin, which contains 24 carbon atoms in the skeleton. The synthesis of the natural (9Z)-methylbixin (270) was accomplished by application of the Cy + Cio + 67 = C24 strategy [108,109]. Condensation of the lactone 271 with the phosphonate 272 led to the (E/Z) mono ester 273 which was converted, with thionyl
chloride,
into
the
acid
chloride;
subsequent
reduction
with
lithium
tributoxyaluminium hydride resulted in the Cy-aldehyde 274 in an overall yield of 22% referred to 271 {Scheme 56). P0(0Et)2 \
HO ^
272
MeO,C
o o 271
273
GOGH
274
Scheme 56
The (a\\-E)'Cj building block 275 was synthesized from methyl 4-bromo-3methylcrotonate (276). The Krohncke reaction of 276 with p-nitrosodimethylaniline gave the aldehyde 277 which was reacted with ethyl orthoformate and reduced with LiAIH4 to give the alcohol 278. Treatment of 278 with acetic anhydride and tartaric acid resulted in the acetate 279 which was reacted with phosphorane 280 in a Wittig reaction to give the ester 281. The acetoxy group was converted into a phosphorane group by reaction with triphenylphosphine hydrobromide and NaOH. The Wittig reaction of the product 275 with the Cio-dialdehyde 45 and sodium methoxide gave the Ciy-aldehyde 282 which was reduced with NaBH4. The phosphorane 283 was then obtained by treatment with triphenylphosphine hydrobromide and sodium methoxide. The Wittig reaction of 283 with aldehyde 274 and sodium methoxide resulted in natural (9Z)-methylbixin (270) in an overall yield of 0.4% referred to 276 (Scheme 57).
603 OEt 1^16020-^"^
Br
_•
1^6020-^ ^ ^ ^ ^ O 277
276
->
->
AcO^
- - T O
PhjP ^
\ ^ ^ ^ ^ Y ^ OEt 278
COjMe 280 AQO
CO,Me
279 ,C02Me
Cio-clialdehyde45
O^ 282
275
MeOX CO.Me
274
^O
283
5.
TECHNICAL SYNTHESES Synthetic carotenoids have been produced on an industrial scale since 1954 by F.
Hoffmann-La Roche Ltd. and since 1960 by BASF and the field has recently been reviewed [30,110]. It was estimated that in 1995 the sales of synthetic nature-identical carotenoids approached in total US $ 500 million. Of the more than 600 naturally occurring carotenoids [4,35] only six are produced industrially. Figure 7 gives the formulae of these carotenoids and their main application. p.p-Carotene (2)
margarine, juice, health food (antioxidant) fertility (cattle)
Canthaxanthin (77)
poultry (egg yolk and broiler skin pigmentation)
604
Astaxanthin (83)
aquaculture (salmonoidae, crustaceae)
Ethyl 8'-apo-p-caroten-8'-oate (286)
poultry (egg yolk and broiler skin pigmentation)
v:^.^ N>s^ v ^ ^ > ; s ^ s > ^ ^ s ^ ^^^O
8'-Apo-p-caroten-8'-al (287)
cheese, dressings
^^/^^vv/^:^//^^/^^^ <^^ <^ o
Cltranaxanthin (292)
poultry (egg yolk pigmentation)
Figure 7
5.1
Synthesis
offi^Carotene
The first industrial synthesis of p,p-carotene (2) followed the C19 + C2 + Cig = C40 strategy with a Grignard reaction for the coupling of the building blocks (see Scheme 13). Other processes have been developed, following the strategy C15 + C10 + C15 = C40 and C20 + C20 = C40 applying the Wittig reaction for the construction of the carbon skeleton [111-114] (Scheme 58).
Scheme 58 {continued...,)
605
XPh,P
285
Scheme 58
The Ci5-phosphonium salt 284, a key intermediate also in the industrial synthesis of vitamin A (29), is reacted in a double Wittig olefination with the Cio-dial (45) to give p,pcarotene (2) in an overall yield of ca. 80% [111,112]. Alternatively also the Wittig reaction of retinal (33) with retinyltriphenylphosphonium salt (285) gives 2 in yields up to 85% [113,114]. 5.2.
Synthesis of apo-j3-carotenoids The commercial apo-p-carotenoids ethyl 8'-apo-p-caroten-8'-oate (286) and 8'-apo-
p-caroten-8'-al (287) may be prepared from the Cig-aldehyde 53, used in the synthesis of p,p-carotene (2). By reaction of 53 with the Ce-acetal 288 the C25-aldehyde 15,15'didehydro-12'-apo-p-caroten-12'-al (289) is obtained [115]. This compound can be transformed into the Cso-aldehyde 287 by consecutive enol ether condensations first with vinyl ethyl ether (17), to give the C27-aldehyde 290, and then with prop-1-enyl ether (18), followed by partial hydrogenation and isomerization [116] (Scheme 59 OEt ,0
606
Ethyl 8'-apo-p-caroten-8'-oate (286) can be synthesized by Wittig olefination of 15,15'-didehydro-C27-aldehyde 290 with 291 [117] {Scheme 60 o PhgP
291
In another approach to the technical synthesis of the apocarotenoids 286, 287 and 292 [118] the C25-aldehyde 12'-apo-p-caroten-12'-al (293) is the key intermediate. Several ways to synthesize this compound have been developed, applying the Wittig reaction to couple the building blocks. By the reaction of the C25-aldehyde 293 with the protected Cs-phosphonium salt 294 the Cao-aldehyde is obtained [119,120], and this can be transformed by a base-catalysed aldol condensation with acetone (295) to give the C33-ketone citranaxanthin (292) [121]. Alternatively the Cas-aldehyde 293 can be reacted in a Horner-Emmons reaction with the Cs-phosphonate 296 to give 292 [122] {Scheme 61).
286
Scheme 61 (continued...,)
607
5.3.
C4(rXanthophylls Although total syntheses of canthaxanthin (77) have been developed, no industrial
implementation of these methods has followed and currently 77 is produced by oxidation of p,p-carotene (2). For this transformation several procedures have been elaborated, such as reaction of 2 with NBS [123], followed by hydrolysis of the resulting intermediate or oxidation with oxyhalo acids [124,125] (Scheme 62).
J
NBS CHCI3/HOR
CJC^
RO
OR
NBS. CHCI3/HOAC
i^ f
>60%
j NaCIOJNal] CH^CIj/HjG > 65 %
C
(DAc
hydrolysis dehydrogenation
hydrolysis
1r
0 II
Scheme 62
In the 1970s, F. Hoffmann-La Roche developed a new strategy for the synthesis of a great number of xanthophylls, starting from the readily available 6-oxoisophorone (56) [16]. Since 1984 the synthesis of astaxanthin (83), as a mixture of the three stereoisomers, has been carried out industrially. First, the starting material 6oxoisophorone (56) was transformed to the ketal rac-86 and the subsequent reaction with the acetylide 297 gave the Ci5-building block rac-91 which was aftenA^ards transformed to the phosphonium salt rac-92. The Wittig olefination with the Cio-dial 45, followed by thermal isomerization, gave astaxanthin (83) [26,126] {Scheme 63).
608
Acknowledgements: We are grateful to F. Hoffmann-La Roche Ltd., Basel and the Swiss National Foundation for the support of our research in carotenoid synthesis. The authors thank Dr. G. Britton, University of Liverpool for linguistic help and BIrkhauser Verlag, Basel for the copyright of parts of the manuscript. References [1] P. Karrerand E, Jucker, Carotinoide, Birkhauser, Basel 1948; English Translation, (Carotenoids, translation E. A. Braude), Elsevier, Amsterdam, 1950. [2] Carotenoids, ed. O. Isler, Birkhauser, Basel, 1971. [3] Key to Carotenoids, ed. O. Straub, Birkhauser, Basel, 1976. [4] Key to Carotenoids, 2nd Edition, ed. H. Pfander, Birkhauser, Basel, 1987. [5] Carotenoids Vol. 1A, Isolation and Analysis, ed. G. Britton, S. Liaaen-Jensen and H. Pfander, Birkhauser, Basel, 1995. [6] Carotenoids Vol. IB, Spectroscopy, ed. G. Britton, S. Liaaen-Jensen and H. Pfander, Birkhauser, Basel, 1995. [7] Carotenoids Vol 2, Synthesis, ed. G. Britton, S. Liaaen-Jensen and H. Pfander, Birkhauser, Basel. 1996. [8] O. Isler, R. Ruegg and U. Schwieter, Pure Appl. Chem., 14 (1967) 245. [9] B. C. L Weedon, Pure Appl. Chem., 14 (1967) 265. [10] B. C. L. Weedon, Pure Appl. Chem., 20 (1969) 531. [11] B. C. L Weedon, Pure Appl. Chem., 35 (1973) 113. [12] B. C. L Weedon, Pure Appl. Chem., 47 (1976) 161. [13] S. M. Makin, Pure Appl. Chem., 47 (1976) 173. [14] F. Kienzle, Pure Appl. Chem., 47 (1976) 183. [15] H. J. Bestmann, Pure Appl. Chem., 51 (1979) 515. [16] H. Mayer. Pure Appl. Chem., 51 (1979) 535. [17] H. Pfander, Pure Appl. Chem., 51 (1979) 565. [18] R. K. Mulier, K. Bernhard and M. Vecchi, in Carotenoid Chemistry and
609
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
613
Structure Elucidation of Phenylpropanoid Wood Extractives Shuji Ozawa, Takashi Sasaya and Sansei Nishibe 1. INTRODUCTION Woody
plants
are
capable
of
synthesizing
a wide
range
of
phenylpropane derivatives including lignans, neolignans, coumarins and flavonoids. Among them, lignans can be generally characterized as
stereospecifically
oxidative
coupling
phenylpropane units, linking them by lignan
structures
can
dibenzylbutyrolactones, naphthalenes neolignans bonds,
and show
biphenyls
as being
varied
as
two
dibenzylbutanes,
furofurans,
(ref.
linked via
structures
of
8, 8' bonds. The principal
recongnized
tetrahydrofurans,
bridged
are known
and
be
products
such
1).
tetrahydro-
Alternatively,
3.3', 8.3' as
and
8-0-4'
dihydrobenzofurans,
biphenyls, and phenylalkylethers. Although a number of reviews of lignan and neolignan structures are available (refs. 1,2,3,4,5) the recent
book
edited
by
Ayres
and
Loike
gives
a
comprehensive
description of the chemistry of lignan and neolignan (ref. 6 ) . In many woody plants, gymnosperms, and angiosperms, lignans and neolignans
are
important
constituents
(refs.
7-10)
exhibiting
various biological activities (ref. 11). This review deals with the structural
elucidation
of
phenylpropanoid
derivatives,
neolignans and sesquilignans from woody plants, Abies
Salix
sachalinensis,
Chosenia
arbutlfolia
lignans, sachallnensis,
and Eucommia
ulmoides,
which has been carried out by our laboratory.
2. Studies on Lignans, Neolignans and Sesquilignans from
sachalinensis Abies
Abies
Masters
sachalinensis
(Japanease name: todomatsu) has long been a
major forest species and widely planted in Hokkaido. The timber is largely used for pulp and in housebuilding. In previous studies on extractives of Larix
leptolepis
(Japanease name: karamatsu) (refs.
12-16), various lignans, neolignans, and higher oligomeric forms of phenylpropanes were isolated. They were in good agreement with the hydrolysis products of lignins. In connection with our interest in
614 the relationship between lignans and lignin, we recently focused our attention on the chemical studies of lignans and neolignans from A. sachalinensls.
Thus^
fourteen
lignans,
three
neolignans
and
two
sesquilignans were isolated from the ethanol extract of the wood of
sachalinensls.
A.
2.1 Lignans (refs. 17,18,19-22) The wood of A. sachalinensls Repeated
silica-gel
column
was extracted with 95 % EtOH.
chromatographies
of the diethyl ether
solubles of the EtOH extract resulted in the isolation of fourteen lignans(1-14). Among them, four compounds (1-4) were represented in the examples of naturally occurring lignan lactols and acetals, and sachalinensls.
were characteristic lignans of A.
Compound 1, todolactol B, was obtained as colorless crystals, mp 181-182*'Cf [a]j5 +38.8'' (c=0.33, acetone). The molecular formula, ^20^22^6("^/z 358.1449), was based on the result of high-resolution mass spectrometry (HR-MS). The ^H- and ^-^C-NMR analyses of 1 and its triacetate la epimers
indicated that compound
having
a
1 existed as a mixture of
hydroxytetrahydrofuranyl
structure.
The
^H-NMR
spectral data of 1 and la are summarized in Table 2.1. In the ^H-NMR spectrum
of
1,
broad
doublets
at
6 5.34
and
5.04, which
were
integrated
in a total of one proton, were assigned to a hydroxy
proton
a
in
compound
hydroxyfuranyl
structure,
and
this
suggested
that
1 existed as a mixture of epimers. This observation was
further supported by the appearance of broad double doublets at 6 5.20 and 5.41 due to a hemiacetal proton (H-9). The interpretation of the proton spin-spin coupling pattern of la was made with •^H--^H COSY (ref. 18). Thus, the correlation between H-9 (6 6.09 and 6.39) and and H-8 (6 2.10-2.36) was observed, and also H-7 (6 2.86-3.24) was correlated with only H-8. H-8' (6 2.46) was correlated with H-8, H-7', and H-9'. The ^^C-NMR spectral data of 1 are listed in Table 2.3. The
carbons
confound
1
gave
existed
rise partly as
a
mixture
to
a set of
of
epimers.
signals
because
The
absolute
configuration of 1 was determined by CD spectrum. Compound 1 had a negative Cotton effect at 292 nm, suggesting the S-configuration at the 7'-position (ref. 23). Also, CD curve of 1 exibited positive
615
MeO
OMe 0R1
OMe
2 : Ri=H, R2=Me 2a: Ri=Ac, R2=Me 3 : Ri=H, R2=Et 3a: Ri=Ac, R2=Et
1 :R=H la: R=Ac
4 :R=H 4a: R=Ac
H O f ^ O H .^N.^^^^OMe HlH.)
HI.-)—(••"•H
HO
MeO HO
(H.IH
OMe
OMe
R0H2Cv_^^^^v,^,^^v:.
HOH2C
OMe HO
OMe 8 :R=H 9 : R=p-coumaroyl 10: R=feruloyl
OMe
OMe 11
12
616 Cotton
effects
at
275
and
238
nm.
This
confirms
that
the
stereochemistry at 8- and 8' -positions was in an R configuration by comparing
it
with
the
CD
curve
of
the
corresponding
tetrahydronaphtalene-type lignan (ref. 24). From the above results. Compound 1 was identified with structure 1, and named todolactol B (refs. 17,18). In the investigation of the extractives of the same souce, an optically active lignan, todolactol B
(1) was obtained
from the hydrolysis products of the solvent-soluble polymer, Brauns lignin (ref. 25). Compound 2, todolactol C, was isolated as colorless crystals, mp 195-196*'C, [a]j5 +49.0° (c=0.51, MeOH), and its molecular formula, ^21^24^6 ("^/2
372.1555), was based on the result of HR-MS. The EI-MS
of 2 exhibted a M"*" at m/z 372 and a base ion peak at m/z 340 attributable to [M^ - MeOH]. The ^H- and ^^C-NMR analyses of 2 and its acetate 2a
indicated that compound
2 was a lignan having a
methoxytetrahydrofuranyl structure. In the ^H-NMR of 2 (Table 2.2),
Table 2.1
^H-NMR data for compounds 1 and la 1^)
7 8 9 7' 8' 9' arom. OMe OH
alc-OAc ph-OAc
la^
2.74-3.08 m 2.00 m and 2.29 m(lH) 5.20 br dd(6.0, 6.0) and 5.41 br dd(4.4, 4,4)(1H) 3.56-3.84 m 2.55 m 3.48 dd(8.0, 10.0), 3.56-3.84 m 6.23 s(lH), 6.60-6.82 m(4H) 3.78 s and 3.79 s(3H), 3.81 s(3H) 5.04 br d(4.8) and 5.34 br d(6.0)(lH), 7.21 br s and 7.25 br s(lH), 7.44 br s(lH) -
(270 MHz) a ) : In acetone-dg.
b ) : In CDCI3.
2.86-3.24 m 2.10-2.36 m 6.09 d(5.9) and 6.39 d(4.4) (IH) ^) 2.46 m 4.03 pt(8.1), 3.66 dd(8.1, 9.9) 6.50 s(lH), 6.62-6.78 m(3H) 3.74 s and 3.76 s(3H), 3.81 s and 3.82 s(3H)
2.09 s and 2.12 s(3H) 2.23 s(3H), 2.31 s(3H)
c ) : Overlapped with OMe signals.
617 ^H-NMR data for compounds 2, 2a and 3a
Table 2.2 H
2a)
7a
2.91 m
7b
2.91 m
8 9 7' 8' 9'a
2.03 m 4.94 d(4.4) 3.65 d(11.4) 2.76 m 3.55 dd (9.9, 7.9) ca 3.76°) 6.22 S(1H), 6.73 m(4H)
9'b arom.
OMe OH OAC CH2 in Et Me in Et
3.31 s, 3.78 3.80 s 7.23 s, 7.45 -
2a^)
Sr
S
3ai^)
2.93 dd (16.1, 5.1) 3.10 dd (16.1, 12.5) 2.12 m 5.00 d(4.8) ca 3.77°> 2.49 m 3.60 dd (9.9, 7.9) 3.90 pt(7.9) 6.47 S(1H), 6.70 m(3H) 6.96 d(8.1)(lH) 3.38 s, 3.74 s, 3.81 s 2.23 s, 2.31 s -
(270 MHz) a ) : In acetone-dg. b ) : In CDCI3.
2.92 dd
(16.1, 5.1) 3.13 dd (16.1, 12.5) 2.10 m 5.11 d(4.8) 3.77 m 2.49 m 3.58 dd (9.9, 8.1) 3.90 pt(8.1) 6.48 S(1H) 6.71 m(3H) 6.96 d(8.1)(lH) 3.75 s, 3.81 s 2.23 s, 2.31 s 3.46 m, 3.77 m 1.19 t(7.0)
c ) : Overlapped with OMe signals.
a singlet at 6 3.31 was due to an acetal methoxyl group. The signal for an acetal methine was observed at 6 4.94 as a doublet (J'=4.4 Hz) which must exist in the axial
orientation because the chemical shift
of C-9 (6 105.71) in the ^^C-NMR spectrum (Table 2.3) suggested that the acetal methoxyl group was attached to the 9-position in the eguatrial orientation (ref. 17). Furthermore, the NOESY experiments were performed to show the spatial relationships of protons.
The
existence of a weak NOE effect between H-9 and H-8' requires the orientation of the axial
H-9 consistence with the above assingnment.
Moreover, there was no NOE effect between H-8 and H-8* which implies a
trans
configuration
for
them.
The
absolute
configuration
of
Compound 2 was determined on the chiroptical properties which agreed with those of todolactol B (1). Thus, compound 2, named todolactol
618 Table 2.3
I^C-NMR spectral data for compounds 1, 2 and 3
c
la)
1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6' 7' 8' 9' arom.-OMe 9-OMe Me in Et CH2 in Et
128.59 128.17 112.55 148.50 146.20 146.23 116.43 116.53 137.53 137.00 c) 32.15 47.28 50.39 99.02 104.35 133.67 113.10 113.06 146.88 146.91 145.38 145.58 115.70 122.01 51.36 50.15 46.51 72.32 71.20 56.31 56.23 -
(270 MHz) a) In acetone-dg.
2^) 128.39 112.51 148.52 146.23 116.43 137.38 c)
47.08^) 105.71 133.65 113.06 146.89 145.44 115.76 122.01 51.43 46.95^) 72.58 56.31 56.23 54.70 -
3b) 129.03 112.50 149.04 146.79 117.26 137.77 30.63 47.78^) 105.11 134.63 113.48 147.41 145.94 116.51 123.11 52.30 47.73d) 73.29 56.85 56..82 16.34 63.84
b) In dioxane-dg.
c) Hidden under solvent signal,
d) Signals may be reversed.
C, was concluded to be structure 2 (ref. 18). Compound
3, todolactol D,
[a]jj +56.1° (c=0.46, acetone), was
isolated as yellowish amorphous. The molecular formula was found to be C22H260g (m/z 386.1726) from the HR-MS. The EI-MS had a M"^ peak at m/z 386 and a base ion peak at m/z 340. The latter was caused by the elimination of a mole of water from the M"*". Interpretation of the ^HNMR and ^^C-NMR indicated that compound 3 had an acetal structure. Acetylation of 3 yielded diacetate 3a. In the ^H-NMR of 3a
(Table
2.2), the triplet at 6 1.19 originated from the methyl protons in the ethoxy group. This signal was coupled with two multiplets (6 3.46 and 3.77) which can be assigned to the non-equivalent methylene protons in the ethoxy1 group. The other peaks were very similar to
619 those of 2a. Comparison of the ^^C-NMR spectra of 3 with that of 2 showed their close structural resemblance (Table 2.3). Moreover, the chiroptical features of 3 were compatible with those of todolactol B (1) and C (2). Consequently, compound 3 was to be the structure 3, and named todolactol D (ref. 18). The possibility exists that the ethoxy acetal
3
may be artifacts since ethanol was used as the
extraction solvent. Thus, the status of this compound
as a true
natural product is uncertain. Compound
4, todolactol A,
[a]^
-80.6** (c=1.02, dioxane) was
isolated as colorless crystals, and its molecular formula, C20H24O7 (m/z 376.1555), was determined by HR-MS. The ^H-NMR spectrum of 4 suggested the presence of six aromatic protons groups.
Also
the
structure
of
4
was
and two methoxyl
suggested
to
be
a
dibenzylbutyrolactol derivative by the •'^H-NMR spectrum of 4 and its acetate 4a. The wood of A. sachallnensis such as todolactol B
contains some cyclolignans
(1) together with todolactol A
(4), and it
seems that the former arises from todolactol A by dehydration in the wood. Thus, to investigate the conversion of 4 into todolactol B (1), Compound 4 was treated with trifluoroacetic acid in dioxane at room temperature. The resulting product yielded only todolactol B (1). It seems that acid-catalyzed cyclization of 4 presumably takes place through an electrophilic reaction of the resultant carbocation onto the aromatic ring and results in the exclusive formation of the more stable 4S configuration with a quasi-equatrial
aryl substituent
rather than a 4i? configuration with a quasi-axial
aryl substituent.
Cosequently, compound 4 was concluded to be structure 4, and named todolactol A (ref. 19). Todolactol A, B, C and D have not been reported as naturally occurring lignans, but similar types of lignans have been isolated
from Carissa
edulls
(ref. 26), Piper
(ref. 28), Dacrydlum intermedium and A. koreana sachalinensis
clusli
(ref. 27), P. cubeba
(ref. 29), Abies
mariesii
(ref. 30)
(ref. 31). It seemed that lignans from the wood of A. were characterized
by
a preferential
occurrence of
lactol and acetal types of lignans. Several other types of lignans have been isolated from the wood of
A.
sachalinensis.
These
were
(+)-pinoresinol
(5),
(+)-
620 epipinoresinol (6), (-)-a-conidendrin (7), (+)-lariciresinol (8), (+)-lariciresinol p-coumarate ( 9 ), (+)-lariciresinol ferulate (10), ( + )-tetrahydro-a^, 2-bis-(4-hydroxy-3-methoxyphenyl)-3,4furandimethanol (11), (-)-tetrahydro-2-(4-hydroxy-3-methoxyphenyl)4-(4-hydroxy-3-methoxybenzoyl)-3-furanmethanol
(12 ),
resinol
di-p-coumarate
(13), and
(+)-secoisolariciresinol
( + )-isolarici(14)
(refs. 19-22). 2.2 Neolignans (refs. 20,21) Three neolignans (15, 16 and 17) were isolated from the wood A.
of
sachallnensis,
and
their
structure
was
elucidated
by
spectroscopic analyses, they were identified with 2,3-dihydro-2-(4hydroxy-3-methoxyphenyl)-3-hydroxymethyl-5-(2-formylvinyl)-7hydroxybenzofuran (15), 2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3(p-coumaroyloxymethyl) -5-w-hydroxypropyl-7-hydroxybenzof uran and
(16)
1-(4-hydroxy-3-methoxyphenyl)-2-[4-(2-formylvinyl)-2-methoxy-
phenoxy]-propane-l,3-diol (17). The absolute configurations of the former two neolignans (15) and (16) have been demonstrated, using CD data and ^H-NMR shifts (ref. 20).
o
CHO HO//
II
\^
;ax^'
COH2C //,,
OMe
CHO
Q ^ O H HO
OMe
17
^x^^^^:^.^-^''"'^^^^^ CH2OH
16
621 2.3 Sesquilignans (ref. 32) Repeated silica-gel column and prep.TLC of the diethyl ether insoluble fraction of the EtOH extract from the wood of Abies sachallnensis have resulted in the isolation of two new sesquilignans, abiesol A (18) and B (19). The findings from the spectral analysis indicated that abiesol A had dihydrobenzofuran and tetrahydrofuran structures, and abiesol B was shown to have dihydrobenzofuran and butyrolactol structures which are considered to exist as an equilibrium mixture of the tautomeric forms.
1>v^8' 8 ^ CH2OR
ROHgCv^s' R0^v^2" OMe
18 :R=H
18a: R=Ac
?^
9"
ROH2C
ff
\ 8"
^^-^p^^^ySUAQ' OMe [9 RO
R0^^v^2" OMe
19b, 19d: R=Ac 20*: R=H
ji r ^ 2
W^OMe OR 19c, 19e: R=Ac 22*: R=H
*: Compound 19, abiesol B, existed as a mixture of tautomeric four isomers which were comprised of the two diastereomers of structures 20 and 22.
Compound 18, abiesol A, [o.]^ +15.0"* (c=1.04, MeOH) was isolated as being amorphous. The molecular formula, ^2o^24p9' ^^^ based on the result of HR-MS. In the ^H-NMR spectrum of tetraacetate 18a
(Table
2.4), the two multiplets at 6 2.56 and at 6 2.70, and one doubledoublet, at
6
2.85
were
confirmed
to
be
two
protons
of
a
tetrahydrofuran ring and two protons at the 7' position, which was further confirmed by the signals at 6 49.05, 42.49 and 33.40 in the
622 Table 2.4
^H-NMR data for compound 18a 18a
H 7 8 9 7' 8' 9'a 9'b 7" 8" 9" arom. OMe alc-OAc ph-OAc
4.86 2.56 4.21 4.37 2.56 2.85 2.70 3.78 4.09 5.52 3.78 4.37 6.63 3.82 2.04 2.31
d(5.5) m d d ( 7 . 7 , 11.4) m m d d ( 4 . 9 , 13.0) m m d d ( 6 . 4 , 8.6) d(6.6) m m s ( 2 H ) , 6 . 9 5 m(6H) s , 3.84 s , 3.90 s s(6H) s(6H)
(In CDCI3, 270 MHz)
OMe 18b (m/z315) 18 (m/z538)
— H2O
H2C<^
fVT K^ OMe
a
^0-"^
'CH2
OMe
18c (m/z297)
Fig. 2.1.
Important ion peaks in the MS spectra of abiesol A (18)
623 ^^C-NMR Of 18a with
a
part
(Table 2.6). A doublet at 6 4.86 was found to couple of
the
signal
at
6
2.56
by
a
double
decoupling
experiment, and which was assigned to a methine proton at an arylsubstituted
tetrahydrofuran
ring.
The
spectrum
further
showed
a
doublet at 6 5.52 which was due to a methine proton (H-7") at an aryl-substituted suggested
the
dihydrobenzofuran presence
of
a
structure.
These
aryldihydrobenzofuran
results
ring
and
a
tetrahydrofuran structure in compound 18. The mass spectrum of 18 exhibited an important fragment at m/z 315 (18b) due to the cleavage between C-7' and C-8' and a base ion peak at m/z 297 can be assigned to cation 18c formed by the elimination of a mole of H2O from cation 18b
(Fig. 2.1). The ^^C-NMR spectral data of 18 and its tetraacetate
18a
are listed in Table 2.6. Signals due to an aryl-substituted
dihydrobenzofuran
moiety
and
a
tetrahydrofuran
structure
were
observed. Thus, compound 18, named abiesol A, was concluded to be structure 18. Biogenetically, abiesol A would be derived from the oxidative coupling of lariciresinol and coniferyl alcohol. Compound 19, abiesol B, [a]^ -2.4** (c=1.66, MeOH) was isolated as being
amorphous. The HR-EI-MS yielded
predicting
a
molecular
a M"^ at m/z 554.2112,
formula, C3QH340^Q.
The
EI-MS
spectrum
of
confound 19 showed a prominent ion peak at m/e 297 attributable to cation 18c which was shown by the fragmentation of abiesol A (18). In
the
^H-NMR
spectrum
of
acetate
19 a,
resolved
singlets
were
observed at 6 6.14, 6.12, 6.09, and 6.06 and integrated in the total of one proton. These singlets were assigned to an acetal proton (H9'). Moreover, resolved doublets at 6 5.81, 5.74, 4.97 which
were
integrated
in
suggested that compound 19
the
total
of
one
proton.
we
were
unable
4.90
These
data
existed as a mixture of four isomers.
Although we attempted to separate each isomer by methods,
and
to
identify
the
HPLC
chromatographic condition
which
permitted the separation of all four isomers. This difficulty was overcome by coverting the 19 into their corresponding acetates 19a, and separating the acetates by chiral column (Daicel Chiralcel OD) chromatography. The isolated isomers, 19b, 19c, 19d and 19e, were analysed by means of ^H- and ^^C-NMR spectroscopies and their
624 Table 2.5
iH-NMR d a t a f o r compounds 19b, 1 9 c , 19d and 19e
H
19b
7 8 9a
5.81 d (9.9) 2.68 m 3.80 m
9b 7'a
3.80 m 2.68 m
7'b 8' 9' 7"
2.68 m 2.68 m 6.06 s 5.54 d (7.0) 3.80 m 4.32 dd (8.1, 11.0) 4.45 dd 4.45 dd (5.1, 11.0) (5.1, 11.0) 6.66 s(2H) 6.69 s(2H) 6.96 m(6H) 6.94 m(6H)
8" 9"a 9"b arom.
OMe alc-OAc
ph-OAc
3.82 3.83 3.91 1.99 2.04 2.05 2.30
19c
s s s s(3H) s(3H) s(3H) s(6H)
4.97 d (9.5) 2.84 m 4.22 dd (6.6, 10.1) 4.35 m 2.58 dd (10.3, 13.2) 2.84 m 2.84 m 6.12 s 5.54 d (7.0) 3.84 m 4.35 m
3.82 3.84 3.92 1.94 2.04
s s s s(3H) s(6H)
2.31 s(6H)
19d 5.74 d (10.3) 2.71 m 3.79 m 3.79 m 2.71 m
19e 4.90 d (9.5) 2.85 m 4.19 dd (5.9, 11.4) 4.31 m 2.63 dd (10.3, 13.2) 2.85 m 2.85 m 6.14 s 5.56 d (7.0) 3.84 m 4.31 m
2.71 m 2.71 m 6.09 s 5.55 d (7.0) 3.79 m 4.33 dd (7.7, 11.2) 4.43 dd 4.44 dd (5.1, 11.0) (5.0, 11.2) 6.80 s(lH) 6.88 m(8H) 6.83 S(1H) 6.88 S(1H) 6.98 m(5H) 3.83 s 3.81 s 3.85 s 3.84 s 3.91 s 3.91 s 1.93 s(3H) 1.99 s(3H) 2.05 s(3H) 2.04 s(3H) 2.07 s(3H) 2.05 S(3H) 2.31 s(6H) 2.31 s(6H)
(In CDCI3, 270 MHz)
assignments are summarized in Tables 2.5 and 2.6, respectively. The first, isomer 19b, had [a]^ -13.1° (c=0.35, CHCI3). The ^HNMR spectrum of 19b
(Table 2.5) confirmed the presence of three
alcoholic acetoxyl groups, two phenolic acetoxyl groups and three methoxyl groups. A singlet at 6 6.06 was assigned to an hemiacetalic proton
(H-9'). A doublet at 6 5.81 was attributed to a benzylic
methine proton at the C-7 position, indicating the presence of a secondary benzylic alcohol structure in 19b. A doublet at 6 5.54 was assigned to H-7" on the dihydrobenzofuran ring. The ^^C-NMR spectrum
625 Table 2.6
c 1 2 3 4 5 6 7 8 9 1' 23' 4' 5' 6' 7' 8' 9' 1" 2" 3" 4" 5" 6" 7" 8" 9" arom.-OMe
I^C-NMR spectral data for compounds 18, 18a, 19b, 19c, 19d and 19e 18*) 135-03 110.72 148.63 147.70 115.84 119.74 83.75 54.43 60.91 130.70 118.35 137.09 146.96 145.44 114.56 34.23 44.05 73.64 135.54 110.96 148.83 148.01 116.11 120.07 88.73 55.45 65.19 56.92 56.75 56.71
18ab)
19bb)
139.68«=) 109.80 151.14 139.04 122.66 117.84 82.92 49.05 62.72 127.30 116.47 133.77 146.53 144.33 113.03 33.40 42.49 72.85 141.58 110.12 151.30 139.65°) 122.90 118.24 87.79 50.75 65.44 56.21 55.92
139.59°) 111.50 151.27 137.62°) 123.11 119.33
CO in Ac
170.92 170.75 169.09 168.96
Me in Ac
20.89 20.81 20.68
(270 MHz) a) In acetone-dg.
19cb)
139.68°) 110.57 151.21 139.55°) 122.89 118.69 84.20 d) 48.23 47.17 69.95 61.28 127.31 127.43 117.02 116.76 132.68 132.16 146.75 146.76 144.36 144.39 113.40 113.20 38.60 31.58 50.03 48.39 102.76 101.19 139.94°) 139.68°) 110.08 110.13 151.33 151.31 139.71°) 139.55°) 122.92 122.73 118.20 118.35 87.80 87.90 50.73 50.71 65.43 65.33 56.25 56.23 55.98 56.02 55.97 55.79 170.82 170.75 170.69 170.19 169.64 169.73 168.99 168.95 168.81 168.95 21.30 21.35 20.81 21.11 20.78 20.68 20.66
b) In CDCI3.
d) Hidden under solvent signal.
19db)
19eb)
138.40°) 111.30 151.18 137.58°) 122.99 120.87 84.47 d) 47.28 48.42 61.19 70.03 127.18 127.35 115.09 115.43 134.32 132.88 148.04 148.20 144.46 144.32 113.07 113.19 31.69 38.67 48.22 50.09 101.21 102.80 139.78°) 139.76°) 110.12 110.05 151.34 151.33 139.32°) 139.42°) 122.95 122.88 118.31 118.21 88.10 88.13 50.72 50.65 65.11 65.24 56.07 56.30 56.01 55.98 138.52°) 112.05 151.17 137.86°) 122.95 121.05
170.67 170.23 169.64 169.04 168.96 21.30 21.18 20.75 20.66
170.76 170.66 169.70 169.09 168.95 21.37 20.82 20.68 20.65
c) Signals may be reversed.
626 (Table 2.6) showed signals at 6 87.80, 50.73 and 65.43 which were attributed to C-1",
8" and 9", respectively, in an aryl-substituted
dihydrobenzofuran structure. On the basis of these findings, isomer 19b was concluded to be structure 19b. The second, isomer 19c, had [a]^
+14.5° (c=0.40, CHCI3). In the ^H-NMR spectrum of 19c
(Table
2.5), no signal was observed at about 6 5.8, indicating the absence of a secondary acetate group in the benzyl position. However, 19c showed
an
additional
benzylic
methine
(6
4.97,
d,
^=9.5
Hz)
substituted by oxygen. This interpretation indicated the presence of an aryl-substituted tetrahydrofuran methine in 19c. Also, a double doublet at 6 4.22 for two protons, a multiplet at 6 4.35 for one proton, and a double doublet at 6 4.45 for one proton was assigned to two -CH2OAC groups. This was further confirmed by the appearance of signals at 6 65.33 and 61.28 in the ^^C-NMR spectrum (Table 2.6). Isomer 19c therefore was presumed to have structure 19c. The third, isomer 19d, had [a]^ -16.3° (c=0.43, CHCI3). The close similarity of the ^H-NMR and ^^C-NMR spectral data between 19b
and 19d suggested
that they had a diastereomeric relationship with each other (Tables 2.5
and
respectively).
2.6,
However,
stereochemical
differences
between 19b and 19d could not be determined. The forth, isomer 19e, had [a]j5 +9.5° (c=0.40, CHCI3). Comparison of its NMR spectral data with that of 19c
suggested that they were a diastereomer for each
other (Tables 2.5 and 2.6). The stereochemistry of 19e could not be deduced. On
the
basis
isomers, 19b,
of
these
19c, 19d
spectral
examinations
of
acetylated
and 19e, the mixture of four isomers in
compound 19 is comprised of the two diastereomers of structures 20 and
22,
respectively.
chromatographic
behavior
interconvertible
via
the
Furthermore, indicated
the
that
open-chain
above
these
aldehyde
evidence
isomers 21
and
could
(Fig.
be
2.2).
Consequently, compound 19, named abiesol B, existed as a mixture of the tautomeric four isomers, comprised of the two diastereomers of structures 20 oxidative
and
22. Considering the likely course of
couplings,
the
formation
of
abiesol
B
(19)
attributed to the couplings of todolactol A (4) and coniferyl
natural may
be
627
HOH2C
OMe Fig.
2.2.
Tautomerism of Abiesol B (19)
alcohol. Salix
3. Studies on Lignans, Neolignans and Sesquilignans from
sachallnensls The
Fr.Sch.
family
temperate
and
Taxonomically,
Populus,
Salix,
salicaceae subarctic
the
very
zones
family
Tolsusu
is
is
widely
of
the
usually
and Chosenla.
distributed northern
divided
into
in
the
hemisphere. four
genera,
A number of studies on the
extractives of members of Salicaceae have been published to date. The
studies
biological Especially,
have
discussed
activity much
of
of
the
the
the
structure,
chemical
distribution,
constituents
phytochemical
(ref.
investigation
on
and 33). the
Salicaceae has been concerned with the phenolic glycosides such as salicin
(ref. 34). Recently, intense interest has focused on some
species in the Salicaceae as rapidly growing hardwoods with regard to
forest
biomass
Fr.Schm., Salix
resources.
pet-susu
Among
Salix
sachalinensis
maximowiczii
Henry have
them,
Kimura and Populus
been studied for their production in Hokkaido (ref. 35). To obtain basic information on their chemical features, alcohol extractives
628 from
the
leaves,
investigated
(ref.
twigs
and
wood
36). Thus,
of
many
these
phenolic
species
have
compounds
been
such
as
flavonoids, lignans and phenolic glycosides were isolated. It is S.
worthy to note that the extractives of the leaves and twigs of sacallnensis
were comprised mainly of flavonoids and the content of
ampeloptin was estimated to be about 2.5 % of dry matter (ref. 36). This section deals with the structural elucidation of lignans, neolignans
and
sachallnensis
sesquilignans
obtained
from
the
wood
of
S.
(refs. 37,38,39).
3.1 Lignans (refs. 37,39) The
EtOH
fractionated silica-gel
extracts
with
column
diethyl and
of
the
wood
ether
and
preparative
of the
sachallnensis
S.
solubles
thin-layer
was
subjected
to
chromatographies
to
give (+)-syringaresinol and two lignan esters 23 and 24.
23 '. R-|=R2=H 23a: Ri=R2=Ac 24 : Ri=H, R2=Et 24a: Ri=Ac, R2=Et Compound
2 3 , [a]^ +3.52
(c=0.30, MeOH), was isolated as a
colorless powder. The FD-MS spectrum showed a [M"*"] at m/z 556. Also, the EI-MS of compound 23 showed prominent peaks at m/z 121 and 138 implying
p-hydroxybenzoyl
and
p-hydroxybenzoic
acid
moieties,
respectively. Table 3.1 presents the ^H-NMR data for compound 23 and its
acetate
23a
and their
assignments. The presence
of a p -
hydroxybenzoic acid moiety was further confirmed by two doublets of A2B2 patterns at 6 7.22 (2H, J=8.6 Hz) and 6.73 (2H, J=8.6 Hz) in the ^H-NMR spectrum of 2 3 . Also, the ^H-NMR spectrum of 23a confirmed the presence of one alcoholic acetoxyl group, two phenolic acetoxyl
629 Table 3.1 ^H-NMR data of compounds 23^ 23a and 24a H 2 and 6 7
23a
23a)
8 9
6.49 s 4.90 d (3.4) 2.62 m 4.46 m
2' and 6* 7'
4.77 dd (3.9, 10.8) 6.60 s 4.46 m
8' 9'
2.69 m ca3.72^)
6.51 s 5.17 d (3.0) 2.84 m 4.41 dd (8.8, 11.0) 4.73 dd (4.7, 11.0) 6.57 s 5.70 d (11.0) 3.00 m ca3.72^)
ca3.58^)
ca3.82^)
7.22 d (8.6) 6.73 d (8.6) 3.65 s(6H) 3.72 S(6H) -
8.07 d (8.6) 7.19 d (8.6) 3.72 S(6H) 3.82 s(6H) 2.05 S(3H) 2.33 s(9H) ~
2" and 6" 3" and 5" OMe OAc CH2 in Et
~
Me in Et
—
—
24a 6.55 s 5.02 d (4.8) 2.78 m 4.50 pt (10.5) 4.74 dd (5.4, 10.5) 6.52 s 4.45 d (5.0) 2.60 m 4.20 d (8.4) 4.20 d (8.4) 8.00 d (8.5) 7.17 d (8.5) 3.73 S(6H) 3.80 S(6H) 2.32 s(6H) 2.33 s(3H) 3.35 m(lH) 3.43 m(lH) 1.22 t(6.9)
(In CDCI3 , 500 MHz) pt: pseudotriplet.
a ) : In DMSO-d^ + D2O.
b ) : Overlapped with OMe signals. groups and four methoxyl groups. The ^-^C-NMR spectrum of 23a
is
summarized in Table 3.2. The ^^C-NMR spectrum of 23a, which was coupled with a DEPT experiiaent, also showed the existence of a phydroxybenzoyl moiety and a tetrahydrofuran
structure in compound
23. The binding position on the tetrahydrofuran lignan structure of a p-hydroxybenzoic acid moiety was established by a comparison of ^H-NMR spectra between 23 and 23a. Methylene protons (H-9) were
630 ^^C-NMR spectral data for compounds 23a and 24a
Table 3.2
c
23a
1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6' 7' 8' 9' 1" 2" 3" 4" 5" 6" 7" Me in Et CH2 in Et OMe Me in Ac CO in Ac
24a
127..61^) 101,.54 151..71 137..62 152..41 101..54 83..68 48..34 63..34 128,.73^) 102..99 152..17 141..19 152,.17 102..99 73..75 45..64 70..05 127..31^) 131,.19 121,.90 154,.63 121..90 131,.19 165,.49 56..10, 56..26 20..49, 20..51 21,.14, 21 .16 168..62, 168.,76 168..93, 169,.85
127 .68^) 102..03 152..07 141..08 152,.07 102,.03 84,.22 47,.14 64,.06 127..39^) 102,.83 152,.34 139,.44 152,.34 102..83 79,.84 48..60 69..18 127..99^) 131..08 121,.83 154..51 121..83 131..08 165..64 15..40 64..43 56 .06, 20 .43,
56.18 21.14
168 .74, 168.76
(In CDCI3, 500 MHz) a ) : Signals may be revearsed. observed at 6 4.46 and 4.77 for 23 and at 6 4.41 and 4.73 for 23a. Thus, the absence of a significant downfield shift after acetylation for H-9 indicated that the p-hydroxybenzoic acid moiety was attached to the methylene carbon at the 9-position as an ester. Furthermore, the
relative
spectral
configuration
measurements
of
(ref.
23
was
37). On
seen the
by basis
NOE of
differential the
above
631 spectroscopic studies, compound 2 3 was deduced to have structure 2 3. Compound
24 was obtained
spectrum of 24
revealed
as colorless crystals. The FD-MS
an M"^ at m/z
molecular formula C3^H3gO^^. The ^H-and derivative 24a
584 corresponding
to the
^^C-NMR spectra for acetyl
are summarized in Tables 3.1 and 3.2, respectively.
The NMR spectral data for 24a were very similar to those of compound 23a. The main differences observed were readily explained on the basis of the existence of an ethoxyl group in 2 4 instead of an alcoholic hydroxy group in 23. Furthermore, the ethoxyl group must be at the 7'-position because the HMBC spectrum of 24a
showed a
long-range correlation between the methylene carbon (6 64.43) of the ethoxyl group and the benzyl methine proton
(6 4.45) at the 7'-
position. On the basis of these results, compound 2 4 was concluded to be Structure 24, which corresponded to an ethyl ester of compound 23. Compounds 23
and 24 have not been reported to date. However,
the possibility exists that the benzyl ethoxyl compound 24 from the wood of S. sachallnensis
may be an artifact since ethanol was used
as the extraction solvent. 3.2 Neolignans (ref. 38) Repeated
silica-gel
column
and
preparative
thin-layer
chromatographies of the EtOAc soluble fractions of the EtOH extracts from the wood of S. sachallnensis
have resulted in the isolation of
two neolignan esters 25 and 26. Compound 25 was isolated as amorphous, m.p. 87-89°C, [al^ -14.4 (c=0.50, MeOH). The FD-MS spectrum showed a M+ at m/z 728. The EI-MS showed prominent ions at m/z 194, 179, 150(base ion) and 135. An ion peak at m/z 194 corresponded to ferulic acid. The ^H- and ^^C-NMR spectra of 25a are summarized in Tables 3.3 and 3.5, respectively. Compound
25
was
a mixture
consisting
of the
isomers. The ratio was found to be about 2.1
erythro
and
threo
: 1.0 by the ^H-NMR
spectrum of the acetate 25a, which clearly showed distinguishable peaks of the benzyl methine proton {H-7') at 6 6.12 erythro)
and 6.17
(d, J=6.4 Hz, threo).
(d, J=5.4 Hz,
In the dowen field, four
doublets at 6 7.68 (IH, J=15.9 Hz), 7.53 (IH, J=15.9 Hz), 6.42 (IH, J=15.9 Hz) and 6.35 (IH, J=15.9 Hz) were due to olefin protons of
632
7
9 II . 8 . CHgOC-ziA Q'" O
^
\\
OMe 25 : R=H 25a: R=Ac
OMe 26 : R=H 26a: R=Ac
two feruloyl groups. A doublet at 6 6.62 double doublet at 6 6.20
(IH, J=15.8 Hz) and a
(IH, J"=6.3, 15.8 Hz) were derived from
olefin protons of the coniferyl ether moiety. These assingments for 25a
were
chemical
substantiated structure
of
by 25
^H-^H was
COSY
experiments.
established
through
A
detailed
the
^^C NMR
measurement of 25a. The resonances were assigned to the protonbearing
carbons
quaternary
directly
carbons
using
from the
the
HMQC
HMBC
spectrum,
spectrum.
and
The
for
the
long-range
connectivities observed in the HMBC spectrum enabled the connection between
the
guaiacylglycerol-p-coniferyl
ether
skelton
and
two
feruloyl groups. Methylene protons at 6 4.21, 4.43, and 4.53 (H-9') were correlated with a carbonyl carbon at 6 166.42 (9"-C=0) which showed a connection with the olefin proton at 6 6.35
(H-8") (Fig.
3.1). Another carbonyl carbon at 6 166.56 (9'''-C=0) was correlated to the methylene protons at 6 4.85 proton
at 6 6.42
(H-8''')
(H-9) and also to the olefin
(Fig. 3.1)
Thus, the HMBC
experiment
permits the complete assignment of two feruloyl groups in structure
633 Table 3 . 3
^H-NMR data of compound 25a 25a
H 7 8 9 7' 8' 9' 7" 8" 7"' 8'" arom. OMe alc-OAc ph-OAc
6.62 d(15.8) 6.20 dd(6.3, 15.8) 4.85 d(6.3) 6.12 d(5.4, e ) , 6.17 d(6.4, th) 4.70 m(th), 4.74 m(e) 4.21 dd(5.6, 11.9, th), 4.43 m(th, e ) , 4.53 dd(5.7, 12.0, e) 7.53 d(15.9) 6.35 d(15.9) 7.68 d(15.9) 6.42 d(15.9) 6.81-7.13 m 3.79 s, 3.82 s, 3.85 s, 3.86 s 2.08 s(th), 2.10 s(e) 2.29 s, 2.31 s, 2.32 s
(In CDCI3, 500 MHz)
e: erytro.
th:
threo.
7
9
O 110 2'"
OMe
OMe
25a
Fig. 3.1. Important ^H--^^C long-range correlations observed for 25a and 26a
634 Table 3.4
^H-NMR data of compound 26a
H
26a
2 and 6 7 8 9 2' and 6' 7' 8' 9' 2" and 6" 3" and 5" 2 ' •' and 6 " ' 3 • " and 5' " OMe alc-OAc ph-OAc
6.56 6.62 6.29 4.97 6.69 6.18 4.69 4.26 4.64 7.88 7.11 8.12 7.18 3.67 2.04 2.29
s d(15.8) dd(6.0, 15.8) d(6.0) s d(4.8, e ) , 6.21 d(6.2, th) m(th), 4.77 m(e) dd(5.2, 12.0, th), 4.53 m(th, e ) , dd(6.1, 11.9, e) d(8.5) d(8.5) d(8.5) d(8.5) s, 3.71 s, 3.74 s, 3.79 s s(th), 2.16 s(e) s, 2.32 s
(In CDCI3, 500 MHz)
e: erythro,
25.
In
th:
threo.
conclusion,
the
structure
of
compound
25
was
guaiacylglycerol-p-coniferyl alcohol ether diferulate. Compound 26 was isolated as a pale yellowish powder, m.p. 14014rC,
[a]j5+2.9° (c=0.50, MeOH). The FD-MS showed M^ at m/z 676. The
^H-and
^^C-NMR spectral data for the acetylated compound 26a
are
summarized in Tables 3.4 and 3.5, respectively. The ^H and ^^C NMR spectra of 26a
suggested
the existence
of
a
syringylglycerol-p-
sinapyl alcohol moiety and two p-hydroxybenzoyl
groups, and also
indicated that compound 26a was thought to be a mixture of threo erythro
isomers. The ratio of threo
and erythro
and
isomers was found to
be 1.0 : 2.8 by the relative intensities of signals for H-7' in the arylglycerol benzoyl
structure. Complete NMR
groups
experiments.
The
was
established
^H-^H
COSY
by
assignment of two p-hydroxy ^H-^H
spectrum
of
COSY,
HMQC
compound
and
26a
HMBC showed
significant correlations between the aromatic protons at 6 8.12 (H2 • ' • and 6' ' ') and 7.18 (H-3' ' ' and 5' ' ' ) , and between the aromatic protons at 6 7.88 (H-2" and 6") and 7.11 (H-3" and H-5"). The long-
635 Table 3.5
^^C-NMR spectral data for compounds 25a and 26a
c 1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6' 7' 8' 9' 1" 2" 3" 4" 5" 6" 7" 8" 9" 1" 2" 3" 4" 5" 6" 7" 8" 9" OMe Me in Me in CO in CO in
25a
ph-OAC alc -OAC ph-OAC alc -OAc
26a
131.91 110.24 151.00°) 147.10(e), 147.30(th) 122.65^) 119.84 133.95 122.29 65.21 135.38(th), 135.20(e) 111.96 151.10°) 139.78 122.92^) 119.33 73.83(e), 74.53(th) 80.45(th, e) 62.83(e), 63.34(th) 133.15 111.13 151.40°) 141.49^) 123.25^) 121.58 144.73 117.56 166.42 133.31 111.24 151.40°) 141.57^) 123.28^) 121.26 144.40 118.09 166.56 55.82, 55.90, 55.93 20.64, 20.65, 20.68 21.07(th), 21.11(e) 168.77, 168.82 169.53
132.27 103.57 153.16 135.46 153.16 103.57 134.11 122.83 65.45 128.63 103.87 151.96 135.57 151.96 103.87 74.70(e), 75.80(th) 81.01(th), 81.11(e) 63.62(e), 64.62(th) 127.36 131.23 121.59 154.36 121.59 131.23 165.46 127.68 131.21 121.50 154.29 121.50 131.21 165.55 55.87, 55.92, 56.14 20.45 21.11(th), 21.12(e) 168.58, 168.76, 168.83 169.52
(In CDCI3, 500 MHz)
e: erytro.
th: threo.
a ) , b ) , c ) : Signals may be reversed.
636 range connectivities between the carbonyl carbon at 6 165.46 (7"CH2COCO) and the protons at 6 7.88 (H-2" and 6"), 4.26, 4.53 and 4.64 (H-9'), and between the carbonyl carbon at 6 165.55 (7' ' '-CH2COCO) and the protons at 6 4.97 (H-9) and 8.12 (H-2''' and H-6' ' ' ), were established by the HMBC experiments (Fig. 3.1). Thus, these results allowed us to complete and verify the assignments of the two phydroxybenzoyl
moieties.
syringylglycerol-p-sinapyl
Consequently, alcohol
Compound
ether
26
was
to
be
di-p-hydroxybenzoate.
Syringylglycerol-p-sinapyl alcohol ether and its acylated derivative 26 have not been previously reported. Although extensive studies on cinnamic acid derivatives in woody plants have been previously reported (refs. 8,9,11) and that benzoylated Salicaceae
compounds
occur
characteristically
(ref. 9 ) , Compounds 25
and 26
in
the
family
are only known at this
time as neolignan esters from Salicaceae. 3.3 Sesquilignans (refs. 38,39) Chromatographic separation of the EtOAc soluble part of the EtOH extracts from the wood of S. sachalinensis compounds
27, 28
and
2 9. These
three were
afforded three new comprised
of
three
phenylpropane units acylated with p-hydroxybenzoic acid. Compound 27 was isolated as a colorless powder, m.p. 108-110 **Cf [a]j3 -1.06(c=0.50, MeOH). The FD-MS of compound 27
showed an M^
at m/z 702. Also, the EI-MS of compound 27 showed a base ion at m/z 121 implying a p-hydroxybenzoyl moiety, which was further confirmed by two doublets of A2B2 patterns at 6 7.98 (2H, J=8.6 Hz) and 7.17 (2H, J"=8.6 Hz) in the ^H-NMR spectrum of acetate 27a
(Table 3.6).
Also, the spectrum showed two doublets at 6 9.66 (IH, J=7.7 Hz) and 7.41 (IH, J=15.8 Hz) and a double-doublet at 6 6.60 (IH, J=7.7, 15.8 Hz), suggesting the existence of a formylvinyl group as the partial structure. A doublet at 6 5.63 was assigned to a methine proton (H7) at an aryl-substituted dihydrobenzofuran ring. The doublet at 6 6.14 was assigned to a methine proton at an arylglycerol structure as a threo
isomer, and another one(d, J"=5.4 Hz) at 6 6.10 which
corresponded to that of an erythro
isomer (ref. 43). These results
suggested that compound 27 existed as a mixture of threo
and
erythro
637
RiO
OMe
27: Ri=H, R2=CH0 27a: Ri=Ac, R2=CH0 28 : Ri=H, R2=CH20H 28a: Ri=Ac, R2=CH20Ac
;^0R OMe
OMe
29 : R=H 29a: R=Ac
isomers. The ^^C-NMR spectrum of 27a
was assigned with the aid of
DEPT and HMQC spectra (Table 3.7). Also, the connecting position of p-hydroxybenzoic acid moiety was determined by the HMBC spectrum. Thus,
the
methylene
protons
at 6 4.60
and
4.72
(each
m,
H-9)
correlated with carbonyl carbon at 6 165.38, indicating that the phydroxybenzoic
acid
moiety
was
attached
to
C-7
in
the
dihydrobenzofuran ring. These results supported that compound 27 was to be structure 27 . Compound 28 was isolated as colorless crystals. The spectral data of compound 28 closely resembled those of compound 27. The FDMS of compound 28
showed an M^ at m/z 704 and the EI-MS spectrum
furnished a significant ion at m/z 370. These ion peak were 2 amu more than the corresponding peaks in the mass spectrum of compound 27.
^H-NMR spectral details for the acetylated compound 28a
are
given in Table 3.6. The general appearance of the ^H-NMR spectrum of
638 Table 3.6
^H-NMR data of compounds 27a, 28a and 29a
H 2 and 6 7 8 9 2' 5' 6' 7' 8' 9' 2",5"and 6" 7" 8" 9"
27a
28a
6.54 s 6.55 s 5.63 d(7.0) ca3.9^^ 4.60 m 4.72 m 6.90-7.13 m 6.90-7.13 m 7.41 d(15.8) 6.60 dd (7.7, 15.8) 9.66 d(7.7) 6.90-7.13 m 6.10 d(5.4, e) 6.14 d(6.3, th) 4.50 m(th) 4.60 m(e) ca3.9^)(th)
4.23 m(e) 4.34 m(th) 4.48 m(e) 2"* and 6'" 7.98 d(8.6) 3 " ' and 5 " ' 7.17 d(8.6) 7'-OMe arom.-OMe 3.65 s, 3.66 3.67 s, 3.81 3.96 s (12H) alc-OAc 1.97 s, 1.99 2.00 s, 2.04 (6H) ph-OAc 2.29 s, 2.30 2.33 s(6H)
s. s. s. s s
29a 6.49 s
6.55 s 5.57 d(7.0) ca3.9^> 4.50 m ca4.7^) 6.88-7.13 m 6.88-7.13 m 6.60 d(16.4) 6.16 dd (6.6, 16.4) 4.71 d(6.6) 6.88-7.13 m 6.07 d(5.0, e ) . 6.13 d(6.3, th) 4.50 m(th) 4.58 m(e) 4.25 m(e) 4.33 dd (4.7, 11.6, th) 4.50 m(e, th)
8.00 7.16 3.64 3.66 3.81 1.96 2.00 2.13 2.29 (6H)
d(8.6) d(8.6) s, 3.65 s, 3.81 s (12H) s, 1.98 s, 2.10 s (9H) s, 2.33
s. s. s. s. s
4.93 d(4.8) 2.54 m 4.45 m 4.69 m 6.84-7.02 m 6.84-7.02 m 6.84-7.02 m 4.48 d(5.0) 2.80 m 4.20 m 6.84-7.02 m 6.08 d(4.9, e) 6.14 d(6.3,th) 4.45 m(th) 4.57 m(e) 3.91 dd (4.4, 11.8, th) 4.20 m(e) 4.33 dd (4.6, 11.8, th) 4.45 m(e) 7.99 d(8.6) 7.17 d(8.6) 3.23 s 3.67 s, 3.68 s. 3.80 s, 3.81 s (12H) 1.96 s, 1.98 s. 2.04 s, 2.13 s (6H) 2.29 s, 2.31 s 2.33 s(9H)
(In CDCI3, 500MHz) e: erythro. th: threo. a ) : Overlapped with OMe signals. b ) : Overlapped with H-9' signal.
639 Table
^^C-NMR spectral data for compounds 27a, 28a and 29a
3.7
c
27a
7 8 9 7' 8' 9' 7" 8" 9"
89.23 50.32 65.73 152.54 126.78 193.39 75.17(th) .73 .90(e) 80.83(th,e) 62.53(e), 63. 34(th) 165.38
•7111
29a
28a 89.01 50.69 66.16 134.18 121.41 65.16 75.13(th) ,73.93(e) 80.79(e), 80..90(th) 62.58(e), 63,.42(th) 165.43
84.14 48.44 63.71 81.34 47.22 69.22 75.30(th),73.99(e) 80.64(e),80.82(th) 62.57(e),62.61(th) 165.55
(In CDCI3, 500 MHz)
e: erythro. 28a
was
th: threo.
very
similar
to
those
of
compound
27 a.
A
conspicuos
difference was the presence of signals for a propenol moiety instead of a propenal moiety. Also, the presence of a propenol structure in the compound 28 was further supported by signals at 6 134.18, 121.41 and 65.16 in the ^^C NMR specrum of compound 28a
(Table 3.7). On the
basis of these results, and in conjunction with two dimensional NMR experiments, the stucture of compound 28 was ascertained. Compound 29 was isolated as yellowish crystals, mp 156 **C. The FD-MS spectrum of compound 29 exhibited an M"^ at m/z 736. Analysis of the ^H-NMR spectrum of 2 9 and its acetate 29a
established the
presence of a substituted tetrahydrofuran moiety, an arylglycerol structure
and
a p-hydroxybenzoyl
details for 29a
group.
^H- and
^^C-NMR
spectral
are summarized in Tables 3.6 and 3.7, respectively.
The binding site of p-hydroxybenzoyl group was clearly determined by HMBC
spectrum of 29a. The
benzyl
carbonyl
carbon
long-range connectivities
at 6 165.55
(7*"'-CO)
and
between the
the
methylene
protons at 6 4.45 and 4.69 (H-9), and also olefin protons at 6 7.99 (H-2''' and 6''') were observed. Thus, a p-hydroxybenzoyl group was linked to the 9-position. Consequently, the structure of compound 2 9 was identified. Three sesquilignans 27, 28 and 29 are the first confounds to be reported from Salicaceae. The occurrence of these sesquilignans
640 suggest the existence of a higher polymer of phenylpropane units, which related with soluble polymers such as Brauns lignin (refs. 25,44). 4. Studies on Neolignans and Sesquilignans from Chosenla
arbutifolla
A.Skvortz. Taxonomically, the family Salicaceae is usually divided into
four genera, Populus,
Salix,
Toisusu,
about 35-45 species, and Salix 500
species.
Chosenla
and Chosenia.
including Tolsusu
arbutifolla
A.
Populus contains
contains about 300-
Skvortz
(Japanease
name:
Keshoyanagi), the only plant in the genus Chosenla,
is mainly found
in Hokkaido, Japan. A
extractives
Salicaceae
number
of
studies
on
the
have been reported to date. However, little
about the extractives of C. arbutifolla.
of
is known
We have now investigated
the constituents of the wood of C. arbutifolla
and isolated four
neolignans and three sesquilignans including
five novel compounds
(refs. 45,46). This section reviews the structural elucidation of arbutifolla•
neolignans and sesquilignans from the wood of C. 4.1 Neolignans(ref. 46) The wood of Chosenla the
extract
was
arbutifolla
fractionated
by
was extracted with EtOH, and LPE,
Et20
and
EtOAc.
Repeated
silica-gel column and prep.TLC of the Et20 solubles resulted in the isolation of four neolignans 30, 31, 32 and 33. The EI-MS spectrum of 30 showed an M* at m/z 386 and a base ion at m/z 368 caused by the loss of H2O from M"^. In the ^H-NMR of 3 0 (Table 4.1), two doublets at 6 9.58 (J=7.8 Hz) and 6 7.61 (J=15.8 Hz) and
a
double-doublet
at
6 6.68
(J=7.8,
15.8
Hz)
suggested
the
presence of a formylvinyl group. A doublet at 6 5.61 was assigned to a
benzyl methine proton at an aryl-substituted
ring.
Also,
signals
for
one
alcoholic
dihydrobenzofuran
acetoxyl,
one
phenolic
acetoxyl and three methoxyl groups were discernible in the ^H-NMR of diacetate 30a
(Table 4.1). In addition, a singlet at 6 6.63
(2H)
derived from aromatic protons in a syringyl ring as well as two singlets
for
aromatic
protons
in
the
1,3,4,5-tetrasubstituted
benzene structure were observed. From the spectral data described above, compound 30 was deduced to have structure 30. Although this
641 98>CH0
r-x ?\ Ro^CpH2C
OMe
OMe
30 : R=H 30a: R=Ac
31 :R=H
31a: R=Ac
CHO
RO
CHO
OMe
OMe
32 : R=H 32a: R=Ac
33 : R=H 33a: R=Ac
compound has been obtained from the hydrolysis products of hardwood lignin (ref. 47), the isolation as a naturally occurring neolignan has not been reported. The FD-MS of Compound 31 exhibited
a
prominent
ion
showed an M^ at m/z 506. The EI-MS
peak
at
m/z
138
implying
hydroxybenzoic acid moiety. The ^H-NMR data of compound 2
the
p-
and its
acetate 31a are summarized in Table 4.1. The NMR spectral data for compound 31 were very similar to those of compound 3 0 except for signals derived
from a p-hydroxybenzoyl
spectrum of diacetate 31a
moiety. Also, the ^H-NMR
showed the presence of the two phenolic
acetoxyl groups. No signal was observed for the alcoholic acetoxyl group. Moreover, the absence of a significant downfield shift after acetylation for the methylene protons attached to the 7-position, indicated
that
the p-hydroxybenzoxyl
group
was
attached
to
the
methylene carbon at the 7-position. Cosequently, compound 31
was
concluded to be structure 31. The isolation of this compound as a naturally occurring neolignan has not been previously reported. Compound
32
showed very similar
spectral characteristics
to
compound 30. The EI-MS of compound 32 exhibited an M"^ at m/z 338 and a base ion peak at m/z 338, both of which were 30 amu less than the corresponding ions in compoud 30. This could be due to the lack of a methoxyl
group
in
the
molecule.
The
•'•H-NMR
spectrum
of
the
642 Table 4.1
^H-NMR spectral data for compounds 30, 30a, 31 and 31a
H
30a^)
30^)
2 and 6 7 8 9
6.63 s 5.56 d (7.1) ca 3.95^) 4.38 dd (11.3, 7.1) 4.46 dd (11.3, 5.7) 7.07 s 7.10 s 7.42 d (15.8) 6.60 dd (15.8, 7.7) 9.67 d (7.7) —
6.67 s 5.61 d (6.5) ca 3.95^) 3.57 m ca 3.85^)
2'and 6'
7.23 s 7.28 s V 7.61 d (15.8) 8' 6.68 dd (15.8, 7.8) 9' 9.58 d (7.8) 2" and 6" ~ 3" and 5" OMe
—
• "
3.81 3.95 2.07 2.33
3.81 S(6H) 3.95 s(3H)
OAc
(500 MHz) a ) : In MeOH-dg.
s(6H) s(3H) s s
b ) : In CDCI3.
31a^)
31°) 6.80 s 5.74 d (7.3) ca3.95^) 4.57 dd (11.1, 7.6) 4.73 dd (11.1, 5.0) 7.35 s 7.36 s 7.63 d (15.8) 6.70 dd (15.8, 7.7) 9.64 d (7.7) 7.80 d (8.8) 6.88 d (8.8) 3.76 S(6H) 3.95 s(3H)
6.61 s 5.68 d (6.8) ca3.95^) 4.59 dd (11.3, 7.6) 4.73 dd (11.3, 5.1) 7.09 s 7.12 s 7.40 d (15.8) 6.60 dd (15.8, 7.7) 9.66 d (7.7) 7.98 d (8.7) 7.17 d (8.7) 3.72 s(6H) 3.96 S(3H) 2.33 s(6H)
-
c ) : In acetone-dg.
d ) : Overlapped with OMe signals. acetylated
derivative
32a
showed
the
presence
of
an
alcoholic
acetoxyl group, a phenolic acetoxyl group, two methoxyl groups and five
aromatic
protons
(Table
4.2). The
resembled those reported for Balanophonin Balanophora
japonica
spectral
data
closely
(ref. 48) isolated from
Makino. Cosequently, compound 32 was identified
as the known compound, Balanophonin. The syntheses of this compound and the analogous derivatives previously have been reported (ref. 49). Compound
33
showed
spectral characteristics
very
similar to
compound 31 and so they have the same basic type of structure. The FD-MS of compound 33 exhibited an M"^ at m/z 476. The EI-MS showed a
643 Table 4.2
^H-NMR spectral data for compounds 32a and 33a 32a
H 2, 5 and 6
7 8 9
2'and 6'
7' 8' 9'
33a
6.81-7.09 m 5.61 d (6.7)
6.97-7.32 5.71 d (7.7)
ca3.95^)1 4.35 dd (11.5, 7,.0) 4.45 dd (11.5, 5..6) 6.81-7.(39 m 7.41 d (15.8) 6.61 dd (15.8, 7..7) 9.66 d (7.7)
ca3.96^) 4.58 dd (11.3, 7.5) 4.71 dd (11.3, 5.2) 6.97-7.32 m 7.41 d (15.8) 6.60 dd (15.8, 7.7) 9.66 d (7.7) 7.98 d (8.7) 7.17 d (8.7) 3.73 s 3.96 s 2.30 s 2.33 s
2" and 6" 3" and 5" OMe
3.82 3.95 2.07 2.31
OAc
s s s s
m
(In CDCI3, 500 MHz) a ) : Overlapped with OMe signals* large
peak
at
m/z
338
suggesting
the
elimination
of
a
p-
hydroxybenzoic acid moiety from M"^. These ions are 30 amu less than those
for compound
31. The
^H-NMR of
diacetate
33a
showed
the
presence of two phenolic acetoxyl groups, two methoxy groups and nine aromatic protons including four protons on the p-substituted aromatic ring (Table 4.2). The other ^H-NMR signals were comparable with those of 31a. Thus, compound 33 was concluded to be structure 33.
The isolation of this compound
33
has
not been
previously
reported. Compounds 30-33 belong to a dihydrobenzofuran type of neolignan. These compounds have been obtained for the first time in the family Salicaceae, in spite of (refs. 2,50).
the widespread
occurrence
of
neolignans
644 4.1 Sesquilignans (ref. 45) Repeated silica-gel column and prep^TLC of the 'E>t,^0 solubles of the
EtOH
extracts
from
the
wood
of
Chosenia
arbuti
folia
have
resulted in the isolation of three sesquilignans 34, 35 and 36. Two of them are p-hydroxybenzoates (35 and 36) and the remaining one is a known compound, buddlenol A (34).
OMe
34 : R=H 34a: R=Ac
j®>CHO
OMe
35 : R=H(f/7reo) 35a: R=Ac(threo) 36 : R=H{erythro) 36a: R=Ac(erythro)
Compound 34 was isolated as being yellowish amorphous. The FDMS spectrum of 34 exhibited an M"^ at m/z 582. In the EI-MS spectrum of 34, peaks at m/z 386, 180, 151 and 137 were observed as principal ions. A prominent peak at m/z 386 could be assigned to neolignan structure 30. A large peak at m/z 180 could be due to a coniferyl alcohol ion derived from an arylglycerol moiety in structure 34. The "^H-NMR spectrum of the acetate 34a mixture of threo
and erythro
(Table 4.3) showed it to be a
isomers. Two doublets at 6 6.08 (J=5.5
Hz) and 6.15 (J=6.1 Hz) were attributed to H-7" in an arylglycerol structure for erythro
and threo
isomers, respectively. The relative
645 Table 4.3
^H-NMR spectral data for compound 34a 34a
H 7 8 9 7' 8' 9' 7"
8" 9" arom. OMe alc-OAc ph-OAc
5.51 d (7.1) 3.90^) 4.26-4.52 m 7.42 d (15.8) 6.61 dd (15.8, 7.6) 9.66 d (7.6) 6.08 d (5.5, e) 6.15 d (6.1, th) 4.26-4.52 m(e, th) 4.26-4.52 m(e, th) 6.56 s(2H) 6.91-7.10 m(5H) 3.70-3.95 s(12H) 1.98-2.07(9H) 2.30 s(3H)
(In CDCI3, 500 MHz) e: erythro. th: threo. a): Overlapped with OMe signals. intensities of these signals indicated that the ratio of the and erythro
threo
isomers was 2 : 1 . All of the other spectral data agreed
with those of the known compound, buddlenol A isolated from the stems
of buddleja
davldli
(ref. 51) . Consequently, compound 34 was
identified as buddlenol A. Compound 35 was isolated as amorphous. The ^H-NMR data for 3 5 and its acetate 35a are summarized in Table 4.4. Compound 3 5 showed spectral characteristics very similar to compound 27 isolated from Salix
sachalinensis.
sachalinensls,
In
compound
the 27
chemical
was
obtained
investigation as
a
of
S.
mixture
of
diastereoisomers. On the other hand, comparison of the values of the chemical shift and the coupling constant of H-7" and H-8" with values for acetylated compounds 27a and 35a indicated that compound 35 belongs to the threo
series (Table 4.4) (refs. 52,53).
646 Table 4.4
^H-NMR spectral data for compounds 35, 35a, 36 and 36a
H 2 and 6 7 8 9
2'and 6' V 8' 9' 2" 5" 6" 7" 8" 9"
3'"and 5' "
2'"and 6' "
OMe
OAc
35^) 6.65 s 5.62 d (8.0) 3.98 m 4.54 dd (8.8, 11,.2) 4.77 dd (5.4, 11. 2) 7.08 s 7.14 s 7.42 d (15.8) 6.61 dd (7.8, 15. 8) 9.65 d (7.8) 6.94 m 6.87 d (8.1) 6.94 m 5.02 d (8.6) 3.98 m 3.33 d (12.7) 3.54 dd (2.8, :L2. 7) 6.82 d (8.6) 6.84 d (8.6) 7.76 d (8.6) 7.79 d (8.6) 3.77 S(6H) 3.86 S(3H) 3.96 S(3H)
35a
36^)
6.55 s 5.63 d (7.1) ca3.95^) 4.60 dd (11.2, 7.6) 4.71 dd (5.4, 11.2) 7.08 s 7.13 s 7.42 d (15.8) 6.60 dd (7.6, 15. 8) 9.67 d (7.6) 7.02 m 7.02 m
6.65 s 5.66 d (7.0) 4.05 m 4.54 dd (8.6, 11 .1) 4.77 dd (5.1, 11,.1) 7.09 s 7.16 s 7.43 d (15.8) 6.61 dd (7.8, 15 .8) 9.65 d (7.8) 6.83 m 6.83 m
36a 6.54 s 5.64 d (6.8) ca3.96^) 4.59 m 4.71 dd (5.4, 11 .2) 7.09 s 7.14 s 7.42 d (16.1) 6.60 dd (7.8, 16 .1) 9.66 d (7.8) 6.94 m 6.94 m
6.94 m 7.02 m 6.83 m 6.08 d 6.14 d 4.96 d (4.4) (6.4) (3.5) 4.59 m 4.50 m 4.10 m 4.24 dd ca3.95^) 3.52 dd (7.8, 11..7) 4.35 dd (2.5, 12. 1) 4.47 dd (7.0, 11.;9) ca3.92^) (4.2, 11..7) 7.17 d 7.17 d 6.83 m (8.3) (8.7) 7.99 d (8.7) 8.00 d (8.7) 3.67 s(6H) 3.81 s(3H) 3.96 s(3H) 1.97 2.01 2.30 2.33
7.79 d (7.2) 7.81 d (7.2) 3.74 s 3.75 s 3.86 s 3.96 s
s s s s
(In CDCI3, 500 MHz) a ) : CDCI3 + D2O. b ) : Overlapped with OMe signals.
7.99 d (8.3)
3.65 3.70 3.80 3.96 1.97 2.14 2.29 2.33
s s s s s s s s
647 Consequently, compound 35
was concluded to be the threo
isomer of
structure 3 5. Confound 36
was isolated as amorphous. The spectral data of
compound 36 and its acetate 36a suggested that the structure of 36 was close to those of conpound 35. Remarkable differences were seen in the ^H-NMR spectrum where the coupling constant between H-7" and H-8" was smaller (J=3.5 Hz) and the chemical shift of H-7" was in a higher field respectively)
(6 4.96) than in compound 35 (Table
4.4). Thus,
compound
(J"=8.6 Hz and 6 5.02, 36
was
erythro-
the
diastereomer of compound 35. Three sesquilignans 34, 35 and 36 are the first compounds to be reported from Salicaceae. Also, the occurrence of these compounds 34, 3 5 and 36, along with neolignans 3 0 and 31, lends support to the biosynthetic hypothesis of their formation by the sequiential coupling of individual phenylpropane units. 5. Studies on Neolignans and Sesquilignans from Eucommia
ulmoldes
01 iv. The bark of Eucommia
ulmoides
has been used as a tonic and
antihypertensive medicine in Japan and China, which is available on the market as the Chinese Crude Drug "Cortex Eucommia". The bark
contains
many
lignans
and
their
glucosides, which
consist of derivatives of pinoresinol, medioresinol, syringaresinol and olivil (ref. 54). In these lignans, ( + )-pinoresinol-di-0-p-D-glucoside syringaresinol-di-0-p-D-glucoside
are
reported
active principles of the bark of Eucommia
to
be
ulmoldes
and
( + )-
biologically
(refs. 55,56).
( + )-Syringaresinol-di-0-p-D-glucoside
showed anti-stress activity on
swimming
tendency
exercise
in
rats
and
the
to
increase
the p-
endorphin content in plasma. This section reviews the structural elucidation of neolignans and sesquilignans from the bark of Eucommia
ulmoldes.
5.1 Neolignans (ref. 57) The air-dried bark of Eucommia
ulmoldes
was extracted with 50%
methanol in water. After extracting the extract solution with ethyl acetate and then 1-butanol, the butanol extract was subjected to
648
37 ! Ri=R2=H 37a: Ri=Ac, R2=H 38 : Ri=H, R2=0H (erythro) 38a: Ri=Ac, R2=0Ac 39 : Ri=H, R2=0H (threo) 39a: Ri=Ac, R2=0Ac silica gel chromatography with chloroform-methanol-water (100:10:1, 80:20:3) to give three neolignans, 37, 38 and 3 9. Compound 37 was isolated as a colorless syrup,
[a]j^ +5.5°
(MeOH). Acetylation of 37 gave a triacetate (37a), which gave peaks at m/z 486 (M+), 426 (M'^-CH3C00H) and 348 in MS spectrum. Compounds 37 and 37a were identified as dihydrodehydrodiconiferyl alcohol and its triacetate, respectively. Compound 3 8 was obtained as a colorless syrup, [a]jj 0° (water). The IR spectrum showed the presence of hydroxyl groups and aromatic rings. The UV spectrum of 3 8 showed an absorption maxima at 228, 283 and 288 nm. The bathochromic shift of the absorption maxima in the presence of the base was very similar to that of 37. Compound 3 9 was obtained as a colorless syrup, [ajjj 0° (water). The IR and UV spectra of 39 were very similar to those of 38. Acetylation of 3 8 and 3 9 gave pentaacetates, 3 8a and 3 9a, respectively. The MS of 38a and 39a were almost identical with each other, showing peaks at m/z 602 (M"*") and 542 (M^-CH3C00H). The ^H-NMR spectrum of 38a showed signals due to four alcoholic
acetyl groups
(6 2.00, 2.05
and 2.13), a
phenolic acetyl group (6 2.30), two aromatic methoxyl groups (6 3.82 and 3.91), and five aromatic protons (6 6.83 and 6.99). That of 39a showed
similar
guaiacylglycerol
signals. tetraacetate
It and
was its
reported threo
that isomer
erythrocould
be
distinguished on the basis of the chemical shifts and the coupling constants of the benzylic protons (refs. 58,59). The benzylic proton
649 signals of 38a and 39a appeared at 6 5.93 (d, J=5.72 Hz) and 6 5.89 (d, J=7.48 H z ) , which showed threo
38a
and 3 9a
to be the erythro
and
isomers, respectively. From these results, compounds 38 and 39
were established
to be
erytiiro-dihydroxydehydrodiconiferyl
alcohol
and its threo isomer, respectively. 5.2 Neolignan and Sesquilignan Glucosides (refs. 60,61). The air-dried bark of Eucommia
ulmoides
was extracted with hot
water. After shaking the aqueous extract solution with ethyl acetate and then
1-butanol,
the aqueous
chromatography
on
solvent
with
system
increased.
This
Daiaion the
was
solution was
HP-20,
eluting
proportions
followed
with
of
subjected
with
a
methanol
purification
to
column
water-methanol gradually by
being
silica
gel
chromatography, to give two neolignan glucosides, 40 and 41 and two sesquilignan glucosides, 42 and 4 3 .
CH2OR1
MeO 9'
R20H2Cv..^8;0
RiO 40 : Ri=glc, R2=H 40a: Ri=glc(Ac)4, R2=Ac 40b:Ri=R2=H 40c: Ri=R2=Ac
41 : R^=gic, R2=H 41a: Ri=glc(Ac)4, R2=Ac 41b:Ri=R2=H
Compound 40 was isolated as a white powder, mp 134.7**C, [o.]^ -62.3° (MeOH), whose FD-MS gave peaks at m/z 683 (M"^ + 1 ) , 521 {it
-
CgH^QOj + 1) and 359 (M+ - 2 x CgH^QOg -»- 1 ) . The IR spectrum showed the presence of hydroxyl groups and aromatic rings. The ^H-NMR spectrum showed
signals
at
6 3.74
and
3.80
due
to
two
aromatic
methoxyl
groups, at 6 5.52 (IH, d, J=6.15 Hz) due to a benzylic proton, at 6 6.19 two
(IH, dt, J=15.83, 6.59 Hz) and 6.59 (IH, d, J=15.83 Hz) due to olefinic
protons,
protons. Acetylation
of
and
at
40
6
gave
6.80-7.20
due
a nonaacetate
to
five
(40a).
aromatic
The
^H-NMR
spectrum showed signals at 6 1.96 and 1.98, due to nine alcoholic acetyl
groups. The
enzymatic
hydrolysis
of
40
with
p-glucosidase
gave an aglycone (40b) and glucose. Acetylation of 40b
afforded a
650 triacetate
(40c), whose MS gave peaks at m/z 484 (M"*"), 424 (M"^ -
CH3COOH) and 382. The ^H-NMR spectrum showed signals at 6 2.05 and 2.10 due to two alcoholic acetyl groups, and at 6 2.30 due to a phenolic acetyl group. Compounds dehydrodiconiferyl
40b and 40c
were identified as
alcohol and its triacetate, respectively. From
these results, compound 40 was determined to be dehydrodiconiferyl alcohol diglucoside in which one glucose moiety is attached to the phenolic
group.
This
conclusion
was
supported
by
the
^^C-NMR
glucosylation shifts of C-1 (+3.0) and C-3 (+1.6 ppm), in going to 40 from 40b; they are identical with the 4-0-p-D-glucopyranosylation shifts of the guaiacyl group (ref. 62). The position of the other glucose linkage was determined from the ^-^C-NMR glucosylation shifts of the C-8' and C-9' atoms. As shown in Table 5.1, the signals of 40 at 6 123.6 and 68.7 were assigned to C-8' and C-9', respectively. The shifts of the corresponding carbons in going to 40 from 40b were
-4.3
and
+7.2 ppm,
respectively, which
indicated
that one
glucosyl group in 40 was linked to the C-9' atom. Thus, compound 40 was
established
as
dehydrodiconiferyl
alcohol
4,9'-di-O-p-D-
glucopyranoside. Compound 41 was isolated as an amorphous powder, [a]jj -16.3° (MeOH).
The
UV
and
IR
spectra
suggested
the
lignan
structure.
Acetylation of 41 gave a heptaacetate (41a), whose ^H-NMR spectra showed the presence of seven alcoholic acetyl groups (6 1.97, 2.03, 2.10 and 2.13). The enzymatic hydrolysis of 41 with p-glucosidase afforded an aglycone (41b) and glucose. The MS of 41b gave peaks at m/z 406
(M"^), 358 (M+ - CH2OH - OH) and 210. The ^H-NMR spectrum
showed signals at 6 3.74 and 3.77 due to three aromatic methoxyl groups, at 6 6.15 (dt, J=15.83, 5.71 Hz) and 6 6.58 (d, J=15.83 Hz) due to two trans
olefinic protons, and at 6 6.57 and 6.75-7.20 due
to five aromatic protons. The ^^C-NMR signals of 41 and 41b were assigned as shown in Table 5.1. From these results, compound 41b was determined to be guaiacylglycerol-p-O-4-synapyl
ether. The ^^C-NMR
chemical shift differences of the corresponding carbons in going to 41 from 41b were +3.1
(C-1') and +1.6 ppm
(C-3'), respectively,
which indicated that a glucose moiety in 41 was attached to the
651 Table 5.1
I^C-NMR
and 41b
c
spectral data for compounds 38, 39, 40, 40b, 41
38 and 39 ^) 134.7 110.7 148.9 147.6 116.1 119.7 89.1 55.2 64.8 136.8 112.9 145.2 149.1 129.8 116.6 77.4 7 5 . 5 , 76.1 6 4 . 3 , 64.6 56.4 56.8
1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6* 7' 8' 9' OMe
40
40b
135.3 110.6 149.1 146.3 115.6 118.1 86.9 53.1 63.0 130.3 110.6 143.7 147.4 131.8 115.2 129.3 123.6 68.7 55.8
132.3 110.5 147.5 146.3 115.3 118.4 87.1 52.9 62.9 129.5 110.5 143.6 147.1 130.5 114.9 128.9 127.9 61.5 55.6
100.2 101.9 73.5 76.8 69.7 70.2 76.8 60.8 61.1
glc- • 1 glc- -2 glc- -3 glc- -4 glc- -5 glc- -6
41b
41
132.2 103.8 152.6 134.9 152.6 103.8 129.9 128.4 61.1 133.1 111.2 146.8 145.2 114.5 119.2 72.0 85.9 59.6 55.5 55.8 56.0
132.3 103.7 152.7 134.9 152.7 103.7 130.0 128.4 61.3 136.2 111.6 148.4 145.4 115.0 119.1 71.9 85.9 59.6 55.7 55.9 100.3 73.2 76.7 69.7 76.7 60.6
(In DMSO-dg, 60 MHz) a): In MeOH-d4.
phenolic
group. The
^H- and
^^C-NMR
chemical
shifts
of
41
were
identical with those of citrusin B. Thus, compound 41 was identified as citrusin B. Compound 42 was obtained as an amorphous powder, [a]^ -11.5° (MeOH), which gave peaks at m/z 908 (M+), 746 (M+ - C^E^^O^), 584 (M^ - 2 X CgH^QOg) and 388 in the FD-MS spectrum. The IR spectrum of 42 showed
the
presence
of
hydroxyl
groups
and
aromatic
rings. The
652 •^H-NMR spectrum of 42
showed
signals
at 6 3.76
(s) due to
four
(s) and 6.70-7.16 (m) due to
aromatic methoxyl groups^ and 6 6.64
eight aromatic protons. Acetylation of 42
with acetic
anhydride-
pyridine gave a decaacetate (42a), which gave signals at 6 1.86 (s), 2.00 (s) and 2.11 (s) due to ten alcoholic acetyl groups, 6 3.75 (s) (s) and 6.70-7.16
due to four aromatic methoxyl groups, and 6 6.63
(m) due to eight aromatic protons in the ^H-NMR spectrum. Hydrolysis of
42
with
p-glucosidase
Acetylation
of
42b
tetraacetate(42c), whose 2.15 2.31
gave
with
an
aglycone
acetic
(42b)
and
anhydride-pyridine
^H-NMR showed
signals at 6 1.99
(s) due to two alcoholic acetyl groups, and at 6 2.29 (s)
due
identified
to
as
medioresinol
two
phenolic
hedyotol
C
acetyl
tetraacetate
tetraacetate).
ether
groups.
From
these
Compound
glucose. gave
a
(s) and (s) and 42 c
was
(guaiacylglycerol-presults,
42
was
considered to be hedyotol C diglucoside in which the two glucosyl groups are attached to two phenolic groups. The ^^C-NMR data of 42 and
42 b
supported
this
conclusion. As
shown
in
Table
chemical shifts of corresponding carbons in going to 42 were
+3.0
(C-l*
and
C-1")
and
+1.5
ppm
indicated that the two glucosyl groups in
(C-3' and
5.2,
the
from 42b
C-3"),
which
42 were linked to two
guaiacyl rings at the C-4' and C-4" positions. Furthermore, the ^-^C-
MeO 9"
R40H2C\8"0
OMe
OR4
42 : R-|=R2=R4=H, R3=glc 42a: Ri=R2=H, R3=glc(Ac)4, R4=Ac 42b: Ri=R2=R3=R4=H 42c: Ri=R2=H, R3=R4=Ac 43 : Ri=R2=0Me, R3=glc, R4=H 43a: Ri=R2=0Me, R3=glc(Ac)4, R4=Ac
653 Table 5.2
c 1 2 3 4 5 6 7 8 9 1' 2' 3* 4' 5' 6' 7' 8' 9' 1" 2" 3" 4" 5" 6" 7" 8" 9" OMe
g l c --1 g l c - -2 g l c - -3 g l c - -4 g l c - -5 g l c - -6 Me i n Ac
I^C-NMR s p e c t r a l d a t a f o r compounds 4 2 , 4 2 a , 4 2 b , 43 and 43a 42
42a
42b
43
43a
134.8 103.5 152.6 136.9 152.6 103.5 84.8 53.7 71.2 135.3 110.9 149.0 145.9 115.6 118.1 85.1 53.7 71.2 136.2 111.6 148.5 145.5 115.0 119.3 72.0 85.9 59.8 55.9 56.0 100.4 101.9 73.3 73.5 76.8 69.8 76.8 60.7 61.1
132.6 103.0 152.6 137.6 152.6 103.0 84.7 53.7 71.2 137.6 111.1 149.9 145.2 118.1 118.3 85.1 53.7 71.2 133.9 111.8 149.5 145.5 118.1 119.3 73.3 79.8 62.3 55.9 56.1 98.8 99.1 70.9
135.0 103.5 152.7 137.0 152.7 103.5 85.2 53.5 71.2 132.3 110.6 147.5 146.0 115.2 118.7 85.2 53.8 71.0 133.2 111.2 147.0 145.4 114.7 119.4 72.2 86.1 59.9 55.6 56.0
134.8 103.4 152.5 137.0 152.5 103.4 84.9 53.5 71.1 133.6 104.3 152.5 137.0 152.5 104.3 84.9 53.5 71.1 133.8 105.3 151.9 138.1 151.9 105.3 72.1 85.7 59.7 55.9 56.3 102.7 102.9 74.0
34.1 103.4 153.1 137.5 153.1 103.4 86.0 54.6 72.4 134.1 103.8 153.1 137.5 153.1 103.8 86.0 54.6 72.4 134.4 105.5 153.6 138.2 153.6 105.5 72.2 80.8 62.6 56.3 56.7 101.2
76.4 69.8 76.9 60.9
72.2 72.2 73.4 62.6
CO i n Ac
( I n DMSO-dg, 60 MHz) a ) : I n MeOH-d4
72.0 68.3 71.2 61.7 20.3 20.6 168.8 169.1 169.4 169.8
69.9
20.7 169.3 169.5 169.6 170. 170, 170,
654 NMR signals of the glucose moieties were consistent with the pglucopyranosyl form. Thus, compound 42 was concluded to be hedyotol C
4' ,4"-di-0-p-D-glucopyranoside
(guaiacylglycerol-p-medioresinol
ether 4 ', 4 "-di-0-p-D-glucopyranosi-de). Compound 43 was obtained as an amorphous powder, [a]^ -11.1° (MeOH), which gave a spot a little higher than that of 42 on TLC with chloroform-methand-water
(70:30:5) as a developing solvent.
The IR spectrum of 43 showed the presence of hydroxyl groups and aromatic rings. The ^H-NMR spectrum showed signals at 6 3.74 due to six aromatic methoxyl groups, and at 6 6.64
due to six aromatic
protons on three syringyl rings. The FD-MS spectrum gave peaks at m/z 1007 (M+ + K ) , 991 (M+ + Na), 829 (M+ - CgH^oGs + Na), 667 (M+ - 2 X CgH^QOg + Na) and 418. These peaks are 60 mass units larger than the corresponding peaks of 42. These findings suggest that compound 43 is syringylglycerol-p-syringaresinol ether glycoside. Acetylation of 43 with acetic anhydride-pyridine gave a decaacetate(43a), whose ^H-NMR showed signals at 6 1.99, 2.03, 2.15 and 2.18 due to ten alcoholic
acetyl
groups, at 6 3.76,
aromatic methoxyl groups, at 6 5.04
3.80
and
3.83
due
to
six
(2H, d, J=6.0 Hz) due to two
anomeric protons of glucose, and at 6 6.53, 6.56 and 6.60 due to six aromatic protons in three syringyl rings. Except for the glucose moieties,
the
^H-NMR
spectrum
syringylglycerol-p-syringaresinol
of
43 a
ether
was
similar
tetraacetate.
to
that
of
Furthermore,
the ^"^C-NMR signals of 43 and 43a were assigned as shown in Table 5.2, in comparison with those of 42, 42a and liriodendrin
[( + )-
syringaresinol-di-0-p-D-glucoside]. From these results, compound 43 was determined to be syringaresinol-p-syringaresinol ether 4',4"-di0-p-D-glucopyranoside.
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S. Ozawa and T. Sasaya, Mokuzai Gakkaishi, 33 (1987) 747-748.
18
S. Ozawa and T. Sasaya, Mokuzai Gakkaishi, 34 (1988) 169-175.
19
S. Ozawa and T. Sasaya, Mokuzai Gakkaishi, 34 (1988) 851-857.
20
S. Ozawa, T. Sasaya and Y. Tabei, Mokuzai Gakkaishi, 34 (1988)
21
T. Sasaya, T. Takehara and T. Kobayashi, Mokuzai Gakkaishi, 26
(1980) 633-636.
942-946. (1980) 759-764. 22
T. Takehara, T. Kobayashi and T. Sasaya, Mokuzai Gakkaishi, 26
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R. J. Swan and W. Klyne, Chem. Ind., (1965) 1218.
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J. Sakakibara, I. Hiroji, H. Ina and M. Yasue, 94 (1974) 1377-
(1980) 274-279.
1383. 25
S. Ozawa and T. Sasaya, Mokuzai Gakkaishi, 37 (1991) 847-851.
26
H. Achenbach, R. Waibel and I. Addae-Mensah, Phytochemistry, 22
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S.K. Koul, S.C. Taneja, K.L. Dhar, C.K. Atai, Phytochemistry^ 22 (1983) 999-1000.
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B.R. Prabhu, N.B. Mulchandani, Phytochemistry, 24 (1985) 329-
29
R.C. Gamble, G.R. Clark, P.A. Craw, T.C. Jones, P.S. Rutledge
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S. Omorl, S. Ozawa and K. Taneda, Mokuzal Gakkalshl, 40 (1994) 1107-1118.
31
Y.-G. Kim, H. Lee, S. Ozawa, T. Sasaya and C.-K. Moon, Mokuzal Gakkalshl, 40 (1994) 414-418.
32
S. Ozawa and T. Sasaya, Mokuzal Gakkalshl, 37 (1991) 69-75.
33
R.T. Palo, J. Chem. Ecol., 10 (1984) 499-520.
34
R. Hegnauer, Chemotaxonomle der Pflanzen, Vol.IV, Blrkhauser Verlag Basel, (1973) 241-258.
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S. Chlba, Y. Nagata, Blomass Henkan Kelkaku Hokoku(Report of blomass Conversion Project, In Japanease), Ojl Inst. Forest Tree Improv., Ojl Paper Co. Ltd., (1990) 2-38.
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H. Lee, N. Watanabe, T. Sasaya and S. Ozawa, Proceedings of 7th International Symposium on Wood and Pulping Chemistry (Beijing), Vol 3 (1993) 560-565.
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H. Lee, N. Watanabe, T. Sasaya and S. Ozawa, Mokuzal Gakkalshl, 39 (1993) 1409-1414.
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H. Lee, S. Ozawa and T. Sasaya, Mokuzal Gakkalshl, 40 (1994) 620-626.
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K. Herrmann, Fortsch. Chem. Org. Naturst., 35 (1978) 73-132.
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P.J. Houghton, Phytochemistry, 24 (1985), 819-826.
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Y. Sano and A. Sakakibara, Mokuzai Gakkaishi, 16 (1970) 81-86.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
659
Chemical and Biological Investigations of Salvia Species Growing in Turkey Ayhan Ulubelen and Giilacti Topcu *Faculty of Pharmacy, University of Istanbul, 34452 Istanbul, Turkey; **Tubitak, Marmara Reseach Center, Department of Chemistry, P.O. Box 21,41470, Gebze- Kocaeli, Turkey
1. INTRODUCTION The name Salvia is derived from the latin word salvare (healer). Salvia species have been used as folk medicine since ancient times in order to cure tuberculosis^ cancer^, diabetes^, coronary heart diseases, angina pectoris, myocardial infarction'^'^ skin diseases such as psoriasis and eczama^ and have oestrogenic activity^. Salvia (Lamiaceae) species are 30-100 cm tall, herbaceous, suflfruticose, perennial, rarely biennial or annual plants, growing in calcareous sunny lands in southern Europe and other parts of the world. There are about 500 Salvia species grown naturally in the world, Turkey has 90 species, 44 of them being endemic. In this review article we have dealt with the chemistry and biology of 16 Salvia species which were studied since 1992. In the previous review articles^'^ , the data of about 20 other Turkish Salvia species were given. 2. CHEMICAL STUDIES Until now, we have isolated over 50 triterpenoids, about 100 diterpenoids, more than half being new compounds, a total of 15 sesquiterpenoides and sesterterpenoides, 40 flavanoides and several aromatic compounds. Recently we have studied quite a number of Salvia species and isolated various group of compounds. The structures of the new compounds are discussed below.
