Studies in Natural Products Chemistry
Volume 33 Bioactive Natural Products (Part M) M)
Chemistry Studies in Natural Products Chemistry edited by Atta-ur-Rahman
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Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidation (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure and Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bioactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bioactive Natural Products (Part B) Bioactive Natural Products (Part C) Bioactive Natural Products (Part D) Bioactive Natural Products (Part E) Bioactive Natural Products (Part F) Bioactive Natural Products (Part G) Bioactive Natural Products (Part H) Bioactive Natural Products (Part I) Bioactive Natural Products (Part J) Bioactive Natural Products (Part K) 1-30 Studies in Natural Products Chemistry: Cumulative Indices Vol. 1-30 Bioactive Natural Products (Part L) Bioactive Natural Products (Part M)
Studies in
natural Products Chemistry Natural Volume 33 Bioactive Natural natural Products (Part M)
Edited by
Atta-ur-Rahman H.E.J. H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, 75270, Pakistan
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FOREWORD Natural products present in the plant and animal kingdom offer a huge diversity of chemical structures which are the result of biosynthetic processes that have been modulated over the millennia through genetic effects, including those triggered by environmental stresses. With the rapid developments in spectroscopic techniques and accompanying advances in high-throughput screening techniques, it has become possible to isolate, determine the structures and biological activity of natural products rapidly, thus opening up exciting new opportunities in the field of new drug development to the pharmaceutical industry. Even if the bioactive natural products cannot themselves be used in medicine directly because of the difficulties in obtaining them in sufficient quantities by isolation or synthesis, they may still offer potential new pharmacophores. These pharmacophores may comprise the part of their structures responsible for the activity and they may be more accessible synthetically. The present volume contains 22 articles written by leading experts in natural product chemistry on biologically active natural products. It includes researches on a variety of different classes of natural products including sesquiterpenes, quassinoids, diterpenoids, lignans, oligostilbenes, phenylethanoids, phenylpropanoid glycosides, curcumin analogues, glycosphingolipids etc. Many of these have been found to be active in a number of different disease conditions. I hope that the present volume will provide a large material of interest to natural product chemists, medicinal chemists, and pharmacologists as well as other researchers, particularly those in academia and in the pharmaceutical industry. We would like to express our thanks to Mr. Liaquat Raza and Ms Qurat-ul-Ain Fatima for their assistance in the preparation of the index. We are also grateful to Mr. Wasim Ahmad for composing and typing and to Mr. Mahmood Alam for secretarial assistance.
Prof. Atta-ur-Rahman Federal Minister/Chairman Higher Education Commission/ Director, International Center for Chemical Sciences Karachi, Pakistan
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vii Vll
PREFACE One of the most fertile areas of chemistry is the interface between chemistry and biology. It is there that chemists, armed with modern tools for separation and spectroscopic analysis, bring to light the structural and mechanistic workings of nature at the molecular level. This basic knowledge drives further investigations in diverse areas such as chemical synthesis, protein structure and function, and cell biology, and ultimately leads to advances in the treatment of diseases of humans, animals, and plants, all of which impact upon the quality of our lives. The editors and publishers of "Studies in Natural Products Chemistry" have again done a great service to the natural products community by assembling this 33rd volume in the series, the 13th devoted to Bioactive Natural Products. Here the reader will find timely reviews written by outstanding scientists from around the world on topics ranging from the purely chemical to the very biological. As a contributor to the first volume of "Studies in Natural Products Chemistry" some 18 years ago, it is a special pleasure to thank Professor Atta-ur-Rahman for his leadership and commitment to excellence that in no small part have been responsible for the success of this series.
Eugene A. Mash Department of Chemistry University of Arizona Tucson, Arizona, USA
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IX ix
CONTENTS Foreword
v
Preface
vii
Contributors
xi
Focus on fluorescent proteins GUIDO JACH AND JOCHEN WINTER
3
Structure, function and mode of action of select arthropod neuropeptides GERD GADE AND HEATHER G. MARCO
69
Natural products as modulators of apoptosis and their role in inflammation JOSE LUIS RIOS AND M. CARMEN RECIO
141
Sesquiterpenes classified as phytoalexins A.K. BANERJEE, M.S. LAYA AND P.S. POON
193
Bioactive triterpenes and related compounds from celastraceae NELSON ALVARENGA AND ESTEBAN A. FERRO
239
Structure-activity relationships of sesquiterpene lactones THOMAS J. SCHMIDT
309
Synthetic investigations in the field of drimane sesquiterpenoids PAVEL F. VLAD
393
Quassinoids: Structure diversity, biological activity and synthetic studies IVO J. CURCINO VIEIRA AND RAIMUNDO BRAZ-FILHO
433
The diterpenoids from the genus Sideritis FRANCO PIOZZI, MAURIZIO BRUNO, SERGIO ROSSELLI AND ANTONELLA MAGGIO
493
Recent developments in the asymmetric synthesis of lignans GIUSEPPE DEL SIGNORE AND OTTO MATHIAS BERNER
541
Natural oligostilbenes MAO LIN AND CHUN-SUO YAO
601
Isolation, structure elucidation and bioactivities of phenylethanoid glycosides from Cistanche, Forsythia and Plantago plants T. DEYAMA, H. KOBAYASHI (Late), S. NISHIBE AND P. TU 645 Pharmacological activities of phenylpropanoids glycosides MARINA GALVEZ, CARMEN MARTIN-CORDERO, MARIA JESUS AYUSO 675
x Development of tubulin inhibitors as antimitotic agents for cancer therapy S. MAHBOOBI, A. SELLMER AND T. BECKERS
719
Cholesterol biosynthesis inhibitors of microbial origin HYUN JUNG KIM, IK-SOO LEE AND SAM SIK KANG
751
Structure-activity relationships of curcumin and its analogs with different biological activities LI LIN AND KUO-HSIUNG LEE
785
The Vinca alkaloids: From biosynthesis and accumulation in plant cells, to uptake, activity and metabolism in animal cells MARIANA SOTTOMAYOR AND ALFONSO ROS BARCELO
813
The chemistry of Olea Europaea ARMANDODORIANA BIANCO AND ALESSIA RAMUNNO
859
The chemistry of the genus Cicer L. PHILIP C. STEVENSON AND SHAZIA N. ASLAM
905
New research and development on the Formosan annonaceous plants YANG-CHANG WU
957
Structural and functional aspects of fungal glycosphingolipids ELIANA BARRETO-BERGTER, MARCIA R. PINTO, MARCIO L. RODRIGUES
1025
Phytochemical studies and pharmacological activities of plants in genus Hedyotis/Oldenlandia NORDIN HJ. LAJIS AND ROHAYA AHMAD
1057
Subject Index
1091
xi
XI
CONTRIBUTORS Rohaya Ahmad
Faculty of Applied Science, University Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
Nelson Alvarenga
Departamento de Fitoquimica, Facultad de Ciencias Quimicas, Universidad Nacional de Asuncion, P.O. Box 1055, San Lorenzo, Paraguay
Shazia M. Aslam
Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, United Kingdom
Maria Jesus Ayuso
Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
A.K. Banerjee
Instituto Venezolano de Investigaciones Cientificas, (IVIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela
Alfonso Ros Barcelo
Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain
Eliana Barreto-Bergter
Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil
T. Beckers
Therapeutic Area Oncology, ALTANA Pharma AG, D78467, Konstanz, Germany
Otto