660 2.1. SESQUTTERPENOIDS Although sesquiterpenoids are rather rare in Salvia species, three sesquiterpene lactones, an eremophilanolide and two eudesmanolides as well as caryophyllene oxide and (-)-glechomafiiran were isolated from S. palaefolia^^'^^. Recently, we have obtained several sesquiterpene lactones from S. yosgadensis^^,
Istanbulin
D,
lp-acetoxy-8p-hydroxyeudesm-4(15),7-dien-8,12-olide
(isolated
previously from S. potentillifolia^^) and 13-acetoxyeudesm-4(15),7-dien-8,12-olide as well as sesquiterpene spathulenol. We have also isolated the sesquiterpenes caryophyllene oxide and spathulenol from S. sclarea^^. From S. divaricata^^, a new linear sesquiterpene was isolated and named salvinine (1). The EIHR mass spectum of 1 indicated the molecular formula C15H26O3 Qn/z 254.1888, calc. 254.1877). The ^H NMR spectrum showed the structure clearly. A terminal ethylene group was present at 5 5.85 (lH,dd, J=10 and 17 Hz), 5.17 (IH, dd, J=1.5 and 17 Hz) and 4.99 (IH, dd, J=1.5 and 10 Hz), the signals at 5 6.40 (IH, d, J=16 Hz, H-9) and 6.90 (IH, d, J==16 Hz, H-10) showed a ^awis-ethylenic group. Methyl signals were at 5 1.37 (6H, 2xMe at C-11), 1.28 (3H, s, Me at C-3) 1,17 (3H, d, J=7 Hz, Me at C-7), hydrogen geminal to the latter methyl group was at 5 4.10 (IH, m, H-7), three adjacent methylene groups at 5 3.03 (2H, t, J=7Hz, H2-4), 1.60 (4H, m, H2-5 and H2-6). Mass degradation of 1 fully supported the assigned structure. The base peak at m/z 71.0779 (a) (C4H7O) showed the ethylenic group attached to carbon atom carrying a methyl and a hydroxyl groups, while the fragments at m/z 85.064 (b) (C5H9O), m/z 113.0063 (c) (C6H9O2), m/z 141,009 (d) (CgHisOz) were consistent with the tail part of the new sesquiterpene, salvinine (1).
OH
HO 10
2.2. DITERPENOIDS 2.2.1 Abietane Diterpenoids Turkish Salvia species contain mainly abietane type diterpenoids^^ while the majority of Salvia from the American continent have clerodane type diterpenoids^^. From Salvia candidissima Vahl subsp. occidentalis^^ Hedge, six abietane diterpenoids were isolated, five of which were known compounds ferruginol, horminone, 7-acetylhorminone, cryptanol.
661 montbretyl-12-methyl ether and one was a new compound 11-hydroxy-12-methylabieta-8,l 1,13-triene (2). Compound 2 has the molecular formula C2iH3202( w/z 316.2494, calc. 316.2514) as decided from its HR mass spectrum. The UV max at 276 nm indicated an aromatic ring system. The IR spectrum showed a hydroxyl at 3510 cm'^ and aromatic absorbances at 3080, 3050, 1605, 1520 cm"\ The structure was deducedfrom^H and ^^C NMR spectral data. The signals at 5 0.82 (3H, s), 0.85 (3H, s) (Me-18 and Me-19), 1.20 (3H, s, H-20), 1.22 (3H, d, J= 6.8 Hz) and 1.24 (3H, d, J=6.8 Hz) (Me-16 OMe HO^ . ^ ^
0^
^.^
HOOC 3 R=R'=H, R^=0 4 R=Me, R'=H, R^O 5 R=R2=H, R * = 0 H
and Me-17), together with the methine proton at 5 3.16 (IH, septet, J=6,9 Hz, H-15) indicated an abietane skeleton. Other signals were observed at 5 3.75 (3H, s, OMe), 6.46 (IH, s, H-14), 6.00 (IH, s, OH) (D2O exchangeable). The H-lp proton at 5 3.20 (IH, ddd, J=3.5, 5, 13 Hz) indicated a hydroxyl group at C-11 rather than at C-14. The ^^C NMR (APT) spectrum showed six methyl, five methylene, three methine and seven quaternary carbon signals (Table 1). Three new abietane diterpenoids were isolatedfromS. divaricata^^, which were 6-oxoroyleanone 18-oic acid (3), 6-0x0-12-methyh-oyleanone-18-oic acid (4) and horminone-18-oic acid (5). Compound 3 indicated the molecular formula C20H24O6 (m/z 360.1566, calc. 360.1572). The UV spectrum having maxima at 405 and 343 nm indicated a quinoid type structure rather than an aromatic abietane diterpene. The IR spectrum at 1680 and 1660 cm"* revealed the p-quinoid structure, while the carboxyl group and the carbonyl signals were observed at 1700 and 1720 cm'\ respectively. The *H NMR spectrum of 3 showed a resonance at 5 12.88 (IH, br s) (acid proton), while the isopropyl group was evident with the signals at 5 1.32 (6H, d, J=7 Hz, Me-16 and Me-17) and 3.54 (IH, septet, J=Hz, H15). Other signals were at 6 1.27 (3H, s, H-19), 1.37 (3H, s, H-20). The sequence of Ci- C3 was
662 decided from spin decoupling experiments with the signals at 5 2.35 (IH, ddd, J=15, 5 and 1 Hz, HIp), 1.60 (IH, ddd, J=15,13 and 5 Hz, H-la), 2.12 (IH, m, H-2p), 1.72 (IH, dddd, J=13,12, 7.5 and TABLE 1. ^^C NMR Data of Compounds 2,3, 4 and 5
c
2
3
1
41.6t
35.401
35.791
35.901
2
19.21
19.851
20.011
20.401
3
39.41
41.851
40.911
39.721
4
34.4 s
36.77 s
36.80 s
36.70 s
5
52.91
47.94 d
48.25 d
45.62 d
6
19.41
200.90 s
201.70 s
41.20 t
7
36.51
29.60 d
29.60 d
65.40 d
8
133.5 s
142.80 s
142.75 s
143.40 s
9
137.7 s
148.40 s
149.10 s
148.00 s
10
32.0 s
38.66 s
39.01 s
39.80 s
11
142.4 s
182.30 s
183.60 s
184.20 s
12
147.0 s
150.20 s
150.80 s
151.40 s
13
133.0 s
124.20 s
123.90 s
124.40 s
14
117.5 d
186.70 s
187.00 s
184.50 s
15
26.4 d
28.85 d
29.02 d
27.75 d
16
19.9 q
21.50 q
21.45 q
21.80 q
17
22.7 q
21.50 q
21.55 q
21.80 q
18
33.4 q
177.90 s
178.20 s
19
22.1 q
16.28 q
15.30 q
16.10 q
20
26.0 q
23.18 q
23.00 q
26.20 q
OMe
62.8 q
57.73 q
-
-
5
4
181.70 s
4.2, H-2a), 1.56 (IH ddd, J=14, 12.7 and 3.9 Hz, H-3P) and 1.22 (IH, m, H-3a). The ^^C NMR spectrum (Table 1) was in agreement with the given structure.
663 The *H NMR spectrum of compound 4 was quite similar to that of 3 with the exception of a methyl group at C-12. Although the stmcture of compound 5 was quite similar to those of 3 and 4, the 6-0X0 group was missing and there was a hydroxyl group at C-12. The spectral data resembled that of horminone, except for the additional acid group at C-4 instead of Me-18. From the aerial parts of Salvia pomifera^^ in addition to two known diterpenes ferruginyl 12methyl ethef^ and 18-hydroxyabieta-8,11,13-triene-7-one(6)^^ five new abietane diterpenes were isolated, pomiferin A-E (7-11). The ^H NMR spectrum of the new compounds clearly indicated their structures which are given in Table 2. Spin decoupling experiments were run to show the ^H-*H relations. The ^^C NMR spectra correlated with the given structures (Table 3).
Rl
R'
6 H
^ CH2OH 0
7 H
CH2OH
H
10 OH
Me
H
Rl
Ri
8 H
R' H
CH2OAC
11 OH
Me
H
9 Me
OH
COOH
12 OH
Me
0
13
CH2OAC H
Rl
H
Bl
R3
Another study^ with the same plant has yielded two more of the same group of compounds pomiferin F and G (12, 13). The ^H NMR spectrum of pomiferin E (12) exhibited an additional hydroxyl group at C-15, as decidedfi'omthe lack of H-15, and the presence of Me-16 and Me-17 as singlets at 5 1.29 (3H), and 1.33 (3H) instead of doublets. A signal at 5 4.16 (IH, dddd, J=4, 4, 11.5, 11.5 Hz, H-2P) indicated the presence of a secondary hydroxyl group at C-2 which was also observed in compounds 10 and 11. The C ring protons were at 5 7.38 (IH, d, J=8 Hz, H-11), 7.75 (IH, dd, J=2 and 8 Hz, H-12), 8.08 (IH, d, J=2 Hz, H-14), the latter signal being indicative of an 0x0 group at C-7. Pomiferine G (13) showed the methyl signals at 5 1.26 (6H, d, J=7 Hz, Me-16 and Me17), 1.02 and 1.26 (3H, each) (Me-19, Me-20) and isopropyl methine signal was at 5 2.94 (IH, septet, J=7 Hz, H-15). A methylene pair appeared at 6 3.78 and 3.86 (each IH, J=12 Hz,
664
TABLE 2. ^H NMR Data of Compounds 7-11 H
7
8
9
1
2.28 ddd
2.19 ddd
3.54 3.54ddd ddd
10
11
2.62 ddd 2.62 ddd
2.68 ddd
1.33 ddd 1.33 ddd --
4.05 4.05ddd ddd
3
--
1.85 m -
4
—
1.28 ddd
2
-
-
4.05
5 6 7 8 9 10 11
7.17 d
6.85 s
--
7.15 d
7.30 d
12
6.90 dd
-
~
6.98 dd
7.42 d
14
6.80 d
6.65 s
6.51s
6.90 br s
7.89 d
15
2.88 sept
3.12 sept
3.16 sept
2.83 sept
2.94 sept
16
1.22 d
1.23 d
1.18d
1.22 d
1.16d
17
1.22 d
1.23 d
1.21 d
1.22 d
1.16d
18
3.22 d
3.63 d
-
0.97 s
1.01s
18'
3.47 d
3.95 d
-
-
--
19
1.23 s
1.22 s
0.96 s
0.99 s
1.06 s
20
0.85 s
0.90 s
0.86 s
1.20 s
1.20 s
C=0
-
CH3
-
2.02 s
OMe
-
--
13
3.72 s
665 H2-18) while the aromatic protons were at 5 7.31 (IH, d, J=8 Hz, H-11), 7.42 (IH, dd, J=2 and 8 Hz, H-12) and 7.89 (IH, d, J=2 Hz, H-14). The ^^C NMR data of 13 are given in Table 3. TABLE 3 ^^C NMR Data of compounds 6 - 10,13
c 1
6 38.51
7
8
9
38.21
38.31
41.51
10
13
51.4t
37.41
2
18.71
18.61
18.51
20.01
65.8 d
18.lt
3
35.lt
35.lt
35.51
38.81
47.91
36.01
4
37.4 s
30.5 s
36.7 s
41.6 s
36.1s
31.5s
5
43.9 d
43.9 d
44.0 d
47.6 d
50.3 d
43.9 d
6
19.01
18.lt
18.91
20.01
19.21
19.61
7
29.41
30.01
30.21
31.9t
20.01
192.6 s
8
127.0 s
135.6 s
127.2 s
125.4 s
134.7 s
132.6 s
9
148.3 s
147.9 s
148.6 s
129.5 s
147.1s
138.5 s
10
37.9 s
38.0 s
37.4 s
34.1s
39.1s
38.3 s
11
lll.Od
123.9 d
lll.Od
118.1s
127.2 d
123.6 d
12
150.8 d
123.7 d
150.6 s
147.8 s
124.0 d
124.9 d
13
131.6s
145.9 s
131.4s
134.6 s
145.8 s
152.0 s
14
126.6 d
126.7 d
126.7 d
142.4 d
124.0 d
134.5 d
15
26.8 d
33.5 d
26.3 d
32.7 d
33.7 d
33.6 d
16
22.6 q
24.1 q
23.8 q
23.1 q
33.7 q
23.8 q
17
22.7 q
24.1 q
23.8 q
23.5 q
23.8 q
24.0 q
18
72.2 t
72.2 t
72.3. t
180.9 s
33.6 q
71.6t
19
17.4 q
17.3 q
17.4 q
18.4 q
22.1 q
18.1 q
20
25.2 q
25.9 q
25.3 q
26.5 q
24.5 q
24.3 q
--
-
171.8 s
--
--
20.6 q
54.8 q
--
—
C=0
-
-
170.8 s
CHs
-
~
21.6 q
OMe
-
~
666 Salvia monthretii?^'^^ yielded two new compounds, one of which is a derivative of salvinolone (14). The compounds isolated were salvinolonyl 12-methyl ether (15) and 14-hydroxyferruginol (16) in addition to the known compounds ferruginol, ferruginyl 12-methyl ether, taxodione, hypargenin F and demethylcryptojaponol. The *H NMR spectrum of the new compounds showed typical signals
OMe
15
16
for an abietane-type structure. H-ip in 15 was observed at 6 3.27 as a double triplet (J=3 and 13 Hz), indicating the presence of a hydroxyl group at C-11, while the signal at 7.72 (IH, s, H-14) showed the presence of an oxo group at C-7. The rest of the ^H NMR signals were typical for the abietane structure. The ^^C NMR spectra of both compounds are given in Table 4.
17
18
In a recent study with a fresh collection of the same plant^, we have isolated 6-hydroxysalvinolone (17) and 7-hydroxytaxodione (18). The structures were determined by ID and 2D NMR techniques. The HRCI mass spectrum of 17 indicated the molecular formula C20H26O4 {m/z 331.1906, M+1). The chemical shift of the C-4 methyl groups at 5 1.42 and 1.43 (3H each, s) indicated a double bond at C-5 and a hydroxyl group at C-6. The signal at 6 7.71 (IH, s, H-14) revealed the presence of a carbonyl group at C-7. The C-11 hydroxyl group was deducedfromthe chemical shift of H-lp at 5 3.05 (IH, septet, J=7 Hz). The ^^C NMR and HETCOR experiments
667 TABLE 4 ^^C NMR Data of Compounds 15,16 and 19
c
15
16
19
1
40.51
37.81
214.6 s
2
18.71
17.lt
42.81
3
34.21
38.81
33.91
4
38.2 s
31.2s
47.4 s
5
175.4 s
51.4d
44.9 d
6
123.5 d
17.81
20.91
7
185.4 s
30.41
30.71
8
124.4 s
114.4 s
134.5 s
9
136.7 s
133.5 s
146.9 s
10
42.2 s
38.8 s
50.2 s
11
145.8 s
109.2 d
122.6 d
12
148.2 s
141.8 s
124.6 d
13
139.7 s
121.5 s
145.5 s
14
115.8d
143.2 d
126.8 d
15
26.8 d
66.5 s
29.6 d
16
23.6 q
22.2 q
24.1 q
17
23.6 q
22.2 q
24.1 q
18
24.8 q
31.3 q
182.5 s
19
29.3 q
21.8 q
16.2 q
20
33.2 q
23.8 q
26.1 q
OMe
61.9q
-
gave the correlations between the carbons and protons (Table 5). A new abietane diterpene l-oxo-abieta-8,1 l,13-triene-18-oic acid (19) and the known compounds horminone and ferruginol were isolated from the roots of S. tomentosc^. The molecular formula of 19 C20H26O3 was derivedfromits HRMS (m/z 314.1887). The ^H NMR spectrum of 19
668 TABLE 5 *H and ^^C NMR Data of Compounds 17 and 18 17
1 2 3
3.0 ddd, 1.50 m 1.90 ddd, 1.68 m 2.0 m, 1.30 m
18
36.61
*
42.3
17.91
*
18.71
30.41
*
32.91
4
-
37.6 s
-
32.9 s
5
-
170.8 s
2.90 s
62.2 d
6
-
141.0 s
-
182.0 s
7
-
180.0 s
-
142.3 s
8
-
123.2 s
-
126.5 s
9
-
132.9 s
-
141.9 s
10
-
38.3 s
-
41.8 s
11
-
143.4 s
-
145.4 s
12
-
145.6 s
-
183.4 s
13
-
138.3 s
-
138.4 s 133.1 d
14
7.72 s
116.6d
6.45 s
15
3.04 sept
27.4 d
2.92 sept
26.9 d
16
1.28 d
22.4 q
0.95 d
21.4 q
17
1.32 d
22.4 q
0.85 d
21.4 q
18
1.42 s
22.7 q
1.32 s
32.5 q
19
1.43 s
28.0 q
1.28 s
21.8 q
20
1.67 s
27.9 q
1.10s
21.9 q
Overlapped signals indicated the aromatic signals at 5 7.17 (IH, d, J=8.5 Hz, H-11), 7.02 (IH, dd, J=2 and 8.5 Hz, H-12), 6.9 (IH, d, J=2 Hz, H-14) and the other signals were at 5 2.86 (IH, sept J=7 Hz, H-15), 1.24 (6H,d, J=7Hz, H-16andH-17), 1.23 (3H,s,H-19), 1.28 (3H, s, H-20). The location of
669 an 0X0 group was deduced at C-1 due to the chemical shift of H-11 as well as the chemical shifts of A ring protons. The *^C NMR spectrum of 19 showed the C-1 carbonyl at 5 214.6, the carboxyl group was at 6 182.5 (C-18) and the rest of the signals were in agreement with the suggested structure (Table 4). The structure of nemorosine (20) was established as 2a,14-dihydroxydehydroabietic acid which was isolated fi*om S. nemoroscF. Its structure was deduced fi-om the ^H NMR spectrum, spin decoupling experiments and by comparison with structurally resembling compounds. The C-ring signals were observed at 5 7.12 (IH, d, J=8.5 Hz, H-11), 6.8 (IH, d, J=8.5, H-12), and 5.82 (IH, br s, C-14 OH) (D2O exchangeable). The signal at 5 4.10 (IH, dddd, J=4,4, 9.5,10 Hz, H-SP) indicated an a-OH group at C-2 . The methyl signals were at 6 1.04 (3H, s), 1.09 (3H, s) (Me-18 and Me-20), 1.22 (6H, d, J=7 Hz, Me-16 and Me-17) together with an isopropyl methine signal at 5 3.80 (IH, sept. J=7 Hz, H-15). The plant material also yielded the known diterpenoids pachystazone, horminone and acetylhorminone.
Another new abietane diterpenoid was isolatedfi-omS. sclared^. Its structure was established as 7-oxoferruginol-18-al (21) on the basis of ^H NMR spectral data. The signal at 5 7.84 (IH, s, H-14) indicated the presence of an 0x0 group at C-7, while another signal at 5 9.78 verified the presence of an aldehyde group in the molecule. The aldehyde group could be placed either at C-4 or C-10. When the aldehyde group is placed at C-10 the two methyl signals at C-4 (Me-18 and Me-19) would be
close
together in their ^H NMR spectrum^' ^ while in the present case, the methyl groups were at 5 1.02 and 1.20 (each 3H, s) (Me-19, Me-20) showing the placement of the aldehyde group at C-18. Other signals were at 5 1.28 (6H, d, J=6.8 Hz, Me-16 and Me-17), 6.90 (IH, s, H-11) and 3.16 (IH, sept. J=6. 8 Hz, H-15). Two known compounds ferruginol and 7-oxoroyleanone were also isolated.
670 A new abietane diterpene heldrichinic acid (22) was isolated from .SI heldreichiana Boiss.^^. The molecular formula of 22 was calculated from its HREI mass spectrum {m/z 332.1980). The ^^C NMR spectmm of 22 indicated the presence of three methyl quartets, seven methylene triplets, three methine doublets and seven quaternary carbon singlets for 20 carbon atoms. The signals of the carbonyl group was at 5 214.2 while that of the carboxyl group was at 5182.3. Two downfield signals at 5 148.7 s and 110.3 t indicated the presence of an exo methylene group. The double bond was verified from the signals at 5 111.2 s and 110.5 d, whereas a tertiary hydroxyl was indicated by the signal at 5 76.5 s. The ^H NMR spectrum together with mass degradation signals indicated the structure of 22 clearly. The ^H NMR spectrum showed signals at [5 4.81 (2H, br s, C-16 exo-CH2), 1.72 (3H, s, Me-17), and 5.12 (IH, dd, J=4 and 7 Hz, H-11) (the vinylic proton)]. Two other methyl signals were at 6 1.27 (Me-19) and 0.94 (3H, s, Me-20). The mass fragmentation indicated that the double bond should be at ring C and since no conjugation was observed in the UV spectrum (207 nm), it should be situated between C9 and C-11. Spin decoupling experiments established the vicinal relations between the olefinic H-11 and H2-I2. The location of the hydroxyl group at C-8 was similar to that of compactone^V The ^^C NMR
COOH 22
23
24
data of 22 are given in Table 6. S. nc^ifolict^
yielded five new abietane diterpenoids, 6,12,14-trihydroxyabieta-6,8,ll,13-
tetraene (23), 7,20-epoxyroyleanone (24), 1-oxoferruginol (25), 6-oxoferruginol
(26), 11,12-
dioxoabieta-8,13-diene (27) together with seven known abietane diterpenes horminone,
acetyl-
horminone, ferruginol, pachystazone, cryptanol, cryptojaponol and sugiol. The molecular formula of 23 was C20H28O3 as indicated by its HREI mass spectrum {jn/z 316.2311). The UV spectrum showed a maximum at 332 nm indicating a conjugated aromatic system as observed in cryptanol^^. The ^H NMR
671 spectrum showed signals at 5 6.87 (IH, s, H-7), 6.20 (IH, s, H-11) and 5.28 (IH, br s, OH) (disappeared with D2O), while the protons for the isopropyl group of the abietane skeleton were observed at 6 1.14 (3H, d, J=7 Hz), 1.16 (3H, d, J=7 Hz) (Me-16 and Me-17), and at 5 3.07 (IH, sept, J=7 Hz, H-15). Other methyl signals were at 5 1.09 (3H, s, Me-20) and 1.24 (6H, s, Me-18 and Me19). The observation of H-ip at 6 2.92 (IH, dt, J=1.5 and 10 Hz) indicated the lack of a hydroxyl group at C-11. In the latter case H-ip shifts to ca 3.1-3.8 ppm^'*'^^ The lack of a proton doublet in the lowerfieldindicated that the third hydroxyl group should be at C-6. Compound 24 had the molecular formula C20H26O4 {m/z 330.1818) as evidentfi'omits HRMS. Its ^H and *^C NMR showed its structure clearly. The other ^H NMR signals were shown at 5 4.41 (IH, dd, J=1.5 and 4.0 Hz, H-7a), 3.73 (IH, d, J=7 Hz), 3.65 (IH, d, J=7 Hz) (oxymethylene protons), 3.16 (IH, sept, J=7 Hz, H-15), 2.68 (IH, dt, J=3, 4 and 12 Hz, H-lp), 1.18 and 1.22 (each 3H, d, J=7 Hz) (Me-16 and Me-17), 0.92 and 0.88 (each 3H, s) (Me-18 and Me-19). The ^^C NMR spectrum (Table 6) is in agreement with the given stnicture. The HREI mass spectrum of compound 25 indicated the molecular formula C20H28O2 {m/z 300.2104). The ^H NMR spectrum showed two proton singlets in the downfield area at 5 7.40 (H-11) and 7.18 (H-14), the methyl protons were at 5 1.28 (3H, s, Me-20), 1.17 and 0.87 (each 3H, s) (Me-18 and Me-19), and the isopropyl group was at 1.24 (6H, d, J=7 Hz, Me-16 and Me-17), 3.28 (IH, sept, J=7 Hz; H-15). The lack of H-lp signal and the downfield appearence of H-11 indicated the 1oxoferruginol structure for compound 25. Another similar compound (26) with the molecular formula C^nOiQn/z 300.2110) was also isolatedfi-omthe same extract. Its ^H NMR signals were quite similar to those of ferruginol, resonating at 5 6.92 (IH, s, H-11), 6.71 (IH, s, H-14), 3.17 (IH, sept, J=7 Hz, H-15), 1.18 and 1.22 (each 3H, d, J=7 Hz) (Me-16 and Me-17), 0.88, 0.91 and 1.27 (each 3H) (Me18, Me-19 and Me-20). The isolated doublet signals at 6 3.02 and 2.57 (each IH, J=14 Hz, H2-7) and the singlet signal at 5 2.61 (IH, H-5) as weU as the slightly downfield shift of Me-20 (5 1.27) indicated that the 0x0 group should be at C-6. The ^^C NMR spectrum of 26 was in accordance with the suggested structure (Table 6). The last new compound 27 obtainedfi'omthis plant had the molecular formula C20H28O2 {ni/z 300.2070). Its UV spectrum with a maximum at 395 nm indicated a quinonoide structure rather than aromaticity in the C ring. The IR spectrum was consistent with an orthoquinonoide structure with the signals at 1685 and 1645 cm"\ The *H NMR spectrum showed the signals at 5 6.31 (IH, d, J=1.5 Hz, H-14), 2.98 (IH, sept, J=7 Hz, H-15), 2.70 (IH, dt,Hz, H-lp), 1.28 (3H, s, Me-20),
672 1.10, 1.01 (each 3H, d, J=7 Hz) (Me-16 and Me-17), 0.91 and 0.89 (each 3H, s) (Me-18 and Me-19). The *^C NMR spectrum verified the quinone structure with the signal at 182.0 ppm for two carbonyl groups (Table 6).
25 R=0, R1=H 26 R=H. R1=0
27
A new abietane diterpenoid was isolated fi-om Salvia forskahlei L. syn. S. hierosolymitana Boiss. var. pontica Freyn and Bomm^^. The new compound forskalinone (28) had the molecular formula C21H28O6 as establishedfi-omits HREI mass spectrum {m/z 376.1880). The UV [ 369, 400 (sh) ] and the IR (1680,1650,1622 cm'^) spectra indicated a quinonoid structure for 28. The ^H NMR spectrum of forskalinone (28) was consistent with the given structure. A sharp singlet at 5 13.40 indicated a hydrogen bond between the hydroxyl at C-14 and the carbonyl at C-7^^"^^. Another signal at 5 5.67 also showed a hydrogen bond between a hydroxyl at C-11 and a methoxyl at C-12. The presence of the hydroxyl group at C-11 was correlated with the signal at 5 3.24 (IH, br d, J=12 Hz) for H-ip'^^'^^V Two singlets at 5 3.64 and 3.60 (each IH) indicated the presence of a hydroxymethylene group which could be placed either at C-4 or at C-10. When it is at C-4, the methyl group at C-10 is shifted upfield to 6 0.80-0.85, while at C-10, the two methyl groups would have a chemical shift difference of 0.1-0.2 ppm""^ as observed in compound 28. Other signals were at 5 3.78 (3H, s, OMe), 3.30 (IH, sept, J=7 Hz, H-5) 1.38 (3H, d, J=7 Hz) and 1.36 (3H, d, J=7 Hz) (Me-16 and Me-17), 1.34 (6H, s, Me-18 and Me-19). The singlet at 5 2.61 (H-5) indicated the presence of an 0x0 group at C-6, thus supporting the ring B is an a-diketone. The ^^C NMR spectrum was in agreement with the given structure (Table 6). The ^H-^H COSY experiment confirmed the connectivity between C-1, C-2, C-3.
673 TABLE 6 *^C NMR Data of Compounds of 22, 24,26, 27 and 28
c
22
24
26
27
28
1
43.1
35.6
35.4
37.0
41.1
2
19.6
18.5
18.7
18.2
19.0
3
41.2
40.9
41.9
40.8
35.8
4
35.4
32.9
32.2
33.4
35.8
5
58.0
45.4
58.0
46.4
49.6
6
46.1
22.9
207.2
23.7
186.0
7
214.2
69.2
42.5
33.4
184.2
8
76.5
134.2
135.8
148.3
138.0
9
111.2
147.8
149.3
154.0
152.1
10
39.8
39.1
41.4
39.3
41.1
11
110.5
184.2
118.9
182.0
161.0
12
30.0
150.6
151.2
182.0
152.1
13
40.0
124.6
132.8
136.0
134.1
14
43.1
182.4
126.5
118.3
153.2
15
148.7
24.2
26.7
25.0
26.0
16
18.3
19.6
21.6
20.4
20.3
17
110.3
19.8
21.1
20.6
20.3
18
182.3
33.1
32.3
33.1
32.8
19
29.7
22.9
22.8
22.0
21.5
20
15.6
65.4
22.5
20.1
70.5
OMe
-
-
-
-
62.0
Salvia limbatct^ yielded two known abietane diterpenoids ferruginol and abieta-8,11,13-triene. Salvia multicaulis Vahl"*^ syn. S. acetohulosa has yielded six new abietane and norabietane diterpenoids in addition to 1-oxoferruginol, 3-oxo-ferruginol, horminone, pisiferal, 18-oxoferruginol and sempervirol.
674
HQ HOH2C
28 Thefirstnorditerpenoid compound multicaulin (29) gave the molecular formula C20H22O (/w/z 279.1678, M+1) as observedfi-omits HRCI mass spectrum. The ^H NMR spectrum of 31 indicated a fiilly aromatized structure giving the signals at 6 8.39 (IH, d, J=9 Hz, H-1), 7.95 (IH, s, H-11), 7.88 (IH, d, J=8.7 Hz, H-6), 7.70 (IH, d, J=9 Hz, H-2), 7.67 (IH, s, H-14), 7.41 (IH, d, J=8.7, H-7). The ^^C NMR (APT) spectrum verified the aromatic structure of compound 29. The carbons and protons were correlatedfi'omthe HETCOR experiment (Table 7). Compound 30 was found to be the demethyl derivative of 29, its ^H and ^^C >MR spectra being quite similar to those of 29 with the exception of a methoxyl group. The HETCOR data of 30 are given in Table 7. Compounds 31 and 32 possess an orthoquinonoid ring C with aromatic A and B rings. Compound 31 afforded the molecular formula C20H20O3 (m/z 308.1422) as calculatedfi"omits HREI mass spectrum. The ^H NMR spectrum showed the aromatic protons at 5 7.50 (IH, d, J=9 Hz,H-6), 7.17 (IH, s, H-2), 7.10 (IH, d, J=9 Hz, H-7), and 7.05 (IH, s, H-14). The chemical shifts of H-6 and H-7 were characteristic for the orthoquinonoid system in ring C as observed in saprorthoquinone'*^. The HETCOR experiment (Table 8) showed the correlation between carbons and protons.
29 R=Me
31 R=Me
30 R=H
32 R=H
TABLE 7 'H and 13CNMR data of compounds 29, 30, 31 and 32 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
OMe
30
'H
I3c
'H
8.39 7.70
120.9 d 128.0 d 140.0 s 135.6 s 122.7 s 125.5 d 127.9 d 126.1 s 147.8 s 133.4 s 110.6 d 151.4 s 130.3 s 125.5 d 25.5 d 22.7 q 22.7 q 20.9 q 18.3 q 55.4 q
8.30 7.60
-
7.88 7.41
-
7.95
-
7.67 3.46 1.32 1.32 2.65 2.52 4.06
7.85 7.40
7.91 7.57 3.39 1.38 1.38 2.64 2.52
31
"c 121.0 d 128.2 d 140.0 s 135.5 s 123.0 s 125.6 d 127.9 d 126.3 s 147.8 s 133.4 s 109.9 d 148.9 s 130.0 s 124.9 d 26.0 d 22.8 q 22.8 q 21.0 q 18.4 q
'H
7.17
7.50 7.10
7.05 3.30 1.22 1.22 2.33 2.25 3.88
32
l3c 133.9 s 152.8 d 140.6 s 133.4 s 127.6 s 125.5 d 132.4 d 128.5 s 126.0 s 152.4 s 183.3 s 181.4 s 135.3 s 126.6 d 26.3 d 23.0 q 23.2 q 20.8 q 17.9 q 56.0 q
'H
7.12
7.42 7.10
7.03 3.21 1.27 1.26 2.32 2.25
l3c 133.2 s 153.0 d 141.0 s 133.5 s 127.0 s 122.6 d 133.0 d 128.9 s 127.0 s 150.1 s 183.2 s 183.0 s 134.9 s 126.5 d 25.8 d 22.9 q 23.0 q 21.0 q 17.6 q
675
676 Compound 34 was the demethyl derivative of 33. Both compounds have spectral similarities with the lack of a methoxyl group in compound 34. The molecular formula of 32 was found to be C19H18O (m/z 294.1260)fromthe HREI mass spectrum. Table 7 shows the ^H and ^^C NMR data of 32. Other new abietane diterpenoids obtained from S. multicaulis^^ were 12-methyl-5-dehydrohorminone (33), 12-methyl-5-dehydroacetylhomiinone (34). The HREI mass spectrum of 33 indicated the molecular formula C21H28O4 {m/z 344.1992). The ^H and *^C NMR spectra of 33 clearly showed its structure giving the characteristic H-7 signal for horminone at 5 4.70 as a broad singlet together with other similar signals at 5 3.30 (IH, septet, J=7 Hz, H-15), 1.21 and 1.18 (each 3H, d, J=7 Hz) (Me-16 and Me-17), 1.09 (3H, s, Me-20) and 1.05 (6H, s, Me-18 and Me-19). Additional signals were at 5 6.91 (IH, br s, H-6) and at 5 3.82 (3H, s). The latter (OMe) could only be placed at C-12. Compound 34 was found to be the 7-acetyl derivative of 33. Its HRMS gave the molecular formula C23H35O5 {m/z 386.2122). The signal for H-7 was shifted to 5 5.35
OMe
33
R=H
34
R=Ac
(IH, br s) thus indicating the placement of the acetyl group. Other signals were similar to those of compound 33 appearing at 5 6.91 (IH, br s, H-6), 3.83 (3H, s, OMe), 3.30 (IH, septet, J=7 Hz, H-15), 1.19 and 1.21 (each 3H, d, J=7 Hz, Me-16 and Me-17), 1.08 (3H, s, Me-20), 1.05 (6H, s, Me-18 and Me-19), 2.03 (3H, s,OAc). The *^C NMR data of both compounds are given in Table 8. S.tchihatchefftf^ yielded two new abietane diterpenoids tchitatine (35) and salvitchitatine (36). The molecular ion peak at m/z 284.2147 of 35 indicated the molecular formula C20H28O. The ^H NMR spectrum indicated two downfield signals as narrow doublets at 5 7.20 (IH, d, J=1.5 Hz, H-12), 6.20 (IH, d, J=1.5 Hz, H-14), two broad singlets at 5 4.78 and 4.72 (each IH, CHr 16), at 5 1.85 (3H, s,
677 TABLE 8 ^^C NMR data of 33,34 and 38
c
33
34
38*
1
37.41
37.51
42.31
2
18.21
18.21
18.61
3
40.81
40.71
36.7 s
4
33.4 s
33.4 s
33.5 s
5
127.7 s
127.6 s
62.1 d
6
106.7 d
108.0 d
181.1s
7
77.8 d
76.9 d
141.0 s
8
124.7 s
124.8 s
126.7 s
9
142.6 s
142.6 s
145.5 ss
10
41.2 s
41.2 s
41.7 s
11
183.2 s
183.2 s
136.8 s
12
154.5 s
154.4 s
200.4 s
13
129.4 s
129.5 s
144.8 s
14
181.4 s
182.0 s
133.1 d
15
26.4 d
25.9 d
27.0 d
16
21.4 q
21.5 q
21.4 q
17
21.6 q
21.5 q
21.2 q
18
21.6 q
22.0 q
32.8 q
19
33.4 q
33.0 q
21.5 q
20
20.1 q
20.2 q
21.0 q
OMe
56.4 q
55.9 q
-
CO
-
172.6 s
-
Me
-
22.3 q
-
*A11 assignments correspond to C and C\ Me-17) and three other methyl singlets at 5 0.96, 0.72 and 0.70 (Me-18, Me-19, M&-20). Spin
678 decoupling experiments showed the relation between the C-1 - C-3 and C-6 - C-7 protons and between the H-12 and H-14. The ^^C NMR displayed 20 C atoms consisting of four methyl quartets, six methylene triplets, three methine doublets and seven quaternary carbons. The assignment of carbons
35
36
and protons were made by the observation of their direct correlations in the HETCOR experiments (Table 9). The hydroxyl group should be at C-11 due to following reasons; the J values of the two aromatic protons showed their meta positions. There is no hydrogen geminal to the hydroxyl group and the observation of H-ip at 5 3.16 (IH, br d, J=12 Hz) established the presence of a C-11 hydroxyl group. The structure of tchitatine was concluded to be as 1 l-hydroxyabieta-8,ll,13,15-tetraene. Salvitchitatine (36) had the molecular formula C20H28O {m/z 284.2148). The *H NMR spectrum showed the presence of aromatic signals similar to those of 35, at 5 7.20 (IH, d, J=1.5 Hz, H-11), 6.21 (IH, d, J=1.5 Hz, H-14) and an olefinic proton signal at 6 6.59 (IH, t, J=4 Hz, H-6). An isopropyl side chain was observed at 5 2.88 (IH, sept. J=7 Hz, H-15) and 1.14 (6H, d, J=7 Hz, Me-16 and Me-17). Other methyl signals were at 5 1.10, 1.18, 1.27 (each 3H, s) (Me-18, Me-19, Me-20). Spin decoupling experiments showed the relations between the C-1 - C-3, and C-6 - C-7 protons. The ^^C NMR (APT) and HETCOR experiments made possible the assignment of the protons and carbons (Table 9). The structure of salvitchitatine (36) was deduced as 1 l-hydroxyabieta-5,8,11,13-tetraene. From a Turkish Salvia species S. montbretii^, two abietane type dimeric diterpenoids were isolated for thefirsttime. One of them, 7,7-bistaxodione (37) had the molecular formula C40H50O6 (m/z 626.3730). The *H NMR spectrum of 37 resembled to those of taxodione and 7-hydroxytaxodione (18), with the methyl signals resonating at 5 1.41, 1.24 and 1.05 corresponding to Me-18, Me-19 and Me-20; two methyl doublets resonated at 5 1.08 and 1.10 (each 3H, J=7 Hz, Me-16, Me-17),
679 TABLE 9 *H and ^^C NMR data of Compounds 35 and 36
c
35
36 ^H
^'C
'H
^^C
1
3.16 brd, 1.60 m
38.71
3.10 brd, 1.80 m
40.3 t
2
2.55 m
18.61
2.50 m
20.21
3
2.25 m, 1.60 m
41.2t
2.30 m, 1.80 m
40.81
4
-
31.6s
-
33.0 s
-
147.1 s
5
2.601
46.2 d
6
1.58 m, 1.28 m
20.21
6.591
135.2 d
7
3.60 m, 2.25 m
31.8t
2.25 m
34.61
8
-
126.1s
-
126.5 s
9
-
147.0 s
-
146.5 s
10
-
38.8 s
-
36.4 s
11
-
151.2 s
-
150.9 s
12 13 14
140.9 d
7.20 d -
131.4 s 106.1 d
6.21 d
-
144.2 s
2.80 sept
28.6 d
110.31
1.14d
23.6 q
6.20 d
15
140.9 d
7.20 d -
130.8 s 106.3 d
16
4.72 brd, 4.78 brd
17
1.85 s
16.1 q
1.14d
23.6 q
18
0.72 s
33.4 q
1.10s
32.1 q
19
0.70 s
23.5 q
1.18s
24.0 q
20
0.96 s
24.5 q
1.27 s
24.6 q
methine proton appeared at 5 2.98 (IH, d septet, J=1.2 and 7 Hz, H-15), while H-5 occured at 6 as a sharp singlet at 5 2.71. Two downfield signals were at 5 6.60 (IH, d, J=1.2 Hz, H-14) and 7.63 (IH, s, C-11 OH, D2O exchangeable). The presence of afragmentpeak at m/z 313 in its mass spectrum assignable to half of the structure indicated that the compound could be a dimeric taxodione. In the case of a symmetrical dimer the linkages of the two taxodione molecules could be either at C-14, C-11, C-7
680 or if the compound is asymmetric the linkage could be between C-7 - C-14, C-11 - C-7 or C-11 - C-14. The signals of the *H NMR spectrum indicated that the substance was a symmetrical dimer otherwise more signals should have been observed for the olefinic protons. In the *H NMR spectrum H-11 and H14 signals were clearly seen as in taxodione, so the only plausible way of joining the two units together wasC-7-C-r.
The second dimeric compound, 11,1 r-didehydroxy-7,7-dihydroxytaxodione (38), had the molecular formula C40H50O6 (m/z 626.3722). The *H NMR spectrum showed methyl signals at 6 1.02, 1.20, and 1.26 (each 3H, s) for Me-18, Me-19, Me-20, respectively. Two other methyl signals were at 5 0.78 and 0.88 (each 3H, d, J=7 Hz, Me-16 and Me-17), a methine signal appeared at 5 2.96 (IH, d septet, J=1.2 and 7 Hz, H-15) and a singlet resonated at 6 2.92 (IH, s, H-5). Downfield signals were observed at 5 6.37 (IH, d, J=1.2 Hz, H-14) and at 6 7.18 (IH, s, D2O exchangeable, C-7 OH). The spectral data including ^^C NMR (Table 8) indicated a symmetrical dimeric compound with the presence of a C-7 hydroxyl group (5H 7.18; 5 C 141.0 ) as well as a H-14 signal (6H 6.37 ; 5C 133.1). The two parts of the molecule were therefore joined together at C-11 - C-IT to form ll,ir-didehydroxy-7,7'dihydroxytaxodione (38).
2.2.2. Rearranged Abietane Diterpenes Salvia candidissima Vahl. subsp. occidentalis^^'^'^^ yielded six rearranged abietane diterpenes, three of them being new. The known compounds were 1-oxoaethiopinone, salvipisone, microstegiol and the new compounds, 1-oxosalvipisone (39), 3-oxosalvipisone (40) and candidissiol (41). The structures of
681 three new compounds were established by spectral data including the HETCOR, HMQC and HMBC correlations. The HREI mass spectrum of compound 39 indicated the molecular formula C2oH2204( m/z 326.1532 ). The UV maximum at 432 and 355 nm showed extended conjugation in the molecule. Some of the *H NMR signals were identical to those of salvipisone at 5 7.98 (IH, d, J=8 Hz, H-7), 7.58 (IH, d, J=8 Hz, H-6), 2.43 (3H, s, Me-20), 1.29 (6H, d, J=7 Hz, Me-16 and Me-17), 3.38 (IH, septet, J=7 Hz, H-15), 1.77 (3H, br s, Me-19). The signals which showed differencesfromthose of salvipisone were at 5 7.30 (IH, br s, C-12 OH) (D2O exchangeable) and 4.82 (2H, s, H-18 and H-18'). The signals for the side chain were observed at 5 2.75 (2H, dd, J=6 and 7 Hz) and 1.60 (2H, m) (C-2 and C-3 protons). The lack of the benzylic protons at 5 3.13, the presence of an 0x0 group at 1705 cm"^ in its IR spectrum and the signal at 5 195.5 in the ^^C NMR spectrum indicated the structure 39 (1-oxosalvipisone). The ^^C NMR data of 39 are given in Table 10. Compound 40 was found to have the same molecular formula as 39, C20H22O4 (/w/z 326.1522) as evident from its HREI mass spectrum. The ^H NMR spectrum had many similarities in both compounds, but also exhibited some chemical shift differences. However, TLC comparison showed that the two compounds were different. The signals at 6 8.06 (IH, d, J=8 Hz, H-7), 7.59 (IH, d, J=8 Hz, H-6), 4.77 and 4.22 (each IH, br s, H-18 and H-18'), 1.79 (3H, s, Me-19), 2.33 (3H, s, Me-20) showed the main differences between the compounds 39 and 40. The 0x0 group should therefore be either placed at C-2 or at C-3. Because of the lack of the isolated methylene signals for the C-1 and C-3 protons it cannot be placed at C-2 and it should therefore be at C-3. The HMBC experiment verified this conclusion (Table 10).
39
40
43
The third new compound candidissiol (41) may arised by the possible rearrangement of aethiopinone (42) as shown in Scheme 1. The structure of 41 was determined from ID and 2D NMR techniques including *H-^H and ^H-^^C COSY as well as NOE and spin decoupling experiments. The ^H NMR
682 spectrum of 41 indicated aromatic signals 5 7.12 (IH, d, J=7.8 Hz, H-6), 7.02 (IH, d, J=7.8 Hz, H-7) and 7.09 (IH, d, J=l Hz, H-14) and exomethylene signals at 5 4.92 and 4.97 (each IH, br s). Table 10 presents the ^H, ^^C and HETCOR NMR data. The HREI mass spectrum of 41 indicated the molecular formula to be C20H24O2 (/w/r 296.1437). TABLE 10 ^^C NMR Data of 39, ^H and ^^C NMR Data of 41
c
39
41 ^H
^^C
^^C
1
195.5 s
2.77,4.08
25.81
2
36.51
1.68, 1.82
30.01
3
36.51
1.33,2.24
32.41
4
145.6 s
-
152.3 s
5
135.4 s
-
137.9 s
6
131.0d
7.12
130.2 d
7
128.3 d
7.02
127.9 d
8
133.4 s
-
125.8 s
9
130.7 s
-
139.1s
10
153.5 s
-
144.1s
11
183.6 s
-
80.1s
12
156.8 s
-
209.2 s
13
130.3 s
-
142.4 s
14
184.3 s
7.09
140.7 d
15
25.3 sept
3.00
26.8 d
16
19.7 q
1.21
21.7 q
17
20.3 q
1.13
20.2 q
18
110.2t
2.48,2.25
54.31
19
22.4 q
4.92,4.97
117.7t
20
26.2 q
2.30
20.0 q
683
42
41 Scheme 1
A new compound 43 similar to that of salvipisone was also isolatedfromS. sclarea^^ together with microstegiol and candidissiol. The molecular formula of 43 was found to be C20H22O3 (jn/z 310.1560). From the ^H NMR and spin decoupling experiments its structure was determined as 2,3dehydrosalvipisone. The ^H NMR spectrum exhibited resonances at 5 7.97 (IH, d, J=10 Hz, H-7), 7.58 (IH, d, J=10 Hz, H-6), 7.64 (IH, s, C-12 OH) (D2O exchangeable), 2.42 (3H, s, Me-20), 1.84 (3H, s, Me-19), 4.81 (2H, br s, CH2-I8). The isopropyl protons resonated at 5 1.28 (6H, d, J=7 Hz, Me-16 and Me-17) and 3.37 (IH, septet, J=7 Hz, H-15). The signals for the side chain (C-1 - C-3 ) protons were quite different than those of salvipisone, resonating at 5 6.82 (IH, d, J=16 Hz, H-3), 5.10 (IH, dt, 7, 7, 16 Hz, H-2) and 3.02 (2H, br d, J=7 Hz, CH2-I) and indicating the presence of a double bond at A''^ or A^. The UV spectrum as well as tlie presence of the benzylic CHrl group and spin decoupling experiments proved that the double bond was located between C-2 and C-3. Salvipisone was also found in S. nemoroscP. Six new rearranged abietane diterpenes were isolated from S. limbata C.A. Meyer'*^. These compounds werel2-hydroxysapriparaquinone (44), 3,12-hydroxysapriparaquinone (45), 2-hydroxysaprorthoquinone (46), limbinol (47), salvilimbinol (48) and dehydrosalvilimbinol (49). The structures of
44 R=H 45 R=OH
46
684
TABLE 11 'H and 13CNMR Data of Compounds 44,45,46 and 47 44
45
46
'H
l3C
'H
I3c
'H
1 2 3 4
3.19 2.18 5.29 brt -
8.37 d 7.57 d
5
-
7.50d 7.96 d -
129.6 d 128.8 d 150.0 s 126.5 s 142.2 s 127.9 d 118.3 d 134.0 s 132.7 s 143.7 s 184.8 s 153.1 s 132.7 s 184.0 s 24.1 d 20.9 q 20.9 q 29.7 q 29.7 q 18.4 q
1.18,3.08 4.88 d 5.50 d
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
30.2 t 27.8 t 123.7 d 126.3 s 144.4 s 136.2 d 126.4 d 136.3 s 132.5 s 143.2 s 184.6 s 153.2 s 132.5 s 183.3 s 24.4 d 19.8 q 19.8 q 26.9 q 17.6 q 20.3 q
-
-
3.38sept 1.29d 1.29d 1.72s 1.60s 2.44s
7.45 d 8.15 d
3.45 sept 1.34 d 1.34 d 1.79 s 1.79 s 2.81 s
7.07 d 7.50 d
7.20 d 2.97 sept 1.32 d 1.36 d 1.82 s 1.77 s 2.35 s
47
'H
l3C
32.3 t 73.0 d 124.4 d 136.2 s 137.8s 124.4 d 124.1 d 120.1 s 129.0s 137.8s 183.0 s 184.3 s 136.2 s 115.7 d 26.9 d 25.6 q 25.6 q 27.7 q 18.3 q 22.5 q
5.35 d 5.40 d 7.00 d 7.12 d
7.10 d 6.95 d 2.97 sept 1.15 d 1.13 d 1.74 s 1.76 s 2.22 s
l3C
195.5s 65.6 d 124.9 d 136.5s 136.0 s 126.5 d 130.5 d 127.7 s 133.7 s 139.6s 136.0d 139.6 s 133.7s 136.5 d 26.9 d 21.4 q 21.5 q 25.8 q 18.1 q 18.8 q
685 all six compounds were decided from the ID and 2D spectral data. The chemical shift of the protons at C-6 and C-7 indicated a paraquinonoid structure at ring C for compounds 44 and 45 while in 46 the signals for H-6 and H-7 were relatively upfield, thus indicating its orthoquinonoid structure at ring C. Diterpenes limbinol (47), salvilimbinol (48) and dehydrosalvilimbinol (49) do not have quinonoid C rings as seenfromtheir NMR data (Table 11). The UV, IR and ^^C NMR spectral data showed the quinonoid (44-46) and aromatic (47) characters. The COSY and HETCOR as well as spin decoupling experiments supported their structures (Tables 11 and 12). Compounds 48 and 49 showed differences from the other four compounds, especially from the signals for the C-1, C-2 and C-10 protons at 6 1.86 (IH, m, H-lb), 1.94 (IH, dd, J=5 and 11 Hz, H-la)
47
48
49
2.57 (IH, ddd, J=5.5, 10, 10 Hz; H-2b), 2.89 (IH, ddd, J=2, 5.5, 11 Hz, H-2a), 2.42 (IH, dd, J=5, 10 Hz, H-10) for compound 48 and the signals at 5 1.80 (IH, m, H-la), 2.30 (IH, ddd, J=3, 11 and 12 Hz, H-lb), 2.67 (IH, dd, J=9, 16 Hz, H-2a), 2.90 (IH, m, H-2b) and 2.44 (IH, dd, J=4 and 12 Hz, H-10) for compound 49, indicating saturated C-9 - C-10 positions. The structures of these two compounds were established by the COSY, HETCOR and COLOC experiments (Table 12). OH
OHO
CCr 50
AcO.
51
686
TABLE 12
'H, I3CNMR and COLOC Correlations of 48 and 49 49
48
'H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.86, 1.94 2.57,2.89
6.92 7.05 2.42
6.78 2.90 1.12 1.17 1.37 1.42 2.24
l3c 23.5 t 25.3 t 210.7 s 74.2 s 138.7 s 126.6d 130.0d 135.5s 76.2 s 45.4 d 136.3 s 141.0 s 128.1 s 134.9 d 27.4 d 20.7 q 21.6 q 26.5 q 32.6 q 19.8 q
COLOC
'H
c-3, c-5, c-9 c-3, c-4, c-10
1.80, 2.30 2.67, 2.90
c-8, c-9, c-10, c-20 c-5, c-9
6.98 7.10
c-2, c-3, c-9, c-1 1
2.44
c-7, c-9, (2-12 C-13, C-14 C-12, C-13, C-14 C-13, C-14 c-2, (2-3, c-4 c-2, c-3 C-6, C-7, C- 10
6.89 2.98 1.11 1.11 4.90,4.91 2.01 2.25
I3c
24.4 t 25.1 t 201.7 s 149.1 s 137.3 s 126.5 d 130.1 d 135.6 s 74.5 s 45.8 d 137.3 s 141.9 s 126.6 s 135.5 d 26.6 d 21.4 q 21.4 q 111.7t 24.4 q 19.8 q
COLOC c-3, c-9 c-1, c-4, c-10
C-8, C-10, C-14, C-20 C-5, C-14 c-2, c-5, c-11
c-7, c-8, c-12 C-14 C-13, C-15 C-13, C-15 C-3, C-4, C-19 c-3, c-18 c-6, c-7
687 TABLE 13 ^H and *^C NMR of 50 and 51 501 ^H
51 ^'C
'H
^'C
1
2.95 m
30.21
-
196.0 s
2
2.30 dd
27.81
5.38 d
65.3 d
3
5.45 brd
123.7 d
5.50 dt
125.2 d
4
-
126.3 s
-
126.3 s
5
-
144.4 s
-
144.6 s
6
7.52 d
136.2 d
6.96 d
135.6 d
7
7.82 d
126.4 d
7.12 d
130.8 d
8
-
136.3 s
-
135.6 s
9
-
132.5 s
-
130.8 s
10
-
143.2 s
-
125.2 s
11
-
184.2 s
7.10 d
127.1 d
12
-
153.2 s
-
136.3 s
13
-
132.5 s
-
136.3 s
14
-
183.8 s
6.90 d
126.3 d
15
2.90 m
24.4 d
2.95 sept
27.2 d
16
10.69 brs
201.9 d
1.15d
19.8 q
17
1.32 d
19.8 q
1.15d
20.1 q
18
1.78 s
26.9 q
1.77 s
26.8 q
19
1.83 s
17.6 q
1.80 s
17.6 q
20
2.40 s
20.3 q
2.20 s
20.3 q
protons and carbons (Table 14). Extensive selective INEPT experiment allowed the unambiguous assignment of the groups. Irradiation of H-15 (5 3.35) enhanced C-12, C-14, C-16, while irradiation of Me-18 (5 1.15) enhanced C-4 and C-19, and irradiation of Me-19 (5 1.62) enhanced C-4 and C-18. All the rest of the carbons which were irradiated indicated the structure clearly.
688
Hydration of olefin
w
Reduction of double bond
52 R=H 53 R=0 Scheme 2 1-Oxosalvipisone (53) had the molecular formula C20H24O3 (m/z 312.1744). The ^^C NMR spectrum showed the presence of two carbonyl groups at 5 210.8 and 202.3. The lack of a typical signal for H-1 at 5 3.85 as ddd indicated that the second 0x0 group should be at C-1 which was verified by the SINEPT experiment. All other signals are in agreement to those of 52. 2.2,3 Pimarane Type Diterpenoids Salvia wiedemannif^'^^ yielded pimarane type diterpenoids together with abietanes. 14-Oxoisopimaric acid (54) was found to be a new compound. It was obtained along with two known compounds, isopimaric acid^^ and 7P-hydroxysandracopimaric acid^^. Compound 54 had the molecular formula C20H30O3 {rn/z 318.3201) deducedfi'omits HREI mass spectrum. The IR spectrum indicated the presence of an acid group (a large shoulder between 2500 and 2800 cm'* and carboxyl group at 1700 cm'*), and a carbonyl signal at 1725 cm'*. The *H NMR spectrum clearly indicated the presence of an ethylene side chain in the isopimarane skeleton at 6 5.88 (IH, dd, J=l 1 and 17 Hz, H-16), 5.14 (IH, dd, J=1.5 and 17 Hz, H-17), 4.92 (IH, dd, J=1.5 and 11 Hz, H-IT) in addition to three methyl signals at 5 1.28 (3H, s, Me-15), 1.22 (3H, s, Me-18), 0.87 (3H, s, Me-20). The latter signal indicates the presence of the acid group at C-4 rather than at CA(f^. The position of the carbonyl group at C-14 was
689 concluded from the downfield shift of the signals of the ethylene protons as well as from the mass degradation pattern of 54 (Scheme 3). The diagnostic massfragmentsat m/z 182 (a), 138 (b) as well as 137 (c) indicated the presence of the C-14 carbonyl group. The same compound was also isolated from S. candidissimd*^. TABLE 14 ^H and ^^C NMR of 52 and 53 52
53
-
1
3.83 ddd, 3.02 ddd
26.51
2
2.20 ddd. 1.72 m
23.01
2.19 ddd, 1.65 dt
35.lt
3
1.60 ddd, 1.20 m
33.31
3.04 ddd, 2.67 dt
41.21
210.8 s
4
-
85.0 s
-
84.3 s
5
-
132.7 s
-
132.2 s
6
7.08 d
126.7 d
7.11 d
126.3 d
7
7.48 d
125.8 d
7.62 d
128.1 d
8
-
134.1s
-
126.8 s
9
-
129.7 s
-
130.4 s
10
-
147.3 s
-
147.4 s
11
-
41.6 s
-
41.2 s
12
-
202.9 s
-
202.3 s
13
-
134.9 s
-
136.6 s
14
7.33 s
120.7 d
7.41s
121.1 d
15
3.35 sept
27.6 d
3.35 sept
27.8 d
16
1.31 d
22.3 q
1.28 d
22.3 q
17
1.35 d
22.4 q
1.33 d
22.3 q
18
1.15s
26.3 q
1.22 s
25.9 q
19
1.62 s
27.4 q
1.67 s
26.2 q
20
2.42 s
19.1 q
2.32 s
18.7 q
690 Another new pimarane diterpenoid salvipimarone (7P-hydroxy-3,ll-dioxo-pimara-8(14),15dien) (55) was isolated from S. multicaulis Vahl."*^. The structure of salvipimarone was deduced from ID and 2D NMR spectra. The compound was found to have the molecular formula C20H28O4 {m/z 332.1991). The ^H NMR spectrum indicated its ethylenic side chain by signals at 5 5.79 (IH, dd, J=l 1
HOOC
CO2 HOOC
Scheme 3 and 16.5 Hz), 4.96 (IH, dd, J=2 and 16.5 Hz), and 4.92 (IH, dd, J=2 and 11 Hz). The broad singlet at 5 5.50 assigned to an olefinic proton (H-14) while a narrow triplet at 6 4.20 (IH, t, J=1.5 Hz, H-7a) was indicative of the presence of a P-hydroxyl group at C-7. Similar compounds with C-7p hydroxyl group and a double bond between C-8 - C-14 have been isolatedfromArelia cordatc^'^^ with similar chemical shifts. The IR spectrum of 55 showed the carbonyl signals at 1737 and 1720 cm"^ which were detected in the *^C NMR (DEPT) spectrum by the resonances rat 6 214.0 and 210.1. The two 0x0 groups were assigned to C-3 and C-11 on the basis of HMBC experiment (Table 15). S.heldrichiancf^ yielded three known pimarane diterpenoids, isopimaric acid, 7P-hydroxy sandracopimaric acid and 7-oxo-13-e/?/-pimara-8,15-dien-18-oic acid.
N ^ ^ ' ' ^
55
691 2.2.4. Labdane Type Diterpenoids Labdane and pimarane type diterpenoids are rather rare in Turkish Salvia species, and only a few of these compounds have been obtained. Sclareol and manool were isolated from S. sclared^. Manool was also obtainedfromS.limbatcf', while manoyl oxide was reportedfromS. candidissima^'^^ together with 1 ip-hydroxymanoyl oxide^^ and 8,13-di-^/?/-manoyl oxide^^. Although these two compounds were TABLE 15 ^^C NMR and HMBC Data of 55
c
13(.
1
39.lt
H-7
C-5, C-6, C-9, C-14
2
34.3 t
H-15
C-11,C-12
3
214.0 s
H-16
C-13
4
42.1s
H-17
C-11,C-13,C-14,C-15,C-16
5
46.2 d
H-18
C-2, C-3, C-5, C-6
6
29.3 t
H-19
C-3, C-5, C-6
7
73.4 d
H-20
C-1, C-2, C-9
8
144.1s
9
78.0 s
10
39.1s
11
210.1s
12
42.lt
13
37.3 s
14
134.0 d
15
148.3 d
16
110.6t
17
25.6 q
18
33.4 q
19
21.9 q
20
14.2 q
Irradiated H
Correlated C
692
56 already known the spectral data given in the literature was not sufficient. We have recorded the HMQC (inverse phase HETCOR) and HMBC (inverse phase COLOC) spectra of 56 (Table 16). Norlabdane diterpenoids were isolated from S. yosgadensi^^.
These included two known
compounds, ambrenolide and norambrenolide and three new compounds 6a-hydroxyambrenolide (57), 6a-hydroxynorambrenolide (58) and 6a-hydroxy-8a-acetoxy-13,14,15,16-tetranorlabdane-12-oic acid (59). 0 ^0
57
58
59
The ^^C NMR (APT and DEPT) spectra of 6a-hydroxyambreinolide (57) displayed four methyl, six methylene, three methine and four quaternary carbons (Table 17). The HREI mass spectrum showed the molecular ion peak at m/z 280.2031 analyzing for the molecular formula C17H28O3 which was consistent with a hydroxyl derivative of ambrenolide. The IR spectrum indicated the presence of a six membered lactone at 1716 cm"^ and a hydroxyl at 3454 cm'\ The ^H NMR spectrum of 57 exhibited a methine proton at 5 3.90 (J=4, 11 and 12 Hz). The multiplicity and the J values indicated the presemce of a secondary hydroxyl group located between the methine and a methylene groups possibly either at C-6 or C-11 positions of ambrenolide. The location of the hydroxyl group at C-6 was decided from the chemical shift of C-7 at 5 51.87 with a downfield shift ca \1 ppm relative to ambrenolide, and the chemical shift of C-5 which was shifted to 5 61.14, also indicative the presence of the hydroxyl group at C-6. Spin decoupling and phase sensitive COSY experiments confirmed the relationship between H-6 (5 3.90) and H-7a (6 1.78), H-7p (5 2.33) as weU as between H-5 (6 1.10) and H-6.
693
TABLE 16 HMQC and HMBC Data of 56
c
^^C
Direct correlated protons
Long range correlated protons
1
39.22
H.la,H-lp
H-20
2
18.42
H-2a,H-2p
H-la
3
41.92
H-3a, H-3P
H-la,H-18
4
33.19
-
5
57.03
H-5a
H-la, H-18, H-19, H-20
6
20.14
H-6a, H-6p
H-5, H-7a, H-7p
7
44.56
H-7a, H-7p
H-9, H-17
8
74.80
-
9
56.45
H-9a
10
37.76
-
11
65.24
H-lla
H-12a, H-12P
12
44.20
H-12a, H-12P
H-15b,H-16
13
72.38
-
14
147.77
H-14
H-12a,H-12p,H-15a,H-16
15
110.47
H-15a,H-15b
-
16
29.73
H-16
H-12a,H-12p
17
27.44
H-17
H-7, H-9
18
33.50
H-18
H-3a
19
21.38
H-19
H-3a, H-5, H-18
20
17.12
H-20
H-la, H-5, H,9
H-2a,H-2p,H-19
H-9, H-17 H-la, H-7a, H-12a, H-12P, H-17,H-20 H-9, H-20
H-12a, H-12P, H-14, H-15a, H-15b
The stereochemistry of the hydroxy! group at C-6 was decided as a on the basis of the coupling constants and NOESY results (Fig 1). All carbons were assigned by HMQC (Table 17) and HMBC (Table 18) experiments.
694 TABLE 17 *H and ^^C NMR Data of Compounds 57, 58 and 59 58
57
59
^H
^^C
^H
^^C
^H
1
1.59,0.98
39.28
1.42, 1.09
39.46
1.5,1.10
40.44
2
1.61, 1.48
18.18
1.68, 1.47
17.89
1.62,1.45
19.15
3
1.22, 1.39
43.42
1.32, 1.43
43.65
1.22,1.32
44.81
^'C
4
-
33.67
-
33.82
5
1.10
61.14
1.24
62.10
1.17
61.38
6
3.90
68.45
3.99
69.82
3.80
69.53
7
2.33, 1.78
51.87
2.42, 1.82
49.90
2.93, 1.94
50.40
8
-
82.53
-
84.25
-
86.65
9
1.53
53.09
2.02
58.81
2.26
55.82
10
-
37.71
-
35.51
-
39.76
2.25, 2.44
-
34.66
11
1.76, 1.67
15.93
12
2.68, 2.54
28.71
-
176.39
-
171.94
-
-
-
28.80
2.34, 2.42
31.55
13
-
171.40
-
17
1.45
24.01
1.37
22.90
1.56
21.80
18
1.19
36.47
1.18
36.20
1.19
37.00
19
1.02
21.96
1.02
21.54
0.99
22.40
20
0.89
16.22
0.95
16.39
0.88
17.30
1.92
22.60
-
170.00
CHs C=0
-
-
-
-
-
-
-
The HR mass spectrum of 6-hydroxynorambrenolide (58) exhibited the molecular ion peak at m/z 266.1869 indicating the molecular formula C16H26O3 consistent with a hydroxyl derivative of norambrenolide. The ^H NMR spectrum displayed a signal at 6 3.99 (IH, ddd, J=4, 11, 12 Hz) showing a secondary hydroxyl group between a methine and a methylene groups. After acetylation
695
Figure 1 TABLE 18 HMBC Data of Compounds 57, 58 and 59 Irradiated H
Long Range Correlated Carbons 57
1
58
59
C-20
2
C-1, C-10
3
C-19 C-19
5 7
C-6, C-8, C-17
C-5, C-6, C-8, C-17
9
C-8,C-10,C-ll,C-17,C-20
C-8, C-10, C-11, C-17, C-20
11
C-8, C-9, C-12
C-8, C-9, C-12
C-8, C-9, C-10
17
C-7, C-8, C-9
C-7, C-8, C-9
C-7, C-8, C-9
18
C-3, C-4, C-5, C-19
C-3, C-4, C-5, C-19
C-3,C-4, C-5,C-19
19
C-3, C-4, C-5, C-18
C-3, C-4, C-5, C-18
C-3,C-4, C-5, C-18
20
C-1, C-5, C-9, C-10
C-1, C-5, C-9, C-10
C-5, C-9, C-10
this peak was shifted to 5 5.19. The *H and ^^C NMR spectra are given in Table 17. The spectra of compounds 57 and 58 were quite alike. However in compound 58, the lactone carbonyl appeared at 1785 cm'^ indicating afivemembered lactone ring. Unambiguous assignment of ^H and *^C NMR data was made possible by a combination of HMQC (Table 17) and HMBC (Table 18). The structure of 58
696 was established as a tetranorlabdane, 6a-hydroxynorambrenolide. Compound 59 had the molecular ion peak at m/z 326.2088 (HREI mass spectrum) analyzing for the molecular formula CigHsoOs. The IR spectrum showed a large hydroxyl band at 3240-3400 cm'^ and two carbonyl absorptions at 1685 cm'^ (carboxylic acid) and at 1725 cm'^ (acetyl). These carbonyl groups were substantiated by two signals present at 6 171.9 and 170.0 in its ^^C NMR spectrum. A methine proton at 5 3.80 (IH, ddd, J=4,l 1,12) was consistent with H-6, as in compounds 57 and 58. Connectivity of H-9 and H-11 as well as H-5, H-6 and H-7 was confirmed by COSY experiment, and the location of the 6a-hydroxyl group was deduced fi-om the NOESY results (Figure 1). The HMQC and HMBC experiments allowed the assignments to all the carbons and protons. The structure of 59 was deduced as 6a-hydroxy-8a-acetoxy-13,14,15,16tetranorlabdane-12-oic acid.
2.3. SESTERTERPENOroS Sesterterpenes and norsesterterpenes are not common in Salvia species. Rustaiyan et af^'^ isolated some sesterterpenes fi-om Iranian Salvia species. This is the first isolation of two norsesterterpenes^^ and two sesterterpenes^^ fi-om S.yosgadensis. Two new 19,20-dinorsesterterpenes, yosgadensonol (60) and 13-ep/yosgadensonol (61), were isolated together with compounds 57-59. The HREI mass spectrum of yosgadensonol (60) gave the molecular ion peak at m/z 362.2895 corresponding to the molecular formula C23H38O3. Its ^^C NMR spectrum indicated the presence of six methyl, seven methylene, five methine, and five quaternary carbons. The presence of a secondary hydroxyl group was shown by a methine signal at 5 3.87 (IH, ddd, J=4, 11,12 Hz). The location of the hydroxyl group at C-6 and the relation between H-5, H-6 and H-7 were deduced fi-om the COSY spectrum. Acetylation yielded a mono acetate 60a in which H-6 was shifted downfield to 5 5.12. The presence of an ether ring in 60 was suggested by two quaternary carbon signals at 5 73.03 and 74.75. When the *^C NMR spectrum of 60 was compared with those of manoyloxide and its derivatives, it was deduced that compound 60 possesses the same A, B and C rings. The rest of the molecule carries a side chain containing five carbons, the presence of a conjugated ketone system could be assignedfi-omthe IR band at 1625 cm'^ (C=C) and trans coupled two vinylic protons at 5 6.25 and 6.77 (J=16 Hz) indicating the presence of a CH=CH group. This was confirmed by two olefinic carbons at 6 125.31 and 154.17. The downfield shift of one of the olefinic carbons fiirther suggested a conjugated system v^th a ketone carbonyl group (5C 195.31; IR max 1695 cm"^). The HMQC (Table 19) and HMBC (Table 20) experiments allowed the assignment of all protons and carbons in compound 60. The HMBC technique was particularly usefiil in determining the partial structure of the side chain. Thus H-14 showed long
697 range couplings with the C-16 carbonyl and C-21 methyl carbons. Compound 60 was therefore deduced as 19,20-dinorsesterterpene containing the partial structure of manoyl oxide, and it was named yosgadensenol. Compound 61 exhibited spectral data similar to those of 60. The *H and *^C NMR spectra of both compounds (Table 19 and 20) resembled each other closely. In the ID NOE difference spectrum obtained by irradiation of H-6P, the signals for H-22, H-24 and H-25 and H-7 eq. were clearly enhanced, suggesting a P-orientation for these groups in both compounds. However a difference was detected by NOE irradiation of H-22 which is the most downfield signal in both compounds, which led to enhancements on H-21 and H-25 in compound 60 indicating a P-orientation of these groups. However, when H-22 of compound 61 was irradiated, only H-25 was enhanced. The lack of NOE between H-22 and H-21 suggested the a-dispositions of H-21 at C-13. The results of ID NOE experiments of both compounds indicated a stereochemical difference at C-13. Compound 61 is therefore 13-epimer of 60 and it was named 13-e/?/-yosgadensonol.
"23 OH
60 13P-Me, 61 13 a-Me The sesterterpenes obtainedfromthe same plant material^^ were 6a,14-dihydroxymanoyl oxide15,17-dien-16,19-olide and 6a,16-dihydroxymanoyl oxide-14,17-dien-16,19-olide were named as yosgandensolide A (62) and yosgandensolide B (63). Compound 62 afforded the molecular formula C25H38O5 (m/z 418.2700). Its ^^C NMR spectrum consisted of 25 carbon atoms as given in Table 21. The IR spectrum showed a five member lactone ring by a band at 1780 cm'^ and unsaturation by the absorption at 1615 cm'^. The UV spectrum exhibited a maximum at 274 nm consistent with an a,Punsaturated five member lactone ring. The IH NMR spectrum showed five methyl singlets at 5 0.81, 1.00, 1.18, 1.24, 1.38 and a vinylic methyl doublet at 5 2.16 (J=1.5 Hz). A carbinol methine was at 5 3.88 (IH, ddd, J=3.8, 11.2, 12 Hz) indicating a secondary hydroxyl group situated between a methine
698 TABLE 19 ^H and ^^C NMR Data of 60, 60a and 61 60 ^H
61
60a ^'C
^H
^'C
^H
^^C
1
1.60, 1.34
39.91
1.58, 1.28
38.80
1.60, 0.91
39.23
2
1.52
18.20
1.40
18.11
1.47
18.33
3
1.42, 1.27
43.54
1.38, 1.21
43.30
1.39, 1.16
43.60
33.70
-
33.28
-
31.68
4 5
1.00
61.69
1.12
58.85
1.02
61.69
6
3.87
69.15
5.12
70.72
3.84
69.13
7
2.20, 1.58
54.29
2.12, 1.60
49.69
2.13,1.48
53.97
74.75
-
74.47
-
75.64
8 9
1.19
54.86
1.14
54.88
1.25
57.65
10
-
37.70
-
37.80
-
32.92
11 1.62, 1.36
15.39
1.62, 1.34
15.38
1.60, 1.38
16.05
12 2.08, 1.56
34.86
2.02, 1.58
34.85
2.20, 1.57
36.44
13
73.03
-
73.07
-
72.84
14 6.77
154.17
6.97
154.17
6.98
154.62
15 6.25
125.31
6.22
125.38
5.99
125.18
16
195.31
-
201.68
-
201.51
17 2.58
33.96
2.58
33.90
2.61
35.13
18 1.10
8.11
1.08
8.20
1.11
8.08
21 1.29
28.81
1.29
28.78
1.22
25.26
22 1.25
26.87
1.40
26.68
1.18
33.78
23 1.16
36.44
1.00
36.00
1.15
37.55
24 0.99
21.86
0.84
21.98
0.97
21.80
25 0.80
16.58
0.86
16.50
0.74
17.23
-
CHs -
-
2.04
21.89
-
-
c=o .
-
-
170.13
-
-
699 TABLE 20 HMBC Data of Compounds 60 and 61 Irradiated H
5 6
Long Range Correlated Carbons 60
61
C-4, C-7
-
-
C-5
7
C-5, C-6, C-8, C-9, C-22
C-6, C-8
14
C-16, C-21
C-16, C-21
15
C-13,C-17
C-16, C-17
17
C-18
C-16, C-18
18
C-16, C-17
-
21
C-12, C-13
-
22
C-7, C-8, C-9
C-7, C-8
23
C-3, C-4, C-5, C-24
C-24
24
C-3, C-4, C-5, C-23
C-6, C-23
25
C-1, C-9, C-10
C-1, C-5, C-10
and a methylene groups which could be either at C-6 or at C-11. The ^^C NMR spectrum helped to differentiate between these two locations. In the case of a C-11 hydroxyl group, C-12 should be shifted to 40-45 ppm while the hydroxyl at C-6 induces a chemical shift at C-7 of more than 50 ppm. In the present case C-7 was observed at 5 54.1. No other methylene carbon which could be shifted to around 50 ppm was present in the molecule. The second carbinol methine proton was at 5 4.45 (IH, d, J=9.5 Hz, H-14) which was coupled with a vinylic proton at 6 5.20 (IH, d, J=9.5 Hz, H-15). Another vinylic proton was observed at 5 5.96 as a narrow doublet (IH, d, J=1.5 Hz, H-18). Spin decoupling experiments showed the relations between H-6 and H-5, H-6 and H2-7, as well as H-14 and H-15. The HETCOR and HMBC experiments allowed the unambiguous assignment of protons and carbons. Long range correlations were observed between H-14 and C-15, C-16 as well as between H15 and C-16, C-17, between Me-20 and C-16, C-17, C-18 and between Me-21 and C-15. Acetylation
700
62
63
of 62 yielded two derivatives which were separated by preparative TLC, indicating that compound 62 was a mixture of two isomers. The spectral data of both isomers 62aAc and 62bAc (Table 22) were quite similar, only the protons and carbons for C-14, C-15, C-18 and C-20 showing differences, indicated a possible epimerization at C-15 to form the E and Z isomers. Based on the spectral data the structures of 62a and 62b were established as 6a,14-dihydroxymanoyl oxide-15,22-dien-15(Z),19-olide and 6a,14-dihydroxy-15,22-dien-15(E),19-olide. Another epimer of this group was isolated separately (62c). Its ^H NMR spectrum was found to be quite similar to that of 62 (Table 22), the main differences observed being at H-14 and H-15 as well as in some methyl group (Me-21 and Me-22), indicating a possible epimerization at C-13. However, the small quantity of 62c did not allow further investigation of its stereochemistry. Yosgadensolide B (63) had the molecular formula C25H38O5 (m/z 418.2705). The IR spectrum showed afive-memberedlactone ring at 1780 cm'* and its UV spectrum exhibited a shoulder at 274 nm. The *H NMR spectrum (Table 23) showed a carbinol methine at 5 3.85 (IH, ddd, J=3.8, 11.2, 12 Hz) which was assigned to H-6P, as also found in (62a-62c). There were three olefinic protons, one being at 5 5.79 (IH, d, J=1.5 Hz, H-18) similar to that of compound 62, and the other two protons appearing were at 6 6.15 and 5.72, indicating a trans double bond with a J value of 16 Hz. These were placed on the side chain. The placement was deduced to be between C-14 and C-15. The *^C NMR spectrum (Table 21) indicated a sesterterpene structure derivedfrommanoyl oxide like yosgadensolide A (62), but its side chain differedfromthat of 62. Compound 64 which is an epimer of compound 63 was isolated separately. The UV, IR, and MS spectra of the new compound 64 were exactly similar to those of 63, while the *H and *^C NMR data showed chemical shift differences particularly for H-14 (5 6.35), H-15 (5 5.52) and for some methyl groups. The two methyl groups next to the oxygenated carbons were observed at 5 1.22 (Me-21) and
701 TABLE 21 ^^C NMR Data of Compounds 62,62aAc and 63 62
62aAc
63
1
39.0
38.8
39.4
2
18.3
18.2
18.7
3
43.5
43.3
43.3
4
30.7
33.3
33.2
5
61.6
61.7
59.6
6
69.0
70.7
70.6
7
54.1
49.4
53.3
8
73.2
74.1
73.6
9
58.0
58.8
60.4
10
33.8
33.3
34.0
11
14.9
14.8
14.2
12
37.6
36.0
37.6
13
75.3
75.0
75.2
14
72.9
75.4
147.2
15
109.3
106.6
119.7
16
151.0
152.2
107.0
17
154.7
154.7
164.0
18
117.3
117.7
118.2
19
176.7
172.2
171.3
20
11.7
11.9
12.8
21
25.9
25.9
30.9
22
24.6
23.9
24.0
23
36.4
36.0
35.9
24
21.8
21.1
21.6
25
17.2
16.8
14.9
C=0
-
170.1
-
CHs
-
21.8
-
C=0
-
169.8
-
CHs
21.9
702
TABLE 22 *H NMR Data of Compounds 62, 62a Ac, 62b Ac and 62c H
62(62a+62b)
62a Ac
62b Ac
62c
6
3.88 ddd
5.08 ddd
5.08 ddd
3.88 ddd
14
4.45 d
5.57 d
5.68 d
4.31 d
15
5.20 d
5.32 d
5.51 d
5.30 d
18
5.96 d
5.97 d
6.04 br s
5.96 d
20
2.16d
2.18 brs
2.08 d
2.17d
21
1.38 s
1.37 s
1.38 s
1.36 s
22
1.24 s
1.28 s
1.28 s
1.17s
23
1.18s
1.00 s
1.01s
1.17s
24
1.00 s
0.85 s
0.84 s
1.01s
25
0.81s
0.85 s
0.84 s
0.82 s
OAc
-
2.10s
2.08 s
-
OAc
-
2.02 s
2.05 s
-
1.15 (Me-22) in compound 64, while in compound 63, they were observed at 6 1.36 and 1.28 indicating a possible epimerization at C-13. The NOE experiment verified the epimerization at C-13 (Table 23). Based on spectral data, compound 63 was established as 6a,16-dihydroxymanoyl oxide-HjHdien- 16,19-olide and 64 as 6a, 16-dihydroxy-13-epi-manoy\ oxide-14,17-dien-16,19-olide. 2.4. TRTTERPENOroS Triterpenoidal compounds are common in Salvia species. The recently isolated triterpenes fi'om Turkish Salvia species have mainly the oleane, ursane and lupane-type skeletons and some steroids which are abundant compounds. These include oleanolic acid its methyl ester, a-amyrin, lupeol, taraxasterol and p-sitosterol fi^om S. pomifera^^, ursolic, oleanolic acids and P-sitosterol fi'om S. divaricata^^, vergatic acid, a-amyrin, stigmasterol fi'om S. limhata^^^ . S. nemoroscP yielded a new triterpene salvinemorol (65) in addition to a-amyrin, ursolic and oleanolic acids and steroidal compounds stigmast-7-en-3-one, 24-methylenecycloartenol, stigmast-4-en-3-one and stigmast-7en-3-ol and P-sitosterol..
703
TABLE 23 ^H NMR Assignments of Compounds 63, 64, 63 Ac, 64 Ac 63
64
63 Ac
64 Ac
6
3.85 ddd
3.85 ddd
5.08 ddd
5.08 ddd
11
2.18 dd
2.11 dd
2.12 dd
2.12 dd
14
6.15 d
6.35 d
6.19 d
6.17 d
15
5.72 d
5.52 d
5.49 d
5.40 d
18
5.79 qd
5.82 brs
5.90 d
5.90 s
20
2.04 d
2.01 brs
1.99 s
1.97 s
21
1.36 s
1.22 s
1.19s
1.17s
22
1.28 s
1.15s
1.32 s
1.24 s
23
1.17s
1.15s
1.26 s
1.14s
24
1.00 s
0.98 s
1.22 s
1.00 s
25
0.82 s
0.74 s
0.74 s
0.85 s
OAc
-
-
2.10 s
2.03 s
H
The HREI mass spectrum of the new compound (65) indicated the molecular formula C30H50O3 {m/z 458.3753). The ^^C NMR (APT) spectrum substantiated the presence of 30 C atoms corresponding to eight methyl quartets, eight methylene triplets, seven methine doublets and seven carbon singlets. The HETCOR experiment showed the relation between the carbons and protons. In the ^H NMR spectrum of salvinemorol (65) eight methyl signal were observed at 6 0.78, 0.82, 1.05, 1.20 (each 3H, s) and 0.88, 0.98 (each 6H, s). The three hydroxyl groups present in the molecule were decided to be at C-3, C-11 and C-21. The relations between the signals at 6 5.28 (IH, d, J=4 Hz, H-12) and 5 4.31 (IH, dd, J=4 and 8.5 Hz, H-1 ip) were shown by spin decoupling experiments, as well as between H-11 and H-9 (5 1.70, IH, d, J=8 Hz, H-9). Other significant signals were at 5 3.17 (IH, dd, J=5 and 12 Hz, H-3a) and 3.41 (IH, dd, J=5 and 11 Hz, H-2ip). Spin decoupling experiments showed the relations between H-3a and H-2p (6 1.97, IH, dd, J=5 and 10 Hz). The stereochemistry of the hydroxyls at C-3 and C-11 were decided by measuring the J values and studying a Dreiding model. The C-21 position of the third hydroxyl group was decided by comparing its ^^C NMR values to those reported in the literature^^^^.
704
65 When there is a-hydroxyl group at C-21 it appear ca 74.3-74.5 ppm, C-20 resonates around 34-36 ppm and C-22 resonates at 46.6 ppm. In compound 65 these signals were observed in similar frequencies at 5 32.2, 76.0 and 46.5 respectively. The relation between H-21p and H-22a (5 1.82, IH, dd, J=5 and 11 Hz) was shown by spin decoupling experiments. Its stereochemistry was deduced based on J values and studying of a Dreiding model. The ^^C NMR values of 65 were given in Table 24.
66 R=H 67 R=//-aw5-coumaryl 68 R=cw-coumaryl
S. montbretii^ also yielded ursolic and oleanolic acids, a-amyrin, lupeol and P-sitosterol as well as another known triterpene monogynol A (66) together with its two new esters SP-O-trans-pcoumroylmonogynol A (67) and 3P-0-c/5-coumaroylmonogynol A (68). Although monogynol A was first isolated in 1959, its ^H and ^^C NMR data were not reported in the literature, therefore its spectral data were recorded and its X-ray single crystal analysis was performed . The ^H NMR spectrum of 66
705 consists of eight methyl singlets at 6 0.75, 0.80, 0.82, 0.94, 0.96, 1.05, 1.10 and 1.22. The signal at 5 3.20 (IH, dd, J=5 and 11 Hz) indicated the axial hydrogen at C-3. No vinylic signal was present. The ^^C NMR (APT) spectrum (Table 24) showed two signals at 5 78.9 (d) and 73.2 (s) indicating the hydroxyl group-carrying carbon atoms at C-3 and C-20. The structure of 66 was established as 3P,20lupandiol. Compound 67 aflforded additional signals in its *H NMR spectrum, at 5 7.60 (IH, d, J=16 Hz, H-3'), 6.27 (IH, d, J=16 Hz, H-2'), 7.42 (2H, d, J=8.5 Hz, H-3" and H-5"), 6.82 (2H, d, J=8.5 Hz, H-2" and H-6") indicating a //-oAw-p-coumaroyl moiety. H-3 was shifted to 5 4.60 (IH, t, J=8 Hz) indicating the ester formation at C-3. The rest of the signals were similar to those of 66. Compound 68 showed a d5-p-coumaroyl moiety with the signals at 5 6.80 (IH, d, J=12.5 Hz, H-3'), 5.82 (IH, d, J=12.5 Hz, H2'), 7.64 (2H, d, J=8.5 Hz, H-3" and H-5"), 6.78 (2H, d, J=8.5 Hz, H-2" and H-6").
CH2OH 69 SalviapomifercP yielded a new and three not so common triterpenes, 23-hydroxygermanicone (69), erithrodiol, moradiol and moronic acid, respectively. The HR mass spectrum of 69 gave the molecular ion peak at m/z 440.3671 corresponding to C30H48O2. The IR spectrum showed hydroxyl (3450 cm'*), carbonyl (1710 cm"*) and unsaturation (1640 cm'*) absorbances. The *H NMR spectrum showed seven methyl signals at 6 0.77, 0.93, 0.94, 1.04, 1.07, 1.10 and 1.11 (each 3H, s). The lack of a C-3 hydrogen geminal to a hydroxyl group indicated the presence of a possible 0x0 group at C-3. A proton signal was observed at 5 4.87 as a broad singlet, its chemical shift being typical for olean-18-en without an acid group at C-17. In case of the presence of the substituent other than a methyl group at that position, the H-19 shifts to ca 5.15-5.20 as observed in case of moronic acid^^ Other *H NMR signals were at 5 3.61 (IH, d, J=l 1 Hz, H-23) and 4.17 (IH, d, J=ll Hz, H-23') indicating the presence of a hydroxymethylene group at C-23. The chemical shift of these two groups showed showed that they were deshielded by the 0x0 group at C-3. The *^C NMR
706 Spectrum of 69 is given in Table 24. The signal at 6 76.2 in its *^C NMR spectrum confirmed the presence of the hydroxymethylene group, which wasfiirthersupported by the downfield shift of the MeTABLE24 ^^C NMR Data of Compounds 65, 66, 67 and 69 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
c-r
C-2' C-3' C-l" C-2",C-6" C-3",C-5"
65 36.71 26.61 76.9 d 38.9 s 56.7 d 17.71 34.61 43.8 s 52.3 d 36.8 s 66.9 d 123.8 d 150.3 s 44.1s 32.81 26.41 31.1s 46.3 d 35.81 32.2 s 76.0 d 46.51 28.5 q 13.0 q 18.0 q 15.1 q 26.2 q 28.0 q 33.2 q 23.6 q
-
66 38.71 27.51 78.9 d 38.7 s 55.2 d 18.3 t 34.51 41.3 s 50.2 d 37.0 s 21.41 27.41 37.4 d 44.6 s 27.71 35.51 43.4 s 48.3 d 49.9 d 73.1s 29.01 40.21 28.0 q 15.4 q 16.1 q 16.1 q 14.8 q 19.2 q 24.7 q 31.5q
-
67 38.3 t 23.61 80.71 38.0 s 55.2 d 18.21 34.41 41.3 s 50.1 d 37.3 s 21.3 t 27.51 36.9 d 44.6 s 28.81 35.51 43.4 s 48.3 d 49.9 d 74.0 s 29.lt 40.lt 28.0 q 16.2 q 16.7 q 16.2 q 14.8 q 19.2 q 24.5 q 31.5 q 167.2 s 116.1 d 144.0 d 127.2 s 115.9d 129.8 d
69 37.81 36.61 208.1s 50.6 s 53.2 d 18.21 34.61 42.3 s 52.1 d 37.3 s 22.lt 27.lt 37.4 d 28.6 s 28.41 35.51 34.2 s 142.0 s 134.1 d 32.2 s 28.41 40.lt 76.21 15.4 q 16.1 q 17.2 q 14.3 q 24.7 q 31.5 q 28.4 q
-
707
24 signal to 5 15.4. Due to the a-eflfect of the oxo group and the p-eflfect of the hydroxyl group, the C-4 signal was observed at 5 50.6, the same carbon appears at 5 47.2 in germanicone. The spectral data indicated that compound 69 was 3-oxo-23-hydroxymethylene-olean-18-ene.
70 S. glutinosa^ yielded eleven known triterpenoids and three steroidal compounds a-amyrin, aamyrin acetate, 11-oxo-a-amyrin (3p-hydroxy-ll-oxo-ursa-12-ene), 11-oxo-P-amyrin (SP-hydroxy-lloxo-oleana-12-ene), 3-acetoxy-olean-9,ll-diene, ursolic acid, oleanolic acid, ursolic and oleanolic acid methyl esters, lupeol, erithrodiol 28-acetate, stigmasterol, sitosterol, 7a-hydroxysitosterol along with a new compound l-oxo-7a-hydroxysitosterol (cholest-5-ene-3p,7a-diol-l-one) (70). The HREI mass spectrum of 70 suggested the molecular formula C29H48O3 (rn/z 444.3562). The IR spectrum showed signals for a hydroxyl (3450 cm"^) and a six-membered ring carbonyl (1712 cm"^). The *H and ^^C NMR spectra indicated the structure clearly. The signals at 5 5.55 (IH, br s, H-6), 4.33 (IH, br s, H-7) were typical for A^-7a-0H groups. The relation between H-6 and H-7 were shown by spin decoupling experiments. A p-OH group was evident with the signal at 5 4.19 (IH, m, H-3a). The signals for H-3 and H-7 were somewhat further downfield than expected and when compared to 7a-hydroxysitosterol (5 3.85 for H-7 and 3.66 for H-3). The presence of an 0x0 group at C-1 is the only explanation for these chemical shifts. Other ^H NMR signals were at 5 0.67 (3H, s, Me-18), 0.99 (3H, s, Me-19), 0.82 (3H, d, J=6.5 Hz, Me-27), 0.85 (6H, d, J=6.5 Hz, Me-26 and Me-29), 0.91 (3H, d, J=7 Hz, Me-21). "C NMR (APT) showed a six-membered ring carbonyl signal at 6 208.4 (s). The unsaturated carbon atoms were at 5 142.4 (s) (C-5) and 132.6 (d) (C-6) while the carbon atoms carrying the secondary hydroxyl groups were observed at 5 67.9 (d) (C-7) and 87.0 (d) (C-3) (Table 25). S. tchihatcheffif^ yielded four triterpenoids one of them being a new compound. The known compounds were identified as 3-acelylerythrodiol, 28-acetylerythrodiol and 3-acetyloleanolicaldehyde (3P-acetylolean-12-en-28-al). The new triterpene (71) had the molecular formula C34H54O4 {m/z
708
526.4034). The IR spectrum exhibited acetyl absorbances for the acetyl group(s) at 1737 and 1242 cm'\ The ^H NMR spectrum displayed a signal at 6 2.07 corresponding to the two acetyl methyl protons, and an olefinic proton at 5 5.19 (IH, t, J=3 Hz, H-12) and a methylene pair of protons at 5 3.7 (IH, d, J=l 1 Hz) and 4.03 (IH, d, J=l 1 Hz) indicating the presence of one of the acetyl groups at C-28. The
CH2OAC
AcO'
71 TABLE 25 ^^C NMR Data of Compound 70
1
208.4 s
11
23.01
21
18.7 q
2
39.61
12
39.61
22
33.91
3
87.0 d
13
42.4 s
23
26.01
4
42.41
14
50.8 d
24
45.7 d
5
142.5 s
15
24.lt
25
29.0 d
6
132.5 d
16
28.21
26
19.5 q
7
67.9 d
17
55.lt
27
20.2 q
8
36.1 d
18
11.9q
28
20.71
9
45.8 d
19
18.6 q
29
11.9q
10
37.0 s
20
36.1 d
0.95, 1.16 Oand 1.17 as singlets. Acetylation of erythrodiol yielded 71 as evident from its spectral ( and TLC comparison which established that the new compound (71) was 3.28-diacetylerythrodiol.
709 2.5. AROMATIC COMPOUNDS AND FLAVONOroS Aromatic compounds are not common in Salvia species while flavonoids are abundantly present, especially in the aerial parts and the most abundant flavone is salvigenine in Turkish Salvia species. 2.5.1 Aromatic Compounds From the aerial parts ofS. divaricata^^, the known aromatic compounds 3-methoxysalicylic acid, p-hydroxybenzoic acid, cis- and //"owis-p-coumaric acids and from S, candidissima*^, o,pdimethoxybenzoic acid were isolated. A new aromatic compound di(4,4'hexyloxy- carbonylphenyl) ether (72) was obtained from S. heldrichianc^^. The HREI mass spectrum of 72 indicated the molecular formula C26H34O5 (m/z 426.2411). The UV maximum showed a substituted aromatic system (278 nm). The signals of the IR spectrum at 1730 cm"^ (carbonyl), 3050, 1616, 1558, 1520 cm"^ (aromatic) were consistent with the aromatic structure. The ^H NMR spectrum of 72 clearly indicated the structure by the signals at 5 7.07 (4H, d, J=8 Hz, H-3, H-5, H-3', H-5'), 6.76 (4H, d, J=8 Hz, H-2, H-6, H-2', H-6'), 4.23 (4H, t, J=7 Hz, CH2-8 and CH2-8'), 2.85 (4H, t, J=7 Hz, CHr9 and CH2-9'), 2.28 (4H, br t, J=7 Hz, CHrlO and CH210'), 1.58 (4H, br t, J=7 Hz, CH2-II and CHrll'), 1.15 (4H, br t, J=7 Hz, CH2-I2 and CH2-I2'), 0.9 (6H, t, J=7 Hz, Me-13 and Me-13').
3 2 13
2' 3'
8-12
, 8'-12'
13'
•0-HCH2)5—CH3
CH3—(CH2)^5 6
_ai
72
The ^^C NMR (Table 26) spectrum is in agreement with the given formula. The mass spectrum showed important ions at m/z 340 [M-C6Hi3-H]+ (a), 323 [M-C6H13O-2HI+ (b), 221 [M-Ci3Hi702]+ (c) which corraborated with the given formula.
710 S. forskahlef^ yielded two other dimeric aromatic compounds, which were octanol esters of cis- and /ira^w-cinnamic acids. They were separated by preparative TLC plates. The cis isomer 73, had the molecular formula C36H50O7 (m/z 594.3550). The UV spectrum showed a conjugated aromatic
MeO
MeO
H=CH-(CH2)7-CH 7
8
(1-7)"
H=CH-(CH2)7-CH3 7
8-
(1-7)"
73 7,8,7,8' cis 74 7,^,7,S'trans system giving the maximum at 5 325 nm. The IR spectrum indicated the aromatic character by the signals at 1605, 1585, 1520 cm"\ and the presence of an ester was indicated by the signals at 1705 and 1275 cm"\ The ^H NMR spectrum displayed signals at 5 6.78 (2H, d, J=12 Hz, H-8 and H-8'), 5.82 (2H, d, J=12 Hz, H-7 and H-7), 7.77 (2H, d, J=2 Hz, H-3 and H-3'), 7.10 (2H, dd, J=2 and 8 Hz, H-5 and H-5'), 6.87 (2H, d, J=8 Hz, H-6 and H-6'), 4.10 (4H, t, J=7 Hz, CH2-l"and CHz-l'"), 3.92 (6H, s, C-1 OMe and C-l'-OMe), 1.68 (4H, m, 2XCH2), 1.35 (4H, m, 2xCH2),1.28 (12H, m, 6XCH2), 1.15 (4H, m, 2XCH2), 0.87 (6H, t, J=7 Hz, Me-8" and Me-8'"). The ^^C NMR spectrum also supported the structure (Table 26). The trans isomer 74, showed ^H NMR spectral differences only in cinnamic acid part, particularly for the protons of C-7,8 and C-7',8'. These signals were displayed at 5 7.61 (2H, d, J=16 Hz, H-8 and H-8'), 6.29 (2H, d, H-7 and H-7), 6.92 (2H, d, J=8 Hz, H-6 and H-6'), 6.88 (2H, dd, J=2.5 and 8 Hz, H-5 and H-5'), 4.18 (4H, t, J=7 Hz, H-1" and H-1'"), which was consistent with the presence of the />*am'-cinnamic acid. Other NMR signals and mass degradation were similar in both isomers. From S. yosgaciensis^\ two aromatic compounds p-hydroxyquinone and p-acetylphenol were isolated.
711 2.5.2. Flavonoids Almost all Salvia species naturally grown in Turkey contain salvigenin as a marker flavonoid which was first isolatedfi"omS. triloba^^ by our group. S. heldrichianc?^, S.wiedemannii^'^^ yielded only salvigenin while other Salvia species yielded additional flavonoids, these were 6-hydroxyluteolin6,7,3',4'-tetramethyl ether, 6-hydroxyapigenin-7,4'-dimethyl ether, 4'-methylapigenin, apigenin and TABLE 26 *^C NMR Data of Compounds 72 and 73 C
72
73
1, r
154.2 s
147.5 s
2, 2'
130.0 d
152.3 s
3, 3'
115.3 d
115.7d
4, 4'
142.2 s
129.5 s
5, 5'
115.3 d
114.7d
6, 6'
130.0 d
109.3 d
7, T
174.0 s
144.8 d
8, 8'
64.91
123.2 d
9, 9'
34.3 t
168.4 s
10, 10'
31.0t
-
11,11'
24.91
-
12, 12'
22.61
-
13, 13'
14.0 q
-
1",1"'
-
2", 2'"
-
31.9t
OHI
64.61
-
29.51
4" ,4'"
-
29.51
5" ,5'"
-
28.71
6" ,6"'
-
25.91
7" ,7'"
-
22.71
8" ,8'"
-
14.1 q
2xOMe
-
55.9 q
OH
712 luteolin from S. sclarea^^, crysoeriol and diosmetin from S. candidissima^^, luteolin, eupatilin, pectolinarigenin and quercetin-3-methyl ether from S.limbata*^^, eupatilin, apigenin and luteolin from S. nemoroscF', luteolin, apigenin, apigenin-7-methyl ether, apigenin-4'-methyl ether, apigenin-7,4'dimethyl ether, apigenin-6,4'-dimethyl ether and kaempferol 3-methyl ether from S. yosgadensi^^. S. montbretif^ did not yield salvigenin, however, apigenin, luteolin and drsiliol were isolated. So far, in the investigated Turkish Salvia species, mainly apigenin and luteolin-type flavones were found while flavonol derivatives were rather rare. 3. BIOLOGICAL A C n v m E S Turkish Salvia species have been used as folk medicine for their antiseptic, antibacterial, diuretic, hemostatic, spasmolitic, carminative and wound healing properties'^. Due to these folklorixc uses a number of diterpenoids were tested against standard Gram-negative and Gram-positive bacteria, such as Bacillus subtilis ATCC 6633, Stcq?hylococcus aureus ATCC 6538P, Staphylococcus epidermidis ATCCC 12228, Proteus mirabilis ATCC 14153, Escherichia coli ATCC 8739, Klebsiella pneumonia ATCC 4352, Pseudomonas aureginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Candida albicans ATCC 10231 using Disc-diflRision method'^'^^. When the inhibition zones are greater than 7 mm, the tube dilution tests^^ were performed to determine the antibacterial activity quantitatively for inhibition concentrations (MIC).
Table 27 shows the results of 14 diterpenoids. Among them,
salvipisone was found to be highly active against B. subtilis, S. aureus and S. epidermidis, pisiferic acid 12-methyl ether was active against 5. subtilis while 2,3-dehydrosalvipisone and 7-oxoroyleanone were most active against the yeast, Candida albicans. The latter two abietane diterpenes were isolated from S. sclarea and in the same study, labdane diterpenes sclareol and manool as well as sesquiterpenes spathulenol and caryophyUene oxide were also isolated and tested for their activity and found to be active against S. aureus with the MIC values of 48.25, 13.75, 13.75 and 136.00 ng/ml, respectively. In fact, sclareol is an important bioactive diterpene having different pharmacological and microbiological effects. Ferruginol, taxodione, horminone and their derivatives were very common diterpenes of Salvia species which were also isolated from numerous Turkish Salvia species possessing antimicrobial^"*'^^^ and antitumour activities^'*'^^ In a recent study, with S. multicaulis, four of the new compounds (29-32) showed structural resemblence to tanshinone-type compounds. Therefore, they and the other new compounds (33-34 ) isolated from the same plant, were tested against bacterial strains (Table 28). All the compounds from this plant were also tested in the Mycobacterium tuberculosis H37Rv test system. Their antituberculous activity was established according to broth microdilution method^^'^. When the MIC results were
TABLE 27.
54.00
-
NT
6.75
713
7-Oxoroyleanone
MIC vaIues of the Diterpenoids against Standard Bacterial Strains ( p g h l )
714
compared to known tuberculostatic agents^* such as streptomycine (2-10 |ig/ml), kanamycine (5-10 ^g/ml), rifampicine (0.5 |ig/ml), PAS (p-amino saliyclic acid) (5-10 ^g/ml), INH (isonicotinic acid hidrazide) (0.2-5 ^g/ml), ETA (ethionamide) (5-10 ^ig/ml) and ethambutal (5-10 ^ig/ml), almost all the tested compounds given in Table 29 were found to be more active. The least active compound was salvipimarone (55) which is a pimarane diterpene. TABLE 28 Antibacterial Activity of the Diterpenes from Salvia multicaulis (ng/ml) E.
Compound S.
P.
Kl
Enl.
p-hem.
Ps.
aureus coli mirabilis.pneumon Streptococcusfaecalis aeruginosa ia 29
0.17
0.7
1.4
NA
0.17
NA
NA
30
NA
NA
NA
15.6
1.95
NA
15.6
31
0.123 NA
NA
NA
0.125
2.03
0.5
32
NA
4.6
NA
NA
NA
NA
NA
33
NA
NA
NA
7.15
NA
NA
NA
34
NA
NA
3.6
NA
NA
3.6
NA
NA=not active We have isolated two 3,7-dihydroxysteroidal compounds from S. glutinosa^ , one known 7-ahydroxysitosterol and one new l-oxo-7a-hydroxysitosterol (70) . According to the literature^'^^ , cholest-5-ene-3P,7p-diol derivatives showed activity in the P-388 murine lymphocytic leukemia test TABLE 29 Antituberculous Activity of Compounds 29-34 and 55 Compound M C (|ng/ml)
29
30
31
32
33
34
55
5.60
0.49
2.03
1.17
6.15
0.89
73.00
system while 3P,7a-dihydroxy derivatives has little activity. Therefore, both compounds obtained from S. glutinosa were tested in P-388 and KB system and showed marginal activity, verifying the literatuire findings. However, one of our testedrearranged diterpenes, microstegiol from S, microstegia^^ was
715 found to be active in P-388 with ED50 value of 3.0 ng/ml. Also, a number of diterpenoids, isolated during these studies are being investigated for their cytotoxic and antiviral activities.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
719
The Chemistry of Some Natural Colourants J.H.P. Tyman 1.
INTRODUCTION This account is concerned with colourants and pigments mostly
with those having E numbers in the European permitted range which are associated with manufactured edible products. The natural diet of human beings may contain a great variety of colourant substances although
in
manufactured
products
the
range
is
considerably
restricted. Until 1973 a wide spectrum of synthetic dyestuffs was allowed in foods so that for example in the mid fifties (1957) 32 synthetic dyes were permissible whereas by 1973, 19 of these had been removed and in that year the Colouring Matters in Food Regulations were introduced restricting the number to 14 with the provisional addition of 4 others which are under closer examination (ref.1). Many amendments have been made since that time and for instance by 1979 the list of synthetic dyes had been reduced to eleven (ref s .2, 3) . The position on natural colourants has remained more open although also as with synthetic dyes specified in 1973, the number of compounds or natural mixtures has diminished over the intervening years. Natural botanic families contain a vast number of organic chemical structures so that in gastronomy and in diet many of these are encountered gratuitously and assimilated. Their
entry or non-entry
into the human food chain with different peoples of the world has occurred by a process of experience, legend and
prohibition. From
the time when food processing and preservation moved largely from the home to the manufacturer there has always been an aim to maintain the colour of the original raw material.
In manufactured foods the
extent of usage of natural products is smaller than with synthetic products. Remarkably, some of the present colourants are obtained by extractive preparation while others are of synthetic origin and have come to be termed nature-identical. The usage of substances
sometimes
arose
from
emergency
certain coloured
situations
in
which
legislation was involved. Thus in the case of the plant pigments, the
720 carotenoids for example came into more extended usage through the vitaminisation of margarine with p-carotene because of its usefulness as a source of vitamin A. Apart from the
derivatives of p-carotene, other pigments are the
anthocyanins, the chlorophylls, cochineal, the betalaines, turmeric, caramel and riboflavin. In this account the historical background in brief, the chemistry of these pigments, their extraction
from
natural sources and their synthesis will be discussed. The present ^permitted'
natural
substances
and
nature-identical
synthetic
materials (also described by the Food and Drugs Administration, the FDA, as colorants ^exempt from certification') is quite small in number. Reference is also made to colourants no longer listed but which have an historical organic chemical significance such as for example brazilin and its relative haematoxylin. 2
THE PERMITTED NATURAL COLOURANTS Table 1 shows alphabetically the name, botanical origin (or
occurrence) the production method and the E number of some of the principal permitted natural colourants. TABLE 1 Some Permitted Colourants of Natural (N) and /or Synthetic(S)Origin Compound(s) Anthocyanins (Malvin)
Biological origin
Widespread pigments
Vltls
Beta Betanine (Beetroot red) p-Carotene
vinlfera)
N
vulgaris
Widespread in plants, insects
Bixin(Annatto) Bixa orellana 'Apocarotenal' p-Carotene Canthaxanthin Mushroom source Capsanthin
Production
Capsicum annum
Chlorophyll All plants Chlorophyllin (copper complex)
Official 'E' Number E 163 E 162
S,N
E 160(a)
N S S
E 160(b) E 160(e) E 161(g)
N
E 160(c)
N Semi-S
E 140 E 141
N Semi-S
E 120
Carminic acid (Cochineal)
Dactylopius coccus Costa
Curcumin (Turmeric) Riboflavin
Curcuma longa
N
E 100
Widespread vitamin
S
E 101
721 With reference to the materials in Table 1, the structures of which are shown in the following section, the anthocyanins are a large family of glucosides whose members constitute the pink, red, mauve violet and blue colouring matter of flowers, fruit and vegetables. Perhaps for economic considerations those from the red grape skin of the common species, Vitis' vinifera^ comprising delphinin, cyanin, malvin, pelargonin and peonin have become the compounds of commercial interest as food colourants. The blackberry (Rubus fructicosus), the blackcurrant (Rihes nigrum), the raspberry {Rubus ideaus), the cherry (Prunus cerasus) and strawberry (Fragaria genus of the Roseacae)also each contain more resticted numbers of anthocyanins. The red-purple pigment of beetroot {Beta vulgaris) comprises the betalaines the principal member of which is betanine, together with a very small amount of isobetanine and the vulgaxanthines. The carotenoids are a very large family consisting of C40 hydrocarbons, typified by a,Pand ycarotene (most commonly in the carrot, Daudas carota, Apiaceae), and the oxygen-containing xanthophylls. These are generally a lesser occurring group, with * permitted' members such as canthaxanthin (in the mushroom, Cantharellus clnnabarlnus) [E161(g)], capsanthin and capsorubin (Paprika oleoresin) [E160(c)], cis-bixin, a major component of the colourant annatto, a resinous material which coats the seeds of Blxa orellanar and norbixin, its corresponding dicarboxylic acid. The apocarotenoids are also in this category, for example, 'apocarotenal', (p-apo-8'-carotenal) [E160(e)], and ethyl p-apo-8'carotenate [E160(f)] Chlorophyll (E140) itself which exists as the compounds a and b (where the 3-methyl group in a has been replaced by a formyl substituent) is a less satisfactory colourant for commodity purposes than one of its derivatives lacking the phytyl group prepared by coppering), namely copper semi-synthesis (hydrolysis and chlorophyllin. Carminic acid, the active colourant principle of cochineal, is employed in the form of its derivatives, notably, the aluminium chelate which is obtained by semi-synthesis. Curcumin is the yellow pigment of the root of the tumeric plant, a spice material of legendary importance. Riboflavin (lactoflavin) enjoys a place as a vitamin rather than as a somewhat little used colourant. The carotenoids in Table 1, p-carotene, 'apocarotenal and
722 canthaxanthin have also achieved chemical significance through their commercial
production
by
total
synthesis,
while
the
inorganic
derivatives of carminic acid and the transformation of chlorophyll to sodium copper chlorophyllin are obtained by straightforward onestep semi-syntheses. Numerous xanthophylls, although not commercially prominent, in the listed 'permitted' range are lycopene [E160(d)](tomato), flavoxanthin [E161(a)],
lutein
[E161(b)](egg
yolk,
orange),
cryptoxanthin
[E161(c)] (the mango, orange), rubixanthin, [El61(d)], violaxanthin [E160 (e)], (the materials
orange),
having
rhodoxanthin
a secondary
[E161(f)]
colouring
effect
and
the
natural
such as paprika,
turmeric, saffron and sandalwood which are also included in the list. It
should
be
emphasised
that
many
xanthophylls
for
example,
zeaxanthin (egg yolk, mango), antheraxanthin (orange), neoxanthin and astaxanthin (lobster) are encountered in the diet or gastronomy and are not in the listed permitted colourant matters or commercially extracted for food colourant purposes. The occurrence of some of these xanthophylls is shown in Table 2 Table 2 The Occurrence of Some Xanthophylls permitted as Food Colourants (1973 amended regulations) Compound
Botanical Origin
Structure
Lycopene Flavoxanthin
Lycoperslcon Taraxacum
5,8-Epoxy-3,3'-dihydroxy-
Lutein
many plants,egg yolks
escalentum officinale
a-carotene
(dandelion) (tagetes) a-Cryptoxanthin Capsicum annum^
'Openchain p-carotene'
maize
3,3'-Dihydroxy-a-carotene 3'-Hydroxy-a-carotene
(red pepper) Rubixanthin
Rosa
ruhinosar
3-a-Hydroxy-Y-carotene
ripe fruit Rhodoxanthin Violaxanthin
Taxus
baccata
3,3'-diketoretrodehydro-
(yew, red fruit)
p-carotene
Viola
5,6,5',6'-diepoxy-p-
trichloris,
green leaves
carotene-3,3'-diol
It should be emphasised that carotenoids and xanthophylls are a vast family of materials and three of the most naturally abundant are fucoxanthin, violaxanthin and neoxanthin (ref. 4) although they are not
723 in evidence as food colourants. Lutein, the fourth is permitted and is commercially available by extraction. The chemistry and biochemistry of the whole group have been described in great detail (refs.5,6,7). 3
THE STRUCTURES OF THE MAJOR FOOD COLOURANTS The structures of the substances of interest in the permitted range together with some associated structures are shown in the following sections 3.1 The Anthocyanidins and Anthocyanins Anthocyanidins (aglycones), flavyliun salts (chlorides), are shown generally by (1), the anthocyanins (3-monoglucosides) by (2), and 3, 5-diglucosides by (3) . The major anthocyanidins are cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin and the substituents in their structures are shown below for the formula (1) . Table 3 The Structures of some anthocyanidins Anthocyanidin Cyanidin Delphinidin Malvidin Pelargonidin Peonidin Petunidin
-w OH
R^
OH
OH
H
OMe
OMe
H
H
OMe
H
OMe
OH
Malvin , a major component of the grape skin (Vitis vinifera) depicted by (4) as a 3,5-diglucoside structure which has been Rl i^j^^OH
Cl~
HO^
iT 11 ^ II
1T
NJCgHiiOg
OH (2)
Rl Cl"
s:?^^ Malvin (4)
(CgHiiOs)
J s ^ OH
-6H1105 (3)
is
724 synthesised (ref.8)and compared with a sample of natural origin from Primula
viscosa
vinifera,
Vitls
(ref. 9) . The structural type malvidin in
referred to earlier, is listed (ref.10) as
all but pelargonidin, with
accompanied by
all present as 3-glucosides
although
another source (ref. 11) mentions the 3-glucosides of only malvidin, peonidin
and
Diglycosidyl
petunidin
as
the
components
(of
the
fruit).
3-
(rutinosides, sophorosides and sambusiosides) and 3-
triglycosides are also
present. The instability of the glucosides
to
may
acidic
conditions
account
for
reported
compositional
differences. Thus in their synthetic work on malvin 3,5-diglucoside the
authors
reported
monoglucoside which chloride
its
hydrolysis
in
from its properties
(5-glucoside)
and
not
acidic
(4) (ref. 8)
solution
to
a
'was certainly malvenin 'oenin
chloride'
(3-p-
glucosidylmalvidin chloride) which was synthesised (ref .12) . The name oenin chloride was used for this anthocyanin and oenidin for the anthocyanidin in an early historical summary by a pioneer in the chemical investigation of flower pigments (ref. 13). 3.2 The Betalaines The betalaines comprise two groups, the betacyanines (the major colourant
constituents
of beetroot)
and
the
lesser
group,
the
betaxanthines. The former constitute the majority of the compounds in beetroot and only much smaller proportions of isobetalaines , (5%) and vulgaxanthines are present. The betanidines are the aglycones and the betanines contain alcohol
a 5-0-glucosidic group, in which the primary
is present to a small extent as a sulphate. The structures
of betanidine (5) and its corresponding glycoside betanine (6), the stereoisomeric aglycone at C^s , isobetanidine (7), and its glycoside (8) are depicted.
«°ac; s-."o°^"
H
725 The minor compounds shown are the amide, vulgaxanthine I (9; R = NH2) and the aldehyde, vulgaxanthine II (9; R = H ) . The compound (10), indicaxanthin, although not present in the betalaines, is structurally related and occurs in other colouring matters.
H ^ : : ^ ; ^ : ^ " ,
H''
N-^ ^COoH
C02H (10)
HOoC' ^ N ' ' ' ^ C 0 2 H H
RO2C'
^N^ H
CO2R
3.3 The Carotenoids The formulae of the carotenoids, from which the yellow, orange and red colours of vegetable and fruit derive, comprise hydrocarbon types
and the oxygen-containing
xanthophylls.
namely, for the hydrocarbons, p-carotene
They
are depicted
(11), a-carotene (12),
(12) y-carotene
(13), lycopene (14); for the xanthophylls, canthaxanthin
726 (15),
capsanthin
apocarotenoids
(16)
and
capsorubin
(16a),
and
for
the
(P-apo-8'-carotenal), (17; X = H) , ethyl p-apo-8'-
carotenate (17; X = OEt).
The structures are shown of cis-bixin (18; R = Me), norbixin (18; R = H) and crocetin (19), the parent acid of the colourant principle of saffron, sometimes described as a natural substance having a secondary colouring effect, which exists as the bis-gentibiose ester, crocin,
although crocetin and crocin do not have E numbers and
are
727
not permitted colourants.
HO2C
;02R .CO2H
HO2C (19)
The formulae of some less well-known xanthophylls, flavoxanthin (20), [E161 (a)], lutein (21), [E161 (b)], a-cryptoxanthin (22), [E161(c)], rubixanthin (23), [E161(d)], violaxanthin (24), [E161(e)]
(24)
728 and rhodoxanthin (25), [E161(f)] are also represented.
3.4 The Chlorophylls Chlorophyll (E 140) exists as chlorophyll a (26) and chlorophyll jb (27) and although it is not widely used as a commercial
colourant,
a derivative, copper chlorophyllin (29) is employed.
<26)Chlorophyll
COgBfe
H MG
H
a
Me
In this the magnesium is replaced by a copper atom and the phytyl chain has been removed. Chlorophyll c (28) is of marine origin.
729
CX)«Me ^
NaOsC (28)ChlorqphYll c C2 , R = CH=CH2 Ci , R = Et
(29)Sodium salt of copper chlorophyllin
3.5 Cochineal The colourant p r i n c i p l e o f cochineal w h i c h is used as carmine metallic
chelates
is carminic acid (30)
in the form of its aluminium and other
(calcium-aluminium)
in m u c h
t h e same w a y that
alizarin w a s formerly employed as a m o r d a n t d y e b y chelation of the 1,2-dihydroxy
system.
HO2C.
Me
O
OH
(30)
3.6 Curcumin Curcumin
(31, t h e active p r i n c i p l e in turmeric, is used b o t h
for its colour and flavour. It exists as an intramolecular keto-enol chelate containing a six-membered ring.
»Me
MeO (31)
730 3.7 Riboflavin Riboflavin (32) (R = H) or lactoflavin [and its 1° phosphate (33)] is more familiar as vitamin B2 than as
a food colourant.
^N"^ ^^5!;^ ^Me CH2[CH(0H) IgCHgOR [32, R = H ; 33, R = P(0)(OH)2]
3.8 Sandalwood The red colourant component of sandalwood (in the 'insoluble^ red woods group) has attracted interest for its colourant potential although this application does not appear to be active. Polyphenols (ref.l4) together with less complex compounds are present such as pterostilbene, (34), (4'-hydroxy-3,5-dimethoxystilbene), pterocarpin (35) and homopterocarpin (36),the formulae of which are shown. The nature
of
the
colourant
structure
does not
seem
to have
been
established.
MeO
MeO
(36)
The natural materials referred to have E numbers which relate to European (EEC) practice. They also have universal CI (Colour Index) numbers and in the USA, numbers Practice (ref. 15).
listed in the Code of Federal
731 4
THE EXTRACTION of NATURAL FOOD COLOURANTS As with many natural products for pharmaceutical or medicinal
use, extraction of ^permitted* natural food colourants takes priority over synthesis except in the instance of the carotenoids. In this class a remarkable
synthetic activity makes the three
important
compounds p-carotene, 'apocarotenal', and canthaxanthin commercially available. 4.1 Anthocyanins In general, anthocyanins can be obtained from natural sources by aqueous acidic or alcoholoc extraction. Specifically, grape colour extract (El63)is a dehydrated water-soluble powder (obtained by spray drying) or the itself
can
be
aqueous solution from which it is prepared. This obtained
by
extraction
of
the
pigments
in
the
precipitate resulting from the storage of, for example. Concord grape juice. Alternatively in the case of grape skin extract the purple skins of Vitis
vinifera
consisting of the deseeded residues from
pressing the grapes to obtain wine or grape juice, are steeped, during which some fermentation also occurs with formation of ethanol. An extract is produced termed, containing inorganic
anthocyanins, salts. Sulphur
evaporation
in vacuo
enocianina, a purplish red liquid
tartaric dioxide
gives
acid, is
tannins,
added
a concentrate
in
sugars,
and
process
and
the
containing
ca.
1% of
colourant material. Analysis by the uv absorption of citrate buffered solutions enables the % anthocyanin to be determined distribution
of
component
from the maximum at 520nm. The
anthocyanins
can be
found by
TLC on
cellulose with various strongly acidic solvents (eg. cone HCl, formic acid, water, 19:19.5:61.5) of extracts of plant material obtained with 1% hydrochloric acid in methanol. The Rf values found for 3monoglucosides of the anthocyaninidins of Vitis the petunidin compound
vinifera
(0.13), for that of malvidin
were, for (0.22) and of
peonidin (0.25) (ref. 11). 4.2 Betalaines The red betacyanines, the major components and the minor yellow betaxanthine pigments of the red table beet. Beta
vulgaris,
present
to 0.12% in the natural product, are obtained from aqueous extraction at mild temperatures, as a concentrate by evaporation of the solution in
vacuo,
or as a powder by spray drying of the concentrate. The
product is stable to heat below 50°C at pH 4-7 and if not exposed to light.
732 Betanine can be analysed from its uv absorption maximum at 535nm (at pH
3)
while
betaxanthine
absorbs
at
480nm.
Preparation
for
chromatographic examination (ref. 11) by TLC on cellulose is effected by firstly homogenisation of the beetroot sample (50g) with aqueous methanol (1:1), filtration and concentration at less than 40°C. The residual solution is applied to the plate as a streak and doubly developed with isopropanol-ethanol-water-acetic acid (30:35:30:5). After drying of the plate under nitrogen, betanine appeared as a violet zone at RpO.27 with four other bands present. 4.3 Carotenoids Carotene was first extracted from the carrot by Wackenroder in 1831 and later from green leaves, although the identical nature of the material from the two sources was not established until 1907 by Willstatter. The former source comprises in fact a mixture of a- and p-carotenes which was later separated by Kuhn in the early thirties by the then new technique of column chromatography although this procedure was first described in 1906 by Tswett for the separation of carotene and chlorophyll. For the recovery of p-carotene, vegetable sources are largely used. The material is macerated in a blender with methanol-acetone (2:1), filtered and reextracted until a colourless filtrate results. The combined filtrates are evaporated at less than 40°C in
vacuo^
residue
10% sodium
is dissolved
in diethyl
ether, washed with
the
hydroxide solution and then with water, dried and concentrated. isolated
plant
materials
are
stored
at
low
temperature
The
under
nitrogen. For the isolation of carotenoids from animal sources, the tissue is extracted with propanone and a similar work-up procedure adopted as for plant carotenoids. Xanthophylls often occur as lipidic materials esterified with fatty acids and a hydrolysis step of the isolated extract, (obtained by ethereal extraction) is required. Treatment with methanolic sodium hydroxide pigment
at
which
ambient is
temperature
recovered
by
during
ethereal
6 hours
liberates
extraction,
washing
the to
neutrality, drying and concentration under reduced pressure. Sterols and
proteinaceous
material
are
removed
by
filtration
of
the
concentrate dissolved in propanone and kept at low temperature. Several different solvent systems have been described for TLC of carotenoid hydrocarbons Kieselguhr G
(refs. 16,17).
Thus on magnesium oxide-
(1:1) layers with light petroleum(40-60°C)-propanone
733 (85:15), the Rf values found for the carotenes were a- (0.86), p(0.82), Y- (0.33), 6- (0.65)and for lycopene (0.04). Reversed-phase systems have found use with hydrocarbon/xanthophyll mixtures. For example on RP-18 silica gel F254 layers with light petroleum(40-60°C)acetonitrile-methanol (20:40:40) as developing solvent the Rf values shown
(ref.ll)
canthaxanthin violaxanthin
were
for
(0.51), (0.68).
(0.13),
p-carotene
lutein
(0.55),
lycopene
capsanthin
(0.23),
(0.64)
and
Prior to the synthesis of p-carotene, early
recoveries were examined of carotenoid-rich fractions from palm oil species (ref. 18) with a view to potential large-scale chromatography of the unsaponifiable material recoverable after alkaline hydrolysis of the oil. For the commercial preparation of Annatto (E160b) , the fruit from the tree Bixa
orellandr
a fast-growing tropical shrub indigenous to
India, East Africa and the Caribbean is used. The fruit contains seeds which are coated is
extracted
with
with a coloured resinous layer or marc which
various
materials
such
as
edible
vegetable
triacylglycerols or with aqueous alkaline solvents. In the latter case acidification affords the annatto pigments (ref.20) comprising predominantly, water-soluble norbixin and oil-soluble bixin. Annatto extract is one of the oldest known pigments used for food , textiles and cosmetics. Saffron,
from the dried stigma of Crocus
sativa^
the
colourant
principle of which is crocetin is produced in several sub-tropical countries, expensive
for yet
example. North Africa, quite
stable
material
Spain, although
France. not
a
It
is an
permitted
colourant with an E number. Paprika and its oleoresin in the red and yellow varieties owe their colour to carotenoids such as capsanthin and capsorubin. They are obtained
from the sweet red pepper by solvent
extraction which
affords an oleoresin containing mainly the two carotenoids. They are more important as flavour materials
and illustrate, as do turmeric
and its oleoresin, the feature that flavour is often the property of greater interest than colour alone. 4.4 Chlorophyll and derivatives A simple laboratory recovery of chlorophyll can be achieved by the maceration of the green plant source with twice its weight of propanone at ambient temperature, a process which is repeated until the filtrate from the operation is colourless. One convenient green source is dried alfalfa, more popularly
known as the perennial
734 lucerne,
Medicago
sativa,
although
nettles
and
grass
are
also
employed. The combined filtrates from the extraction are evaporated under reduced pressure at less than 40°C. The residue is treated with 10% sodium chloride solution, extracted with diethyl ether, the solution
dried
and
concentrated
and
the
residue
stored
under
nitrogen. Chlorophyl a and b [and their transformation products
a'
and jb' respectively (stereoisomers at CIO) which result from heating pyridine solutions at 60°C for 1 hour in sealed tubes] have been separated (ref. 11, 19) on sucrose layers (for example, icing sugar) with
light
petroleum
(40-60°C)-isopropanol (99:1) .
The
various
chlorophyll (chl) members have the Rf values, chl a (0.56), chl a' (0.72), chl jb (0.30) and chl jb' (0.44) while the pheophytins (lacking an Mg
atom)
a
and
jb respectively
have
Pheophorbides (the free acids without the
values
0.84
and
0.72.
phytyl group and Mg atom)
a and b possess considerable polarity with R^ values 0.05 and 0.02 respectively. Cellulose can be used with the solvent light petroleumpropanone-isopropanol(90:10:0.45) on an analytical and a preparative basis. For deriving copper chlorophyllin (E141), chlorophyll is hydrolysed under mild alkaline conditions and the product ^copperised* to obtain the coordinated copper derivative. 4.5 Cochineal Cochineal extract
(Colour Index 75470, E 120)is the final
alcohol-free material obtained after aqueous ethanolic extraction of the dried bodies of the female scale insect Dactylopius which lives
on cacti such as Opuntia or Nopalea
coccus
Costa
coccinelllfera
a
species indigenous to Peru and Mexico, although also found in the Canary Islands. In practice a simpler procedure consists of aqueous alkaline extraction. The colourant principle is carminic acid which is more well-known in the form of carmine, an aluminium
chelate of
carminic acid, a material insoluble in water and stable on the acidic side. A uv spectrophotometric study has been made
(ref .21) . Thin
layer chromatography of cochineal has been examined on acetylated cellulose with the solvent system, ethyl acetate-tetrahydrofuranwater (6:35:47)
in which the Rf was 0.94 (refs.22,23). The history
of the chemistry of cochineal has been discussed (ref.24). 4.6 Curcumin Curcumin is the colourant principle of turmeric (Colour Index 75300, E 100) and tumeric oleoresin. The dried and ground powder from the rhizome or root of Curcuma
longa
of the Zingiberaceae family
735 constitutes extracting
turmeric the
purification
powder
while
turmeric
with
various
oleoresin organic
is
obtained
solvents.
by
Further
is adopted to obtain curcumin. Turmeric powder can
contain as much as 90-95% curcumin. 4.7 Miscellaneous materials Caramel (Colour Index natural brown, 10,E 150) , the dark brown colour additive resulting
from the controlled heat treatment of
various carbohydrate sources is in effect a complex semi-synthetic mixture rather than an extract. As indicated earlier many colourants are encountered gratuitously in domestic practice as for example in the surface darkening observed in cut fruit or vegetables due to the oxidative action of the enzyme phenolase upon polyphenolic compounds. Reactions involve o-quinone
formation, and polymerisation of the
substrates chlorogenic acid (a structural relative of caffeic acid), and 3,4-dihydroxybenzoic acid. Participation of tyrosine residues in enzyme proteins results in melanin formation. Enzymic oxidation of polyphenolic compounds in tea. Camellia
sinensiSf
gives a range of
coloured products, the theaflavins and the thearubigins all of which are desired in the process of tea-making. The extraction processes described in the first part of this section are based upon very traditional methods. However in recent years a number of new approaches have been developed in which the properties of the natural
ingredients
are more
faithfully preserved.
Thus
supercritical fluid extraction has become adopted in the flavour and perfumery industries (ref.25) 5
THE SYNTHESIS OF THE NATURAL PERMITTED COLOURANTS Although only three carotenoids are commercially synthsised for
use as permitted food colourants, namely p-carotene, 'apocarotenal* and canthaxanthin, the availability of nature-identical counterparts of the natural products has always attracted interest. For the study of
biological properties and for chromatographic studies access to
synthetic
versions
is
highly
desirable.
In
the
course
of
the
isolation of the natural product its total synthesis finally always remained historically as a target. Such chemical work had
invariably
been carried out well before the introduction of the
'permitted'
range concept focussed attention on certain natural products 5.1 Anthocyanins The naturally-occurring anthocyanins are complex mixtures and classical synthesis was aimed at the construction of the 3-glucosides
736 and the 3, 5-diglucosides. The first approach to the formation of the parent aglycone, the anthocyanidin pyrylium salt was made by the reaction of resorcinol with benzoylacetaldehyde in the presence of hydrogen chloride (ref.26), a process that was improved upon (ref.27) by employing 2,4-dihydroxybenzaldehyde and acetophenone in glacial acetic acid, ethyl acetate or dioxan containing hydrogen chloride at 0°C. An alternative earlier approach to pyrylium salts had involved the
reaction
of
5,7-dihydroxy-3-methoxycoumarin
with
Grignard
reagents followed by dehydration of the resultant chromenol by the action of hydrogen chloride (ref.28) . Much of the first investigative work has been summarised (ref.29). The development of methods for anthocyanidins, by the preferred use of aromatic aldehydes and co-hydroxyacetophenones led by way of their 0-(D)-glucopyranosyl analogues to the synthesis of the anthocyanins themselves. Although pelargonin chloride is an anthocyanin which does not occur in the natural prepared
sources used
sources
(eg
grape
for the extraction of skins),
it
represents
commercially the
simplest
structural member and the methodology for its synthesis (ref.30) was intrinsic to the preparation of its five analogues. The route is shown in Scheme 1. Scheme 1
/ ^ OH
OMe
U
^ ° v ^ "-^ "• AcO'
CI
OAc
Pelargonln-S-glucoside H
H Glucose (G) 2
3 OH'I
^„ OH
G(Ac)4 _ * - AcO
OAc OAc
R e a g e n t s : ( i ) CICH2COCI, AICI3, (ii)KOAc, AcOH, ( i i i ) d i l . N a O H , (iv)Ac20, NaOAc, (v)Ag2C03 , a - B r , 2 , 3 , 4, G-CgH-^O (OAc) 4, ( v i ) 2-BzO-4, 6-(OH) 2C6H2CHO, HCl, ( v i i ) d i l . NaOH; H C l ; p u r i f n . t h r o u g h p i c r a t e .
737 Anisole
was
converted
to
4-(S)-chloracetyl) phenol
and
thence
in
glacial acetic acid containing potassium acetate and some ethanol to 4-(co-acetoxyacetyl) phenol.
Hydrolysis
with
10% sodium
hydroxide
afforded the monosodium salt of o, 4-dihydroxyacetophenone acetylation of which
(a method under aqueous alkaline conditions termed the
Chattaway method) gave 4-hydroxyacetylphenyl
acetate. Reaction of
this with 2,3,4,6-tetra-O-acetyl-a-(D)-glucosyl bromide in benzene containing silver carbonate afforded the p-glucoside. With the 2-0benzoyl derivative of phlorglucinaldehyde in ether and chloroform containing hydrogen chloride this p-glucoside gave the pyrylium salt shown. Hydrolysis with aqueous sodium hydroxide, acidification and exhaustive
purification
afforded
3-p-(D)-glucosidylpelargonidin
chloride which proved to be identical with callistephin chloride isolated from the purple-red aster Callistephus
chinensis
Reference
the
has been made
to oenin
malvidin, which was synthesised
chloride,
(ref.31).
3-glucoside
of
(ref.l2) by an analogous type of
route to that shown in Scheme 1, from the 2-benzoyl derivative of phloroglucinaldehyde
and
co-tetra-O-acetyl-p-(D)-glucosyloxy-4-
acetoxy-3,5-dimethoxyacetophenone hydrogen
chloride.
acidification
and
Hydrolysis extensive
in with
ethyl
acetate
containing
sodium
hydroxide,
dilute
purification
afforded
3-p-
glucosidylmalvidin (oenin chloride). For the synthesis of malvin (4), the 3, 5-diglucoside of malvidin, the same p-(D)-glucosidyloxyacetophenone was required and in addition a 2-p-(D)-glucoside of phloroglucinaldehyde. This was obtained in low yield from phloroglucinaldehyde and 2,3,4,6-tetra-O-acetyl-a-(D)glucosyl bromide. The
synthesis {ref.8)is depicted in Scheme 2.
Scheme 2 (G = Glucose; GAC4 = Glucosetetraacetate as Scheme 1)
OH
OH
738 Reagents: (i) a-Br-2,3,4,6-(OAc) 4C6H,0, MeCN, aq.KOH, (ii)4-OAC-3,5(OMe)2C6H2COCH20G(OAc)4, EtOAc, HCl, (iii) 10%NaOH; HCl. Malvin chloride has been described as forming dark reddish-brown needles having a grenish lustre. The colour in solution is dependent upon the pH. Cyanin chloride (ref. 32) (3, R^ = OH^R^ = H) , its methyl ether, peonin chloride (ref.32) (3, R^ = OMe, R^ = H), petunin chloride (3, R^ = OMe, R2 = OH) and delphinin chloride (3, R^ = R^ = OH), were synthesised similarly. OMe HO O^^ Petunin (chloride)
Cyeuiin (chloride)
OMe OH
O" O^
G
Peonin (chloride)
Delphinin (chloride)
The a c e t y l a t e d glucosyloxyacetophenones shown were used in a s i m i l a r methodology, t o g e t h e r with in a l l c a s e s , the 2 - p - g l u c o s d i d e of phloroglucinaldehyde. OAc
OMe y.-w
T -
OAc Y^*-
OMe urae
^Y^^OAc O"
O
GAc.
GAC4
I
I
(For peonin chloride)
OAc O
o I
I
GAC4
(For cyanin chloride)
OAc
(For delphinin chloride)
GAc, (For petunin chloride)
The generation of the required (o-hydroxyacetophenone for the preparation of the respective tetraacetylglucosyl derivative was
739 found
to
need
a modified
procedure
involving
the
use
of
the
corresponding (o-diazoketone, prepared from the acid chloride (ref .33) , and thence the formyloxy compound which was selectively
and more
easily hydrolysed than the corresponding acetate to the necessary (ohydroxy intermediate for glucosylation. Although the formation of the acetylated pyrylium salts in the acidic condensation of 2-glucosyloxyphloroglucinaldehyde and the acetylated glucosyloxyacetophenone
generally
proceeded
in
good
yield,
for
example malvin 65%, and for pelargonin-3-glucoside of the same order, the initial yield of the glucosylphloroglucinolaldehyde
component
was low. It seems likely that the liberation of two molar proportions of water in the acidic reaction could have resulted in partial hydrolysis. This might now be
avoidable by
the
inclusion of a
molecular sieve in the reaction medium. Removal of the protective groups
leading
to more
soluble
products
and
also
the
possible
hydrolysis of sugar groups under the final acidic conditions were some of the difficulties encountered. Chromatographic monitoring of a typical reaction mixture during all the stages would be of interest to establish the total pathway. Both anthocyanidins and anthocyanins undergo structural and thus colour changes with alteration of pH. At very low values the pyrylium salt exists while at high values the oxyanion of a quinonoid form is present leading finally to a ring-opened structure. Scheme 3
740 These transformations, typically given by cyanine chloride, are reversed upon lowering the pH as shown in
Scheme 3. At any given pH
it appears that three or four structural entities may be present in varying proportions. 5.2 Betalaines Beta
It was thought originally that the pigments of beetroot vulgaris
were
related
to
the
anthocyanins,
and
were
indeed
nitrogenous members of that series. However many studies showed their complete structural dissimilarity with that group and although their colour is similar it is constant over a wide pH range unlike the wide variability
found with
anthocyanins. They have been classed
as
alkaloids and reviewed (refs. 34,35). The betacyanines, comprise the indoles, betanine (6) the major red pigment component and its stereoisomer at C^s, isobetanine
(8), a
minor material. Both possess mono-p-(D)-glucopyranosyl groups at C5. The corresponding aglycones are betanidine (5) and its stereoisomer at Ci5, isobetanidine
(7) . The betaxanthines
are
pyrrole-based,
unlike the indole-based betacyanines, and are minor yellow pigments
of Beta
vulgaris.
After studies of the structure and stereochemistry of betanidine and isobetanidine indole
(ref. 36) the former was synthesised
component,
the
methyl
ester
of
(ref.37). The
L-cyclodopa,
dihydroxyindoline-2-carboxylic acid, has been prepared
5,6-
(ref. 38) and
the piperidine moiety, betalamic acid (ref.39) has been synthesised by another distinct route. The reactions used
for the synthesis of betanidine (5) (ref. 37) are
depicted in Scheme 4. Chelidamic acid (obtained from chelidonic acid available from propanone and diethyl oxalate by Claisen condensation and then amination) , was reduced in 42% yield to the
meso compound
cis,cis-4-hydroxy-1,2,3,4-tetrahydropiperidine-2,6-dicarboxylic and
the
derived
dimethyl
ester
oxidised
in
90% yield
to
acid the
corresponding piperidone. The semicarbazone of 2, 3-dihydrobetalamic acid was obtained (44%) through the use of a Horner-Wittig reagent and thence the semicarbazone of dimethyl betalamate itself in 41% yield
through oxidation with dimethyl sulphoxide in trifluoroacetic
acid containing dicyclohexylcarbodiimide. Reaction with the methyl ester of L-cyclodopa
in methanolic hydrogen chloride afforded a
diastereoisomeric mixture, (the trimethyl ester of betanidine and the iso compound)
in 87% yield.
Hydrolysis with warm
hydrochloric acid gave betanidine (5)
concentrated
and some of the iso compound
741 (7) which were characterised chromatographically and spectroscopically. Betanidine is partly isomerised to the iso compound under alkaline and acidic conditions (ref.36). The reaction of L-cyclodopa appeared to proceed unambiguously, that is with the double bond in the piperidine ring at the 2,3-position. The final conversion to the glucoside betanine(6) was not described and does not appear to have been yet achieved. Scheme 4
OH
C. ^
OH -^ HOsC^N-^COgH
MeOgC"
NNHCONH2
MeOgC'"" N H
'COsMe
N'^ '"COgMe
MeOgC"
N
'COgMe
NNHCONH2
MeOgC'"' ^N^'^COgMe H
MeOsC"
CO2H
N
COgMe
CO2H
(5)[+ some (7)] Reagents: (i) 5%Rh/Al203, H2O, E^,A atm., 75°C; MeOH,/HCl, (ii)C6HiiN=C=NC6Hii on polymer, DMSO, CgHgN^CFsCOs", (iii) (EtO)2P(0)CH2CH=NNHCONH2, NaH, (MeOCH2)2, (iv) C6HiiN=C=NC6Hii, DMSO, CF3CO2H, PhH, (v) L-cyclodopa Me ester, MeOH/HCl, (vi)Conc. HCl,(vii)not effected. By reaction of the dehydropiperidine compound with L-proline
in
propanone/methanol containing hydrogen chloride the betaxanthine (10/ R = Me) resulted, together with its diastereoisomer, in low yield. L-Cyclodopa methyl ester hydrochloride was prepared (ref.38) from LP-(3,4-dihydroxyphenyl)alanine methyl ester by the route shown in
742 Scheme 5a. L-p-(3, 4-Dihydroxyphenyl)alanine itself was derived from L-tyrosine either
by the action of tyrosinase (ref.39)or by means
of silver oxide oxidation. Scheme 5a
,cn:;^ Zyyr: "°XXir - -IX):^-
HO-^ - ^ (C)
^ CI
^
I
T\4.y-C02Me
Reagents: (i) Tyrosinase, or Ag20 (ii) SOCI2/ MeOH, (iii) K^FeCNg^pHS, (iv)pH8,Na2S204; HsO". A
different
approach
to
cyclodopa
(alkyl
dihydroindole-2-carboxylates has been devised described process
5,6-dihydroxy-2,3(ref. 40) since the
(ref.38) only results in acceptable yields when
operated in very dilute solution. In the modified method, Scheme 5b, racemic 3,4-dihydroxyphenylalanine (dopa) methyl ester hydrochloride (C) was iodinated with potassium iodate in a two phase system, then treated with sodium dithionite and acetylated to afford methyl 5,6diacetoxy-7-iodo-2, 3-dihydroindole-2-carboxylate. Reductive treatment of this with Pd-C, and hydrogen in ethanolic solution containing triethylamine and conversion to the hydrochloride (methyl
gave the product
ester diacetate hydrochloride)in 40% overall yield.
Scheme 5b
=' • * nW-^°^ "" IID>"=" R e a g e n t s : ( i ) K I 0 3 , C H C l 3 , H2O; Na2S204; ACjO, EtOH; H C l ( g ) , E t 2 0 .
Py.,
(ii)
Pd-C,H2,
NEts,
743 Another approach to the
synthesis of betalamic acid was effected
from N-benzylnorteloidinone (ref. 41) a structure prepared earlier as the N-Me analogue (ref.42). 2,5-Dimethoxy-2,5-dihydrofuran, obtained from furan by bromination in methanol containing potassium carbonate, was converted
in ethanolic solution at -5°C with aqueous potassium
permanganate containing magnesium sulphate heptahydrate to cis-3,4dihydroxy-2, 5-dimethoxytetahydrofuran. This was hydrolysed with warm M hydrochloric acid to afford meso-tartaraldehyde which in buffered solution at pH 5 was reacted with acetone dicarboxylic acid in a Robinson-Schopf type synthesis and then with aqueous benzylamine hydrochloride
to
give
benzylnorteloidinone.
with
With
carbon
methyl
dioxide
orthoformate
evolution, this
diol
Nin
refluxing dichloromethane containing trifluoroacetic acid yielded a 10:1
epimeric
mixture
of
the
ortho
esters.
Hydrogenolysis
in
methanol-trifluoroacetic acid in the presence of palladium-carbon gave the secondary amine which was reacted at 0°C in diethyl ethertetrahydrofuran with allylmagnesium bromide to produce the carbinol. The
N-benzoyl
derivative
of
this
compound
was
obtained
by
condensation with dibenzoyl peroxide in dimethylformamide containing potassium carbonate. After prolonged acetylation in refluxing diethyl ether with acetic anhydride containing
4-dimethylaminopyridine this
0-benzoylhydroxylamine with aqueous oxalic acid gave the monoformate which was hydrolysed to the diol by treatment with aqueous sodium bicarbonate. The a-diketone was then derived by oxidation of the diol in
toluene
solution
at
-30°C
with
N-chlorosuccinimide-dimethyl
sulphide followed by treatment with triethylamine. Ozonolysis of the diketone
in ethyl
acetate-methanol
at -78°C and
reduction
with
dimethyl sulphide resulted in the aldehyde from which with lead tetraacetate in benzene-methanol at 0°C racemic dimethyl betalamate was obtained. The final product in the characterised form of its dimethyl
ester semicarbazone was condensed under aqueous
conditions
with
racemic
0,0-diacetylcyclodopa
hydrochloride to give betanidine. 6.
no o
y
MeO-L
o
V-OMe
acidic ester
The route is depicted in Scheme
Scheme6
I
methyl
OH
MeO-L VoMe"^ ^o
744
NOCOPh
ix
^^^^^^
Meo-
1 ) _ > ' \ ^ \ ^ OHCOOH NOCOPh X
HO. ^v^Av^^ HO
41
O^^/k. "^
OAc
OAc NOCOPh
xii O^ OAc
OAc
NH
NNHCONHo
MeOsC MeOgC
xiii MeOgC"" ^ N ^ "COgMe
AcO.,^x%,^^
I MeOgC"
xiv
N-'^^COaMe H
HO.
AcO
MeOgC"
N
C02Me
HOoC
N
^COoH
Reagents: (i) Br2-MeOH, Na2C03, (ii) KMn04, (iii) H30% {iv)pH5, CO(CH2C02H)2; aq. BnNH3^Cl, (v) HC (OMe) 3, CH2CI2-CF3CO2H, A , (vi) Pd-C,H2, MeOH/CF3C02H, (vii) CH2=CHCH2MgBr, Et20-THF, (viii) (BzO)2/DMF, K2CO3, (ix)Ac20, Py., 4-DMAP; aq.(C02H)2, (x)aq. NaHCOj, (xi) NCS-Me2S, PhMe,30°C; NEt3 , (xii)03 , EtOAc-MeOH, -78°C, (xiii) Pb (OAc) 4 PhH-MeOH, 0°C/ NH2NHCONH2HCI, NaOAc , (xiv), (xv) as in Scheme 4. The total
synthesis of betanine does not yet seem to have
been
achieved and a sequence whereby this might be effected is shown in Scheme 7. 0-Benzoylvanillin would be asymmetricaly converted to L-p(4-0-benzoyl-3-methoxyphenyl)alanine methyl ester and thence to the 4-O-benzoyl-3-0-methyl
derivative
of
L-cyclodopa
methyl
Reaction with the semicarbazone of betalamic acid diester
ester. (perhaps
745 also N-protected) would afford the 5-0-benzoyl,6-inethyl ether of betanidine/isobetanidine dimethyl esters. Selective demethylation with aluminium triiodide and reaction with 2,3,4,6-tetra-O-acetyl-a(D) -glucosylbromide (BrAc4G) followed by mild hydrolysis/deprotection might be expected to lead to betanine/isobetanine. Scheme 7
»- rxn:'^- ixx)-MeO,^^^*,,,^
I
HO,
I >C02Me
5.3 Carotenoids The commercial development of synthetic routes to carotenoids of interest as food colourants must be renowned as one area where synthetic organic chemical methodology has proved of outstanding manufacturing
significance.
An
integrated
approach
based
on
acetylenic chemistry embraced the syntheses of vitamin A, the Cio perfumery
alcohols, linalol, geraniol/nerol,
certain
of
the C15
members for example, nerolidol as well as that of the permitted colourants p-carotene, canthaxanthin, p-8'-apocarotenal, and ethyl p-apo-8'-carotenate. These four colourants could be obtained in a (nature-identical)
pure
state
unlike
the
mixtures
normally
encountered with natural carotenoids and enabled the colour range from yellow to orange and red to be met (ref.43) . The first synthesis
746 of p-carotene methodology
(ref. 44) was also based on p-ionone and acetylenic but
was
not
developed
industrially.
Numerous
intermediates are involved in the synthetic array of the diversified acetylenic methodology. p-Ionone was a key intermediate in several diferent approaches. 5.3.1 p-Carotene The route from propanone to p-ionone is shown in Scheme 8, which depicts at the end, the classical route to pseudoionone citral (C)
(P) from
which was originally produced from lemon grass oil but
is now also derived from p-pinene by way of myrcene. In one of several ways to p-ionone, propanone affords by nucleophilic addition of the acetylide ion the acetylenic alcohol which can be selectively hydrogenated
in
the
presence
of
Lindlar
catalyst
(Pd-BaS04,
controlled by lead acetate) to the vinylic alcohol. Alternatively this compound is accessible by direct reaction of propanone with vinylmagnesium bromide in tetrahydrofuran. The acetoacetyl derivative results
from
reaction
with
diketene
and
rearrangement
of
the
resultant 'allylacetoacetate* with loss of carbon dioxide gives the Cg compound *methylheptenone' (a natural component of of oil of rue) . A repitition of acetylide addition, and of acetoacetylation followed by thermal rearrangement
affords pseudoionone
(?) by way of an
allenic intermediate. Cyclisation under selective conditions gives mainly p-ionone with some of the a-isomer. Scheme 8
I
i^ \y^
Ji
O
l/'^'iii {(""="
X- % ^
- v ^ -^ojj
^
^o Reagents: (i)CsC', solvent,KOH, {CH2CO) 2/solvent, (iv) A,
(c> (ii) Pd-BaS04, Pb(0Ac)2, H2, (iii)
747 BFg Et20, solvent. For the technical synthesis of p-carotene (11), p-ionone is converted to a Ci4 aldehyde by the Darzens reaction, thence first to a Cig and finally a C19 aldehyde two moles of which with a C2 reactant
affords
the required C40 structure. p-Ionone undergoes the Darzens reaction with ethyl chloracetate in the presence of sodium ethoxide to afford the p-hydroxy-a-chloroester and thence an a,p-epoxyester hydrolysis of which followed by acidification and decarboxylation gives a C14 a,p-unsaturated
aldehyde. Conversion to the diethyl acetal by the
action of ethyl orthoformate and reaction of this
with ethyl vinyl
ether in the presence of boron trifluoride-zinc chloride followed by hydrolysis and deethanolation gives a C^g a, P unsaturated aldehyde. Submission of the diethyl acetal of this aldehyde to chain extension with ethyl prop-l-enyl ether by a similar procedure as for the C14 compound results in a conjugated C^g aldehyde. Two moles of this react
with
acetylene
dimagnesium
bromide
to
give
a
C40 diol,
dehydration of which followed by Lindlar hydrogenation and final thermal isomeristion of the cis-product yields p-carotene
(11). It
is a red-brown crystalline air-sensitive material, m . p . 181°C, X^^^ 416 and 485nm.
The route is depicted in Scheme 9 .
Scheme 9
>OHJv^^^2Et
rU.
^o
*'-
OEt [
11
OEt
IV
r T
OEt OEt OEt
748
iW -i^ (ii) HO"; H3O"; (i) ClCH2C02Et, NaOEt, NH, BF3,ZnCl2, (v) CO2, {iii)HC(OEt) 4-TSA, (iv)CH2=CH0Et, HCl, (vi)HC(OEt)3,4-TSA/ MeCH=CHOEt, BF3, ZnCl2, (vii)HCl, (viii) C19 aldehyde, BrMgC=CMgBr, (ix) HCl, {x)H2 , Lindlar, (xi)A, (80^C) light petroleum.
Reagents:
Acetylenic routes do not represent the only approach to a p-carotene synthesis.
Thus
the
Wittig
reaction
with
the
employment
of
phosphonium salts has been technically exploited by BASF (ref. 45). Vitamin A acetate can be used for this purpose synthesised from a Grignard
reaction
of
p-ionone
with
vinylmagnesium
bromide,
isomerisation and formation of a C15 phosphonium salt. Reaction of the intermediate phosphoran with the C5 a,p-unsaturated
aldehyde, 3-
formylcrotyl acetate, then gave vitamin A acetate as shown in Scheme 10a. From this by hydrolysis, vitamin A is obtained which was then used to obtain the phosphonium
salt. This salt
(A) can undergo
autoxidation to yield p-carotene (ref. 46), but the specific use of hydrogen
peroxide
in
aqueous
solution
containing a base affords a higher yield
on
the
phosphonium
salt
(ref. 47) of p-carotene
through oxidative coupling of the phosphoran as depicted in Scheme 10b. The customary Wittig reaction involving the use of the isolated aldehyde is thereby avoided. Procedures have been developed for converting triphenylphosphine oxide to triphenylphosphine for reuse (ref. 45). The two stages are depicted in Schemes 10 (a and b ) . Scheme10 (a)
5^° 5rH 5r^::
P(Ph)3
OAc
P"^^^
749 (b)
Reagents: (a) (i) CH2=CHMgBr, THF(ii) PPh3.HCl,EtOH,RT, (iii) OHCC (Me) =CHO Ac, NaOMe, MeOPH, -15°C. (b) (i) H2O2, HO". There are of course many other approaches to the p-carotene structure (ref.48) such as one based on the combination of 2Ci6 and a Cg intermediate, another on the combination of 20^4 with a C12 component and also by the use of 20^^ reactants with a C^o dialdehyde. However these routes have not apparently been
developed technically as that
given in Scheme 9. 5.3.2 Canthaxanthin Canthaxanthin (15) which forms purple crystals, m.p. 218°C, X^^^AlOnm, was
the
last
of
the
carotenoids
to
be
brought
to
technical
production. A simple commercial approach to this compound has been based on the bromination of p-carotene with N-bromosuccinimide in acetic
acid
solution
containing
chloroform
(ref. 49)
to
give
isozeaxanthin diacetate. This was hydrolysed to the the diol which submitted to the Oppenauer oxidation afforded canthaxanthin in good yield as depicted in Scheme 11.
OAc
750 Reagents: (i) BrNiCOCHjls, AcOH, CHCI3, (ii) HO"; Al (OBu^ 3, Me2C0. A route to canthaxanthin using Wittig reaction methodology has been (ref. 50) . In this
described oxidised
in
dichloromethane
approach
with
retinol
manganese
(vitamin A)
dioxide
to
was
retinal
(vitamin A aldehyde) which was converted to 4-acetoxyretinal
by
bromination with N-bromosuccinimide in dichloromethane containing acetic
acid.
hydrogensulphate
By
reaction
derived by
retinol hydrogensulphate, Treatment
of
this
dehydroretrocarotene
with
retinyltriphenylphosphonium
reaction of
triphenylphosphine
isocryptoxanthin
with which
68%
hydrobromic
with
with
acetate was obtained. acid
formed
N-bromosuccinimide
in
chloroform/acetic acid afforded isozeaxanthin diacetate. Alkaline hydrolysis and Oppenauer oxidation with aluminium isopropoxide in propanone
then gave canthaxanthin
(15).
Scheme
12 depicts the
sequence. The Wittig reaction with the acetoxy derivative of the phosphonium salt could give isozeaxanthin diacetate directly. Schemee 12
OH
j.^^^^Y'^^'***''^^^
C2O
>0>»x^V'''vxk^,P
-^
VI
Vll
(Pha)
751 Reagents: (i) Mn02, CH2CI2, (ii) NBS, CHCI3/ACOH, (iii) L, NaOMe, PhH, (iv)CH2Cl2, HBr, -45°C; Na2C03/A , (v) NBS, CHCI3/ACOH; PhNEt2 / (vi)KOH, (vii)Al(0Pr^)3, Me2C0. Although invariably routes to carotenoids have been based on the use of p-ionone, in one early synthesis of canthaxanthin the cyclohexenyl ring
was
formed
from
an
acyclic
component
obtained
from
isobutyraldehyde (ref. 51) which provided a C4 chain and thence the Cg component necessary for the ring was derived in the following way. Acrylonitrile and isobutyraldehyde underwent Michael addition in the presence of the basic resin Amberlite IRA-400 (OH) and the product 4-cyano-2,2-dimethylbutanal nitric/sulphuric
acids
was both hydrolysed and oxidised with
to
afford
Esterification with methanol
2,2-dimethylglutaric
containing
acid.
sulphuric acid gave the
dimethyl ester which was then selectively semi-hydrolysed at the less sterically hindered ester group with methanolic potassium hydroxide to give the half
ester. The acid chloride produced under mild
conditions with thionyl chloride was then reacted in benzene with diethylcadmium to produce methyl 2^ 2~dimethyl-5-oxoheptanoate. After protection of the keto group by ketalisation with ethane-1,2-diol, the
ester
was
dimethylheptanoic
hydrolysed
to
give
5~ethylenedioxy-2,2-
acid which was converted with ethereal methyl
lithium to 6-ethylenedioxy-3,3-dimethyloctan-2-one
(K) . These stages
are shown in Scheme 13a Scheme 13a
i
iv
ii
V
COCl
^^
iii
^V"*^^ O
^
^
^^^ ^ ^ ' ' ^ ' ' ^ ^ 0 0
viii
Two moles of this ketone (K) were then condensed by Claisen reaction in 5% ethanolic potassium hydroxide with crocetindial
(ref.52) and
the terminal ketal groups in the product then removed by refluxing with
acetone
containing
4-toluenesulphonic
acid.
The
resultant
752 tetraketone was transformed into canthaxanthin (15) by refluxing in methanolic potassium hydroxide containing benzene. These steps are illustrated in Scheme 13b. Scheme 13b
(Vo.
•°(0
Reagents: (i)Amberlite IRA-400, OH, 50-55^C, ( i i ) 65%HN03, H2SO4,60°C, (111) MeOH, H2S04,A (iv)MeOH,KOH,20°C; Yi-^O^ (v)S0Cl2, 20°C, (vi)
PhH, Et2Cd, H3O" , (vii) MeCH2C (OCH2CH20)Me, 4-TSA,A (viii)5MNaOH,MeOH,A; ^^0^ , (ix)MeLi,Et2) , (x) Crocetindial, 5%KOH,EtOH, 20°C, (xi)4-TSA, MesCO, (xii) 10%KOH,MeOH,A. 5.3.3 p-Apo-8'-carotenal (p-Cgo-apocarotenal)
Many
routes
crystalline
to
this
C30 colourant,
substance, m.p.l39°C,
which
is
X^^y. 462
and
a
violet-coloured
488nm,
have
been
described. A technical pathway to P~apo-8'-carotenal (17/ X = H)(ref. 53) involves the use of the same C19 aldehyde as for the technical synthesis of p-carotene (Scheme 9 ) . Thus reaction of this compound with a Cg acetylenic acetal led to a C25 intermediate which was then submitted to firstly a C2 and then a C3 chain extension to give the dehydro C30 structure. The required Cg acetal was prepared from the monodiethylacetal of malonaldehyde which was first converted by deethanolation to the conjugated
enol
ether.
This was
reacted
with
acetylide
ion
in
anhydrous ammonia, followed by mild acidic dehydration and then diethylacetal Scheme 14a.
formation with ethyl orthoformate, as depicted in
753 OH OEt
OEt
OEt
OEt OEt OEt C6 The Ci9 conjugated aldehyde component was reacted with the lithium derivative of this Cg acetal in ammonia to afford an hydroxy compound which was dehydrated under mild acidic conditions to the fully conjugated C25 substance.
Chain extension with ethyl vinyl ether in
the presence of boron trifluoride-zinc chloride and mild acidic deethanolation gave the C27 acetal which was then converted with prop1-enyl ethyl ether under the same conditions to the required C30 structure
of
dehydro-p-apocarotenal.
Lindlar
partial
reduction
followed by isomerisation afforded the final product. The route is shown in Scheme 14b Scheme 14b
(17,X = H)
754 Reagents: (a) (i)NaC=CH, NH3 , (ii) HC(0Et)3, H3P04,4-TSA. (ii) 4-TSA, (iii)CH2=CH0Et, BF3,ZnCl2; HCl; (b) (i)Li, NH3 HC(OEt)3,4-TSA, {iv)MeC=CHOEt, BFs^ZnClz/HCl, (v) Lindlar, H2;A. Apart
from the technical route described
readily
available
vitamin
A
alcohol
to p-apo-8'-carotenal,
(C20)
has
served
as
an
intermediate in the form of the phosphonium salt by reaction with the monodiethyl
acetal
of
a
Cio dial
monodiethylacetal was obtained mono
aldehyde-protected
54) . The
(ref.
required Cio
(ref.5, p409) by the reaction of the
derivative,
the
enol
ether
of
methylmalonaldehyde, (C4) with the acetylenic Grignard reagent from trans
3-methyl-2-penten-4-yn-l-ol (Cg) followed by acidic dehydration
and partial reduction with Lindlar
catalyst to give
hydroxy-2,6-dimethylocta-2,4,6-triene-l-al
firstly 8-
(Cio). Protection of the
hydroxyl
group by acetylation
in pyridine
chloride
and formation of the diethyl acetal with ethyl orthoformate
solution with
acetyl
followed by hydrolysis of the acetyl group and oxidation afforded the final CIO aldehyde component (D)shown in Scheme 15a. The reverse approach, illustrated in Scheme 15b was also used (ref. 54, ref.247, p446) by employing vitamin A aldehyde
(A) and the
phosphonium salt formed from the Cio component (D) after its reduction to the allylic hydroxy monodiethylacetal. Scheme 15 OEt
Cl + o. P (Pha)
x^K,^.-'^^
OEt (D)
Cio
C30
t
Et
(Ph) 3P >
OEt
Br ClO
^ v / \ (Vitamin A aldehyde) Clearly a great number of combinations are possible, the viability of which is dependent on the ready availability of intermediates. Thus for example the use of vitamin A aldehyde by
reaction with a
C5 phosphonium component (ref. 5, p400) to afford a C25 compound and
755 then repetition of the process to give p-apo-8'-carotenal, has been described as depicted in Scheme 15c. Scheme 15c ci (Ph) 3P
^^--^
(A)
C20
(Ph)3P,
p-Cyclocitral, obtained in one procedure together with the a-isomer by the cyclisation in sulphuric acid of the Schiff's base of citral with aniline, by reduction with aluminium isopropoxide afforded pcyclogeraniol which was then converted to the triphenylphosphonium bromide with triphenylphosphine in dimethylformamide containing hydrogen chloride. Wittig reaction with the C20 comoponent, crocetin dial afforded another approach to p-apo-6'-carotenal, as depicted in Scheme 15d ci-
Although not used as a commercial colourant, crocetindial is important as an intermediate for several carotenoid syntheses and for obtaining bixin compounds. It was originally derived from 2,7dimethylsuberate (Cg dimethyl ester) by a lengthy process involving formation of 2,7-dimethylocta~2,4,6-triene-l, 8-dial and its reaction with two moles of a C5 phosphonium salt (ref. 55) . It can be obtained (ref.56) more easily from the Wittig reaction of the two
756 related Cio components as depicted in Scheme 16a. Both these C^o reactants were used for the synthesis of p-apo-S'-carotenal (Scheme 15a,b). Alternatively reaction of the bis diethyl acetal of the C^o dial can be used (Scheme 16b), firstly with vinyl ethyl ether (C2) to produce a C14 intermediate and secondly with prop-1-enyl ethyl ether (C3), (as in the methodology for the synthesis of p-carotene) (Scheme 9) followed by hydrolysis of the bis-acetal. Several other routes to the important C^Q dial and its bis diethylacetal have been listed (ref. 5, pp.431,433). Schemes 16a,b
CH(0Et)2
(EtO)2CH
OEt
CIO
5.3.4 Ethyl p-apo-8'-carotenate The ester, ethyl p-apo-8^-carotenate (17; X = OEt) corresponding to p-apo-8*-carotenal is an important colourant which has been referred to earlier. Technically it has been derived from the C27 compound, dehydro-p-C27-carotenal, an intermediate in the synthesis of p-apo8'-carotenal (Scheme 14b) by Wittig reaction with the phosphoran formed from the triphenylphosphonium salt of ethyl a-bromopropionate with sodium ethoxide (ref. 5,p459). The resultant dehydro compound was partially reduced with Lindlar catalyst and the product then themally isomerised to ethyl p-apo-8'-carotenate as shown in Scheme 17.
C27
757 COgEt
rPh3Ps^C02Etj
COgEt (17, X = OEt) Reagents: (i) HCl, (ii)NaOEt, (iii) Pd-BaS04, H2,Pb(OAc)2/A. 5.3.5 Citranaxathin In addition to p-carotene, canthaxanthin, p-apo-8'-carotenal ethyl p-apo-8'-carotenate, the ketone citranaxanthin occurs in certain citrus species,
has been cited
and
(C33), which
(ref. 57) as a
compound produced on a manufacturing scale. It is obtained by the condensation
of
C30,
p-apo-8'-carotenal
with
propanone
in
the
presence of aqueous potassium hydroxide (Scheme 18) . Its use does not appear to be applicable to the UK. Scheme 18
5.3.6 Capsanthin and Capsorubin Capsanthin
(16) and capsorubin
{16a), the
colourants in paprika
oleoresin, although not produced by commercial synthesis have been prepared in the course of carotenoid studies (ref. 58). Capsanthin has
been
synthesised
from
p-citraurin
('3-hydroxy-p-apo-8'-
carotenal') which is available from zeaxanthin (3R,3RM"P-carotene3,3'-diol), by oxidation with potassium permanganate (ref. 59). The required cyclopentane component was derived
(ref. 60) by the
sequence of reactions depicted in Scheme 19. The diketo ester shown is available from the cyclisation with sodium ethoxide of the Michael adduct of diethylmethylmalonate with mesityl oxide. The less hindered
758 keto group was first ketalised and the remaining keto group and the ethoxycarbonyl group then reduced with lihiuiti aluminium hydride. The product was
dehydrated and deketalised under acidic conditions to
the unsaturated keto alcohol. Catalytic reduction of the ring double bond
and oxidation of the methylol group afforded the keto acid,
autoxidation of which gave the diosphenol (1,2-diketo structure) .This underwent
ring
addition
of
contraction phosphoric
by
alkaline
acid/sodium
trimethylcyclopentanone-4-carboxylic reduction produced
the
alcohols. The racemic
racemic trans
els
treatment, bismuthite,
acid.Sodium (X) and
compound
trans
followed to
by
3,3,4-
borohydride (Y) epimeric
(Y) was then converted by
methyllithium to the hydroxy ketone. From Claisen condensation of two moles of the hydroxy ketone with crocetindial, racemic capsorubin was obtained which was chromatographically (TLC) similar to the natural product (Scheme 19a). The cis-alcohol afforded a racemic epimer Scheme 19a
CO2H
Reagents:
(i) EtOH, 4-TSA;LiAlH4; HsO",
(ii) Pd-C,H2;Cr03 , (iii)02.
759 KOBu% ( i v ) 5 % KOH; c r o c e t i n d i a l , 5%K0H,
H3PO4, EtOH
(v)NaBH4
NaBi02,
(vi) MeLi,
(vii)
With p-citraurin from natural sources or obtainable semisyntheticallly by the oxidation of zeaxanthin with permanganate, the same racemic hydroxy ketone derived from (Y) afforded by Claisen condensation, capsanthin as a mixture of diastereoisomers (ref. 61). The process is shown in Scheme 19b. Both capsanthin and capsorubin have yet to be synthesised in optically active form. Scheme 19b
Capsamthin 16
5.3.7 Norbixin derivatives Bixin, present in Annatto, is an important natural colourant which is obtained commercially from Bixa orellana. It is unusual in not having a typical isoprenoid structure composed of multiples of the fundamental C5 unit. All trans diethyl norbixin has been synthesised readily from crocetindial (ref.55,p221) by the Wittig reaction with two moles of the phosphoran from the triphenylphosphonium salt of ethyl chloracetate as shown in Scheme 20. The natural product is the form in which the colourant is used. Scheme 20 Et02C'''''*^Cl i ^
EtOoC'"^^ P'^PhoCl"
EtOoC
O
PPh,
C20
COgEt
EtOgC C24
760 Reagents: (i) PPhj , (ii) NaOEt, (iii) condn. The technical syntheses described for p-carotene, canthaxanthin, papo-8'-carotenal and the corresponding ethyl ester, represent only a small part of the synthetic work carried out (ref. 5) by the Swiss group and others. Some of the many methodologies for the construction of carotenoids have been discussed
(ref. 57). The range of colour
of aqueous suspensions/solutions varies from a greenish yellow to a deep bluish red. Typically
p-carotene gives a yellow, p-apo-8*-
carotenal an orange and canthaxanthin a tomato-red colour. Conformational studies by a number of techniques have established (ref.62)that the ring double bond in carotenoids is clsoid respect to the exterior linear unsaturated chain
with transold,
and not
5.4 Chlorophylls Chlorophylls {E140) have been described as dark green greasy (lipidic) powders and are only used to a minor extent. They have been largely replaced by copper chlorophyllin which is obtained by semisynthesis.
The
biochemical
colour
interest
chemistry
in
of
haemoglobin,
the
phthalocyanines
and
and
in
had
vitamin
B12
concentrated attention on the remarkable properties exhibited by five-membered and related heterocyclic structures having centrally a
metal
in
synthetic
chelation.
studies
on
the
structural
chlorophyll,
However
following
elucidation early
and
work
by
Willstatter, occupied Fischer and coworkers for three decades (ref. 63)
culminating in the synthesis of inactive chlorphyll a
successors
(ref.
accomplished
64)
work
preparation
which
of
achievement was overshadowed
had
been
phaeporphyrin
dependent d^
by his on
(ref.65).
the This
by the simultaneous publication of the
total synthesis of the optically active form of chlorophyll a (ref. 66) . Full experimental details of this landmark in synthesis were not published until 1990 (ref.. 66) more than ten years after the death of the originator. The absolute configuration of chlorophyll a was established (ref. 67) as 7S,8S,lOR,7»R,ll'R through a correlation of a
degradation
product
from
ring
C
of
phaephorbide
a
with
a
transformation product of the terpenoid lactone (-)-a-santonin The
construction
of
the
chlorophyll
a
molecule
availability of four different pyrroles. A, B, C,
required
the
and D, all of
which were obtained from 2,4-di(ethoxycarbonyl)-3,5-dimethylpyrrole synthesised originally by Knorr (ref. 68) . This had been prepared historically from ethyl acetoacetate by conversion to the nitroso
761 derivative, the reduction of this to the corresponding amino derivative and its condensation with ethyl acetoacetate. The formulae of these four pyrroles are depicted and the route to the fundamental intermediate, 'Knorr's pyrrole*.
>i°^" ^.
NC
>
H02C-(^/-C02H
(C)
(B)
(A)
EtOsC
CHO
NH
NH
(D)
COjBt co,
--^ EtOgC—( (i)HN02,(ii)Zn
2 NHo
COgEt EtOgC-
o^_^ AcOH,NaOAc
NH 'Knorr's pyrrole'
The synthetic pathways leading to these four structural building molecules A, B, C, and D are depicted in Scheme 21. Scheme 21 .COgEt
Et02c4 V ^
CO2H
"2304 Et02c4 V
NH
^
Et
COMe
AcCl ^1^13 EtOsC v^rn.rJ
V -
EtOgC-
NH (J)
N2H4
r \ Ni
Vilsmeier
K
^COgEt EtOpC
i
y_
jR
\ Et \ — v ' CH2(CN)2
'^OgEt
Br2_^2Cl2
^NH
CO2H
NH
C02Et
J-y
EtOgC-^^^
Aq_^tOH, K5k
HO2C
II \ NE
(B)
762
Scheme 21{contd.) CHO (J)
—^
Et02C-<
>•
Vilsmeier
EtOgC-
NH
(I) CH2(CN)2
i.
CH=C(CN)2
\
^
EtOgC
Ah V-<
^
y
rH-r/rM\
CH-C (CN) g HQ-
\
4>c
EtOjC-C, >-C02Me ^ NH
CO2H
CH2(C02H)2 \
(I)
,
^ Br^eOH
^
H2^i \ _ / ^
CO2H SO2CI2
(D)
CO2H \ y - ^
EtO,C-4 > NH
COgMe NaOH^heat
vilsmeier
;CH2N2
NH
COgMe
^COaMe
("VcHO NH
+
OHC-<> NH
(C)
The strategy of the approach to the synthesis of chlorophyll a was to react A and B to form one dipyrrylmethane {A-CH2-B) and C with D to form another
{C-CH2-D) . The A-CH2-B component was then reacted
with the C-CH2-D molecule through formation of a flexible methylenic link to a Schiff^s base group enabling neighbouring groups to react. The * square* structure of a hydroporphyrin was thus derived from the electrophilic reaction of a protonated carbonyl group at the aposition of the 'B pyrrole' with the a-position of the 'pyrrole
C
and of the protonated Schiff's base at the a-position of 'pyrrole A' with the a-position of 'pyrole D'. The sequences leading to A-CH2-B and to C-CH2-D are depicted in
the
Schemes 22a and 22b.
Formation of Component A-CHg-B (X) : Pyrrole A, 2-(p,p-dicyanovinyl)-3,5-dimethyl-4-ethylpyrrole, 69) , was converted with sulphuryl chloride to the
(ref.
5-chloromethyl
derivative and then reacted in aqueous ethanolic hydrochloric acid with
pyrrole
afford
B
(3-ethoxycarbonyl-4-methylpyrrole),
(ref.70),
to
3',4-dimethyl-3-ethyl-4'-ethoxycarbonyl-5-(P,p-dicyanovinyl)-
dipyrrylmethane which with 3-methoxycarbonylpropionyl chloride gave 3', 4-dimethyl-3-ethyl-4 ' -ethoxycarbonyl-5- (P, p-dicyanovinyl) -5' - {3-
763 methoxycarbonylpropionyl)-dipyrrylmethane. This was hydrolysed with concentrated diazomethane
aqueous to
yield
alkali,
and
the
acid
methylated
formyl-5'-(3-methoxycarbonylpropionyl)-dipyrrylmethane. condensation
with
with
3 \ 4--dimethyl-3-ethyl-4'-methoxycarbonyl-5
ethylamine, the ethylformimino
derivative
By was
obtained, the hydrobromide of which with hydrogen sulphide in the presence of sodium methoxide gave the thioaldehyde (A-CHj-B) (X) . Scheme 22a
(X) COgMe COgMe
:02Me
lOgMe
^OgMe
A-CH2-B
Reagents: (i) S02Cl2,AcOH, 550C;B, Aq.HCl, EtOH, (ii)Me02C {CH2) 2COCI, ZnCl2 CH2CI2, (iii) Aq.NaOH;CH2N2, (iv) EtNH2, AcOH; HBr;H2S, PhH^MeOH^MeO". Formation of Component C-CH2-D: Pyrrole D, 2,5-Dicarboxy-3-methyl-4-formylpyrrole (ref. 71) was reacted with nitromethane in the presence of diethylamine to give 2,5-Dicarboxy-3-methyl-4-{2-nitrovinyl)pyrrole,
reduction
of
the
external double bond of which with aqueous sodium borohydride gave the
corresponding
2-nitroethyl
derivative.
Decarboxylation
and
hydrogenation of the nitro compound in methanol in the presence of Adams* catalyst afforded 3-methyl-4- (2-aminoethyl) -pyrrole which with pyrrole C (2-formyl-3-methyl-4-(2-methoxycarbonylethyl)-pyrrole (ref 72)
in methanolic hydrogen bromide resulted in 3,3'-dimethyl-4-(2-
aminoethyl-4'- (2-methoxycarbonylethyl) -dipyrromethene dihydrobromide. Reduction of the methene with aqueous sodium borohydride gave the corresponding highly sensitive dipyrrylmethane forthwith reacted with the thioformyl
compound
(C-CH2-D)which was (X) to give the
764 Schiffs base (H) (shown in Scheme 23) Scheme 22b •^N02
CHO . A . CO2H _v
X^NHg
^V^COsH
vi
H02C
OHC
(H) MeOsC
Reagents: (v)MeN02,EtOH,Et2NH, A; NaBH4,aq, NaHCOs, (vi)NaOAc,KOAc, 120°C; PtO2,H2, MeOH,(vii)D, MeOH,HBr, (viii)Aq.NaBH4, (ix)X,CH2Cl2, Reaction of A-CHg-B with C-CHg-D The key step of double ring closure by treatment of the readily formed
Schiffs
base compound
(H) with
12M methanolic
hydrogen
chloride gave the hydroporphyrin salt (I) oxidation of which with iodine followed by acetylation with acetic anhydride/pyridine gave the porphyrin (J)in 50% overall yield from (X). Aereal oxidation in acetic acid afforded (K) which upon equilibration in the same solvent led
to
the purpurin
Hydrolysis
of
the
(L) having
acetyl
a semi-saturated
compound
chloride, exhaustive methylation
with
M
pyrrole
methanolic
ring.
hydrogen
and Hofmann elimination
led to
formation of the vinyl compound (M). By oxidation with air in the presence of light, cleavage occurred of the bond indicated resulting in the purpurin aldehyde (N) which underwent with dilute methanolic potassium hydroxide reverse condensation leading to loss of the oxalyl
ester
group
and
cyclisation
of
the
formyl
and
the
6-
methoxycarbonyl groups to give (racemic) isopurpurin 5 methyl ester (0) which was spectrally identical with a sample from a natural source
(ref.73).
Hydrolysis
with
very
dilute
sodium
hydroxide
afforded chlorin 5 (P) which was resolved with quinine to give the
765
(+) salt from which (+)-chlorin 5 was obtained. Scheme 23
MeOgC
COgMe (K) \
(L)
COsMe
Me02C
Me02C (• Purpurin')
( • Porphyrin')
COgMe (M)
\ COsMe
\ COgMe
(N)
MeOgC
:H0C02Me CO ) COoMe Me02C
(P) HO2C Reaction of (P) with diazomethane then produced active purpurin 5
766 dimethyl ester, (Q) brief reaction of which with hydrogen cyanide in dichloromethane containing triethylamine resulted in the cyanolactone (R) . This
was
treated
with
zinc
and
acetic
acid
followed
by
diazomethane to produce chlorin eg dimethyl ester nitrile (S) which was
hydrolysed
under
mild
conditions
with
chloride to give chlorin eg trimethyl ester
methanolic
hydrogen
(T)identical with a
natural sample. Scheme 23(contd.)
xviii
ff
HO2C
^yj
QT^O^
(2 6)Chlorophvll a
The conversion
to phaeophorbide a (U) and thence by esterification
with phytol to afford first phaeophytin, lacking the magnesium atom, then to chlorophyll a (26) had been described (ref 74).
767 Reagents: (x) 12MHCl,MeOH, (xi) I2; ACjO/py., (xii) 02,AcOH, warm, (xiii) 02,hi), AcOH,N2,110°C, (xiv)MHCl,MeOH,hot;Me2S04,MeOH,NaOH, (xv) (xvi)ciil.MeOH,KOH, (xvii) M/25 NaOH,H20. dioxan; quinine, (xviii) { + )salt,H30% CH2N2, (xix)HCN, CH2Cl2,Et3N, (xx) Zn, AcOH; CH2CI2 ,(xxi)MeOH,HCl,ambient,(xxii)Dieckmann,(xxiii)phytol; Mg^\ Optically active phytol has been synthesised methodology
is
depicted
in
Scheme
24. In
(ref. 75) and the
this
route
(R)-(+)-
citronellol was converted to 4(R),8-dimethylnonanoic acid which was then
anodically
methylglutarate
cross-coupled to
give
with
methyl
hydrogen
3(R),7(R),11-trimethyldodecanoic
3(R) acid.
Similar cross-coupling with levulinic acid afforded 6(R),10(R),14trimethylpentadecan-2-one and reaction with methoxyacetylene followed by
acidic
treatment
gave
a
mixture
of
cis
and
trans
phytenoates which were separated. Reduction of the trans
methyl
ester with
lithium aluminium hydride resulted in phytol identical with the natural product. Scheme 24
CO2H
COgMe HO2C
VA„ COgMe
Reagents: (i) Pd-C,H2;PBr3;CN";H30% (ii) 'O2CCH2CH (Me) CH2C02Me,-e, (iii) -02CCH2CH2COMe, -e, (iv)MeOC2C-/H30% (v)LiAlH4. The phytyl ester at C7 in chlorophyll can be removed enzymically by chlorophyllase
to
afford
phaeophytin,
or
chemically,
without
affecting the methoxycarbonyl group at CIO under mild acidic or alkaline conditions in an inert atmosphere. In acidic treatment removal of the magnesium atom occurs, thus the semi-synthesis of
768 sodium copper chlorophyllin
(29) requires, in the final step, the
insertion of the copper(II) atom and neutralisation. Chlorophyll b does not appear to have been synthesised. 5.5 Cochineal The colourant principle of cochineal is carminic acid (30) and although carminic acid was
first obtained as a red
crystalline
substance in 1858 (ref.76) having been in use as a dye for several centuries, its structure was not fully elucidated for many years until the side-chain at C7 was found to be linked as a C-glucoside (ref. 77) and the carboxyl group was relocated from the 4- to the 2(ref.78).
position
The
p-configuration
of
the
glucoside
was
established later as the final refinement (ref.79). Carminic acid, (30) (7p-D-glucopyranosyl-l-methyl-3,5,6,8-tetrahydroxy-9,10-anthraquinone-2-carboxylic acid) has been synthesised (ref. 80)although it is unlikely that the route will challenge the traditional extraction process which has been operated for very many years. The cochineal carmine of commerce is a hydrated calcium complex of carminic acid useful
for
its high
colour
yield,
pigmentation
properties
and
photostability. The aglycone of carminic acid is kermesic acid itself an ancient dyestuff, obtained from a European source, Kermes
illiciSf
which was gradually displaced historically by carminic acid imported from Central and South America. by two routes
Kermesic acid has been synthesised
(ref. 81, 82) and together with isokermesic acid
(3,5,7,8-tetrahydroxy-l-methyl-9,lO-anthraquinone-2-carboxylic
acid)
by a non-regiospecific method (ref .83) . This procedure and others has recently been reexamined (ref.84). Me
O
OH (Kermesic acid)
Due
to
the
synthesis
of
spiraling carminic
price acid
of
cochineal
appeared
to be
in
the mid
a viable
eighties, commercial
proposition. This seemed at first to be readily soluble simply by the glucosylation of kermesic acid,
a process conceived to be the
probable biosynthetic pathway. However, numerous studies of this potential route had no useful outcome (ref. 85) . Nevertheless in view of the
failure of
several
routes
from different
retrosynthetic
769 schemes it appeared probable from theoretical considerations that a related structure, 6-deoxykermesic acid, could be selectively glycosylated at the 7-position to provide a route to 6-deoxycarminic acid whence carminic acid itself had been derived earlier (ref. 86). This total pathway proved to be feasible and 6-deoxykermesic acid was synthesised by the Diels-Alder addition reaction of 2chloronaphthazarin (E) with E/Z-3-alkoxycarbonyl-2,4bis(trimethylsilyl)-penta-1,3-dienes (D). Alkyl acetoacetates (R^ = Me,Et,Bn) were acetylated to produce the alkyl dialkyl acetoacetates(ref. 87) which were converted to bis(trimethylsilyl) derivatives (D) as depicted in Scheme 25a. 2-Chloronaphthazarin (E, X = CI) was prepared from 1,5-dinitronaphthalene by treatment in fuming sulphuric acid with sulphuric acid resulting first in the formation of naphthazarin (ref. 88) which was then dichlorinated and the product dehydrochlorinated (ref. 89) (Scheme 25b). An alternative route was from the acylation of 2-chlorohydroquinone with maleic anhydride in a sodium/aluminium chloride melt (ref.90)(Scheme 25c). 2-Bromonaphthazarin (E, X = Br) was obtained from the acylation of hydroquinone with 2-bromomaleic anhydride under similar conditions. 2-Halogenonaphthazarins can be equally represented 2-halogeno-l,4dihydroxynaphth-5,8-quinones or 2-halogeno-5,8-dihydroxynaphth-l,4quinones. Scheme 25a,b,c
(a)
X.CO.R. v "°^Y^° ii o
OH
O
OH
'"°'';xf'°™'o, TMSO
•" ^ f 1 ^ CO O
3.3.3.
OH
OH
OH
(E)
on
O
OH
::w
6
^
770 Reagents: (a)(i)Mg,AcCOCl, PhH.A; NaHCO, , (ii) Method 1, N,0-bisMe3SiMeCONH2, Et20;Method 2,Et3N, TMSCl.PhH. {b){i)H2S04, SO3,S,60°C, {ii)Cl2,AcOH, (iii) EtOH, A, (iv) AlCls^NaCl, 180°C. By
the
Diels-Alder
reaction
of
3-alkoxycarbonyl-2,4-
bis(trimethylsilyl)-1, 4-pentadienes (D) with 2-halogenonaphthazarins (E)
in hot toluene
followed by desilylation, the methyl,ethyl and
benzyl esters (F) respectively were formed in high yield by addition at the chlorine-containing ring. Methylation with dimethyl sulphate afforded the trimethyl ethers (G) reductive methylation of which (by sodium dithionite pentamethyl
followed by methylation)
ethers
(H)
91) .
(ref.
gave the
3,5,8,9,10-
Glucosylation
occurred
regiospecifically at the 7-position with 2,3,4,6-tetra-O-benzyl-a-ltrifluoroacetyl-D-glucopyranose in dichloromethane containing boron trifluoride etherate to afford, for example with the benzyl ester of (H) , the p-glycoside (I, R^ = Bn) . Oxidation of this with pyridinium chlorochromate
then
selectively to give
restored
the
9,10-anthraquinone
structure
(J, R^ = Bn) . Hydrogenolysis with palladium-
carbon in THF containing hydrochloric acid gave the parent glycosidic acid
(K)
containing
which
by
acetylation
with
4-dimetylaminopyridine
acetic
anhydride/pyridine
afforded the tetraacetate
(L) .
Demethylation of the trimethyl ether with boron tribromide gave the trihydroxy compound (M), and acetylation produced the heptaacetate (N)
which
was
identical
with
natural
6-deoxycarminic
acid
heptaacetate prepared from carminic acid. Hydrolysis resulted in 6deoxycarminic acid (0), acetylation of which under relatively mild conditions
to
preserve
the
3 , 2 \ 3',4',6*-pentaacetoxy tetraacetate
resulted
in
5,8-dihydroxy
compound the
(P) .
sensitive
system
Oxidation
bis-quinone
gave
the
with
lead
(Q) , which
underwent Thiele acetoxylation to give the octaacetate (R) identical with
natural
carminic
acid
octaacetate,m.p.189-90°C
.
Acidic
hydrolysis with ethanolic hydrochloric acid then produced carminic acid (30). The route is depicted in Scheme 26. Scheme 26
o
'-* xJk VA0 (E)
0
OH
000 Me
OH
R^OoC^ ' - H O ^
r ^
(F)
0
^
OH
/ ^
II
OH
R^OgC
0
OMe
(G) 0
OMe
Me
-^V-NA^
COV
MeO-^
771
Scheme 26
(contd.
Me
Me
OMe OMe
OMe OMe
jOBn
R^O
,OBn
iv
iii
MeO'"^-s^'''^N|^^^ C^eOMe
OMe OMe
OBn
(H) RiOgC
Me o ™= o
OMe owe
pBn .OBn
HOgC
OMe
O (J) HOgC
Me ra«
o O
Me Me
OMe UMe
OH
oo
PAc .OAc
(M)
(L) HOgC
«Me e
o O
OAc OAc
.OAc
Me
o
OH
Me Me oo
^
^ T
nr
Me
9^
Y"
If
T \ , A
O
OH
o
O
^--^\
/xi PAc ,OAc
AcO'X.AyA^^ *
^OAc _^
OH
OAc
^ i
(P)
xiii
(Q) Me
o
Me ™
OAc
HOgC AcO
AcO'\^«!*^^\jX'^^
o O
OH Ott
^.OH -OH
bAc
."•'HO'''^^^^**^^!^''^^
OH
Carminic acid (30)
R e a g e n t s : ( i ) P h M e . IIO^'C; aq.THF, (ii)Me2S04, Me2CO,K2C03, ( i i i ) Bu4N^Br, Na2S204,HO"; Me2S04, ( i v ) s t e p l , (CF3CO) 2O, 2 , 3 , 4, 6 - t e t r a - O - B n (D) g l u c o s e ; s t e p 2 , H , CH2Cl2/BF3.Et20,reagent from s t e p l , (v) PCC,CH2Cl2/ ( v i ) P d - C , H 2 , THE, HCl, (vii)AC20, p y . , DMAP, ( v i i i ) B B r 3 , CH2CI2, ( i x ) AC2O, H2SO4 ( c a t . ) , (x)MeOH, HCl, A, (xi)Ac20, lOO^'C, ( x i i ) AC2O, Pb(0Ac)4, ( x i i i ) H2SO4, ( x i v ) EtOH, HCl.
772 5.6 Curcumin The turmeric root is the underground stem or rhizome of Curcuma longdr
the active colourant principle of which is curcumin (31), an
orange crystalline compound, m.p. 183°C . The compound was obtained crystalline and the structure was elucidated by a number of separate studies (refs. 92,93,94,95). An early history of its chemistry has been described
(ref.96). A number of investigators attempted to
synthesise
parent
the
compound,
dicinnamoylmethane.
Thus
the
reaction of ethyl cinnamate with cinnamoylacetone was unsuccessful although this ester reacts readily with acetone (ref. 97) to give the latter. However, the parent compound was eventually prepared by the alkylation
of
sodio
cinnamoylacetone,
obtained
from
ethyl
cinnamoylacetoacetate (ref. 98), with cinnamoyl chloride (ref. 99). By the use of appropriately substituted reactants and the employment of the methoxycarbonyl group for phenolic group protection, this methodology was then applied to the synthesis of curcumin itself successfully (ref.100) from vanillin by firstly its conversion to 4hydroxy-3-methoxycinnamic
acid. The 4-hydroxy
group in this was
protected by reaction with ethyl chloroformate and the acid chloride of
the
product
then
obtained.
methoxycarbonyloxycinnamoyl
The
chloride
resultant
3-methoxy-4-
(P) reacted with
the
sodio
derivative of ethylacetoacetate prepared in diethyl ether from sodium ethoxide,to
afford ethyl
3-methoxy-4-methoxycarbonyloxycinnamoyl-
acetoacetate. Mild 'ketonic' hydrolysis with decarboxylation gave 3methoxy-4-methoxycarbonyloxycinnamoylacetone
which by reaction in
anisole
with
containing
sodium
methoxycarbonyloxycinnamoyl
sand
chloride
(P) produced
3-methoxy-43,3'-dimethoxy-
4, 4 '-di (methoxycarbonyloxy) cinnamoylmethane. Mild hydrolysis with dilute potassium hydroxide followed by precipitation with carbon dioxide
gave
3-methoxy-4-hydroxycinnamoylmethane,
which
exists
predomonantly in the H-bonded form (31) as shown in Scheme 27. Scheme 27
MeCCoV OMe
M e O , C o V '"' oMe
MeO.CoV OMe
773 0 .^sw Ill
MeOgCO
.x^ J L
0
O
O
J^ .^^f^''^.^'^'*''^:^^^^^^^^^.^^
^^^^^•''^^X^^N.X^ ^ ^
f^
i-V
L._
Y^
^"^
^'^
^"^
O
I
OMe
AM^ OMe
OMe
H
OCOgMe
Q
OMe
OMe
(31) OMe
OMe
R e a g e n t s : ( i ) S 0 C l 2 , ( i i ) AcCH2C02Et, NaOEt, Et20 , ( i i i ) d i . NaOH, -CO2 , (iv)PhOMe, Na s a n d , 3-MeO-4-MeOC02C6H3CH=CHCOCl ; H,0% (v) 2M KOH, 50°C; CO2.
5.7 Riboflavin Riboflavin or vitamin B2 is obtained entirely by synthesis and its usage and role is predominantly as a vitamin rather than as a permitted colourant. The synthesis by Karrer was later improved by Tischler and proceeds from 3,4~xylidine (available through the nitration of o-xylene and reduction of the product), the Schiff^s base of which with D-ribose is catalytically reduced. Benzenediazonium chloride is coupled with the hydrogenation product and the resultant azo compound is then condensed with barbituric acid in acetic acid (with loss of aniline) to give the final product. The process is illustrated in Scheme 28. Scheme 28
CH2 [CH (OH) ] 3CH2OH
I
I
I
(I
CH2 [CH (OH) ] 3CH2OH
Riboflavin (32, R « H)
O^^^^^N-^V-^e CH2CCH(OH)]3CH20H
774 5.8 Sandalwood Red sandalwood Pterocarpus
santalinus
has attracted interest
as a colourant and contains the colourless components, pterostilbene (34), pterocarpin (35) and homopterocarpin (36) the occurrence of which (ref. 101) and the chemistry (ref. 102) have been reviewed. The nature
of
the
possible
involvement
of
these
compounds
in
the
development of colour in sandalwood is not known although quinone and quinone dimer structures seem highly probable. Pterostilbene is the dimethyl ether of resveratrole, a natural widely-occurring
phenolic
component of many plants including grapes, mulberries and peanuts. The
anticancer
properties
(ref.
103)
and beneficial
effect
on
coronary heart disease of this have been intensively studied recently (ref 104). The structures of pterocarpin and of homopterocarpin were elucidated (ref.105) and the synthesis
described of pterostilbene (ref.106) by
the reaction of 3,5-dimethoxybenzaldehyde with 4-hydroxyphenylacetic acid in hot acetic anhydride, followed by hydrolysis of the acetyl derivative formed and decarboxylation in quinoline containing copper as depicted in Scheme 29. Scheme 29 MeO
MeO
MeO
MeO
HO2C
A—\
(Pterostilbene)
MeO Reagents: (i) AC2O, A, ;H0' , (ii) Cu, quinoline, A . Racemic chalcone
homopterocarpin has been obtained
by
the
synthesised
(ref. 107)
condensation
of
from a
2-hydroxy-4-
methoxyacetophenone with 2, 4-dibenzyloxybenzaldehyde. Conversion to an
isoflavone
with
thallium
(III)
nitrate
was
followed
by
hydrogenation to generate the racemic furanochroman (C)in low yield together
with
two
other
products
(A) and
(B). Chromatographic
separation of (C) and methylation afforded the final product as shown in Scheme 30.
775
Scheme 30
x^
MeO.
Bn0^^x%^^OBn
txpo MeO,
MeO. MeO. OBn HO^Xx-^OH MeO.
MeO.
(Homopterocaurpin)
OMe
R e a g e n t s : ( i ) EtOH, HO" s e p n . , ( i v ) M e l , K2CO3.
(ii) T1(N03)3, MeOH, (iii) Pd-C, H2; SiO,.
Racemic pterocarpin was synthesised (ref. 108) and later by other improved methods (ref. 109, 110). In the first of these latter methods (ref. 109) 2-benzyloxy-4,5-methylenedioxyphenylacetaldehyde was converted with pyrrolidine to an enamine which underwent reaction with 2-acetoxy-4-methoxybenzoyl chloride. Cyclisation led to an isoflavone acidic treatment of which followed by borohydride reduction and ring closure effected formation of the required pterocarpin as depicted in Scheme 31. Scheme 31
<xi::.r <::^^ ^-...^x'^^OBn
'
-
"
^
o
OMe
776
Reagents: (i) HN{CH2)4, A, (ii) (iii) 2-OAc-4-MeOC6H3COCl,A, (ivHCl, NaBH4 , AcOH A remarkably simple approach to racemic pterocarpin is indicated by a
two-step
readily
synthesis
formed
by
(ref.llO)
in which
dehydration
methoxychroman-4-one,
was
of
the
reacted
in
7-methoxy-2H-chromen,
reduction the
product
presence
of
of
7-
lithium
chloropalladite with 2-chloromercuri-4,5-methylenedioxyphenol. This reactant
was
itself
obtained
from
3,4-methylenedioxyphenol
and
mercuric acetate followed by treatment with sodium chloride. The process is illustrated in Scheme 32. Scheme 32
"-"X^O ^ZO:} ^ Reagents: (i) Li2PdCl4 , MeCN; SiOs sepn. 5. 9 Miscellaneous conqpounds Like sandalwood, brazilwood Caesalpinia
braziliensis
has had
a long history of use for dyeing purposes and although as with logwood {haematoxylon of colourants colourant
principle
haematoxylin.
campechianum),
it is not in the permitted range
it has an association with food colourants. The Both
of brazilwood structures
are
is brazilin closely
and
related
of to
logwood, those
of
pterocarpin and homopterocarpin and in the fundamental structural unit a -CH2_ simply replaces the - 0 - of the furan ring and a thydroxyl group is present at the cis
bridgehead at the 3-position of
the chroman ring. The structure of these two compounds has been discussed at length (ref.29,96) and although the mode of synthesis
777 of brazilin was apparent
from early work
(ref. Ill, 112), the
completion of this was achieved much later (ref. 113).
.OH
^ HO
OH
HO
Brazilin
In
the
total
Haematoxylin
synthetic
methoxychromanone,
OH
route
obtained
(ref. from
113)
the
to
(+)-brazilin,
cyclisation
of
7-
3-(3-
methoxyphenoxy)propionic acid with 80% sulphuric acid in 60% yield, was
condensed
with
3,4-dihydroxybenzaldehyde
containing hydrogen chloride to give the
in
acetic
acid
3,5-dihydroxybenzylidene
compound(J). 3-(3, 4-Dihydroxybenzyl)-7-methoxychromanone was obtained from this by catalytic hydrogenation in ethanol with palladium. Methylation in potassium hydroxide solution with dimethyl sulphate followed by cyclisation of the trimethyl ether through
dehydration
in hot benzene containing phosphorus pentoxide afforded a low yield of
deoxytrimethylbrazilone
(B) a
known
transformation
product
obtainable from natural brazilin. Catalytic hydrogenation of (B) in acetone
solution
containing
Pd-SrCOj gave
trimethylbrazilane,
demethylation of which in acetic acid with boiling hydrobromic acid followed by acetylation of the product gave 0-triacetylbrazilane. Upon oxidation in aqueous acetic acid with chromium trioxide at 50°C, the diketone, 0-triacetylbrazilone resulted which was reduced to the pinacol with zinc dust
in ethanol solution containing acetic acid.
This compound was hydrolysed in aqueous ethanolic potassium hydroxide and upon acidification with acetic acid was converted by dehydration to
brazilein
which
in warm
methanol
borohydride was transformed to brazilin
by
addition
of
potassium
(R) . This was resolved by
acylation in pyridine solution with 1-menthoxyacetyl chloride and the separated
(-)-diastereoisomer
hydrolysed
with
aqueous
ethanolic
potasium hydroxide to give (+)-brazilin identical with the natural product. The
synthesis is depicted in Scheme 33.
778 Scheme 33
rxX) ^
MeO.
MeO.
"CO2H
MeO,
MeO.
(B) OMe
-0^ ^
V^ ^
MeO
OMe
MeO
OMe
1' ^ ° 1
AcO^
w
HO
OMe
OH
vii
w
«
AcO
OAc
AcO
OAc
AcO.
Reagents: (i) 80%H2SO4 , (ii) 3,4-diOHC6H3CHO, AcOH, HCl, (iii) Pd,H2, EtOH; Me2S04, KOH, (iv) PhH, P2O5, (v) Pd-SrCOj, Me2C0, (vi)HBr, AcOH, (viiJACjO, NaoAc , (viii) CrOj, aq.AcOH, (ix)Zn, aq EtOH, (x)KOH, EtOH;aq.AcOH , (xi) KBH4, MeOH; 1-menthoxyacetyl chloride,py.; diastereoisomer sepn.; KOH,EtOH. A difficulty with this synthesis is the low yield in the cyclisation leading to (B). With the objective of finding another route to this ring, the Pechmann condensation of resorcinol with 5, 6-dimethoxy-2itiethoxycarbonylindan-1-one was examined
(ref.114). The latter was
779 prepared by the cyclisation of 3-(3,4-dimethoxyphenyl)propionic acid and methoxycarbonylation of the resultant 5,6-dimethoxyindan-l-one with dimethyl carbonate or by reaction with dimethyl oxalate, followed in this latter case by decarbonylation. Lithium hydride reduction of the coumarin carbonyl group afforded (B) as shown in Scheme 34.
CO2H
MeO,
ii
MeO
MeO'
MeO.
CO2H
MeO.
i
P
MeO
HO, COgMe
MeO.
MeO.
O^^O
^
MeO
MeO
OMe
Reagents: (i) Na/Hg, NaOH, (ii) PhH, P2O5 , (iii)method 1, NaOEt, C0(0Et)2 , method 2, NaOMe, {ZQ^^t) 2 / Glass, A , 180^C, (iv)l,3diOHC6H4, HCl, EtOH, (v) Me2S04, K2CO3, Me2C0, (vi) LiAlH4, THE; Yi^O'Haematoxylin was also synthesised
(ref. 113) in optically active
form.
key
However
the
required
intermediate,
deoxy-0-
tetramethylhaematoxylone was obtained from the oxidation of tetramethylhaematoxylin
(from
the
methylation
of
0-
natural
haematoxylin) rather than by synthesis from 7,8-dimethoxychromanone as for the brazilin synthesis from the 7-methoxy Haematoxylin was obtained with 1-menthoxyacetyl
compound.
( + )-
chloride, as for
racemic brazilin. The route is shown below in Scheme 35.
OAc MeO.
HO
OH
780 Reagents: (i) Pd-SrCOs, H2, (ii)HBr, AcOH, (iii) Ac20,NaOAc, (iv),Cr03, aq.AcOH, (v) Zn, EtOH, (vi KOH, aq.EtOH, (vii) KBH4 , MeOH; resolution; hydrolysis. Prior to this publication the syntheses of racemic brazilin (ref. 115) and of racemic haematoxylin alternative
methodology.
In
(ref. 116) were described by an
the
approach
to
brazilin
the
dihydroxybenzylidene derivative (J, Scheme 32) was methylated to form the trimethyl ether (1; R = Me) and the analogous tribenzyl ether (1; R = Bn) was synthesised from appropriate starting materials. (I; R = Me was then epoxidised by treatment with hydrogen peroxide in alkaline solution. From treatment with sodium
borohydride and then
catalytic hydrogenation in acetic acid in the presence of Adams' catalyst
a
mixture
of
dihydroxy
products
resulted.
By
mild
cyclisation with perchloric acid, racemic brazilin trimethyl ether (T , R = Me) was derived. From the tribenzyl ether (I; R = Bn) by a similar series of reactions, racemic brazilin tribenzyl ether (T; R = Bn) was obtained and from hydrogenolysis, racemic brazilin was derived. The process is depicted in Scheme 36. Scheme 36
.
^0^:^
Reagents: (i) H2O2, NaOH, {ii)NaBH4, iPrOH ; PtOj, H2, (iii) AcOH, HCIO4, (iv) (for,H), Pd(OH)2-BaS04, H2, AcOH. By related reactions racemic haematoxylin was synthesised (ref. 116) by first formation of 7,8-dihydroxychromanone from reaction of
3-
chloropropionic acid with pyrogallol in the presence of hydrofluoric acid. Claisen condensation with 3, 4-dihydroxybenzaldehyde in ethanol
781 containing hydrogen chloride and perbenzylation of the product resulted in 1, 8-dibenzyloxy-3-(3,4-dibenzyloxybenzylidene)-chroman-4one. Epoxidation of a propanone solution with hydrogen peroxide in methanolic sodium hydroxide gave the 3a-epoxide and reduction of the chromanone with sodium borohydride in isopropanol afforded a mixture of the cis and trans compounds, 4-hydroxy-7, 8-dibenzyloxy-3a-epoxy-3(3,4-dibenzyloxybenzyl)-chroman. Reductive cleavage of the epoxide with lithium aluminium hydride in tetrahydrofuran gave the cis and trans 3,4~dihydroxy compounds (two possible racemates) which by treatment in acetic acid with perchloric acid produced haematoxylin tetrabenzyl ether. Hydrogenolysis with palladium-carbon and hydrogen in ethyl acetate containing methanol at ambient temperature and pressure afforded racemic haematoxylin. The reactions are shown in Scheme 37. Scheme 37
- "°txxa:; OH
OH
U OBn OBn
BnO,
OBn
OBn OBn BnO.
BnO.
iBn
OBn
HO
OH
Haematoxylin
Reagents: (i) CICH2CH2CO2H, HF, (ii)EtOH, HCl, ( i i i ) BnBr, K2CO3, Me2C0, (iv)H202, NaOH, (v) NaBH4,iPrOH; LiAlH4, THF, (vi) HCIO4, AcOH, (vii)Pd-C, H2, EtOAc. The chromane and indane r i n g s i n both s y n t h e t i c b r a z i l i n and haematoxylin a r e c o n s i d e r e d t o be cis fused ( r e f . 1 1 5 , 1 1 7 ) . However
782 it would appear that the absolute configuration of both (+)-brazilin and (+)-haematoxtlin has not been established although it seems to be suggested (ref. 115) that in (+)-brazilin the t-OH group has the a-configuration and at the ring junction the designations would be (R) for the C-OH group and (S) for the C-H. The majority of the permitted colourants discussed in this account are natural products
with
the
exception
of
the
four
synthetic
carotenoids. The only semi-synthetic materials used appear to be copper
chlorophyllin
and
caramel. Although
materials discussed in this whole account
in
all
the
natural
mixtures are undoubtedly
present, the composition of caramel (E150, CI natural brown 10) must be even more complex, probably justifying the expression describing it as 'a composition of matter'. It is made
by heating sugar to around 115°C with incorporation of
other materials and reactants such as milk , fats or starches as desired
according
to
the
flavour
or
other
requirement.
Hardly
surprisingly, the nature of both the composition and the origin of the resultant colour are largely unknown (ref. 118). Several colourant materials for example those in fruit, tea and in wine which have been used historically world-wide and bear no E numbers
have
more
recently
been
judged
to
have
useful
health
properties probably through their role as antioxidants (ref. 119). Thus in this class can be instanced the flavonol, epicatechin, the flavanoid quercitin, the stilbene referred to earlier, resveratrol. and theaflavin, the colourant of black tea (ref. 120), (the absolute configuration of this is not known). OH
OH Epicatechin
OH
HO Resveratxole
783 The present colour range of the permitted natural colours (Appendix 1) enables by the use of either single materials or mixtures the whole visible spectral region to be matched and much is now known about their other properties. By contrast there is a vast range of natural colourants which may be encountered gratuitously in the diet or in gastronomy about which comparatively little is known. Conclusions The synthetic chemistry of some of the E number permitted food colourants
has
been
described
with
respect
to members
of
the
anthocyanins, the betalaines, the carotenoids, to chlorophyll a, cochineal, curcumin and to sandalwood components. Although it is only with the four commercial carotenoids that synthesis has been adopted and for the rest the extraction of the natural product remains the source for use, the potential availability by synthesis of very pure nature-identical materials is important. The products of nature are complex and it seems probable that analytical techniques are only now revealing their nature and complete structure. Nevertheless although it appears unlikely that synthetic versions, other than carotenoids, would ever be employed it is important that their structural basis should be understood.
Moreover structure/property studies require
the availability of pure standards. Synthesis has provided this basis and simultaneously
evolved a vast methodology for application in
other fields of organic chemical endeavour. It is perhaps significant that during the last hundred years concerned with the elucidation of the structure and the synthesis of the natural colourants discussed in this account the work partially
led to the awards of Nobel
laureates to R. Willstater (1915), H. Fisher(1930), P. Karrer, joint with W. Haworth, (1931), R. Kuhn (1938,but declined), R. Robinson (1947), and R.B. Woodward (1965). Involved also indirectly through theory and experimentation is the work of many others similarly honoured as for example that of G. Wittig (1979). References 1
2 3
The Colouring Matters in Food Regulations 1973,S.I. 1973,1340. Food Additives and Contaminants Committee, Interim Report on the Review of the Colouring Matter in Food Regulations 197(FAC/REP/29,HMSO 1979). J.N. Counsell, (Ed.), Natural Colours for Food and other Uses, Applied Science Publishers Ltd., London, 1981 G.A.F. Hendry and J.D. Houghton, (Eds.), Natural Food Colourants, Blackie, Glasgow, 1992.
784 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
B.C.L. Weedon, in Carotenoids, (Ed. 0. Isler)^Birkhauser, 29-59,1971. 0. Isler, in Carotenoids, (Ed. 0. Isler), 11-29, Birkhauser, 1971. N.I. Krinsky, M.M. Micheline-Roth and R.F. Taylor, (Eds.),Carotenoids, Chemistry and Biology, Plenum Press, New York,1989. T.W. Goodwin, (Ed.), Chemistry and Biochemistry of PLant Pigments, Vols.,1 and 2, Academic Press, London, 1976. R. Robinson and A.R. Todd, J. Chem. Soc, 1932, 2299-2305. P. Karrer and A. Widmer, Helv. Chim. Acta, (10),1927, 32. T.P. Coultate, in Food, The chemistry of its components. The Royal Society of Chemistry, Cambridge, 2nd. Edn., 1996,pl50. O.M. Andersen and G.F. Francis, in The Natural Pigments, in Handbook of Thin Layer Chromatography, Eds. J. Sherma and B.J. Fried, M. Dekker, New York, 2nd. Edn., 1996, p724. L.F. Levy, T. Posternak and R. Robinson, J. Chem. Soc, 1931, 2701. R. Willstatter and H. Zollinger, Annalen, (408),1915,83-109; (412), 1916, 195-216. T.R. Seshadri, Phytochemistry, (11), 1975, 881-98. D. Marmion, in 'Colorants for Foods, Drugs and Cosmetics*, in Kirk-Othmer Encyclopedia of Chemical Technology,4th Edn. Vol.6, p925. F.H. Foppen, in Chromatographic Reviews, (14), (Ed. M. Lederer) , Elsevier, Amsterdam, 1971, pl33. R.F. Taylor, in Advances in Chromatography, (22), (Eds. J.C. Giddings, E. Grushka, J. Cazes and P.H. Brown), M. Dekker, New York, 1983, pl57. R.F. Hunter, A.D. Scott and N.E. Williams, Biochem. J., (38),1944, 209-11. A.H. Jackson, in Chemistry and Biocheistry of Plant Pigments, (Ed, T.W. Goodwin),2nd. Edn., Academic Press, London, 1976, Vol, 1, p6. D.A.V. Dendy, East Afr.Agric. Forest J., (32), 1966, 126-132. M.J. Schwing-Weill, Analusis, (14), 1986, 290-95. L. Massehelein-KLeiner, Mikrochim Acta, 1967, 1080-85. P. Wollenweber, J. Chromatogr.,(7), 1962, 557-560; see also K. Jentzsch, P. Spiegel and R. Kamitz, Sci. Pharm., (36), 1968, 251-6. F.L.C. Baranyovits, Endeavour, (2), 1978, 85-9. D.A. Moyler, Chem. and Ind., (London), 1991, 11-14. C. Bulow and W. von Sicherer, Ber., (34), 1901,3889-3897. W.H. Perkin and R. Robinson, Proc. Chem. Soc, (23),149, 1907. R. Willstatter and L. Zechmeister, Sitz.-Ber. Preuss. Akad. Wiss. Physik-Math. Kl., (34), 1914, 886-893. K.W. Bentley, (Ed. K.W. Bentley),The Natural Pigments,in Pyran Pigments II,Interscience, New York, 1960, 24-51. A. Robertson and R. Robinson, J. Chem. Soc, 1928, 1460-72. R. Willstatter and Ch. L. Burdick, Annalen.,(412), 1916, 149164. R. Robinson and A.R. Todd, J. Chem. Soc, 1932, 2488-2496. W. Bradley, R. Robinson and G. Schwarzenbach, J. Chem. Soc, 1930, 793-817. T.J. Mabry, in Chemistry of the Alkaloids, Ed.S.W. Pelletier, Van Nostrand Reinhold, New York, 1970, Chap. 13; M. Piatelli, in Chemistry and Biochemistry of Plant Pigments, Ed. T.W. Goodwin, Academic Press, London, 1976, 2nd edn., vol.1, chap. 11.
785 36 M.E. Wilcox, H. Wyler and A.S. Dreiding, Helv. Chim. Acta, (48), 1965, 1134-1147. 37 K. Hermann and A.S. Dreiding, Helv. Chim. Acta, (58), 1975, 1805-1808; (60), 1977, 1805. 38 H. Wyler and J. Chiovini, Helv. Chim. Acta, (51), 1968, 14761488. 39 H.S. Raper, Biochem. J., (20), 1926, 735-42. 40 G. Biichi and T. Kamikawa, J.Org. Chem., (42), 1977, 4153-4. 41 G. Biichi, H. Fliri and R. Shapiro, J. Org. Chem., (42), 1977, 2192-4. 42 J.C. Sheehan and B.M. Bloom, J. Am. Chem. Soc, (74), 1952, 3825-28. 43 0. Isler, R. Ruegg and U. Schwieter, Pure Appl. Chem.,(14), 1967, 245-264. 44 P. Karrer and C.H. Eugster, Compt. rend., (230), 1950, 19201. 45 H. Pommer, Angew.Chem. Int. Ed. Engl., (16), 1977, 423-492. 46 H.J. Bestmann, 0. Kratzer, Chem. Ber., (96), 1963, 1899-1908; H.J. Bestmann, L. Kiesiowski and W. Distler, Angew. Chem. Int. Ed. Engl., (15), 1976, 298-9. 47 B. Schulz, J. Paust and J. Schneider, DOS, 2505869, (1976). 48 Ref.29, 257-271. 49 0. Isler and P. Schudel,in Advances in Organic Chemistry, Methods and Results, Eds. R.A. Raphael, E.C. Taylor and H. Wynberg, Interscience, New York, 1963, vol. IV, pll5. 50 J.D. Surmatis, A. Walser, J. Gibas, U. Schwieter and R. Thommen, Helv. Chim. Acta, (53), 1970, 974-990. 51 C.K. Warren and B.C.L. Weedon, J. Chem. Soc, 1958, 39863993. 52 0. Isler, H. Gutmann, H. Lindlar, M. Montavon, R. Rtiegg, G. Ryser and P. Zeller, Helv. Chim. Acta, (39), 1956, 463-473. 53 R. Ruegg, M. Montavon, G, Ryser, G. Saucy, U. Schwieter and 0. Isler, Helv. Chim. Acta, (42), 1959, 854-64. 54 U. Schwieter, H. Gutmann, H. Lindlar, R. Marbet, N. Rigassi, R. Riiegg, S.F. Schaeren and 0. Isler, Helv. Chim. Acta, (49), 1966, 369-89. 55 L.F. Fieser and M. Fieser, in Topics in Organic chemistry, Reinhold, New York, 1963, p221. 56 0.Isler, H. Gutmann, M. Montavon, R. Ruegg, G. Ryser and P. Zeller, Helv. Chim. Acta, (40) , 1957, 1242-1249. 57 F. Kienzle, Pure and Appl. Chem., (47), 1976, 183-190; see also H.Pfander, Advances in the Synthesis of opticaly Active carotenoids, in Carotenoids, Chemistry and Biology, (Eds. N.I. Krinsky, M.M. Matthews-Roth and R,F, Taylor,Plenum Press, New York, 1989, pl25. 58 B.C.L. Wedon, Pure and Appl. Chem., (14), 1967, 265-78. 59 P. Karrer, A. Ruegger and U. Solmssen, Helv. Chim. Acta,(21), 1938, 448-51. 60 R.D.G. Cooper, L.M. Jackman and B.C.L. Weedon, Proc. Chem. Soc, 1962, 215. 61 R.D.G. Cooper and B.C.L. Weedon, (unpublished work). 62 C. Sterling, Acta Crystallogr., (17), 1964, 1224-1228. 63 H. Fischer, Naturewissenschaften, (26), 1940, 401-3. 64 M. Strell, A. Kalonjanoff and H. Roller, Angew. Chem., (72) 1960, 169-70. 65 H. Fischer, E. Stier and W. Kanngiesser, Annalen, (543), 1940, 258-70. 66 R.B. Woodward, W.A. Ayer, J.M. Beaton, F. Bickelhaupt,R. Bonnett, P. Buchscacher, G.L. Closs, H. Dutler, J. Hannah, S. Ito, A. Langemann, E. Le Goff, W. Leimgruber, L. Lwowski, J.
786
67 68
69 70 71 72 73 74
75 76 77 78 79 80
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Sauer, Z. Valenta and H. Volz, J. Am. Chem. Soc, (82), 1960, 3800-2; Tetrahedron, (46), 1990, 7599-7659. I. Fleming, J. Chem. Soc, 1968, C 2765-2770. L. Knorr, Annalen, (236), 1886, 318; see also A.H. Jackson, The total Synthesis of Pyrrole Pigments, in The Total Synthesis of Natural,Products, (ed. J. ApSimon) ,Voll,1973, pl49,J. Wiley, H. Fischer and M. Neber, Annalen, (496),1932,1-26. A. Treibs, R. Schmidt and R. Zinsmeier, Chem. Ber., (90), 1957, 79-84. H. Fischer and K. Zeile, Annalen, (483), 1930, 251-71. H. Fischer and Z. Csukas, Annalen, 508, 1934, 167-84. H. Fischer and M. Strell, Annalen, (540), 1939, 232-49. R. Willstatter and A. Stoll, Annalen, (380), 1911, 148-154; H. Fischer and A. Stern, Annalen, (519), 1935, 244-253; H. Fischer and S. Goebel, Annalen, (524), 1936, 269-284; A. Stoll and E. Wiedemann, Fortschr. Chem. Forsch., (2),1952, 538. J.W.K. Burrell, L.M. Jackman and B.C.L. Weedon, Proc. Chem. Soc, 1959, 263-4. P. Schutzenberger, Ann. Chim. Phys., (54), 1858, 52. M. Ali and L.J. Haynes, J. Chem. Soc, 1959, 1033-35. J.C. Overeem and G.J.M. Van der Kerk, Rec Trav. Chim., (83), 1964, 995-1004; A.R. Mehendale, A.V. Rama Rao, I.N. Shaik and K. Venkataraman, Tetrahedron Lett., (18), 1968, 2231-2234. A. Fiecchi, M. Anastasia, G. Galli and P. Gariboldi, J. Org. Chem., (46), 1981, 1511. P. Allevi, M. Anastasia, P. Ciuffreda, A. Fiecchi, A. Scala, S.J. Bingham, M.Muir and J.H.P. Tyman, J. Chem. Soc. Chem. Commun., 1991, 1319-20; J.H.P. Tyman and A, Fiecchi, EP 0 602027/91, USP 5 424 421/92; S.J. Bingham, Ph.D. thesis, Brunei Univ. 1992;J.H.P. Tyman, Synthetic and Natural Phenols, Elsevier Science, Amsterdam, 1996, p624. P.Allevi, M. Anastasia, P. Ciuffreda, A. Fiecchi, A. Scala, S.J. Bingham, M.Muir and J.H.P. Tyman, J. Chem. Soc. Perkin Trans I (submitted for publication). G. Roberge and P. Brassard, J. Chem. Soc, Perkin Trans I, 1978, 1041-46. D.W. Cameron, D.J. Deutscher, G.I. Feutril and P.G. Griffiths, Aust. J. Chem., (34), 1981, 2401-21. A.V. Rama Rao and K. Venkataraman, in Recent Progress in Natural Product Chemistry, Prentice Hall of India, 1972, p341; D.D. Gadgil, Ph.D. thesis, Univ. Poona. J.H.P. Tymam and S.J. Bingham, (unpublished work). A. Fiecchi (unpublished work). 0. Dimroth and H. Kammerer, Chem. Ber., (53), 1920, 471-80. M. Viscontini and N. Merckling, Helv. Chim. Acta, (3) 1952, 2280-82. R. Roussin, C.R. Seances Acad. Sci., (52), 1861, 1033. D.B. Bruce and R.H. Thomson, J. Chem. Soc, 1955, 1089-1096. K. Zahn and P. Ochwat, Annalen, (461), 1928, 72-97. G.A. Kraus and T.O. Man, Synth. Commun., (16), 1986, 1037-42. G. Ciamician and P. Silber, Chem Ber., (30), 1897, 192-195, C.J. Jackson and A.E. Menke, Amer. Chem. Amer. Chem. J., (6),1884,78-88. W.H. Perkin, Chem. Soc. Trans., (85), 1905, 63. J. Milobedzska, S. von Kostanecki and V. Lampe, Chem. Ber., (43), 1910, 2163-2170. A.G. Perkin and A.G. Everest, The Natural Colouring Matters, Longmans Green, New York, 650pp., 1918; see also, P.J.
787
97 98 99 100 101 102 103
104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
Roughly and D.A. Whiting, J. Chem. Soc.,Perkin I Trans., 1973, 2379-88. H. Ryan and J.M. Dunlea, Proc. Roy. Irish Acad., (32), 1913, 9. E. Fischer, Chem. Ber., (16),1883, 166. V. Lampe and J. Milobedzka, Chem. Ber., (46), 1913, 2233. V. Lampe, Chem. Ber., (51), 1918, 1347-55; see also C. Kashima, N. Mukai, Y. Yamamoto, Y. tsuda and Y. Omote, Heterocycles, (7), 241-246, 1977. F.E, King, Chem. and Ind.(London), 1953, 1325-28. F.M. Dean, The Synthesis of Oxygen Ring Compounds, in The Total Synthesis of Natural Products, ed. J. ApSimon, Vol.1, 1973, p513, J. Wiley. M. Jang, L. Cai, G.O. Udeauni, K.V. Slowing,C.F. Thomas, C.W.W. Beecher, H.H.SD. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon and J. Pezzuto, Science, (275), 1997, 218-20. E.H. Siemann L.L. Creasy, J. Enol. Vitic, (43), 1992, 49; A.L. Waterhouse, Chem. and Ind.,(London), 1995, 338. A. McGookin, A. Robertson and W.B. Whalley, J. Chem. Soc, 1940, 787-95. E. Spath and K. Kromp, Chem. Ber., (74), 1941,189-196. A.V. Prasad and R.S. Capil, J. Chem. Soc, Perkin Trans I, 1986, 1561-63. K. Fukui and M. Nakayama, Bull. Chim. Soc. Japan, (42). 1969, 1408-1411. M. Uchiyama and M. Matsui, Agric. Biol, Chem., (31), 1967, 1490-1498. H. Horino and N. Inoue, J. Chem. Soc. Chem. Commun., 1976, 500-501 W.H. Perkin Jnr. and R. Robinson, Proc. Chem. Soc, (28), 1912, 7. R. Robinson and F. Morsingh,(unpublished work, referred to in ref.29, p90). F.Morsingh and R. Robinson, Tetrahedron, (26), 1970, 281-289. J.H.P. Tyman and M. Patel, (unpublished work); Abstract E15, lUPAC Symp.Chem.Nat.Products, Dunedin, N. Zealand 1976. 0. Dann and H. Hofmann, Annalen, (667), 1963, 116-25. 0. Dann and H. Hofmann, Chem. Ber., (98), 1965, 1498-1504. J.C. Craig, A.R. Naik, R. Pratt and E. Johnson, J. Org. Chem., (30), 1965, 1573. W.R. Eichenberger, in. Caramel Colours: Manufacture, Properties and Composition, Amer. Chem. SocMeeting, Aug.29th.,1972. A.L. Waterhouse, Chem. and Ind. (London), 1995, 338-341. D.D. Collier, T. Bryce, R. Mallows, P.E. Thomas, D.J. Frost, 0. Koever and C.K. Wilkins, Tetrahedron, (29), 1971, 125-142.
Appendix 1 The report referred to (ref.l) contains an Appendix V which lists the whole range of 'Colouring Matter permitted in food by the colouring matter in food regulations 1973' (as amended). The natural materials given in that appendix are as follows. Curcumin (ElOO), riboflavin or lactoflavin (ElOl), riboflavin-5'phosphate [ElOl(a)], cochineal or carminic acid (E120),
788 chlorophyll (E140), copper complexes of chlorophyll and chlorophyllin (E141), caramel (E150), alpha-, beta- , gammacarotene, [El60{a)], annatto, bixin, norbixin, [E160(b)],capsanthin or capsorubin (Paprika oleoresin), [E160(c)], lycopene,[E 160(d)], beta-apo-8'-carotenal, [E160 (e)], ethyl
beta-
apo-8'-carotenate, [E160(f)], flavoxanthin, [E 161(a)], lutein [E161(b)], cryptoxanthin, [El61(c)], rubixanthin, [E161(d)], violaxanthin, [E161(e)], rhodoxanthin, [E161(f)], canthaxanthin, [E161{g)], and beetroot red or betanin, (E162). Natural substances listed having a secondary colouring effect are (a) paprika, (b) turmeric, (c)saffron and (d) sandalwood.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
789
Bioactive Natural and Synthetic Acronycine Derivatives Modified at the Pyran Ring Sylvie Michel and Francois Tillequin 1.
INTRODUCTION
Acronycine (1.) is a natural alkaloid which was first isolated in 1948 from the bark of a small Australian Rutaceous tree, Acronychia baueri Schott (1). Since that time, the status of this plant within the Rutaceae family has been revised several times, in the course of successive taxonomic studies (25). It is now widely accepted that the plant belongs to the genus Sarcomelicope and should be named Sarcomelicope simplicifolia (Endl.) Hartley ssp. simplicifolia (4).
The presence of an acridone basic skeleton and of a dimethylpyran ring in the acronycine structure was demonstrated fairly early (6-9). Tremendous effort was then necessary to determine whether the pyran ring was fused linearly or angularly on the acridone skeleton. In 1966, the regioselective synthesis of l,3-dimethoxy-2-methoxycarbonyl-10-methyl-9(10//)-acridinone (2j permitted Macdonald and Robertson (10) to assign unambiguously the angular structure 3,12-dihydro-6-methoxy-3,3,12-trimethyl-7//-pyrano[2,3c]acridine-7-one to acronycine. This structure was confirmed later on, both by nmr study of various acronycine derivatives (11) and by X-ray crystallographic analysis of 5-bromo-l,2-dihydroacronycine (33 (12).
790
COOCH3
OCH.
Acronycine was first synthesized in 1967-68 by Beck et al. who published simultaneously three interrelated syntheses of that alkaloid (13, 14). Around ten other total syntheses have been reported up till now (1526). Most of the synthetic strategies developped are sufficiently versatile to permit access to acronycine structural analogues as far as substitution on the A ring is concerned. Syntheses of analogues modified at B, C and D rings have been comparatively less explored. Most of them rely on chemical modifications performed on a preformed pyranoacridone skeleton, at the latest steps of the synthesis. The biological interest of acronycine was revealed in 1966 by Svoboda at the Eli-Lilly Laboratories (27, 28). Acronycine was found to be active against a broad spectrum of solid tumors including sarcoma, myeloma, carcinoma and melanoma. In contrast, it only exhibited poor activity against leukemias. The activity against X-5563 myeloma was of particular interest, since this plasma cell tumor has several properties that relate to those of multiple myeloma in human patients. This is the reason why Scarffe in 1983 (29) performed phase I-II clinical evaluation of acronycine in patients with refractory multiple myeloma. Oral acronycine capsules produced one clear response in sixteen patients. The remission was maintained 72 weeks, using daily dose of 300 mg/m^. The limited success of that trial is probably related to the moderate potency of acronycine and to its very low water-solubility {ca. 2-3 mg per liter of water) which do not permit an efficient parenteral formulation of the drug. Despite the broad antitumor activity of acronycine, the mechanism of its action at both cellular and molecular levels has not yet been clearly established. First observations suggested that the drug did not interact with DNA but acted primarly by alteration of subcellular organelle membranes and that its delayed effects were due to interference with the structure, function, and turnover of cell-surface components (30-33). More recently Dorr and Liddil reinvestigated the DNA-binding property of acronycine.
791
using solutions obtained with a solvent which did not contain dimethylsulfoxide (34). Under such conditions, acronycine was shown to display classic non-covalent binding patterns on DNA thermal degradation. Those experiments strongly suggest that acronycine should interact with DNA either by intercalation or by some other non-covalent process in order to stabilize the double helix against thermal denaturation. This hypothesis seems in good agreement with the approximately flat structure of 5-bromo1,2-dihydroacronycine previously established by X-ray diffraction (12). It is also consistent with recent experiments which gave evidence for the DNA binding activity of acronycine azine (4), a dimeric analogue of acronycine which exhibits increased cytotoxic activity (35).
2.
STRUCTURE ACTIVITY RELATIONSHIPS IN THE ACRONYCINE SERIES
Since the discovery of the broad experimental antitumor spectrum of acronycine in 1966, more than one hundred compounds derived from the 3,12-dihydro-7//-pyrano[2,3c]acridin-7-one basic skeleton have been synthesized or isolated from natural sources. Unfortunately, many of these compounds have not been studied from a biological point of view. Only a few have been tested in vitro for cytotoxic activity. Even fewer have been studied in vivo for antitumor properties. In addition, the experimental conditions and the cell lines used for the tests greatly vary from one compound to another. It nevertheless seems possible to draw some limited
792
conclusions in the field of structure-activity relationships in the acronycine series. 2.1 Modification at the A-ring Some acronycine derivatives substituted at 9, 10 or 11-position have been tested for cytotoxic and/or antitumor activity. 9-Hydroxyacronycine (5_) prepared by addition of acronycine to a metabolizing culture of Aspergillus alleaceus (36) or Cunninghamella echinulata (37) was devoid of antitumor activity when tested in mice implanted with X-5563 plasma cell myeloma or C-1498 myelogenous leukemia (36). In a similar way, 9-chloroacronycine (6.) synthesized by Fryer et al. (38) exhibited no significant antitumor activity when tested in mice bearing the solid form of sarcoma 180 or injected with Ehrlich ascites.
11-Methoxyacronycine (7J was first obtained by total synthesis (39) and was later isolated from various Citrus species (40, 41). It was tested for human promyelocytic leukemic cell line HL-60 growth inhibition. Under such conditions, it was shown to be significantly active (IC50 = 17.2 p-M), as was 10,11-dimethoxyacronycine (8.) (IC50 = 15.5 |iM), when compared with acronycine (IC50 = 26.2 |iM) (42, 43). In contrast, it should be noted here that 11-azaacronycine ( = 6-methoxy-3,3,12-trimethyl-3,12-dihydrochromeno[5,6b][l,8]naphtyridin-7-one) (£) synthesized by Reisch et al (44, 45) was found inactive when tested against P388 leukemia transplanted in mice.
793
O
OCH.
R=H R = OCH3
2.2 Modifications at the B-ring Informations available on the role of the substituent on the nitrogen atom at 12-position are very limited. In fact, A^-desmethylacronycine (10) was the only compound modified at that position to be studied from a biological point of view. The inhibitory effect of 1 ^ against HL-60 cell growth in vitro (IC50 = 17.6 |i.M) (42, 43) was of the same order of magnitude as that reported for acronycine itself.
M 2.3 Modifications at the C-ring The substituent at C-6 on the C-ring has a dramatic influence on the antitumor activity in the 3,12-dihydro-7//-pyrano[2,3c]acridin-7-one series. Noracronycine (11). bearing a phenolic OH group instead of a methoxy group at C-6, was shown not to possess any antitumor activity (27, 28). It is also devoid of significant activity against HL-60 cell growth in vitro (42, 43). A series of 6-alkoxy analogues of acronycine 12-16. was obtained by Fryer et al. (38) upon treatment of noracronycine (11) with an appropriate alkyl sulfate or alkyl halide under basic conditions. Among the derivatives 1 2 - 1 6 . only 15_ exhibited significant antitumor activity causing a 52% reduction in tumor size in mice bearing the solid form of sarcoma 180 and a 60% prolongation of the lives of mice injected with Ehrlich ascites (38).
794 11 R = H 12 R = C2H5 1 2 R = CH2-CH=CH2 1 4 R = CH2-0-CH3 1 5 R = CH2-CH2-N(CH3)2 16 R = CH2-COOC2H5 Both active compounds l_ and H bear an alkoxy substituent at C-6, whereas inactive noracronycine (11) bears a free OH group at that position. The lack of antitumor activity of noracronycine should be therefore related either with the reduction of the steric hindrance at C-6, or with the dramatic change in the electronic density within the aromatic structure, due to chelation of OH-6 by the neighbouring carbonyl group. In order to determine which of those two parameters was the most important in terms of structure activity relationships, we considered the synthesis and biological evaluation of 6-demethoxyacronycine (17) as highly desirable. Coppola previously prepared that compound in 1984 (46) through reaction of iV-methylisatoic anhydride with the enolate derived from 2,6,7,8tetrahydro-2,2-dimethyl-5//-l-benzopyran-5-one, but no biological data was published at that time. Our synthetic approach (Scheme 1) (47) was completely different and potentially more versatile. Alkylation of 3-nitrophenol (18) with 3-chloro-3-methylbut-l-yne (19) gave the propargylic ether 20.. The nitro group of 20. was selectively reduced by iron powder to yield 3-(3-aminophenoxy)-3-methyl-but-l-yne (21). Claisen rearrangement of 2 1 was highly regioselective and provieded the required 5-amino-2,2-dimethylchromene (22) in 60% yield. Ullmann condensation of 22. with 2-bromobenzoic acid (23) gave the carboxylic diphenylamine 24. in 75% yield. The last steps of the synthesis were essentially similar to those described by Loughhead for the synthesis of acronycine itself (18). Cyclization of 24 using trifluoroacetic anhydride gave rise to 6-demethoxy-12-demethylacronycine (25) in 85% yield. Finally methylation of 2J_ with methyl iodide under phase-transfer catalyzed conditions afforded H in 83% yield.
795
r^^=^
r ^ ^ ^
12
H,N
.COOH
The cytotoxic activity of 6-demethoxyacronycine (17) was determined in vitro in comparison with acronycine, against wild type (DC-3F) and actinomycine D-resistant (DC-3F/ADX) Chinese hamster fibroblastic lung cell lines and against murine L1210 leukemia cells (47, 48). 6-Demethoxy acronycine (12.) was found to have a potency within the same order of magnitude as acronycine itself against the different cell lines (Table 1). Table 1 Cell lines DC-3F
Acronycine 1 IC50= 11-25 |iM
6-Demethoxyacronycine 17
DC-3F/ADX
IC50 = 3.5^M
IC50 = 2.75 ^iM
L1210
IC50 = 27^M
IC50 = 29.9 ^M
IC50 = 8.75 ^iM
796 In contrast with noracronycine, some of its derivatives substituted on the A ring by hydroxy or methoxy groups at 10 and/or 11-position, such as compounds 26-28. have been claimed to exhibit cytotoxicity against HL-60 cells in vitro (42, 43). O OH
26 Ri=R3 = H 2 7 Ri=H
R2 = 0H
R2 = 0H
R3 = CH3
2 8 Ri = OH R2 = OCH3 R3 = CH3 9-Nitronoracronycine ( 2 9 ) . 10-nitronoracronycine (30) and 11nitronoracronycine (31) were synthesized by Reisch et al. (49, 50) and tested by the N.C.I, against P388 leukemia transplanted in mice. At lower doses, no significant antitumor activity could be detected whereas higher doses proved to be toxic.
1 9 Ri = N02
R2 = R3 = H
^
R2 = N02
Ri=R3=H
3 1 R3 = N02
Ri=R2 = H
2.4 Modifications at the D-ring The two geminal methyl groups at C-3 appear as an important structural requirement for antitumor activity in the series. Indeed, 3hydroxymethylacronycine (32.), obtained by addition of acronycine to a metabolizing culture of Streptomyces spectabilicus, was devoid of antitumor activity when tested in mice implanted with X-5563 plasma cell myeloma or C-1498 myelogenous leukemia (36). More recently, Reisch et al. prepared
797
acronycine analogs 33-35 by total synthesis (51). Compounds 33-35 were tested in vitro on sixty human tumor cell lines of various types of cancer by the N.C.I. (52). Except 3 3 , all substances were determined inactive. Compound 3_3. was also inactive against most of the sixty cell lines, but showed weak cytostatic activity against the renal cancer UO-31 cell line (51).
OCH.
Only little chemical and biological investigations have been conducted on acronycine derivatives modified at the 1 and/or 2 positions on the pyran ring. From a chemical point of view, previously described compounds modified at those positions only include bromo (7), nitro (8, 27), dihydro (36) (7, 27), and dihydronitro (8, 27) derivatives. The 1,2-double bond has been so far generally considered as an essential structural requirement to observe antitumor activity, since 1,2dihydroacronycine (36) and dihydronitroacronycine were determined inactive in the course of early in vivo experiments performed at the Eli-Lilly Laboratories (27). Effect of substitution on the 1,2-double bond remains unclear. Nitration of acronycine using nitric acid leads to a mononitroacronycine, whose structure was reported as 1-nitroacronycine by Drummond and Lahey (8) and as 2-nitroacronycine by Svoboda (27). This compound was described as biologically inactive by Svoboda (27). It has been more recently evaluated by Cordell et al. (35) who found that nitroacronycine demonstrated a greater
798
activity than acronycine itself against a battery of cultured mammalian tumor cells and selected it as a promising agent in that series (35). In summary, only a few compounds modified at positions 1 and 2 have been prepared and studied from a biological point of view. This is the reason why we have been interested in the preparation of such compounds, in order to determine unambiguous structure activity relationships and to be able to prepare novel active acronycine derivatives. OCHa
3. NATURAL AND SYNTHETIC ACRONYCINE DERIVATIVES MODIFIED AT THE PYRAN RING 3.1 Nitro. amino and amido derivatives The nitro and dihydronitro derivatives of acronycine appeared to us of particular interest, since water soluble amino and amido compounds should be easily obtainable from them. Treatment of acronycine (1) with fuming nitric acid in acetic acid at 0°C gave mononitroacronycine in 90 % yield, as previously described (8, 27). Observation of a strong cross-peak between the singlet of the pyran proton and the signal of the N-CH3 group at the 12-position in the 2D NOESY iH NMR spectrum permitted us to unambiguously establish the structure of this compound as 2-nitroacronycine ( 3 7 ) . Similarly, nitration of 6demethoxyacronycine (17) afforded 2-nitro-6-demethoxyacronycine (38) in 50 % yield (53).
799
NOo 1 12
R = OCH3
2 7 R = OCH3
R=H
2S R = H
Reduction of 37. using sodium borohydride afforded the known 2-nitro1,2-dihydroacronycine (39). which could be reduced into 2-amino-l,2dihydroacronycine ( 4 0 ) by hydrogenation using Pd/C as a catalyst. Alternatively, 40L was obtained more easily from 3JL by direct reduction using sodium borohydride in the presence of cupric acetate. This latter reaction also afforded small amounts of 2-oxo-l,2-dihydroacronycine oxime (41) which was more conveniently prepared in high yield by reduction of 2nitro acronycine (37.) with tin and hydrochloric acid in methanol (53). OCH
H2/Pd-C/MeOH
800
2-Amino-l,2-dihydroacronycine (40) could be easily converted into variously substituted amines and amides. Treatment of 4 0 with formaldehyde and sodium borohydride afforded 2-dimethylamino-l,2dihydroacronycine (42.). Both aliphatic and aromatic amides were obtained from 40_ upon treatment with acid anhydrides in pyridine. This latter reaction is exemplified by the preparation of 2-acetylamino-l,2dihydroacronycine (43) and 2-benzoylamino-l,2-dihydroacronycine (44) using acetic anhydride and benzoic anhydride, respectively (53). OCHa
4 2 R = N(CH3)2 4 3 R = NHC0CH3 4 4 R = NHCOC6H5 Compounds 3 7 - 4 4 were tested for inhibition of L-1210 cell proliferation in vitro, in comparison with acronycine (1_) and 6demethoxyacronycine ( 1 7 ) . The results are summarized in Table 2. Considering the structure-activity relationships, it appears that only compounds bearing both a methoxy substituent at the 6-position and a 1,2double bond, such as 3 7 and 4 1 (as its tautomeric 2hydroxylaminoacronycine form) (54) exhibit a significantly more potent activity than acronycine itself.
Table 2 - Inhibition of L-1210 cell proliferation by compounds 37-44 in comparison with acronycine (1) and 6-demethoxyacronycine (17) Compound
1
11
12
il
1^
4J1 11
41 11
±A
IC50 M-M
27
29.9
0.09
13.7
32.7
12.5
>50
47.9
2.8
>50
The very high cytotoxicity of 2-nitroacronycine (37) in vitro and the discrepancies in the litterature data as to its biological activity (27, 35)
801
prompted us to re-evaluate this compound in in vivo experimental tumor models, in comparison with acronycine (JJ. As shown on Table 3, 2nitroacronycine (37) was devoid of antitumor activity against P 388 leukemia and C 38 colon adenocarcinoma in mice, unlike acronycine (IJ which is moderately active against P 388 leukemia, but markedly active against C 38 colon cells (53). Table 3 - Antitumor activities of 2-nitroacronycine (37) in comparison with acronycine (1) against P 388 leukemia and C 38 colon carcinoma in mice.
1
17
P388 T/C %
125 (200mg/kg)
113 (50mg/kg)
C38 T/C %
4 (200mg/kg)
137 (25mg/kg)
3.2. Hydroxydihydro derivatives and glycosides The alcohols resulting from the addition of water to the double bond of the pyran ring of acronycine, 1-hydroxy-1,2-dihydroacronycine (45) and 2hydroxy-l,2-dihydroacronycine (46J were prepared in our laboratory (55). Our final aim was to combine those alcohols with various sugar units, since numerous anticancer agents possess a 0-glycosidic moiety which strongly influence both their bioavailability and their selective toxicity towards tumor cells.
4 5 Ri = OH R2 = H 46 R i = H
R2 = 0H
The benzylic alcohol 45. was prepared in two steps, using acronycine (JD as starting material. Treatment with N-bromosuccinimide in aqueous
802
tetrahydrofuran led to racemic r r f l n 5 - 2 - b r o m o - 1 - h y d r o x y - 1 , 2 - d i h y d r o acronycine (47). In a second step, the bromohydrin 47_ was smoothly debrominated to 45_ using tributyltin hydride (55). O
OCH.
PCH3
4 7 (relative configuration) OCH^ Bu^SnH/AIBN Toluene/Rx
45 Unfortunately, 1-hydroxy-1,2-dihydroacronycine (45.) proved rapidly to be very unstable in organic solutions, and all our attemps towards its glycosidation have remained unsuccessful (56). A good precursor of 2-hydroxy-1,2-dihydroacronycine (46) was cis1,2-dihydroxy-1,2-dihydroacronycine (48), which was first prepared as a racemate by osmium tetroxide oxidation of acronycine (57). This reaction was difficult to apply for a large scale preparation of 48., due to the cost and toxicity of osmium tetroxide and to the difficulties encountered during the work-up under such conditions. Catalytic osmium tetroxide oxidation of 1, using iV-methylmorpholine-N-oxide to regenerate the oxidizing agent proved recently more convenient for the preparation of 48. (58). A first reaction sequence involving sulfuric acid dehydration of the diol £8_ to the homobenzylic ketone 49., followed by borohydride reduction of 49^ to 46. was successfully accomplished but the overall yield remained very low (55). From a quantitative point of view, better results were obtained when converting 48. into the corresponding cyclic thiocarbonate 50. by use of N,N'-
803
thiocarbonyldiimidazole. Benzylic reduction of 5j0 with tributyltin hydride smoothly afforded 46. in a second step (55). O
O
OCH.
OCH.
OsO.
(relative configuration)
N^'-thiocarbonyl diimidazole
H2SO4/H2O
JButanone O
O
OH
OCH3
OCH.
ill
(relative configuration) O
O
Bu3SnH/AIBNCH3^ Toluene/20°C>
MeOH
OH In contrast with 45., the homobenzylic alcohol 46. was stable enough to give satisfactory glycosidation reactions (56). Condensation of 46. with 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide under Konigs-Knorr conditions only led to the diasteroisomeric
804
orthoesters 51a. 51b which could not be separated. In contrast, the use of 3,4-di-O -acetyl-2,6-dideoxy-a-L-araZ7/no-hexopyranosyl bromide as glycosylating reagent under the same conditions afforded the expected a-Lglycosides 52a. 5 2 b . together with trace amounts of the corresponding p anomers (56).
AcO 51a 2R 51b 2S When 46. was treated with l,3,4-tri-0-acetyl-2,6-dideoxy-a-L-araZ7/n^hexopyranose in the presence of tin tetrachloride in acetonitrile, the reaction was stereoselective, and only the a-L-glycosides 52a. 52b. were obtained (56). In a similar way, bromoglycosides 53a. 53b (59), chloroglycosides 54a. 5 4 b (59), and azidoglycosides 5 5a. 5 5 b (56) and 56a. 5 6 b (56) were prepared using 1,4-di-O-acetyl-3-bromo-2,3,6-trideoxy-L-araZ7/«6>-hexopyranose, l,4-di-0-acetyl-3-chloro-2,3,6-trideoxy-L-ar<3Z7/«6>-hexopyranose, l,4-di-0-acetyl-3-azido-2,3,6-trideoxy-L-araZ7/n6>-hexopyranose, and 1,4-diO -acetyl-3-azido-2,3,6-trideoxy-L-/>'X6>-hexopyranose, respectively, as glycosylating reagents. Catalytic hydrogenation of the azidoglycosides 56a. 56b afforded the corresponding aminoglycosides 57a. 57b. whereas catalytic hydrogenation of 55a. 5 5 b followed by deacetylation gave the fully deprotected aminoglycosides 58a. 58b. The diastereoisomeric glycosides (2R and 25) could be separated by column chromatography in the cases of 52a. 52b. 53a. 53b. 54a. 54b and 58a. 58b (56, 59). The absolute configuration at C-2 on the aglycone part of each individual glycoside was deduced from ^H and l ^ c NMR data, compared with those of related angular hydroxydihydropyranocoumarin glycosides of known configuration (60).
805
Finally, reaction of 46. with di-0-acetyl-L-rhamnal in the presence of A^iodosuccinimide, furnished the two diastereoisomeric iodoglycosides 59a. 59b which were easily separated by column chromatography and whose configurations were determined as previously (56). OCH.
AcO ^
£ l a 2/?
52a IR
n
51b 25
AcO ]
^
££b 2S
AcO
AcO
R=
Q
N
K"
5 i b 25
Br AcO
Q
R=
O^ , S l a 27^
51a IR
S l a IR S i b 25
AcO I NHo
-p HO
Q^
Sib 25
i8.a 2R SS.b 25
NHo
AcO
_Q ^
AcO
^
IR
IR
£5.b 25
£2.b 25
^a
2-Hydroxy-l,2-dihydroacronycine glycosides were tested for inhibition of L-1210 cell proliferation in vitro in comparison with acronycine (1_). The results are summarized in Table 4. Compounds 53a. 53b. 54a.b and 59a.b including a halogenated sugar moiety displayed cytotoxic activities of the same order of magnitude as acronycine itself. Azidoglycosides 55a.b and 56a.b were significantly more
806
potent than acronycine in inhibiting cell proliferation. More hydrophilic aminoglycosides 57a.b and 58a.b only exhibited marginal activity. The cytotoxic activity of 2-hydroxy-l,2-dihydroacronycine glycosides therefore appears to be related with the lipophilicity of the sugar unit (56). Table 4 - Inhibition of L 1210 cell proliferation by 2-hydroxy-l,2dihydroacronycine glycosides in comparison with acronycine (56).
L
Sia i2.b ila ii.b £4.a,b 55a.b 56a.b
il.a,b 58a.b
£±a,b
IC50 ^M 24.1 20.8 82.5 11.3 19.8 13.4 3.0 2.8 77.2 40.7 5.6
3.3. Dihydroxydihydro derivatives and esters In the course of a systematic study of the alkaloid contents of Rutaceae species belonging to the genus Sarcomelicope (57, 61-71), we isolated several new acronycine derivatives modified at the pyran ring. Indeed, cisl,2-dihydroxy-l,2-dihydroacronycine (48) and r r a n ^ - l , 2 - d i h y d r o x y - l , 2 dihydroacronycine (60) were obtained as optically active compounds from the bark of Sarcomelicope glauca Hartley (57) and Sarcomelicope dogniensis Hartley (69).
4£
OH
807
Acronycine epoxide 6X was isolated in minute amounts from Sarcomelicope agyrophylla Guill. and from Sarcomelicope simplicifolia (Endl.) Hartley ssp. neo-scotica (P.S. Green) Hartley (68). This compound should be considered as a biogenetic intermediate between acronycine itself and the cis and fran^'-dihydrodiols 48. and 60. in Sarcomelicope species. It is a highly unstable compound whose reactivity most probably explains the difficulties encountered by us (72) and by others (73, 74) in attempts towards its synthesis. Reisch et al. finally succeeded in synthesizing 6JL in 14 % yield by treatment of acronycine with dimethyldioxirane in the presence of potassium carbonate (75).
The high reactivity of acronycine epoxide (61) led us to speculate that this compound should be the active metabolite of acronycine in vivo and that it should be responsible for the alkylation of nucleophilic targets within the tumor cell (68). This hypothesis was in good agreement with the mode of action previously established (76) for the insecticidal chromenes precocenes I (62.) and II (63), whose structures share a dimethylbenzopyran unit in common with acronycine (1).
R=H R = OCH3
The pyran double bond plays a crucial role in the insecticidal activity of precocenes, since the corresponding epoxides, formed in situ through cytochrome P 450 mediated oxidation, are the active metabolites of these compounds (76-78). These extremely reactive epoxides are responsible for the alkylation of nucleophiles present in biological matrices and ultimately
808 for irreversible damage and cell death (76, 79). From a chemical point of view, the alkylating p r o p e r t i e s of e p o x y p r e c o c e n e II ( 6 4 ) towards nucleophilic agents were unambiguously demonstrated by reaction with various thiols, leading to adducts with the sulfur atom linked to the benzylic position of the benzopyranyl skeleton (80). OCH H3CO
H3CO
OH
M The great chemical instability of acronycine epoxide, for instance its fast reaction with water to yield the corresponding diols (72), exclude its possible use as an anticancer agent. This is the reason why we synthesized a series of c i \ y - l , 2 - d i h y d r o x y - l , 2 - d i h y d r o a c r o n y c i n e
and
l,2-dihydro-6-demethoxyacronycine
order
esters,
in
c/\y-l,2-dihydroxyto
obtain
new
anticancer candidates with better stability than acronycine epoxide, but with similar reactivity towards nucleophilic agents (58).
Racemic
4S
Ri = OCH3
R2 = R3 = OH
65
Ri = H
R2 = R3 = OH
66
Ri = OCH3
R2 = R3 = OCOCH3
67
Ri = H
R2 = R3 = OCOCH3
^
Ri = OCH3
R2 =0H R3 = OCOCH3
69
Ri = OCH3
R2 =0H R3 = OCOC6H5
70
Ri = 0CH3
R2= OCOCH3 R3= OCOC6H5
cf5"-diols 48. and 65. were prepared as previously described by
catalytic osmic oxidation of acronycine (V) and 6-demethoxyacronycine respectively. Treatment of those diols with excess acetic anhydride the
corresponding
equivalent
of
diesters
anhydride
was
6_6_ and 6 7 . used,
respectively.
monoesters
at
the
When less
(17).
afforded
only hindered
one 2-
position, such as acetate 68. and benzoate 6 ^ were obtained. Treatment of the monobenzoate
69. with excess acetic anhydride furnished the mixed ester 70..
809
Finally, reaction of the c/\s'-diols 48. and 65. with A^,A^'-carbonyldiimidazole 2-butanone led to the cyclic carbonates XL and 22. (58).
21 72
in
R = 0CH3 R=H
In order to ensure that the reactivity of c z \ s - l , 2 - d i h y d r o x y - l , 2 dihydroacronycine diesters towards nucleophilic agents was similar to that of acronycine and precocene epoxides, the diester 6 ^ was reacted with benzylmercaptan (73) in acidic medium. The cis and trans adducts 74 and 7.5 with the sulfur atom linked to the benzylic position of the pyranyl ring were obtained in almost quantitative yield under these conditions (58). OCHcr
H3COCO OCOCH3
OCOCH3
810
Compounds 4 8 . 6 5 - 7 2 were tested for inhibition of L-1210 cell proliferation in vitro, in comparison with acronycine (V), dihydroacronycine (36) and 6-demethoxyacronycine (17). The results are summarized in Table 5. Table 5 - Inhibition of LI210 cell proliferation dihydroacronycine derivatives (58).
1 IC50 M^M
24.1
by
l,2-dihydroxy-l,2-
lA
LI
4^
6^
M
6-2 6S. 6_9 2-fi 1 1 2-2
34.4
29.7
80.6
>50
5.8
>50
8.9
7.1
5.0
0.32
>50
Compounds 66. 68 and 70. were slightly more potent (3-5 fold) than acronycine, while the most cytotoxic derivative, cyclic carbonate 7X, was 75 fold more potent than acronycine in inhibiting L 1210 cell proliferation. All these active compounds bear a methoxy group at position 6 and at least one ester group at C-2. The presence of the methoxy group markedly increased the cytotoxicity, as shown by 7 1 . which is one hundred fold more potent than 22. These preliminary results prompted us to test some 1,2-dihydroxy1,2-dihydroacronycine derivatives for antitumor activity in vivo. We used two standard experimental models, the i.p. P 388 leukemia and the s.c. colon 38 adeno-carcinoma. The results are summarized on Table 6, in terms of percent T/C (survival or tumor volume) obtained at the optimal dosage, i.e. the dose giving the best therapeutic effect without toxicity. Against P 388 leukemia, acronycine was moderately active while compounds 66., 69L, 70. and 7_L were markedly active, at doses 4 to 16 fold lower. Against the colon 38 adenocarcinoma, compounds 66., 6£ and 70. were highly efficient. C/\s-l,2-diacetoxy-l,2-dihydroacronycine (66) was the most active, all treated mice being tumor-free on day 23. As previously reported (81), acronycine was also markedly active on this model, but less than 66., and at a 16 fold higher dose.
811
Table 6 - Antitumor activity of l,2-dihydroxy-l,2-dihydroacronycine derivatives in comparision with acronycine (58). P 388 leukemia Compound
1 L6.
LI lA II
4.
dose T/C % survival 200mg/kg ip 125 25mg/kg ip 289 25mg/kg iv 220 12.5mg/kg ip 258 50mg/kg ip 201 12.5mg/kg ip 202
Colon 38 adenocarcinoma dose T/C tumor volume
200mg/kg ip 4 12.5mg/kg 0
ip
6.25mg/kg ip 18 12.5mg/kg ip 13 6.25mg/kg ip
68
1
1
CONCLUSION
Only little was known about the structure activity relationships in the acronycine series, as far as modifications at positions 1 and 2 were concerned. A rational study based on naturally occurring models has led for the first time in that series, to the synthesis of compounds more active in vivo than acronycine itself as antitumor drugs. Indeed, l,2-dihydroxy-l,2dihydroacronycine esters exhibit promising antitumor properties with a broadened spectrum of activity and an increased potency when compared with acronycine, on several tumor strains in vivo. C / 5 ' - l , 2 - d i a c e t o x y - l , 2 dihydroacronycine appears of particular interest in this respect, due to its high activity against P 388 leukemia and against the highly resistant solid tumor colon 38 adenocarcinoma.
ACKNOWLEDGEMENTS The authors wish to thank the many colleagues and students who have stimulated their interest in the acronycine series for the past few years, and
812
more particularly Pr. M. Koch and Drs G. Baudouin, M. Brum-Bousquet and A. Elomri (Universite Paris V, France), Prs. A.-L. Skaltsounis, S. Mitaku and E. Mikros (University of Athens, Greece), Drs J. Pusset and T. Sevenet (Mission CNRS in New-Caledonia), and Pr Gh. Atassi and Dr A. Pierre (Institut de Recherches Servier, Issy-les-Moulineaux, France).
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814
47 48 49 50 51 52
53 54 55 56 57 58
59 60 61 62 63 64 65 66 67 68 69
A. Elomri, S. Michel, F. Tillequin, and M. Koch, Heterocycles, 34 (1992) 799-806. F. Tillequin, Actual. Chim. Ther., 23 (1996), in press. J. Reisch and W. Probst, Arch. Pharm. (Weinheim), 322 (1989) 31-34. J. Reisch and P. Dziemba, Arch. Pharm. (Weinheim), 324 (1991) 67-71. J. Reisch and A.A.W. Voerste, Sci. Pharm., 62 (1994) 255-260. A. Monks, D. Scudeiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, and M. Boyd, J. Natl. Cancer Inst., 83 (1991) 757. A. Elomri, A.-L. Skaltsounis, S. Michel, F. Tillequin, M. Koch, Y. Rolland, A. Pierre, and Gh. Atassi, Chem. Pharm. Bull., 44 (1996) in press. H. Erlenmeyer and H. M. Weber, Helv. Chim. Acta, 21 (1938) 614-615. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, and M. Koch, Planta Med., 54 (1988) 24-27. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, M. Koch, Y. Rolland, A. Pierre, and Gh. Atassi, Pharm. Res., 13 (1996) 939-943. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, M. Koch, J. Pusset, and G. Chauviere, J. Nat. Prod., 49 (1986) 1091-1095. A. Elomri, S. Mitaku, S. Michel, A.-L. Skaltsounis, F. Tillequin, M. Koch, A. Pierre, N. Guilbaud, S. Leonce, L. Kraus-Berthier, Y. Rolland, and Gh. Atassi, J. Med. Chem., 39 (1996) in press. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, and M. Koch, Synthesis, (1992) 1068-1070. A.-L. Skaltsounis, S. Mitaku, G. Gaudel, F. Tillequin, and M. Koch, Heterocycles, 34 (1992) 121-128. M. Bert, M. Koch, and M. Plat, Phytochemistry, 13 (1974) 301-302. F. Tillequin, G. Baudouin, M. Koch, and T. Sevenet, J. Nat. Prod., 43 (1980) 498-502. B. Couge, F. Tillequin, M. Koch, and T. Sevenet, PI. Med. Phytother. 14 (1980) 208-212. G. Baudouin, F. Tillequin, M. Koch, M.E. Tran Huu Dau, J. Guilhem, J. Pusset, and G. Chauviere, J. Nat. Prod. 48 (1985) 260-265. M. Brum-Bousquet, F. Tillequin, M. Koch, and T. Sevenet, Planta Med., 51 (1985) 536-537. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, M. Koch, J. Pusset, and G. Chauviere, Heterocycles, 26 (1987) 2057-2063. S. Mitaku and J. Pusset, PI. Med. Phytother., 22 (1988) 83-87. M. Brum-Bousquet, S. Mitaku, A.-L. Skaltsounis, F. Tillequin, and M. Koch, Planta Med., 54 (1988) 470-471. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, M. Koch, and J. Pusset, Ann. Pharm. Fr. 47 (1989) 149-156.
815
70 71 72 73 74 75 76 77 78 79 80 81
A.-L. Skaltsounis, L. Sedrati, F. Tillequin, M. Koch, J. Pusset, and T. Sevenet, Nat. Prod. Lett., 5 (1995) 281-287. S. Mitaku, A.-L. Skaltsounis, F. Tillequin, M. Koch, J. Pusset, and T. Sevenet, Nat. Prod. Lett. 7 (1995) 219-225. M. Brum-Bousquet, S. Mitaku, A.-L. Skaltsounis, F. Tillequin, and M. Koch, unpublished results. J. Reisch and A. Wickramasinghe, Monatsh. Chem., 121 (1990) 709-712. J. Reisch and M. Top, Pharmazie, 46 (1991) 745. J. Reisch and K. Schiwek, Liebigs Ann. Chem., (1994) 317-318. W.S. Bowers, Amer. ZooL, 21 (1981) 737-742. G.E. Pratt, R.C. Jennings, A.F. Hamnet, and G.T. Brooks, Nature, 284 (1980) 320-323. D.M. Soderlund, A. Messeguer, and W.S. Bowers, J. Agric. Food Chem. 28 (1980) 724-731. W.S. Bowers, P.H. Evans, P.A. Marsella, D.M. Soderlund, and F. Bettarini, Science, 217 (1982) 647-648. A. Conchillo, F. Camps, and A. Messeguer, J. Org. Chem. 55 (1990) 17281735. R.T. Dorr, J.D. Liddil, D.D. Von Hoff, M. Soble, and C.K. Osborne, Cancer Res. 49 (1989) 340-344.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
817
Chiral Synthons by Selective Redox Reactions Catalysed by Hitherto Unknown Enzymes Present in Resting Microbial Cells Helmut Simon and Helmut Gunther Institut fur Organische Chemie iind Biochemie, Technische Universitat Miinchen D 85747 Garching, Germany Abstract: Resting cells of various strict anaerobic or anaerobically grown facultative microorganisms are useful biocatalysts for redox reactions in a preparative scale. This will be shown for Clostridia and Proteus species. Electron donors may be hydrogen gas, the cathode of an electrochemical cell, often formate, but in some cases also carbon monoxide. In addition to well-known reactions catalysed by yeasts, hitherto unknown reactions are also catalysed very effectively. These reactions are performed with substrate concentrations between 0.1-0.6 M. The productivity numbers are usually 10-500 times higher than with yeasts. For maximum productivity and long stability of up to 600 h under operational conditions, artificial electron mediators such as viologens or quinones and others in 1 mM concentration are essential. The stereo- and regioselectivity is excellent. In a few cases special conditions have to be observed which can be understood biochemically. The enzymes catalysing the reductions of many different 2-enoates, 2-enals, the oxo group of 2-oxo carboxylates, carboxylates without activation to aldehydes have been isolated and partially characterized. The enzymes are reversible and are not dependent on pyridine nucleotides. They are able to transfer single electrons. For dehydrogenations very different electron mediators can be used. For example anthraquinone-2,6-disulphonate is very effective. But useful NAD(P)H dependent enzymes are also present in Clostridia. Some of them contain high enzyme activities by which NAD^ or NADP^ can be reduced with the above mentioned electron donors and mediators to NAD(P)H. Because these enzymes are reversible, selective NAD(P)^ dependent dehydrogenations are also possible. By reduction many carboxylates carrying a chiral carbon atom in a-and/or in B-position, (R)hydroxy carboxylates especially those with additional functional groups and chiral alcohols have been prepared. 2-Oxo aldonates and aldarates and other 2-oxo carboxylates have been obtained by dehydrogenation. Preparative redox reactions under pyridine nucleotide regenerating conditions will be demonstrated with different substrates. Since all reaction sequences contain single electron transferring steps they can be carried out in electrochemical cells. Their over-all reaction rate can be continuously monitored by measuring the electrical current. The systems are very useful for the preparation of stereospecifically deuterated products.The optimal growth conditions of the commercially available microbial cells are indicated. 1.
INTRODUCTION
Many stereo- and regioselective redox reactions have been carried out on a preparative scale by the catalysis of enzymes or microorganisms. The majority of these reactions involves the reduction of keto groups to chiral secondary alcohols or derivatives thereof Not only the reductions of CX-double bonds, but also selective dehydrogenations are of interest. A well-known example of a regioselective biocatalytic dehydrogenation is the formation of L-sorbose from D-sorbitol which is actually the key step in the synthesis of vitamin C. In the last two decades many new redox enzymes have been isolated, characterized and also applied for preparative work (1-4). The use of isolated enzymes for redox reactions requires the regeneration of reduced or oxidized coenzymes, e. g. the pyridine nucleotides NADH, NADPH, NAD^ or NADP^. This has been studied intensively
818
(Section 5-5.2). In most cases an additional enzyme has to be applied for this regeneration. If microbial cells are used for redox reactions instead of isolated enzymes, one can differentiate between growing or resting cells and crude cell extracts. In the latter case cells are broken for instance by freezing and thawing or by another procedure. For microbial transformations it may be an advantage to work with resting or broken cells instead of growing cells. Actually the cells do not have to be alive. The general aspects for using cells and the advantages or disadvantages of applying growing or resting cells are discussed in the literature (1,4-6). Applying microbial cells instead of isolated enzymes may have advantages (Section 1.1). The number of organisms available from the various collections of microorganisms exceeds the number of commercially available enzymes by far. For a preparative organic chemist it may be relatively easy to grow a suitable microorganism. In contrast, the isolation of an enzyme can be more troublesome especially if an isolation procedure for the necessary enzyme has not been published. Using whole cells often, but not always, solves the problem of coenzyme regeneration. A microbial transformation is different from a fermentation. For a more detailed discussion see references (1,3,4,6). 1.1 Some differences of the here described systems to those usually applied The microbial redox reactions described in this article are mostly conducted with strict or facultative anaerobic, prokaryotic cells. They have been grown in volumes of up to 300 litres and stored in the form of wet packed cells under an atmosphere of nitrogen in gas tight containers at about -15 °C for weeks or even years. After thawing of the frozen cells a suitable part can be applied for redox reactions and the residual material can be frozen again. That means that resting cells or actually mostly crude cell extracts are used because the applied organisms break rather easily by freezing and thawing. Freeze dried cells can be stored for a long time at room temperature. All operations are conducted under an atmosphere of nitrogen. In special cases enzymes, coenzymes and/or the crude extract of another cell type can be added to these broken cells. By such measures the efficiency of the systems can be increased by 1-2 orders of magnitude. For examples see Section 5.2. The reactions described here show typical differences from the well-known reductions carried out by yeasts especially Baker's yeast. The efficiency of the conversion of substrates (educts) using microorganisms can be characterized by a productivity number PN (7,8): PN =
amount of product (mmol) dry weight of catalyst [kg] x time [h]
Bioconversions with a high productivity number compared to a low one mean that less biocatalyst is necessary and/or a better volume-time yield can be reached. If, for example, 50 mmol of a product is to be synthesized in 24 h, approximately 100 g of catalyst is required, if its productivity number is 20 which may be the case if yeasts are
819 applied in a classical way. Only 2 g biocatalyst are needed if the productivity number of the applied microorganism is 1000. Moreover, it is much simpler to separate for instance 10 g of a product from 2 g of a microorganism than from 100 g. With high productivity numbers the reaction volume can also be often drastically reduced. In the presence of catalytic concentrations of artificial electron mediators (Section 1.2) the majority of the preparations described here show productivity numbers from 500 to 20 000 or more. Yeast (9) usually only shows productivity numbers in the range from 0.5-50. The main reason for the low productivity numbers of yeast can be seen from Scheme 1. In reductions catalysed by yeasts carbohydrates are normally used as reducing agents (source for electrons). NADH is formed as a catabolic intermediate. The reaction is accompanied with the formation of acetaldehyde most of which is reduced with NADH to ethanol. This unwanted reaction may consume as much as 95-99 % of the NADH, and the intended reduction of a CX-double bond takes place only to an extent of < 1-5 %. XH
carbohydrate
CH3CHO CU.CU.OU
SCHEME 1: Flow of reducing equivalents in Baker's yeast from carbohydrates to NAD^ forming NADH which is consumed in a competitive way. Often 100-1000 molecules of ethanol are formed for each molecule ofRR 'CHXH According to Reactions [l]-[3] we showed the possibility of stereospecific reduction with various yeasts which were supplied with electrons from continuously electrochemically reduced methylviologen (MV^*) instead of carbohydrates (10). The following reactions take place if a ketone such as hydroxyacetone is reduced. 2 MV^^ + 2e 2MV^* + H^ + NAD^ H"^ + NADH + CH3COCH2OH Sum: CH3COCH2OH + 2H* + 2e
cathode yeast •
yeast ^
NADH + 2MV"" + CH3CHOHCH2OH CH3CHOHCH2OH
[1] [2] [3] [4]
As will be shown in Sections 5 and 6 methylviologen can be reduced at the cathode of an electrochemical cell to the mono cation radical MV^* which transfers electrons by the action of an enzyme type which we call AMAPOR for artificial mediator accepting pyridine nucleotide oxidoreductase. Finally the NADH is used for reducing the substrate by the catalytic action of an NADH dependent reductase. By this procedure (10) the productivity number of a yeast was about 60 times higher than by the classical procedure involving the application of a carbohydrate as an electron donor (11).
820
Yeasts, especially Baker's yeast can be bought at many places but it may cause a series of problems: Cells bought at different times or from different places may vary in their activity for various reductions. A well-known example is the reduction of ethyl 3oxobutyrate or other 3-oxo esters. It is often described in the literature together with very empirical measures to improve the enantiomeric excess of ethyl (5)-3-hydroxybutyrate (7). Baker's yeast may contain altogether 4 reductases which reduce 3-oxo esters. Two of them form the R- and two the iS-enantiomer (12). The enantiomeric excess of the product depends on the activity of the various enzymes under the reaction conditions, such as substrate concentration, pH value, temperature, storage conditions of the yeast before its application, etc. Since two of the enzymes are NADH and the other two are NADPH dependent (12) the ratio of the products also depends on the relative concentrations of both reduced pyridine nucleotides. The concentrations of NADH and NADPH again depend generally on the metabolic state of the yeast and on the enzyme activities forming them. In addition resting cells strongly differ in their capability to degrade pyridine nucleotides (13). According to our experiences the concentration of pyridine nucleotides is often too low especially if a reaction runs over 1-2 hours. See for example Table 29. These simple facts clearly show that it is a disadvantage if cells contain various enzymes catalysing the same reaction with different stereochemistry of the products. If the yeast strain, the growth and reaction conditions are not strictly defined, the results often cannot be reproduced. Therefore the procedures have to be described exactly. In this respect the existing literature is sloppy in many cases. In the following reductions are described with cells which do not show the briefly mentioned problems of yeasts applied in the classical way. All the strictly anaerobically grown cells of anaerobic or facultative organisms are able to accept hydrogen gas, many accept formate and some carbon monoxide as electron donors and contain redox enzymes in often high activities which take up the electrons and deliver them to viologens and other artificial mediators which transfer them further (Section 1.2). The standard redox potential E°' of the electron delivering reactions H2 HCOO" CO +
H2O
• 2H^ ^ H^ • 2H^
+ + +
2e CO2 CO2
+ +
[5] [6] [7]
2e 2e
are -420, -420 and -560 mV, respectively. Therefore the equilibrium constants for the reduction of CO- or CC-double bonds are very favourable and there is no by-product formed which consumes electrons. The enzymes hydrogenase, the non-pyridine nucleotide dependent formate dehydrogenase and carbon monoxide dehydrogenase transfer electrons produced by Reactions [5]-[7] to viologens or other mediators (Reactions [5a]-[7a] and Scheme 2a). H2 + 2V^^ HCOO" + 2V^^ CO + H2O + 2V^^
• 2H' + • H^ + ^ 2H^ +
2V CO2 CO2
+ +
2V^* 2V^*
[5a] [6a] [7a]
821
The application of enzymes detected by us in recent years together with the above indicated electron donors in the presence of catalytic concentrations of artificial electron mediators will be described. Clostridium tyrobutyricum, C. kluyveri, C. formicoaceticum or C. thermoaceticum are strict anaerobes. Proteus species are facultative microorganisms since they are able to grow in the complete absence of oxygen as well as in its presence. The expression of some redox enzymes in facultative anaerobes is quite different depending on aerobic or anaerobic growth conditions of the cells. As an example the application of two anaerobically grown Proteus species will be given. The mentioned electron donors have the following advantages: (i) The equilibrium of the reductions of CC- or CO-double bonds with hydrogen gas, formate or carbon monoxide is far on the product side, (ii) In resting cells the electron donor can be used solely for the desired reaction since there is no other competing, unsaturated compound present. Side reactions, which can occur, for example with acetaldehyde (Scheme 1) or other products that are formed from carbohydrates, do not take place, (iii) The consumption of hydrogen gas can be monitored manometrically. Also the progress of a reduction with formate can roughly be estimated by the carbon dioxide formed. The reaction can also be carried out in electrochemical cells (Section 6). The microorganisms applied by our group are well defined and available from internationally known collections of microorganisms. All of them transfer electrons from hydrogen gas to the unsaturated substrates. Under anaerobic conditions in the presence of an antibiotic the reductions can be performed for periods of up to several hundred hours. However, most reactions can be carried out within 5-24 h. More than 99 % of the protons which are fixed to the substrate together with two electrons are derived from the water. Therefore the here described methods are effectively suitable for preparing compounds stereoselectively labelled with deuterium if ^H20 is used instead of ordinary water during a reaction. The applied microorganisms are freeze dried before suspending them in ^H20 (14,15). This will be shown in Section 2.5.3. Finally the redox enzymes in anaerobes seem to be less regulated, show less substrate or product inhibition than enzymes from yeasts, and often the enzymes possess a broad substrate specificity. Nevertheless the regio- and stereoselectivity is usually very strict. 1.2. Artificial electron mediators According to our experience the productivity numbers obtained with the aforementioned microorganisms can be enhanced by the use of catalytic concentrations (0.5-2 mM) of artificial electron mediators. Their properties and aspects of their use have been described in a review article (6). Table 1 lists some mediators with their redox potentials. The cells or crude cell extracts which will be described proceed by ways revealed by Schemes 2a and 2b, respectively. Electrons supplied by hydrogen gas, formate, carbon monoxide or the cathode of an electrochemical cell are channelled via an artificial electron mediator such as a reduced viologen (V^*) or others to the enzymes reducing
822 TABLE 1 Structure and standard potentials of some artificial mediators Structure
Abbreviation
Standard potential EO'^CmV)
CH3—N(
))—(C
)N—CH,
HN m i NH
.Al-XLlll -443/-772 ®)
^MV v
C''^°J^
Co-sep
H2N(CH2)3-NQ)—(0^(CH2)3-NH2
DAPV
a)
-440
b)
-302
c)
-377/-686 ^) b)
^»'-^'Q)-Q^<^^ H2NOC-CH2-N( ) ) — ( C )N-CH2-CONH2
^^ CAV
-331
•»"
o
CI a) b) c) d) e)
Cobalt-sepulchrate Phosphate buffer, 0.1 M, pH 7.0 Tris/HCl buffer, 0.1 M, pH 7.0 Average of different values in the literature Standard potential of a second electron transfer
")
-3027-632®^
O
DCPIP
c)
+217
823
med^ electron donor
*ox
NAD(P)H^
^ ^
:x
or 2e
E2 NAD(P) •^red med,red
XH
SCHEME 2a: Regeneration of reduced artificial mediators for reductions with nonpyridine nucleotide dependent or pyridine nucleotide dependent reductases. IfES is a pyridine nucleotide independent reductase accepting electrons from artificial mediators, only this enzyme is necessary, if the mediators are reduced electrochemically. If they are reduced enzymicly El has to be included. Reduced pyridine nucleotides are formed by E2. These artificial mediator accepting pyridine nucleotide oxidoreductases (AMAPOR) catalyse Reaction [8] and [8a] forming NAD(P)H. Further examples are given in the text. Artificial mediators are indicated by „med". Y are natural mediators except pyridine nucleotides. They may assist the electron transfer, but they are not necessary.
Anode electron acceptor
SCHEME 2b: Regeneration of oxidized mediators for dehydrogenations with nonpyridine nucleotide dependent or pyridine nucleotide dependent dehydrogenases. If EI is a pyridine nucleotide independent dehydrogenase reducing medox to medred it can be reoxidized by an anode of an electrochemical cell or by an enzyme E3, which delivers the electrons to an acceptor such as dimethyl-sulphoxide or others. Pyridine nucleotide dependent enzymes El produce NADH or NADPH by the dehydrogenation of their substrates. The reduced pyridine nucleotides are reoxidized by the AMAPOR E2, which in turn reduces an artificial mediator medox to medred- Medred delivers electrons to E3 or an anode. For the reoxidation ofE3 see text.
824
CX-double bonds. If the reductase is pyridine nucleotide dependent, the electrons are used to regenerate NADH or NADPH by rather ubiquitous AMAPORs according to Reactions [8] and [8a]. NAD"" NADP^
+
medred
+
r
medred
+
H^
•
4
NADH
+
medox
[8]
NADPH +
medox
[8a]
2. REDUCTION OF THE CC-DOUBLE BOND OF a,B-UNSATURATED CARBOXYLATES, ALDEHYDES OR a,B-UNSATURATED METHYL KETONES. Cells of Clostridium tyrobutyricum (DSM 1460) from Deutsche Sammlung ffir Mikroorganismen (16) grown on (£')-2-butenoate contain enoate reductase (EC 1.3.1.31). We detected this enzyme some time ago and characterized it partially in respect to its kinetics and reaction mechanism (17,18). Structurally it is a dodecamer of 73 kDa subunits containing an Fe/S cluster together with FAD and FMN (19-21). It reduces 2enoates, 2-enals and 3-enones according to Reactions [9] to [10] (Table 2). Reactions [9] and [9a] show that the enzyme accepts electrons from NADH or from reduced methylviologen (8,17,20). Whereas the reduction of enoates is not reversible, saturated aldehydes can be dehydrogenated by enoate reductase as shown by Reaction [12] (18). Additional reactions catalysed by this enzyme are also shown in Table 2. In earlier papers we termed this microorganism in which we found enoate reductase as Clostridium La 1. For details and for differences between this organism and C. kluyveri see (22,23). 2.1 Reduction of 2-enoates Table 2 reveals the multitude of catalysed reactions and Table 3 shows the surprising broad substrate specificity of enoate reductase from Clostridium tyrobutyricum DSM 1460. For a series of substrates Table 3 shows kinetic data (6,8,18,20,24). Only a few of these substrates can not be prepared in a sterical pure form with whole cells or crude extract from C. tyrobutyricum without additional measures. There are two aspects which have to be emphasized: Whole cells contain a 2-enoyl-CoA reductase (EC 1.3.1.8) besides enoate reductase (EC 1.3.1.31) (Scheme 3). After offering (£')-2-butenoate, (£)2-pentenoate or (£')-2-methyl-2-butenoate to the cells a part of these enoates is converted to the CoA-esters which are reduced to the saturated acids. As shown by Scheme 3 the trans-diMiiion of the hydrogen occurs by this 2-enoyl-CoA reductase in the opposite way as that catalysed by enoate reductase. But there are only a few enoates which are activated by C. tyrobutyricum to the CoA-esters and then reduced. As the above mentioned cases show, they have to be rather similar to (£')-2-butenoate. If C. tyrobutyricum is grown on glucose instead of crotonate it forms no enoate reductase but only 2-enoyl-CoA reductase. With cells of C. tyrobutyricum grown on crotonate the above mentioned substrates can be reduced in a stereochemically defined way when
825 TABLE 2 Reactions catalysed by enoate reductase. The stereochemical course of reactions [9a]-[10b] is the same as that indicated for reaction [9]. COO
COO
+ NADH +
+ NAD^
W
• ^R^RCH - CH^RCOO' + 2MV"" •^RCH = CHCOO" + NAD^ + HX
[9]
^RCX = CHCOO-
+ 2MV'* + 2 ^ 4- NADH + H^
^R^RC = C^RCHO ^R^RC = C^RCHO
+ NADH + H" 4=^ 'R'RCH - CH'RCHO + 2V^* + 2H^ ^=^ ^R^RCH - CH^RCHO
+ +
NAD^ 2Y'
[10a] [10b]
fs[AD^
+ 2V^*
+
2V^^
[11]
+ +
DCPIPred
[12a] [12b]
'R^RC = C'RCOO"
^R^RCH-CH^RCHO + DCPIPox ^R^RCH-CH^RCHO -f O2 CHO
+
H^ ;—• NADH • ^R^RC = C^RCHO — • 'R'RC = C'RCHO CHO
R
H2O
[9a] [9b]
[13]
R
the formation of the enoyl-CoA is inhibited. This can be achieved by the presence of arsenate and only low concentrations of phosphate. Section 2.5.3 gives an example of the preparation of sterical pure (2iS',3iS)[2,3-^H]-pentanoate. According to Scheme 3 it is possible to prepare sterical pure (iS)-2-methylbutyrate or (/^)-2-methylbutyrate from (E)-!methyl-2-butenoate, or the pure enantiomers of (2,3)-[2-^H]-butyrate or valerate. O 11 C-X
H-C—^R
X=0 ,H
H-C—^R
enoate reductase 2
4
C—X
X=SCoA enoyl-CoA reductase
,
O II C-X I
'R-C-H ^R-C-H
SCHEME 3: A few l-enoates (see text) are reduced by enoate reductase or after activation to a 2'enoyl-CoA-ester also by enoyl-CoA reductase. The stereochemical course of both reactions is different (25). In resting cells ofC. tyrobutyricum the reaction ofenoylCoA reductase can be blocked (Section 2,5,3).
826
TABLES Substrates of enoate reductase that are reduced according to the reactions shown in Table 2. All CC-double bonds in addition to the a,P position are E configured. The relative rate 100 % corresponds to 22 U*mg'^ of purified enoate reductase at pH 6.0 and 0.25 mM NADH. The K^ value for NADH is 0.012 mM and for reduced methylviologen 0.4 mM (8). With reduced methylviologen the F^ax values are about 2 times higher than with NADH. In two cases (No. 39 and 49) X is CHO instead of COO". The substrates 45 and 46 are a-enones. Other enoates which have been reduced by cells only but not by isolated enzyme are given in Table 4. Residues R correspond to those in Table 2. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
R'
R^
R^
CH3 H H H H H H CH3 CH3 H H CH3 H CH3 H H H H H H NHCHO F CI Br I F Br CI CI CH3 H H H H
CH3 CH3 C2H5 CH3 (CH3)2CH CH3CH=CH COOCH3 COOCOOCH3 COOCH3 CeHs CeHs CftHs CeHs p-ClCeHt P-O2NC6H4 ;7-CH30C6H4 /7-(CH3)2NC6H4 o-HOCfiHi 0,W-(CH30)2C6H3 CfiHs CH3 H CH3 H CgHs CfiHf P-CIC6H4 H HOCH2CH2 HOCH2CH2 CH3 CH3 (CH3)2C=CHCH=CHCH2
H H CH3 C2H5 H H H H H CH3 H H CH3 CH3 H H H H H H H H CH3 H CH3 H H H /j-ClCsHt H CH3 HOCH2CH2 CH3OCH2CH2 H
V ' max
Km
100 1.5 280 0.8 11 11 130 3 1 ' 0.15 9 26''-'^55 250 1.7 190 16 125 0.2 167 0.03 9 0.04 8 0.08 88 30 44 29 35 8 21 150 6.2"^ 180 5.2° 150'' 28° 180° 21° 30 0.10 76 0.50 20 0.10 0.03 120 10 10 9 15 5 14 T
827 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
H H H H H H
(CH3)2C=CHCH=CH(CH2)3 CH3 (CH3)2C=CH(CH2)2(CH3)C=CH C6H5(CH2)9 (CH3)2C=CH(CH2)3 CH3(CH2)2C=C-
(CH2)I4 CH3 H C3H7-S-(CH2)5 CH3 CH3(CH2)6
CH3 (H3)2C=CH(CH2)3
H H H H H
C6H5CH=
H H
63 H 64 H
(CH3)2C= CH3 5' '-CH3(CH2)6-2 '-C6H4N2-4-C6H4' HOC2H4 HOC2H4 C2H5 H p-nOCeYU CaHsCH^C C6ri5CH2CH=C (CH3)2C=CH CH3(HOCH2)C=CH C2H5CH=CH
H CeHs (CH3)2C=CH(CH2)2 CH3
0.19 16'^'' 3.3 97 0.01 60^^ 0.06 163'-'' 1.4 17.5 0.26 20' 115 0.03 140 0.05 a.h
H
CaHs
i.r
ga,hI
Cyclohex-2 enone 2-CH3-cyclohex-2-enone H CH3 H CH3 CH3 H H H H H H H H H CH3 Br
19a 0.01
H H H CH3
H H H
-
83"f 0.04 1.1" 58f 0.09 'JQb.g
0.13
110 15 i
a a
H H H H H CH3 (CH3)2C=CH(CH2)2
a a a
0.5 0.014 0.1
Only the a,P-double bond is reduced. Reaction could not be performed under saturating conditions. ^ 90 % (E)' and 10 % (Z)-isomer. Marked substrate inhibition above 40 mM. ^ Marked substrate inhibition above 10 mM. X = CHO. ^ For increased substrate solubility the reaction solutions contained 7 % ethanol (v/v) for No. 8 and 10 % methanol (v/v) for No. 50. The complete substrates are shown. ^ Reduced in the presence of arsenate. ^ (£)-2-methyl-3-[4-(5heptylpyrimidine-2-yl)-phenyl]-propenoate. Table 3 (No. 16) shows that (jE')-p-mtro-cimiamate or derivatives can be reduced by enoate reductase, too. However, this reaction can be performed only with isolated enoate reductase using NADH as electron donor. Reduced methylviologen spontaneously reacts with nitro groups. Aliphatic as well as aromatic nitro compounds are also reduced by ferredoxins present in Clostridia (26). We prepared many different saturated acids carrying a chiral centre at carbon atom 2 and/or 3 from 2-enoates. Many of these acids have been carefiiUy checked for their opti-
828
TABLE 4 Stereospecific hydrogenation of 2-enoates with whole cells of Clostridium tyrobutyricum on a preparative scale. Productivity numbers (PN) are given for 1 atm of hydrogen gas and 1 mM methylviologen. Cobalt sepulchrate (2mM) instead of methylviologen shows « 85 % of these rates. Residues R correspond to those in Table 2. Productivity numbers may be higher for cells grown under optimised conditions (Section 2.4). No. R' 1 2 3 4 5 6 7 8
H H H H H H H H
9
H
10 H 11
H
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
H H H H H CHj CH3 CH3 CH3 CH3 CH3 CH3 OCH3 OCH3
CN Br Br C2H5 C2H5 C2H5
R^
K'
H H CH3 H CH3
H CH3 H p-CfiHiBr
C2H5 C3H7 ^0 CH2
9^ or
CeHs (C2H5)CftHs ;p-C6H40H p-CfiRiBr p-C6H2-(3,5-Br)-NH2
H H CH3 CH3 CeHs 5' '-(CH3(CH2)6)-2 '-C6H4N2-4-C6H4'' C2H5CH= ;?-ClC6H4 p-C6H4Br
210 380 1900 1700 1900
H
2470
H
1900
H
380 -
H H H CH3 H CH3 H CH3 H H
1630 1000 3800
730 5700
570 2800
400 560 180°
H H OC2H5
CeHs
Br CeHj CH3CH= C6H5CH = CeHs (C2H5)=
6100 3200 3100
CH3 H H H
H Br
PN
1700 1400
380 1700
450 1500
-
^ Reduction equivalents were supplied by formate in the presence of Proteus vulgaris cells (Section 2.5.2). (^-2-methyl-3-[4-(5-heptylpyrimidine-2-yl)-phenyl]-propenoic acid.
829
cal purity. Such analyses have also been carried out in other laboratories which used the products. In all cases only one enantiomer could be detected (27-30). Regarding the enoates which can be readily reduced by cells of C tyrobutyricum or by the isolated enzyme the following rules have been observed: Residue ^R (Table 2) should not be too large; enoates in which ^R is a phenyl ring, NHCOCH3 or an OC2H5 residue are not accepted in place of NHCHO and OCH3, respectively. The halogens F, CI, Br and I are tolerated in the a-position, but are reductively eliminated in the 6-position. In this case the following reaction takes place: RCX=CHCOO-
+
4[H]
• RCH2-CH2COO-
+
HX
[14]
For a discussion of this mechanism see (20,24). The choice of ^R (Table 2) is subject to the least number of restrictions. It can be extremely large, i. e. enoate No. 22 in Table 4 . From this substrate almost 100 g have been reduced in a strictly enantiomeric fashion. If ^R and ^R are interchanged (i. e. if the {£)and (Z)-isomers of substrates of the type ^R^R=CXCOOH are used) different enantiomers or diastereomers are produced. Therefore, ElZ mixtures of substrates in which the 6-carbon becomes chiral by reduction cannot be employed if enantiomerically pure products are expected. As can be seen by comparison of substrates 12 and 13 or 30 and 32 in Table 3, branching in the 6-position diminishes the rates without much influence on the A^m value. In the case of mono-methyl esters of 2- or 3-methylfumarate, substrate 9 and 10, the Fmax is diminished, too, if the methyl group stands in the 6- instead of the aposition. In this case the ^m value for the 6-branched substrate increases about tenfold. A negative charge in y-position increases the K^a and decreases Fmax drastically as can be seen by the comparison of substrates 8 and 9 (Table 3). If a long chain in a 2-enoate is rigid due to additional double bonds, the K^^ value increases and that of Kmax decreases. This can be seen by comparing substrates 37 and 38. Double and triple bonds in conjugation to the double bond in 2-position do not render an enoate a non-substrate (substrates 37 and 40.) The double bond can also be part of a ring as is shown by the fact that cyclohexene-1-carboxylate 41 is a suitable substrate. From a practical point of view very selective reactions are possible. As aheady mentioned the CC-double bond of/>-nitro-cinnaniic acid can be reduced with enoate reductase and NADH without attacking the nitro group. The following formally a,P-unsaturated acids are not substrates of enoate reductase: benzoic, orotic, nicotinic, and shikimic acid. Aliphatic acylates show no measurable inhibition, if they are present at concentrations of 100-fold the K^ values of the corresponding enoates. Measurable inhibitions can be observed with phenyl group-containing acylates. 3-Phenylpropionate in concentrations of 38 mM shows about 86 % inhibition. Fumarate, which is only a poor substrate, inhibits the reduction of enoates by NADH as well as by reduced methylviologen (Reactions [9] and [9a]). However, the reduction of NAD^ by reduced methylviologen (Reaction [11]) is not inhibited by fumarate. Interestingly, inhibitors such as morin or dicoumarol, which probably bind to the flavin domain of enoate reductase, do not impair the reduction of enoates by reduced methylviologen, but all reductions with NADH are inhibited.
830
Reaction [9] is inhibited by 0.4 mM morin or dicoumarol to 80 and 60%, respectively. The same concentration of dicoumarol inhibits Reaction [9a] by less than 5 % (17). These facts and other results (20) are indicative of three binding domains of enoate reductase: one for NADH which can be blocked by dicoumarol or morin, another for enoates which can be occupied by fumarate, and a third one for reduced methylviologen. For studies on the iron/sulphur centres see (21). Enoate reductase exclusively splits off the (4iS)-hydrogen atom from NADH. There is no direct hydrogen transfer from NADH to the products. If (45)-[^H]-labelled NADH with a total tritium activity of 4.67*10^ decays per minute was dehydrogenated with an excess of enoate, the isolated product showed a tritium content of less than 0.005 % of that of the NADH. Almost all the tritium was in the water. In the absence of an enoate as acceptor, the tritium exchange from (45)-[4-^H]-NADH catalysed by enoate reductase is very slow. Depending on the substrate concentration, the isotope effect of the reduction of (£')-2-methyl-2-butenoate with (45)-[4-^H]-NADH varies from 6.8 to 1.3. The presence of NAD^ decreases the isotope effect (17). From a mechanistic point of view, the behaviour of the P-halogenated enoates is of interest (24). (Z)-3-Chloro-cinnamate and (Z)-3-chloro-butenoate as well as (Z)-3-bromocinnamate consume in the presence of enoate reductase 2 mol NADH per mol instead of 1 mol as other enoates do. The products are the saturated halogen-free carboxylates (Reaction [14]). Most of the substrates shown in Table 3 have also been hydrogenated with resting cells of C. tyrobutyricum. Further examples are shown in Table 4 with productivity numbers between 180 to 6100. Two consecutive reactions are taking place. The first is the reduction of MV^^ by hydrogen gas or an other electron donor (Scheme 2a) and the second is Reaction [9a]. By observing the colour of the suspension at the beginning of the hydrogenation one can judge which reaction is rate limiting. If the suspension is not or hardly coloured Reaction [9a] is faster than the reduction of MV^^ by hydrogen gas catalysed by hydrogenase (Reaction [5a], Scheme 2a). When the reaction comes to its end the suspension shows the blue to violet colour of MV^*. The reason is the decrease of the substrate concentration leading to a decrease of the rate of Reaction [9a]. If the suspension is blue right from the beginning of the hydrogenation Reaction [9a] is slower than the reduction of the viologen. If neither hydrogen consumption takes places nor a blue colour appears the system was more or less inactivated by oxygen or due to another reason. The influence of H2-pressure is shown in Table 8. Table 5 reveals the products of substrates which have been deuterated in ^YiiO. In other cases deuterated enoates were reduced in H2O. The reductions in ^H20 lead to acylates with two chiral carbon atoms due to stereospecific deuteration of methylene groups. Most of these have been used in studies of biosynthetic pathways of natural products. For examples see (29,30). Reactions in ^H20 proceed usually slower than in H2O. The differences may be less than a factor 2 if optimal pH and p^H values are determined and applied. The p^H and pH value for optimal reaction rates are not the same (31).
831
2.2.1 Reduction of fumarates and derivatives with Clostridium formicoaceticum (32) (5)-2-Methyl-, (iS)-2-ethyl- and (iS)-2-chlorosuccmate were prepared in concentrations of 1 M or higher with Clostridium formicoaceticum grown on fiiictose and fiimarate (Section 5.3). The enantiomeric excess of the products was higher than for most of the homogeneous chemical catalyses (33,34). Some time ago we observed with Proteus vulgaris a higher productivity number for the reduction of 2-methylfumarate (1500 instead of 1080), but we tested no additional substrates (35). The stereoselective dideuteration of the substrates is possible by using freeze-dried C. formicoaceticum cells, which show for reductions nearly the same rate in ^H20 and H2O. Fructose and fructose/fumarate grown C formicoaceticum cells contain a dimethyl maleate and a dimethyl fumarate reductase. To our knowledge both enzymes have not been described in the literature so far. Besides the artificial mediators mentioned so far safi'anine T in 0.75 mM concentration shows about 80 % of the activity observed with 1 mM methylviologen or 1 mM anthraquinone-2,6-disulphonate for the reduction of 2-methylfumarate. The absence of a mediator decreases the reduction rate to 3-4 %. C formicoaceticum is also usefiil for stereoselective hydrations of fumarate and maleate derivatives (36). 2.2 Reduction of 2-enals and allyl alcohols As shown in Table 6 enoate reductase or Clostridia containing this enzyme (C tyrobutyricum or C kluyveri) catalyse the reduction of a,P-imsaturated aldehydes (enals) (Table 2 Reactions [10a] and [10b]). In contrast to saturated carboxylates, saturated aldehydes can be dehydrogenated to a,P-unsaturated aldehydes (enals) by enoate reductase in the presence of electron acceptors such as oxygen or dichlorophenol-indophenol, (Table 7, and Table 2 Reactions [12a] and [12b]), (18). In low concentrations the reduction of 2-enals proceeds with similar rates as those of 2enoates. At concentrations above 1-5 mM the rate decreases to about 30-50 %. There are other marked differences in the reactions with carboxylates and aldehydes, respectively. All enoates tested so far have been reduced with enantiomeric excess >96 %. No racemization could be observed with saturated carboxylates, even by very sensitive methods using tritium-labelled compounds. The difference between aldehydes and carboxylates can be understood, if one takes into account that the acidity of the a-protons of aldehydes is orders of magnitude higher than that of carboxylates. On the basis of the aforementioned halogen elimination from 3- halogeno-2-enoates by enoate reductase, we suggested an 1.4-addition mechanism for enoates, in which during the reduction the Pcarbon atom picks up a hydride ion followed by the stereospecific fixation of a proton in a-position (20, 24). If this is the case, one can assume that for the dehydrogenation or racemization of a saturated carboxylate or aldehyde, an a-proton has to be split off in the
832
TABLE 5 Various stereoselectively a-, a,P- or P-deuterated carboxylates prepared form deuterated substrates in H2O or by reduction of nonlabelled substrates in ^H20 buffer and freeze dried microorganisms. No.
Product
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
(25',3i^)[2,3-^H,^H]- Propanoic acid (i^)[2,3-^H]-2-Methylpropanoic acid (2S,3^)[2,3-^H]- 3-Phenylpropanoic acid (2i?,3/?)[2,3-^H]- 2-Methyl-3-phenylpropanoic acid (25,3i?)[2,3-^H]-3-(3-Hydroxyphenyl)-propanoic acid (2S,3/?)[2,3-^H]-3-(4-Hydroxyphenyl)-propanoic acid (2R,3S)[2,3-^n]-2' Methyl butanoic acid (2S)[2,3-^H]-4-Phenylbut-3-enoic acid (2S,35)[2,3-^H]-Pentanoic acid (2i?)[2-^H, l-^^C]-4-Phenyl-(Z)-pent-3-enoicacid (25,3i^)[2,3-^H]-5-Methylhex-4-enoic acid (2/?,3S)[2,3-^H]-5-Methylhex-4-enoic acid (2S,3/^)[2,3-^H]-6 Hydroxy-5-methylhex-4-enoic acid (2S,3i^)[2,3-^H]-hept-4-enoic acid (25,35)[2-^H]-Oct-4-inoic acid (3^[3-^H]-Oct-4-inoic acid (27^)[2-^H]-Oct-4-inoic acid (2S,3i^)[2,3-^H]-Oct-4-inoic acid (35)[2-^H]-5,9-Dimethyldec-4,8-enoic acid (25',3i^)[2,3-^H]-5,9-Dimethyldec-4,8-enoic acid (2i^)[2-^H]-12-Phenyldodecanoic acid (2S,35)[2,3-^H]-12-Phenyldodecanoic acid (27?,3i^)[2,3-^H]-12-Phenyldodecanoic acid (3/^)[3-^H]-9-Thiadodecanoic acid (25',3S)[2,3-^H]-9-Thiadodecanoic acid (IS) [2-^H] -Succinic acid monomethyl ester {2S,3S)[2-^UM', 3-^H]-Succinic acid monomethyl ester (2/?,35)[2,3-^H]-2-Succinic acid" (25,3S)[2,3-^H]-2-Methylsuccinic acid" (25,3S)[2,3-^H]-2-Ethylsuccinic acid" (25,35)[2,3-^H]-2-Methylsuccinic acid dimethyl ester"
^ The compounds were reduced with C formicoaceticum.
Amount (mMol) 1 7 45 8.3 7.4 18 4.6 1 50 1.7 8.6 17 2 32 30 2 11.6 25 0.35 3.5 2.5 1.6 1.9 1.3 2.6 2.4 2.4 2.4 2.4
833 first step. This is no problem with an aldehyde, but obviously it cannot be achieved with a carboxylate. Not as easily understood is the racemization of saturated aldehydes and the fact that both a-protons of 3-phenylpropanal are exchanged by enoate reductase. The reason may be an unspecific Schiffs base mechanism (18). Nevertheless enoate reductase can be used to prepare chiral alcohols from a,B-unsaturated aldehydes or alcohols (allyl alcohols) (37,38). TABLE 6 Reductions of unsaturated aldehydes and unsaturated alcohols with enoate reductase. All of the here mentioned products possess (i^)-configuration (18). No. Substrate 1
^CHO
Biocatalyst
Other additives
C. kluyveri
H2 or Ethanol
Ph CHO
2^
Enoate reductase
Electron donor
MV"^
Cathode
NADH^
Ethanol
CHzCH C. kluyveri
^CHO ,,^=\ Ph
Enoate reductase andHLADH'
NADH
^CHO
Enoate reductase
MV^'/hexane
Ethanol
10-33"
^CHO
21
CH2OH
74
CH2OH
53
CH2OH
95
Ph H2
P h ^ 6'
^CHO \
Ph
Ph
5"'^
p/
ee%
Ph"\
Ph
4'
Product
Ph
CHjCM Enoate reductase ^/=< andHLADff Ph ^
NADH'^
CHjOi Enoate reductase /*^\^ from C. kluyveri, Ph andHLADff
NADH^
Ethanol Ph Ethanol Ph
^ More than 10 experiments. Values of enantiomeric excess depend on temperature, reaction time etc.. The ee values decrease with increase of temperature and reaction time. A total volume of 50 ml contained: 50 mM substrate, 1 mg (~ 12 U) enoate reductase. After 17 h at 18 °C > 95 % product. ^ A total volume of 4 ml contained: 70 |Limol ethanol, 1.4 fimol NADH, 100 mg C. kluyveri or 1 U enoate reductase. After 22 h at 25 °C > 95 % product. ^ The NADH contained about 5 % NAD, which is necessary for the dehydrogenation of the substrate to 2-methyl-3-phenyl-enal which is then reduced to the (i?)-2-methyl-3-phenyl-propanol. ^ Horse liver alcohol dehydrogenase, 2.5 U. After 22 h at 25 °C > 95 % product.
834
Clostridium tyrobutyricum or C. kluyveri cells are able to reduce allyl alcohols according to Reaction [15]: RCH=CHCH20H
+ NAD(P)H
• RCH2-CH2CH2OH
+
NAD(P)H
[15]
The following sequence of reactions was observed (38): RCH=CHCH20H -*• RCH=CHCHO -•RCH2CH2CHO -* RCH2CH2CH2OH
[16]
The first step is catalysed by the pyridine nucleotide dependent alcohol dehydrogenase (NAD^-dependent in C kluyveri and NADP^-dependent in C. tyrobutyricum) leading to the 2-enal which in turn is reduced by enoate reductase to the saturated aldehyde (Reactions [10a] and [10b]). The saturated aldehyde is fiirther reduced to the alcohol. The rate of the reduction depends not only on the activity of the involved enzymes but also on the concentration and on the ratio of NAD(P)^/NAD(P)H. In the presence of MV^* which is formed by the reduction of MV^^ by the system H2/hydrogenase (Reaction [5a]), the ratio NAD(P)^/NAD(P)H is too small for the fast and complete dehydrogenation of an allyl alcohol since the first step of the reaction sequence [16], which needs NAD(P)^, is too slow. It turned out that ethanol is a better electron donor than hydrogen gas in this case. For the reduction of 50-70 mM (£')-2-methyl-2-butenol to {R) -2-methyl1-butanol by C kluyveri the optimum concentration of ethanol was 1-2 M and the pH was kept at about 8. For C. tyrobutyricum the optimal conditions have not been determined yet. They should not be too different. 2.3 Dehydrogenation of aldehydes The rate of the dehydrogenation of saturated aldehydes (Table 7) depends to a great extent on the pH. It rises fi-om pH 6 to pH 9 and probably has not reached its maximum. However, at pH 10, enoate reductase is inactivated within a few minutes. The K^a values of saturated aliphatic aldehydes are rather high, i. e., about 66 mM for {R,S) 2-methylbutanal. That of aromatic aldehydes such as 3-phenylpropanal is 1.4 mM, which is about 15 times higher than that observed for the corresponding unsaturated aldehyde. It has not been determined yet, whether the step reducing enoate reductase by the saturated aldehyde or its reoxidation is rate limiting. Rather surprising is the relatively high stability of enoate reductase in the presence of oxygen for the dehydrogenation of aldehydes (1. exp. Table 7). In the presence of NADH, enoate reductase can be inactivated by oxygen within a minute. Even after 30 min, the dehydrogenation of 2-methyl-3-phenylpropanal with oxygen continues, and about 50 % of the activity for the reduction of an enoate is left, if oxygen is carefiiUy eliminatedfi*omthe system before the test (18). Reaction [13] in Table 2 is actually a consequence of the ability of enoate reductase to dehydrogenate saturated aldehydes with NAD^. Kinetic and analytical studies showed that the apparent isomerisation is actually a redox reaction needing traces of NADH besides enoate reductase. The 2-methenyl-aldehyde is reduced by NADH and the NAD^
835
formed dehydrogenates the intermediate saturated aldehyde to the a,P-unsatiirated 2methylaldehyde, which is probably thermodynamically more stable. TABLE? Dehydrogenations, racemizations, exchange of a-protons and rearrangements of aldehydes catalysed by enoate reductase. Depending on the rate of reaction different amounts of enzyme have been used. The racemizations have been studied at pH 7.0, the H/^H-exchange and the dehydrogenations at pH 8.0 (18). Electron acceptor ^ m (mM) (mM)
Substrate
(R,s) ,—
CHO
^ CHO
Relative rate (%)"
Air
3.6
1.5
DCPIP *" (0.05)
1.4
12
Ph
Product CHO Ph
CHO Ph
(R.S) /
CHO (^
DCPIP *" (0.05)
66
9
CHO
none
3.6
2-3
none'
-
3-4
NADH (0.5)
~
(R) / — < Ph ^
CHO Ph CHO
0.4
^
^
CHO CHO (R,S) , ^ Ph ^ CHO ^ ^ H Ph ^H
CHO
^ Compared to reductions. T)ichlorophenol-indophenol. Reaction studied in ^H20 at p'^H 7.0.
2.4 Growth and harvest of Clostridium tyrobutyricum up to 300 litres In C tyrobutyricum (DSM 1460) the specific activities of hydrogenase and enoate reductase depend on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased, and then dropped to about 10 % of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase/enoate reductase changed fi'om 120 to 1 (23). Depending on the growth conditions of the cells the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase. The specific activities of ferredoxin-NAD oxidoreductase and butyryl-CoA dehydrogenase (enoyl-CoA reductase. Scheme 3) increased 3-4 fold during growth on crotonate
836
instead of glucose. Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities, and others which cause the reverse effect (23). When the Fe^^ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity, and very low enoate reductase activity were obtained in the stationary phase. At Fe^^ concentrations above 3*10"^ M enoate reductase increased, and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulphate was studied (23). Growth of Clostridium tyrobutyricum DSM1460 on crotonate The following procedure is a reasonable compromise of enzyme activity and simplicity of growth: One litre of medium contains 70 mM sodium crotonate, 150 mg (NH4)2HP04, 100 mg K2HPO4, 33 mg MgCl2*6H20, 0.6 mg MgS04*7H20, 40 mg CaCl2*2H20, 0.4 mg MnS04*2H20, 0.4 mg FeS04*7H20, 50 mg NH4CI, 10 mg (NH4)6Mo7024*H20, 0.04 mg biotin, 0.8 mg p-aminobenzoic acid, 1 mg resazurin, and 8 ml of a 50 % K2CO3 solution. The resazurin is an indicator for oxygen. As long as the solution is not pink its oxygen content is rather low. Starting from 100 ml volumes, 2, 20 and up to 300 litres of C. tyrobutyricum cultures have been grown. The growth time is usually 15-20 h. The culture should stay for 4-5 h in the stationary phase. 2.5 Practical aspects and examples of enoate reductase application Wet packed cells of C. tyrobutyricum grown as described in Section 2.4 are used. They were grown in 200-300 1 and after centrifiigation stored in gas tight containers under an atmosphere of nitrogen at -15 °C. If not mentioned otherwise the indicated weights are given for wet packed cells. The dry weight is about 20 % and the protein content of the wet packed cells is about 10 %. The sensitivity of the enzymes in the cells against oxygen can be diminished by adding to the cell suspension about 10 ^M hexacyanoferrate III which oxidizes enoate reductase and probably some other enzymes. This treatment is especially recommended if the cells are lyophilised for deuterations. The cells can be lyophilised without loss of hydrogenase and enoate reductase activity. Without this treatment usually 60-80 % of the enzyme activity can get lost during freeze drying, even if contact with oxygen is tried to omit. Freeze dried cells are used for reductions in ^H20. Cells of C tyrobutyricum can be immobilised and stored. In operation at 35 °C the enzymes have a half-live of about 10 days. The catalyst can be reisolated and used again. The presence of an organic solvent such as decalin does not diminish the enzyme stability (39).
837
As shown in Table 8 the addition of 1 mM methylviologen and the increase of the pressure of hydrogen gas enhances the rate of the reduction of (£')-2-methyl-2-butenoate. One mM methylviologen under 1 atmosphere of hydrogen gas causes an increase of the productivity number of about 110 %. Increasing the concentration of the dissolved hydrogen by increasing the pressure of hydrogen gas leads to a further enhancement of the enoate reduction rate by about 4.5. Often the hydrogenase reaction is limiting. As explained in Section 2.1 the reductions in +•
progress will be coloured by the violet MV only faintly or not at all. That means Reaction [5a] is rate limiting. Since the A'm value for dissolved H2 of hydrogenase from C. tyrobutyricum is even higher than the solubility of hydrogen gas under atmospheric pressure in water the rates of hydrogenations can be increased by using higher hydrogen pressure. The regeneration of MV^* can also proceed in an electrochemical cell by Reaction [1]. For practical aspects see Section 6. If larger amounts of enoates are reduced the regeneration of MV^' can also be carried out by Reaction [6a]. However, this reaction is not catalysed by C tyrobutyricum. Therefore cells oi Proteus vulgaris, P. mirabilis or C thermoaceticum have to be added. As an example the reduction of 286 mmol (£')-2methyl-3-[4-(5-heptylpyrimidine-2-yl)-phenyl]-propenoate will be described (Table 3 No. 50 and Table 4 No. 22). Depending on the substrate the productivity number for the reduction of many enoates with cells of C. tyrobutyricum is in the range of 400-1500. The optimal temperature is 35-37 C, the optimal pH is 6.0-6.2. Purified enoate reductase shows a pH maximum at 5.2 for reductions with methylviologen. At pH 4.5 and 6.2 the rate is two thirds of that at pH 5.2. However, at pH values of 5.0 or lower enoate reductase is labile. When resting cells are used together with hydrogen gas the pH optimum for two consecutive reactions has to be considered, i. e. Reaction [5a] followed by the reactions as indicated by Table 2. Stereospecific deuterations of 2-enoates (14,15,29,30,40) have to be carried out in ^H20 buffer with freeze dried cells (Table 5). Freeze drying under exclusion of oxygen leads to cells without loss of enzyme activity if again under anaerobic conditions 1 g wet packed cells is slowly stirred or shaken with 0.5 ml of a 10 JLIM solution of potassium hexacyanoferrate-III for 20 minutes at 35 C. Under these conditions enoate reductase and probably also hydrogenase are transferred into the oxidized state. It is also possible to reduce enoates which are only sparingly soluble in water. This was shown by reducing 286 mmol of (£)-2-methyl-3-[4-(5-heptylpyrimidine-2-yl)-phenyl]propenoate in about 3 litre buffer. The ATm value estimated by extrapolation is about 130 ^M but its solubility only about 30 |iM. Since it is difficult to shake or stir a volume of 3 litres effectively in order to saturate it with hydrogen gas, Proteus vulgaris cells were used as a second microorganism possessing a viologen dependent formate dehydrogenase. This enzyme catalyses Reaction [6a]. Another possibility would have been to conduct the reaction in a stirred autoclave with increased pressure of hydrogen gas (Table 8).
838
Reductions of enoates with enoate reductase or with resting cells in an electrochemical cell allows to study the influence of various parameters from one single experiment (Section 6). TABLE 8 Dependence of the productivity number on the pressure of hydrogen gas for the reduction of (£')-2-methyl-2-butenoate by Clostridium tyrobutyricum with and without methylviologen. Pressure (atm)
Methylviologen
Productivity number (mmoUkg'^h'^)
normal 80 normal 20 40 80
_ + + + +
730 1250 1540 4300 6630 7080
The stirred mixture contained in 30 ml 0.1 Mpotassium phosphate buffer, pH 7,0, 0.2 g wet packed cells of C. tyrobutyricum, 0.5 M (E)-2-methyl-2-butenoate and, if indicated above, 1 mM methylviologen. At pH 6.0 the productivity numbers should generally be higher. 2.5.1 Hydrogenations in a scale from 0.1 to about 20 mmol For planning a hydrogenation experiment in a larger scale it is useful to know roughly the productivity number which can be expected. To get an idea for a hitherto not applied substrate one can do an experiment in a scale of 50-100 jxmol. Warburg vessels combined with mercury filled manometers which can be shaken in a thermostated water bath are very useftil for such a hydrogenation. The volume for the hydrogen gas should be about 4-6 times that of the volume of hydrogen gas which will be consumed by a quantitative reduction. The amount of wet packed cells applied for a hydrogenation depends on the time in which the reduction shall proceed. It is no problem to run an experiment for 50-100 h. All experiments lasting longer than 8 hours should be carried out in the presence of an antibiotic, which does not contain reducible functional groups. Tetracycline in a concentration of 30-40 ^M can be recommended. Standard experiment for determining the hydrogenation conditions of a 2-enoate A two-necked flask of 20-50 ml connected to a mercury filled manometer is flushed with nitrogen or argon and filled with a suspension of 0.4 g wet packed cells of C. tyrobutyricum in 3 ml 100 mM potassium phosphate buffer, pH 6, containing 1 mM methylviologen and 50-300 ^mol of an enoate. If it is expected that the experiment runs longer
839
than 6-8 h about 0.4 mg tetracycline per 10 ml suspension should be added. The protective gas is substituted by hydrogen gas, the system closed and the flask immersed in a thermostated bath of 35 "C. After thermal equilibration a slight over-pressure of about 20-25 mm mercury is applied. The system can be shaken or stirred. If the volume of the system is known the rate of dihydrogen uptake can be followed by the attached manometer. If the hydrogenation proceeds slowly and for a longer time changes of the atmospheric pressure should be included calculating the dihydrogen consumed. If the second neck of the hydrogenation flask is closed with a suitable air tight septum a small sample of the solution can be withdrawn with a syringe and analysed. Another possibility is to open the flask under a stream of hydrogen gas and withdraw a small sample which can be analysed by GLC or HPLC. The results of such an experiment provide information for experiments on a larger scale. 2.5.2 Reduction of 0.286 mol of sparingly heptylpyrimidine-2-yl)'phenyl]-propenoate
soluble
(E)-2-methyl'3'f4'(5'
A saturated solution of the sodium salt of the substrate forms a concentration of 30 |LiM in a 0.05 M potassium phosphate buffer, pH 6.5, at 35 °C. The substrate was reduced as a suspension. Less than 1 % of the material was in solution. In 3 litres 0.05 M potassium phosphate buffer 286 mmol substrate was suspended together with 270 g wet packed cells of C. tyrobutyricum and 33 g oi Proteus vulgaris. The reduction was carried out with 0.5 M sodium formate in the presence of 1 mM methylviologen under slight stirring. After 74 h 95 % of the substrate was reduced. The ee value of the product was >98 %. 2.5.3 Stereospecific deuteration of two enoates (i) 5,9-Dimethyl-deca-2,4,8-trienoate in ^HjO to (25',3i?)-[2,3-^H]-5,9-dimethyl-deca4,8-dienoate. Twenty five mmol (4.88 g) of the trienoate were dissolved in 250 ml potassium phosphate buffer, p^H 7.0, together with 1 mM methylviologen, 25 mg tetracycline*HCl and 27.7 g of freeze dried cells of C. tyrobutyricum. The system was hydrogenated under H2 (not ^Yii)- After finishing the reduction the suspension was acidified to pH 1.5 and extracted with ether. After purification by chromatography on a silica gel column with hexane/diethyl ether 3:2 (v/v) the yield was 87 %. (ii) Deuteration of (£)-2-pentenoate to (2iS',3iS)-[2,3-^H]-pentanoate. Under exclusion of oxygen 10 g freeze dried cells of C. tyrobutyricum were suspended for 30 min in 300 ml 50 mM arsenate in ^H20 at 35 °C. Afterwards to this solution 50 mmol (6.1 g) sodium E-pentenoate and 1 mM methylviologen were added. The shaking flask was connected with a volume of 4 litres H2 and a manometer. After the disappearance of the substrate (18 h) the suspension was acidified, centrifiiged and the aqueous layer extracted with ether. To the washed ether extract some water was added and the pentanoic acid titrated. After the evaporation of the ether the yield was 81 %.
840
3. REDUCTION OF 2-OXO CARBOXYLATES TO (jR)-2-HYDROXY CARBOXYLATES AND DEHYDROGENATION OF (^)-2-HYDROXY CARBOXYLATES TO 2-OXO CARBOXYLATES Chiral 2-hydroxycarboxylic acids are valuable synthons (41,42). This is especially true if they contain additional functional groups which by chiral induction can be converted to further chiral centres in diastereoselective chemical reactions. From this point of view chiral 2-hydroxy-3-en carboxylates or 2-hydroxy-4-oxo carboxylates are of interest. With anaerobically grown cells oi Proteus mirabilis or P. vulgaris we prepared according to Reaction [17] many (i^)-2-hydroxy carboxylates including those mentioned before. Proteus spec.
RCOCOO"
+
2YC +
2e
•
(i?)-RCHOHCOO-
[17]
Also very different (/?)-2-hydroxy carboxylates could be dehydrogenated on a preparative scale. Scheme 4 summarises the unusually broad range of applications. With {K)-2hydroxy-3-enoiccarboxylates (43) we performed a series of reactions which led to highly functionalised acids with 3 chiral centres (44,45) (Table 13). The product of 22 (Table 10) was used to prepare various 5-methoxy-pentano-l,4-lactones (45). Tables 10, 11, 15 and 16 give an impression and suggestions for applications oi Proteus species with other substrates. For preparative work the so far applied 2-hydroxy carboxylate oxidoreductases are pyridine nucleotide dependent (l,2a,3). The substrate specificity of the individual representatives of the various oxidoreductases is rather narrow. For thermodynamical reasons the quantitative dehydrogenation of 2-hydroxy carboxylates by pyridine nucleotide dependent redox enzymes is not so easy. But such a reaction is interesting, too. For details see Section 3.2. It was of interest to purify and characterize the (7?)-2-hydroxy carboxylate oxidoreductase fi-om Proteus vulgaris since it turned out that it does not react with NAD(H) or NADP(H) (46). Enzymes catalysing reversible reductions of carbonyl groups have been characterizedfi^omarchaea, eubacteria and eukaryotes. The enzymes fi-om eubacterial or eukaryotic sources depend on pyridine nucleotides; the enzymes from archaea also use pyridine nucleotides or deazaflavin F420. This coenzyme has a structure related to that of NAD(P)^. Since viologens are suitable artificial electron mediators for this previously unknown enzyme we named it (/^)-2-hydroxy carboxylate viologen oxidoreductase (HVOR). In the purified form the membrane bound enzyme shows a specific activity of 1800 U*mg"^ protein for the reduction of phenylpyruvate (46). (One U (unit) reduces 1 fimol 2-OXO carboxylate per minute).
841
DMSO
HO R
w
H2 and/or HCOO" or electrochemical cell
DMS + H,0
Other electron acceptors may be amine N-oxides
CO.
COO-
a) Many different mediators are possible with redox potentials E°' between -440 and +217 mV
Types of residues R
Examples
(i)
imbranched and branched alkyl, aryl
R =fromCH3 to at least C6H13 R = (CH3)3C; fR,5>C2H5CHCH3, C6H5
(ii) OH groups in 3 and other positions
R = (7?,5>C3H7CHOH; various aldonic acids can be dehydrogenated
(iii) One or two conjugated CC-double bonds
R = R' ( / V / ) ^^j ^^ 2^ith alipathic or aromatic R\ also R"0CH2O
(iv) Additional CO group (v) -00C(CH2)n
R = C6H5COCH2.
and others
n = 1 to 3 at least
So far no satisfactory reactions with R = R'CHX-(X = CI or Br) Scheme 4: Reductions of2-oxo carboxylates to (R)-2-hydroxy carboxylates and dehydrogenations of (R)-2'hydroxy carboxylates to 2-oxo carboxylates. Properly grown Proteus cells contain hydrogenase and formate dehydrogenase which can be used to deliver electrons carried by viologens or anthraquinone-2,6-disulphonate to HVOR. The latter carrier also transfers electrons from HVOR to DMSO reductase during dehydrogenations of (R)-2'hydroxy carboxylates.
842
TABLE 9 Kinetic parameters determined for reactions catalysed by hydroxy carboxylate viologen oxidoreductase and dimethyl-sulphoxide reductase in Proteus vulgaris. Substrate
K, (mM)
Methylviologen,d Methylviologen,d Methylviologend Benzylviologen,d b Benzylviologen,d b Benzylviologen,d b Carbamoylmethyl viologend Anthraquinone-2,6-disulphonated Pyruvate Pyruvate Pyruvate Pyruvate Phenylpyruvate Phenylpyruvate Phenylpyruvate 4-Methyl-2-oxopentanoate 4-Methyl-2-oxopentanoate 4-Methyl-2-oxopentanoate 3-Methyl-2-oxobutanoate 3-Methyl-2-oxobutanoate 2-Oxobutanoate 2-Oxobutanoate 2-0~0-3,3-dimethylbutanoate 2-Oxoglutarate
0.0 16 0.033 0.2 0.06 0.0 12 0.0 1 <0.07 1.8 c,e 0.6 0.44 0.72 22.0 0.14 0.13 0.04 0.68 0.25 0.20 0.74 4.0 0.70 1.82 7.5 2.5
V,,
mM"
2.0 1.o 5.0 2.5 50.0
0.04 4.6
3.6
4.0 10.0 0.72 0.4 0.8 10.0 0.35 0.08 0.34 0.05 0.08 0.36 0.35 0.08 0.08 0.35
2"d Substrate Phenylpyruvate 4-Methyl-2-oxopentanoate 3 ',4'-Dihydroxyphenyl-2-oxo-propanoate 4-Methyl-2-oxopentanoate 3-Methyl-2-oxobutanoate Phenylpyruvate Phenylpyruvate Pyruvate Methylviologen,d Carbamoylmethylviologen,d Benzylviologen,d Anthraquinone-2,6-disulphonate,d Methylviologen,d Carbamoylmethylviologen,d Benzylviologen,d Methylviologen,d Benzylviologen,d b Carbamoylmethylviologen,d Methylviologen,d Benzylviologen,d Benzylviologen,d b Methylviologend
Phenyl-CH=CH2-Fql-CH=CHPhenyl-CH=CH-CH=CH-
(cyclohexan-2-one-yl)-CH=CHFCH2HOCH1(CH3)2CH-
0.8 0.4 0.5 0.7 0.7 0.7 2.3
(R)-Lactate (R)-Lactate @)-Lactate (R)-Phenyllactate (R)-Phenyllactate (R)-Phenyllactate (R)-2-Hydroxy-4-methylpentanoate
0.76 2.2 390 ' 0.42 0.65 0.4 0.58
Anthraquinone-2,6-disu1phonate0, Carbamoylmethylviologerb, Methylene blue Methylviologe%, Thionine Toluidine blue
0.9 0.05 0.02 6.6 0.02 0.03
Dimethyl-sulphoxide 1.4 c,d Anthraquinone-2,6-disulphonate~ 0.14 c,d
2.9 2.3 2.1 3.6 0.7 1.5 2.6 2.48 1.4
1.o 1.o 1.o 1.o 1.o 1.o 1.o 1.o 10.0 10.0 10.0 2.0 10.0 1.o
2.3 1.6 1.3 8.8 0.85 1.00
50.0 15.0 15.0 15.0 15.0 15.0
Benzylviologen,d b Benzylviologen,d b Benzylviologen,d b Benzylviologen,d b Benzylviologen,d b Benzylviologen,& Benzylviologenr& Carbamoylmethylviologerb, Anthraquinone-2,6-disulphonateox Anthraquinone-2,6-disulphonate, Carbamoylmethylviologeq,, Cyanomethylviologeh, Anthraquinone-2,6-disulphonate, Carbamoylmethylviologerb,
@)-Lactate (R)-Lactate (R)-Lactate (R)-Lactate (R)-Lactate (R)-Lactate
saturating Anthraquinone-2,6-disulphonatered saturating Dimethyl-sulphoxide
Concentrations of the second substrate. Benzylviologen was reduced to an extend of = 2/3. The apparent K,,, values for the substrates of the enzymes were determined with crude extracts at pH 8.5. Values for dimethyl-sulphoxide reductase. This K,,, value of (@lactate was determined in the presence of 500 mM pyruvate (pyruvate inhibition). a
843
844
The oxidoreductase contains 1 molybdenum cofactor and 4 iron and 4 sulphur ions per subunit of 80 kDa (46). The natural electron mediator is not known yet. The enzyme reacts not only with viologens, but also with many other artificial mediators. Their redox potentials are in the range from -440 to +220 mV (Section 3.2, Table 14). Depending on the redox potential of the mediator in use the equilibrium constants of Reaction [17] varies by more than 20 orders of magnitude. That means reductions as well as dehydrogenations are possible very effectively. The yields of product are greater than 98 % in almost all cases studied so far. The ee values are also >0.98. As a matter of fact the (S)-enantiomer could never be observed in products. The (iS)-enantiomers become available by the dehydrogenation of racemic 2-hydroxy carboxylates with P. vulgaris (7a). By using special growth conditions extraordinary high enzyme activities in Proteus mirabilis or P. vulgaris can be achieved (Table 18) (47). Rather different examples of substrates and reaction products are shown in Scheme 4 and Tables 10 and 11. The residue R of 2-oxo carboxylates can be aliphatic unbranched or branched. No size limitation has been observed yet. Also aromatic residues are accepted. Phenylglyoxylate as well as phenylpyruvate and homologues can be reduced. The presence of various functional groups as part of R are tolerated. As already mentioned these may be additional carbonyl groups or CC-double bonds. They are not reduced by Proteus mirabilis or P, vulgaris cells. That means the HVOR is extremely regio- and stereospecific. Other enzymes which form the opposite enantiomers or by-products are not present in these cells. Unsaturated hydroxy carboxylates of the type (/^)-R-CH=CH-CHOH-COO" which were prepared from the corresponding 2-oxo carboxylates could be converted to various products with additional chiral centres with reasonable or excellent diastereoselectivity (Table 13) (44,45). A series of kinetic data for reductions and dehydrogenations are shown in Table 9. The mechanism of HVOR is not known yet. It seems not to be a ping pong mechanism since the K^ values of 2-oxo carboxylates depend on the applied mediator. The temperature dependence of HVOR with reduced methylviologen and 3phenylpyruvate shows an increase from 24-39 °C of about 2 fold per 10 "^C (48). In the a-position deuterated (7^)-2-hydroxy carboxylates have been prepared in ^H20. Examples are (/?)(2-^H)-3-dimethyl-2-hydroxypropanoic acid, (i^)(2-^H)-(2)-hydroxybutanoic acid, (/^)(2-^H)-3-phenyl-2-hydroxypropanoic acid. 3.1 Preparation of (J?)-2-hydroxy carboxylates In the presence of a catalytic concentration (0.5-2.0 mM) of an artificial mediator resting cells of both Proteus mirabilis or P. vulgaris reduce 2-oxo carboxylates to sterically pure (/^)-2-hydroxy carboxylates at the expense of formate and/or hydrogen gas. The reductions can also be carried out in an electrochemical cell where the regeneration of the reduced mediator proceeds at the cathode (Section 6, Fig. 6). Under these condi-
845
tions the productivity numbers can be extremely high. For the reduction of 2-oxo-4methyl-pentanoate we observed a productivity number of about 120 000 (unpublished) with cells grown as described in Section 3.3. The reductions shown in Table 10 would show about 2-3 times higher productivity numbers if cells grown according to this procedure would have been used. Before conducting dehydrogenations we grew cells on glucose instead of lactate. The cells used for the reductions described in Tables 10 and 11 have lower HVOR activities. Details for the reduction of various 2-oxo carboxylates have been published (7,7a,43,45,49). The 2-0X0 carboxylates which were not commercially available were prepared by several routes according to Reactions [18]-[21]. ^RCHO +
^RCHsCOCOOH
'
^RCH=C^RCOCOOH
[18]
^RCH=C^R-MgBr + EtOOCCOOEt
'
^RCH=C^RCOCOOEt
[19]
^RC0CH2^R
•
^RCOCH^RCOCOOEt
[20]
+
RCHCl-COCOOR
EtOOCCOOEt +
OH "-
•
RCHOHCOCOOR +
CI"" [21]
2-0x0 acids (12-17, 19, 21) (Table 10) were obtained in analogy to (50-52), by condensing aromatic aldehydes or derivatives such as cinnamic aldehyde with pyruvate or 2oxobutyrate (Reaction [18]). A rather general method seems to be the condensation of the Grignard products of 1-bromo-l-alkenes with diethyl oxalate (Reaction [19]) (53). The 2,4-dioxo carboxylates were prepared from suitable ketones and diethyl oxalate (Reaction [20]) (54) and the 2-oxo-3-hydroxy acids such as 10 by hydrolysing the corresponding ethyl 3-chloro-2-oxo carboxylates (Reaction [21]) which in turn were prepared from epoxides formed by condensing aldehydes with ethyl dichloroacetates (55,56). Esters of 2-oxo acids are not converted by the HVOR. If esters were the products of the 2-0X0 acid synthesis the hydrolysis was carried out as a separate step by alkaline hydrolysis or by Lipozyme® at neutral pH. Especially unstable carboxylates such as the 2,4dioxo carboxylates 18 and 20 (Table 10) were formed enzjnnically by Lipozyme® from the esters during the bioreduction. If ethyl 2,4-dioxo carboxylates are used for bioreductions the ratio of Lipozyme® activity and amount of P. vulgaris cells has to be optimised. If too little lipase is present, the reaction proceeds slowly. If too much lipase is used, labile carboxylate accumulates, and side reactions may occur. The racemic mixtures of the 2-hydroxy-3-enoic acids used for analytical studies were prepared by reduction of the oxo acids with sodium borohydride in the presence of equimolar concentrations of eerie trichloride in analogy to (57). Ce-III inhibits the reduction of the CC-double bond adjacent to the carbonyl group. This method did not work for the regioselective reduction of 2,4-dioxo carboxylates or esters. The bioreductions were performed on scales from 0.3 up to 120 mmol in the presence of 1 mM benzylviologen. Usually 0.1 g wet packed cells, corresponding to -20 mg dry weight, were used for the reduction of 0.3 mmol 2-oxo carboxylate in 3 ml buffer at the
846 expense of hydrogen gas in order to compare various substrates or to study parameters such as pH dependence etc.. The substrate concentrations was mostly 0.1 M. TABLE 10 Reductions in a preparative scale of various 2-oxo-carboxylates (RCOCOO) by Proteus vulgaris (43,45). No. R 1 2 3 4 5 6 7 8 9 10 11 12 12 12 13 14 15 16 17 18 19 20 21 21 22 23
CH3C2H5CH3-CH=CH(CH3)2CH(CH3)3C(7?,5)-C2H5-CHCH3CH3(CH2)5HOCH2FCH2(/?,5)-n-C3H7-CHOH-' C6H5-CH2 CgHs-CH^CHC6H5-CH=CHC6H5-CH=CHC6H5-CH=C(CH3)p-Cl-C6H5-CH=CHp-Br-C6H5-CH=CHC6H5-CH=CH-CH=CHC6H5-CH=CH-CH=C(CH3)C6H5-CO-CH22-Furyl-CH=CH(Cyclohexaii-2-one-yl)2-Thienyl-CH=CH2-Thienyl-CH=CHCH30CH2-CH=CHHOCH2C(CH3)2-
Scale (mmol) 0.3 10-120 0.3 0.3 0.3 10-45 0.3 0.3 10 up to 16 100 0.3 0.3 10-100 10 10 10 10 10-45 10 10 10 10 19 f 3«
Electrondonor H2orHCOOH2 H2 H2 H2 H2 HCOOH2 H2 H2 H2 or HCOO HCOOH2 H2 + HCOO' HCOO" H2 H2 H2 H2 HCOOH2 HCOOH2 HCOOHCOOHCOO"
Yield ^
PNso'
(%) 19 000 10 500 70-90 11500 10 000 2 400 8 450 90 26-35 000 8 550 7 000 6 250 20 000 * >90 17 800 >90 9 800 >90 68 600 >90 11 700 87 9 200 96 7 600 90 8 300 98 4 000 95 _d 87 78 13 800 _d 74' 90 11000 31000 90 63 98 740 >90
Isolated products; bioconversion >97 %. Sometimes the reduction of the last 20 % occurred over night without observing the rate. Therefore the productivity number after 80 % product formation is given. "" Synthesized according to RCHCl-COCOOR -^ RCHOHCOCOOR. ^ Ethyl ester combined with an esterase. The carboxylates were formed by hydrolysis from the ethyl esters by Lipozyme®.The hydrolysis rate was limiting. PNs of 1 000 - 3 000 were usually observed. ^ Yield determined by weight of the residue of the ether extract. ^ The total amount of 19 mmol in 10 ml was added in 4 portions to a starting volume of 30 ml with 1 g wet packed cells (see for comparison Fig. 1). Optimising the system in an electrochemical cell PNs of >150 000 have been reached (see also Section 6). ^ Ketopantoate (49).
847
TABLE 11 Relative initial rates of reductions of 2-oxo carboxylates to (i^)-2-hydroxy carboxylates with partially purified 2-hydroxy carboxylate viologen oxidoreductase (HVOR) fi-om Proteus vulgaris and P. mirabilis (58,59, unpublished). Relative activity (%)' P. mirabilis P. vulgaris
No.
Substrate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Phenylpyruvate Pyruvate 2-Oxobutyrate 2-Oxo-4-(methylthio)-butyrate Indolylpyruvate 5-Benzyloxyindolylpyruvate 3-Fluoropyruvate 2-Oxo-4-methylpentanoate (i^)-2-Oxo-3-methylpentanoate (/^,iS)-2-Oxo-3-methylpentanoate 2-Oxo-2,3-dimethyl-4-hydroxybutanoate Phenylglyoxalate 2-Oxononanoate Oxalacetate 2-Oxoglutarate 2-Oxoadipate 2-Oxo-(4-hydroxy-methyl-phosphinyl)-butanoate 3-Oxoglutarate Hydroxyacetone
^ In the presence of MV^*orBV^*.
100 92 _ b _ b
35 30 22 81 28 _ b
100 85 65 85 67 _ b
20 _ b
25 46 4 5
7 16 _ b 83 73 50 78 62 _ b 70 _ b 24 not aI substrate not a substrate
Not measured.
In order to obtain higher product concentrations on a preparative scale the substrate can be added repeatedly after most of the original 0.1 M substrate is reduced (43,45). An example is shown by Fig. 1. The bioreductions in a preparative scale were conducted by various protocols: (i) With hydrogen gas at atmospheric pressure, (ii) with sodium formate (Table 12) under an atmosphere of nitrogen and (iii) by the combination of hydrogen gas and sodium formate. In order to judge the dependence of reduction rates on various factors one has to be aware of the fact, that the reduction of a 2-oxo acid according to Scheme 2a consists of a series of consecutive reactions. First the oxidized viologen is reduced by hydrogenase or formate dehydrogenase at the expense of hydrogen gas or formate. In the second reaction the reduced viologen transfers electrons to the HVOR which in turn reduces the 2-oxo acid. Usually the first reaction is rate limiting.
848
Fig. 1. Time course of the reduction of altogether 0.1 mol 12 (Table 10) in a final volume of 450 ml 0.2 M phosphate buffer, pH 7.0 by 9.2 g wet packed cells of P. vulgaris, 1 mM BV at the expense of formate. Start: 0.033 mol 12 and 0.025 mol formate after about 1.5 and 3 h additional 0.033 mol 12 were added. The pH was kept at 7.0 by adding formic acid. T ^=^-~r 5 10 15 Time Ch]
Fig. 2. pH dependence of the reduction of 12 by P. vulgaris in the presence of hydrogen gas. For pH 6.0 8.0 potassium phosphate buffer and for 8.0-8.5 Tris buffer was applied.
Fig. 3. Reaction progress for the reduction of 12 with P. vulgaris at the expense of 1 atm of hydrogen gas m-^ formate o-o, and the combination of hydrogen gas and formate 4-^.
n
0.5
1
1.0
r
1.5
Time Ch]
2.0
849
Generally the commercially available benzylviologen can be used for such reductions. If 1 mM benzylviologen or another viologen is used as a mediator the reaction mixture is usually not or only faintly blue until the reaction is almost completed. Then it turns to an intensive blue colour, indicating that the reduction of the viologen by the hydrogenase or formate dehydrogenase at the expense of hydrogen gas or formate is faster than the consumption of the reduced viologen by the HVOR catalysed reaction. In a few cases methylviologen can lead to spontaneous reactions (see later and (48)). TABLE 12 Productivity numbers (PN) observed for the reduction of 2-oxo-4-phenyl-(3£)butenoate in a concentration of 0.1 and 0.2 M using hydrogen gas, formate or their combination. Electron donor 0.1 M H2 HCOOH H2 + HCOOH
10 000 30 000 91000
PN after product formation of 50% 80% 0.2 M 0.1 M 6 400 3 000 10 000
9 800 25 900 68 600
100% 0.1 M 8 600 20 000 30 000
The use of hydrogen gas for the reduction has several advantages. The progress of the reaction can be followed by the manometrically measured hydrogen consumption. The pH value of the reaction solution does not change during the reduction. However, hydrogen gas under normal pressure is less effective than formate and the larger the volume of the liquid phase the more difficult it is to saturate it with hydrogen gas, if no increased pressure is applied. The use of formate also has several advantages. A homogeneous system can easily be enlarged. The reaction rate does not depend on the shaking frequency or stirring rate as it is the case for larger volumes with hydrogen gas as electron donor. Gentle stirring is sufficient. It is more convenient to handle sodium formate than hydrogen gas. Disadvantages of the use of formate are that the progress of the reaction cannot be observed directly, and that for the reduction of a 2-oxo carboxylate with formate, an additional proton is necessary leading to an increase of the pH value during the reaction. The use of high buffer concentrations increases costs and means less elegant product isolation. Therefore, an automatic pH control system was applied which added formic acid during the reaction. Under these optimised conditions phosphate buffer concentrations of 20 mM and 0.1 g wet packed cells of P. vulgaris for the reduction of 1 mmol substrate in 0.1 M concentration were used. Conditions for conversions in a preparative scale (see above) were optimised with 2oxo-4-phenyl-3-butenoate. Reaction rates depended on pH and reached their maximum at pH 7 (Fig. 2). Table 12 shows the productivity numbers for the reduction of 0.1 and 0.2 M 2-oxo-4-phenyl-3-butenoate with hydrogen gas, formate or a combination of both
850
TABLE 13 Products with 3 c h a l carbon atoms synthesized fiom various (R)-2-hydroxy-(3E)-en-carboxylates (R)-’RCH=C’R-CHOHCOOH (44).See notes a-c on the next page.
No. Substrates
‘R
Products ’R
Yield a
de
(%)
Bromolactonization
H H p-c1c6& 2-thienyl H CH3 C6HS 5 C~HSCH=CHH
1 2 3 4
C6HS
(2S,3R,4S)-2-Hydroxy-3-bromo-4-phenyl-y-butyrolactone (2S,3R,4S)-2-Hydroxy-3-bromo-4-(p-chlorophenyl)-y-butyrolactone (2S,3R,4s)-2-Hydroxy-3-bromo-4-(2’-thienyl)-y-butyrolactone (2S,3R,4S)-2-Hydroxy-3-bromo 3-methyl-4-phenyl-y-butyrolactone (2S,3R,4s)-2-Hydroxy-3-bromo-4-(2’-E-phenylethenyl)-y-butyrolactone
55 60 83 71 85
80 88 90 90 80
Br2addition ~
~~~
6 C6H5
C6HsC 8 C6Hsd 9 CH3 7
~~~~~~
H H H H
(2S,3R,4S)-2-Hydroxy-3,4-dibromo-4-phenyl-y-butanoic acid Methyl (2S,3R,4s)-2-hydroxy-3,4-dibromo-4-phenyl-y-but~oate (2S)-5-( 1 ‘,2’-dibromo-2’-phenylethenyl)-l,3dioxolanone-4 Methyl (2s)-2-hydroxy-3,4-dibromo-pentanoate
45 98 82 87
>96 90 60 50
H H H H
(2S,3R,4S)-2-Hydroxy-3-iodo-4-phenyl-y-butyrolactone (2S,3R,4S)-2-Hydroxy-3-iodo-4-(p-chlorophenyl)-y-butyrolactone (2S,3R,4~-2-Hydroxy-3-iodo-4-(2’-thienyl)-y-butyrolactone (2S,3R,4S)-2-Hydroxyy-3-iodo-4-methyl-y-butyrolactone
71 58 64 53
>96 >96 >96 >96
Iodolactonization 10 C6Hs 11 p-clc6& 12 CH3 13 CH3
851
reducing agents. Formate causes a 2.5-3 times faster reduction than hydrogen gas. This could be different if a higher hydrogen pressure was used. We did not check this problem. The formate dehydrogenase activity can be rather high in Proteus species (Table 18). Up to a substrate conversion of 80% the reduction rates achieved with a combination of hydrogen gas and formate exceeded those calculated from the sum of the rates observed with hydrogen gas or formate, respectively (Table 12). The reduction of the last 20 % of the substrate also proceeds relatively fast. This can be explained as a consequence of low K^ values (Table 9) and low enzyme inhibition by the product. On the other hand the reduction shows a rather pronounced substrate (educt) inhibition by 2oxo-4-phenyl-3-butenoate in the region from 0.1 to 0.2 M (Table 12). In the presence of hydrogen gas and a 0.2 M solution of the substrate the rate is reduced resulting in a productivity number only 64 % of that for a 0.1 M solution. When formate is applied as reducing agent the productivity number for a 0.2 M solution of the substrate is only 10 % of that of a 0.1 M solution. This indicates that the formate dehydrogenase is more inhibited by increased 2-oxo acid concentrations than the hydrogenase. Fig. 1 shows the time course of the reduction of the substrate, if it is added in portions to a proceeding reaction and gives a protocol if high productivity numbers, high product and low buffer concentrations are aimed at. As can be seen from Table 10 most of the different 2-oxo acids can be reduced with productivity numbers in the region from 2 400 to more than 30 000. The conditions were not optimised in all cases. Cells grown as described in Section 3.3 show probably 3-5 times higher productivity numbers. The indicated yields correspond to the isolated material if more than 0.3 mmol substrate was converted. The product isolation is rather simple since relatively low amounts of biocatalyst are necessary. The conversion of substrate is complete, and no side products could be detected. In general there is no drastic effect of the residue adjacent to the 2-oxo group on the rate of reduction. The rates are almost all in the range of one order of magnitude. One or two CC-double bonds conjugated to the 2-oxo group are tolerated (Table 10, substrates 3, 12-17, 19, 21). An additional oxo group in 4-position is not reduced (products 18,20). Carbon atom 3 may be secondary, tertiary or quartemary. In the latter case (5) (Table 10) the reduction rate is relatively low. Also fluorine or hydroxy groups at carbon atom 3 (8, 9) are accepted. However, as observed with 3-bromopyruvate and 2-oxo-3-halo-4methylpentanoate chlorine as well as bromine in 3-position lead to an inhibition of the HVOR and products cannot be detected. On the basis of the broad substrate specificity of HVOR it is not surprising that the two enantiomers of 2-oxo carboxylates with a chiral centre at carbon atom 3 are reduced with similar rates Table 10 (10). The time course of the reduction of 10 mmol of (i?,5)-3hydroxy-2-oxohexanoate with hydrogen gas was studied. Taking small aliquots and anaNotes for Table 13 ^ Yields are those of the recrystallised products. The de values were obtained by HPLCanalyses of the reaction solution after disappearance of the starting material. ^ The substrate was used as methyl ester. The l,3-dioxolanone-4 derivative was used.
852
lysing them, reverse phase HPLC shows a de-value of 69 % after 19 % conversion. At the end of the reaction both diastereomers were present in equal amounts. The course of reduction of (i^,iS)-2-oxo-3-methylpentanoate was similar to that of the 3-hydroxy-2oxohexanoate (43). Almost all of the 2-hydroxy carboxylates prepared were analysed on a Chiral-1/HPLC column by ligand exchange chromatography using a dilute copper sulphate solution as eluent. As already shown a-values for both enantiomers of aromatic as well as for aliphatic 2-hydroxy acids are in the range of 1.3-1.8 with higher retention times for the (Ryi-hydroxy acids (60). For details see (43). Spontaneous reaction between 2-oxo acids and reduced viologens Concentrations of 5 mM phenylglyoxylate or 2 mM 2-oxo-4-phenyl-3-butenoate show in the presence of 0.5 mM reduced benzylviologen and in the absence of the biocatalyst in oxygen fi-ee cuvettes a slow spontaneous reaction by which reduced benzylviologen seems to disappear irreversibly. 2-Hydroxy acids as products could not be observed. Such a reaction does not seem to play an essential role in the here described biological reductions. During microbial reductions the total concentration of benzylviologen was always 1 mM but the solutions were hardly coloured. That means, the steady state concentration of reduced benzylviologen was rather low. At the end of a preparative reaction there was always a strong colour from the reduced benzylviologen indicating the presence of much of the original 1 mM benzylviologen. No spontaneous reaction was observable with the above mentioned 2-oxo carboxylates and reduced carbamoylmethylviologen which has an E° of -302 mV, about 40 mV less negative than benzylviologen. For more details regarding various viologens see (6). General procedure for reductions Oxygen was excluded from the Proteus cells. Before use, buffers were boiled and cooled under an atmosphere of nitrogen. Bulbs and other containers were flushed with nitrogen. In order to judge a new substrate and to compare its reaction rate with that of the reduction of another 2-oxo carboxylate the following standard procedure was applied: A total volume of 3.0 ml containing 0.1 M substrate, 1 mM oxidized benzylviologen, 0.1 M potassium phosphate buffer, pH 7.0, and 0.1 g wet packed cells (-20 % dry weight) of was shaken in a water bath at 35 C in little bulbs connected to mercury filled Warburg manometers under an atmosphere of hydrogen gas. The rate of dihydrogen consumption indicating the rate of product formation was registered by reading the dihydrogen pressure in suitable time intervals. If necessary besides measuring the dihydrogen consumption the concentrations of substrates and products were analysed by taking small aliquots via suitable syringes from the reaction mixture, heating the samples in closed Eppendorf caps for protein precipitation and injecting 5 or 10 fxl on a HPLC-column. Reductions up to 10 mmol of substrate at the expense of hydrogen gas were usually carried out by shaking the reaction mixtures in suitable vessels to which a manometer and a storage vessel for hydrogen gas was connected. As long as less than 1-2 mmol sub-
853
strate were applied reductions with formate as electron donor or with hydrogen gas and formate in combination were conducted in the same manner. Substrate amounts up to 0.12 Mol were reduced with formate by stirring the reaction mixture in 3-necked flasks containing a pH electrode connected with the formic acid reservoir of an automatic pH control system. Examples for preparative reductions with formate (45) Variation A without using a pH-control system. Under an atmosphere of nitrogen in a two-necked round bottom flask closed with rubber septums 1.5 equivalents of sodium formate and 1 equivalent of 2-oxo carboxylate as sodium or potassium salt, making the final concentration 100 mM, were dissolved in 80 % of the total amount of 0.1 M potassium phosphate buffer, pH 7.0. Wet packed cells of P. vulgaris were suspended in the residual oxygen fi*ee buffer and injected into the flask through the septum. After warming to 35 °C a solution of benzylviologen was injected to reach a 1 mM concentration. The suspension was stirred under a slow stream of nitrogen, passing a water trap in order to moisten the nitrogen, through an inlet and outlet syringe needle. For 10 mmol substrate 2.0-2.6 g wet packed cells of P. vulgaris were applied. The reaction rate slows down due to a pH increase. Variation B. Instead of 0.1 M phosphate buffer a 20 mM concentration is sufficient. Into the reaction mixture a pH electrode was immersed and by an automatic pH control system set to pH 7.0, 1 M formic acid was automatically added. Variation C. The set up was as described for variation B. But after the conversion of the substrate present at the beginning at 0.1 M a second and a third or even fourth portion was added in form of a concentrated solution (Fig. 1). By this procedure in 8.5 h, 0.12 mol of 2-oxo-4-phenyl-3-butenoate was converted by 9.2 g wet packed cells. Reduction of2y4-dioxo acids under in situ hydrolysis of the ethyl esters Variation A was applied. The cell suspension which was added to the reaction mixture contained per mmol of substrate 1 ml of Lypozyme®. Per mmol substrate 2 g wet packed cells of/*, vulgaris were used. 3.2 Dehydrogenation of (J?)-2-hydroxy carboxylates to 2-oxo carboxylates Also dehydrogenations of 2-hydroxy carboxylates can be rather usefiil. Nevertheless much fewer biocatalytic dehydrogenations than reductions are described. As already mentioned, HVOR in Proteus mirabilis or P. vulgaris reacts with many different artificial electron carriers (mediators) as artificial cosubstrates, varying in their redox potential by more than 600 mV (Table 14) (47,61). Therefore, depending on the redox potential of the artificial mediator the equilibrium constants {K) of the reactions shown by Scheme 4 can be changed by more than 20 orders of magnitude. Taking the relation
for the mediators methylviologen (E"" = -440 mV) and dichlorophenol-indophenol (E'' '= +217 mV) the equilibrium constants K of Reaction [17] are 7.5*10^^ and 4.6*10"^ respectively, calculated for n = 2 at pH 7 and 298 K. If the mediators are not protonated
854
in the reduced and oxidized form as it is the case with viologens, also the pH value under which the reaction is conducted has a very pronounced influence on the actual equilibrium constant K'= ^/[H^]^. The situation becomes more complicated, if the reduced form of the mediator is dissociated partially into an anionic form and protons. Reactions up to 0.6 M substrate concentrations can be carried out (Table 15) and (62). TABLE 14 Relative initial rates of the dehydrogenation of (i^)-lactate with various mediators determined with crude extracts of Proteus vulgaris (6,62). For preparative purposes the redox potential of the applied mediator should be not more negative than -200 mV. E°' Mediator (mV) -440 -360 -320 -302 -289 -252 -225 -184 -87 -8 +11 +27 +64 +217
Methylviologen Benzylviologen NAD(P)^ Carbamoylmethylviologen Safranine T Phenosafranine Anthraquinone-2-sulphonate Anthraquinone-2,6 -disulphonate Cresyl violet Meldola blue Methylene blue Toluidine blue Thionine Dichlorophenol-indophenol
(mM) 5.00 2.50 2.50 2.50 0.10 0.10 2.50 2.50 0.10 0.10 0.10 0.06 0.05 0.10
Relative activity (%) 30 117 0 100" 62 23 83 91 125 70 96 76 76 225
The reaction rate with carbamoylmethylviologen (2.5 mM) is defined as 100 %. Some electron acceptors are soluble rather sparingly. It was not determined whether the concentrations were saturating. Other redox mediators and their half wave potentials are listed in (63). If the reduction is conducted with benzylviologen (E°' = -330 to -360 mV (6)) as the mediator at pH 6 the 2-oxo carboxylates will be completely reduced since the equilibrium constant for the reduction of pyruvate as an example is 8*10^. At pH 8.5 with anthraquinone-2,6-disulphonate (E°' = -184 mV) the equilibrium constant of this reaction is about 6 orders of magnitude smaller than with benzylviologen and dehydrogenations of (i^)-2-hydroxy carboxylates can be conducted quantitatively when the reduced quinone (AQ-2,5-DS-H2) is effectively reoxidized for instance by Reaction [22]: DMSO + AQ-2,6-DS-H2
'
DMS
+
H2O +
AQ-2,6-DS
[22]
The pair dimethyl-sulphoxide/dimethyl-sulphide (DMSO/DMS) has an E'"= +160 mV. The AG of Reaction [22] is -25.1 kJ*mor^ at pH 8.5 and 25 °C corresponding to a AT
855
= 2.5*10'*. The use of anthraqumone-2,6-disulphonate as mediator is easily possible since P. mirabilis and P. vulgaris possess a DMSO reductase (Scheme 4). Its activity depends on the growth conditions (Table 18), (47,62). (i) Preparation of pyruvate from (R)-lactate as an example This reaction may not be very interesting for a research laboratory. The procedure was developed for industrial use. But there are aspects which may be interesting for the production of other 2-oxo carboxylates. From racemates of 2-hydroxy carboxylates the (i^)-enantiomer can be dehydrogenated leaving the (iS)-enantiomer. Pyruvate is an interesting building block since its 3 carbon atoms represent 3 different reactive groups accessible to various reactions. On an industrial scale (i?)-lactate is much cheaper than pyruvate. Abstracts of numerous patents published in recent years indicate that most biocatalytic pyruvate preparations show space time yields of less than 0.5 mol*l ^d"\ Only two of 18 procedures achieved values near 1. Based on experiment 7 of Table 15 we showed that up to 100 g wet packed cells oi Proteus can be applied per litre in concentrations of 0.65 M (i^)-lactate which was converted to 94 % in about 1 h to pyruvate corresponding to about 300 fold higher space time yields as those reported before. Often the productivity numbers of published procedures cannot be calculated, because the amount of cells used is not indicated. The concentration of formed pyruvate ranges from 12-590 mM. The highest published concentrations could be reached by the here described methods, too. A single enzyme from the microbial cell is sufficient for the dehydrogenation of lactate to pyruvate, if the electron mediator is regenerated by a nonenzymic method, e. g., by an electrochemical process (62). The success of pyruvate production from lactate depends also on the stability of pyruvate in the presence of Proteus mirabilis or P. vulgaris cells. In fact the cells degrade pyruvate rather effectively if the enzyme pyruvate:formate-lyase (EC 2.3.1.54) is active. Since it is known that for the activation of this enzyme ferrous ions are necessary (64) we blocked the ferrous ions by 5 mM EDTA. Under these conditions pyruvate is stable for more than 100 h (62). Another problem was to find a stable and cheap mediator and an effective regeneration system for the oxidized mediator. Table 14 shows the activity of different mediators. Under various aspects anthraquinone-2,6-disulphonate was especially usefiil. It is completely stable under the reaction conditions and can easily be reisolated if necessary. It works not only very well with HVOR but also with dimethyl-sulphoxide reductase present in Proteus mirabilis or P. vulgaris (Scheme 4). Suspensions of cells were prepared by adding 1 g wet packed cells to 1 ml of 0.1 M Tris/HCl buffer, pH 8.5, which was previously boiled and cooled under an atmosphere of nitrogen. The reactions were started by the addition of this cell suspension to the reaction mixture.
856
TABLE 15 Various methods of pyruvate formation from (R)-lactate. The procedures are applicable to other (R)-2-hydroxy carboxylates, too (62).
dry Cells No.
Volume
Mediator
weight (mmol)
(g)
1 2 3 4
P. vulgaris P. vulgaris P. vulgaris P. vulgaris 5 P. mirabilis 6 P. mirabilis 7 P. mirabilis
0.09 0.72 0.18 0.36 0.18 0.17 1.0
50b 50 70 70 50b 50 50
CAVd AQDS' AQDS' AQDS" AQDS" AQDS" AQDS"
8 P. mirabilis
0.18
70
AQDS" 0.35
0.15 0.15 0.35 17 0.15 0.15 0.15
Final electron (R)- Conver- Space time acceptor lactate sion yield (mmol) (mmol) ( % ) (moltl-'*d-')* Fumarate DMSO N-MMNO AQDS ' Fumarate DMSOf DMSO
17" 27.5 32.5 35
N-MMNO'
45
15 26
13 26 36.4 17 25 32.5 32.5
42
>99 99 >99 99 >99 >99 94 95 96 >97
PN (mmol*kg-' (dry cells)*h')
10.2 9.1 17.2 8.0 13.3 11.5 14.7 9.9 7.5 11.0
2 1200 18900 50200 23300 28100 24000 30410 (1.0 h) 20550 (1.5 h) 15650 (2.0 h) 32200
Space time yield based on 1 g P. VuIguris cells (dry weight). In 0.1 or 0.3 M TriskICI-buffer, pH 8.5. ' Due to the solubility of AQDS higher concentrations are not suitable. Carbamoylmethylviologen. Anthraquinone-2,6-disulphonate. Dimethyl-sulphoxide. N-MethylmorpholineNoxide.
a
If not mentioned otherwise, the reactions were conducted in deionized water containing 5 mM EDTA. The pH was kept constant by adding 2 or 4 Msodium hydroxide. P. vulgaris cells usedf o r experiment I were grown on glucose. All others on lactate.
857
Practical working conditions for the preparation of pyruvate with dimethylsulphoxide (DMSO) as electron acceptor A volume of 50 ml contained 26.0 mmol (/^)-lactate, 0.15 mmol anthraquinone-2,6disulphonate, 0.25 mmol EDTA, 26.0 mmol DMSO and 3.7 g wet packed cells of P. vulgaris at 40 °C. The pH 8.5 in the slowly stirred suspension was kept constant with 2 M sodiimi hydroxide. The dimethyl-sulphide formed from DMSO (Scheme 4; Reaction [22]) was removed actually completely by a slow stream of nitrogen gas. The boiling point of the dimethyl-sulphide is 38 °C, and its solubility in water is rather low. The formed dimethyl-sulphide was condensed in a cold trap. The same procedure was used with N-methyl-morpholine-N-oxide as electron acceptor instead of dimethyl-sulphoxide. However, the formed amine does not leave the system. Formation of pyruvate with stoichiometric amounts of anthraquinone-2,6-disulphonate as electron acceptor A volume of 70 ml contained 17 mmol (i^)-lactate, 17 mmol anthraquinone-2,6disulphonate, 0.35 mmol EDTA and 1.8 g wet packed cells of P. vulgaris at 40 °C. The pH 8.5 was kept constant with 4 M sodium hydroxide. After the reaction 12 mmol reduced anthraquinone-2,6-disulphonate (70 %) was isolated by centrifiigation at 4 ''C. For the isolation of pyruvate the reaction mixture was acidified with perchloric acid to pH < 2, centrifiiged, and the supernatant continuously extracted with diethyl ether. After the ether was evaporated the pyruvic acid was isolated as sodium pyruvate (84). A series of various other experiments are shown in Table 15. Experiment 7 resulted in a space time yield of 294 mol*l "^d"^ with 94 % conversion in 1 h. It was conducted in deionized water without any added buffer. That means crude pyruvate solutions are obtained after the mediator, EDTA and cell components had been eliminated. The pH value in the experiments without buffer was kept constant by automatically adding small amounts of 2 or 4 M sodium hydroxide or 1 M hydrochloric acid if an N-oxide was used as an electron acceptor. For fiirther discussions of the various experiments together with those not mentioned here see (62). (ii) Selective dehydrogenation of aldonates and aldarates with R-conJigurated acarbon atom to 2-oxo carboxylates Some methods are known for the preparation of 2-glyculosonates (2-oxoaldonates) (65,66,67). The catalytic dehydrogenation of a-carbon atoms of aldonates from the hexose series to 2-glyculosonates shows a pronounced time optimum and up to 5-6 byproducts (68). A fermentative process is described by which a Pseudomonas species converts glucose to D-araZ>/>20-hex-2-ulosonate (2-oxo-D-gluconate) in a 0.32 M solution, which was converted to iso-vitamin C (70). As recently shown by the group of Wong (66) with the synthesis of 3-deoxy-D-/waA2«o-2-octulosonic acid (KDO), 2glyculosonates can also be prepared by aldolase catalysed reactions. Important is the
858
preparation of L-xy/o-hex-2-ulosonate (2-oxo-L-gulonate), which is converted to Lascorbic acid. We prepared pent- and hex-2-ulosonates according to Scheme 4 (61). The apparent K^ values of the substrates are mostly < 1 mM (Table 16). They were determined with crude extract of P. mirabilis to which the substrates were added as salts at pH 8.5. The apparent Fmax values are 2-13 % of that for (i?)-lactate dehydrogenation (47,62). The absolute values are still rather high when compared with redox enzyme activities of many other microorganisms for rather simple substrates. Two examples with P. mirabilis cells grown on racemic lactate and dimethyl-sulphoxide will be briefly described. TABLE 16 Dehydrogenations of aldonates and aldarates with resting cells of Proteus mirabilis. These cells contained 2-hydroxy carboxylate viologen oxidoreductase (HVOR) with a specific activity of 5.1 U*mg'^ protein for the dehydrogenation of (i?)-lactate (61). Substrate
^m
(mM) D-Gluconate D-Gulonate D-Galactonate L-Mannonate Lactobionate 6-Phospho-D-gluconate L-Arabinonate D-Ribonate D-Xylonate D-Glucarate Galactarate L-Arabinuronate AQDSox C A V
BV"" DMSO'
0.98 0.64 0.48 0.92 0.66 0.64 2.03 0.30 1.27 1.68 0.42 1.46 0.19 0.025 0.042 0.66
V ' max (U.mg-') 0.20 0.15 0.25 0.20 0.15 0.20 0.30 0.65 0.15 0.40 0.10 0.15° 0.50' 0.25° 0.35° 0.45°
Conversion ( % )
Yield'' (%)
Productivity number"
96 97 99 98 99 99 98 99 99 99 79
85 95 98 88 70 98 65 71 78 91 70
760 740 650 210 480 250 280 690 270 270 140
The apparent ^m values were determined with crude extracts of P. mirabilis at 38 °C and pH 8.5. The data were calculated according to Eadie and Hofstee (69). The substrates were tested in the presence of 6 mM anthraquinone-2.6-disulphonate (AQDS). ^ Yield as isolated product. *" Kinetic data for the dimethyl-sulphoxide reductase were determined with 0.6 mM reduced anthraquinone2.6-disulphonate. The ^m values for the mediators were determined with 50 mM D-arabino-hex-lulosonate as substrate. ^ mmol*kg"^(biocatalyst)*h'\ mediators were determined with 50 mM D-araZ>/>70-hex-2-ulosonate.
859
D-Arabino-hex-2-ulosonate sodium salt monohydrate Ten mmol sodium D-gluconate, 15 mmol dimethyl-sulphoxide, 0.05 mmol anthraquinone-2,6-disulphonate, 0.25 mmol EDTA and 5*10"^ mmol tetracycline were dissolved in 50 ml deoxygenated, deionized water, and 2.0 g wet packed cells of P. mirabilis cells were added. The pH of the buffer-free solution was held constant at 9.0 during the reaction by a pH-controUing system delivering 2 M sodium hydroxide. The mixture was stirred under an atmosphere of nitrogen gas at 38 °C. The course of the reaction was monitored by HPLC. At the end of the reaction ('-98 % conversion) the pH of the mixture was brought to 3.5 with perchloric acid, the protein precipitate was removed by centrifiigation, the supernatant was treated with active charcoal and filtered. The free acid was isolated by ion exchange chromatography on a 10*210 mm Dowex-50W (20-50 mesh H^ form) column. The pale yellow solution was then brought to pH 8.0 with sodium hydroxide and lyophilised. The residue was taken up in 20 ml methanol containing 2 % water and stirred for 1 h at room temperature. The sparingly soluble sodium salt was collected by filtration and washed with cold methanol and acetone. The resulting white amorphous hygroscopic product (8.8 mmol, 88 % from D-gluconate) was dried and stored over potassium hydroxide at 4 °C. The NMR spectrum in ^H20 is identical with that of an authentic sample from Aldrich. A small portion was converted to methyl 2,3,4,5-tetra-0-acetyl-D-araZ7/>2o-hex-2-ulosonate (71). Starting from D-gulonic-y-lactone the salt was formed and a 0.5 M solution converted to D-Jcy/o-hex-2-ulosonate sodium salt monohydrate in the same manner as described for the preparation of sodium D-amZ?/>7o-hex-2-ulosonate. Part of it was converted to methyl-D-xy/o-hex-2-ulosonate, and this fiirther to D-//zr^o-hex-2-enono-l,4-lactone (Dascorbic acid) as described (72). The ^H and ^^C-NMR spectra in ^H20 are identical to those of an authentic sample of L-ascorbic acid. 3.3 Growth oi Proteus mirabilis (DSM 30 118) or Proteus vulgaris (DSM30115) (16) Proteus spp. were grown on a medium containing in gJ'^ 5.0 tryptone (Oxoid), 5.0 yeast extract (Difco), 5.1 K2HPO4, 1.0 HCOONa. In mg*r^ were added: 25 MgS04 *7H20, 170 NH4CI, 40 CaCl2*2H20, 13.7 Na2Mo04*2H20, 0.4 MnS04*H20, 0.4 FeS04*7H20, 0.4 /7-aminobenzoic acid, 0.26 Na2Se03*5H20, 0.02 biotin. As carbon source were added 7.2 g sodium fK,5y)-lactate containing 32 % (R)- and 68 % fS^-lactate per litre. External electron acceptor was 50-60 mM dimethyl-sulphoxide. The initial pH was adjusted to 7.5. Growth was strictly anaerobic under an atmosphere of nitrogen gas at37°C. Cells were harvested by centrifiigation, washed and suspended in 10 mM Tris/HCl buffer, pH 7.5. Crude extract was prepared by a French press at 130 Mpa. It was centrifiiged at 25 000 g for 10 min (4 °C) to remove cell debris. The enzyme assays were carried out at 37 °C. For enzyme activities see Table 17 and for additional details (47).
860
The enzymes necessary for the reduction of 2-oxo carboxylates apphed in form of resting Proteus cells are rather stable under operational conditions. They were used repeatedly or for up to 600 h. They can also be immobilised (47, 73). TABLE 17 Activities of hydroxy carboxylate viologen oxidoreductase (HVOR) and dimethylsulphoxide reductase in P, vulgaris grown on glucose or (7^,5)-lactate with or without different electron acceptors (47). Growth condition
HVOR as (i?)-lacWet DMSO^ tate weight reductase dehydrogenase of cells (U» mg"' protein) (U. mg'' protein) (g-r')
Glucose Glucose + HCOa' Glucose + DMSO Glucose + DMSO + HCOs" (i?,5)-Lactate (7?,5)-Lactate + DMSO (S)-Lactate + DMSO (i^)-Lactate + DMSO (i^,5)-Lactate + TMANO^ (i?,5)-Lactate + N-MMNO' (i?,S)-Lactate + nitrate (i?,S)-Lactate + ftimarate (i^,*S)-Lactate + pyruvate (i?,iS)-Lactate + pyridine N-oxide
2.3 3.9 3.5 4.2 <1.0 2.5 2.1 1.6 2.9 2.2 1.2 2.5 1.4 1.4
2.6 1.6 0.8 0.6 n.d. 3.8 4.1 3.6 1.6 0.7 <0.01 1.4 8.0 0.9
0.3 0.3 0.7 0.5 n.d. 0.7 0.5 0.9 0.5 0.4 <0.01 0.7 0.6 1.1
(i?,iS)-Lactate + tetramethylene sulphoxide
<1.0
n.d."
n.d.
^ DMSO: dimethyl-sulphoxide, ^ TMANO: trimethylamine N-oxide, *" N-MMNO: 7V-methylmorpholine N-oxide, *^ n.d.: not determined. The specific enzyme activities (U*mg'^ protein) of the crude extract of P. vulgaris were tested with reduced benzylviologen for dimethyl-sulphoxide reductase and oxidized benzylviologen for (R)-2-hydroxy carboxylate viologen oxidoreductase dehydrogenating (R)-lactate.
861
TABLE 18 Enzyme activities of (Ryi-hydroxy carboxylate viologen oxidoreductase (HVOR), dimethyl-sulphoxide reductase, formate reductase and hydrogenase in crude extracts of P. vulgaris and P. mirabilis (47). Substrate for enzyme tests (i^)-Lactate Pyruvate ^ Diethyl-sulphoxide Dimethyl-sulphoxide Ethylmethyl-sulphoxide (S)-Methionine-sulphoxide Tetramethylene-sulphoxide A^-Methylmorpholine N-oxide Pyridine N-oxide Trimethylamine N-oxide Formate ^ Proton (Hydrogenase) ^'^
P. vulgaris ^ (U*mg'^ protein) 2.7 11.1 0.2 0.7 0.6 1.3 1.6 2.4 4.0 3.6 8.5 0.5
P. mirabilis ^ (U*mg"^ protein) 3.5 16.2 0.6 1.9 1.5 4.1 3.7 5.3 8.3 7.7 18.4 1.0
^ The (i?,*S)-lactate for the growth of P. vulgaris was {R) : (5) = 32 : 68 and for P. mirabilis 56 : 44. ^ Measured at pH 7.0. " Reduced methyl viologen (SSTS = 9.0 mM'^cm'^) was used as electron donor. The organisms were grown on (R,S)4actate with dimethyl-sulphoxide. Reductase activities were tested with reduced benzylviologen. If not indicated otherwise all reductions were carried out at pH 8,5 since it was intended to couple them with the dehydrogenation of (R)-lactate. 4. REDUCTION OF CARBOXYLATES AND DEHYDROGENATIONS OF ALCOHOLS WITH CLOSTRIDIUM THERMOACETICUM A previously unknown enzyme activity reducing non-activated carboxylates in Clostridia has been found by our group (74-77). Although this activity is present in several anaerobic bacteria and archaea (77a), its physiological role is unknown. The reaction is remarkable because the reduction of carboxylic acids in aqueous solution is not possible with chemical reducing agents. These agents reduce the protons of water rather than an acylate. The truly reversible aldehyde oxidoreductases from Clostridium thermoaceticum and one from C. formicoaceticum depend strictly on tungsten. The tungsten is complexed to a pterin cofactor which is identical or at least similar to the pterin moiety of the molybdenum cofactor present in most molybdenum containing enzymes (77,78). The pH optima for carboxylate reductions with the purified enzyme from C. thermoaceticum and CAV^* (Reaction [23]) shift to lower values with lower pK values of the substrates. However, at pH 4.0 and lower the enzyme is not stable enough for kinetic measurements. Probably the non-dissociated acids are the real substrates of the tungsten containing aldehyde oxidoreductase of C thermoaceticum. That means the non-dissociated form is bound to the enzyme, or an acylate bound to the enzyme is protonated before it is
862
reduced. The redox potential of a non-negatively charged species, i. e., an non-dissociated carboxyhc acid, is much less negative than that of a carboxylate. This fact may explain why the enzyme from C. thermoaceticum is able to reduce carboxylates with a redox potential of about -580 mV (E"" = -581 mV for acetate/ethanal at pH 7) (79) at the expense of reduced carbamoylmethylviologen with a redox potential of only E°'= -302 mV (Table 1). The tungsten containing aldehyde oxidoreductase of C, formicoaceticum is rather different from that of C thermoaceticum. This holds for the amino acid sequence of the N-terminus, the pH dependence of the carboxylate reduction and the redox potential of the artificial mediators necessary for reducing carboxylates. The pH optimum of 6.0 for the carboxylate reduction is independent of the pK value of the acids. This was shown for propionate, 4-fluorobenzoate, benzoate, and 4-methoxy-benzoate. Reduced carbamoylmethylviologen is not able to transfer electrons to this enzyme. Reduced benzylviologen, methylviologen and l,r-2,2'-tetramethylviologen show relative reduction rates of 12, 30 and 100 %, respectively. The E°'-values of these viologens are -340, -440 and -550 mV (Table 1). In addition to the tungsten dependent enzyme C. formicoaceticum forms a molybdenum containing aldehyde oxidoreductase, too. This enzyme reduces carboxylates only with the tetramethylviologen mentioned above (80). The purified aldehyde oxidoreductase from C thermoaceticum reduces carboxylates to the aldehydes only (Reaction [23]). No alcohols were present among the products of these reductions (77). The same is probably true for the enzyme from C. formicoaceticum. Before preparative reductions of carboxylates with cells will be described some hints about the purified enzyme will be given (75-77). Besides the purification and partial structural characterisation we conducted some kinetic measurements (77). In addition to the carboxylate reduction the dehydrogenation of aldehydes was studied. There is a broad substrate specificity of the enzyme from C. thermoaceticum for aliphatic and aromatic aldehydes. The K^ values for ethanal, propanal and butanal are in the region of 0.0060.01 mM. The K^ values for acetate and propionate are about 5 and 6 mM that means 3 orders of magnitude larger. The ratio of ^cat / ^m for aldehydes and the corresponding carboxylates is about lO'^-lO^. The K^ values of aromatic aldehydes are < 0.02 mM. An exception is 4-hydroxybenzaldehyde. 4-Hydroxybenzoate the corresponding carboxylate has an extremely high K^n value, too. Table 19 shows fiirther examples of carboxylates being reduced. Additional structural and kinetic data of the tungsten and molybdenum containing aldehyde oxidoreductases from C. formicoaceticum are described (78,81). When the aldehyde oxidoreductase of C. thermoaceticum reduces the non-dissociated acids bound to the enzyme, esters, which are not charged, could also be substrates. However, esters such as methyl propionate are no substrates at all. Lactones seem to be reduced only after the rate-limiting opening of the ring (Table 19). 4.1 Reductions of non-activated carboxylates to alcohols Table 19 shows the productivity numbers for the reduction of various carboxylates to alcohols with resting cells of C. thermoaceticum and carbon monoxide as an electron donor. Four consecutive Reactions [7a] and [23]-[25] catalysed by four enzymes present
863
in C. thermoaceticum lead to the observed mono- and di-alcohols. CO-Dehydrogenase reduces at the expense of two carbon monoxide molecules four molecules V^^ to V^* (Reaction [7a]). These are consumed by Reactions [23]-[24] AldOR
RCOOH
+
2V^- +
2H^
NADP"
+
2V"- +
H"
^ ^ ^
+ NADPH +
H^
^^^
RCHO
^^=^
RCHO +
2V^^ +
H2O
NADPH + 2V"" RCH2OH
+
[23] [24]
NADP^
[25]
For the term AMAPOR see Sections 1.2 and 5 (6,82,83). According to Reaction [23] 2 mol carbon monoxide deliver 4 electrons that are necessary for the reduction of a carboxylate to the corresponding alcohol. Reaction [26] summarises these reactions. 2C0
+
RCOOH +
H2O
^^==^
2CO2
+
RCH2OH
[26]
Under an atmosphere of carbon monoxide cells of C thermoaceticum reduce propionate (pK 4.87) at pH 5.5 at an optimal rate and lactate (pK 3.86) at pH 4.5 (not shown). For the reduction of propionate with reduced carbamoyhnethylviologen the pH optimum of the purified enzyme was 4.65. At pH 5.5 the purified enzyme reduced propionate at about 20 % of the maximal rate (77). In cuvettes only the initial rate of Reaction [23] is measured. Within a microorganism Reactions [7a] and [23]-[25] take place in a consecutive manner. The pH optimum of this reaction sequence is different fi-om that of Reaction [23] alone. For instance the pH optimum of carbon monoxide dehydrogenase is 8.4 (85). At pH 5.5, carbon monoxide dehydrogenase and carboxylate reductase showed similar activities in cells grown according to Section 5.3. At pH 4.5 CO-dehydrogenase activity was limiting. At this pH the solutions of reductions with whole cells were not blue, because Reaction [7a] which forms V^* is slower than Reactions [23] and [24] in consuming V^*. At pH 7.0 the aldehyde oxidoreductase does not any longer reduce carboxylates to aldehydes but the enoate reductasefi^omC. tyrobutyricum is active (Section 2). Starting fi-om from a- and/or ^-substituted enoates the chiral saturated acids can afterwards be reduced fiirther with carbon monoxide and C thermoaceticum to the corresponding chiral alcohols. An isolation of the chiral acid is not necessary. After reducing the 2enoate (Reaction [9]) the pH has to be shifted to pH 5.0-5.5. Cells of C. thermoaceticum are added and the chiral carboxylate is reduced with carbon monoxide (74,77). The enoate reducing system of C thermoaceticum shows a low activity and seems not to be as stereospecific as that of C. tyrobutyricum (unpublished). Lactones (Table 19) were slowly reduced to di-alcohols. The rate limiting step in this reaction was probably the hydrolysis of the lactone ring, which was slow at pH 5.5. 2Hydroxypyridine-5-carboxylate was converted to 3-hydroxymethylpyridine. That means the 2-hydroxy group was reductively eliminated. Racemates of chiral substrates carrying a chiral carbon atom in a-position were reduced, too, but in no case could marked stereospecificity be found. Reactions with (K,5j-2-(4-chlorophenoxy)-propanoate were stopped after 50 % reduction. Both enantiomers of the 2-(4-clilorophenoxy)-propan-l-ol
864
TABLE 19 Productivity numbers and relative rates for the reduction of various carboxylates by C. thermoaceticum at the expense of carbon monoxide. The substrates (33 mM) were supplied as their sodium salts in 0.3 M potassium phosphate buffer, pH 5.5. One mM methylviologen was used as mediator. The productivity numbers are calculated for one pair of electrons consumed after a reaction time of one hour. ~~~
Substrate
Product
Acetate Propanoate Butanoate Pentanoate 3-Methylbutanoate (R,S)-2-Methylbutanoate 2-Methyl-(E)-2-butenoate 2-Methyl-(E)-2-butenoate (R,S)-2-Methylpentanoate (R,S)-3-Methyl-pentanoate (R)-Lactate (S)-Lactate 4-Methoxyphenylacetate (R,S)-2-phenylpropanoate (R,S)-2-phenoxypropanoate (R,S)-2-(2-chloro-phenoxy)-propanoate (R,S)-2-(4-chloro-phenoxy)-propanoate (R,S)-2-(2,4-dichloro-phenoxy)-propanoate (R,S)-2-(2,4,5-trichloro-phenoxy)-propanoate Benzoate 4-Hydroxybenzoate 4-Methoxybenzoate
Ethanol Propanol Butanol Pentanol 3-Methylbutan-l-ol (R,S)-ZMethylbutan-1-01 2-Methyl-(E)-2-buten- 1-01 and (R,S)-2-Methylbutan-l-ol (R,S)-2-Methylpentan-1-01 (R,S)-3-Methylpentan-l-ol 1,2-Propandiola 1,2-Propandiola 2-(4-Methoxyphenyl)-ethanol (R,S)-2-phenylpropan-l-ol (R,S)-2-phenoxypropannl-ol (R, S)-2-(2-chloro-phenoxy)-propan-1-01 (R, S)-2-(4-chloro-phenoxy)-propan- 1-01 (R,S)-2-(2,4-dichloro-phenoxy)-propan-l-ol (R,S)-2-(2,4,5-trichloro-phenoxy)-propan-l-ol Benzyl alcohol 4-Hydroxybenzyl alcohol 4-Methoxybenzyl alcohol
Productivity number 630 1140 1180 1110 840 1030 685 1255 870 990 115" 80" 880 1025 0 730 580 470 150 725 150 1300
4-Methylbenzoate 4-Fluorobenzoate 4-Chlorobenzoate Pyndine-2-carboxylate Pyndine-3-carboxylate Pyndine-4-carboxylate 6-Hydroxypyridine-3-carboxylate 6-Chloropyridine-3-carboxylate Succinate Glutarate Hexane dicarboxylate (R,S)-Methylsuccinate 3-Methylglutarate (R,S)-3-Methylhexane dicarboxylate y-Butyrolactone 6-valerolactone E-caprolactone (R,S)-4-Methyl-6-caprolactone
4-Methylbenzyl alcohol 4-Fluorobenzyl alcohol 4-Chlorobenzyl alcohol 2-Hydroxymethylpyridine 3-Hydroxymethylpyridine 4-Hydroxymethylpyridine 3-Hydroxymethylpyridine 3-Hydroxymethyl-6-chloropyridme Butane- 1,4-diol Pentane- 1,5-diol Hexane- 1,6-diol (R,S)-2-Methylbutane-1,4-diol 3-Methylpentane-1,5-diol (R,S)-3-Methylhexane-1,6-diol Butane- 1,4-diol Pentane- 1,5-diol Hexane- 1,6-diol (R,S)-3-Methylhexane-1,6-diol
625 675 575 550 360 580 60 340 1355 1125 1220 300 315 1160 5 150 25 30
This rate increases at pH 5.0. The enantiomeric excess of the product was not determined. The product formed is 3hydroxymethylpyridine instead of 3-hydroxymethyl6-hydroxypyridinedue to GUMS-analysis. After one hour depending on the substrate 25-75 % of the formed diol are present in form of the lactone. The lactones react rather slowly. After a reaction time of 19 hours. a
865
866
had been formed in about equal amounts. As shown earher, (R)- and (*S)-lactate were reduced to 1,2-propandiol with similar rates (74). Reductions of carboxylates up to at least 400 mM are possible. During the reduction the pH of such solutions with 100-400 mM carboxylate may drop considerably. This is probably due to the reduction of carbon dioxide to formate at the expense of carbon monoxide. Mechanistically carbon monoxide dehydrogenase (Reaction [7a]) delivers electrons from carbon monoxide to formate dehydrogenase which adds them to carbon dioxide yielding formate (Reaction [27]). Carbon dioxide is formed by oxidation of carbon monoxide (see above). CO2
+
2e
+
2H^
•
HCOO'
+
H^
[27]
The pH of such solutions has to be kept constant by adding sodium hydroxide with a pH controlling system. Reduction of 6-chloropyridine-3-carboxylate to 3-hydroxymethyl-6-chloropyhdme With chemical reagents this reduction causes problems because the bound chlorine is labile and may get lost. The pH optimum for the microbial reduction with methylviologen as electron mediator was 5.0, leading to a productivity number of 190 in an electrochemical cell (in analogy to Section 6). The optimum for the working potential was -750 mV versus SCE (saturated calomel electrode) (Table 20). The electromicrobial reduction leads also to the undesired by-products pyridine-3-carboxylate and 3hydroxymethylpyridine. These were even the main products when tetramethylviologen was used as a mediator, with a working potential of -840 mV versus SCE. The lower the pH, and the more negative the working potential, the higher was the amount of the chlorine free by-products. So a compromise had to be found between optimal productivity and minimal formation of the by-product by increasing the pH to 5.6 and applying a working potential of -750 mV versus SCE. The product/by-product ratio was 3.4 under these conditions. Better results were obtained by the microbial reduction of 6-chloropyridine 3-carboxylate using carbon monoxide. The product/by-product ratio was about 80, if a carbon monoxide pressure of 20 bar was applied. In another experiment ethylene glycol diethyl ether was used as a second phase in order to transfer the product to the organic solvent (Table 20) (86). A solution of 30 ml 0.1 M potassium phosphate buffer, pH 5.6, containing 270 mM 6chloropyridine-3-carboxylate and 8 g wet packed cells of C. thermoaceticum was stirred under an atmosphere of about 1.1 bar CO. In another experiment 30 ml ethylene glycol diethyl ether was used as the second phase. Before the reaction was started, carbon monoxide was flushed through the solution. For the experiment with 20 bar carbon monoxide a glass flask with a magnetic stirrer containing the above indicated ingredients was put in an 200 ml autoclave.
867
TABLE 20 Electromicrobial and microbial reduction of 6-chloropyridine-3-carboxylate. The productivity numbers (PN) are given after 7 and 24 h for the uptake of 4 electrons. The organic phase was 30 ml ethylene glycol diethyl ether. Working potential or CO pressure
Mediator Ratio product/by-product ^ (mM) PN(7h) PN(24h) (7h) (24 h)
- 750 mV versus SCE - 840 mV versus SCE 1.1 bar {+ organic phase) 20 bar (+ organic phase) 20 bar (- organic phase)
MV(5) 150 TMV(5) 40 MV(4) 200 MV(4) 240 MV(4) n.d.
130 20 100 150 130
n.d. n.d.
24 82 n.d.
3.4 0.03
10 80 80
^ Product: 3-hydroxymethyl-6-chloropyridine. By-product: 3-hydroxymethylpyridine 4.2 £lectromicrobial dehydrogenations of primary alcohols C. thermoaceticum was investigated for the dehydrogenation of primary alcohols with the application of different electron mediators (86). The final electron acceptor was an anode and the reaction proceeded in aqueous media. Unexpectedly the mere choice of the artificial electron mediator determined whether aldehydes or acids were formed. When cells of C. thermoaceticum were used, some mediators did not react with aldehyde oxidoreductase. If aldehyde was the final product, an aldehyde scavenger had to be applied, otherwise the enzymes were inhibited. Table 21 gives a summary of the results of the dehydrogenation of propanol. With anthraquinone-2-sulphonate as mediator besides n-propanol «-butanol, 2-methylpropanol, fK,S^-2-methylbutanol, benzyl alcohol, and allyl alcohol were converted to acids showing productivity numbers between 400 and 1200. The dehydrogenation of fjR,iS^-2-methylbutanol showed no enantioselectivity. 3Aminopropanol, ('jR,iS>)-2-aniinobutanol and 5-aminopentanol as well as «-octanol and ethylene glycol could not be dehydrogenated. The dehydrogenation of a primary alcohol to the aldehyde seems to depend on the E''' of the mediator and its reactivity with the aldehyde oxidoreductase. The more positive the E°' of the mediator, the lower is its reactivity with the enzyme. If thionine or methylene blue is applied, having the most positive E°' values among the applied mediators, only the aldehyde is formed. If resorufin was used, with an E°' between that of the phenothiazine-dye-type mediators (E°' >0 mV) and the mediators used for acid production (E°' < -180 mV), the aldehyde formation was only slightly preferred over acid formation. Carbamoylmethylviologen and anthraquinone sulphonates showed only acid production; even when an aldehyde scavenger was applied, only small amounts of aldehyde were formed (86).
868
TABLE 21 Electromicrobial dehydrogenation of propanol, using different electron mediators. Mediators (mM) 1 Thionine 1 Thionine' 1 Methylene Blue ' 1 Thionine" Resorufin 0.25 3 AQ-2-S AQ-2-S 3 AQ-2,6-DS 3 CAV 3
E"' Hydrazine (mM) (mV)
After 1 h reaction After 20 h reaction PNald. PNacid PN aid. PN acid
+ 60 + 60 + 11 + 60 -51 -225 -225 -184 -302
1200 1200 2500 1200 n.d. n.d. <50 <50 <50
100 100 100 0 100 100 0 0 0
<50 <50 <50 <50 n.d. n.d. 1500 1250 2500
400 1400 1500 330 730 300 <50 <50 <50
<50 <50 <50 <50 420 1300 1500 1200 1650
The buffer also contained 6 mM FeEDTA. A sample of 50 ml 0.1 M Tris/HCl buffer contained 50 mM propanol and 0.2 g wet packed cells ofC. thermoaceticum. The reaction temperature was 40 ^C and the working potential differed depending on the applied mediator. Using carbamoylmethylviologen as mediator the pH was 8.0; with the other mediators it was 7.0. The applied dyes, which have low solubility in water, can be used in an electrochemical cell only in connection with a better water-soluble co-mediator like 6 mM Fe-EDTA. This co-mediator can readily be reoxidized at an anode and reacts quickly with the reduced dyes. 5. USE OF ARTIFICIAL MEDL\TOR ACCEPTING PYRIDINE NUCLEOTIDE OXIDOREDUCTASES (AMAPORs) FOR THE REGENERATION OF PYRIDINE NUCLEOTIDES The role and the reactions of AMAPORs for the regeneration of pyridine nucleotides are generally introduced in Section 2.1. Many different systems have been suggested for the regeneration of (mostly) one of the coenzymes NAD^, NADH, NADP^ or NADPH. Only a few publications which appeared after the review articles of the Whitesides group (87-89) shall be mentioned (90,91). For the photochemical and electrochemical regeneration of nicotinamide cofactors see (92,93). Many experiences exist for the regeneration of NADH by use of an NAD^-dependent formate dehydrogenase from a Candida yeast (94) which catalyses Reaction [28]. NAD(P)^
+
HCOO-
•
NAD(P)H
+
CO2
[28]
For the production of this enzyme a continuous computer controlled production and isolation procedure on a pilot scale has been developed (95). Carbon dioxide as the product of the electron donor formate, leaves the solution and does not interfere with the purification of the product. An important progress is a formate dehydrogenase obtained by engineering of an enzyme from the methylotrophic bacterium Pseudomonas sp. 101
869
which accepts NAD^ and NADP^. StabiHty measurements show no decrease of activity over a period of 7 days incubation at a temperature of 25 °C (96). Usually formate does not inhibit other oxidoreductases. The disadvantage of most other systems for NADPH regeneration is the formation of a by-product in stoichiometric amounts which does not leave the system. An example is 2-oxoglutarate from .S-glutamate and glutamate dehydrogenase. These problems and others are discussed in the above mentioned reviews. Our findings and experiments on AMAPORs are described in a series of papers (6,7, 82,83,97-100). For some time we called these activities viologen accepting pyridine nucleotide oxidoreductases (VAPORs). They are present in many aerobic and anaerobic microorganisms (7,97). Surprisingly high activities are present in mitochondria from beef heart or yeasts (100). Since we observed that these enzymes accept also artificial mediators such as cobalt complexes (101-104), anthraquinones, thiazine dyes and others we changed the name of such enzyme activities into artificial mediator accepting pyridine nucleotide oxidoreductases AMAPORs (82). They reversibly transfer electrons between various artificial electron mediators and a pyridine nucleotide. The thermophilic Bacillus DSM 406 (16) and other related species (Table 22) contain an AMAPOR which reduces NAD^ and NADP^ at the expense of reduced methylviologen. The enzyme from the species DSM 406 was purified and studied fiirther. Some kinetic data are given in Table 23. The seven indicated K„, and six K\ values for this enzyme show that it is suitable for the regeneration of all four forms of pyridine nucleotides. The stability in presence of oxidized and reduced methylviologen at 35 °C or 60 °C is satisfying for preparative work (98). May be that this enzyme reacts with more artificial electron mediators than viologens. That was not studied by us. The NADH dehydrogenation with CAV^^ shows its pH optimum at 10. The activity at lower pH values is sufficient for preparative work. For the reduction of NAD^ with MV^* the pH optimum is about 6.5. The astonishingly high AMAPOR activities present in crude extract of C. thermoaceticum are summarised in Table 24 (82). In the form of crude extract the rather oxygen insensitive activities are useful for the regeneration of all four forms of pyridine nucleotides on a preparative scale. Reductions via NADH or NADPH dependent reductases can be conducted with reduced methylviologen according to Reaction [29] and [30]. 2MV^'
+
NAD(P)H +
NAD(P)^
+
H^ AMAPOR 2MV^^
)C=X
+
H^
+
^ NAD(P)^ -f
NAD(P)H
[29]
H-)C—XH
[30]
In crude cell extracts of C. thermoaceticum the specific activity of AMAPORs for NADP^ reduction with reduced MV is extraordinarily high and more than twice of that for NAD^. NADH and NADPH dehydrogenation for the regeneration of NAD^ and NADP^ is achieved with the same enzyme activities and anthraquinone-2,6-disulphonate (Reaction [31a]). The activities are about the same as for carbamoylmethylviologen (CAV) (Table 24).
870
TABLE 22 Specific activities of AMAPOR in crude cell extracts of different thermophilic bacilli measured at 30 T (98). Specific Strain activity ^
Strain Bacillus Bacillus Bacillus Bacillus
stearothermophilus sphaericus 461 sp. DMS 405 sp. DMS 406
0.09 0.27 0.14 0.25
Specific activity ^
5acz7/w5sp. DMS411 Bacillus sp. DMS 465 Bacillus sp. DMS 466 Bacillus sp. DMS 730
0.18 0.40 0.57 0.18
^Reoxidation of 2 jimol MV^**mg"^ proteimmin'^ with NAD^. Table 23 Kinetic parameters of AMAPOR from Bacillus spec. 406. For the formation of reduced pyridine nucleotides MV^' and for their reoxidation CAV^^ were used (98). A^m-values (mM) MV NAD" NADP"^ CAV"' CAV^^ NADH NADPH
0.24 0.06 1.82 0.52'' 0.29° 0.22 1.43
Product NADH NAD^ NADPH NADH
Rel. rate (%)" 100
no"
50 37
/Tj-values (mM) MV** NADH NADPH CAV"* NAD^ NADP*
100 50 33 1.8 19 13.5
^ 100 % corresponds to 58 U for the purified enzyme. ^ For the reoxidation of NADH. *" For the reoxidation of NADPH. ^ At the pH optimum for the reoxidation of NADH the rate is about 260% of that of the NADH formation with MV^* at pH 7.0. In crude extract of C. formicoaceticum the activity for the reduction of NAD"^ with MV^* is about 6 U*mg"^ and that for NADP^ 3.5 U*mg"^ protein. The dehydrogenation of NADH and NADPH proceeds with CAV^^ as acceptor with a specific activity of 14 and 8 U*mg"* protein (unpubhshed). For the AMAPORs from C thermoaceticum and C. formicoaceticum the K^ values for the artificial electron mediators and those for pyridine nucleotides are in a reasonable range for preparative transformations. Table 24 shows some ^m values for enzymes present in C. thermoaceticum.
871
TABLE 24 Kinetic data of AMAPOR activities in crude extracts of Clostridium thermoaceticum at 37 "C.
Mediator
Pyridine nucleotide
3.9 9.8 1.6 2.0 1.6 1.7
NAD^ NADP* NADH NADPH NADH NADPH
MV"* MV"* CAV^^ CAV** AQDSox AQDSox
Specific activity (U.mg'' protein)
Mediator (mM) 0.38 0.44 0.35 0.25 0.7 1-2
^m
Pyridine nucleotide (mM) 0.88 0.20 n.d. 0.02 0.07 0.12
pHOptimum 6.5 6.5 8.5 8.5 8.5 8.5
We demonstrated the application of reactions with AMAPOR activities from C thermoaceticum in electrochemical cells and in other forms for the preparative reduction of various substrates (82,83,86). One NAD^/NADH dependent AMAPOR from C. thermoaceticum has been purified and partially characterized (86a). 5.1
NAD^ and NADP^ regeneration with AMAPOR from Clostridium thermoaceticum and anthraquinone-2,6-disulphonate using oxygen as final electron acceptor
In contrast to viologens reduced anthraquinone-2,6-disulphonate formed by Reaction [31a] can be reoxidized in a non-enzymicly catalysed reaction with oxygen as final electron acceptor (Reaction [31b]). The formed hydrogen peroxide can be split by the extremely cheap catalase (Reaction [31c]). The sum of Reactions [3la]-[31c] leads to Reaction [31]. NAD(P)H +
H^
+
0.5 O2
• NAD(P)^ +
H2O
[31]
This NADP* regeneration consists of the three consecutive Reactions [31a]-[31c]. The reduction of anthraquinone-2,6-disulphonate at the expense of NAD(P)H catalysed from AMAPORs in form of crude cell extract of C. thermoaceticum, the reoxidation of the reduced mediator in a spontaneous reaction with oxygen (112) and the splitting of the hydrogen peroxide by catalase. NAD(P)H + AQ-2,6-DSox + H ' ; = ^ NAD(P)^ + AQ-2,6-DSred H2O: 2'-'2
+
O2
AQ-2,6-DSo H2O
AQ-2,6-DSred
[31a]
H2O: 2»-'2
[31b]
0.5 O2
[31c]
872
TABLE 25 Dehydrogenation of glucose-6-phosphate (G-6-P) to 6-phosphogluconate using AMAPORs from C. thermoacetjcum, glucose-6-phosphate dehydrogenase (G-6-P DH), anthraquinone-2,6-disulphonate and oxygen for NADP^ regeneration (82). G-6-P (mM)
AMAPOR U
G-6-P DH" U
20 20 20 100 300
23 23 23 46 69
28 28 28 28 42
Catalase SOD" kU kU 0 130 130 130 150
Yield (%)
Reaction time (h)
63 92 92 84 41
5 5 5 23 23
0 0 2.2 0 0
Mass balance (%) n.d. 95 95 88 60
Number of cycles forNADP* 130 180 180 810 830
^ EC 1.11.1.6,^ SuperoxidedismutaseEC 1.15.1.1 A total volume of 40 ml 0.1 M Tris/HCl buffer, pH 7.5, at 33 ^C, contained in addition to the indicated substrate concentrations and enzyme activities 1 mM anthraquinone2,6-disulphonate, 3.3 mMMgCh and 0.1 mMor 0.15 mMNADP\ (i)
Preparation of 6-phosphogluconate and (2S,3R)-isocitrate under regeneration ofNADP^ (82) For the preparation of 6-phosphogluconate from glucose-6-phosphate with commercially available glucose-6-phosphate dehydrogenase from yeast the results are summarised in Table 25. Electrochemical experiments using the anode as the electron acceptor are shown in Section 6. Without catalase the yield of 6-phosphogluconate was only 63 % after 5 h and almost the same after 71 h (not shown for 71 h). Hydrogen peroxide formed during the regeneration of antliraquinone-2,6-disulphonate with oxygen (Reaction [31b]) seems to be the reason. The presence of catalase considerably increased the yield. The addition of superoxide dismutase together with catalase did not improve the results. With higher substrate concentrations NADP^ cycle numbers of above 800 were obtained. Recently NAD^ regeneration with a cycle number of less than 20 was described for the formation of 6-phosphogluconate with an NAD^ depending dehydrogenase (105). Using pyruvate and (iS)-lactate dehydrogenase for NAD^ regeneration led to cycle numbers of about 1000, but this system is not suitable for NADP^ regeneration (88). Another problem of all NADP(H) containing systems is the low stability of NADP(H) in the presence of high concentrations of phosphorylated compounds such as glucose-6phosphate and 6-phosphogluconate (87,89). The synthesis of (2iS',3i^)-isocitrate from (2/^,3S;25,3i?)-isocitrate by enantioselective oxidative decarboxylation of (2/?,3iS)-isocitrate to 2-oxoglutarate is shown in Table 26. The latter compound can easily be separated from the remaining (2iS',3/?)-isocitrate by the different solubilities of the barium salts of these acids in water. (2iS',37^)-Isocitrate is not commercially available and might be an interesting chiral building block. A cycle number
873
of 1250 without a by-product was observed for NADP^ regeneration. This is a satisfying value compared to methods mentioned before. The originally (2iS',3/^)-isocitrate present in the applied racemate completely reappeared on HPLC. The isolated crystalline mono potassium salt of isocitrate gave the expected optical rotation of [a]2o^= -20.4° and the correct elemental analysis. In a control assay with (2i?,35)-isocitrate dehydrogenase (EC 1.1.1.42) the (2i^,35)-enantiomer could not be detected. TABLE 26 Oxidative decarboxylation of (2i^,3tS)-isocitrate from (2i?,3*S';2»S',3i?)-isocitrate leading to (2iS',3i^)-isocitrate with NADP^ regeneration using isocitrate dehydrogenase (IC-DH, EC 1.1.1.42), AMAPORs in form of crude extracts of C thermoaceticum, anthraquinone-2,6-disulphonate and oxygen. The reaction mixture contained --^140000 U catalase. rac. threo isocitrate (mM)
AMAPOR
IC-DH
Yield
Reaction time
(U)
(U)
(%)
(h)
Number of cycles forNADP^ regeneration
50 360
23 70
22 45
100 100
4 33
260 1250
A total volume of 40 ml 0.1 M Tris/HCl buffer, pH 7A, at 33 ^C, contained 1 mM anthraquinone-2,6-disulphonate, 3.3 mM MgCh, 0.1 mM NADP^ and the indicated concentrations of (2R, 3S;2S, 3R)-isocitrate. (ii) Dehydrogenation of the (S)-enantiomer from racemates of 3-hydroxycarboxylates with NADP^ regeneration for preparation of (i^)-3hydroxycarboxylates For the enantioselective dehydrogenation of the (»S^-enantiomer from a racemic mixture of 3-hydroxybutyrate three different methods were used to regenerate NADP^ (Table 27) (83). The first method worked with catalytic concentrations of NADP^ and anthraquinone-2.6-disulphonate. NADPH was reoxidized by oxidized antliraquinone-2.6disulphonate catalysed by AMAPOR (Reaction [31a]). The reduced anthraquinone-2.6disulphonate was electrochemically reoxidized. In experiment 2 a method described by the Whitesides group (106) was applied for NADP^ regeneration. Oxidized anthraquinone-2.6-disulphonate could also be used in stoichiometric concentrations (exp. 3). In such a case its electrochemical reoxidation was not necessary. Obviously in experiment 1 the product isolation is especially simple. Using the purified (5)-3-hydroxybutyrate oxidoreductase from C tyrobutyricum (107) together with an electromicrobial NADP^ regeneration system with AMAPOR of C. thermoaceticum crude extracts and anthraquinone-2.6-disulphonate, the (iS)-enantiomer of 1.6 mmol (/?,5)-3-hydroxybutyrate was dehydrogenated (exp. 1, Table 27). The remaining (i?)-3-hydroxybutyrate showed 98 % ee. Application of the NADP^-regenera-
874
tion system described by Chenault and Whitesides (87) with the purified (iS)-3hydroxybutyrate oxidoreductase also led to (i^)-3-hydroxybutyrate with 98 % ee but in the presence of 1 equivalent 2-oxoglutarate. In the microbial dehydrogenation (exp. 3) 0.8 g C. tyrobutyricum and 0.3 g C. thermoaceticum cells together with 110 mM anthraquinone-2.6-disulphonate were used. The (.SJ-enantiomer from 4.0 mmol racemic 3-hydroxybutyrate was enantioselectively dehydrogenated. Part of the 3-oxobutyrate formed was converted into acetone and CO2. It was proven that a crude extract of C. tyrobutyricum is not able to transfer electrons to the mediator anthraquinone-2.6-disulphonate, and that C. thermoaceticum lacks (iS)-3-hydroxycarboxylate oxidoreductase. TABLE 27 Enantioselective dehydrogenation of the (5)-enantiomer of (i?,iS)-3-hydroxybutyrate to a mixture of (i^)-3-hydroxybutyrate, 3-oxobutyrate and acetone. NADP^ was regenerated in three different ways (83). No.
Substrate concentr. (mM)
Amount of Reaction Yield* Enantiome- Pyridine nucleotide substrate time ric excess regeneration system (mmol) (h) (%) (%)
1
40
1.6
16
95
98
Electromicrobial NADP^ regeneration with C. thermoaceticum
2
40
1.6
16
95
98
2-Oxoglutarate/glutamate dehydrogenase
3
200
4.0
30
90
96
AQDS(!10mM),NADP" (1 mM), C. thermoaceticum, C. tyrobutyricum
Yield of (i?)-enantiomer.
5.2 NADH and NADPH regeneration with crude extract of Clostridium thermoaceticum and other cells Using the purified fS^-3-hydroxycarboxylate oxidoreductase (107) and two forms of NADPH regeneration led to the chiral 3-hydroxy carboxylates shown in Table 28 (83). All products of the bioreduction procedures showed ee values greater than 98 %. On the basis of the optical rotation value reported (108) the synthesized 3-hydroxybutyrate was the (5)-enantiomer. It was assumed that the configuration of the other prepared 3hydroxycarboxylates was the same. As checked with racemic mixtures of the derivatives of the four 3-hydroxy acids mentioned in Table 28, the applied chiral gas/hquid chromatography columns separated the enantiomers. For details see (83).
875
TABLE 28 Preparation of various (iS)-3-hydroxy-carboxylates by reducing 130 mM solutions of the corresponding 3-oxo carboxylates with purified (iS)-3-hydroxy carboxylate oxidoreductasefi*omC tyrobutyricum (109) and the regeneration^^ of NADPH (83). Enantiomeric excess ( % )
Yield (%)
Pyridine nucleotide regeneration system
3-Hydroxybutyrate 3-Hydroxybutyrate
>99 >99
98 98
Electrochemical cell/AMAPOR FDH/formate
3-Hydroxyvalerate 3-Hydroxyvalerate
>99 >99
98 98
Electrochemical cell/AMAPOR FDH/formate
3-Hydroxycaproate 3-Hydroxycaproate
>98 >98
95 95
Electrochemical cell/AMAPOR FDH/formate
3 -Hydroxyisocaproate 3-Hydroxyisocaproate
>99 >99
80 95
Electrochemical cell/AMAPOR FDH/formate
Product
NADPH was regenerated under anaerobic conditions by crude extracts of C. thermoaceticum containing formate dehydrogenase (FDH) as well as artificial mediator accepting pyridine nucleotide oxidoreductases (AMAPORs) and formate as electron donor. For electromicrobial redox reactions see Section 6. Also the microbial reduction of 3-oxobutyrate with cells of C. tyrobutyricum occurred enantioselectively (Table 29). The (5)-3-hydroxybutyrate showed ee values above 96 %. Without addition of NADP^ to C tyrobutyricum cells 3-oxobutyrate was reduced very slowly (exp. 1). The addition of 1 mM NADP^ to broken cells of C. tyrobutyricum led to an almost 16 fold productivity number (exp. 2). This is the simplest method. By addition of cells of C thermoaceticum the quantitative reduction of 200 mM 3-oxobutyrate could be performed at the expense of hydrogen gas with a productivity number of 1520 (exp. 3). The acceleration fi'om experiment 2 to 3 is caused by the higher AMAPOR activities in the reaction mixture. Cells of C. tyrobutyricum possess 0.80 U*mg'^ protein NADP(H)-dependent AMAPOR activity (97), whereas in cells of C. thermoaceticum this activity is about 8-9 U*mg'^ protein (82). Further examples of the combination of enriched or even purified enzymes together with a crude extract of C. thermoaceticum in the presence of added small concentrations of pyridine nucleotide are shown in fiirther experiments. The increased concentration of NADP^ in the reaction system when additional 0.06 g wet packed cells of C. thermoaceticum were used resulted in a productivity number of 890 (exp. 4). This indicates that the amount of NADP^ in cells of C. thermoaceticum was sufficient for a reasonable reduction rate. We determined about 50 ^g NADP^ per g C. thermoaceticum cells. The conditions applied in experiments 3 and 4 were also usefiil for the reduction of 400 mM solutions of 3-oxobutyrate (exp. 5 and 6). Using formate instead of hydrogen gas as electron donor, under conditions otherwise identical to those used in experiment 4, decreased the productivity number to 420 (exp.7). Starting with 10 mmol 3-oxobutyrate in a 200 mM concentration, obtained
876
by hydrolysis of the ester, led in the presence of 0.3 mM NADP^ with C. tyrobutyricum to a productivity number of 350 (exp. 8). This seems to be the most economic procedure, since ethyl 3-oxobutyrate is much cheaper than the lithium salt of 3-oxobutyrate (83). Table 29 Preparation of (/S)-3-hydroxybutyrate from 3-oxobutyrate by resting cells of C. tyrobutyricum and various additions for the regeneration of the pyridine nucleotide NADPH. For methods A, B and C see text. Substrate No. concentr. (mM)
Reaction PN time * (h) mmol*kg^h-^
Yield Electron donor (%)
Pyridine nucleotide regeneration system
1
200
72
37
15
H2
MV (1 mM)''
2
200
30
590
88
H2
MV (1 mM), NADP"^ (1 mM), method A
1520
95
H,
MV (1 mM)^ NADP* (ImM), 60 mg C. thermoaceticum
200
4
200
25
890
90
H2
MV (1 mM)^ 60 mg C thermoaceticum
5
400
24
890
90
H2
MV (1 mM)^ NADP^ (ImM), 60 mg C thermoaceticum
6
400
48
450
80
H2
MV (1 niM)\ 60 mg C. thermoaceticum
7
200
40
420
82
8
200
48
350
80
^ Time for quantitative reduction.
Formate Formate (250 mM), 900 mg C. tyrobutyricum, 600 mg C. thermoaceticum, method B H2
MV (1 mM), NADP^ (0.3 mM), 3000 mg C tyrobutyricum, method C
Variationof method A.
With purified (5)-3-hydroxybutyrate oxidoreductase yields of 80-98 % for reductions and dehydrogenations are quite acceptable (Tables 28,29). The use of formate as an electron donor together with crude extract of C thermoaceticum is simple and effective. NADH regeneration systems A-C mentioned in Table 29 (83) Method A: A solution containing 1.6 ml 100 mM Tris/HCl buffer, pH 8.0, 0.09 g wet packed cells of C tyrobutyricum, 1 mM NADP^, 1 mM methylviologen and 50 jug tetracycline was placed in a 25 ml glass vessel combined with a mercury filled Warburg
877
manometer. The reduction was started by adding 200 mM 3-oxo-butyrate, produced by hydrolysis of 200 mM ethyl 3-oxo-butyrate by 50 U esterase to the mixture and shaking it under an atmosphere of hydrogen gas at 35 °C. The progress of reduction was indicated by the decreasing hydrogen pressure. Method B: A solution containing 25 ml 0.1 M Tris/HCl buffer, pH 8.0, 0.9 g wet packed cells of C. tyrobutyhcum, 0.6 g wet packed cells of C thermoaceticum, 1 mM NADP^, 250 mM formate, 0.5 mg tetracycline and 200 mM 3-oxo-butyrate, by hydrolysis of 200 mM ethyl 3-oxobutyrate by 100 U esterase, was placed in a 50 ml glass vessel at 35 °C under an atmosphere of nitrogen gas. The mixture was magnetically stirred. Method C: A solution containing 50 ml 0.1 M Tris/HCl buffer, pH 8.0, 3.0 g wet packed cells of C tyrobutyricum, 0.3 mM NADP^, 1.5 mg tetracycline and 200 mM 3oxobutyrate, produced as mentioned above by hydrolysis of 200 mM ethyl 3-oxobutyrate by 100 U esterase, was placed in a 500 ml glass vessel under an atmosphere of hydrogen gas at 35 °C. The reduction started after adding 1 mM methylviologen and shaking. The progress of the reduction was followed by measuring the decreasing dihydrogen pressure. 5.3 Growth of Clostridium thermoaceticum and Clostridium formicoaceticum The cells of C. thermoaceticum DSM 521 (16) were grown in volumes up to 300 1 at 65 °C according to (82a) with the following change and additional supplements: sodium thioglycolate was omitted, 10 g NaHCOs, 0.1 g Na2S204 and 0.134 mg NiCl2*6H20 were added per litre growth medium. The yield on wet packed cells was 10-14 g*r^ (76). Cells of C. formicoaceticum DSM 92 (16) were grown on DSM medium 14 (16, p. 280) with some modifications (81). The medium contained the following trace elements additionally or in differing amounts: 100 ^M Fe(NH4)(S04)2, 100 ^iM C0CI2, 10 ^iM Na2W04, 10 ^MNa2Mo04*2H20, 1 iaMNa2Se03, l^MNiCl2, and 280 ^MNa2S204. A modified medium containing 100 mM fiimarate instead of NaHCOs at a pH value between 8.0-8.5 delivered cells with high activities of fiimarate reductase and fimiarate hydratase. The cells were grown in a range from 0.2-300 1, harvested at the beginning of the stationary phase (A578 = 4.5) and stored at -18 °C under nitrogen.
6. FURTHER EXAMPLES OF ELECTROMICROBIAL AND ELECTROENZYMIC REDOX REACTIONS Enoate reductase, 2-hydroxy carboxylate viologen oxidoreductase (HVOR) and AMAPORs (Section 5) are enzymes able to accept reversibly single electrons from artificial mediators such as viologens and others. These mediators transfer electrons from or to electrodes. Therefore by the presence of the aforementioned enzyme activities biocatalytic redox reactions can be carried out in electrochemical cells. As already mentioned in Section 1.2 AMAPORs catalysing Reactions [8] and/or [8a] are rather ubiquitous. Electromicrobial reductions can also be carried out with yeasts (Section 1,2). Since the potential of the working electrode can be chosen at will, reductions as well as
878
dehydrogenations can be performed if suitable mediators are applied. We demonstrated that in various papers (6-8,10). For reductions in aqueous solutions cathodes with a high overpotential for hydrogen formation must be applied in order to reduce only the artificial mediator. This can be checked by means of cyclovoltammetry (110). Moreover the artificial mediator must not react spontaneously with the substrate. Gold, silver, carbon and mercury are suitable cathode materials when working in a neutral pH range and at working potentials higher than -450 mV versus SCE (saturated calomel electrode). In case of more negative working potentials, lead, bismuth antimony and tin cathodes become also suitable. The electrochemical cell is a standard three electrode arrangement with the reference electrode as the third electrode. Schematic drawings of electrochemical cells used are depicted in (7,8) together with more detailed information. When reductions are performed, oxygen is formed at the anode which may oxidize the reduced artificial mediator forming highly reactive species of oxygen which in turn destroy other components of the catholyte. Therefore catholyte and anolyte must be separated by a diaphragm, which has to meet several requirements: (i) It has to allow proton transfer from the anolyte where protons are formed to the catholyte where protons are consumed (Scheme 2a). (ii) It has to be sufficiently conductive, (iii) It has to prevent oxygen from passing through. Nafion® ion exchange membranes with a thickness of 0.5 mm have proofed to be suitable. In addition anaerobic conditions are maintained by flushing buffer and cathode compartment of an electrochemical cell with an inert gas. For dehydrogenations the working electrode must be inert to the positive potentials applied, therefore noble metals have to be used in order to avoid the eventually negative influence of heavy metal cations on enzymes. Also carbon electrodes are very suitable working electrodes since on oxidation no inhibiting heavy metal cations are formed. For optimisation purposes of the described redox systems an electrochemical cell may be especially useful, since the current is a measure of the reaction rate. For a reaction consuming two moles of electrons per mol of substrate a current of 3.2 mA reduces 1 jimol substrate per minute. The influence of all measures taken to optimise the system as varying pH, concentrations, temperature etc. can be followed by recording the current. It is easy to supply 10^-10"* U of enzyme activity contained in wet packed cells of a microorganism or especially when the enzyme is already enriched in 100 ml electrolyte. Under these conditions, the over all reaction rate in the electrochemical cell is often limited by the reduction of the artificial mediator. Sufficiently large electrodes and efficient stirring are helpfiil measures to avoid such limitations. When a microorganism is used for the catalysis of a pyridine nucleotide dependent redox reaction the reaction rate may be limited by the concentration of the pyridine nucleotide. If broken cells are used the concentrations of the necessary nucleotide such as NAD"^ or NADP^ can be added (see Table 29 for an example). If the reaction rate is limited by AMAPOR activity crude extracts of C. thermoaceticum or that of another microorganism can be added. An example for such an optimisation is shown in Fig. 4 (97). This experiment was performed in order to demonstrate what is going on. As examples for preparative purposes see Fig. 4-6 and Tables 30, 31.
879
mA 1.5 1.0 0.5 3
7 Time [h] Fig. 4. Combination of Candida utilis and Alcaligenes eutrophus cells in an electrochemical cell for the reduction of hydroxyacetone. The C. utilis cells contained 0.63 U of a carbonyl group reductase and the A. eutrophus cells 0.62 U AMAPOR. The cells were suspended in 40 ml 0.1 M Tris buffer, pH 7.0, containing 3.3 mM methylviologen. The applied cathode potential was -700 mV versus SCE. A: Addition of 1.4 mg disrupted cells (dry weight) of C. utilis; B: Addition of 32 jjmol NAD^; C: Addition of 400 jjmol: hydroxyacetone; D: Addition of 0.65 mg disrupted cells (dry weight) of A. eutrophus. The C. utilis cell extract contained about 0.12 U AMAPOR for NADHformation and that of A. eutrophus < 0.003 U hydroxyacetone reductase. 1
4
5
6
Two typical examples for an electroenzymic and an electromicrobial reduction of 6 mmol (£')-2-metliylcinnamate and 24.6 mmol 2-oxo-5-methylpentanoate, respectively, are given in Fig. 5 and 6(111). Examples for electroenzymic dehydrogenations combined with electromicrobial regeneration of pyridine nucleotides with C. thermoaceticum are presented in Tables 30 and 31. A graphite working electrode (35-40 cm^, Sigraflex 72010 from SIGRI Electrographite, Germany) was applied. The counter electrode compartment contained a platinum electrode dipped in 3 ml 3.0 M H2SO4. Details are given in Tables 30 and 31. In Section 5 Table 25 the preparation of 6-phospogluconate from glucose-6-phosphate is described using oxygen as the final acceptor. For HVOR oxidized methylviologen is a positive effector but reduced methylviologen becomes strongly inhibiting already in concentrations <0.1 mM, therefore the electrochemical reaction (Reaction [1]) must always be the limiting reaction (48). For the reduction of 2-0X0 carboxylates the rates increase with the decreasing ratio MV^*/MV^^. Benzylviologen does not show these effects. In Fig. 6 the current does not drop to the the starting value but to -50 mA when all phenylpyruvate is reduced after -12 h. At this rate hydrogen gas is formed via hydrogenase also present in P. vulgaris since the concentration of MV^* was <1/10 the K^ of the hydrogenase until the HVOR reaction stops. Using cells of P. vulgaris grown on lactate instead of glucose productivity numbers of up to 450 000 have been achieved (unpublished).
880
i
imA
40 30 • 20 10
11 1
•
i
4
^
h
10
12 * Time [h]
Fig. 5. Electroenzymic reduction of {E)-2'methylcinnamate. The cathode compartment of an electrochemical cell contained 85 ml 0.1 M potassium phosphate buffer pH 7, 6.0 mmol 2-methylcinnamate and 0.28 mmol methylviologen. The working potential was set to -760 mM versus SCE and after the reduction of the methylviologen the reaction was started by adding 15 U enoate reductase. A maximum current of -40 mA was recorded. The drop of the current to almost zero was indicative for the complete conversion of the substrate to {R)-2-methyl-3-phenylpropanoate. Fig. 6 demonstrates the preparative electromicrobial reduction of of 2-oxo-4-methylpentanoate to (i^)-2-hydroxy-4-methylpentanoate. ^mA
^^Time[h] Fig. 6. Scan of the current during the electromicrobial reduction of phenylpyruvate. The cathode compartment contained 200 ml 0.1 Mpotassium phosphate buffer, pH 7.0, 20 mmol phenylpyruvate and 3 mM methylviologen. The working potential was set to -760 mV versus SCE. After adding 0.075 g wet packed cells of Proteus vulgaris and starting to stir at a rate of ^400 rpm a current of ^35 mA was obtained. The amount of P. vulgaris was too much since the electrochemical reaction Reaction [1]) was limiting (48) (catholyte not blue). Therefore at A the starting concentration of 3 mM methylviologen was doubled to 6 mM. According to HPLC, 96 % of the expected quantity of (R)-phenyllactate was formed after 12 h. The productivity number was approximately 125 000.
881
Table 30 Oxidative decarboxylation of 6-phosphogluconate to ribulose-5-phosphate with 6phosphogluconate-dehydrogenase and electromicrobial NADP^ regeneration with partially purified AMAPOR. Forty ml 0.1 M Tris/HCl buffer, pH 7.2, contained 20 mM 6-phosphogluconate, 3 mM CAV, 0.5 mM NADP^, 3.3 mM MgCl2 and 6-phosphogluconatedehydrogenase (15 U). The reaction temperature was 30 °C and the working potential was -200 mV versus SCE. NADP^ regenerating system
AMAPOR (U)
Yield (%)
C. thermoaceticum^ C. thermoaceticunf AMAPOR^
20 7 20
80 80 98
Reaction time
(h) 2.3 n.d. 2.5
^ Used as crude extract. ^ Partially enriched by heat treatment of crude extract at 61 °C for 30 min, ammonium sulphate precipitation (45 % saturation), resolubilisation of the precipitated protein in 50 mM TEA-maleate buffer, pH 7.5, and chromatography on a Sepharose G 200 column. Table 31 Electromicrobial NADP^ regeneration for the oxidative decarboxylation of (2R,3S)isocitrate fi-om racemic isocitrate leading to (2iS,3i?)-isocitrate, using different mediators. Forty ml 0.1 M Tris/HCl buffer, pH 7.4, contained the indicated concentration of racemic isocitrate, 3 mM mediator, 0.5 mM NADP^, 3.3 mM MgCl2, 0.1-0.3 g wet packed cells of C thermoaceticum and 20-40 U isocitrate dehydrogenase EC 1.1.1.42 fi-om porcine heart. The reaction temperature was 37 °C, and the working potential was -200 mV versus SCE. Substrate concentration (mM) 400 50 50
Mediator
After I h
CAV CAV AQ-2,6-DS
n.d. 15 000 17 000
Productivity number After the end Reaction time of reaction (h) 2 600 13 000 14 000
37.3 3.8 3.6
The relatively low productivity number in the experiment with 400 mM substrate is probably due to an inhibition of one or both enzymes by isocitrate .
882
ACKNOWLEDGEMENTS We gratefully acknowledge financial support by Deutsche Forschungsgemeinschaft (SFB 145), EG Programmes BAP 250 (D) and BRIDGE BIOT-CT-90-0157, Fonds der Chemischen Industrie, by BASF and other chemical companies. The extraordinary engagement of co-workers mentioned in the references and of the technical staff is thankfully appreciated.
REFERENCES 1 2 2a 3 4 5 6 7 7a 8 9 10 11 12 13 14 15
16 17 18 19 20
K. Drauz and H. Waldmann (eds.) Enzyme Catalysis in Organic Synthesis. A Comprehensive Handbook., VCH, Weinheim, 1995. J.M.S. Cabral, D. Best, L. Boross and J. Tramper (eds.) Applied Biocatalysis. Harwood Academic pubhshers, Chur, 1993. C. H. Wong and G. M. Whitesides, Enzymes in Synthetic Organic Chemistry. Pergamon 1994. K. Faber, Biotransformations In Organic Cheniistry.2nd Edition Springer-Verlag, Berlin, 1995, pp. 1-356. H. G. W. Leuenberger, in: H.-J. Rehm, G. Reed (eds.) and K. Kieslich (Volume editor). Biotechnology Vol. 6a: Methodology, Verlag Chemie, Weinheim 1984, pp. 5-29. H. Yamada and S. Shimizu, Angew. Chem. Int. Ed. Engl., 27 (1988) 622-642. H. Giinther and H. Simon, Biocatalysis & Biotransform., 12 (1995) 1-26. H. Simon, J. Bader, H. Giinther, S. Neumann and J. Thanos, Angew. Chem. Int. Ed. Engl, 24 (1985) 539-553. H. Skopan, H. Giinther and H. Simon, Angew. Chem. Int. Ed. Engl., 26 (1987) 128130. I. Thanos, J. Bader, H. Giinther, S. Neumann, F. Krauss and H. Simon, Meth. Enzymol., 136 (1987) 302-317. P. D'Arrigo, H.-E. Hogberg, G. Pedrocchi-Fantoni and S. Servi, Biocatalysis, 9 (1994) 299-312 (see here earlier literature). H. Giinther, C. Frank, H.-J. Schiitz, J. Bader and H. Simon, Angew. Chem. Int. Ed. Engl., 22 (1983) 322-323. P. A. Levene and A. Walti, Org. Synth., 10 (1930) 84-86. K. Nakamura, Y. Kawai, N. Nakajiama and A. Ohno, J. Org. Chem., 56 (1991) 4778-4783. H. J. Schiitz and H. Simon, Z. Naturforsch., 41c (1986) 172-178. H. Giinther, H. Simon and H.-L. Schmidt, in: T. A. Baillie and J. R. Jones, (Eds.), Synthesis and Applications of Isotopically Labelled Compounds. Proceedings of the Third International Symposium, 1988, pp. 323-328. R. Eck, H. Giinther and Simon, in: J. Allen and R. Voges (Eds.), Synthesis and Applications of Isotopically Labelled Compounds. Proceedings of the Fifth International Symposium. Strasbourg, France, 1994, J. Wiley & Sons, Chichester, 1995, pp. 735-746. Catalogue of Strains 1993, German Collection of Microorganisms and Cell Cultures. Fifth Edition, Mascheroder Weg, Braunschweig, Germany M. Biihler and H. Simon, Hoppe-Seyler's Z. Physiol. Chem., 363 (1982) 609-625. I. Thanos, A. Deffiier and H. Simon, Biol. Chem. Hoppe-Seyler, 369 (1988) 451460. S. Kuno, A. Bacher and H. Simon, Biol. Chem. Hoppe-Seyler, 366 (1985) 463-472. H. Simon, in: F. Miiller (Ed.), Chemistry and Biochemistry of Flavoenzymes Vol. II, CRC Press Inc., Boca Raton, Florida, 1991, pp. 317-328.
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21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
J. Caldeira, R. Feicht, H. White, M. Teixeira, J. J. G. Moura, H. Simon and I. Moura, J. Biol. Chem., 271 (1996) 18743-18748. J. Bader, H. Giinther, E. Schleicher, H. Simon, S. Pohl and W. Mannheim, Arch. Microbiol., 125 (1980) 159-165. J. Bader and H. Simon, Arch. Microbiol., 127 (1980) 279-287. H. Sedhnaier, W. Tischer, P. Rauschenbach and H. Simon, FEBS Letters , 100 (1979) 129-132. H. Hashimoto, B. Rambeck, H. Giinther, A. Mannschreck and H. Simon, HoppeSeyler's Z. Physiol. Chem., 356 (1975) 1203-1208. L. Angermaier and H. Simon, Hoppe Seyler's Z. Physiol. Chem., 364 (1983) 961975. R. Leinberger, W. E. Hull, H. Simon and J. Retey, Eur. J. Biochem., 117 (1981) 311-318. A. R. Battersby, A. L. Gutman, C. J. R. Fookes, H. Gunther and H. Simon, J. Chem. Soc. Chem. Comm., (1981) 645-647. A. Gabler, W. Boland, U. Preiss and H. Simon, Helv. Chim. Acta, 74 (1991) 17731789. M. Veith, M. Lorenz, W. Boland, H. Simon,and K. Dettner, Tetrahedron, 50 (1994) 6859-6874. P. Egerer and H. Simon, Biochim. Biophys. Acta, 703 (1982) 158-170. R. Eck and H. Simon, Tetrahedron, 50 (1994) 13631-13640. H. Brunner, W. Zettlmeier, in Handbook of Enantioselective Catalysis with Transition Metal Compounds, VCH Weinheim, Weinheim - New York. A. Togni, C. Breutel, A. Schnyder, F. Spindler, H. Landert and A. Tijani, J. Am. Chem. Soc, 116 (1994) 4062-4066. E. Krezdom, S. Hocherl and H. Simon, Hoppe-Seyler's Z. Physiol. Chem., 358 (1977)945-948. R. Eck and H. Simon, Tetrahedron, 50 (1994) 13641-13654. L. Angermaier, J. Bader and H. Simon, Hoppe-Seyler's Z. Physiol. Chem., 362 (1981)33-38. J. Bader, M.-A. Kim and H. Simon, Hoppe-Seyler's Z. Physiol. Chem., 362 (1981) 809-820. P. Egerer and H. Simon, Biotechnol. Lett., 4 (1982) 501-506. G. Gorgen, W. Boland, U. Preiss, and H. Simon, Helv. Chim. Acta, 72 (1989) 917928. M.-J. Kim and G. M. Whitesides, J. Am. Chem. Soc, 110 (1988) 2959-2964. M. A. Luyten, D. Bur, H. Wynn, W. Parris, M. Gold, J. D. Friesen and J. B. Jones, J. Am. Chem. Soc, 111 (1989) 6800-6804 and literature cited there. A. Schummer, H. Yu and H. Simon, Tetrahedron, 47 (1991) 9019-9034. H. Yu and H. Simon, Tetrahedron, 47 (1991) 9035-9052. D. Bonnaffe and H. Simon, Tetrahedron, 48 (1992) 9695-9706. T. Trautwein, F. Krauss, F. Lottspeich and H. Simon, Eur. J. Biochem., 222 (1994) 1025-1032. C. Schinschel and H. Simon, Appl. Microbiol. Biotechnol., 38 (1993) 531-536. H. Gunther, S. Neumann and H. Simon, J. Biotechnol., 5 (1987) 53-65. R. Eck and H. Simon, Tetrahedron Asym., 5 (1994) 1419-1422. M. Reimer, J. Am. Chem. Soc, 53 (1931) 3147-3149. E. Friedmann, Helv. Chim. Acta, 14 (1931) 783-793. T. Anke, G. Schramm, B. Schwalge, B. Steffan and W. StegUch, Liebigs Ann. Chem., (1984) 1616-1625. M. Rambaud, M. Bakasse, G. Duguay and J. ViUieras, Synthesis, (1988) 564-566. G. M. Ksander, J. E. McMurry and M. Johnson, J. Org. Chem., 42 (1977) 11801185.
884
55
A. Takeda, S. Wada, M. Fujii and H. Tanaka, Bull. Chem. Soc. Jpn., 43 (1970), 2997-2998. 56 S. Tsuboi, H. Furutani, M. Utaka amd A. Takeda, Tetrahedron Lett., 28 (1987), 2709-2712. 57 J.-L. Luche, J. Am. Chem. Soc, 100 (1978), 2226-2227. 58 S. Neumann and H. Simon, FEES Letters, 167 (1984) 29-32. 59 H. Simon, H. Giinther, J. Bader and S. Neumann, in : Enzymes in OrganicSynthesis, Ciba Foundation Symposium, Pitman, London, 111 (1985) 97-111. 60 G. Gubitz and S. Mihellyes, Chromatographia, 19 (1984) 257-259. 61 C. Schinschel and H. Simon, Bioorg. & Med. Chem., 2, (1994) 483-491. 62 C. Schinschel and H. Simon, J. Biotechnol., 31 (1993) 191-203. 63 M. L. Fultz and R. A. Durst, Anal. Chem. Acta, 140 (1982) 1-18. 64 J. Knappe and G. Sawers, FEMS Microbiol. Rev., 75 (1990) 383-398. 65 O. Theander, in: W. Pigman and D. Horton (eds.) The Carbohydrates. Chemistry and Biochemistry Vol. IB., Academic Press, New York, 1980, pp. 1013-1085. 66 T. Sugai, G. J. Shen, Y. Ichikawa and C. H. Wong; J. Am. Chem. Soc, 115 (1993) 413-421. 67 H. Roper, VCH Verlagsgesellschaft Weinheim 1991 267-288, in: Carbohydrates as Organic Raw Materials, ed. F. W. Lichtenthaler. 68 P. C. C. Smits, EP-A-0 152 498 (Int. CI. C0H7/027(1984)) Chem. Abstr., 103 (1985) 160805q. 69 (a) G. S. Eadie, J. Biol. Chem., 146 (1942) 85; (b) B. H. J. Hofstee, J. Biol. Chem., 199(1952)357-364. 70 V. F. Pfeifer, C. Vojnovich, E. N. Heger, G. E. N. Nelson and W. C. Haynes, Ind. Eng. Chem., 50 (1958) 1009-1012. 71 M. A. Ghalambor, E. M. Levine and E. C. Heath, J. Biol. Chem., 241 (1966), 32073215. 72 K. Gatzi and T. Reichstein, Helv. Chim. Acta, 21 (1938) 456-463. 73 G. Karsten and H. Simon, Appl. Microbiol. Biotechnol., 38 (1993) 441-446. 74 H. Simon, H. White, H. Lebertz and I. Thanos, Angew. Chem. Int. Ed. Engl., 26 (1987)785-787. 75 H. White, G. Strobl, R. Feicht and H. Simon, Eur.J. Biochem., 184 (1989) 89-96. 76 G. Strobl, R. Feicht, H. White, F. Lottspeich and H. Simon, Biol. Chem. HoppeSeyler, 373 (1992) 123-132. 77 C. Ruber, H. Skopan, R. Feicht, H. White and H. Simon, Arch. Microbiol., 164 (1995)110-118. 77a H. White and H. Simon, Arch. Microbiol., 158 (1992) 81-84. 78 C. Ruber, J. Caldeira, J. A. Jongejan and H. Simon, Arch. Microbiol., 162 (1994) 303-309. 79 P. A. Loach in: G. D. Fasman (Ed.), Handbook of Biochemistry and Molecular Biology: Physical and Chemical Data (3'^* Edition). CRC Press, Cleveland, 1976, pp. 122-130. 80 H. White, C. Huber, R. Feicht and H. Simon, Arch. Microbiol., 159 (1993) 244-249. 81 H. White, R. Feicht, C. Huber, F. Lottspeich and H. Simon, Biol. Chem. HoppeSeyler, 372 (1991) 999-1005. 82 M. Schulz, M. Bayer, H. White, H. Giinther and H. Simon, Biocatalysis, 10 (1994) 25-36. 82a L. G. Ljungdahl and J. R. Andreesen, Methods Enzymology, 53 (1978) 360-372. 83 M. Bayer, M. Schulz, H. Giinther and H. Simon, Appl. Microbiol. Biotechnol., 42 (1994)543-547. 84 E. V. Price and L. Levintov, Biochemical Preparations, 2 (1952) 22-24. 85 S. W. Ragsdale, J. E. Clark, L. G. Ljungdahl, L. L. Lundie and H. L. Drake, J. Biol. Chem., 258 (1983) 2364-2369.
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86 86a 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112
M. Schulz, H. Leichmaim, H. Giinther and H. Simon, Appl. Microbiol. BiotechnoL, 42 (1995) 916-922. M. Bayer, K. Walter and H. Simon, Eur. J. Biochem., 239 (1996) 686-691 H. K. Chenault and G. M. Whitesides, Appl. Biochem. Biotech., 14 (1987) 147-197. H. K. Chenault and G. M. Whitesides, Bioorganic. Chem., 17 (1989) 400-409. H. K. Chenault, E. S. Simon and G. M. Whitesides, Biotech. Genetic Eng. Rev., 6 (1988)221-270. J. Peters and M.-R. Kula, Biotechnol. Appl. Biochem., 13 (1991) 363-370. D. Westerhausen, S. Herrmann, W. Hummel and E. Steckhan, Angew. Chem. Int. Ed. Engl., 31 (1992) 1529-1531. I. Wilhier and D. Mandler, Enzyme Microb. Technol., 11 (1989), 467-483. A.-E. Biade, C. Bourdillon, J.-M. Laval, G. Mairesse and J. Moiroux, J. Am. Chem. Soc, 114(1992)893-897. U. Kragl, D. Vasic-Racki and C. Wandrey, Chem.-Ing.-Tech., 64 (1992) 499-509. D. Weuster-Botz, H. Paschold, B. Striegel, H. Gieren, M.-R. Kula and C. Wandrey, Chem. Eng. Technol, 17 (1994) 131-137. K. Seelbach, B. Riebel, W. Hummel, M.-R. Kula, V. I. Tishkov, A. M. Egorov, C. Wandrey and U. Kragl, Tetrahedron Lett., 37 (1996) 1377-1380. J. Bader, H. Giinther, S. Nagata, S.; H.-J. Schutz, M.-L. Link and H. Simon, J. Biotechnol, 1(1984)95-109. S. Nagata, R. Feicht, W. Bette, H. Giinther and H. Simon, Appl. Microbiol. Biotechnol, 26 (1987) 263-267. H. Giinther and H. Simon, Appl. Microbiol. Biotechnol, 26 (1987) 9-12. S. Nagata, H. Giinther, J. Bader and H. Simon, FEBS Letters, 210 (1987) 66-70. H. Giinther, A. S. Paxinos, M. Schulz, C. van Dijk and H. Simon, Angew. Chem. Int. Ed. Engl, 29 (1990) 1053-1055. A. S. Paxinos, H. Giinther, D. J. M. Schmedding and H. Simon, Bioelectrochem. Bioenerg., 25 (1991) 425-436. H. Giinther, C. Frank and H. Simon, in: D. Behrens (ed.), DECHEMA Biotechnology Conferences 4, VCH, Weinheim, 1990, pp. 107-110. H. Giinther, A. S. Paxinos, M. Schulz and H. Simon, in: D. Behrens (ed.), DECHEMA Biotechnology Conferences 4, VCH, Weinheim, 1990, pp. 281-285. O. Miyawaki and T. Yano, Enzyme Microb. Technol, 14 (1992) 474-478. C.-H. Wong, A. PoUak, S. D. McCurry, J. M. Sue, J. R. Knowles and G. M. Whitesides, Methods Enzymol, 89 (1982) 108-121. M. Bayer, H. Giinther and H. Simon, Appl. Microbiol Biotechnol, 42 (1994) 4045. A. Kramer and H. P. Pfander, Helv. Chim. Acta, 65 (1982) 293-301. M. Bayer, H. Giinther and H. Simon, Arch. Microbiol, 163 (1995) 310-312. J. C. Hoogvliet, L. C. Lievense, C. van Dijk and C. Veeger, Eur. J. Biochem., 174 (1988)273-280. H. Simon, and H. Giinther, in: W. Crueger, K. Esser, P. Praeve, M. Schlingmann, R. Thauer and F. Wagner (Eds.), Jahrbuch der Biotechnologie 1986/87, Carl Hanser Verlag, Miinchen, pp. 359-367. H. E. J. Hendriks,B. F. M. Kuster and G. B. Marin, Carbohydr. Res., 214 (1991) 7185.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 20 © 1998 Elsevier Science B.V. All rights reserved.
887
Microcystins and Nodularins Hepatotoxic Cyclic Peptides of Cyanobacterial Origin Luis Moroder and Sabine Rudolph-Bohner Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, D-82152 Martinsried Germany
The harmfull effects of cyanobacteria (blue-green algae) that grow worldwide in eutrophic fresh and brackish water as well as in marine environments, were originally noted more than a century ago, but only recently was it shown that these organisms produce a variety of bioactive compounds. Among these the microcystins are one important group of cyanobacterial toxins that are involved in poisoning of animals and in human health problems around the world. The microcystins are cyclic heptapeptides characterized by a common invariable structure containing a few variable sections, and all are potent toxins that inhibit the action of type-1 and type-2A protein phosphatases, two of the major serine!threonine specific protein phosphatases in eukaryotic cells, and thus are potent liver tumour promoters. While there is ample information as to the toxicity of these cyanobacterial peptides, their mechanism of action at molecular level as well as their three-dimensional structure in solution and in the enzymebound state have only recently been disclosed. Because of their profound cellular effects, their use as pharmacological probes for enzyme functions may facilitate the understanding of a large diversity of physiological processes that are under the control of reversible phosphorylation. Similarly, knowledge of their mode of action may possibly lead to the design of new classes of phosphatase inhibitors as potential anti-tumor agents, anti-inflammatories and immunosuppressants.
1.
Introduction
Reversible phosphorylation of proteins on serine, threonine and tyrosine residues by protein kinases and phosphatases represents the principal mechanism of signal transduction events that control a multitude of cellular processes (see ref. 1 and 2 for detained reviews). Phosphorylation is a posttranslational chemical modification that is used by prokaryotic and eukaryotic cells to define the properties of a large
888
variety of proteins including enzymes, receptors, ion channels and regulatory and structural proteins.
A^P
Protein kinase
Protein
^P^
Phosphoprotein
Phosphoprotein Phosphatase
^ Q
Fig. 1. Reversible protein phosphorylation From the thermodynamic point of view, phosphorylation and dephosphorylation are both favorable reactions which occur efficiently in the presence of the right catalysts, i.e. of protein kinases or protein phosphatases. Protein kinases are phosphotransferases which catalyse the transfer of the y-phosphoryl group of ATP to the side-chain hydroxyl group of serine, threonine and tyrosine in the presence of Mg2+. Protein phosphatases are phosphotransferases which catalyse the transfer of the phosphoryl group from the side-chain of an amino acid residue to water molecules (Fig. 1). Protein kinases recognize specific substrates and much is known and understood about their mode of regulation in vivo. Conversely, the broad substrate specificity of the protein phosphatases suggests the need of particular mechanisms to restrict the actions of these enzymes. It is therefore likely that higher orders of protein structure are involved in dictating substrate specificity for the phosphatases, since the activities of both protein kinases and phosphatases acting on a particular target molecule are tightly regulated (3). Protein phosphatases, unlike protein kinases, belong to several protein/gene families. They were classified on the basis of their substrate specificity, dependence on metal ions, and sensitivity to inhibitory agents (see Table 1). The activities of the type 1 and type 2A protein phosphatases (PP-1 and PP-2A) are independent of metal ions (4,5). The catalytic subunit of protein phosphatase-1 is bound to the regulatory
889
targeting proteins that determine the subcellular localization and activity of the enzyme (6). Protein phosphatase-2A is inactivated by phosphorylation of tyrosine residues on the molecule (7). The type 2B protein phosphatase (PP-2B), also known as calcineurin, consists of a catalytic subunit (A-subunit) and a regulatory subunit (B-subunit). It is dependent on Ca^^-calmodulin complex for complete activation (3).
Table 1. Classification of the phosphatases PP-1/PP-2A/PP-2B Family of Serine/Threonine Phosphatases - all are sensitive to okadaic acid - PP-1\ dephosphorylate preferentially the p subunit of phosphorylase kinase - inhibited by inhibitor 2 and by the phosphorylated form of inhibitor 1 - PP-2'. dephosphorylate preferentially the a subunit of phosphorylase kinase - insensitive to inhibitor 2 and the phosphorylated form of inhibitor 1 - PP-2 A: divalent cations are not required for activation - PP-2B (calcineurin): activated by Ca^"^ PP-2C Family of Serine/Threonine Phosphatases insensitive to okadaic acid • activated by Mg2+ PTP Family of Tyrosine Phosphatases with transmembrane domains without transmembrane domains with dual specificity, i.e. dephosphorylation of serine/threonine and tyrosine
Over 40 protein tyrosine phosphatases have been characterized so far. They possess a catalytic domain and a range of other domains that appear to be essential for subcellular localization and regulation of enzymatic activity (4).
2.
Natural Inhibitors of Phosphatases
In many protein kinases, particularly those which are under the control of second messengers, the regulatory domain contains a pseudo-substrate sequence which binds to the catalytic site and thus prevents access of external substrates to the catalytic site. Binding of the second messengers to the regulatory domain relieves this
890
internal inhibitory effect. For other protein kinases phosphorylation of the catalytic domain is required. This phosphorylation can be either constitutive (autophosphorylation) or provided by a regulatory kinase. Conversely, little is known, so far, about the regulation of protein phosphatases by endogenous modulators. However, a large number of natural toxins (see Table 2) has been identified as powerful and specific inhibitors of the type 1, 2A and 2B family of serine/threonine phosphatases (Fig. 2) (8).
I
stimuli, e. g. Pliorbol Esters, cAMP, cGMP
^ .
/
^
^ fee^H
( (rhr)-OH
ATP V^
Protein Ser/Thr Kinases
0-po:
Protein Phosphatases 1 and 2A
t
toxins, e. g. microcystins
Fig. 2. Inhibition of enzymatic dephosphorylation of serine- and threonine-0phosphate residues in proteins by toxins. Among these natural inhibitors of most diverse chemical structure there are two functional classes (9). First, there are the endogenous inhibitors 1 and 2 (heat-stable proteins) that regulate phosphatases within eukaryotic cells (10-12). The second group are secondary metabolites of bacteria, fungi, plants, dinoflagellates and insects whose natural roles may be in signalling, attack or defense interactions among the various organisms (Fig. 3). Unknowingly, the scientific study of natural substances that inhibit protein phosphatases dates back over one hundred years (13) with early reports on toxins produced by cyanobacteria (blue-green algae). In the last decade it could be shown that cyanobacteria produce a variety of bioactive compounds (4) among which microcystins represent the most important and dangerous group of cyanobacterial toxins (15). Since the signalling pathways based on phosphorylation/dephosphorylation play a key role in essential in vivo regulatory mechanisms, such as hormone action and cell growth control (2,16-17), inhibiton of the enzymes involved by exogeneous substances, present in food and water supply, may represent serious hazards not only to livestock, wild animals, birds and fish, but also to human health.
891 Microcystin-LR
Hs^H^
^ r
H
HNv^/N
HCH3H COOH'
I
NHo
Nodularin COOH
HX
Okadaic acid
Tautomycin
^^
CHo CH«
892
Calyculin-A OH
A °"
H3C
CH3
OH OH
OCH3
Cyclosporin A CH3 CH3
^'^IH^
I
\H3CHO
H^^HCHa
^
H3C
H
CH3
Ri = CH(OH)CH(CH3)CH2CH=CHCH3
FK506
OCH, CH3 CH3 ^ ^ - - - O H
I
H
..
H
893
Cantharidin
<5^ Endothall
Fig. 3. Chemical structures of exogenous natural inhibitors of type 1, 2A and 2B serine/threonine-protein phosphatases.
Table. 2. Properties of serine/threonine-protein phosphatase inhibitors Inhibitor
Chemical Structure
Potency
phosphatases 1 and 2A - okadaic acid - acanthifolicin - dinophysistoxin
polyether carboxylic acids
PP-2A>PP-1»PP-2B
- tautomycin
polyketide
PP-1>PP-2A»PP-2B
- microcystin - nodularins - mutoporins - calyculin A
cyclic peptides
PP2A~PP-1»PP-2B
phosphorylated polyketide
PP2A~PP-1»PP-2B
- cantharidin
terpenoid
PP2A>PP-1»PP-2B
- endothall
terpenoid
PP2A>PP-1»PP-2B
- inhibitors 1 and 2
heat-stable proteins
PP-1
- cyclosporin
cyclic peptide
inhibition as cyclosporin/ cyclophilin complex
- FK506
macrocycHc lactone
inhibition as FKBP/FK506 complex
phosphatases 2B
894
Such exogenous inhibitors, however, may also be of great benefit as was found to be the case for the cycHc peptide cyclosporin A and the macrolide FK506 from fungal origin, which are excellent inhibitors of calcineurin, the protein phosphatase 2B (18,19). Both are presently used as potent immunosuppressants in human medicine for the prevention of graft rejection. Cyclosporin A and FK506 interact with the endogenous proteins (immunophilins) cyclophilin and FK506-binding protein (FKBP), respectively, and the resulting complexes bind to the activated form of calcineurin and block its phosphatase activity with an IC50 < 10 nM. The physiological relevance of these pharmacological effects is unknown since no endogenous equivalent of cyclosporin or FK506 has yet been identified.
3,
Microcystins and Nodularins
Cyanobacteria grow in nutrient-enriched fresh and brackish waters as well as in marine environments around the world (20,21). The ability of the common bloomforming genera of cyanobacteria to produce low molecular weight hepatotoxins is well documented (22-24), although individual genera can include both toxic and nontoxic species. These toxins have been implicated worldwide in poisonings of domestic livestock, pets, and wildlife after the animals have consumed toxin-containing water and/or cyanobacterial cells from lakes, ponds and farmdugouts (25-28). Accidental ingestion of the toxins may also compromise human health (23,29-31). Cyanobacteria known to produce hepatotoxins include species of Microcystis (32-36), Anabaena (37-40), Nostoc (41,42), Oscillatoria (43,44) and the brackish water Nodiilaria spumigena (45,46). These cyanobacteria produce a wide range of toxins including neurotoxic alkaloids, lipopolysaccharides, phenolic compounds and most importantly, the cyclic hepatotoxic peptides microcystins and nodularins. Since both microcystins and nodularins were recently discovered even in shellfish (47) and tropical fish species (48), and a nodularin variant was isolated in Papua New Guinea also from the tropical marine sponge Theonella swinhoei (49), marine prokaryotes are apparently producing identical toxins as the cyanobacteria. However, since this nodularin variant was isolated from a marine sponge it may well be produced by a microbial symbiont. 3.1.
Chemical Structure of Microcystins and Nodularins
Microcystins constitute a large family of cyclic heptapeptides with a high degree of homology in the amino acid sequence. As shown in Fig. 4, they are composed of DAla in position 1, two variable L-amino acids at positions 2 and 4, the p-linked Deryr/zro-P-methylaspartic acid (MeAsp) in position 3, the novel C20 p-amino acid (2S,3S,85,9S) - 3 - amino - 9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid
895
D-iso-Glu H COOH
R O
H3C
H
HCH3
CH2KJ'
H >R
4Y
7 Mdhaor O ^^^
H
D-Ala
H3(f)=0 HN
3 D-Me-iso-Asp or D-iso-Asp
Fig. 4. Chemical structure of microcystins D-iso-Glu H
H3C
HCH3
COOH
CHI 3 N'
Mdhb CH.
»VVV' f
Arg
H
Fig. 5. Chemical structure of nodularins
COOH
H CO
D-Me-iso-Asp
HN HgN^
5
NH
896
(Adda) in position 5 followed by D-glutamic acid (D-Glu) y-linked to N-methyldehydroalanine (Mdha) (50-52). The main structural variations of the more than 50 variants isolated so far, are related to mutations at the two L-amino acids labeled with X and Y in Fig. 4 (53); correspondingly, microcystin variants are named according to the residues X and Y (54). Among all the variants identified so far the most abundant and, correspondingly, the most studied is the microcystin-LR. Nodularins which are produced by Nodularia spumigena in brackish water habitats (52,55,56) may be considered as natural analogs of microcystins. In fact, the chemical structure of nodularins is that of a cyclic pentapeptide (Fig. 5) containing the residues 3-7 of microcystin, i.e. the C20 amino acid Adda, the two D-amino acids Diso-G\M and D-z^o-MeAsp, and the most common residue L-arginine in the variable position Y, whereas the Mdha residue is replaced by the homologous Nmethyldehydro-a-aminobutyric acid (Mdhb) (52,57). It lacks therefore the D-Ala^-LLeu^ dipeptide of microcystines. A variant was isolated from the marine sponge Theonella swinhoei in Papua New Guinea (49) where the variable residue Y (Arg) is replaced by the hydrophobic L-Val; this [L-Val]5-nodularin was trivially named mutoporin. 3.2.
Biological Activities of Microcystins and Nodularins
As mentioned before, microcystins are lethal to a diverse group of animals including dogs, livestock, wild mammals, birds and fish, and intoxications in humans were reported. In rats and mice death occurs approximately 1-3 hr after i.p. injection of the lethal dose of microcystin-LR (37,58,59) and nodularin (56) (LD50 of 50-100 |ig/kg). The toxin causes disruption of the liver leading to death possibly due to liver failure and hemorrhagic shock (59). Histologic lesions of acute microcystin toxicosis progress from "rouding up" of hepatocytes with loss of normal cell-to-cell adhesions, to disruption of hepatic cords, loss of sinusoidal integrity, and death of hepatocytes. These changes in hepatic architecture result in severe intrahepatic hemorrhage. Hepatocytes are thus the target cells in vivo due, at least in part, to a specific uptake of the amphipathic toxin via a multispecific organic anion transporter (60-62). The alterations in the morphology of hepatocytes have been attributed to marked disorganization of intermediate filaments (63) and actin microfilaments (64-66). These cytoskeletal effects are thought to be initiated by the microcystin-induced inhibition of cellular phosphatases. In fact, microcystins and nodularins are the most potent and specific inhibitors of the Ser/Thrprotein phosphatases type 1 and 2A in vitro (67-69), as shown in Table 3, and in vivo (70). The cyclic peptides bind to the same site of the protein phosphatases as okadaic acid, since they inhibit [3H]-okadaic acid binding with similar IC50 values (67,69).
897
Protein phosphatase inhibition has been correlated with the onset of acute microcystininduced hepatotoxicosis in mice (70) and with microfilament reorganization and cell deformation in isolated hepatocytes (65). Inhibition of these enzymes causes hyperphosphorylation of numerous cytosolic and cytoskeletal proteins in isolated hepatocytes exposed to microcystin (63,65,71). It has recently been shown that at higher concentrations similar morphological effects are produced also in nonhepatocytes (72). Table 3. Binding affinities of natural inhibitors of protein phosphatases 1 and 2A Inhibitor
ICso (nM) for type 1 protein phosphatase
ICso (nM) for type 2A protein phosphatase
okadaic acid
20
0.2
tautomycin
0.2
1.0
calyculin-A
0.5-2
0.1-1
microcystin-LR
0.15
0.15
mutoporin
0.6
0.6
inhibitor 1
0.45
-
inhibitor 2
0.8
-
Like the diarrethic shellfish toxin okadaic acid (73), microcystins and nodularins function as potent liver tumour promoters (71,74-76) and therefore they represent a very serious health risk for humans (77). This tumour promotion activity has been associated with the hyperphosphorylation of cytokeratin (71). 3.3.
Structure-Activity Relationship of Microcystins and Nodularins
Since a large number of microcystins has been already isolated and chemically characterized, mutations and modifications in the structure have allowed the identification of essential pharmacophores. The variable L-amino acid residues X and Y are most commonly Leu and Arg, respectively. But microcystin variants with both positions occupied by the basic Arg residue or by hydrophobic residues like Leu and Ala or Leu and Tyr are also produced by various Microcystis strains. While substitution of the Arg residue in position Y with hydrophobic residues or with Met(O) is without significant effect on the toxicity, replacement of the Leu residue in position X with Arg or Met(O) leads to significantly reduced toxicity (53). Variants with
898
homotyrosine or homoisoleucine in position X retain fiill activity, whereas the related Ala, Phe or Trp mutants show a 3- to 4-fold reduced potency. Additional variants containing 1,2,3,4-tetrahydrotyrosine, homotyrosine and homophenylalanine in position X and aminoisobutyric acid and homoarginine in Y have been isolated and structurally characterized, but their toxicity was not reported (53). Additional common differences include demethylation of the amino acid residues 3 and/or 7 (41,78) whereby the dehydroalanine^-variant was found to be 5fold less toxic than the related methylated microcystin (39). Replacement of the Mdha'7 residue with the precursor amino acid residue N-methyl-L-Ser is again minimally affecting the toxicity (34), but the variant with lanthionine in this position shows very weak toxicity probably due to the bulky modification (79). This indicates that the dehydro-amino acid residue itself is unimportant, a fact which is further supported by the observation that dihydro-microcystins, obtained by reduction of Mdha with sodium boron hydride, are fully active both as the stereoisomeric mixture or as isolated isomers, i.e. microcystin-L-Ala^ and microcystin-D-Ala^ (80). The stereochemistry of the Adda residue seems to be essential for the activity and toxicity of microcystins (41,78), since altering the configuration of the double bond from (4E,6E) to (4£',6Z) leads to loss of toxicity (78,81). Hydrogenation or ozonolysis of the diene system also gives rise to non-toxic derivatives (82). Conversely, some other variations of the Adda residue are without effect on the toxic properties; e.g. by replacing the methoxy group at the Adda C-9 position with the acetoxy group the toxicity is fully retained (40). A hydroxy variant at the same position of the Adda residue has also been isolated and toxicity was fully retained (34). The amino acid Adda itself is a non-toxic substance (83,84) and similarly biologically inactive are linear microcystin- and nodularin-related peptides (85). From structure-function studies it is also known that esterification of the acarboxyl group of the D-z^o-Glu^ residue generates nontoxic compounds (34,53), probably as a result of the lack of inhibition of the protein phosphatases. In this context it is interesting to note that the methyl ester of okadaic acid is inactive as well (86). The identical hepatotoxicity and binding affinity for protein phosphatases of microcystins and nodularins clearly show that the dipeptide D-Alai-L-Leu(X)2 is unimportant although the D-Ala unit is almost invariant. In fact, only one mutant with D-Ser in this position has been identified so far (40). However, as mentioned above, the cyclic forms of the penta- and hexapeptides are essential. Considerably less variants have been reported for nodularin, but the structurefunction relationships appear to be similar to those of the microcystin series of toxins. Again the [6Z-Adda3]-variant is essentially inactive, whereas the demethylated Addaanalog and the dihydronodularins were found to be as active as the parent compound
899
(53). Interestingly, substitution of the L-Arg residue in nodularin with L-Val (trivially named mutoporin) leads to biological properties which are somewhat different from those of other nodularins. This variant exerts cytotoxicity against tumour cell lines (49), a property not shown by nodularin. Derivatization of the Arg residue in nodularin with acetylacetone to produce the dimethylpyrimidyl-analog led to similar results as the new compound was found to be cytotoxic to L1210 cells (53). Probably this cytotoxicity is also exerted by the microcystin mutants with both the X and Y position occupied by hydrophobic residues. In summary, the toxicity of microcystins and nodularins require that the peptides are in the cyclic form, that the y-linked D-Glu residue contains a free a-carboxyl group, and that the Adda residue has a double bond 6E configuration. 3.4
Biosynthesis of Microcystins and Nodularins Extensive studies have been performed to disclose the biosynthetic pathway of microcystin and their lower mass analogs, the nodularins (53,85). One of the major questions was the origin of the Adda residue. The methyl substitution pattem was indicative of incorporation of either propionate or acetate followed by methylation via S-adenosylmethionine. Although both propionate and methionine were found to be incorporated, the pattem of labelled metabolites was clearly indicative of an acetateplus-methionine sequence for CI through C8. The remainder of Adda presumably derives from phenylalanine via phenylacetic acid. The other subunits are for the most part derived from predictable pathways. According to the biosynthetic intermediates isolated the assembly of the linear penta- and octapeptides occurs with the Adda unit as N-terminal residue and Arg as C-terminus. Cyclization apparently represents the last step, since conversion of N-methyl-serine and -threonine to Mdha and Mdhb, respectively, occurs in earlier steps. 3.5.
Synthetic Approaches for Microcystin and Nodularin
Various syntheses have been reported so far for the p-amino acid Adda (83,8791). In most of these synthesis the E^E-dienic system was prepared by the Julia-Wittigtype chemistry which, however, did not allow for the complete control of the stereochemistry of the double bonds (83,87-89). More recently an alternative synthesis has been proposed (91) in which the fragments C1-C5 and C6-C10 were joint together under Stille conditions (92). Starting from the suitably protected P-methyl-aspartic acid (1) its reduction to the aldehyde and conversion to the trans vinyl iodide followed by stannylation with hexamethyldistannane in the presence of freshly prepared Pd(PPh3)4 led to the trans vinylstannane 2 (Fig. 6). The commercially available (S)-phenyllactic acid (3) was converted to the Weinreb amide which was methylated at the secondary alcohol function and then converted to the propargylic ketone (4). 5j/t-stereoselective
900
reduction of the carbonyl group produced the propargyUc alcohol which was converted to the bromoallene. This intermediate was alkylated with the organocopper reagent MeCuCNLi to afford the alkylalkyne 5 which was subjected to hydrozirconation followed by quenching with iodine to install the halogen at the less hindered side of the triple bond and thus, to define the geometry of the double bond in compound 6 for the Stille coupling with compound 2, Saponification of the methyl ester afforded the NBoc protected Adda amino acid with the correct E,E stereochemistry for its use in the synthesis of nodularin or microcystin.
O
MeO
O
NHBOC ,0H
NHBOC
MeO
'SnBu3
Me
2+6
HO2C. Me
Me
Fig. 6. Synthesis of N-Boc-Adda-OH, according to D'Aniello et al. (91), as suitably protected intermediate for the preparation of nodularin or microcystin.
901
Synthesis of (i-alkylaspartates have been reported previously (93-96), but in the very elegant synthesis of mutoporin Valentekovich and Schreiber (89) used D-Thr as a chiral building block which was converted to a y-lactone in five steps through an intermediate tosylaziridine. The y-lactone served then to produce both the backbone portion (C1-C4) of the Adda residue and a differentially protected p-MeAsp. Assembly of the different units to the pentapeptide with N-methyl-D-threonine as precursor for Mdhb was performed by standard procedures of peptide chemistry and final cyclization via the pentafluorophenylester led at high dilution to the desired protected mutoporin. Interestingly, saponification of the ester groups with Ba(0H)2 led concomitantly in unexpected an manner to the quantitative dehydration of the Nmethyl-threonine. O
COOH ^MBHA resin
1. MesI 2.HF
O
COOH NH2
DBU
Fig. 7. Synthesis of the microcystin fragment Ac-D-Y-Glu-Mdha-D-Ala-Leu-NH2 by solid phase peptide synthesis (100).
902
Although the dehydroalanine is not essential for the biological activities of mycrocystin, the synthesis of the most abundant variant-LR requires incorporation of dehydroalanine. The synthesis of a,P-dehydroamino acids or dehydroamino acidcontaining peptides has been extensively studied (97-99), mainly due to the isolation of numerous biologically active a,P-dehydropeptides from natural, particularly microbial sources. Dehydroamino acid-containing peptides have unique chemical and stereochemical properties which affect both the chemical reactivity and the conformation of the peptides. The methods reported so far are all based on p-elimination of suitable derivatives of serine, threonine or cysteine. Recently the sulfonium salt approach (98,97) has been applied even in the solid phase synthesis of the tripeptide derivative of microcystine Ac-D-Y-Glu-Mdha-D-Ala-Leu-NH2 following the route shown in Fig. 7 (100), but P-elimination was found to occur at extents only slightly higher than 50%. OCH,
base
CH2 11 H2N-C-COOH
Fig. 8. Synthesis of dehydroalanine or dehydroalanine- containing peptides via baseinduced p-elimination of the Se-(4-methoxybenzyl)-selenocysteine residue (101). We have shown that treatment of Se-(4-methoxybenzyl)-selenocysteine or related peptides with DBU in dimethylformamide produces in quantitative manner the related dehydroalanine-compounds as shown in Fig. 8 (101). As this method can be applied to the Fmoc/tBu strategy of solid phase peptide synthesis (102), if Fmoc removal is carried out with due precautions, it should represent a very efficient synthetic approach to microcystins. Recently, syntheses of cyclic peptides modelled on the microcystin and nodularin ring were reported with the dehydroamino acid replaced by Ala and the Adda residue by various a- and p-amino acids which, however, were lacking largesize hydrophobic side-chains (103). Correspondingly, the binding affinities for type 2A protein phosphatase were only in the mmolar range (104). Moreover, the synthesis of a nodularin analog has been reported with the Adda residue replaced by P-alanine or 3hydroxymethyl-P-alanine, Mdhb by sarcosine and MeAsp by Asp (105). The bioactivity of this compound, however, was not reported.
903
4,
Inhibition of Protein Phosphatases by Microcystin and Nodularin
Since microcystins and nodularins (67-69) were recognized as very potent inhibitors of the type 1 and type 2A protein phosphatases, progress in understanding the mechanism of phosphatase inhibition by these toxins was expected from the knowledge of their three-dimensional structures in the free and enzyme-bound state. The cyclic structure and the incorporation of the Mdha or Mdhb residues impose remarkable constraints to the conformational space of these toxins, although one cannot exclude significant conformational changes upon binding of the peptides to phosphatases as was observed for cyclosporin-A upon binding to cyclophilin (106,107). 4.1.
Three-Dimensional Structure of Microcystins in Solution
Conformational analysis of microcystin using NMR spectroscopy and distance geometry or molecular dynamics calculations has been performed in DMSO (108110), DMSO/water (110) and water (111). The biological relevance of the examination of preferred conformations of peptides in DMSO has been discussed (112). Since the receptors for these molecules are composed of both hydrophiUc and hydrophobic portions, DMSO should mimic these properties more properly than does water. Interestingly, the preferred conformations of microcystin in these three media show large similarities regarding the ring portion of the molecule. From NMR data and distance geometry calculations of microcystin-LR in DMSO (108) three families of structures were derived that exhibit a common general feature consisting of a compact planar array of the peptide backbone of Adda^-D-z.y^^-Glu^-Mdha^ ring portion (Fig. 9). In family A the D-Ala^ residue lies in this plane with Leu^-D-Me-Asp^ protruding from the plane and with Arg"^ located at the edge of the plane. In family B the Arg"^ residue is located in the plane and Ala^ at the edge, while in family C Arg"^ becomes part of the protrusion and Leu^ moves to the edge at the opposite side. Thus, the three families exhibit saddle-type structures which differ mainly in small movements of the residues that are shifted out of the plane formed by the residues 5 to 7. This saddle-shaped conformation of the peptide backbone, that was determined by us for microcystin-LR in DMSO, is very similar to the structure derived from NMR experiments in DMSO and molecular dynamics calculations (110). Small differences were revealed in the preferred conformation of this microcystin in DMSO/water which derive from changes of the backbone dihedral angles in the vicinity of the amide bond between Mdha^ and AlaUllO). As discussed above, nodularin corresponds in its chemical structure to residues 3-7 of microcystin LR with concomitant replacement of the Mdha residue with Mdhb,
904
I Mdha
microcystin-LR
I
Mdha
Fig. 9. Superposition of the most convergent microcystin-LR structures subdivided in the three conformational families A, B and C.
905
thus lacking the D-Ala^-Leu^ dipeptide portion of the microcystins. Since microcystinLR and nodularin exhibit identical hepatotoxocities and binding affinities to protein phosphatases 1 and 2A, this sequential insert in the microcystin structure should become a conformational exon to maintain the correct topological array of the rest of the ring structure which has to be responsible for the selective recognition by the receptor molecules. This hypothesis was experimentally confirmed by the NMR and distance geometry analysis of the nodularin variant mutoporin in aqueous solution (111). As well portrayed in Fig. 9, in our distance geometry-derived structures of microcystine-LR the Adda side-chain retains sufficient conformational space for relatively large movements despite the restricted rotational freedom imparted by the double bonds. In family A the Adda side-chain is bent over the ring structure and in family B it is directed away from the ring, an effect even more pronounced in family C. The latter position as well as the relativeflexibilityof the terminal portion of the sidechain compares well with the microcystin-LR structure determined by Trogen et al (110) in DMSO and in DMSO/water. Moreover, this hydrophobic side-chain is at some distance from the sequentially adjacent Arg side-chain. In order to analyze the effect of natural substitutions on the preferred conformation of microcystins we have determined the 3D-structure of microcystin-LY (Fig. 10) in which the variable residue Arg is replaced by the hydrophobic Tyr unit (113). Mdha
Fig. 10. Superposition of the 10 microcystin-LY structures with the smallest distance violations.
906
Although th4 Arg-->Tyr replacement is without significant effect on the toxicity (LD50 = 90 |ig/kg in the rat against 50 |ig/kg for microcystin-LR) (114), incorporation of the Tyr residue adjacent to the Adda unit leads to a surprisingly restricted conformational space for the Adda side-chain most probably as a result of a hydrophobic collapse-type interaction (108). This strong interaction leads to movements in the peptide backbone ring structure which adopts a boat-type array significantly different from the saddle-like array in the LR variant. By examining the structures of the two microcystins as derived from the distance geometry calculations, many of the NOES could not be described using the standard method of applying the NOEs to one conformation. Distance violations approaching 1.0 A were observed, particularly for the residues located around the Mdha residue. The constraint that this residue introduces into the cyclic peptide is significant, and it could very well be the source of dynamics, fast on the NMR timescale. With this in mind, we undertook the calculation of these two inhibitors of the protein phosphatases 1 and 2A using the ensemble approach. The possibility of molecular motions fast on the NMR time-scale must always be kept in mind during structure determination using restraints derived from NMR data. The experimental observations (e.g. NOEs, coupling constants) in the case of such averaging are clearly averaged quantities. Therefore, the restraints will be consistent with the average stmcture, a structure that may not exist in solution or even be physically possible. There are a number of examples in the literature of such dynamics taking place (115-126). Different computational approaches to address fast molecular motion have been proposed (127-131). The approach utilized in our study (109) is very similar to the time-averaged method, but utilizes a large number of the molecules to simulate, and the experimental restraints are applied not to each individual structure but to the entire ensemble as an average, i.e. the penalty function is applied only when the average over the ensemble of structures varies from the experimentally determined value (127,132,133). In theory, the results from the time-average and ensemble methods must be identical, although some differences arising from the practical application of the methods, have been observed (134,135). For the LY variant a rather small conformational family was detected and a clustering of the different members of the ensemble for the residues from Leu^ to Adda^. These residues are therefore well determined and conformational dynamics are not taking place in this portion of the molecule. In contrast, the residues of the other half of the molecule, D-Z^-o-Glu^-Mdha^-D-Ala^ show that dynamics occur. This is especially evident for the Mdha residue, where there are large populations at 0,T values of both +30%-30' and -30\-f-30\ This exchange between these two conformers is necessary to reproduce the NOEs about this residue. It is important to state, that
907
although each member of the ensemble is not planar, the average O,^ values about this residue is 0%0* (i.e. planar). This would suggest that the constraint of the system, induced from the cyclization, forces the Mdha residue out of planarity. The ranges of dynamics of the residues surrounding Mdha'7 are smaller. Therefore, the "flip-flop" process of the Mdha causes some distortions of the adjacent amino acids. The results of the microcystin-LR indicate much more significant dynamics than in the case of the LY analog. The results for the Mdha residue are similar to those observed for the LY analog; there are two large families at approximately -40%-40' and +40%+40\ However, in addition, there is a significant number of conformers that adopt a ^ dihedral angle of 180". The "flip-flop" of the Mdha has a large effect on the conformational features of the preceeding residue and the two following residues, namely D-z^o-Glu^, D-Ala^ and Leu^. The Glu residue adopts a significant number of conformations, but shows a tight clustering of conformations about the central value of -150%+80\ Both of the residues after Mdha adopt two conformational families. The D-Ala has great variance in the O dihedral angle as to be expected (it is adjacent to Mdha) and almost no variation of the ^ torsion. Similar results are observed for the Leu2 residue. The remaining residues show only one significant population, although covering a wide range of dihedral angles, often of ±30°. Clearly the microcystin-LR is less well determined than the conformation observed for the LY analog. Taking into account the results of these ensemble calculations the backbone structure of microcystin-LR reveals a restricted conformational space in part of the ring backbone and significant flexibility in the rest, i.e. mainly involving the residues Mdha'7, D-Ala^ and Leu^. Interestingly, by comparing the 3D-structures of the variant LR in DMSO and in DMSO/water the conformational differences are related to this sequence portion (110). 4.2.
Molecular Dynamics Simulation of Nodularin and Okadaic Acid
From structure-function studies it is known that esterification of the a-carboxyl group of D'iso-G\u^ of microcystin generates untoxic compounds, probably as a result of the lack of inhibition of the protein phosphatases (34,53) and that the methyl ester of okadaic acid is inactive as well (86). Correspondingly, this carboxylic function seems to play an essential role. In order to account for the almost identical potency of microcystins and okadaic acid, we have proposed a location of the carboxyl function of the two diverse compounds at the same position and a wrapping of the okadaic polyether chain around part of the microcystin ring and alignment of its hydrophobic tail with the Adda residue side-chain (108). Recently the computer generated minimum energy conformations of okadaic acid and calyculin have been reported (136) and compared to the minimum energy
908
conformations of nodularin and microcystin-LR (137). In this study a ring structure is proposed for okadaic acid which involves an intramolecular hydrogen bond between the CI carboxyl, attached to the first tetrahydropyran ring, and the C24 hydroxyl group attached to the fourth tetrahydropyran ring. The rest of the molecule would simulate in extended manner the side chain of the Adda residue. The result of our energy minimization for okadaic acid is shown in Fig. 11 (109). A chain reversal is observed in the C-terminal portion of the molecule, probably due to a hydrophobic collapse (138), whilst the rest of the molecule adopts an extended and flexible form.
Fig. 11. Stereoview of the lowest energy conformation of okadaic acid as calculated by free molecular dynamics simulation. Free molecular dynamics simulation performed by Trogen et al. (110) on microcystin led to surprisingly similar results as those obtained with NMR-derived restraints, a fact which confirms the high preference of this cyclic heptapeptide for a defined overall conformation. Since the cyclic pentapeptide stmcture of nodularin should contain even stronger inherent restraints, its lowest energy conformation was derived by us from molecular dynamics simulations (109). This conformation, shown in Fig. 12, displays a backbone array surprisingly similar to that of microcystin-LR, i.e. again a saddle structure, but it is less puckered because of excision of the D-Ala^-Leu^ dipeptide portion of the microcystin ring. Again the Adda residue points away from the ring distal from the Arg side-chain. The overall structure is very similar to the distance geometry-derived solution stmcture of mutoporin in water presented by Bagu et al.
909
(111). Most interestingly, replacement of the Arg by Val was not found to induce a hydrophobic collapse between the Adda and the adjacent Val residue as we have observed to occur for the microcystin-LY variant.
Fig. 12. Stereoview of the lowest energy conformation of nodularin as calculated by free molecular dynamics simulations. The search for structural similarities between microcystin, nodularin and okadaic acid, which apparently all bind to the protein phosphatases at the identical sites, led us to calculate the Connolly surfaces mapped with lipophilic potentials. The compounds show a typical lipid-like character with a large peptide headgroup in the case of microcystin and nodularin and a phospholipid-like headgroup in the case of okadaic acid. The largest degree of similarity is observed between microcystin LR and nodularin with their mushroom-like structures that consist of a slightly bended hydrophobic stalk and a hydrophilic head of similar cross area. The structure of okadaic acid forms again a stalk of dimensions similar to those of microcystin LR and nodularin. A stronger bending of the upper part, possibly induced by the protein binding cleft, would allow for a display of the smaller hydrophilic headgroup in a manner similar to the toxic peptides. Finally, microcystin LY has a significantly larger stalk due to the parallel array of the Adda and Tyr side chains in a hydrophobic collapse-type interaction. If the size of this hydrophobic finger in the enzyme-bound state is conditioned by the size of the binding cleft of the protein phosphatases, disruption of the hydrophobic interaction between the Adda and Tyr side-chain is required for binding. The resulting large
910
enthalpic cost has then be compensated by the enthalpy of binding via additional interactions of the hydrophobic Tyr residue, since the toxicity and thus, most probably, even the binding of this microcystin variant to the protein phosphatase 1 and 2A is very similar to that of microcystin-LR. On the basis of these data a possible picture of the enzyme inhibitor complex was derived which foresees an insertion of the sticky finger of these inhibitor structures into the protein cavity of defined dimensions and a display of the a-carboxyl function of Glu^ in juxtaposition for decisive hydrogen bondings or electrostatic interactions (108,109). However, since the Adda residue itself (83,84) or linear microcystin-related precursor peptides are not toxic (85), it is reasonable to assume that the remainder of the peptide (the planar ring portion) plays an important role in the recognition or in maintaining the proper orientation for the Adda residue. The synergic effect of the head and tail of this structures is also confirmed by the very low inhibitory potency of cyclic nodularin-based peptides lacking the hydrophobic side-chain of the Adda residue (104). 4.3.
Three-Dimensional Structure of Microcystin in the Enzyme-Bound State
The crystal structure of the catalytic subunit of mammalian protein phosphatase1 complexed with microcystin-LR has recently been determined at 2.1 A resolution (139). The metalloenzyme exhibits a fold unrelated to the known structures of catalytic subunits of tyrosine phosphatases. The two metal ions are positioned in a central |3-a-|3 -a-p scaffold at the active site, from which three surface grooves protrude as potential binding sites for substrates and inhibitors. The C-terminus of the catalytic subunit is located at the end of one of these grooves, so that regulatory sequences following this domain may possibly modulate the function. This fold is expected to be closely preserved in the protein phosphatases 2A and 2B. The X-ray structure of the microcystin/enzyme complex reveals an interaction of the toxin with three distinct regions of the surface: i) the metal binding site, ii) the hydrophobic groove that runs between an a-helix and a loop where a cluster of hydrophobic residues, conserved in protein phosphatase 2A and 2B, is exposed to the surface, and iii) the edge of the C-terminal groove near the active site.The hydrophobic Adda side-chain is tightly packed into the hydrophobic groove, the a-carboxyl group of the y-linked D-Glu^ and the adjacent carbonyl group of the Adda residue form hydrogen bonds with two metal-ligand bound water molecules. The carboxyl group of Mc-isO'Asip^ is hydrogen-bonded with Arg-96 and Tyr-134 and the Leu^ side chain is closely packed to Tyr-272. Moreover, a covalent linkage of microcystin via the thiol addition of Cys-273 to the Mdha'^ residue was observed. This covalent hnkage of microcystin was observed previously (140), but it is not essential for inhibition of the
911
Fig. 13. Stereoview of the microcystin-LR structure as determined by X-ray crystallographic analysis of the protein phosphatase- l/microcystin complex.
912
enzyme by microcystin and nodularin as shown by the high toxicity of the dihydrocompounds (80) and of the precursor peptide, the N-methylated L-Ser-variant (34). Additionally, mutagenesis of the cysteine residue (141) or blockade of the Mdha residue in microcystin (140) was not found to affect the inhibition potency. The peptide backbone structure of the enzyme-bound microcystin, shown in Fig. 13, is almost identical to the family B of structures described by us for microcystin in DMSO (107) with Leu^-D-Me-Z^o-Asp^ puckering out of the plane and with Ala^ at the edge and Arg^ in the plane formed by Adda^-D-z^o-Glu^-Mdha^. The Adda residue protrudes from the plane distal from the Arg residue. The strong hydrophobic interaction of the Leu^ residue with Tyr-272 compares well with the observed significantly reduced toxicity of the microcystin variants with hydrophilic residues in the position X (53). Conversely, the residue Y (most abundantly occupied with Arg) is apparently not involved in the binding. Besides the flexibility observed for the sidechains of the Adda (particularly in the terminal portion) and Arg residues, the conformation of the microcystin in solution is retained in the enzyme-bound state. Correspondingly, the entropic cost in the bimolecular recognition and binding process is significantly reduced, thus favoring the observed tight binding.
5.
Conclusion
The surprising similarity of the 3D structures of microcystin in solution and in the enzyme-bound state represents an additional example of how nature evolved highly potent inhibitors for receptor molecules exploiting rigid structures to overcome the entropic penality when two molecules are to associate in tight complexes. This principle is in the present-day dmg research increasingly applied for the peptidomimetic design of small rigid cyclic stmctures containing the pharmacophores responsible for receptor recognition and binding in the correct spatial array. On the other side, the chemically diverse families of protein phosphatase inhibitors discovered in a serendipitous manner in fresh and brackish water as well as in marine environments once more support the view that marine world is an immense source of libraries of unknown compounds (142). Natural products provide most of the world's population with their pharmacopea and represent the source of leading structures for a significant proportion of synthetic pharmaceuticals (143). The success of natural products as drugs, and the fact that the biological resources from which they originate remain underexploited, continues to provide a significant impetus to the dmg discovery process. In this context one has to remember that estimates of macrofauna diversity range from half a million to ten million species and marine microorganisms (viri, monerans and protists) in the range 1 million to 200 million species (143). Among these species already venomous cone snails have generated via a combinatorial library strategy thousands of highly bioactive venom peptides of remarkable specificity and
913
affinity for cell-surface receptors and ion channels (144), which may be used in the future not only as tools for understanding physiological processes, but they may also provide important lessons in designing combinatorial libraries for drug development. The authors would like to thank Hans-Jurgen Musiol for valuable discussions and Norbert Schaschke for his help in the preparation of the manuscript. The coordinates of the X-ray structure of microcystin bound to protein phosphatase-1 were retrieved from the Brookhaven database (lFJM.pdb).
6. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
16 17 18 19
References Coghlan, V.M., Perrino, B.A., Howard, M., Langeberg, L.K., Hicks, J.B., Gallatin, W.M. and Scott, J.D. (1995) Science 267,108-111. Cohen, P. (1989) Ann. Rev. Biochem. 58,453-508. Hunter,T. (1995) Cell 80,225-236. Murray, K.J. and Coates, W.J. (1994) Annu. Rep. M. 29, 255-264. Mumby, M.C. and Walte, G. (1993) Physiol. Rev. 73,673-699. Hubbard, M.J. and Cohen, P. (1993) Trends Biochem. Sci. 18,172-177. Chen, J., Parsons, S. and Brautigan, D.L. (1994) J. Biol. Chem. 269, 79577962. Holmes, C.F.B. and Boland, M.P. (1993) Curr. Opin. Struct. Biol. 3,934-943. MacKintosh, C. and MacKintosh R.W. (1994) Trends Biochem. Sci. 19, 444448. Cohen, P. (1994) BioAssays 16, 583-588. Walsh, D.A. and Glass, D.B. (1991) Methods Enzymol. 201, 304-316. Hunter, T. (1993) Cell 75, 839-841. Francis, G. (1878) Nature 18,11-12. Mundt, S. and Teuscher, E. (1988) Pharmazie 43, 809-815. Codd, G.A. (1992) in: Proceedings of the 4th Disaster Prevention and Limitation Conference. The changing face of Europe: Disasters, pollution and the environment, vol. 4 (Keller, A.Z. and Wilson, H.C., eds) pp. 33-62, University of Bradford. Krebs, E.G. (1986) in: The Enzymes, 3rd edn, XVIIa (Boyer, P.D. and Krebs, E.G., eds) pp. 3-19, Academic Press, Orlando. Fischer, E.H., Charbonneau, H. and Tonks, N.K. (1991) Science 253,401-406. Etzkom, F.A., Stolz, L.A., Chang, Z.Y. and Walsh, C.T. (1993) Curr. Opin. Stmct. Biol. 3, 929-933. Kincaid, R. (1993) in: Advances in Second Messenger and Phosphoprotein Research, vol. 27 (Shenolikar, S. and Naim, A.C, eds.) pp. 1-23, Raven Press, New York.
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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
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39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
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916
55
56 57 58 59 60 61 62 63 64 65 66 67 68 69
70 71 72 73
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921
CUMULATIVE GENERAL SUBJECT INDEX VOLUMES 1-20
Abamectin 12:9 (3^20) Abeataxan 20:80,106 25-(10->9) Abeo-26(S->\5) abeO'9^,l4^,\5a-o\ean-5ene-3(3,29-diol 5:744,748,750 9(10->19) Abeo-diene steroidal alkaloids 2:205,206 11(15^1) Abeataxan 20:80,100,101 Abies koreana 20:619 Abies mariestii 20:619 Abiesen 20:283 Abieta-8,11,13-triene from Salvia limbata 20:673 Abietane 9:297 Abietane diterpenoids 20:660-680 Abietatriene from abietic acid 14:676,677 taxodione from 14:676,677 £«^Abieta-8(14),13(15)-dien-16,12-olide (jolkiolide E) 9:283,284,290 Ent-AhiQimt derivatives 9:265,283-288 Abietatriene 2:402,403 Abietatriene-6-one 14:669,670,673,674,676 Abietatriene-7-one Baeyer-Villiger oxidation of 14:685,687-689 from dehydroabietic acid 14:685,687-689 taxodione from 14:685,687-689 Abietatriene-diol 14:675,676 Abietic acid in (+)-confertifolin synthesis 4:405-416 in (+)-isodrimenin synthesis 4:405-408 in (-)-warburganal synthesis 4:416 taxodione from 14:672-674,676,677,689-692 Abnormal Beckmann rearrangement 16:133 Abramov reaction 6:353 A brus fruticulosus 15:26 Abrus precatorius 7:152,15:25 Abrusgenic acid 7:152 Abruslactone A abrusgenic acid from 7:152 from Abrus precatorius 7:152 Abrusosides A-D from Abrus precatorius 15:25 structure elucidation of 6:559 synthesis of 10:167 Absinthin from Artemisia absinthium 7:215 from Artemisia sieversiana 7:215 Absolute configuration by C D . procedure 9:27 by Horeau procedure 9:25,26 by Nakanishi's method 2:89 in 24-(P-hydorxyethyl)-in 24-(carboxy-methyl) steroids 15:79-81 in 24-ethyl-26-hydroxysteroids 15:84 in 24-hydroxymethyl steroids 15:79 in 24-hydroxysteroids 15:76,77 in 24-methyl-25,26-dihydroxy-steroids 15:84-86 in 24-methyl-26-hydroxy and 24-methyl-26-oic steroidal side-chains 15:81 -84
m 26-hydroxysteroids 15:77,78 of(+)-aristoserratine 11:296 of(+)-monomoriwei 6:449,450 of(-)-hobartine 11:290 of(-)-peduncularine 11:284 of 3 a, 13-dihydroxy-11 -epiapotrichothecenes 13:520 of amphotericin B 6:231 ofaureol 9:31 of benzo [a] pyrene oxide 7:8,14 ofcarotenoids 7:321 of decaprenoxanthin 7:355 of liverwort sesquiterpenoids 18:607-646 of Mosher's esters 13:77 ofpenems 4:435 of phoracantholide I, J 6:546 ofquinocarcin 10:126 ofR, 5-4-hydroxycyclopentenones 6:315 of sclerosporin 6:549-551 of sigmosceptrellins A-C 9:16 ofsolenopsinB 6:430 of sugars saponins 15:190 oftaxol 11:3,7 ofthienamycin 4:433,436 ofurdamycinA 11:134 Acacetine 226 Acacia astringens 7:427 Acacia nilotica I'All Acacia tomentosa 1:427 Acalycigorigia inermis 5:370 Acalycigorigia sp. 5:368,369 Acanthaceae 7:190,193 Acanthacerebroside A 18:481 Acanthaglycosides A,D,F from Acanthasterplanci 7:288 Acanthaster planci acanthaglycosides A-D,F from 7:288 marthasteroside A from 7:288 thomasteroside A from 7:288 Acanthicifoline 7:191 Acanthifolicin 5:384 Acanthella klethra 20:525 Acanthodris nanaimoensis 19:139 Acanthomyops claviger 6:454 A canthoscelides obtectus 10:160 A canthus ebracteatus 7:176 Acanthus illicifolius 7:176,179,181,182,189-191,193 Acarbose asa-amylase inhibitor 13:3 as a-glucosidase inhibitors 7:47 as antihyperglycemic drug 10:505 as antihyperlipoproteinaemic drug 10:505 enzymatic degradation of 10:506 from A ctinoplanes species 10:503 synthesis of 13:49-60 total synthesis of 10:507 Acarviosine 10:503,504,506 Accedine 5:125
922 Accedinine 5:125 Accedinisine 5:125 Acemicoense 17:358,367-368 Aceraceae 17:358,366-367 Acemikoense 17:375 Acerogenin A 17:367,375,20:276 Acerogenin A biosynthesis of 17:375 Acerogenine-E 17:368 Aceroside I 17:367 AcerosidelV 17:367 Aceroside V 17:367 Aceroside VII 17:358 Aceroside XI 17:368 Acesulfame-K 15:4 Acetal 6:336 Acetal derivatives 6:335,336 mechanism of formation 6:335,336 of 3-methylthio-2-oxopropanals 6:335,336 synthesis of 6:335,336 Acetal exchange 1:453 Acetal-ketal formation 4:699 Acetalization 1:584 /ra«5-Acetalization 4:325,326,331,338,339,495 Acetals as chiral auxiliaries 4:327 from (R*,R*)-2,4-pentanediol 14:484 from dimethyl tartrate 14:478 Lewis acid-mediated 14:484 nucleophilic substitution reaction 14:484 reductive cleavage 1:591-595 with dialkylboron bromide and cuprate 14:478 2-Acetamido-1,6 anhydro-2-deoxy-derivative 6:388,389 6-Acetamido-2,3,4-tri-0-allyl-6-deoxy-a-glucoheptopyranosiduronic acid 11:434,435 2-Acetamido-2-deoxy-D-glucopyranose 6:351 2-Acetamido-2-deoxy-D-glucopyranosides 6:389 2-Acetamido-2-deoxy-D-glucose oxazoline derivative of 6:399 2-Acetamido-2-deoxyglucomannans 5:286,287 6-Acetamido-6-deoxy-castanospermine from (+)-castanospermine 12:345,346 synthesis of 12:345,346 p-Acetamidophenyl-1,4,4'-dithiocellotrioside 8:351 /7-Acetamidophenyl-1,4-dithiocellobioside 8:350,351 Acetate-induced decomposition 14:170 Acetoacetic acid ethyl ester 10:410 A cetobacter suboxydans 17:636-637 Acetobromoglucose 10:366,367 P-Acetochloromannose 8:321,322 Acetogenic cyclic peroxides 9:18 Acetogenins 9:385,391,392,395,397-399,403;18:193227;19:81 (-)-Acetomycin absolute stereochemistry of 10:443,444 antimicrobial activity of 10:443 antitumor activity of 10:444,447 biosynthesis of 10:443 Claisen rearrangement by 10:443-448 from Streptomyces ramulosus 10:443 synthesis of 10:443-448 4-ep/- Acetomycin 10:445
(+)-5 -epi-Acetomycin antitumor activity of 10:447 Acetonation 6:279,280 Acetonide 14:160;19:489 Acetonylbutanolides 17:273 Acetophenone 2:169,170 Acetophenone phenylhydroazone 9:581 (+)-exo-5-Acetoxy camphor 4:646 synthesis of 4:656 3-Acetoxy monomethyl phthalates 14:5 19-Acetoxy-11 -methoxytabersonine 2:375 16-Acetoxy-12-O-acetyl-horminone 15:169,170,173 (15/?)-15-Acetoxy-15,20-dihydrocatharanthine 14:815 20-Acetoxy-15,20-dihydrocatharanthine 4:32 (1 S,4R)-4-Acetoxy-2-cyclopenten-1 -ol in prostaglandin synthesis 1:686 16-Acetoxy-7,8-epoxy-3,12-dolabelladien-13-one 20:491 (+)-l- Acetoxy-2-epipinoresinol 5:538-540 la-Acetoxy-20aF-hydroxy-24a,25a-epoxywitha-5-ene22,26-oHde-4p-0-glucopyranoside 20:191 1 P-Acetoxy-8P-hydroxyeudesm-4( 15), 7-diene-8-12olide 20:660 1 - Acetoxy-2-methylbutadiene [2+2] cycloaddition of 12:150,151 with chlorosulfonyl isocyanate 12:150,151 (15'S)-15'-Acetoxy-20'-deoxy vinblastine from (1SR)-15-acetoxy-15,20-dihydro catharanthine 14:815,816 22-Acetoxy-23,24-dinorchola-l,4,6-trien-3-one 11:398 4-Acetoxy-3 [ 1 '-/eA•^butyldimethyl-sily loxy) ethyl] -2azetidinone 4:448 205'-Acetoxy-3p-16-dihydroxylanost-7-en-18-oic acid18,16-lactone 7:269 endo-2-AcQ\.oxyA{exo\ 5-dimethylbicyclo [3,3,1] nonan-9-one 6:65,66 from 4,5-cw-dimethylbicyclo [4.3.0] non-9-en-one synthesis of 6:66 3-Acetoxy-4-hydroxybenzaldehyde 8:166,167 2-Acetoxy-5,6-dihydro-2H-pyrans regioselective alkylation of 10:342 stereoselective alkylation of 10:342 6-Acetoxy-5 -hexadecanolides synthesis of 3:157-160 (+)-l- Acetoxy-6-epipmoresinol 5:538-540 16-Acetoxy-7-0-acetyl-horminone from Rabdosia lophanthoides var. gerardiana 15:173 16-Acetoxy-7a-methoxyroyleanone from Rabdosia lophanthoides var. gerardiana 15:173 from Rabdosia stracheyi 15:175 2-Acetoxy-8,13-dihydroxy-ll-e;?/ APO 13:522 12- Acetoxy-8,13-dihydroxy-l l-e/7/-apotrichothec-9ene 9:20 Acetoxy-abieta-triene 14:675,676 4-Acetoxy-P-lactam from Z)-a//o-threonine 12:160 from 6-aminopenicillanic acid 12:160 from I-aspartic acid 12:160 from Z,-threonine 12:161,162
923 synthesis of 12:160-162 with ester enolate 12:163,164 with imide enolate 12:164-168 with other nucleophiles 12:170-172 with thiol ester enolates 12:168-170 (+)-(^ram-4'-Acetoxy-cinnamoyl)epilupinine from Lupinus hirsutus 15:521 (+)-15-Acetoxy-e«^isocopal-12-en-16-al synthesis of 6:119-122 16a-Acetoxy-holosta-7-en-3b-ol 7:277 16P- Acetoxy-holosta-7,24-dien-3P-ol 7:275 235-Acetoxy-holosta-7,25-dien-3p-ol 7:273 (+)-31 -Acetoxy-Na-benzoylbuxidienine 18-Acetoxy-Na-deacety lisoretuline 1:38-39 5 - Acetoxy arctigenin monoacetate 18:601 6'-Acetoxyavarol 15:301 6'-Acetoxyavarone 15:301 1 -Acetoxybutadiene 1:571 25-Acetoxycholesta-1,4,6-trien-3 -one 11:385 2a-Acetoxycholestanone 18:888 2p-Acetoxycholestanone 18:888 Acetoxycrenulide synthesis of 16:703 (+)-Acetoxycrenulide 18:22-28 20-Acetoxydihydrocatharanthine from catharanthine 14:857,858 vinblastine from 14:857,858 vincristine from 14:857,858 19(/?)-Acetoxydihydrogelse virine from Gelsemium rankinii 15:479 from G. sempervirens 15:479 5 - Acetoxydimethy Imatairesinol 18:602 la-Acetoxyergosteryl acetate synthesis of 11:381-383,388-393 a-Acetoxy fiiranone 12:14 3 -Acetoxy guaiazulene 14:321 235-Acetoxyholost-7-en-3p-ol 7:273 1-Acetoxyisocomene 3:6,60 e«/-3a-Acetoxyisopimar-15-en-8a-ol ^^C-nmrof 15:170 from Rabdosia parvifolia 15:173 'H-nmrof 15:169 6-Acetoxymethyl-2,6-dimethyl-9-methoxy tricyclo [5.3.0^'^] undec-9-en-8,ll-dione 8:163 5-Acetoxymethyltrachelogenin 18:602 5-Acetoxymethyltrachelogenin monoacetate 18:602 13-Acetoxymodhephene 3:6,61 18-Acetoxynorfluorocurarine 1:38-39 (+)-l- Acetoxypinoresinol 5:523,533,538,544 (+)-l- Acetoxypinoresinol-4"-0-methyl ether 5:523,533-535 (+)-l- Acetoxypinoresinol-4"-0-methyl ether-4'-p-Dglucoside 5:523 (+)-l- Acetoxypinoresinol-4'-P-D-glucoside 5:523 3-Acetoxypyrrol-2(5H] -one 13:117,118 (+)-12-Acetoxysinularene from Clavularia inflata 6:11 synthesis of 6:77,78 (±)-5-e/?/-Acetoxy sinularene synthesis 6:78 (+)-16a-Acetoxytabersonine 2:186 5-Acetoxytrachelogenin diacetate 18:601
7-Acetyhorminone from Salvia candidissima 20:660 A^-Acetyl cysteamine 13:496 Acetyl pyrazine 13:320 N-Acetyl a-amino acids asymmetric hydrolysis 1:678,679 P-Acetyl alcohol 14:462 Acetylamino-1,2-dihydroacronycine 20:800 A'-Acetyl amphotericin B methyl ester amphoteronolide B methyl ester from 6:264,265 3:5:9:11-di-0-isopropylidene derivative from 6:264-266 hexaenal from 6:264,265 macrolide from 6:264-268 Acetyl CoA mevalonic acid from 7:322 3-Acetyl deoxynivalenol 13:520,521,536 A^-Acetyl glucosamine 7:31,47,49 P-(l-3 )-A^-Acetyl glucosaminyltransferase 10:484 Acetyl group 4:322,249 Acetyl-horminone 20:670 A^-Acetyl isomuramic acid 6:386,387 TV-Acetyl isomuranic acid lactones circular dichroism spectra of 6:390 O-Acetyl isophotosantonic lactone 14:357 Acetylandromedol 20:27 A^-Acetyl muramic acid alkaline degradation of 6:387 benzyl a-glycoside of 6:390,391 T>{{R)] configuration of 6:387 lactonisation of 6:389,390 synthesis of 6:387,388 X-ray analysis of 6:387 A^-Acetyl muramic acid 7:31,47,49 A^-Acetyl muramic acid lactones 6:389-396 A^-Acetyl muramic acid methyl ester a-benzylglycosides of 6:395 reaction of 6:387 A^-Acetyl muramoylamide derivatives ofMurNAcS-lactones 6:393 N-Acetyl neuraminic acid (NANA) 13:207-210 O-Acetyl normacusine B 13:386 Acetyl oleanolic acid from I-arabinopyranosyl-3P-acetyloleanolate 7:154 1-O-Acetyl oxetanose from cw-2-buten-l,4-diol 10:598,599 glycosidation of 10:603 1-P-isomerof 10:598,599 via nonphotochemical reaction 10:597,598 synthesis of 10:597-604 O-Acetyl preperakine 13:387 0-Acetyl villamine 13:396 2-0-Acetyl-(5)-(+)mandelic acid 13:468,469 1 -5-Acetyl-1 -thiohex-2-enopyranoside regioselective alkylation of 10:342 stereoselective alkylation of 10:342 Na-Acetyl-11 -hydroxyaspidospermatidine 1:40 Na-Acetyl-11,12-dihydroxyaspidospermati-ine 1:40 6-0-Acetyl-2,3,4-tri-O-benzyl-D-glucono-1,5-lactone 12:335 3-Acetyl-2,6-dimethylpyridin-4-one 13:545
924 3 - Acety 1-2-oxazolone from 2-oxazolidinone 12:411,415 preparation of 12:411,415 3-Acetyl-4,6-dimethylpyridm-2-one 13:545 4-Acetyl-6,8-dihydroxy-5-methyl-3,4-dihydroisocoumarin 15:409 3-Acetyl-(3-D-digitoxose 15:362 A^-Acetyl-P-D-hexosainidase 7:416,417 3-Acetyl-(3-lactam 12:152,161 trans-3 -Acetyl-P-lactam from imine 12:173 Acetyl-coenzyme A 7:112 2-0-Acetyl-D-glucal tetraacetate 10:414 //-Acetyl-D-lividosamine synthesis of 14:188,191 a-A^-Acetyl-s-A^-tosyl-L-lysine methyl ester anodic oxidation of 12:309 (3-N-Acety 1-glucosaminidases 12:346 Acetyl-histamines 15:328 A^-Acetyl-muramyl-(L)-alanyl-(D)-isoglutamine (MPD) 13:210-212 N-Acetyl-muramyl-L-alanyl-D-iso-glutaminyl-S-reA•^ butyl-cysteamine 18:927 Nb-Acetyl-Nb-methyltryptamine 9:178 Na-Acetyl-O-methyl strechnosplendine 9:188 3-Acetyl-pyrrolidine-2,4-diones 14:100 17-O-Acetylajmaline 9:183-185 Acetylaminonaphthoquinone 9:437 (-)-A^-Acetylamphetamine synthesis of 14:496,497 Na-Acetylaspidospermatidine 1:40 A^.A/^-Acetylated disaccharide derivative synthesis of 6:401 Acetylation of 5>^« alcohol 19:480 P-Acetylation procedure for acetomycin synthesis 10:447 stereoselective 10:447 2-Acetylbutyrolactone 8:284,285 Acetylcholine receptor 18:863 Acetylcholinesterase 7:18 19-Acetylcorrin 9:600,601 2-Acetylcyclohexanone allylationof 10:412 with allyl acetate 10:412 (+)-Acetyldomesticine 16:519 Acety lenation of acetals 1:611,612 Acetylene mercury catalysed hydrolysis 3:253 reaction with enamines 3:95,96 Acetylene nucleophilicity 1:612 effect on diastereoselectivity 1:612 Acetylene Zipper reaction 8:240,242,469 Acetylene-C-glycosides 10:359 Acetylenes formation formacyl ylides 4:564 formation form carboxyl 4:322, 323 formation from lactones 4:349 Acetylenic a-ketols 6:153 a,P-Acetylenic alcohols 4:493 Acetylenic carotenoids as chemosystematic markers 6:153
biosynthesis of 6:153,154 by chemical conversion 6:142,147 chemical reactions of 6:160 chemosystematics of 6:147 chirality of 6:149 de novo biosynthesis of 6:153 distribution of 6:147 from allenic carotenoids 6:142,147 fimctionof 6:153,154 geometrical isomerism of 6:153 metabolism of 6:153,154 partial synthesis of 6:157-160 spectral data of 6:148,150 stereomutation studies of 6:153,154 steric preference in 6:154 synthesis of 6:157-160 (5)-Y-Acetylenic g-aminobutyric acid (GABA) stereoselective synthesis of 14:473 Acetylenic ketone allylic alcohol from 11:424 asymmetric reduction of 11:424 Acetylenic oxy-Cope rearrangement 8:250 28-Acetylerythrodiol from Salvia tchihatcheffii 20:707 3 -Acety lerythrodiol from Salvia thihatcheffii liS.lOl Acetylexidonin from Rabdosia gaponica var. glaucocalyx 15:171 2'-Acetylglaucarubinone 7:379,380 N-Acetylglucosamine 1:417 P-( 1 -6)-A^-Acetylglucosaminyltransferase in biosynthesis of I-antigens 10:486 iV-Acetylglucosaminyltransferase II 10:500,501 A'^-Acetylglycosides 7:434 (+)-8-Acetylgonoiotril 19:463 3-Acetylgoniotriol 9:394 //-Acetylisocodonocarpine 9:73-75,77 0-Acetylisoretuline 1:38,39 N-Acetyllactosamine (2-acetamido-2-deoxy-4-0-p-Dgalactopyranosyl-a-D-glucose) 10:459,461-467 3-Acetylleptophylline-A 20:486,487 Acetylmelodorenol 9:400,152 A^-AcetyImuramoyl-dipeptide derivatives 6:385 A^-Acetylmuramoyl-dipeptide ester 6:394 A^-Acetylmuramoyl-pentapeptides 6:404,405 7-Acetylneotrichilenone 9:297 A^-Acetylneuraminate lyase from Clostridium perfringes 7:71 Acetylneuraminate pyruvate lyase 11:466,467 A^-Acetylneuraminic acid 10:550 3 P-Acetylolean-12-en-28-al from Salvia tchihatcheffii 20:707 A^-Acetylphenylglycine synthesis of 6:319,320 (+)-Acetylphomalactone antifiingal activity of 19:479 antitumor activity of 19:479 insect antifeedant activity of 19:479 isolation of 19:480 plant growth inhibitory effect of 19:479 synthesis of 19:480 O-Acetylpolyneuridine 5:128 A^-Acetylpipecolate ester 12:309
925 Acetylpipitzol 5:778 O-Acetylretuline 1:38,39 O-Acetylsolasodine 20:489 A^-Acetylsolasodine 20:490 Acetylstrychnovoline 6:523,524 (+)-Acetylthaliporphine 16:519 2-Acetylthiazole (2-ATT) 11:443,444 0-Acetylvallesamine apparicine 5:87,88 ervaticine 5:88 16S-hydroxy-16,22-dihyroapparicine 5:88 pericine 5:88 vallesamine 5:88 0-Acetylvallesamine 9:167,168,171 Acetylvismione D 7:418-420,424 Achantaster planci 15:45 Achillea millefolium 10:151 Achillea nana 10:151 Achillin 7:234 Achiral 2-bromopropionic acid derivatives in Reformatsky reaction 12:166 with 4-acetoxy p-lactam 12:166 Achiral heptanal 12:55 Achiral pyrethroid analogues fromthujone 14:398-401 synthesis of 14:398-401 Achiral thiazolidin-2-thione derivative 12:166 Achiral-a-methoxycycloalkanone chiral co-cyano alcohols from 14:475 with(2/?,4/?)-2,4-bistrimethylsilyl oxime acetates oxypentane 14:475 Aciclovir (9-[2-hydroxyethoxy methyl] guanine) antiviral activity of 10:585 Acid catalyzed arcyrin/arcyrinin model compounds from 12:383 of A^-methylarcyriarubin A 12:383 rearrangement 12:383 Acid chlorides of 2-bromo-3-hydroxybutyric acid 4:473 of 3-hydroxybutyricacid 4:471 Acid-catalysed rearrangement 6:115 of (+)-endo-3 -bromocamphore 16:147 of spiro cyclic lactone 16:225 Acidity ofMT-sulfone 6:329 Acilius sulcatus 5:700 A cinetobacter calcoaceticus 12:103 Acinosolic acids A,B 7:144,145 Aclacinomycine A 4:317,318 Aclacinomycines synthesis of 4:330 Acnistus arborescens 20:241 Acnistus breviflorus 20:234 Aconm 20:22 Aconitin 20:22 Aconitum napellus 20:19 P-Acoradiene 15:261,262 a-Acoradiene 15:262 Acoradienes 5:734 Acoragermacrone synthesis of 8:178
Acorane as sesquiterpene 6:59 a-Acorene 15:260 (-)-Acorenone 16:222 (cf/)-Acorenone B synthesis of 10:315-317 Acosamine synthesis of 4:150 Acrasiomycetes 9:220 Acridin-9-one derivatives 13:348-350,353,361,369, 372-375 synthesis of 13:353-361 Acridone alkaloids 13:347-382 Acridones synthesis of 3:430 ACRL toxins synthesis of 18:178-185 Acromelic acid a potent neurotoxin 19:163 isolation of 19:163 synthesis of 19:163 Acromelic acid A synthesis of 1:329,330 Acronychia baueri 13:347,349;20:789 Acronychia oigophylebeia 13:348,350 oligophylidine from 13:348,350 Acronycin from Acronychia baueri 20:789 structure activity relationship in 20:791-798 Acronycine antitumor activity of 13:365-375 ^om Acronychia baueri 13:347,349 dimerization of 2:127 (±)-Acrorenone B synthesis of 16:223 Acronycine azine 20:791 A croscyphus sphaerophoides 5:310 Acrylamide reagents 4:45,18:320 Acrylate of (5)-ethyl lactate 8:142 titanium tetrachloride reaction 8:142 Acrylic acid hydrogenation of 12:154 stereoselectivity of 12:154 Acrylonitrile 14:551 Aery lonitrile reagents 18:318 Actamycin 9:248,250 Acteoside 5:507 Actinidia polygama Miq. 16:290,291 Actinidine synthesis of 4:614,615 (+)-Actinobolin absolute stereochemistry of 16:3 antineoplastic activity of 16:3 dental cariostatic activity of 16:3 from Streptomyces griseoviridus Var 16:3 immunosuppressive activity of 16:3 microbial antitumor antibiotics 16:3 total synthesis of 16:3-26 Actinogyra muelenbergii 5:311-313 Actinomadura melliaura AT 2433-Ai and A2 from 12:366,368 AT2433-BiandB2from 12:366,368
926
Actinomadura sp. 5:55 Actinomadura sp. SF-2370 K-252a (SF-2370) from 12:366,368 Actinomycetales 19:559 Actinomycetes 5:601,606,77-145 ActinomycinD 20:514 Actinoplanes coloradoensis 17:283 Actinopyga agassizi holothurins from 7:267,269 Actinopyga echinites echinoside A&B from 7:269 Actinopyga mauritiana 7:281 Actinorhodin from4,10-dihydro-3H-naphtho [2,3-c] pyran-10one 11:127,128 synthesis of 11:127,128 Actinostatin 1 7:281 Activating group methoxycarbonyl as 6:540 Active oligosaccharides enzymatic synthesis of 10:479,480 Acuminatopyrone 13:544,545 Acumycin 5:613 Acute leukemia in children 14:805 vinblastine for 14:805 vincristine for 14:805 Acyclic amino acids synthesis of 13:512-516 Acyclic [2,3]-Wittig rearrangements 8:196 Acyclic a,a-dialkylated esters preparation of 10:411 Acyclic amino alcohols from 2-oxazolidinones 12:428-430 Acyclic P-hydroxy ketones asymmetric reduction of 14:183,184 Acyclic bastadins synthesis of 10:634,635 Acyclic compound pyrrolidine derivative from 14:555 with I-proline 14:555 Acyclic lactone 11:345 Acyclic TV-Boc-aminoalcohols from 2-oxazolidinone 12:428-430 hydrolytic cleavage of 12:428-430 5-Acyl meldrum acid 10:60 Acyl migration 6:122 Acyl nitro Diels-Alder reaction 1:281,282 Acyl ylides conversion to acetylenes 4:564 1 -Acyl-1,2-dihydropyridine derivative methylcyanodithioformate reaction with 6:433,434 solenopsin A from 6:433,434 3-Acyl-2-oxazolidinones with 2-exo-methoxy-l-apocamphane carboxylic acid 12:438 3-Acyl-2-oxazolone /A'a«5-5-bromo-4-methoxy adduct from 12:418 rm«5-5-phenyl-seleno-4-methoxy adduct from 12:418 with yV-bromosuccinimide 12:418 with phenylselenyl chloride 12:418
3-Acyl-4-methoxy-2-oxazolidmone iV-deprotection of 12:427,428 l-Acyl-5-benzylpyrrol-2(5//)-ones 13:110,111 N^-Acyl-CC-1065 structure-activity 3:376-379 synthesis of 3:373-376 a-Acylamino radical 12:446 a-Acylamino radical cyclization 1:256,292;12:471 2-Acylamino-2-deoxysugars 14:218 Acylase group 6:551 Acylated amidines 5:574 Acylated flavonol-glycosides 17:142 Acylated guanidines 5:574 Acylating agents enantioselective 14:508 Acylation ofhexaenal 6:264,267 selective 6:283,284 with A^,A^-diisopropylcarbamoyl chloride 10:15 intramolecular 16:18 with Cbz-L-alanine 16:18 with ethy Ichloroformate 16:19 A'-Acylaziridines 13:150 Acylenamines cyclization ot reythrinans 3:460 Acylglycerol 12:392 0-Acylglycosyl bromides 14:172 A^-Acyliminium ion construction of erythrinans 3:458,459 cationic 7i-cyclization of 12:287 intramolecular arylation of 12:447-452 Acyliminium ion cyclization 1:244;12:305,320,335 A^-Acyliminium ion-polyene cyclization 12:464 A^-Acyliminium ion-vinylsilane cyclization 12:453 A^-Acyliminium ions (amido-alkylation reagents) 13:473-518 Acylindole alkaloids 5:80,81 1-Acylindoles photoisomerisation of 1:51 2-Acylindoles 1:51 A^-Acyllactams pyrolysisof 6:430,431 [l,3]-Acylmigration 1:51 A'^-Acylmuramoyl-peptides 6:393 Acylnitrosocycloaddition 19:355 Acylnitroso Diels-Alder reaction 1:386,4:606 Acylnitroso group as dienophile in Diels-Alder reaction 4:606 N-Acylnorreticuline 18:74 Acyloin condensation 8:224,225,12:337 19'-Acy loxfiicoxanthins distribution of 6:134,135 Acyloxonium ion 14:203 P-Acyloxy diazo ketones acid-catalyzed 12:27,28 rearrangement of 12:27,28 to 3-fiiranones 12:27,28 3-Acyloxypyrrol -2(5;^-one 13:116,118,120 15P-Acyloxyquassin fromquassin 11:79,80 Acyloxysulfones reductive elimination of 4:522
927 iV-Acylpyrrol-2(5//)-ones 13:110,111 3-Acylpyrrol-2(5//)-ones 13:112,113,141 3-Acylpyrrolidinone 13:121,122,133,139 3-Acylpyrrolin -2,5-diones 13:114,125,129 A^-Acylsphingosines 18:714 Acylstannanes 10:22-24 Acyltetramic acid biosynthesis of 14:105,106 3-Acyltetramic acids 14:97 Adam's catalyst 4:23;12:152;19:18,70,763 Adamantane 9:116 1-Adamantyl phosphoryl derivatives ADDA Unit 9:496-499 from microcystins 9:496 '^O-NMR of 9:116-118 syn Addition 8:296-298,304 Addition reaction of chiral vinyllithium compounds 12:35-62 to a-methyl-substituted aldehyde 12:35-62 1,4-Addition reaction 14:696,697 Addition to allylic A-systems Felkin-Anh model 3:248 Addition-elimination mechanism for 5-enolpyruvylshikimate 3-phosphate synthase 11:185,187 SE'Additions 10:17-25 Additivity hypothesis 6:149 Adenanthin from Rabdosia adenantha 15:171 from Rabdosia nervosa 15:173 Adenine glycosidation of 10:603,605 with oxetanosyl chloride 10:603,605 Adenocarcinoma of colon 1:276 Adenosine 14:286 Adenosine deaminase 10:587 cv^^/c-Adenosine monophosphate (cAMP) phosphodiesterase 1:422,178 Adenosine-3'-monophosphate 2:47,48 Adenosylhomocy Sterne 19:177 S-Adenosylmethionine 11:201,207 S-Adenosylmethionine dependent methyltransferase 11:208 S-Adenosyl-methionine 1:408 5-Adenosylmethionine (SAM) methylation 9:42,599 S- Adenosylmetionine: caffeate 3-O-metyl transferase 5:468 Adipokinetic hormones 9:487,489 Adiposin-1 and 2 inhibitory activity against a-amylases 10:514 synthesis of 10:514,515 Adiposin-D 10:513 Adiposins synthesis of 13:235-246 Adiposins antibacterial activity of 10:514 from Streptomyces calvus 10:513 a-glucosidase inhibitors of 10:513 Adocia species 7,20-diisocyanoadociane 6:86 Adociane ring system 6:86,87
Adociaquinone A-B 17:33 Adrenaline 15:328 P-Adrenergic blocking agents synthesis of 14:473 p-Adrenergic receptors 18:720 Adriamycin 4-demethoxy analogs of 14:474 synthesis of 14:474,475 Adriamycinone 14:4 Adsorption chromatography 9:450-43,460 chromatographic analysis by 9:450-453,460 Aedes aegypti 9:299 Aegiceras comiculatium 7:176-178,180,185,195 Aegicerasfloridium 7:176-178 Aegicerataceae 7:177,183 Aeridoteres tristis hemogloin components of 5:837 A erobacter aerogenes 11:183 Aerobiosis 9:568 A eromonas hydrophila 4:197 Aeromonas salmonicida 4:197 Aerothionin 10:632 (+)-Aerugin 19:821 Aesculetin from Fraxinus rhynchophyllus 13:660 Aesculetin 7:224 Aesculin 7:224 Aesculosides A,B 7:142,143 A es cuius glabrus 15:191 Aesculus hippocastanum 7:142,143 Aescuius indica aesculosides A,B from 7:142 triterpenes of 7:142,143 A es cuius saponins 15:191 Affmine 16-e/7/-Affmine 5:124 Affmisine 5:123,126;9:179;13:386,429 Affinity column chromatography 16:114 AflatoxinBi 18:711 Aflatoxin M2 synthesis of 14:651-657 via Norrish type II reaction 14:651-657 (+)-Africanol 14:487,488 from chiral dienyl ether alcohol 14:487,488 synthesis of 14:487,488 Africanol 3:93 (±)-Africanol by Claisen rearrangement 6:51 (±)-dactylol from 6:37 fromhumulene 6:37 from Lemnalia africana 6:50 1,2-methyl shift in 6:37 synthesis of 6:50 Afrocurarine 1:126 Afzelia bipendensis bipendensis from 9:256 Agaricales 9:203 a-Agaroftiran (+)-intermedeol from 14:453 Agavaceae 7:427 Agave cantala 7:427 Agel-489 13:650
928
Agelas mauritianus agelasphins from 18:460,467 Agelas nakamurai agelasin-B from 6:28 Agelas species agelasins from 6:28 Agelasin-B from Agelas nakamurai 6:28 from kolavenic acid 6:28 synthesis of 6:28 Agelasins from Agelas species 6:28 Agelasphin-9b synthesis of 18:467-469 Agelasphins from Agelas mauritianus 18:460,467 Agelenopsis aperta 19:675 Ageratina adenophora 5:28 Ageratoriparin 20:282 Ageratum fastigiatum 5:728 Agglutination inhibitors 2:308,309 Aggregation pheromone of Gnathotrichus 1:692 Aggreticin platelet aggregating inhibitor 5:597 Aglaopheniapluma 5:353 Aglycone from cyclohexene imides 12:378 ofAT2433-BiandB2 12:377-379 ofstaurosporine 12:379-381 synthesis of 12:377-381 Aglycone 15:362 a2-Agonists 8:396-298 Agratis ipsilon 7:397 Agrobacterium rhizogenes 15:376,377;17:395,421 Agrobacterium sp. 7:76 Agrobacterium tumefaciens 9:386,388 Agroclavine 11:199,200 Agyl glycosides 7:53-58 AHB (3-amino-5-hydroxybenzoic acid) synthesis of 9:434 Ahydrovinblastine synthesis of 4:31,32 AI-77-AtoG 15:390 AI-77-B 15:393,396,397,402,404,412-418 AI-77-G 15:392 AIBN (2,2*-azobisisobutyronitrile) 6:353;19:340;481 AIDS 2:421-426 Aids virus 12:245 Ailanthes altissima 2:370 Ailanthinone 7:379,380,398 Ailanthone 7:394,398 Ailanthus altissima 7:392,394 Ailanthus grandis 7:369,381 Ailantone conversion to sinjulactone C 5:800 Aizoaceae 0-methyljoubertiamine from 4:3-5 mesembrine from 4:3-5 Aizumycin from Streptomyces aizunensis 12:63 Ajmalicine from Corynantheyohimbe 8:283
Ajmalicine from Rauwolfia serpentina 13:660 (-)-Ajmalicine asymmetric synthesis of 14:563,564 from piperidine 14:563,564 (±)-Ajmalicine 18:332 Ajmalicine-type alkaloids isoreserpiline 5:74 reserpilme 5:74 serpentine 5:74 tetraydroalstonine 5:74 Ajmaline 9:183-185;13:403,404,424-426;15:469 Ajmalinimine 9:183-186 Ajugalactones 20:248 Ajugol 7:455 Akicenin 5:722,723 Akiferidin 5:722,723 Akiferidinin 5:722,723 Akiferin 5:722,723 Aklavinone 4:317,327-329,333,341,342;11:121-123 Akuammicine 1:35:1:45;5:123;9:190,192;14:751 Akuammicine N-oxide 5:123 ;9:190,192 Akuammicine-Nb-metho salt 1:35 Akuammicine-type alkaloids 5:86 (Z)-Akuammidine 15:466,467 19 Akuammidine 5:24,123,127;9:171 (£)-Akuammidine (rhazine) 13:403 Akuammidine-type alkaloids 5:74,76 Akuammigine 9:171 Akuammiline 5:80,126,9:194,195 Akuammiline-type alkalids 5:79,80 ALA (5-aminolevulinic acid) 9:591,593,601,603,606 ALA-dehydrase 9:604 Alane 1:455 L-Alaninals amino sugars from 4:118-127 A^-monoprotected 4:127 ^;A^-diprotected 4:124 synthesis from L-alanine 4:118 D-Alaninals amino sugars from 4:118-127 synthesis from D-alanine 4:118 D-Alanine in synthesis of D-alaninals 4:118 I-Alanine in synthesis of L-alanials 4:118-127 I-serine analogue of 4:127 p- Alaninookadaic acid 5:385,388 I-Alanyl-Z)-isoglutamine benzyl ester 6:398,405 Z-Alanylglycine benzyl ester 6:393,394 Alarm pheromone n-undecane in 6:453,454 Alatol 18:745 9-e/7/-Alatol 18:746 Alatolactone derivatives 7:233 AlbaninFandG 4:618 Albartin 7:224 Albati 17:198 Albazoin 17:19 Albene synthesis of 4:639 Albicanol 17:14 Albicanyl acetate 17:14
929 Albifloranine 9:171 Albomitomycin A from Streptomyces caespitosus 13:434 Aibomycin 9:555 Alcaligenes eutrophus 1:690;4:39;8:299 Alcaligenesfaecalis var.myxogenes 5:314 (+)-Alchomeine 16:439 P-Alcohol 19:248 Alcohol dehydrogenase 17:479 Alcohol inversion 1:456,457,459 Alcohol protection as MEM ether 1:558 Alcohols as chiral building blocks 1:684-688 preparation of optically active 1:6 84-6 8 8 CI(NO) spectrum of 2:4 Alcyonidium gelatinosum 18:695 Aldehyde-ketophosphonate condensation 6:264,265 Aldehydes geminal dialkylation of 8:3,5 CI(NO) spectrum of 2:4 by 1,3-dithiane 6:308 byFAMSO 6:311-313 from carboxylic ester 6:334 synthesis of 6:308,311-313,331 1 -Aldehydofuranosides synthesis by RWG contraction 3:225,226 Alder ene reaction 8:277,278,280 Alderene 1,4-diene 8:278 (£)-Aldimine 11:278,279 Aldimine 18:680 Alditols 9:499 1-Aldo-C-glycosides 10:350,351,389 Aldoheptofuranoses Aldohexofuranose derivatives 10:428-432 asymmetric quatemization of 10:428-432 Johnson-Claisen rearrangement of 10:432-436 Aldohexoses 10:414,415 Aldol condensation 13:448,468 P-elimination 12:13 acid-catalyzed 16:216,220 asymmetric induction in 4:328,329 base-catalyzed 16:220 chelation controlled 10:286 crossed 11:296,297 diastereoselective 4:328,329;12:15,16 intermolecular 10:182,183,318-320;11:113,115 internal 1:363-365 intramolecular 10:303,306,318,329,330;16:141, 216,216,221,228,259,260 of silyl enol ether and acetone 11:296,297 offuranone 12:13 stannic chloride catalysed 4:328,329 TiCU catalysed 1:537,538 Aldol coupling reactions 11:435-439 Aldol cyclization 2-amino alchohol by 12:411,414 asymmetric 4:327 chkal acetals in 4:329 chiral synthons for 4:491 of a-sulfmylesters 4:491 -494 of aldehyde 12:174,411,414 of ascorbic acid 4:699-705
ofenolate 160 stereoselective 12:154 with enolsilyl ether 12:174 with isonitrile 12:411,414 with A^-acylimine 12:160 Aldol stereoselectivity 12:72 Aldol type addition 16:656 Aldol type condensation of aldehydes 8:426,427 with 2-trimethylsiloxy-3-methylfuran 8:426,427 Aldol cyclization 6:49,50 in (+)-spatol synthesis 6:40,41 Aldol reaction intramolecular McMurry approach 13:602 Aldol-type reaction stereocontrolled 12:36 stereoselective 12:166 Aldolase 11:216 Aldolate trapping 4:17,18 Aldolization 6:7,9,10,17 Aldols by kinetic deprotonation 11:337,338 of2-cyclohexen-l-one 11:337,338 with 4-pentenal 11:337,338 Aldonolactones 7:49,50 P-D-Aldopyranosyl-cyanides synthesis of 10:355 Aldose reductase 9:499-503 Aldose reductase inhibition 6:20 by (+)-dysideapalaunic acid 6:20 (+)-Aldosterone enantioselective synthesis of 16:706-707 (1-^1 )-Aldosyl aldoside 1 -thiodisaccharides 8:317-322 (l->2)-Aldosylketoside 1-thiodisaccharides 8:322-326 Alectoria sarmentosa 5:310,311 Alectoria sulcata 5:310,311 Alexa leiopetala (+)-castanospermine from 12:332 (+)-6-e/7/-castanosperime 12:342 (15',65',7/?,87?,8a/?)-tetrahydroxyindolizidine from 12:332 Alexa leiopetale (+)-castanospermine from 11:267 Alexandrium fundyense 18:703 Alexandrium ostenfeldii 18:703 Alexandrium tamarebnsuis 1:703 Alexandrium tamarensis 17:4;18:703 Alexine 7:13,14,10:567,568 Alflavene biosynthesis of 4:618,619 Algae 9:321 Alicyclic polyketide nonaromatic 12:16 Alioline '^C-NMR spectrum of 5:165 COSY.45 spectrum of 5:163,164 *H-NMR spectrum of 5:163 NOE measurements 5:164 UV spectrum of 5:163 Aliphatic acceptors in Michael reaction 4:705,706 Aliphatic isocyanides synthesis of 12:113,114
930 Aliumvineale 15:196 Alkali metals in prenylation methods 4:394,395 Alkaline degradation ofN-acetyl muramic acid 6:387 Alkaline protease 9:412 Alkaloid AG-1 1:127 Alkaloid production in Zenk's medium 2:373,374 Alkaloids P-carboline 17:89 cinchona 7:444 cyclopentanoid monoterpene 7:443,444 from aspidosperma family 2:376,378 from Capparis decidua 9:73-75,77 from corynanthe family 2:376,378 from iboga family 2:378,379 from Prosopis juliflora 9:70-73 from Schweinfurthiapapilionacease 9:25,26,28 fromstrychnos 2:379 indole 7:443,444 indolizidine 6:422,442,443,445-451 ipecacuanha 7:444 isoquinoline 17:89 molluscicidal activity of 7:427 of Strychnos dinklagei 6:503-536 of Tetraponera 6:451 -454 piperidine 6:422-434 production of 17:404,409,411-412 purine 17:89 pyridine 17:89 pyrrolidine 6:434-444 pyrrolizidine 6:422,442,443,445 pyrroloquinoline 7:444 semi-synthesis of 17:631 synthesis of 14:632-639 synthesis of 16:415-452 Alkane-2,6-diones reductive aminocyclization of 6:433,434 Alkanol 7:231 Alkavinone enantioselective synthesis 1:596,597 Alkenals 2:10 Alkene 2-amino alcohol by 12:411,413 asymmetric hydration of 13:451 asymmetric hydroboration of 13:451,452 n-Alkenoic acids 2:9 oxyamination of 12:411,413 photochemical additions to 1:642 A^'^Alkene 12:22 o-Alkenyl dehydroglycosides 3:245 6-Alkenyl-4-oxapyran-2-ones 10:340 by enolate Claisen rearrangement of 10:340 Claisen rearrangement of 3:245 hydroboration of cw-Alkenes 8:471,472 thermolytic cyclization of 6:429 c«-Alkenes 8:471,472 hydroboration of 8:471,472 Alkenols 2:10 (Z)-Alkenones 19:122 Alkenoylcyanide acceptors in Michael reaction 4:709-711
Alkenyl acetates 2:10 Alkenyl azides thermolytic cyclization of 6:429 0-Alkenyl dehydroglycosides Claisen rearrangement of 3:245 (Z)-//-Alkenylinitrone 19:29 Alkenyl phenols molluscicidal activity of 7:427 6-Alkenyl-4-oxapyran-2-ones by enolate Claisen rearrangement 10:340 (3-l-Alkenyl-9-borabicyclo [3.3.1] nonanes 10:156,160 Alkenylcopper-phosphine complex 8:189 Alkenylhydroquinones 9:321,329 Alkenylpyrazines 5:239-246 2-Alkenyltropolones [4+2] cycloadditions of 5:799,800 [6+4] cycloadditions of 5:799,800 A^-Alkenylurethanes 19:29 Alkhanin 7:231 Alkoxides 2-amino alcohol by 12:411,413 Michael addition of 12:411,413 to nitro olefin 12:411,413 2-e;co-Alkoxy derivatives by reduction 12:417 of ketopinic acid 12:417 with L-selectride 12:417 2-e« Jo-Alkoxy derivatives P-Alkoxy diazo ketones 12:27,28 P-Alkoxy ketones from chiral acetal 14:481 pure alcohols from 14:481 2-Alkoxy ketones 14:647 3-[ 15)]-2-exo-Alkoxy-1 -apocamhaneearbonyl]2-oxazolones (45',55)-isomer from 12:420,421 methoxybromination of 12:420,421 methoxyselenylation of 12:421,422 (4/?,5/?)-4-methoxy-5-phenylseleno-2oxazolidinones from 12:421,422 reversed p facial selection in 12:420-422 3-[( 1 S)-2-exo-Alkoxy-1 -apocamphane-carbonyl]-2oxazolone to dialkyl azocarboxylates 12:424,425 a-Alkoxy-a-arylacetates synthesis of 6:331 a-Alkoxy-a-arylacetates esters synthesis of 6:328 2-Alkoxy-butadienes in Diels-Alder reactions 4:581 Alkoxyacrylate by methanolysis 14:563,564 1-Alkoxyalkyl-a-glucosides 7:51 Alkoxyalkyl-P-glucosides 7:51 Alkoxyallenes diastereoselective synthesis of 1:627 (y-Alkoxyallyl) stannanes 11:442,443 (a-Alkoxyallyl)-boronic ester from pinacol (1 -bromoallyl) boronate 11:424 P-Alkoxyboranes elimination 4:116
931 a-Alkoxydimethylhydrazones 2-amino alcohol by 12:411,413 nucleophilic addition of 12:411,413 organolithium reagents to 12:411,413 a-Alkoxymethoxy methyl ketone addition of 12:29,30 3-butenyl magnesium bromide 12:29,30 chelation-controUed 12:29,30 4-Alkoxypyridine as catalyst 4:270 a-Alkoxysulfoxide from monochlorinated sulfoxide 6:310 3-Alkylpthalide alkylation of 5:827,828 hydrogenolysis of 5:827,828 Alkyl 1-thiogalactosides synthesis of 8:342 Alkyl 1-thioglycoside 8:347 Alkyl 1-thioxylosides 8:315 Alkyl 4-thiodisaccharide 8:332,333 a-Alkyl a-amino acids 10:411 Alkyl P-D-galactoside 8:316 5ec-Alkyl boronic ester 11:409,411,412 Alkyl C-glycosides from glycosyl fluorides 10:368 Alkyl ketones 14:645 Alkyl maltotrioside 8:346,347 Alkyl pyrazines mass spectroscopy of 5:259 Alkyl quinones 5:821 2- Alkyl-1,4-naphthoquinones 5:823,824 2-Alkyl-l-piperidine 6:424 in Solenopsis species 6:422 4-Alkyl-2-buten-4-olides 3:166,167 4-Alkyl-2-oxazolidinones from 4-methoxy-2-oxazolidinones 12:426 A^-Alkyl-4-hydroxy-2-pyrrolidinones from p-oxoamides 14:650,651 synthesis of 14:650,651 ^A-a«5-2-Alkyl-5-hydroxypiperidines 13:486 2-Alkyl-6-methyl pyridine from 2,6-dimethylpyridine 6:425 synthesis of 6:424-426 3-Alkyl-8-hydroxy-3,4-dihydroisocoumarins 15:386 a-Alkyl-a-amino acid asymmetric synthesis of 13:95-99 a-Alkyl-a-mino acid derivatives 13:80 Alkyl-P-Z)-glucopyranosides 7:52 Alkyl-p-D-glucosides 7:104,105 Alkyl-P-glucosides 7:50,51 l-iV-Alkyl-deoxynojirimycins 7:46 Alkyl-substituted azulenes oxidation potentials of 14:347 Alkyl-sulfoxides synthesis of 4:490,491 Alkyl-type phosphoramides 14:286 N-Alkylated pyrrolidinones photo-induced annulation of 12:293 24-Alkylated thomasterol A 15:48 5-Alkylation 8:214 intramolecular 13:145
O-Alkylation a-D-glucopyranoside 6:385 glucopyranoside 6:388 A^-substituted amides 10:215 of potassium koj ate 12:261 ofw-haloalcohol 8:195 Alkylation asymmetric 11:367 base (LDA) mediated 10:408,409 diastereoselective 14:491-499 enantioselective 10:412 in basic media 4:389-391 intramolecular 16:125;19:486 of(5)-a-terpinylamine 11:283 of 1,5-acetyl-1 -thiohex-2-enopyranosides 10:342 of l-benzyl-2,6-dicyanopiperidine 6:433 of 1-phenyl-1-methyl 6,7 seleno-1-ethyllithium 8:6,7 of 2-acetoxy-5,6-dihydro-2H-pyrans 10:342 ofacetals 1:613-616 ofbenzyllithium 8:6 ofcarbanion 8:176 ofcarvone 10:408,409 ofchiralacetals 12:489-493 ofcyanohydrin 8:16 of cyanohydrin ethers 8:177-179,183 ofdianion 11:284,285 of dimethyl malonate 8:185,186 ofdioxolaneenolates 1:644 ofFAMSO 6:313 offluoromalonate 13:82 ofmethylmalonate 13:79 ofMT-sulfone 6:334 of A^-nitrosopyrrolidine 6:439,441 of silver cyanides 12:113 of sulfur stabilized carbanion 8:16 of trans-2,5-\)\s (methoxymethoxymethyl) pyrrolidme 10:412 of w-haloalkylphenylthioacetate 8:176 of a-methyl-phenylacetic acid 10:412 of a-phenyl-y-lactone 10:410,411 of P-ketoester 8:181,191 palladium-mediated 16:422 regioselective 10:342 stereoselective 10:342,410,411;11:284,285 synthesis of 3:166,167 TiCU catalysed 1:615 via sequential dianion generation 16:371 with l-TMS-2-pentyne 11:284,285 with 2,5-dibromo-2-methyl pentane 8:6,7 with 3-butynyl p-toluene sulphonate 14:559 with allylic halide 8:178 with benzyloxymethyl chloride 6:561 with di-^butyl dicarbonate 14:559 with House procedure 10:308,309 with indole-protected tryptophyl bromide 11:283 with organometallic reagents 1:613-616 y-Alkylation in (+)-sinularene synthesis 6:76,77 C-Alkylations of phenolates ofcyanohydrin 8:195 with prenyl bromide 4:382-386
932 Alkylcatechols 9:328 3-Alkyldihydroisocoumarins 15:386,387 6-(9-Alkylerythromycins 13:162-165 3-Alkylidene oxepanes synthesis of 10:221 6-(£)-Alkylidene-8-hydroxy-8-methylindolizidines synthesis of 12:295,296 6-(Z)-Alkylidene-8-hydroxy-8-methylindolizidines synthesis of 12:294,295 C-Alkylidenepyraneside alloyohimban 3:406,407 synthesis of 3:220,406 Alkylmalonate fluorination of 13:83 (5)-4-Alkyloxazolidin-2-one derivative 12:164-168 3-Alkylphenols 9:323,329,338,346 5-Alkylresorcinol from Grevillea striata 8:227 Alkylresorcinols 9:328,338 (5)-4-Alkylthiazolidin-2-thione derivative 12:164-168 Alkylxanthates 8:316 Alkyne compound from propargyl alcohol 14:570 trans-o\Qfm from 14:570 Alkynoic acids/esters 2:16 Alkynols 2:14-16 a-Alkynone cyclization 6:45 in (+)-A^^'^^-capanellene synthesis 6:45 P-Alkynyl alcohol 12:24 Aikynyl Grignard reagents reaction with l-methoxycarbonyl pyridinium chloride 6:448 reaction with 2-methylpyridinium salts 6:429 Alkynyl-a-chlorohydrins LAH reductions of 8:247 Alkynylation diastereoselective 1:610-612 Alkynylsilane 14:482 All ^rart5-7,8-renieratene synthesis of 6:156,160 AW-trans (3/?,3/?')-alloxanthin CD spectrum of 6:149 All-trans (35,35')-tetradehydroastaxanthin synthesis of 6:157 AW-trans didehydocarotenoids 6:155 AW-trans geranylgeraniol in taondiol methyl ether synthesis 6:56 AW-trans polyprenols 8:66 All-/A'a«5-7,8-didehydroisorenieratene synthesis of 6:156,160 Allaentus zeylanicus 5:752 Allamanda blanchetii 19:776 Allamanda catharica Linn 16:299 Allamandicin antifungal activity of 16:299 antileukemic activity 16:299 antimicrobial activity 16:299 cytotoxic activity 16:299 synthesis of 16:299-301 £/7/-Allamandicin synthesis of 16:301
Allamandin synthesis of 16:299-301 Allamcin from Allamanda s^p. 16:299 Allamdin antiftingal activity of 16:299 antileukemic activity of 16:299 cytotoxic activity of 16:299 Allamicin from Allamanda sp. 16:299 Allelochemicals 9:387 Allelopathic activities 20:391 Allene 5:370 Allene epoxide rearrangement of 8:36 Allenic acetylenic carotenoids 6:162-164 Allenic carotenoids biosynthesis of 6:139,142 de novo biosynthesis of 6:133-135 chemical reaction of 6:146,147 chemosystematics of 6:133-135 chiralityof 6:137 distribution of 6:133-135 from zeaxanthin (p,P-carotene-3,3- diol) 6:139 fiinction of 6:142,143 isolation of 133,134 metabolic transformation of 6:142,143,155 metabolism of 6:142,143 stereochemistry of 6:138-142 structure determination of 6:135-13 8 synthesis of 6:143-147 (±)-Y-Allenyl-GABA synthesis of 13:514 Allenyltin 12:170,172 Alleochemicals 8:219 Allergenic dermatitis 2:277,280-281 Allergy inhibitors 5:759 (+)-^//o-pumilitoxin 339B synthesis of 16:426 Alloaristoteline (epi-11-aristoteline) ^^C-NMRof 11:314 from Aristotelia australasica 11:317,318 from aristoteline 11:321,322 total synthesis of 11:318-322 (+)-Alloaromadendrane-4,10-diol from Ambrosia peruviana 14:379 (±)-Alloaromadendrane-4a, 1 Oa-diol synthesis of 14:379-384 (±)-Alloaromadendrane-4p, 1 Oa-diol synthesis of 14:379-384 (±)-Alloaromadendrane-4p,10P-diol 14:383 Alloaromadendren-4p-ol 14:381,382 Allocryptopine from canadine methiodide 491,492 synthesis of of 6:491,492 Allocryptopine A^-oxide Meisenheimer rearrangement of 6:494 Z)-Allofuranose C3-P bond analogs of 6:359 D-ribo-hexofiiranose from 6:369 Allohobartine 3-ethyl-allohobartine from 11:318,319
933 5,6,7,8-tetrahydrobenz [b] indolizidine from 11:318,:319 3-trifluoroacetyl-allohobartine from 11:318,319 AUohobartine derivative preparation of 11:316,317 (2/?,35)-Alloisoleucine from (25,3S)-isoieucine 10:277,278 (±)-a-Allokainic acid synthesis of 13:508,509 a-AUokainic acid pyrrolidine derivative from 14:556,557,560,561 2-pyrrolidone from 14:560,561 (-)-Allokhusiol synthesis of 10:410 AUomelanin 9:231 D-Allomethylpyranose 15:190 AUomones 8:219,220 AUomuscarine from 2-deoxy-D-erv^/iro-pentose 10:394 from 2-deoxy-L-ervr/2ro-pentose 10:394 L-(+)-Allomuscarine synthesis of 10:394 Allomyces macrogynus 5:276,6:544 Allomyces sp. 5:276 AUopumiliotoxins 19:52,54 (+)-Allopumiliotoxins 19:60,61-63,65 (+)-Allopumiliotoxin 267 A synthesis of 16:486-487 7-epimer of 12:297 synthesis of 12:297 (+)-Allopumiliotoxin 267A,339A and 339B synthesis of 12:298-300;16:485-487 AUopumitiotoxin synthesis of 12:297-300 D-AUosan from dihydrolevoglucosenone 14:278 D-Allose from 2,3,5-tri-O-benzoyl-p-D-ribofiiranosyl cyanide 10:358 3,4,6-substituted derivatives of 10:358 Allosecurinine 14:657 (±)-Allosedamine 13:474 AUosedamine 10:677 (-)-Allosedamine 13:475,477 AUoserratenone aristolasicone from 11:316,317 AUosorelline 11:320 Allosteric effector 5:835 (3/?,3/?')-Alloxanthin 6:153 Alloxanthin 6:154,155 (-)-Alloyohimban 10:155 (+)-3-£p/-Alloyohimban 18:384 (-)-Alloyohimban 18:384 (±)-Alloyohimbane stereoselective 14:710,711 synthesis of 14:710,711 Alloyohimbine synthesis of 3:407,410 Alloyohimbines allylation 3:135,222 chemical initiation 3:22 Pd-(0)-catalysed 3:135
photochemical initiation 3:22 with allyl-tri-n-butylstannane 3:222 p-Allyldiisopinocamphenylborane 19:14 AUyl 2,3 -di-O-benzyl-a-L-rhamnopyranoside thermal glycosidation of 8:365,366 Allyl2,3-0-benzyl-4-0-rerr-butyldimethylsilyl-a-Lrhamnopyranoside 8:368 AUyl 3,4,6-tri-p-D-glucopyranoside thermal glycosidation of 8:365,366 Ally 13,4,6-tri-O-benzyl-a-glucopyranosides thermal glycosidation of 8:365,366 Allyl3,5-0-isopropylidene-2-0-triflyl-P-Dlyxofiiranoside 8:330 Allyl acetates isomerization of 4:49 allylation 4:21 ofo-vanillin 4:21 (/?)-Allyl aclohols formation of 14:500,501 Allyl alcohol pyrrolidine derivative from 14:571 (5)-Allyl alcohols formation of 14:500,501 Allyl aluminium 8:24 Allyl p-C-glycoside by reduction with EtsSiH-BFj 10:386 from gluconolactone 10:386 Allyl C-glycoside byozonolysis 10:361,371,372 71-Allyl complexes 4:398 exo-2-Allyl derivative of6-methoxyepicamphor 4:657,658 Allyl ether isomerization to vinyl ether 1:453 Allyl glycidyl ether cyclization of 10:588,589 oxetane ring formation by 10:588,589 reaction of 10:588,589 with sec-butyllithium 10:588,589 a-Aliyl glycosides hy anti SN^ displacement 10:348 from D glucal triacetate 10:419 stereochemistry of 10:348 Allyl isocyanide from allyl iodide 12:113 from silver cyanide 12:113 Allyl mangenese reagent 4:34,344 7i-Allyl palladium complex 4:500;10:214,215 Tii-Allyl Pd alkylation intramolecular 10:10-13 AUylphenols 20:271 Allyl sulfones addition to enones 3:23 Allyl sulfoxide anions addition to enones 3:21,22 ahiusenone 3:435 alpine-borane reagent 3:267 chu-al 3:21,22 synthesis of 3:435 Allyl tributylstannane 12:425,426 N,N-Allyl,a-methyl benzylamine 8:403-404
934 (R)-5-Allyl-2-oxazolidinone from(4/?,5/?)-5-allyl-4-phenylthio-2-oxazolidinone 12:434 (45,55)-5-Allyl-4-methoxy-2-oxazolidmone (35,45)-cyclohexylstatine from 12:432,433 (35,45)-statine from 12:432,433 (-)-5-Allyl-4-methoxy-2-oxazolidinone 12:419 (4/?,5/?)-5-Allyl-4-methoxy-2-oxazolidmone 12:431, 432 (4/?,5/?)-5-Allyl-4-phenylthio-2-oxazolidinone (^)-5-allyl-2-oxazolidinone from 12:434 Allyl-9-BBN 14:482 5-Allyl-derivatives from 5-bromo compounds 12:425,426 Allylamines 2-amino alcohol by 12:411,414 cyclocarbamations 12:411,414 Allylation diastereoselective 1:604-610 of2-acetylcyclohexanone 10:412 ofFAMSO 6:315,316 with allyl acetate 10:412 Allylation reaction 12:484,14:474 (9-Allylation-Claisen rearrangement 12:269 AUylboration 8:477,478 AUylboronate stereoselective addition to glyceraldehyder 1:311,312 7c-Allylcation complex 3:82,83 Allylcerium 8:24 Allylchromium species coupling with carbonyIs 3:81 medium ring compounds from 3:81 C-Allylfiiranoside 3:215 a-Allylglucosides synthesis of 3:214,215 (S)-Allylglycine 13:512 Allylic polyprenols synthesis of 8:66,67,72 Allylic (alkyl) ketene acetal [3,3] sigmatropic rearrangement of 10:417 Allylic acetates 8:228 Allylic acetoxylation 16:420 Allylic alcohol allylic aldehyde from 12:46,47 amides from 14:722,723 asymmetric cyclopropanation of 14:490,491 biomimetic cyclization of 14:717-720 by Claisen rearrangement 14:722,723 epoxidation of 10:39,40 2,3-epoxy alcohol from 14:570 from (/?,/?)-2,3-butanediol 14:490 from acetylenic ketone 11:424 from D-glucose 10:428-438 from dimethyl-tartrate 11:267,268 Johnson-Claisen rearrangement of 10:428-438 mesylationof 4:172,173 one-carbon homologation 3:238 oxidation of 12:46,47,16:594 preparation of 11:424 Sharpless epoxidation of 14:570
withMnOz 12:46,47 with VO (acac)2-TBHP 10:39,40 Z-Allylic alcohols cw-hydroxylation of 4:203 Allylic p-hydroxysulfoxides stereoselective hydroxylation 4:503,504 Z-Allylic P-ketosulfoxides synthesis of 4:509,510 Allylic bromide from ethyl acetoacetate 12:269 with4-bromo-l-butene 12:269 Allylic bromination 6:207 Allylic C-0 bond cleavage with dissolving lithium/amine reduction 1:557,558 Allylic carbocation asymmetric epoxidation of 4:172-174 Allylic carboxyl group phytochemical removal 3:487 Allylic displacement reactions palladium catalyzed 16:397 Allylic ethers hydroboration 4:116 Allylic glycolate esters freland-Claisen rearrangement of 10:437 Allylic hydroxylation with Se02/tert-butylhydroperoxide 1:535 Allylic hydroxylation 6:200,201 Allylic A^-benzoyl-a-amino acid esters Claisen rearangement of 11:471 Allylic oxidation 3:448;6:126,159,162;8:197,198; 11:11,40,41;12:311;16:420,669 ofperezinone 5:722 with Collins reagent 11:83,84 with manganese dioxide 11:356,357 withSeOz 1:549,550 Allylic radicals reductive elimination 4:525 Allylic rearrangement 4:556,6:159,14:191,16:305, 617,669 Allylic strain 6:122 Allylic strain (nonchelate-controlled mode) 14:553 Allylic sulfoxide rearrangement 1:560,564 Allylic sulfoxides [2,3] sigmatropic rearrangement of 11:326,327 Allylic trichloroacetimidates [3,3] sigmatropic rearrangement of 10:421 Allylmetal additions 8:16 a-Allyloxy anions [2,3]-Wittig rearrangement of 3:248-250 Allylsilane 14:482 Allylstannane 1:247,256,312,313,10:17-25 Allyltin derivatives nucleophilic addition by 11:442,443 AUysilane 1:314 (Allyloxy) methyllithium rearrangement of 8:200 Alnusfirma 17:359,364 Alnus glutinosa 19:246 Alnushirsuta 17:360-361 Alnus japonica 17:360,368 Alnus rubra 17:359 Alnus serrulatoides 17:359-360
935 Alnus species 17:358-359,368,375 Alnusdiol 17:368 Alnuson 17:368 Alnusonol 17:368 Alnusoxide 17:368,371 Aloperine 14:754 Alpha-D3 9:513 Alpha-tocopherol 14:617 Alpinia katsumadai 17:362,375 Alpinia ojficinarum 17:362-363,375 Alpinia oxyphylla 17:362,375,379 Alpinia sieboldiana 17:362 Alpinia species 17:358 Alpinigenine synthesis of 1:213,214 c/5-Alpinigenine synthesis of 1:214-217 Alsophila pometaria 18:681 Alstocraline 13:395 Alstonamide ^H-NMR spectrum of 5:155 mass spectrum of 5:155 UV spectrum of 5:155 *^C-NMR spectrum of 5:140 COSY-45 spectrum of 5:139,140 ' H - N M R spectrum of 5:138-140 mass spectrum of 5:138 Alstonerine frommacroline 13:398 synthesis of 13:383,408,411,415- 423 Alstonia augustifolia 13:422 Alstonia scholaris 5:135,137,138,13:383 Alstonia constricta 1:125,214-217,219,2:369 Alstonia macrophylla 5:135,154-157 Alstonia venenata 1:125 Alstoniline synthesis of 1:129,132,138 Alstonilinol synthesis of 1:128,129 Alstonine 1:125 Alstonisidine 13:383,393,405-407 Alstonisine 13:388,391,399 Alstophylline 13:388,403,405 Alstopicralamine COSY-45 specgrum of 5:156,157 ^H-NMR spectrum of 5:157 mass spectrum of 5:157 Alstopicralamine 9:185,186 Alstovine 1:36 Altemaria solani 598 Altermaria mali 12:400 Altemaria 9:203 Altemaria cinerariae dehydrocurvularin by 11:194 Altemaria kikuchiana 5:598 A Itemaria kikuchiana tanaka 15:385 Altemaria solani altersolanol A from 15:346 Altemaria tannius 9:300 Altersolanol A from Altemaria solani 15:346 Altholactone (goniothalenol) 9:393 (+)-Altholactone 19:468
Altholactone biological activity of 19:498 isolation of 19:498 AltohyrtinA 19:580 D-Altropyranoside 6:359,360 C3-P bond analog of 6:359,360 D-Altrose 4:203 Aluminium hydride selective reduction with 3:474 Alutera scripta 5:390 Alzheimer's diseases (-)-physostigmine for 14:637 AMAPOR activity 20:875,878 Amadori rearrangement 18:680 Amalgam procedure 14:746 Amanita agaricus 9:203 Amantia phalloides 5:496 Amaouciaxantin 10:153 Amarogentin 7:490 Amarolide synthesis of 11:78,79 Amaroswerin 7:490 Amarouciaxanthin A 6:136,142 Amarouciaxanthin B 6:151,152 Amaryllidaceae alkaloids 4:8,12,27;10:411;11:229; 16:444 Amaryllidaceae buphanisine from 4:3,4 crinine from 4:3,4 galanthamine from 4:3,4 haemanthidine from 4:3,4 lycoraminefrom 4:3,4 pretazetting from 4:3,4 Amastatin [(25,3/?)-3 -amino-2-hydroxy-5-methyl hexanoyl-L-valyl-L-valyl-L-asparticacid)] leucine aminopeptides A inhibitor of 12:434 leucine aminopeptidase inhibitor of 12:434 Amata sp. 5:225,252 Amathamide A-G 17:83 Amathamides 17:82,84,88,93,95.97 Amathamides C,D,E and F 18:715 Amathia altemata 17:85 Amathia convoluta 17:75,18:715 Amathia genus 17:82 Amathia wilsoni 17:92,95,97,101,18:693 Amauromis phoenicurus hemoglobin components of 5:83 AMB (aminomethyl bilane) 9:595,596 Ambergris fragrances synthesis of 14:420-425 Amberlite IR-45 15:460 Amberlite IRA-400 12:317 AmerlystA-21 19:131 (±)-Ambinine cyclization 14:788-791 from 13-methyltetrahydro-protoberberine 14:790, 14:791 synthesis of 14:788-791 X-ray analysis of 14:791 Ambliofriran analogs 1:666-670 Ambrenolide from Salvia yosgadensis 20:692
936 Ambrosanolide 16:140 Ambrosia maritima lAll Ambrosia peruviana 14:379 (+)-Alloaromadendrane-4,10-diol from 14:379 (-)-Ambrox synthesis of 14:420-425 (-)-ep/-Ambrox synthesis of 14:420-425 Ambrucitin from alkynylyl lithium 10:386 synthesis of 10:386 Amebicidal activity of emetine 6:485 of 1,2-secoemetine derivatives 6:485 Amenanthus retroflexus 7:398 American celastraceae triterpenes from 18:757-764 Amethystoidin A from Rabdosia macrophylla 15:173 Amethystonal '^C-nmrof 15:128 from Rabdosia amethystoides 15:171 *H-nmrof 15:121 Amethystonoic acid ^^C-nmrof 15:128 from Rabdosia amethystoides 15:171 ^H-nmrof 15:121 Amicetin 4:234,240,242 Amicoumacin A, B and C 15:388 Amicoumacins biological activities of 15:410-412 ^mm-Amidation 6:409,412 Amide cyclisation 4:546 Amides CD of 2:171 dehydration with chloromethylene 3:212 Amidic penicillins 12:135 Amidoalkylation intramolecular 10:108 Amidoalkylation reagents (A^-acyliminium ions) 13:473-518 Amidocarbonylation 16:408 Amidocyclization 1:382 (±)-Amijitrienol from Dictyota linearis 6:52 synthesis of 6:53 (+)-Amijitrienol by intramolecular cycloalkylation 6:53 by methylenecyclohexane annulation 6:53 synthesis of 6:53 Amination intramolecular 6:429;16:438 Amines formation from ketones 6:429 photosensitized oxidation of 16:604 reductive amination of 6:429 (I-a-Aminoadipoly)-L-cysteinyl-D-valine6(LLD ACV) cephalosporin C from 11:211-213 penicillins from 11:211-213 L-a-Aminouronic acid synthesis of 11:459,460
I-a//o-a-Amino acid synthesis of 11:460,461 Amino acid 16:395-414,604 as chiral synthons 4:625 by a-halo boronic ester 11:417-420 photooxidation of 16:604 synthesis of 11:417-420,13:507-516,16:395-414 a-Aminoacid 12:115,435-438 as chiral building blocks 1:678-684 by hetero Diels-Alder adducts 12:435,436 conversion to amino sugars 4:111-156 synthesis of 12:435-438 Amino acid analysis 9:541 Amino acid composition of avian hemoglobins 5:841,842 Amino acid decarboxylases 10:148 Amino acid derivatives synthesis of 6:318-320 P-Amino acids by hetero-Diels Alder reaction 12:158,159 cyclization by UG1 -reaction 12:116 cyclization of 12:115,116 p-lactamsfrom 12:115 synthesis of 12:155,158,159 a-Amino acids/esters conversion to azomethine ylides 1:331-338 decarboxylation of 1:331-336,337 imines from 1:331 Amino acylase for synthesis of a-amino acids 1:678-680 anti, syn, 5y«-Amino alcohol 11:246,247 from L-threitol derivative 11:246,247 2-Amino alcohols by aldol reaction 12:411,414 by cyclocarbamation 12:411,413 by electrophilic addition 12:411,414 by Michael reaction 12:411,413 by nucleophilic addition 12:411,413,414 by oxyamination 12:411,414 by reduction 12:411,414 by ring-opening of oxirane 12:411,413 from 2-oxazolone 12:411 -444 synthesis of 12:411-444 a-Amino aldehydes in [4+2] cycloaddition 4:111 in Diels-Alder reaction 4:120 metalloorganic addition to 4:124 //,//-diprotected 4:124 7V-monoprotected 125 preparation of 4:113,114 A^,0-protected 4:130 a-Amino aldehydes 2-amino alcohol by 12:411,414 nucleophilic addition 12:411,414 Amino analogues 12:398 Amino carbonylation intramolecular 14:568 palladium (2^) catalyzed 14:568 2-Amino-1,2-dihydroacronycine 20:800
937 a-Amino ketones 2-amino alcohol by 12:411,414 reduction of 12:411,414 a-Amino ketones steroid-pyrazine dimers via 18:885-887 Amino sugar antibiotics 12:411 Amino sugars of anthracyclinones 4:350 synthesis from amino acids 4:111-156 synthesis from glycine 4:115-118 synthesis of 13:190-207 4-Amino-r',6"-anhydro-2",3",2',3',6',2,3-hept-0benzyl-4,6- dideoxy-p-maltotriose 10:508,509 2-Amino-l ,3-azulenedicarboxylate 14:339 2-Amino-2,3-di-deoxyhexoses 14:145 2-Amino-2,5,6-trideoxy-5-phosphinyl-L-galactopyranoses synthesis of 6:369 [2-Amino-2-deoxy D-galactopyranosyl] adenmes 4:238 synthesis of 4:238 2-Amino-2-deoxy-3-0-(/?)-[(hydroxyme-thyl)ethyl]a-D-glucopyranose 6:387 (i?)-l-(hydroxymethyl)ethyl 2-glyceryl-ether from 6:387 2- Amino-2-deoxy-D-galacto-D-galactans 5:298 2-Amino-2-deoxy-D-glucose 1:512 2-Amino-2-deoxynucleosides synthesis of 4:525 {2S,3Ry3-Amino-2-hydroxy-4-phenylbutyric acid (AHPBA) synthesis of 12:433,434 (2iS,3/?)-3-Amino-2-hydroxy-5-methylhexanoicacid (AHMHA) leucine aminopeptidase inhibitor of 12:434 synthesis of 12:433,434 3-Amino-3-deoxy-D-mannose 12:314 3 - Amino-3 -deoxyhexopyranosy 1 purines synthesis of 4:240 3-Amino-3-deoxynucleosides synthesis of 4:240 3-Amino-3-deoxysugars synthesis of 4:150 4-Amino-4-deoxy-D-mannose 12:314 5'-Amino-5'-deoxythymidine synthesis of 4:289 3-Amino-5-hydroxybenzoic acid (CAHB) synthesis of 9:434 2-Amino-5-nitrotoluene 13:442 2-Amino-5a -deoxy-a-DL-glucopyranose 13:203 Amino-5a-carba-deoxyhexopyranoses 13:203-207 synthesis of 13:17-21 6-Amino-6-deoxysugars from a-amino acids 4:111,127 6-Amino-6-deoxyuloses synthesis of 4:124 5-Amino-7-methoxy-2,2-dimethylchroman synthesis of 13:358,359 (3i?)-Y-Amino-(3-hydroxybutyricacid biological activity of 12:434 central inhibitory transmitter 12:434 synthesis of 12:434
Amino-epoxides 12:351 Amino-sugar related alkaloids 10:553 Aminoacyl heptoglycosides synthesis of 11:433-435 Aminoalcohol benzyl-protected 11:298,299 with aldehyde 11:298,299 Aminoalkyl glycosides 7:48 5'-Aminoalkyl oligonucleotides synthesis of 4:294,295 Aminoalkylated oligonucleotides 4:306 (±)-9-Aminoaporphine 16:516-517 (9-Aminobenzylalcohol 18:164 2-Aminobenzy Itetrahydroisoquinoline 18:73 a-Aminobutyrate analogue of LLD ACV 11:212,213 penam product from 11:212,213 y-Aminobutyric acid condensation of 12:287 synthesis of 13:514 with diethyl acetone dicarboxylate 12:287 9-Aminocamptothecin 13:655 Aminocoumarins 18:978 Aminocyclitol 13:212,14:147 Aminocyclitol fortamine 4:118 Aminocyclitol inhibitors 10:517-524 Aminocyclitol oligosides from glucuronide saponins 7:156-158 Aminodeoxyhexosyl-purines synthesis of 4:238-248 Aminodeoxyhexosyl-pyrimidines synthesis of 4:238-248 2-Aminoethanethiol 8:317 2-Aminoethyl phosphonic acid 2:304-309 2-Aminofluorene 8:377 P-Aminofluorene 8:388 Aminoglycopyranoses P-glactosidase inhibition with 7:47 inhibition constants of 7:47 Aminoglycosides hybrimycine 5:615 mutamicins 5:615 Aminohexose derivatives synthesis of 1:25-27 Aminoketal isolation of 11:298,299 Aminoketone irradiation of 14:657-659 preparation of 14:657-659 5-Aminolevulinic acid (ALA) 9:591,593,601,603,606 Aminolysis mtramolecular 12:279 of disaccharide lactone 6:407 ofMurNAcS-lactones 6:393 Aminomethyl bilane (AMB) 9:597 a-Aminonitriles formation of 14:716-718 via Polonovski reaction 14:716-718 2-Aminonucleosides fiision reactions for 4:238 synthesis of 4:238,239 6-Aminonucleosides 4:248
938 4-Ammooctanal diethyl acetal condensation of 6:445,447 with diethyl-3-oxo-glutarate 6:445,447 withethanal 6:445,447 5-AminooUgonucleotides synthesis of 4:294-292 2-Aminooxetanocin A antiviral activity of 10:619,620 6-Aminopencillanic acid (6-APA) 4-acetoxy-P-lactam from 12:160 Aminopentanal 14:739 Aminopeptidase B 10:629,646,652 Aminophenyl dithiocellotrioside synthesis of 8:349,351 /7-Aminophenyl-l ,4,4'-trithiocellotrioside 8:344,346 p-Aminophenyl-1,4-dithiocellobioside 8:351 20a-Aminopregn-5-en-3p-yl- P-D-glucoside 5:128 2-[3-Aminopropylidene]-l,3-dithiane 12:335 Aminoquinone immunosuppressive effect 5:437 Aminotransferase activity 19:650 Aminotriazole oxidation 4:548 (+)-2-Aminotridecanoic acid enzymatic resolution of 1:684 3-Aminoucleosides synthesis of 4:239-241 4-Aminouncleosides 4:241 -247 5- Aminovalerookadaic acid 5:385,388 Aminyl radical heterocyclization ofchloramines 1:292 Amipurimycin 1:398,404-410,4:246,11:430,433 Amisine 20:234 Amiteol ^om Amitermes excellens 14:450-452 synthesis of 14:456-465 Amitermes evuncifer evuncifer ether from 14:452,463 Amitermes excellens amiteol from 14:450-452 Amitermes messinae evuncifer ether from 14:452 Ammania baccifera sesquiterpenes from 9:65 Ammanol 9:65 (+)-Ammodendrine 15:520 Ammonium D-10-camphor-sulfonate 15:425 Ammonium glycyrrhizin 15:5 Ammonium nitrate 2:412,413 Ammonium sulfate precipitation with 2:392,394,399 Ammonolysis with ammoniacal methanol 16:97,98 Ammophilafernaldi 5:224,232,253 Ammophila nigricans 5:224,232,252 Ammophilaprocera 5:225,232,252 Ammophila urnaria 5:223,224,232,252 Amoebicides 7:398 Amorphane 15:247 c-AMP phosphodiesterase inhibitors synthesis of 3:306-319 Amphibian alkaloids 16:426 Amphidinium species 5:396,19:559
Amphidmolide A ^^CNMRdataof 19:562 NOESY experiments of 19:562 relative stereochemistry of 19:562 Amphidinolides 5:396 Amphidinolide B,C 19:564 absolute stereochemistry of 19:564 X-ray analysis of 19:564 Amphidinolide J biosynthesis studies of 19:561 Amphidinolide M cytotoxicity of 19:566 NMR analysis of 19:566 structure elucidation of 19:566 Amphidinolide O structure of 19:561 Amphidinolide Q cytotoxicity of 19:560 Amphiscolops sp. 5:396 Amphotericin B 2:421,422,446;4:513-520;6:261-306; 10:150;17:245 Amphoteronolide 10:153,166 Amphoteronolide B 4:573;6:261-276 Amphoteronolide B methyl ester 6:264,265 Amsonia elliptica 1:125 Amurensosides A-D from Asterias amurensis 7:298 t-Amyl alcohol 12:156 a-Amylase 7:32-35,10:242,243,504-507,514,13:195; 16:86 P-Amylase 7:33,58,59,10:497;16:86 Amylase 6:551,7:32-36,39 a-Amylase inhibitor acarbose as 13:189 Amylases 10:496-498 Amylases 8:343 Amyloglucosidase 7:13,10:567,357,14:150 Amylopectin 7:6,10:496-498 in starch 7:6 Amylose 5:288,291,292,7:6,10:496,497 Amylostatins 7:20,10:509,510-513 as amylase inhibitor 10:509 from Streptomyces diastaticus subsp. amylostaticus 10:509 synthesis of 10:510,511 synthesis of 13:235-246 Amylostatins GXG inhibitory activity of 10:509 Amylostatins GXGG inhibitory activity of 10:509 Amylostatins GXGGG inhibitory activity of 10:509 Amylostatins XG inhibitory activity of 10:509 synthesis of 10:507-511 Amylostatins XGG inhibitory activity of 10:509 Amylostatins XGGG inhibitory activity of 10:509 Amylosucrase from Neisseria per lava 7:69
939 a-Amyrin from Salvia glutinosa 20:707 from Salvia limbata 20:702 from Salvia montbretii 20:704 from Salvia nemorosa 20:702 from Salviapomifera 20:702 P-Amyrin 7:131-145 triterpenes from 7:131-145 a-Amyrin acetate from Salvia glutinosa 20:707 P-Amyrin linoleate 9:461 P-Amyrin palmitate 9:461 Anabsin 7:239 Anabsinthin 7:239 Anacardiacea 5:824,825,826,7:427,9:315,316,318, 321,323,328 (15:0)-Anacardic acid (2-hydroxy-6-pentadecyl benzoic acid) 9:314,316,345,346,356 Anacardic acids 5:824-831;9:313,315,316,328,330, 331,333-341,346,349,355,358,361-368,370,371 Anacardic aldehyde 9:341 Anacardium giganteum 9:316 Anacardium occidentale 7:427,17:645,34,9:75,316, 318,323,329,9:332,9:335,337,338,340,347 Anacardium semecarpus 9:318 Anacyckus pyrethrum pellitorine from 10:162 Anaerobiosis 9:565,572 Anagasta kueniella 9:322 Anagigantic acid (2-hydroxy-6-undecyl benzoic acid) 9:316 (-)-Anagyrine 15:520 Analgesic 17:633 Anaphylactic bronchoconstiction 12:398 Anas platyrhynchos hemoglobin components of 5:836 Anastrepha suspensa suspensolide from 8:222 Anatoxin 18:697,698 (+)-Anatoxin-A synthesis of 13:493,494 Anchusa officinalis 17:126 Ancistrobrevine B from Ancistrocladus abbreviatus 20:447 Ancistrocerus antilope 5:223,232,253 Ancistrocerus campestris 5:224,252 Ancistrocerus sp. 5:251 Ancistrocladaceae 20:407 Ancistrocladidine 20:408,433,434,437 Ancistrocladine 20,408,419,420,422,423,431 (-)-Ancistrocladine synthesis of 16:447 Ancistrocladus abbreviatus ancistrobrevin B from 20:447 Ancistrocladus alkaloids 20:408,437 Ancistrocladus korupensis 20:442,447 korupensamines A and B 20:442 korupensamines C 20:447 michellamines from 20:442 Ancistrocline 20:408,424,425 Ancistrocongine 20:276 Andrena haemorrhoa 19:129,131-132
Ancymidol as cytochrome P-450 inhibitor 4:621 Andalucin ^om Artemisia lanata 7:214 Anderson-Shapiro reagent 4:652 Andrena ocreata 19:129 Andrena ovatula 19:129 Andrena wilkella 19:129 Andromedotoxin 20:17 Andrena wilkella mandibular gland secretion 1:692 Androgens semi-synthesis of 17:626-627 Andrographis paniculata 5:61^,7:117,17:472 Androsace saxifragifolia saxifragifolins A and B from 15:200 Androst-1,4-diene-3,17-dione 9:411 Androst-4-ene-3,17-dione 9:15-17 Androstanolone 18:885 6,(5a)-Androstene-3,17-dione 8:188 6,(5p)-Androstene-3,17-dione 8:188 Androstenedione from p-sitosterol 9:411 17a-hydroxyprogesteronefrom 9:415 Anemia mexicana antheridiogens from 6:195,202,208 Anemia phyllitidis antheridiogen from 6:194,202 Anethol 13:334-337 trans-AnetholQ 5:473,13:332,15:29 Anethum graveolens 7:108,109 Angasiol acetate 17:6 Angelic acid HETCOR spectrum of 5:778 O-Angeloylperezone '^C-NMR studies on 5:778 transformation of 5:794,795 8a-Angeloyloxy-costunolide 20:471 Angiosperms 9:265 Angiotensin! 9:391,10:629,646,652 12,13-ep/-Anguidine 6:234,236 Anguistidine synthesis of 3:405 Angular triquinanes 13:3-25 Angular triquinane synthesis of 3:14 Angusticraline 13:394 Angustiicine 1:36 Angustine 3:405,5:85,126 Angustine-type alkaloids angustine 5:85 Angustoline synthesis of 3:405 (-)-Anhalonine 2:163 14,15- Anhydro-1,2-dihydrocapuronidine 5:125 l,6-Anhydro-2,4-di-0-benzyl-3-0-(tertbutyl-dimethyl silyl)-P-D- glucose 14:255 l,6-Anhydro-2-azido-4-0-benzyl-2-deoxy-p-Z)glucopyranoside 0-alkylation of 6:388
940 3,6-Anhydro-2-deoxy-D-/v:co-hexose fromgalactal 7:59 1,6-Anhydro-2-0-benzoyl-3,4-di-(9-benzyl-P-Dgalactopyranose 14:256 2,6-Anhydro-2-thio sugar 12:53,54 1,6-Anhydro-3,4-dideoxy-a-D-glycero-hex-3 -enopyranose-2-ulose (levoglucose none) 14:267-281 1,6-Anhydro-4'-0-(3,4-anhydro-a-Dgalactopyranosyl)P-maltose 10:515 2,3-Anhydro-4,6-0-benzylidene-p-£>-talopyranoside 14:167 1,5-Anhydro-4,6-0-benzylidene-D-ribo-hex-1 -enitol Claisen rearrangement of 10:420 1,6-Anhydro-4-deoxy-L-ribopyranose 14:255 3,4-Anhydro-a-D-altropyranoside 14:170 2,3-Anhydro-a//o-hexopyranoside 14:149 1,6-Anhydro-P,Z)-glucopyranose 6:287,288 3,4-Anhydro-p-D-galactopyranoside 14:170 1,6-Anhydro-p-D-glucose 10:425 1,5-Anhydro-galactitol 7:65 1,6-Anhydro-galactose 7:65 1,5-Anhydro-Z-iditol peracetate preparation of 6:372 Anhydroalstonatine 1:125,126 Anhydroamarouciaxanthin B 6:151,152 Anhydroascorbic acid 4:718 Anhydroaustricin 7:236 Anhydrobartogenic acid from Barringtonia speciosa 7:132 14,15- Anhydrocapuronidine 5:125 2',3'-Anhydrocyclitol glycoside 14:147 Anhydrodeacyltautomycin 18:271,284 Anhydrodiatoxanthin from Euglene viridis 6:150 Anhydrodimerisation 7:136 Anhydrodimers 7:136,137 Anhydroerythromycin A 13:158 Anhydrogrossmisin 7:236 1,2-Anhydrohexopyranose derivative 14:147 Anhydromacrosalhine-methine 13:396,397 r,6'-Anhydromaltose 10:507,510,511 1 ",6"-Anhydromaltotriose 10:507 1,6-Anhydromuramic acid derivative prepration of 6:388 E- Anhydropodorhizol 5:486 Anhydropodorhizol (nemorosin) 5:484-488 1,4-Anhydrosorbitol 12:29,30 16,17-Anhydrotacamine 5:126,9:179 Anhydrotaxininol 12:216 15 (28)- Anhydrothyrisiferyl diacetate 5:363 16' -e/7/-A' ^ '^^-Anhydro vinblastine from catharanthine 14:854,855 Anhydrovinblastine anhydrovincristine from 14:818,819 biosynthesis of 14:820,821 Catharine from 14:812,871,820,821 catharinine from 14:820,821,871 (15'5)-20'-deoxy-15'-hydroxy leurosidine from 14:814,815 deoxyleurosidine from 14:871 20'-deoxyvinblastine from 14:871
from catharanthine 14:820,821,869-872 (15'/?)-15-hydroxycatharinine from 14:813,814, 14:820,821 leurosidine from 14:871 leurosine from 14:811,820,821,871,872 oxidation of 14:813,814 3-oxoanhydrovinblastine from 14:813,814 via Polonovski reaction 14:869-872 vinblastine from 14:820,821,871 3'4'-Anhydrovinblastine 2:370372,386-389,392,401 Anhydrovinblastine synthesis of 5:184 16'-e/7/-Anhydrovinblastine synthesis of 5:184,185 Anhydrovinblastine Nb-oxide 15'a-hydroxyleurosidine from 14:812,813 15'-hydroxy-3'-oxoleurosidine from 14:812,813 5'-nor and 5',6'-seco derivatives of 14:872 3'-oxoanhydrovinblastime from 14:812,813 Polonovski reaction of 14:872 Anhydrovincristine from anhydrovinblastine 14:818,819 Anhydrovobasindiol 5:123,9:171,15:469 19 (Z)-Anhydrovobasinediol 15:466,467,491 Aniba sp. neolignans from 8:159 Aniba canellila 19:117 Aniba neolignans total synthesis of 8:159-163 Anigozanthos 17:372 Anilido as protecting group 4:286 8-Anilino-l-naphthalenesulphonic acid 9:453 Anils phenanthridines from 4:542 Anion exchange for oligonucleotide purification 4:283 in HPLC 282, 283 Anion mediated alkylation of difimctional acyclic terpenoids 8:229 [3,3]Anionic oxy-Claisen 12:93 Anionic oxy-Cope rearrangement eight-membered rings by 3:77,78 Anionic pinacol rearrangement 14:360 Anisole Firedel-Crafts alkylation of 6:61,62 Anisomelic acid synthesis of 10:13-17 Anisomycin 14:568 /)/-/?-Anisylmethylamine 12:152 Ankinomycin 11:136 Annelated benzazecines from isoquinoline derivatives 6:483 synthesis of 6:483 Annelation ofresorcinol 19:227 of substituted phenol 19:227 regiospecific 19:227 Annelation reaction 14:694,695 Annona bullata 9:396,397;18:221 Annona densicoma annonacinfrom 9:395
941 Annona muricata 17:277;18:213 Annona squamosa 9:398 Annonaceae 9:391,393,395,396,398,399,402;17:251; 19:498 Annonaceous acetogenins 17:251-286 Annonacin 9:395,396;17:266,272,279 AnnonacinA 17:267 Annonacin-10-one 17:272 AnnoninI 17:252 Annotinine 18:341 /ra«5-Annularene cyclisation in (-)-zonarene synthesis 6:15 2p,3P-Annulated oxathiaphospholane 8:126-127 13,16-Annulated oxathiaphospholane 8:126,127 Annulation by Simmons-Smith reaction 6:5 intramolecular 10:407 of 1 methyl-2-tetralone 14:670,671 of a-diazo-P-keto ester 10:407 of cyclohexane ring system 6:5,6,29,30 of cyclopentane ring system 6:6-8,30,31 ofenolate 10:414,415 of methylenecyclohexane 6:21,22,53,54 of piperidone derivative 14:734 Piers annulation 6:21,22 Rh (Il)-mediated 10:407 Robinson annulation 6:17-21,29,30 with 3-methylsilyl-3-butene-2-one 10:414,415 [2+3]Annulation of aldehydes 3:5-58 triquinane synthesis by 3:47,48 annulation methods 3:7 for five-membered rings 3:7 [4+1] Annulation triquinane synthesis by 3:40-45 [4+4]-Annulation Danheiser version 3:78 for 8-membered ring synthesis 3:78-79 intermolecular 3:78 Nickel catalysed intramolecular 3:78 Anochertus sedilloti 5:223-225,238,254 Anodic methoxylation 13:485,494 Anodic oxidation 7:161,163;8:159-172;13:474,475,479 of a-iV-acetyl-E-7V-tosy-L-lysine methyl ester 12:309,310 Anomalous metabolism 7:117 Anomalous ORD 2:160 P,p-Anomer synthesis of 8:326 Anomeric characterisation of disaccharide dipeptides 6:413-416 Anomeric deprotection of disaccharide dipeptides 6:413-416 Anomeric effect 1:448,16:9,11,498 Anomeric oxidation 13:625 a-D-Anomers from 2,3,5-tri-O-benzyl-P-ribosyl fluoride 10:369 stereoselectivity of 10:369 Anopheles 2:11745, 2:295-297 "Ansa-Chain" compounds rifamycinS 12:37 rifamycinW 12:39
Ansachain 5:592 Ansamitocin 9:435 (P-3)-Ansamitocin 9:433,434 Ansamycin 2:424,9:431-445,10:153 Ansamycin lactams 10:149,150 Ansatrienin (mycotrienin) derived from shikimic acid 11:190 from Streptomyces collinus 11:189 from Streptomyces rhishiriensis 11:189 Ansatrienin A 9:434 Ant repellant neoxanthinas 6:142 Antabine biosynthesis of 11:205-207 by nicotine synthase 11:205-207 from Nicotiana sp. 11:205-207 from nicotinic acid 11:205-207 aaAntagonist 8:399,400 Antagonistic activity 20:515 Anthelmintic activity 1:408,5:552 Anthelmintic agents 12:3 Anthelmintics 1:435 Anthelmycin conformation of 4:226 Anthelvencins 5:552,553 Anthemidin 7:233 Anthemis saguramica 10:153,162 Antheridic acid from gibberellin A7 6:197-201 total synthesis of 6:195-201 Antheridiogens biosynthesis of 6:206 from Anemia mexicana 6:195,202 from gibberellms 6:171,194-209 from Lygodiumjaponicum 6:194,204-206 synthesis of 6:194-212 Antheridium-mducing factor 19:303 Anthocerotae (homworts) 2:277 Anthocidaris crassispina ganglioside GM5 from 18:486 Anthocyanidins 7:112,114,723 Anthocyanins 5:646,647,658,7:192,723,731,735 Anthonomus grandis 1:693,7:187,190 Anthopleura elegantissima 2:306,9:493-495 Anthracenes 11:113,277 Anthracenediols 11:125,126 1,99,9'-bianthracene-10,10' (9H,9'H)-diones from 11:125,126 Anthracnose fimgi 9:228,230,240 Anthracycline synthesis of 14:271 Anthracycline antibiotics antitumor activity of 14:492 asymmetric synthesis of 14:492,493 synthesis of 1:498-514 Anthracyclinone intermediates synthesis of 1:627-629 Anthracyclinones biological activity 4:318 cardiotoxicity 4:318 resolution of 4:318
942 synthesis of 3:448,449 total synthesis 4:317-336 Anthranilate synthase 11:187,188 Anthranilic acid by anthranilate synthase 11:187,188 from chorismic acid 11:187,188 Anthraquinone 9:400,403,11:124 Anthraquinone glutarates 6,11 -naphthacenequinones from 11:122,123 with acetoacetate dianion 11:122,123 Anthraquinone mitoxanthrone 14:21 Anthraquinones 3:433,434;7:418-420,427;13:662,663 Anthraquinone sulphonates 20:867 Anthraquinone-2,6-disulphonate 20:857 Anthraquinones 20:277 Anthraserpine 9:174 Anthricin from /i nthricus sylvestris 18:555 Anthricus sylvestins anthricin from 18:555 Anthrobacter citreus 20:234 Anthrobacter simplex 20:234 Anthrobacter ureafaciens 16:87 Anthrones decarboxylation of 11:121 Anthrotaxis s^^. 3:456 1-Anthroylnitrile fluorescent ester from 5:816 ^«//addition 8:298 Anti-AIDS agent (+)-castanospermine \\'261 Anti-Bcnzo [a] pyrene diol epoxide DNA reaction with 7:9 Anti-Bredt compound 13:447 Anti-Cram product 16:337,473 Anti-digoxin antibodies 15:374 Anti-feedant activity 2:277 Anti-fertility action ofplumbagin 2:229-231 of Plumbago zeylanica 2:229,231 of isoshinanolone 2:229-231 Anti-hepatitis B agents 20:527 Anti-HIV-activity 19:511,748 Anti-HIV-assay 20:535,536 Anti-HIV-screening 13:665 Anti-i-antibody 10:480 Anti-inflammatory activity 13:647 Anti-inflammatory agent 7:397,14:315 Anti-inflammatory agents Anti-leukemic activity 1:305 Anti-malarial agents 20:516-521 Anti-Markovinkov product hydride reduction of 16:337 Anti-microbial activity of sesquiterpenes 2:287,288 y4«//-orientation 14:753 .4«^/-products formation of(5)-ethyl-3-hydroxybutyrate 4:439 Anti-stress activity 20:646 Antibacterial activity 20:245,712 cis Anti-trans-anti stereochemistry of steroid 8:188 Anti-tubercular activity 5:752,753
Anti-tubercular agents 5:751 Anti-tumor activity 1:275,316-320-408 Anti-tumor agent 1:180,268,269 Anti-tumor antibiotics synthesis of 1:498-721 Anti-tumor metabolites 1:239 Anti-venom alkaloid oxidation 1:239 Anti-vitamin activity of 25-aza-vitamin D3 9:519 Antiallergic activity 18:674 Antiallergic effect ofK-252a 12:398 of staurosporine 12:398 Antibacterial activity of 3',4'-dideoxykanamycin 14:145 ofacteoside 5:512 of p-hydroxyacteoside 5:512 of benz [a] anthracene antibiotics 11:134 ofcelastrol 5:747 offorsythiaside 5:512 ofphenylpropanoids 5:512 ofsaframycins 10:78 of staurosporine 12:398 ofsuspensaside 5:512 oftetrazomine 10:117 ofthienamycin 12:147 Antibacterial agent aplasmomycin 5:377 aurantinins 5:601 chimeramycin 5:613 Antibacterial macrolide 5:613,747 Antibiotic activity of2-isocephems 12:192 of 2-iso-oxacephems 12:192 ofsemivioxanthin 11:130 ofthienamycin 12:188 Antibiotic activity of macrolide 17:283 Antibiotic potency of imipenem 4:432 of 1-P-methylthienamycin analogue 4:432 Antibiotic T-1384 5:549 Antibiotics ansamycin 9:431-445 bio-organic chemistry of 9:431-445 biosynthesis of 11:207-213 from microorganisms 5:589 isoquinolinequinones 10:77-145 mitomycin 9:431-445 relative configuration of 6:261 shikimic acid derived 11:182-191 sugar components of 11:213-222 Antibiotics A 26771 B by Penicillium turbatum 11:194 Antibodies 2:345-348,7:29 Anticancer activity DNA-damaging natural products with 20:457-500 ofvinblastine 14:805 of vincristine 14:805 Anticancer agents 19:511 Anticancer compounds 13:347-382
943 Anticandidal activity 20:485 Anticaries activity 18:673 Anticholinergic agents 17:395 Anticoccidium agent 4:591 Antidiabetic agents 7:47 Antidote for scorpion sting 5:751 for snake bite 5:751 Antidysenteric agent 5:758 Antiedema activity of staurosporine 12:384 Antifeedant 17:153,234,237 Antifeedant activity of(-)-specionin 10:425 Antifeedant assays 18:771 -774 Antifeedants 7:395-397 Antifertility activity 5:754 Antifouling 17:93,98 Antifungal agents 17:239 activity of 17:378 macrolide 17:16 sesquiterpene dialdehydes 17:233 Antifungal activity of alga 5:342 ofcoelenterate 5:342,368 ofcordigol 7:408 ofcordigone 7:408 offlavolol 3-methyl ether 7:414 offlavonoids 7:413 of garcigerrin 7:423 ofhydroquinoneuncinatone 7:408 of mollusk 5:342 ofnanaomycins 11:127 ofnaphthoxirene 7:423 ofpterocarpans 7:414 of semi vioxanthin 11:130 of sesquiterpenes 2:287 of sponge 5:342,368 of tunicate 5:342 ofurdamycin 11:134 ofxanthones 7:410 Antifungal agents 4:513 Antifungal antibiotic cerulenin 5:613 irumamycin 5:597 phhoamycin 5:607 Antifungal compounds 7:406,408,415 Antifungal flavones 7:413 O-Antigen from Salmonella anatum 6:262 synthesis of 6:262 I-Antigenic determinant 10:483 O-Antigenic polysaccharide from Pseudomonas aeruginosa 14:236 from Salmonella newington 14:233 from Shigella flexnert 14:233 Antigenic specificity 4:197 I Antigens 10:457-461 II Antigens 10:457,461 Antihormones 20:515 Antihepatotoxic activity 17:377 Antiherpes activity 5:565
Antihyperglycemic activity 18:672;19:351 Antihypertensive activity of Forsythia suspensa 5:513 ofK-252a 12:398 of staurosporine 12:384 of suspensaside 5:513 Antihypoxic activity 18:373 Antiinflammatory activity 5:367;17:137,376;18:775 Antiinflammatory agent 5:753 Antiinflammatory drug 17:130 Antiinflammatory effects ofK-252a 12:398 of staurosporine 12:398 Antileukemic activity 4:232;7:382-387,390;17:348 Antimalarial activity ofacetylvismioneD 7:424 ofartemisinin 7:424 of 3-geranyloxy-6-methyl-1,8-dihydroxy anthraquinone 7:424 of 3-geranyloxy-6-methyl-1,8-dihydroxyanthrone 7:424 of quassinoids 7:391-394 of quinine 7:424 ofvismioneD 7:20 Antimalarial agents 8:391 -394,351 Antimetastatic effect of 16:93 Antimicrobial activity against Candida albicans 5:752 against Clostridium difficile 5:601 against Clostridium perfringens 5:601 against Proteus mirabilis 5:752 against Proteus vulgaris 5:752 against Staphylococcus aureus 5:752 of(-)-acetomycm 10:443 of amphibian venoms 15:327-339 ofcryptolepine 5:752 ofdimertriterpenes 18:776-778 ofmedrrhodine A a n d B 5:618 oftriterpenes 18:776-778 Antimicrobial agent 5:753 Antimitotic activity oflignans 5:461 of gigantecin 9:396 Antimycine A antifungal agent 16:694 Antimycoplasmal activity ofnanaomycins 11:127 Antimetastatic agents 19:351 Antineoplastic activity 4:29 Antineoplastic glycoproteins 7:285 Antioxidant 4:495;9:371,372 Antiperiplanar orientation 14:457 Antiphlogistic 17:451 Antipredation 17:93 Antiplasmodial activity 19:587 Antiplasmodial agents 20:525 Antiproliferative activity 7:419,422,245,515;16:93 Antiproliferative effect against HL-60 cells 12:393 against T cells 12:393 BALB/C mouse 3T3 cells 12:393 bovine brain cortex capillary 12:393
944 cis-platin 12:390 endothelial cells 12:393 human breast cancer 2R-75 cells 12:393 human myeloid progenitor cells 12:393 in walker rat carcinoma cell 12:390 L2CB lymphocytes 12:393 mouse isolated microglia 12:393 NIH/3T3 fibroblasts 12:393 of anticancer drug 12:390 ofstaurosporine 12:393 Antipyretic activity 18:775 Antiquorin 9:280 Antirachitic factors 17:623 Antirride 7:465,466 Antirrinoside 7:455,477 Antisense oligonucleotides 257-294 RNA 13:257-294 synthesis of 13:264-281 Antisera 15:368 Antisweet activity of gymnemic acid 18:671,672 Antituberculous activity 20:712 Antitumor 17:17 Antitumor activity of(+)-5-epi-acetomycin 10:447 of(-)-acetomycin 10:444,447 ofacronycine 13:365-382 of benz[a] anthracene antibiotics 11:134 of cyanocycline A 10:103 ofdidemnins 10:254-257 ofdiphyllin 5:462 ofglyfoline 13:375-380 of indolo [2,3-a] carbazole alkaloids 12:390-396 ofK-252a 12:394,395 oflignans 5:461 of naphthocy anidine 10:104 ofnogalamycin 4:335 ofpristimerin 5:747 ofquinocarcin 10:117 ofrebeccamycin 12:394 ofsaframycins 10:78 ofstaurosporine 12:390-394 oftaxol 12:180 oftetrazomine 10:117 ofUCN-01,UCN-02 12:395 ofurdamycin 11:134 ofstaurosporme 5:275 Antitumor agents 3:301,383;9:383,385 Antitumor antibiotic 5:592,593;19:118 Antitumor bioassay 9:383,386,401 Antitumor polyethers 5:377 Antitussive 17:633 Antivu-al activities Herpes simplex virus 17:146 HIV-2 17:146 of(-)-aristeromycm 10:609 of (+)-C-2'-ara-fluoroguanosine 10:609 of 2-amino oxetanocm-A 10:620,621 of3'-azido-3'-deoxythymidine 10:585 of aciclovir (9-[2-hydroxyethoxy)methyl] guanine 10:585
of carbocyclic oxetanocin A and G 10:620,621 ofcarbovir 10:608,609 of D-2'-deoxycarbocyclic guanosine 10:608,609 ofdidenmins 10:253,254 offlavonoids 17:145 of imidazole analogues 5:566 oflignans 5:461 of marine organisms 5:359,364 ofmycalamide A 5:366 of spermidme 5:563 of oleanolic acid derivatives 17:135 of oxetanocin A 10:608,619,620 of oxetanocin G 10:619,620 of quinovic acid derivatives 17:134 of Theonella sp. 5:364 Antiviral agents 19:351,511 Antiviral protein 13:655 Ants chemical defense in 6:421-465 pipeidine alkaloids in 6:422-434 pyrrolidine venom alkaloids in 6:434-444 Apaenogaster rudis 5:254,255 Apergillus oryzae 18:807 monohexosylceramides from 18:807 Aphaenogaster longiceps 5:252 Aphelasterias japonica 15:69 Aphelasteroside A and B 15:68 Aphidicolin 14:583 Aphomia melleus 15:387 Aphomiaoniki 15:383 Aphomia sociella 15:384 Apidae 6:421 Apigenin 5:654,712;7:206,226;9:390 from Salvia mentbretii 20:712 from Salvia nemorosa 20:712 from Salvia sclarea 20:712 from Salvia yosgadensis 20:712 Apigenin-4'-methyl ether from Salvia yosgadensis 20:712 Apigenin-6,4'-dimethyl ether from Salvia yosgadensis 20:712 Apigenin-7,4'-dimethyl ether from Salvia yosgadensis 20:712 Apigenin-7-methyl ether from Salvia yosgadensis 20:712 Apigravin from 8-methoxyumbelliferone 4:378,381 D-Apiofuranosyl 15:7 Apioglycyrrhizin 15:26 Apiosyl glucosides 5:643 Apium graveolens Aplasmomycin biosynthesis of 11:207-209 by phosphoenol pyruvate 11:207-209 from glycerol 11:207-209 hypothetical mechanism of 11:209 ionophoric antibiotic 14:499 from Streptomyces griseus 19:587 Aplasmomycin B 19:587 Aplasmomycin C 19:587 ApliditesA-G 19:617 Aplidium californicum 10:248;18:716 Aplidium cavernosa 10:248
945 Aplidium constellatum 10:248 Aplidium sp. 5:440,617;10:244,248 Aplinia species 17:358 Aplydilactone 17:6 Aplykurodin A 17:5 Aplyronines A-C cytotoxic activities of 19:609 Aplysia brasiliana 6:6;9:249;18:625 Aplysia dactylomela 5:368;6:35;17:7,8,430 Aplysia faciata 17:6 Aplysia Juliana 17:6 Aplysia kurodai 6:24,56;17:4,6 Aplysia punctata 17:9 Aplysia sp. 5:368 Aplysiapyranoid A-D 17:5 Aplysiatoxins 18:294,549 Aplysilla glacialis 17:14,15 Aplysilla rosea 6:107 Aplysilla sulphurea 17:11 12-ep/-Aplysillin 17:12 Aplysillin 6:107,109 (+)Aplysin-20 synthesis of 1:672 (-)-Aplysin-20 from Aplysia kurodai 6:24 from Laurencia species 6:24 synthesis of 6:24,25 Aplysina fistularis 17:103 Aplysinopsine cytotoxic activity of 5:410 (-)-Aplysistatin antineoplastic agent 16:705 synthesis of 16:705 Aplysulphurin 9:7,8 P-Apo-8'-carotenal 20:752-750,760 Apo-8'-lycopenal 20:592,593 12'-Apo-(3-caroten-12'-al 20:606 8'-Apo-P-caroten-8-al 20:605 8'-Apo-P-caroten-8-oate 20:605 Apo-P-carotenoids synthesis of 20:605-607 Apo-euphanes (apo-tirucallanes) 9:297,307 Apo-tirucallanes (apo-euphanes) 9:297,307 Apoaromadendrone 14:362 Apocarotenal 20:582 P-Cso-Apocarotenal 20:275 Apocynaceae 6:506,520,416,383,748 Apodinine 5:127 Apoenzyme 9:593,595;17:487 P-Apolignans synthesis of 17:339 Aporphine alkaloids synthesis of 16:503-546 Aporphine alkaloids ring expansion in 6:469 Aporphinoids 3:437-444 1 l-Ep/'-Apotrichothecence from Fusarium sporotrichiodes 13:522 Apotrichothecene rearrangement 6:237,238 Apotrichothecenes 6:213,244,245,249,250;13:519,520 (+)-Apoverbenone 19:189
Apovincamine synthesis of 14:635,636 (±)-Apovincamine synthesis of 18:331 (+)-Apovincamine 5:127 Apparicine 5:87,88,125;6:503-506,520,521;9:171,173 Apparicme-derived alkaloids 6:503-506,520 Apparicine-type alkaloids 5:87,88 Apsena pubescens 15:384 APT spectrum : of artemisinin 5:26 ofberberine 5:42,43 of 10-demethoxykopsidasinine 5:52 ofeupatorenone 5:29 ofhortensisn 5:15 of 10-hydroxy-11 -methoxydracaenone 5:19 oflarreantin 5:11 ofnirurine 5:51 ofprionitin 5:31 ofquassinoids 5:39 ofsalvinolone 5:34 of sanguinarine 5:45 Apuleia leiocarpa 5:679,680 Apuleidm 5:679 Apulein 5:680 Apuleisin 5:680 Aquafoliacese 9:402 Aquillochin 5:5,6,8 Aquillochin diacetate APT spectrum of 5:7 ^^C-NMR spectrum of 5:9 ' H - N M R spectrum of 5:9 selective INEPT spectrum of 5:9 Arabinose conversion to nogalamycinone 4:355,356 Arabidopsis thaliana 18:721 I-Arabinitol synthesis of 4:506,507 D-Arabinitol phosphorus analogs of 6:355 D-Arabino-2-hexulopyranose 15:190 D-Arabino-hexofiiranose Ce-P bond analogs of 6:376 Z)-Arabinoftiranose phosphorus analogs of 6:352,335 Arabinofuranose nucleoside from mannofuranose 10:388 Arabinogalactin 15:36 Arabinoglucoronopyranosides of oleanolicacid 7:435 L-Arabinopyranosyl 15:7 I-Arabinopyranosyl 3p-acetyl oleanolate acetyl oleanolic acid from 7:155 methyl I-arabinopyranose from 7:155 L-Arabinopyranosyl unit 15:21 Arabinose 7:136,139 I-Arabinose 7:144,181,294,298;11:7,8 P-D-Arabinose derivatives from 1,2,5,6-di-O-isopropylidene-P-Daltrohexofiiranose 10:431-433 Johnson-Claisen rearrangement of 10:432,433
946 Arabinoside gaudichaudioside A from Baccharis gaudichaudiana 15:21 Arabofuranosides 5:657 Araboglycyrrhizin 15:26 Arabopyranosides 5:657 Arabsin 7:231 Aracaceae 9:321 Arachidonamide 9:582 Arachidonic acid metabolism 20:514 Arachidonic acid 9:368,559,561,564,565,568,569,572, 575,582;13:304 from rat alveolar macrophages 12:394 Arachidonic acid methyl ester CI(NO) mass spectrum of 2:12 Arachniodes exilis 15:33 Arachniodes sporadosora 15:33 Araliaceae 7:427,435 Araliopsis tabouensis 2:121 Aranciamycinones by asymmetric epoxidation 4:345 Araneosol 7:226 Araucaria cookii 17:36 Araucaria cunninghamii 17:36 Arbacia lixula 15:104 Arbaciapunctulata 7:303 Arbiglobin 7:234 (+)-Arborescin synthesis of 14:365,366 (+)-l,10-e/7/-Arborescin 14:365,366 Arborescin derivatives 7:234 Arbusculin A-D 7:231,232 ArbusculinE 7:234 Arbusculone from Artemisia arbuscula 7:208 Arbuzov reaction 12:341;14:126;18:236 Arcanose 4:255 Arcapillin 7:226 Archaster typicus 7:304-306;15:74,84 Arctigenin 5:522 Arctigenin 4'-0-glucoside 5:490,491 Arctigenin monoacetate 18:601 Arctiin 5:475,476,489-491,522 Arctium lappa 5:497 Arcyia Z^w-indolymaleimides hypothetical biogenesis of 12:374 Arcyria denudata arcyriaflavin-B from 12:366,370 arcyriaflavin-C from 12:366,370 arcyriarubin A from 12:366,370 arcyriarubin B from 12:366,372 arcyriarubin C from 12:366,372 arcyriaverdin C from 12:366,372 arcyoxepin A from 12:367,373 arcyroxepin B from 12:367,373 arcyroxindole A from 12:367,372 arcyroxocin B from 12:367,372 dihydroarcyriarubin B from 12:366,370 Arcyria denudata 5:55 Arcyria feruginea arcyriarubin C from 12:366,372
Arcyria natans arcyriacyanin A from 12:366,372 arcyriaflavin-A from 12:366,370 arcyrioxocin A from 12:367,472 dihydroarcyriacyanin A from 12:367,372 Arcyria sp. arcyrinAfrom 12:367,373 arcyrinBfrom 12:367,373 Arcyriacyanin A from Arcyria natans 12:366,372 synthesis of 12:382,383 Arcyriaflavin B 5:55,56 ArcyriaflavinC 1:3;5:55,56 Arcyriaflavin-A from Arcyria nutans 12:366,370,372 synthesis of 12:376-379 Arcyriaflavin-B from Arcyria denudata 12:366,370,372 from Metatrichia verparium 12:366,370,372 synthesis of 1:19;12:379 Arcyriaflavin-C from Arcyria denudata 12:366,370,372 from Metatrichia vesparium 12:366,370,372 Arcyriaflavin-D from Dictydiaethalium plumbeum 12:366,370, 12:372 Arcyriarubin A f[om Arcyria denudata 12:366,372 from A^-methylarcyriarubin A 12:375,376 synthesis of 12:375,376 Arcyriarubin B from Arcyria denudata 12:366,372 synthesis of 12:375,376 Arcyriarubin C 1:3 from Arcyria denudata 12:366,372 from Arcyria ferruginea 12:366,372 Arcyriaverdin C from Arcyria denudata 12:366,372 Arcyrin A from Arcyria sp. 12:367,373 Arcyrin arcyrinin model compounds from A^-methylarcyriarubin A 12:383 synthesis of 12:383 Arcyrinin B from Arcyria sp. 12:367,373 Arcyrioxocin A from Arcyria denudata 12:367,372 from Arcyria nutans 12:367,372 Arcyroxepin A 1:3 ArcyroxepmA 12:367,373 from Arcyria denudata 12:367,373 Arcyroxepm B from Arcyria denudata 12:367,373 Arcyroxindole A from Arcyria denudata 12:367,373 Arcyroxocin B from Arcyria denudata 12:367,372 Ardeola ibis hemoglobin components of 5:837
947 Ardisiajaponica 13:660;17:117,127 Areca catechu arecoline from 13:660 Arecoline from Areca catechu 13:660 Arelia cordota 20:690 Arenaria kansuensis 18:721 Arenarol 5:458;15:315 Arenarone cytotoxic activity of 5:438 Arenecarbene 3:324,325 Arene-metal 7c-complexes 20:310-312 Arene-olefin metaphotocycloaddition 3:14 Arenes microbial oxidation of 18:430-432 Arenesulphonyl-5-(pyridine-2-yl)-tetrazolides 13:275 (±)-Argemonine from methanodibenzazocine 6:471 synthesis of 6:471 Argentation chromatography 9:450,460,464,465 Argentation TLC 9:333,338,343 Argentilactone 19:463,494 Argentiolide A 7:211,231 Argentiolide B 7:230 Argiotoxin-638 655 Arglanin 7:232,234 Argogorytesfargei 5:225,250,253 Argogorytes mystaceus 5:225,231,252,253 Arguticin 9:66 Arguticinin 5:202,203 ;9:66 Arguticinin Argutin 9:66 Argutinin 5:203 Argutinin 5:203 ;9:66 Argylioside 7:443 Aricine 9:174 Ariensin synthesis of 17:318 Arigoni's enzymatic method 18:171 Aristea ecklonii 2:224 (-)-Aristeromycin antivu-al activity of 10:609 synthesis of 8:148 Aristeromycm 8:140 Aristlane 18:607 (+)-Aristocarbinol from (+)-20-hydroxy-hobartine 11:323 Aristocarbinol 9:172 (+)-AristofiTiticosine absolute configuration of 11:325 from Aristoteliafruticosa 11:324 synthesis of 11:323-325 Aristolarine from (+)-20-hydroxyhobartine 11:323 (+)-Aristolasene from (+)-20-hydroxyhobartine 11:323 Aristolasicol (aristotelin-19-ol) 9:172;11:305,312 Aristolasicolone fromhobartin-19-ol 11:305 Aristolasicone 9:172 Aristolasicone (aristotelin-19-one) '^C-NMRof 11:314 *H-NMR spectra of 11:313
from Aristotelia australasica 11:312 fromhobartin-19-ol 11:305 from serratenone 11:312 preparation of 11:312 retrosynthetic analysis of 11:306,315 synthesis of 11:315-317 X-ray crystal analysis of 11:315 (+)-Aristolochene biosynthesis of 15:250 Aristolochia argentina 19:494 Aristolone 20:464 (-)-Aristomakine cw-hexalin structure of 11:302,303 from (-) aristomakinine 11:301,302 from anti-aristotelin-15-ol 11:301,302 from Aristotelia serrata 11:301 IRandNMRof 11:301,302 nOe-difference experiments of 11:303,304 relative configuration of 11:301-303 (-)-Aristomakinine (-) aristomakine from 11:301,302 from fl«r/-aristotelin-15-ol 11:301,302 from Aristotelia serrata 11:301 IRandNMRof 11:301 relative configuration of 11:301-303 Aristone 11:277 (+)-Aristoserratenine absolute stereochemistry of 11:293 from Aristotelia australasica 11:293 from Aristotelia serrata 11:293 3 -ep/-Aristoserratenine from Aristotelia australasica 11:293 IR mass and ^H-NMR spectra of 11:293 Aristoserratine 9:171 (+)-Aristoserratine (aristotelin-15-one) absolute configuration of 11:296 from Aristotelia serrata 11:296 from peduncularistine (18,19-dehydroaristotelin15-one) 11:296 retrosynthetic analysis of 11:296 synthesis of 11:296-300 X-ray crystallography of 11:296 Aristotelia alkaloid (-)-peduncularine synthesis of 13:491,492 Aristotelia alkaloids biosynthesis of 11:278,279 nomenclature of 11:331 numbering system of 11:331 synthesis of 11:277-334 Aristotelia australasica allo-aristoteline {epi-11 -aristoteline) from 11:317 aristolasicone from 11:312 (+)-aristoserratenine from 11:293 3-e/7/-aristoserratenine from 11:293 Aristoteliafruticosa (+)-aristofruticosine from 11:324 (+)-fruticosonine from 11:278 Aristotelia serrata (-)-aristomakine from 11:301 (-)-aristomakinine from 11:301 (+)-aristoserratenine from 11:293 (+)-aristoserratine from 11:296 (-)-hobartine from 11:278
948 g-^H-Aristoteline 11:311 a«//-Aristotelin-15-ol (-)-aristomakine from 11:301,302 (-)-aristomakinine from 11:301,302 Grob-fragmentation of 11:301,302 oxidation of 11:299,300 with DMSO/acetic anhydride 11:299,300 (+)-Aristoteline alloaristoteline from 11:321,322 biosynthesis of 11:278,279 '^C-NMRof 11:314 from (-)-hobartine 11:292-295 from (+).makomakine 11:292-295 synthesis of 11:280-283 X-ray crystal analysis of 11:278 Aristotelinine fromhobartin-19-ol 11:305 Aristotelone 11:321,322 Aritason 20:13 Arjimic acid glycosides of 7:133,134 from Terminalia alata 7:134 Arjunosides I-IV from Terminalia arjuna 7:133 Arlatin 7:234 Armefolin 7:232 Armexifolin 7:232 Armillaria mellea 5:289,290 Armin 7:232 Army worm {Spodoptera frugiperda) 14:451 Amdt-Eistert reaction 13:79,93 Amdt-Eistert homologation 1:237;13:116 Amicenone 3:6,60 Aromadendrane 14:355;15:227;18:607 (+)-4-ep/-Aromadendrene 14:359,362 Aromatic acetylenes 7:222 Aromatic p-carbolines from Nitraria komarovii 14:762 Aromatic chirality method for OH configuration 2:224 Aromatic chromophore Aromatic chromophores 2:171,172;12:46,47 Aromatic compounds oxidation of 6:509 Aromatic erythrinan alkaloids synthesis of 3:456-479 Aromatic isocyanides synthesis of 12:113,114 Aromatic natural products biomimetic synthesis of 11:113-149 v/a polyketides 11:113-149 Aromatic phosphoramidates 14:286 Aromatization 7:360-363;ll:114-119 Array processor 2:113 Arrow poisons 12:233 Arscotin 7:224 Artabortrine 20:480,481 Artabotrys zeylanicus dioxoaporphines of 20:480 Artabsin 7:234 Artabsinolide A-D 7:234 Artabsinolide B 7:209,210,234
Artanin 7:204,205,224 Artanomalolide 7:239 Artausin 7:234 Arteannuin B from Artemisia annua 7:217 Arteannuinic acid 7:238 Arteanoflavone 7:226 Arteanomalactone 7:230 Artecanin hydrate 7:234 Artedouglasia oxides 9:531-533 Artedouglasia oxides A-D 7:217,218 Artefransin 7:234 Arteglasin A,B 7:234 Artelin 7:225 Artemetin 7:206,226 Artemia salina 9:385,387 Artemidin 7:203,223 Arteminisin '^0-NMR spectrum of 17:591 Artemisia abrotanum 7:205,218,240 coumarin-sesquiterpene ethers in 7:205 Artemisia absinthium 7:215,219,220,240 absinthinfrom 7:215 Artemisia alcohol 9:530 Artemisia annua artemisinin from 13:657;20:518 arteannuin B from 7:217 candinane derivatives from 7:217 Artemisia anomala 7:215,240 aurantimide acetate from 7:220 simiarenol from 7:218 Artemisia aragonensis (A. herb alba) 7:212 Artemisia arbuscula 7:208 arbusculone from 7:208 Artemisia argyi 7:218 simiarenol from 7:218 Artemisia aucheri 7:208,241 Artemisia austriaca 7:241 C-methylflavone m 7:207 Artemisia californica 20:4 Artemisia cantabrica 7:216,242 Artemisia capillaris 7:220,242 Artemisia carvifolia 7:206,242 Artemisia compacta 7:205,243 Artemisia douglasiana 7:203,204,217,218,243 Artemisia dracunculus 7:220,243 Artemisia feddei filifolide A from 7:208 filifolone from 7:208 Artemisiafragrans 7:208,244 Artemisia glutinosa 7:220,244 Artemisia gypsacea 7:211,245 Artemisia herba alba (A. aragonensis) 7:212,217, 7:245 Artemisia herba-alba 17:475 Artemisia hispanica elemanolide from 7:216 2a-hydroxyartemorin from 7:211 tamaulipin A from 7:211 Artemisia inculta 7:217,245 Artemisia iridentata ssp. rothrockii 7:208,252 Artemisiajudaica 7:211,216,217,246
949 Artemisia 536 Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia Artemisia
ketone 7:100,101,202,203,208,221;9:530, klotzchiana 7:214,246 koidzumii 7:218,246 laciflora 7:203,218,246 laciniata 7:215,216,246;9:529,531,533-535 lanata 7:214,246 latifolia 1'2\ 9,247 leucodes 7:215,247 longloba 7:218,247 ludoviciana 7:203,247 maritima 7:217,427;13:660 marschalliana 7:220,248 molinieri 7:218,248 monosperma 7:204,220,248 moorcroftiana 9:529,530,534 pallens 7:217,249 fallens 9:531 palustris 7:207,220,249 pectinata 7:214,216,249 persica 7:217,249 roxburghiana 7:219,250 rupestris 7:218,250 rutifolia 7:215,250 salsolodes 9:529-531,533,534 santolina 7:212,250 santonicum 7:212,250 schmidtiana
eudesm-1 l-en-4-ols from 14:450 neointermedeol from 14:450 Artemisia selengensis 7:203,204,215,251 Artemisia S!^. 7:201-263;9:529-536 Artemisia szowitziana 7:216,251 Artemisia toumefortiana 7:212,252 Artemisia umbelliformis 7:215,253 Artemisia valentina 7:213,218 Artemisia vulgaris 7:120,208,215,218,253 Artemisia xanthochroa 7:215,253 Artemisinin 5:24-27,518;7:238,424 from Artemisia annua 13:657 Artemisitene 7:238 Artemorin 7:209,210,230 Artenolide 7:239 Artesieversin 7:239 Artesin 7:212,213 ll-e/7/-Artesin 7:213 Artesovin from Artemisia szowitziana 7:216 Artevasin 7:230 Arthritis 17:137 Arthrobacter globiformis 9:418 Arthrobacter simplex 9:418,427,428 Arthropod alkaloids from Solenopsis sp. 11:231 Arthropod ectoparasites 12:7 Arthropoda 19:627 Arthropods agents active against 1:435 Artidae family 19:125 Artificial electron mediators 20:821-824 Artocarpus nobilis 2:231 Amgomycin 19:118
Aryl P-C-glycosides by condensation with tribenzyloxy benzene 10:377 by Lewis acid assisted condensation of aryl ether 10:375 Aryl C-glycosides from ribofiiranosyl chloride 10:362,363 a-Aryl enamine substrates 18:327 P-Aryl enamine substrates 18:333 Aryl ethano-bridge 11:295 Aryl ethers ^^O-NMR spectrum of 17:586 N-(Arylidene) benzyl amines 1:343 2-azaallyl anions from 1:343 isomerization of 1:343 Arylhalides 4:541,544,545,547 Aryl maltotrioside 8:343,344 Aryl methyl ketone chiral acetal of 14:473 asymmetric cyanation of 14:473 cw-3-A^-Aryl sufonamido bomeol 4:661,662 4-Aryl-1,2,3,4-tetrahydroisoquinoline derivatives synthesis of 12:451 l-Aryl-2-methyl cyclohexane 8:9 l-Aryl-2-methylcyclopentane 8:9 4-Aryl-2-oxazolidiones from 4-methoxy-2-oxazolidinones 12:428 2-Aryl-3-methyl-4-benzyltetrahydrofiirans 17:331 (3-Aryl-p-methylacrylates 12:462 Ary 1-C-glycos ides by condensation 11:139-142 intramolecular 11:139-142 via polyketides 11:13 9-142 regioselecti vity of 11:142 stereochemistry of 11:142 synthesis of 11:139-142 4a-Aryl-cw-decahydroisoquinolin 6-formate 12:462 Arylcycloalkanes 8:6 4a-Aiyldecahydroisoquinolines diastereoselective synthesis of 12:456-463 4a-Aryldecahydroisoquinolines 18:81 Aryldihydronaphthalenes 17:337 N-Arylethylhydroindoles 3:457 3-Arylisoquinoline alkaloid synthesis of 14:796-799 4a-Arylisoquinoline ring system synthesis of 12:457,458 Arybiaphthalenes 5:493;17:332 2-Arylpropanoic acids synthesis of 6:322,323 4-Aryltetralin-type lignan synthesis of 18:586-588 Aryltetralins 17:341,343 Arynes generation of 3:418-421,422-454 natural products via 3:517-454 nucleophilic additions of 3:418,421-437 pericyclic reactions 3:418-437 structure-reactivity 3:417,418 Asadaninl&II 17:369 5-Asarinin 5:753,754 Asatone 8:168
950 Asbcisicacid 2:164 Ascaridol 20:13 Ascidia nigra 10:241 Ascidiacyclamide 4:91-93;5:419,420;10:242 Ascididemin from Didemnum sp. 10:244,245 from Eudistoma sp. 10:244,245 Asclepiadaceae 5:751;18:649 Asclepias curassavica 5:249 Asclepiasfruticosa 5:248,249 Ascobolusfurfuraceus 5:279 Ascomycetes 9:202,203 Ascomycotina 9:202 I-Ascorbic acid 13:70 Ascorbic acid 2,3-0-alkandiyl derivatives 4:720 aldol reaction 4:699-705 biological significance 4:725 deoxygenation 4:715-718 esterification by 4:723 etherification 4:718-720 Mannich reaction 4:715 metal complexes 4:720-722 Michael reaction 4:705-715 oxidation of 4:723 platinum derivatives 4:720-722 structure of 4:723,734 synthesis of 17:636 tautomeric forms 4:724,725 Asebotoxin 20:17 Ashurbin 7:233 Asiaticoside 15:213 Asimicin triacetate 17:262 Asimicin 9:391,392,396 Asimicin triacetate 9:391 Asimina tiloba 9:391 Asinger condensation 12:127,129 Asltonia macrophylla 13:383,422 Asltonia muelleriana 13:383,399,422 Asocainol 6:478 Asparagine-linked oligosaccharides biosynthesis of 10:499-502 Asparagopsis sandfordiana marine sterols from 9:83-85 Asparagus cochinchinensis 17:116-117,130.132 Asparagus curillus 7:427 Asparagus piumosus 7:427 Asparagus species 17:130 Asparenomycines in carbapenems 4:434 L-Aspartic acid 4-acetoxy-p-lactam from 12:160 I-Aspartic acid 6:263;13:63 Asparticacid 6:298,299 A^-Aspartyl-pseudodisaccharides 13:218 Asperdiol 10:18 activity against lymphocytic leukemia 8:15 growth inhibition of KB, PS and LE Cell lines 8:15 Asperentin 15:382,385-387 Aspergillic acid 13:321 Aspergillosis 2:422,425 Aspergillus acylase 1:678
Aspergillus alleaceus 20:792 Aspergillus awamor i 2:322,341,353 Aspergillus brevipus 12:400 Aspergillus caespitosus 19:480 Aspergillus duricaulis 15:343,345 Aspergillusflavus 2:446;5:296;9:300;15:386,245; 18:711 Aspergillusfumigatus 5:295-298,322,326;7:65;9:300; 12:400;18:469,807,809 Aspergillus melleus 15:383 Aspergillus nidulans 5:295,296 Aspergillus niger a-L-rhamnosidase from 7:70 P-glucosides from 7:51,52,55,56,65-67 cycloisomerase from 8:296 glucoamylase from 7:62 isozyme I from 2:322,341 lactone from 8:300 Aspergillus ochraceus ochratoxins from 15:387 Aspergillus ochraceus wilhelm 15:384 Aspergillus orizae 13:304 Aspergillus oryzae 1:697,117;2:322,341;5:295; 6:551,552 Aspergillus parasiticus averufmby 11:194 Aspergillus terreus 19:168 Aspergillus sp. 5:276,278,294,301,325, 5:326,328,368,370 Aspergillus spp. 2:323;9:203,300 glucoamylase from 2:322 Aspergillus stellatus (+)-asteltoxin from 10:439 Aspergillus terreus 5:296,730;15:385 Aspergillus -versicolor 18:807,809 Aspergillus wentii 7:56,64;9:300 Asperline 15:350,351;19:463 Asperuloside 7:439,455,469,470,479,480;16:298 Asperulosidol 7:467,468 Asphaenogaster rudis 5:226,243-245 Aspiculamycin 4:242 Aspidospermatan type alkaloids 1:31,32 Aspidosperma indole alkaloids 13:70,93 Aspidosperma alkaloids synthesis of 19:89-116 Aspidosperma alkaloids 4:27,275,411 Aspidosperma cyclindocarpon 19:112 Aspidosperma marogravianum 1:124 Aspidosperma oblongum 1:124,125 Aspidosperma type amarylidaceae alkaloids 11:229 Aspidosperma 19:114-115 Aspidospermatan-type alkaloids 5:71,86,87,135 Aspidospermatidine 1:40 Aspidospermatine 1:40 (-)-Aspidospermidine 19:143 Aspidospermidine 13:93 ;14:632-636 Aspidospermidine A^-14,15-methyl-14,15-oxido 5:126 Aspidospermidines 4:57-68 Aspidospermidose 5:165-167 Aspidospermine alkaloid 18:338 Aspochalasans 13:108,131,134
951 Aspochalasin B 13:142,143 Aspochalasin C 13:108,131,134 Aspochalasins 15:355 Aspteric acid 5:729,730,732,733 Astaxanthin 6:153;20:577,578,588,607 (35,35')-Astaxanthm 6:158 (3/?,3/?)-Astaxanthin 7:321,361,362 (35,3'5)-Astaxanthin 7:361,362 Asteltoxin from Aspergillus stellatus 16:670 (+)-Asteltoxin 10:439-442 absolute stereochemistry of 10:439,440 Claisen rearrangement by 10:439-442 from Aspergillus stellatus 10:439 synthesis of 10:439-442 Aster saponin A 15:209 Aster saponin H 15:210 Aster saponin U 15:211 Asteraceae 7:411,413,427 Asteria pectinifera 1:307 Asteriarubin 6:162 from Asterias rubens 6:161 Asterias amurensis 7:286-298,303;15:44 Asterias amurensis vesicolor asterosaponin-1 from 7:287 thomasteroside A from 7:287 versicosides (A-C) from 7:287 Asteriasforbesi 7:288;15:58,69 Asterias rubens 6:151,152,156,161;7:288,294,304,306; 15:48 Asterias vulgaris 7:288,291,304;15:59 Asterinapectinifera 7:289;15:44,48,55 Asterinic acid 6:161 Asterogenol (20/?-5a-pregn-9(l l)-ene-3p,6a,20-triol) 7:288,291 Asteroidea 7:265,285-307;15:43-45 Asterone 15:45 Asterone (3p,6a-dihydroxy- 5a-pregn-9(l l)-en-20one) 7:290,291 (+)-Asterosaponin 7:285,288,290,291,294, 7:303,304 Asterosaponin 1 (forbeside C) 7:287,288,293 Asterosaponin A and B from Asterias amurensis 7:286,303 Asterosaponin D1-D3 biosynthesis of 7:299,302,303 from Distolasterias nipon 7:299 Asterosaponin Pi from Oreaster reticulatus 7:297,300 Asterosaponins 15:45-58 Asterosides A-D 7:287,293 Astichoposide C 7:273,281,282 from Astichopus multifidus 1:273 Astichopus multifidus 7:272,273,281 astichoposide C from 7:273 Astragalus 13:660 Astragalus emoryanus (-)-swainsonine from 12:313 Astragalus lentiginosis 7:11,486 Astragalus lentiginosus (-)-swainsonine from 12:313 Astragalus mister 19:118
Astragalus sp. 7:23 Astringent 5:751,754 Astrocytes by staurosporine 12:393 tumor promotor in 12:393 Astropecten aurantiacus 15:104;15:61 Astropecten indicus 5:214;7:299 Astropecten latespinosus 7:288;15:48 Astropectenpolyacanthus 7:306;18:725 Astropecten scoparilis 15:68 Asukamycin 11:189 Asymmetric alkylation 11:367 catalytic epoxidation 11:431,432 catalytic osmylation 11:431,432 cycloaddition 11:358 dihydroxylation of 11:423,424 of /raAM-stilbene 11:423,424 Asymmetric cyclization 10:611,631-633 Asymmetric [3+2] dipolar cycloaddition 13:500 Asymmetric 1,3-dipolar cycloaddition 1:371-375 Asymmetric additions to prochiral carbonyl groups 4:332 to prochiral naphthalene rings 4:332 Asymmetric aldol condensation 12:162;13:63 Asymmetric alkylation of methylmalonic acid 10:411 of (-)-pheny Imenthy 1 ester 10:411 Asymmetric allylboration 8:477,478 Asymmetric aza-annulation reaction 18:378,379 Asymmetric bromolactonization with acetals from tartaric amides 4:338,339 with N-bromoacetamide 4:338,339 with chiral acetals 4:338,339 with unsaturated (5)-proline amides 4:336,337 Asymmetric carbocyclization in (-)-2/?,65,85,95)-2,8-dibromo-9- hydroxy-achamigrene synthesis 6:62,63 Asymmetric cyclization biomimetic 14:506,507 of chiral acetal 14:506,507 Asymmetric cyclopropanation with methyl carbenoid 14:488 Asymmetric Diels-Alder 8:139-157 chiral auxiliaries 4:607 prostaglandin synthesis 4:607 p-santalene synthesis 4:607 tetramycin synthesis 4:607 using chiral boron reagent 4:609 Asymmetric Diels-Alder technology 13:602 Asymmetric dihyroxylation ll:60;19:269-270,274, 278,284 Asymmetric dimethylation 10:412-415 Asymmetric epoxidation of(±)-N-benzyloxycarbonyl-3-hydroxy-4ofallylic alcohol 10:561 ofallylicalkohols 12:323 of allylic alcohols 4:172-174 pentenylamine 12:281 ofswainsonine 10:561 Asymmetric esterification 9:25
952 Asymmetric hydration 13:71 ofalkenes 13:71 Asymmetric hydroboration 8:473,474,478;13:71,72 Asymmetric hydrogenation 12:162;16:411 Asymmetric hydrogenation BINAP-Ru-catalyzed 4:439 of p-keto esters 4:439 Asymmetric hydrolysis by esterases 1:684,685 by lipases 1:684,685 of N-acetyl a-amino acids 1:678,679 Asymmetric hydrosilylation intramolecular 13:72 Asymmetric hydroxylation ofpentadienols 9:572 Asymmetric induction 10:412;12:153,156;14:471, 507,508,524,553 1,2-Asymmetric induction 11:231 -238 1,3-Asymmetric induction 14:529,552 1,4-Asymmetric induction 12:156 1,5-Asymmetric induction 14:499 1,6-Asymmetric induction 14:530 1,4-Asymmetric induction Cram's cyclic model of 4:203,495 in (-)-piperitone 6:15 in aza-annulation reaction 18:373-386 in dienolate addition 3:47 in diyl trapping 3:20 in Ireland-Claisen rearrangement 3:237 reversion using sulfoxides 4:511 self-immolative 3:237 withchiral auxiliaries 4:499-517 Asymmetric ketoester cyclization 4:328-330 Asymmetric metallation of chiral arylaldehyde acetal chromium tricarbonyl complexes 14:511 Asymmetric Michael addition in estrone synthesis 4:501,502 Asymmetric osmylation 19:269 Asymmetric oxidation of prochiral sulfides 14:517,518 of sulfide 4:489 sulfoxides from 14:517,518 with chiral sulfamyloxaziridines 4:489 with chiral titanium complexes 4:489 Asymmetric quatemization by Claisen rearrangement 10:426-428 by consecutive alkylation 10:405,406 of aldohexofuranose derivatives 10:428-432 ofa-carbonof y-lactones 10:405 Asymmetric reduction with Baker's yeast 1:689 Asymmetric reduction by Baker's yeast 10:410 of acetoacetic acid ethyl ether 10:410 of acetylenic ketone 11:424 of acyclic p-hydroxy ketones 14:183,184 of P-keto esters 14:533,534 withlpczBH 8:476 Asymmetric reductions 4:339-345,448 Asymmetric synthesis by diastereofacial selection 13:62-70
by enantiofacial selection 13:70-73 by enantiotopic discrimination 13:60-62 of(-)-ajmalicine 14:563,564 of(-)-sibirine 14:539-544 of (+)-(5,5)-solenopsin A 6:431,432 of (+)-castanospermine 12:346 of(+)-elaeokanineA 12:351,352 of (+)-elaeokanine C 12:351,352 of(/?)-muscone 14:490 of (/?/5)-l,6-dioxaspiro [4.5] decane 14:523,524 of (/2/5)-l,7-dioxaspiro [5.5]-undecane 14:521-526 of [m, n, 1] propellanes 14:490 of 2-deoxy-D-flra6/>jo-hexose 14:176,177 of3-deoxy-D-r/6o-hexose 14:176 of 4-acetoxy-3 -[ 1 '-(/eA•^buty Idimethy 1-silyloxy) ethyl]-azetidinone 4:448 of 4-deoxy-D-/vjco-hexopyranose 14:176 of 5,1-linked naphthylisoquinoline alkaloids 20:420-438 of 5,8-linked naphthylisoqumoline alkaloids 20:442-451 of 7,1-linked naphthylisoquinoline alkaloids 20:438-441 of anthracycline antibiotic 14:492,493 of azetidinone 4:448 ofbuphanisine 4:14,15 of chiral alkaloids 10:671-689 of chiral building blocks 14:551-581 of chiral isoquinolines 10:671 ofchh-alpiperidines 10:671 ofcrinine 4:14,15 ofdesoxydaunomycinone 14:23 of indolizidine alkaloids 6:442,443 of monomorine I 6:449,450 of pyrrolidine alkaloids 6:442,443 of pyrrolizidine alkaloids 6:442,443 of /?,5'-4-hydroxycyclopentenones 6:315 ofsolamin 18:202-206 of talaromycin A and B 14:531-539 propane-1,3-diols by 13:53-105 via chiral organoboranes 8:465-478 with chiral sulfur reagents 10:671-689 Asymmetric transformation crystallization-induced 13:77 of malonic acid derivative 13:88 Asymmetric catalysts hydrogenation 17:323 synthesis of 17:479 Asymmetrization of a-symmetric ketones 11:241,242 AT2433-A, from Actinomadura melliaura 12:366,368 AT2433-A2 from Actinomadura melliaura 12:366,368 AT2433-Bi from Actinomadura melliaura 12:366,368 AT2433-B2 from Actinomadura melliaura 12:366,368 Atalantia ceylanica l,5-dihydroxy-3-methoxy-10-methylacridin-9-one from 13:348,350
953 1 l-hydroxynoracronycine from 13:348,349, 13:356 11-0-methylatalphilidine from 13:348,349 A^-methylatalphyllinine from 13:348,349 Atalantia monophylla atalphyllidine from 13:348,349 atalphylline from 13:350 atalphyllinine from 13:349 A^-methylatalphylline from 13:350 Atalfoline from Citrus bnxifolia 13:350 Atalphyllidine from Atalantia monophylla 13:348 Atalphylline from Atalantia monophylla 13:350 Atalphyllinine from Atalantia monophylla 13:348,349 A telopus chiriquiensis 18:724 Athanasin from Athanasia L. 16:666 Atherosclerosis 11:335;12:399 Atherospermidine 20:480,481 Atisense RNA gene expression by 13:257-261 natural regulation of 13:257-261 £«^Atis-16-ene-3,14-dione 9:68,269,276,277,279,290 £«/-Atisane diterpenoids 9:267-281 £«/-Atisanes 9:267-283 Atisine synthesis of 3:435 Atisu-ene synthesis of 10:180 Atomic orbital coefficients in Diels-Alder reactions 4:586 ATPase 7:282 Atractylis gummifera 20:8 Atractyloside 20:8 Atropa belladona 20:135 AtropagQnvLS 17:395 Atropisomerization 11:49,50 Atropisomers 11:49,50 2-ATT (2-acetylthiazole) 11:443, 11:444 Atta cepholotes 2,5-dimethyl-3-ethyl-pyrazines of 5:222 Atta sexdens rubropilosa 2,5-dimethyl-3-ethyl-pyrazines of 5:222 Atta sexdens sexdens 2,5-dimethyl-3-ethyl-pyrazines of 5:222 Attatexana 5:234,125 Atta-ur-Rahman synthesis of(±)-16-hydroxydihydrocleavamine 14:850 of (±)-16-methoxycarbonyldihydrocleavamine 14:850 of(±)-coronaridine 14:850 of(±)-dihydrocatharanthine 14:850 of(±)-isovincadifformine 14:850 of(±)-a-dihydrocleavamines 14:850 of(±)-p-dihydrocleavamines 14:850 ofanhydrovinblastine 14:857 ofvinblastine 14:850-859 ofvincristine 14:850-859
Attanuatoside C 7:295,296,300 Attenuatodside from Hacelia attenuata 7:295 Attenuatoside A-1 7:295-297,299,300 Attenuatoside A-II 7:295,296,300 Attenuatoside B-I 7:295,296,300 Attenuatoside B-II 7:295,296,300 Attenuatoside C irom Hacelia attenuata 15:61 Aucubigenin 7:478 Aucubin 7:440,455,465,472,473,476,478,479,480, 481-483 6-e/7/-Aucubin 7:472,473 Auguidine 6:225,226,229 Augustilobine A&B 9:171 Aulacohorafemoralis chinensis 18:771 (+)-Auranthioclavine synthesis of 16:438 Aurantiamide acetate from Artemisia anomala 7:220 Aurantinin 5:601,603 AurantininA 5:601,604-607 Aurantinin B 5:601,603-606 Aurapten from Limonia acidissima 20:497 Aureobasidium pullulans 5:307,308,314 Aureobasidium sp. 5:307 Aureol 5:430,431,437,438,444;9:31,32;15:300,314 Aureolic acid antitumor antibiotics 3:173-206 Aureolic acid, 3:173-175 Aureothin 5:551 Auricularia auricula-judae 5:290,316,318,319,733 Auricularia sp. 5:287,317,320 Aurones 7:192 (-)-Austalide B from Aspergillus ustus 18:32 Austalides A-F 18:32,37 Australine amyloglucosidase inhibitor of 10:567 from Castanospermum australe 10:567 oligosaccharide chain inhibitor 10:567 Austrocedru chilensis 19:405 Australine a-glucosidease inhibition by 7:11 amyloglucosidase inhibition by 7:13 from Castanospermum australe 7:13 glucosidases I,II inhibition by 7:13 Autoimmunodeficiency syndrome 18:908 Automated synthesis of oligonucleotides 13:257-294 ofoligodeoxyribonucleotides 4:280 reaction cycle 4:281 Automatic DNA synthesizer 4:304 Autooxidation of 1-methylazulene 14:335,336 of benzylic positions 16:571 ofguaiazulene 14:316-319 ofphenylhydrazine 9:581 Autoxicity selection by 7:116,117 Auxins effect on root culture growth 17:424
954 indoleacetic acid as 7:90 naphthaleneacetic acid as 7:90 Avarol 5:430,431,438,439;9:30,31 ;15:238,252 Avarol monoacetate 15:300 Avarone 5:430,431,434,438,439;15:301,314 Avenasativa 9:220 Avenaciolide 3:259,260;19:485 Avencins Ai,A2,Bi,B2 7:136,137 Avenestergenins Ai,A2,Bi,B2 7:136,137 Average mass 2:26 Avermectin 12:13 Avermectin absolute configuration of 12:6 biological activity of 12:709 biosynthesis of 12:6,7 from Streptomyces avermitilis 12:3 isolation of 12:3-6 stereochemistry of 12:6 structure elucidation of 12:3-6 structure-activity relationship of 12:8 synthesis of 12:9-33 Avermectin A 12:3 Avermectin Aia Dainshefsky synthesis of 12:12,13,15 fromD-robosa 12:12,13 from Streptymyces avermitilis 12:6 Avermectin B 12:3 Avermectin Bi insecticidal activity of 12:9 Avermectin B la synthesis of 1:463-465 Avermectin Bia aglycon White synthesis of 12:15,27,28 Avermectin Bip, B2a and B23 10:177;12:6 conversion to avermectin Bia 1:442,443 synthesis of 1:443-445,468,469 Avermectins from lithium acetylide 10:386 mechanism of action 1:43 5 structures of 1:438 synthetic approaches to 1:435-495 synthesis of 10:386 Avermectins A2a 12:6 from Streptomyces avermitilis 12:6 Avermectins Bia 12:6,12-15,22-24 from Streptomyces avermitilis 12:6 Hanessian's synthesis of 12:12-15 Ley synthesis of 12:22-24 X-ray analysis of 12:6 Averufm biosynthesis of 11:194 in Aspergillus parasiticus 11:194 Avermectin Avian hemoglobin alkaline denaturation 5:841 allosteric regulator binding sites 5:849 amino acid composition 5:841 carbon dioxide transport 5:853 electrophoretic mobilities 5:839-841 heme binding sites 5:869
heterogeneity in 5:856 inositol pentaphosphates in 5:857 oxygen affinity 5:856 oxygen uptake 5:853 physiology 5:854-859 primary structure 5:844-850 structure of 5:835-850 Avicennia alba 7:176,181,182,189,195 Avicennia germinas 7:175 Avicennia marina 7:176,180,183,184,194,195 Avicennia officinalis 7:176,181,182,189,195 Avicennia resinifera 7:193 Avicennia sp. 7:176 Avicennia tomentosa 7:189 Avicenniaceae 7:175 Avicenol 4:372,373 Avicine 14:770 Avicularia 17:193 Avidin 18:919 Avidinphosphatase assay 4:284 Axamide-1 from Axinella cannabina 6:5 synthesis of 6:5,6 Axenomycin 19:555 Axial 2-lithiotetrahydropyrans 10:380 by reduction with lithium di-/err-butylbiphenylide 10:380 from 2-(phenylthio)-tetrahydropyrans 10:380 Axial C-glycopyranosides 10:365,366,382 Axillarin 7:26 Axillaroside 7:26 Axinella canabina axamide-1 from 6:5 axisonitrile-1 from 6:5 Axisonitrile-1 from Axinella cannabina 6:5 synthesis of 6:5,6 Ayani 7:226,411-413 Aza spu-ocycle 10:215,216 Aza-adamantane skeleton 11:295 Aza-annulation of enamine related substrate 18:315-386 Aza-annulation reaction asymmetric induction in 18:373-386 Aza-Claisen rearrangement 16:467 Aza-Cope reaction 16:481 Aza-Cope rearrangement Mannich directed 1:68,69 Aza-Cope-Mannich strategy 16:435 Aza-Diels-Alder approaches 16:456 25-Aza-vitamin D3 anti-vitamin activity of 9:519 Aza-[2,3]-Wittig rearrangement 19:22,45,50 Aza-Wittig reaction 1:168,169 3-Aza[5] (1,7) naphthalenophanes synthesis of 6:477 U-Azaacronycine 20:792 2-Azaallyl anions by deprotonation of imines 1:344-347 by C-Si of C-Sn bond cleavages 1:344-351,353 from imines 1:344-347 from N-lithioimidazolidmes 1:344,349-351 from N-metalloaziridines 1:344,348,349
955 generation of 1:348,350 geometry of 1:348,350 Azaallylic anions with aryl halides 4:547 7-Azabicyclo[2.2.1] heptane 19:66 endo-1'Az2ibicyc\o[2.2.1 ] heptanol 19:71 Azabicyclo[2.2.1]heptanone 19:71 8-Azabicyclo [3.2.1] octan-3-one asymmetric cleavage of 14:573,574 N-protected 14:573,574 9-Azabicyclo [3.3.1] nonan-3-one 14:571-574 trimethylsilyl enolate 14:572,573 with chiral lithium amide 14:571-574 2-Azabicyclo [3.3.1] nonane 11:280 1-Azabicyclo [4.3.0] nonane (octahydroindolizine) 11:229 Azabicyclo ketone system asymmetric cleavage of 14:571-574 cis-a, a'-disubstituted piperdine and pyrrolidines by 14:571-574 3 - Azacephalosporine 12:137 Azadiene Diels-Alder 3:311 1-Azadiene Diels-Alder cycloadditions 16:457 2-Azadienes metalloenamines from 4:7,10-14,17 Azadirachta indica 9:293-311 ;13:644;15:384 Azadirachtin 7:397;9:93,94,96,103-105,297 Azadirachtol 9:308 Azadirol 9:297,298,300,306,308 Azadirone 9:302,304-306 Azafluoranthenes 1:167,169 Azafulvene 9:596 Azafulvene bilane 9:593,595 2-Azahexatriene thermal cyclization of 3:395 Azalides 13:161,162 Azasteroids 4:540 Azasugars 14:179 Azepine ester 19:99 3-Azetidinol from Chara globularis 18:677 Azetidinone 13:87 Azetidinone 4:437,441,448,450,452-454,457,458, 461-466;8:264 1 -Azetidinone acetic acid 12:138 Azetidione derivatives Mori synthesis of 13:500 Azetidone 5:412-414 Azidation reaction asymmetric 19:311 Azides dipolar cycloaddition 3:49 thermolytic cyclization of 6:429 l-Azido-l,2,3-trideoxy-D-arabino-oct-4-ulose 12:350 8-Azido-1 -p-menthene reduction of 11:288 6-Azido-2,4-di-0-benzyl-3,6-di-deoxy-a-D-ribohexopyranosyl chloride 14:145 2-Azido-2-deoxy derivative ofD-glucose 6:388 of muramic acid 6:387,388,402,403
2-Azido-2-deoxy-Z)-glucose derivative 6:388 3'-Azido-3'-deoxythymidine (AZT) antiviral activity of 10:585 3-Azido-3-deoxy-1,2-O-isopropylidene-a-Dglucofliranose 12:325 3-Azido-3-deoxy-/w>'o-inositol2,4,5-trisphosphate 18:411 4-Azido-4-deoxy-D-mannose 12:316 6-Azido-6-deoxy-D-arabino hexulose 10:536 Azido-6-deoxy-L-xv/o-hexulose 10:536 2-Azido-Z)-arabino-hexofuranoses 5-deoxy-5-(phenylphosphinyl) Z,-fucosamine from 6:369 from Z)-ribo-hexafiironose 6:363 Azido-ene reaction 16:473 Azido-olefin cyclization 13:447,448 2-Azidobenzaldehyde 13:448 Azidobutenolides 13:448 a-Azidocinnamates 1:165 Azidodeoxy-/w>'o-inositol 18:411 Azidonitration 1:420,421;10:465 Aziridine from (5)-phenylglycinol 10:138,139 azomethine ylides from 1:328-331 fused 1:189,197 nucleophilic opening 1:201,202 photochemical opening 1:328 synthesis of 1:189,197 thermal opening 1:328 Aziridinometosene Aziridinyltriol Azithromycin 13:161,162,183 Azobisisobutyronitrile (AIBN) 10:204,208 Azocine synthesis of 8:210 Azodicarbonyldipiperidine 19:225 a-Azohydroperoxide 9:580 Azomethine ylide cycoadditions by aziridine thermolysis 1:329,330 intramolecular 1:329,330 Azomethine ylides by 1,2-H shifts 1:331-332 formation of 1:324-344 generation of 1:324,339 preparation by aziridine opening 1:328-331 preparation by desilylation 1:324-328 preparation from a-amino acids 1:331-338 Azomonas macrocytogenes 19:814 4-Azoniaspiro [3,3] heptane-2,6 diol from Chara globularis 18:677 Azospirillum lipoferum 4:197 Azotobactins 19:812 Azoverdin 19:811 Azulenequinones 14:314 Azulenes autooxidation of 14:332 electrochemical oxidation of 14:325 oxidation of 14:332-334 Azulenic hydrocarbons oxidation of 14:313-354
956 B-prostaglandins synthesis of 16:367 B/C-/ra«5-morphinan diastereoselective synthesis of 12:464-471 Babylonia japonica 11:724 BaccatinI 11:4,9;12:179 Baccatin III 11:3,4,179,180;12:179,180;20:81 Baccharis articulata 15:22 Baccharis gaudichaudiana arabinoside gaudichaudioside A from 15:21 Baccharis timer a I'All Bacillarophyceae 6:13 Bacillus antharacis 12:400 Bacillus aurantinus 5:601 Bacillus cereus 7:413 Bacillus megaterium 10:585;12:104 Bacillus pumilis 7:70,308,390 Bacillus sp.TA 12:104 Bacillus sp. 12:103 Bacillus subtilis 5:302,304,368,418,308,353;8:353; 10:117,104,400,345,692,290,556,559;18:778 Bacillus thiaminolyticus baciphelacin from 15:390 Baciphelacin 15:388,390,409,410 Bacitracin 9:413 Backbone rearrangements 7:159,161 -166 Baconipyrones A-D 17:25 Bacteria siderophores from 9:537-557 Bacterial cell wall 6:385,395,397,406 Bacterial growth inhibitors 17:101 Bacterial polysaccharides I-glycero-D-mannoheptose in 4:195 Bacteriorhodopsin 4:526,720,823 Bacterioruberin 7:355-357;20:599 Bacterium pseudomonas 9:322 Bacteroides fragilis 14:107 Bactobolin antibacterial activity of 16:3 antitumor antibiotic 16:3 from Pseudomonas yoshitomiensis Y-12278 16:3 total synthesis of 16:3-26 (-)-Bactobolin 16:22 Baeyer-Villiger rearrangement 4:647,667,139,153; 8:154,597,598 Baeyer-Villiger ring expansion 13:601 Bai-yun-shen baiyuoside from 1:666 Baiyumine A from Citrus grandis 13:348,349 Baiyumine B f^om Citrus grandis 13:350 (+)-Baiyunol synthesis of 1:666-669 thermal glycosidation of 8:362 Baiyunoside synthesis of 1:666-670 from Phlomis betonicoides 15:20 Bakankoside from Strychnos vacacoua 6:503 Baker's conditions 12:46,47
Baker's yeast 4:324,325,340,341,158,263,542,543, 552,553,410;6:13;10:410;13:58,59,309,307;20:573,59 3,818,820 Baker's yeast reduction 13:662,176 Balacnol 5:744,747-749, 7:149,150,152 Balaenonol 5:744,747-749,149,150,152 Balanitaceae 7:426,427 Balanites aegyptiaca 7:426 Balanophonin from Balanophor a japonica 20:642 Balanophora japonica balanophonin from 20:642 BALB/3T3 staurosporine activity against 12:400 Balchanolide 7:230 Baldrinal 7:458,459 Baldwin's rule 14:792,793 Baleabuxidine 2:205 Baleabuxoxazine-C 2:205 Balsamodendron mukul 5:695 Baluchistamine B 20:270 Bamboo shoots GAigfrom 6:187 Bamicetin 4:241,243-245 Ban condensation 1:135 Banerjee and Carrasco synthesis oftaxodione 14:695-699 Banksia grandis 19:246 Bannucine 5:167-169;9:185,186 BAP (6-benzylaminopurine) ascytokinin 7:90,94 Barbilophzia hatcheri 5:730 Barbilophzia lycopodioides 5:730 Barley endosperm assay 6:181 Barnacle settlement inhibitors 17:103 Barrett's approaches for oxahydrindene subunit 12:9-11 ofavermectins 12:9-11 Barrigenic acid from Barringtonia acutangula 7:132 Barrollier reagent 19:755 Ri-Barrigenol 21,22-di-O-angelate 7:139,141 Barringtogenic acid from Barringtonia racemosa 7:132 Barringtonia acutangula barrigenic acid from 7:132 barringtonic acid from 7:132 barrinic acid from 7:132 tangulic acid from 7:132 Barringtonia racemosa barringtogenic acid from 7:132 Barringtonia sp. triterpenes of 7:132,133 Barringtonia speciosa anhydrobartogenic acid from 7:132 bartogenic acid from 7:132 19-e/7/-bartogenic acid from 7:132 Barringtonic acid from Barringtonia acutangula 7:132 Barrigenic acid from Barringtonia acutangula I'AZl Barrollier reagent 19:755
957 Bartlett demethylation 8:119 Bartlett pear ester 10:152 Bartogenic acid from Barringtonia speciosa 7:132 19-6/7/-Bartogenic acid from Barrintgonia speciosa 7:132 Barton oxidation 9-BBN (9-borabicyclo-[3.3.1] nonane) 13:72 Barton radical decarboxylation 18:75,340 Barton reaction 14:160 Barton reduction 12:303 Barton's modification 19:135 Barton's procedure 6:282-288,607 Barton's protocol in free radical deoxygenation 6:21 Barton's reaction 6:228,230,231 Barton-type deoxygenation 20:71 Base promoted cyclisations ofarylhalides 4:541 Base-catalyzed equilibration 14:460 Base-induced rearrangement 12:91 mechanism of 14:372-374 of hydroazulene mesylates 14:22-25 of hydronaphthalene-1,4-diol monosulfonate esters 14:356 of hydronaphthalene tosylates 14:368-370 Basidiomycetes fruiting body formation 1:680 glycosphingolipids of 18:813,814 Basidiomycetes 9:220,317 Basidomycotina 9:202 Bastadin-6 10:630,636-638 Bastadins 10:630,632,633-638 Bastaxanthin 6:150;10:153 Bastaxanthins C,B,D.E,F 9-BBN 6:20 reaction with p-fiiranone 6:20 Batrachotoxins (steroids) 11:244 Batramiaflavone 20:283 Bavachalcone synthesis of 4:378,379 Bayberry 13:660 Bayogenin molluscicidal activity of 7:432 Bazzanene 9:212,213,308,309,520,521 Bazzanenone 10:308,309 Bazzania tridens tridensone from 18:639 BBE (berberine bridge enzyme) 11:204 9-BBN reduction with 4:116,117 trans-BC'X'mg fusion 12:205 stereochemistry of 12:205 BE-13793C 12:366,370,396 antitumor activity of 12:396 from Streptoverticillium mobaraeens 12:366, 12:370 topoisomerase I and II inhibitors of 12:370 Beaucage sulfiirizating agent 13:269 Beckman fragmentation ofanti-oxime 19:486 of erythromycin -9-oxime 13:160,161
ofoxacyclopentanonering 16:221 ofoxime 16:240,330 ofoxime 19:14,498 Beewax 19:246 Beer's law 15:423 Beet armyworm 12:397 Begonia plebeha 19:764 Belemnitella species 13:330 Bellus rearrangement 8:205 Bench-top bioassays 9:383-409 Benz [a] anthracene 11:113 Benz [a] anthracene antibiotics antibacterial activity of 11:134 antitumor activity of 11:134 synthesis of 11:134-144 Benz [a] anthraquinone 11:113 Benz-annulation reaction 1:505,506 Benzal malonates inhibitory activity of 9:224-227 synthesis of 9:224-227 Benzaldehyde acetal chromium tricarbonyl complex 14:477 from (2/?,3/?)-2,3-butanediol 14:477 with trimethyl aluminum and titanium tetrachloride 14:477 Benzamido-2-deoxy-P-D-glucopyranosyl)7-(2theophylline 4:238 3-Benzazepine derivative 6:474,484 Benzazepines 1:169 3-Benzazocine 6:469,471 synthesis of 6:469 2-Benzazocine A^-oxide derivative 6:472 Benzazocines 1:192,193 ;6:468-471,442 3-Benzazomne derivative 6:475,476 Benzene thiol 11:358 1,2-Benzenedithiol 11:357 Benzenesulfenylacetonitrile condensation of 12:287 1,2,4,5-Benzenetetracarboxylate 11:124 5y«-and aw^/'-indenofluorenone from 11:124 N^-Benzensulfonyl CPI synthesis of 3:323,325,326 Benzidine 7:457 Benzilic-type rearrangement 3:226 Benzimidazoles 12:3 Benzofriranes 20:282 Benzo [a] pyrene benzo [a] pyrene oxides from 7:8 carcinogenicity of 7:8 Benzo [a] pyrene oxides absolute configuration of 7:8 from benzo [a] pyrene 7:8 Benzo [a] pyrene-(-)-7/?,8/?-dihydrodiol 7:8,9 Benzo [a] pyrene-7/?,85'-dihydrodiol-95,10i?epoxide 7:8,9 8,13-Benzo [a]naphthacene quinone from naphthacene glutarate 11:123 Benzo [c] phenanthridine alkaloids biomimetic synthesis of 14:769-803 biosynthesis of 14:771-773 from protoberberines 14:769-803 oxygenative conversion of 14:779-784
958
Benzo [c] phenanthridine alkaloids 1:187,542 Benzo[c] phenanthridines 5:42 Benzo[c]naphta [\,2-E] azonine derivative 6:481 Benzochromones 18:978 Benzocyclobutanol 14:685 Benzocyclobutanone synthesis of 14:685 via (2 +2) cycloaddition 14:685 Benzocyclobutenes 3:434-437 2,6-Benzodiazecine derivative 6:486,487 2,5-Benzodiazonin-3-one from keto-imine intermediate 6:480,481 synthesis of 6:480,481 Benzodioxan 8:399,401 1,6,5-Benzodioxazonine from 1,5-benzoxazocineA^-oxide 6:472 4-Benzoloxy-P-lactam from epoxy amide 12:161,162 //^reo(45,55)-4-Benzylamino-5-hydroxy-2-methyl6-phenylhex-l-ene 12:478,479 synthesis of 12:478,479 Benzomorphans 2,6-bridge 3-benzazocine in 6:471 Benzophenanthridine alkaloids synthesis of 3:492,446 Benzoprostacyclins synthesis of 16:386 Benzoquinones 20:273 Benzoquinone derivatives 7:183,184 1,4-Benzoquinones enantiospecific reactions of 16:547-570 stereospecific reactions of 16:547-570 Benzoquinones 5:774-778 [l]Benzothieno [3,2-d] azonine derivative synthesis of 6:476 [l]Benzothiero analogues of hexahydrodibenz [d,g] azecines 6:492 synthesis of 6:492 2,7-Benzoxazacycloundecine derivatives from papavarine 6:495 synthesis of 6:495 X-ray analysis of 6:495 Benzoxazecine from isoquinoline derivatives 6:483 synthesis of 6:483 l//-2,6-Benzoxazecine derivatives 6:486 2//-3,6-Benzoxazecine derivatives 6:486 2,3-Benzoxazepine derivative from c/5-(+)-laudanosme A^-oxide 6:468 2,4-Benzoxazocine 6:469 2,4-Benzoxazocine derivative by ring expansion 6:469 Benzoxazocine derivatives by ring construction 6:468-471 by ring destruction 6:468-471 by ring interconversion 6:468-471 from a-narcotine A^-oxide 6:468 synthesis of 6:468-471 1,5-Benzoxazocine N-oxide 1,6,5-benzodioxazonine from 6:472 thermolysis of 6:472
Benzoxazolidone derivative boron enolate of 12:164,166 CMlkylationby 12:164,166 stereoslective 12:164,166 Benzoxazolin-2-one 7:191,192 2,3-Benzoxazonine from 2-benzazocine A^-oxide derivative 6:472 2,5-Benzoxazonine carbocation intermediate in 6:473 derivatives of 6:476,47 photosolvolysis of 6:475,476 synthesis of 6:473 X-ray analysis of 6:476,477 Benzoxicins 5:798;10:210 N-Benzoyl Cig-phytosphingosine 18:461 A^-Benzoyl phytosphingosine 18:462 3-Benzoyl propionic acid 10:406 4-O-Benzoyl-1,2-cyanoethylidene-a-L-rhamnose 14:242 Benzoyl-1,2:5,6-di-0-isopropylidene-a-Z)-3-Oglucofuranose 6:285,286 (+)-N-Benzoyl-16-acetoxycycloxobuxidine-F 2:200,201 A^-Benzoyl-iso-serinate 12:223,225 Benzoylation 1:415,417,289,290,341 N-Benzoylbaleabuxidine-F 2:205 N-Benzoylbuxidienine 2:205 A^-Benzoylcarbamate cyclization of 14:569 from 2,3-epoxy alcohol 14:569 2-oxazolidinone from 14:569 N-6-Benzoyldeoxyadenosine derivatives 4:278 N-BenzoyIdihydrocyclomicrophylline-F 2:207 5-(l-Benzoylindol-3-ylmethyl)-pyrrolidmone 13:130, 131 0-Benzoylnormacusine 13:386 N-Benzoyhiortropane 1:379,380 synthesis of 1:379,380 1 -Benzoyloxy-3-[(3-guaiazulenyl) methyl]-5-isopropyl8-methylazulene 14:324 iV-Benzoylpyrrolidinone 13:121 A^-Benzoylpyrrolinone synthesis of 8:212,213 Benzyl (-)-P-D-gentosaminide synthesis of 14:50-57 A^-Benzylipiperidine 19:35 (/?)-(+)-Benzyl l-(2-methoxy)-naphthyl sulfoxide 10:684,685 a-Benzyl 1-asparaginate 12:121 (S)-Benzyl 2,3-epoxypropyl ether 10:410,411 from D-mannitol 10:410,411 Benzyl 2-0-methanesulfonyl-b-D-arabinopyranoside 14:50 Benzyl 5-amino-5-deoxy-2,3-0-isopropylidene-a-Dmannofuranoside 10:541 a-Benzyl glycosides of A^-acetylmuramic acid methyl ester 6:395,399 ofMurMAc5-lactones 6:390-395 reactions of 6:390-395 A^-Benzyl lactam (CGP-42700) 12:388 Benzyl sulfmylcarbamate 1:23
959 Benzyl tolyl sulfone 11:349 1 -Benzyl-1,2,3,4-tetrahydro-isoquinolone alkaloids 10:683-685 Benzyl-2,3-anhydro-p-D-ribopyranoside 14:50 4,0-Benzyl-2,3-0-6w (methoxy methyl)-L-threitol 11:233 4-0-Benzyl-2,3-0-isopropylidenetlireose 10:128-130 1 -Benzyl-2,6-dicyanopiperidine alkylation of 6:433 decyanation of 6:433 2,6-dialkylpiperidines from 6:433 6-0-(2-0-Benzyl-3 -deoxy-3 -ethoxy carbonyl-amino4,6-0-isopropylidene-a-D-glucopyranosyl)-A^, A^'diethoxycarbonyl-2-deoxystreptamino 14:145 Benzy 1-3 -oxohexanoate with 3-(l-Ai-pyrroliniumyl) propanal 12:293 Benzyl-4,6-(9-benzylidene-3-deoxy-p-D-r/6ohexopyranoside 14:11,12 synthesis of 14:11,12 Benzyl-5-deoxy-5-(ethyl phosphinothioyl) 3-0compounds 6:374,375 (+)-8-0-Benzyl-6-deoxy-castanospermine 12:346 6-Benzyl-9-(3,4-anhydro-a-l-talopyranosyl)a-denine 4:233,234 A^-Benzyl-L-glutamic acid 12:312 A^-Benzyl-/7-toluensulfonamide 12:323 6-Benzylamino-9-(2-acetamido-2-deoxy-p-Dglucopyra-nosyl) purine 4:238 6-Benzylaminopurine (BAP) 4:227,2387:90,94,95 0-Benzylation 12:327 Benzylation 6:268-270,287,288 A^-Benzylation 7:41-43 of 1,4-dideoxy-1,4-imino-I-allitol 7:41 -43 Benzylbenzoate 9:399 O-Benzyldiscretine fagaronine from 14:776,777 Benzylic hydroperoxide rearrangement 3:316-319 3,5-0-Benzylidenation 12:52,53 of (95)-9-dihydroerythronolide A 12:52,53 Benzylidene glycal 10:344 4,6-0-Benzylidene-a-D-glucopyranoside 14:157 3-Benzylidene camphor 4:663,664 2,4-0-Benzylidene-D-threose 6:354 (l/?)-l-[(dimethoxy) phosphinyl]-Z)-threitol from 6:354 N-Benzylideneaniline 10:679,680 Benzylisoquinoline alkaloids 6:472 Benzylisoquinoline derivatives dibenz[<3(,/]azonine derivatives from 6:477,478,480 Benzylisoquinolines synthesis of 3:422 Benzyllithium alkylation of 8:6 2-Benzyloxy glucals 10:346 (3R,4R)-^A-bis (Benzyloxy) succinic anhydride 12:295 (-)-9-Benzyloxy-9-demethyl-7-methoxynogarene 14:82 (±) A'^-Benzyloxycarbonyl-3 -hydroxy-4-pentenylamine 12:281 Sharpless kinetic resolution of 12:281 Benzyloxycarbonylation 12:478 A^-Benzyloxycarony 1-1 -proline methyl ester 12:295
exo-15-Benzyloxyhobartine 11:299,300 15-Benzyloxyhobartine derivative 11:298,299 Benzyloxymethyl chloride 6:561 a-(2-Benzyloxypropionyl)-butyrolactone 8:287 5-Benzylpyrrol-2(5H)-ones 13:113 Benzyltetrahydroisoquinoline 18:72,76 1 -Benzyltetrahydroisoquinoline system intramolecular oxidative coupling of 6:480 protostephanine from 6:480 rearrangement of 6:480 A^-Benzyltrifluoroacetamide 14:553,554 Benzyne 16:47 Benzyne cyclisation 4:541-551 Benzyne reaction 16:514 Berbamunin 20:292 Berberidaceae 7:416 Berberideine 20:265 Berberine 1:212;13:660 8,14-cycloberbine from 1:211 ^^C-NMR spectrum 5:44 ' H - N M R spectrum of 5:40 APT spectrum of 5:42,43 biosynthesis of 11:201-204 from Berber is sp. 7:88 from Berberis vulgaris 13:660 INEPT spectrum of 5:43,44 Berberme bridge enzyme (BBE) 11:204 Berberis cell cultures j atorrhizine from 11:201 -204 Berberis koetineana 11:202 Berberis sip. 7:88 berberine from 7:88 Berberis stolomifera 20:292 Berberis vulgaris 13:660 Berdy database 19:759 Bergapten from Limonia acidissima 20:497 (+)-P-cw-Bergamotene 16:235 cw-Bergamotene 16:235,236 Bergenin ^omArdisajaponica 13:660 regioselective hydroboration of 13:135 exo-Bervicomin 19:126 Bestatin 12:412,493,434 Beta vulgaris 15:3;20:721,731,740 Betaenone 4:620,621 biosynthesis of 4:620,621 Betaenone B 4:601,602,621,17:475 Betaenone C biomimetic synthesis of 4:620 synthesis of 4:602-604 Betaine 1:89,752,539,92 Betalaines 20:724,740 Betanidine 20:724-740 Betanine 20:724,731 Betula pendula 17:361,366 Betula platyphylla 17:361 5e/M/a species 17:358.361 Betulaceae 17:358,359,364,366 Betulin 7:189,293 Betulinic acid 7:189,507 Betuloside 20:116 Beyerane 15:252
960 BFs rearrangement 5:782,783 BFs-mediated method 11:117 Bharingine 5:174,175 Bhilawainol 9:318 Bhimberine 9:170,172,173 5,5'-Bi(l,3-di-bromoazulene) 14:334 7,7'-Bi(3-bromoazulene)-l,r (7H,7H>dione 14:342 9,9'-Bianthracene-10,10' (9H, 9'H) diones from 1,9-anthracenediols 11:125,126 Bicarbocyclic bridged systems 6:65-74 Bicarbocyclic fused system 6:5-38 Bicarbocyclic lactone 6:80 Bicyclic (3-lactam by Diels-Alder reaction 12:172,173 from cyclic imines carboxylic acid 12:117 from2H-4H, 1,3-dioxin derivatives 12:160,161 synthesis of 12:172,173 Bicyclic C-glycosides from 2-0-Acety 1-3,4,6-(9-benzy 1-a-D-glycosyl bromide 10:338 Bicyclic cis/trans oxazinolactams 11:260,261 Bicyclic cw-glycols 8:245,246 Bicyclic diterpene 8:220 Bicyclic enone taxodione from 14:678,679. from monocyclic ketones 10:311-313 Bicyclic gephyrotoxins (indolizidines) 11:244 Bicyclic keto ester 14:509 Bicyclic keto-acid 12:283 Bicyclic lactones 12:150 Bicyclic olefin 11:371 Bicyclic triene 6:79 Bicyclo [2.2.1] heptanes 8:415 absolute stereochemistry of 8:415 synthesis of 8:415 Bicyclo [2.2.2] octane (±)-sanadaol from 6:70,71 by double Michael addition 8:422 from Li-cyclohexadienolates 8:422 from a,P-unsaturated esters 8:422 synthesis of 8:412-422 Bicyclo [2.2.2] octane ring 8:166 Bicyclo [3.1.0] hexan-2-one (±)-sinularene from 6:79 Bicyclo [3.2.0] heptanone bicyclo [6.3.0] undecanone from 6:36 Bicyclo [3.2.1] octadione synthesis of 8:161,162 Bicyclo [3.2.1] octane 8:420,421,40;12:22 Bicyclo [3.2.1] octanone 6:78,79 Bicyclo [3.2.1] octenes from bicyclo [3.3.0] octanones 10:317,318 Bicyclo [3.2.1] system 12:86 Bicyclo [3.2.1]hemithioacetal 8:205 Bicyclo [3.2.2] nonadienone 1:567 Bicyclo [3.2.2] piperazinedione synthesis of 12:71 Bicyclo [3.2.2] system 12:85 Bicyclo [3.3.0] octane spiro [4.5] decane from 10:315 cw-Bicyclo [3.3.0] octanes 13:4
Bicyclo [3.3.0] octanones 10:310,311,317,318 Bicyclo [3.3.1] nonane 6:65-69,224 Bicyclo [3.3.1] nonanes conformation of 3:95 fragmentation of 3:74 Bicyclo [3.3.1] nonyl transition state 4:447;10:317, 318 Bicyclo [4.2.2] mesylate 12:80 Bicyclo [4.2.2] ring system 12:64,65 Bicyclo [4.3.0] non-l-en-3-one 10:310 Bicyclo [4.3.0] nonane (hydroindane) 11:230 Bicyclo [4.3.0] nonane group 6:5-11 Bicyclo [4.3.1] dec-2-en-7-one by Grobfragmentation6:69 by intramolecular [2 + 2] photocyclo-addition 6:69 synthesis of 6:69 Bicyclo [4.3.1] decane group 6:69-72 Bicyclo [4.3.1] decanone nakafiiran-9 from 6:70 synthesis by four carbon polar annulation 6:17,21 Bicyclo [4.4.0] decane 6:10,235 Bicyclo [4.4.1] undecane 6:72-74,234 Bicyclo [4.4.1] undecane group 6:72-74 Bicyclo [4.4.1] undecanone 12:238,244 Bicyclo [5.1.0] ontanone 12:243 Bicyclo [5.2.2] piperazmedione synthesis of 12:84,85 Bicyclo [5.3.0] decane group 6:11,12 Bicyclo [5.3.1] undecane unit 12:179 Bicyclo [5.4.0] undecane group 6:29-33 Bicyclo [5.4.0] undecanone 12:249 Bicyclo [6.2.1] undecane 12:193 Bicyclo [6.3.0] undecane group 6:33-38 Bicyclo [6.3.0] undecane skeleton 1:568 Bicyclo [6.3.0] undecanone from bicyclo [3.2.0] heptanone 6:36 poitediol from 6:36 Bicyclo [6.4.0] dodecyl structures 12:212 9-Bicycloboranonane 13:135 Bicyclohumulenone 2:278,279 Bicyclohumulenone from Plagischila acanthophylla subsp. japonica 8:183 synthesis of 8:183-186 Bicyclomycin antibiotic activity of 12:63 antimicrobial activity of 12:41 biosynthesis of 12:64,65 from Streptomyces sapporonensis 12:63 relative configuration of 12:64 stereochemistry of 12:64 synthetic studies of 12:65-95 toxicity of 12:63 X-Ray analysis of 12:64 from vicinal tricarbonyl 8:274 Bicyrtes ventralis 5:223,224,253 Biczamycin 12:63 Bidesmosidic saponins 7:429,432 Biellmann coupling 10:6-10 Biflavonoids 5:468,756-758
961 Biflorin from Eremophila latrobel 15:258 synthesis of 3:450,451 Biflustra perfragilis 17:81,89,91 Bifiircanol 20:26 Bifur carta bifurcata 18:713 Bignoniaceae 7:406 Biogenesis ofD:A-friedo-oleananes 7:150-152 of phenolic triterpenes 7:150-152 of triterpene quinone-methides 7:150-152 3,3'-Biguaiazulene 14:315,319 5,5'-Biguaiazulene-3,3' (5H,5'H)-dione 14:320 Bijvoet difference method 13:434 Bile alcohols 17:207,212 Bilirubin 10:153 Billheimer method 9:464 Bilobol synthesis of 9:354 Bimolecular [67i+47t] tropone diene cycloaddition of 12:241 Binandkatsuim A 20:276 ^-BINAL-H 16:377 (5)-BINAP 19:29 (BINAP) 13:72 BINAP-Ru-catalyst in asymmetric hydrogenation 4:439 Binary indole alkaloids 5:140-151 Bio-organic chemistry ofansamycin 9:431-445 of antibiotics 9:431 -445 of mitomycin 9:431-445 Bioactive conformations ofhormones 18:819-866 Bioactive O-heterocyclic compounds 20:494 Bioactive metabolite of gtnus Phomopsis 15:341-356 Bioactive natural products chkal synthesis of 13:84-99 synthesis of 13:473-518 Bioactive polyketides 18:193-227 Bioactive steroids from Pseudobersana mossanibicensis 20:276 Bioactivities 17:253 Bioassay 20:464 KB Bioassay 2:404,405,9:230,231,238,220-222 Bioconversion using Morus alba cell 17:470 Biofilms 17:100 Biogeneration of flavors 13:295-345 Biogenesis by intramolecular Diels-Alder reaction 4:616,618 Diels-Alder reaction in 4:615-617,620 of (±)-16-hydroxy-a//o-ibogamine 6:503 ofacorene 3:30 ofalflavene 4:618,619 ofapparicine 6:520 ofbetaenone 4:620,621 ofbetaenoneB 4:621 ofcadlinolide A 9:9 ofcatharanthine 4:616
of cedranoid sesquiterpenes 3:29 ofchaetoglobocins 4:620 ofchlorothricholide 4:620 ofcompactin 4:620 of cyclostachine 4:618,619 ofcytochalasin 4:620 ofellipticine 6:520 of guggulu steroids 5:709-713 ofheliocideB 4:618,619 of ikarugamycin 4:620 ofilicicolinH 4:620 ofircinianin 4:618,619 ofmevinolin 4:620 ofngouniensine 6:503 ofnodusmicin 4:620 ofpyoverdins 9:552,553 of strellidimine 6:516 oftabersonine 4:616 ofthienamycin 4:620,621 oftubotaiwine 6:503 ofxenocoumacins 15:406-408 ofzoanthaminone 9:11 Biogenetic precursor ofallamandicin 16:301 ofplumericin 16:301 Biogenic amines 15:328 Bioinsecticides 15:381 Biological activities chiralityin 7:3-28 isolation of 4:243 of(+)-compactin 11:335,336 of(+)-mevinolin 11:335,336 of (-)-selin-l l-en-4a-ol 14:450,451 ofamicoumacins 15:410-412 ofavermectins 12:7-9 ofbaciphelacin 15:409,410 of Z)M-indolyl maleimides 12:384-399,133,91 of erythromycin 13:155-185 of indolo [2,3-a] carbazole alkaloids 12:384-399 of intermedeol 14:450,451 of ivermectin 12:8,9 of macrolactone 155-185 ofmicrocystins 20:896,897 of natural products 7:3-29 ofneointermedeol 14:450,451 ofnocamycin 14:110 ofnodularins 20:896,897 of PI turnover inhibitors 15:461,462 of polyene macrolides 6:261 of self-inhibitors 9:222,238 ofspatanediterpenoids 6:39 ofspatol 6:39 ofsphingolipids 18:459,460 of staturosporine 12:399 ofstreptolydigin 14:108,109 oftaxanes 11:4-6 oftaxodione 14:667 of tetramic acid 14:107-110 of tyrosine kinase inhibitors 15:447,449 ofxenocoumacins 15:408,409 Biological Diels-Alder reaction 17:451
962 Biological investigations of S'a/v/a species 20:659-715 Biological properties ofdidemnins 10:241-302 oflipo-gastrin 18:857-863 oflipo-CCK 18:857-863 Biological raw materials 17:601 Biological stereoselectivity 7:8-10 Biologically active compounds 17:153 Biomethylation 9:47 Biomimetic cationic polyene cyclization 12:456 Biomimetic cyclization of allylic alcohol 14:681-684 Biomimetic olefin cyclization 1:671-673 Biomimetic reaction 9:570,328 Biomimetic studies of oligopeptides 10:629-669 Biomimetic synthesis 20:408-410 of aromatic natural products 11:113-149 of(-)-hobartine 11:287-291 of bisindole alkaloids 13:404-407 from protoberberines 14:769-803 of benzo [c] phenanthridine alkaloids 14:769-803 oflupinamine 14:739-741 of g/7/-lupinamine 14:739-741 ofnitramine 14:748-750 ofnitraramine 14:755,756 ofnitrarine 14:761,762 ofbetaenoneC 4:620 ofdiplodiatoxin 4:620 ofmycotoxin 4:620 ofsolanapyrone A 4:620 of polycyclic terpenoids 6:23 of taondiol methyl ether 6:56 into pentacyclic alkaloids 11:292-295 of tetracyclic alkaloids 11:292-295 ofmacroline 13:398 Biomimetic polyene cyclisations 7:131 Biomphalaria glabrata 7:432-435 Biopolymers 17:481 Bioquercetin 7:226 Bioregulators studies with 2:377-386 chiral synthesis of 6:537-566 BIOSIS 19:758 Biosynthesis [2,3-a] quinolizine and its N-oxide 14:759 by aminoethyl phenyl ethers 2:378 enzymes for 2:365 from tetrahydrocorysamine 14:796 of 3,4'-anhydrovinblastine 2:372 of(±)-eremolactone 15:272 of(+)-aristolochene 15:250 of(+)-aristoteline 11:278,279 of(+)-isoeremolactone 15:272,273 of(-)-acetomycin 10:443 of (15 /?)-15-hydroxy catharinine 14:820,821 of(5)-nicotine 11:205-207 of 1,2,3,4,6,7,12,12b-octahydroindolo of 5-0-desosaminylprotylonolide 5:614,618 ofactinorhodin 5:617 ofacyltetramicacid 14:105,106
ofajmalicine 2:376 ofamylopectm 14:148 ofanhydrovinblastine 14:820,821 ofantabine 11:205-207 of antibiotics 11:207-213 ofaplasmomycin 11:207-209 ofarctiin 5:476 of Aristotelia alkaloids 11:278,279 of asparagine-linked oligosaccharides 10:499-502 ofaurantinin A 5:604-607 ofavermectins 12:6,7 ofaverufin 11:194 ofp-lactams 11:207-213 of benzo [c] phenanthridine alkaloids 14:771-773 ofbicyclomycin 12:64,65 ofbisabolanes 8:45 of branching in lactosaminoglycans 10:484,485 of carbohydrates 8:64 of Catharine 14:820,821 ofcatharinine 14:820,821 ofcathenamine 1:90-91 of cephalosporin-C 11:211,212 of cephalosporins 11:211-213 ofchimeramycins 5:614 of cholesterol 1:655 ofcholicacid 17:207 of cinnamic acids 5:468 of cis and trans sabinene 11:221,222 ofcorydalic acid methyl ester 14:796 ofdaunomycinone 11:123 of dihydrokomarovinine 14:763,764 ofelasnin 5:590 ofenniatins 13:535 ofepilupmme 14:738,739 of ergot alkaloids 11:199-207 of Forsythia lignans 5:489-492 ofglucoamylase 2:348-360 of glycogen 14:148 of glycolipids 1:417 of glycoprotein 1:417;14:148 ofglycosphingolipids 10:503 oflgD 10:539 ofIgM 10:539 of indole alkaloids 1:31,34,89-92 of indolocarbazole alkaloid 1:6 of ipomeamarone 15:249 of irumamycin 5:597,598,600,601 ofisonitrarine 14:760 ofkomarovidine 14:763,764 ofkomarovine 14:763,764 ofkomarovinine 14:763,764 ofkuwanon 17:465 of lactose 14:148 ofleurosine 14:820,821 ofleurosine 2:372 of lignans 5:459,474,477 of lignin 5:462-466 oflupinine 14:738,739 ofmellein 15:406 ofmicrocystins 20:899 ofmonolignols 5:467-469,607
963 of morphine 18:51-55 ofmyxopyronin 5:606 of naphthyridinomycins 10:104-106 of natural products 2:365 ofnazlinin 14:759 ofnitramidine 14:760 ofnitraramine 14:754,755 ofnitrarine 14:760,765 ofnodularins 20:899 oformosanine 14:738,739 of penicillins 11:211-213 ofpentalenolactone 13:30 ofphillyrin 5:476 of Podophyllum lignans 5:477-489 ofpolyketides 11:191-207 ofrebeccamycin 12:374 ofribostamycin 11:216-219 ofsaframycinA 10:80 ofschoberidine 14:760 ofschoberine 14:757,758 of seconday metabolites 20:291-293 of shikimic acid 11:182-191 of sinapyl alcohol 5:470 ofsparteine 14:738,739 ofstaurosporine 12:373,374 of strictosidine 1:90,91 of strychnon-type alkaloids 1:31,32 of sugar components of antibiotics 11:213 -222 of terpenoids 2:378 of thienamycin 11:210,211 of thiostrepton 11:209-211 of ubiquinones 8:63 ofvallesiachotamine 1:90,91 ofvindolinine 2:378 ofvineomycins 5:596 of vitamin E 8:63 of yohimbine 2:376 oxidative phenolic coupling in 20:291-293 via hypothetical enamine-aldehyde 14:796 de novo Biosynthesis 17-oxoellipticine 6:521 by cyclopropanone sliding reaction 6:37 from zeaxanthin (P,P-carotene-3,3'- diol) 6:139 of24-propylidenechlosterol 9:37 of acetylenic carotenoids 6:147,155,156 of allenic carotenoids 6:133,134,142 of allenic carotenoids 6:142,143 ofantheridiogens 6:206 ofapotrichothecenes 6:249,250 ofasterosaponins 7:299,302,303 ofbrafouefine 6:521 of C45 carotenoids 7:355-364 of C50 carotenoids 7:354-364 of Cephalotaxus diVi^dXoidiS 6:487 of cyclopropane sterols 9:35-43 of cyclopropene sterols 9:44-47 of eight-membered marine compounds 6:37 officisterol 9:40 offiisarins 6:250 ofgorgosterol 9:45-47 ofhebesterol 9:43 of homoerythrina alkaloids 6:487
ofisobrafouedine 6:521 ofleukotrienes 9:562,576 of lipoxms 9:561,575-578 ofnicasterol 9:43 of norterpene cyclic peroxides 9:23,24 ofpeptidoglycan 6:404 ofpetrosterol 9:41-43 of phenolic lipids 9:341,342 ofprotostephanine 6:480 of sambucinicacid 6:250,251 ofsambucinol 6:249,250 ofsterculicacid 9:45 of sulfidopeptide lipoxins 9:576 oftrichothecene 6:247-259 of vitamin B12 9:591,609 Biosynthetic precursors carotenoid 5,6-epoxides as 6:142 Biosynthetic retroanalysis ofnitraramine 14:754,755 Biosynthetic route of tropane alkaloids 17:314 Biotechnology 13:296,297,299 (+)-Biotin 13:514-516 synthesis of 13:514-516 Biotinylated gastrin 18:920 Biotinylated peptides 18:919 Biotranformations of indole-3-pyruvic acid 12:374 of substrates 2:386 studies of alkaloids 2:386 ofellipticine 6:509 of 24-difluoro-25-hydroxy-vitamin D3 9:517 Bipendensin from Afzelia bipendensis 9:256 Biperezone 20:273 3,3'-Biplumbagin 2:212,214,5:754 ' H - N M R spectrum of 2:227 Bipolaris sorokiniana 4:647;9:238 2,3'- Bipyridyl alkaloids 5:753 Birch reduction ofo-toluidine 11:286 of6-methoxy-l-tetralone 12:235 of4-methoxytoluene 12:22-24 of/7-cresyl methyl ether 6:83,84 BIRD sequence 9:97,98 12,12'- Bis (11-hydroxycoronaridyl) 5:129 3,4- Bis (8-hydroxyquinolin-4-yl)-butyrolactone 5:753 Bis (di-isoamyl) borane reduction 9:366 Bis (EDTA-DISTAMYCIN) phenoxazone 5:570 trans-2,5-B\s (methoxymethoxymethyl) pyrrolidine 10:412 double alkylation of 10:412 Bis (netropsin)-3,6,9,12,15-pentaoxahepta-decanediamideEDTA 5:570 synthesis of 5:570 Bis (trimethylsilyl) ethylene glycol 1,3-dioxolane formation 1:584 reaction with aldehydes 1:584 reaction with ketones 1:584 Bis (trimethylsilyl)-1,2-ethanedithiol 1,3-dithiolanesfrom 1:584
964 reaction with aldehydes 1:584 reaction with ketones 1:584 Bis allylic bromide 8:16 Bis indole alkaloids 2:370371,387,389,390,394,398 3'-Bis(2-cyanoethoxy) phosphoryl group branched RNA from 14:291,292 2'-Bis(2-cyanoethoxy) phosphorylation 14:299,302 Bis(bibenzyls) 2:103,2:113 2,5-Bis(dimethylamino) benzoquinone 13:434 Bis(trimethylsilyl) acetylenes 14:473 Bis(trimethylsilyl) butadiyne 14:473 Bis-(3,7-diisopropyl-l-azulenyl) ketone 14:340 Bis-(EDTA-distamycin) fumaramide 5:569 2,4-Bis-(trimethylsiloxy)-furan 13:451 Bis-diazoketone 6:546,547 intramolecular coupling of 6:546,547 2',4'-Bis-0-(2-methoxy ethoxy methyl)-4-(9-methyl7,8-dihydronogarene 14:87 (±)-2',4'-Bis-0-(2-methoxyethoxymethyl)-7,8dihydronogarene 14:83,87 (±)-2',4'-Bis-(9-(2-methoxyethoxymethyl)-7-con-0methylnogarol 14:88 Bis-THF acetogenins 17:270 Bis-THF structures 17:258 11(15->1), 11(10^9) Bisabeo taxane 20:80,107 5-Bisabolene 17:609 /?-a-Bisabolene 16:232 Bisabolene diterpenes 15:255-257 Bisabolene isoprenologues 15:277 £-5-Bisabolene-8-(/?),(5)-epoxide (-)-(2/?,65',85,95)-2,8-dibromo-9-hydroxy-achamigrene from 6:62,63 glanduliferol from 6:63,64 Bisabololoxide derivatives 7:218 (-)-(R,E)- a-Bisabolone synthesis of 16:231 1-Bisabolones 8:39-44,46,48 Bisabolones absolute configuration of 8:39-59 biosynthesis of 8:44,45 relative configuration of 8:39-59 synthesis of 8:39-59 P-Bisabolon 20:6 Bisacnadinine 20:265 Bisanhydrobacterioruberin 20:601 Bisaryltetrahydrofiirans 17:325 Bisbenzylisoquinoline alkaloids 6:472,657,525 Bisbenzyltetrahydroftirans 17:330 Bisbrusatolyl esters 7:383,384 Bischler - Napieralski reaction 3:460,93,633,790;8:291 Bischler-Napieralski condensation isokomarovine from 14:763 komarovidine from 14:763 oftryptamine 14:763 with quinoline-5-carboxylic acid 14:763 Bischler-Napieralski cyclization 4:20,23,444 Biscyanopropylhenyl siloxane 9:456 Biscyclolobin 20:283 6,10-Bisdeoxyaucubin 7:465,466,474,476,477 Bisdesmosides 15:188
Bisdesmosides 7:155 selective cleavage of 7:155 Bisglutarylidene pentane from triphenylphosphoranylidene glutarimide 14:755 Bishopantholide 7:234 Bishopsolicepolide 7:234 cw-Bishydroxylation 4:162,165,182 Bisindole-type alkaloids (3/?)-ervafolidine 5:120 11 -demethylconoduramine 5:115 12,12'-bis (U-hydroxycoronaridyl) 5:121,129 14,15-dehydrotetrastachyne 5:121 16-decarbomethoxy-19'20'-dihydrovoacamine 5:114 16-decarbomethoxyvoacamine 5:114 19'-hydroxyervafolene 5:120 19'-hydroxyervafoline 5:121 19'-/?-hydroxyervafolidine 5:121 19'5-hydroxy-3-ep/-ervafolidine 5:121 19/?-hydroxytabemaelegantine B 5:117 20'S-19',20'-dihydrotabemamme 5:114 20R-1,2-dihydrocapuvosidine 5:114 205-1,2-dihydrocapuvosidine 5:114 3'-3/2/5-hydroxyvoacamine 5:117 3'-7?/5-hydroxy-7V4-demethylervahanine A 5:115 3'-i?/5-hydroxy-A^4-demethylervahanine B 5:115 3 '-/^'5-hydroxy-A^4-demethyItabemamine 5:114, 5:124 3 '-/?/5-hydroxytabemaelegantine B 5:117 3'-/2/5-hydroxytabemamine 5:114 3 -(2-oxopropyl)-conodurine 5:18 3 -ep/-ervafolidene 5:121 3-ep/-ervafolidine 5:121 3-methoxyltabemacelegantine C 5:118 3-oxo-conodurine 5:116 3 -R/S-hydroxy-16-decarbomethoxyconodurine 5:115 3 -/^5-hydroxyconoduramine 5:117 3-/2/5-hydroxyconodurine 5:118 aceedinine 5:112 aceedinisine 5:112 biomimetic synthesis of 13:404-407 bonafousine 5:122 capuvosine 5:114 conodurine 5:116 dehydroxycapuvosine 5:114 divaricatine 5:118 ervafolene 5:120 ervafolidene 5:121 ervafoline 5:120 ervahanine A 5:115 ervahanineB 5:115 ervahanine C 5:115 gabunamine 5:115 gabunine 5:115 hazuntiphylline 5:120,121 isobonafousine 5:122 isocapuvosine 5:114 moinogagaine 5:119 A'4'-demethylvoacamine 5:115 A'4-demethylcapuvosine 5:114 A'4-demethyltabemamine 5:113
965 pandicine 5:118 pseudo-vobparicine 5:119 tabemaelegantinine A-D 5:175,118,129 taberaamine 5:113 tetrastachyne 5:121 tetrastachynine 5:121 voacamidine 5:116 voacamine 5:116 voacamine-7V4-oxide 5:119 vobtusine 5:120 Bisiridoids 7:443 Bisisodiospyrin 20:276 3,3'-Bisjuglone 20:276 Bisketene 4:349 26,27-Bisnorbrassmolide 19:263-265 26,27-Bisnortyphasterol 19:268 2',3'-Bisphosphoramidite 14:293 2,3'-Bisphosphorylated ribonucleoside 14:284-290 Bisstrictidine 5:140,141 7,7-Bistaxodione from Salvia montbretii 20:678 Bistetrahydrofliran 10:439,440 Bistratamide 10:243 Bistratenes A,B 10:243 BivittosideA 1-210,21 \ Bivittoside A,B,C and D 1:270;15:91 Bixaorellana 20:721,733,59 Blaberus discoidalis 9:488,489 Black vomit 1:679,680 Blackcurrant oil 13:659 Blainvillea acmella 5:728 Blakeslea trispora P-carotene from 7:320 trisporic acid from 7:320 BlasticidinH 4:241,243 Blasticidin S absolute configuration 4:242,243 biosynthesis of 4:243 synthesis of 4:243 (+)-Blastmycinone synthesis of 16:694 Blastodadiella emersonii 5:276 Blastocrithidia 2:298 Blastomyces dermatiditis 5:307 Blastomyces sp. 5:325,328 Blastomycin synthesis of 3:253 Blastomycinone synthesis of 3:253,254 Blastomycosis 2:422 Blattella germanica 6:53;9:488,489 Blatta orientalis 9:488,489 Blechert's construction 12:195 Bledius mandibularis 4:494 Bledius spectabilis 4:494 Bleekeria vitiensis 1:125 Bleekerine oxidation 1:125,158 Bleomycin 16:573;20:458 Blestrin 20:280 Blighia welwitschii 15:205 Block-type polysaccharides 14:248 Blood platelet-aggregation 16:373
Blumea malcomii 5:678 Bmy-41950 from Streptomyces staurosporeus 12:366,368 Boariol 18:743,752 A^-BOC (3/?,45)-statine from (3/?,45)-statine 12:480 BOC anhydride method 12:121 BOC fimctionality deblocking with TMSOT 4:91,92 A^-BOC-1-allo-threonine methyl ester 12:430 A^-BOC-2-amino alcohol 12:480 (i?)-A^-BOC-2-amino alcohols 12:435,436 with thionyl chloride 12:435,436 (5)-iV-BOC-amino alcohols 12:481 0-A^-BOC-amino benzaldehyde 18:163 (3/?,4/?,55)-iV-BOC-dolaisoleucine 12:486 A^-BOC-proline 19:54 A^-BOC-L-threonine methyl ester from methyl trans (4S,5/?)-5-methyl 3>-tert butoxycarbonyl-2-oxo-oxazolidme-4carboxylates 12:430 Boekman-Silver synthesis of(-)-p-gorgonene 6:18 Bohadschia argus bivittoside B,C from 7:270 Bohadschia bivittata bivittosides A, B, C and D from 15:91 Bohadschia graffei holothurins A,B from 7:269 holothurin A2 from 7:269 Bohadschia marmorata bivittoside B,C from 7:270 Bohadschia tenuissima bivittoside B,C from 7:270 Bohadschia vitensis bivittoside B,C from 7:270 Bolbostemma paniculatum (tu-bei-Mu) tubeimoside I from 7:143,144 Boletinus cavipes 20:26 Boletus edulis 5:289 Boll weevil pheromone 1:693 Bombesin-induced phospholipase C activity 15:447 Bombyx mori 5:832;19:647 Bonafousine 5:128 Bonanzin 7:226 Bone calcium mobilization (BCM) 9:514 9-Borabicyclo [3.3.1] nonane (9-BBN) 13:72 Borage oil 13:659 Boraginaceae 7:408 Borane-methyl sulfide complex 11:3 60 Boration 9:366-368 Borealosides A-D 15:71 Bormination of camphor 4:629,633,634,656,657 of camphor derivatives 4:629-632,633,634,636, 656-658 Bom-Oppenheimer approximation 2:155 1-Bomeol 10:52 Bomeol 9:530 (+)-Bomyl acetate 4:645,46,648 (+)/(-)-Bomyl diphosphate formation of 11:219-221
966 from g
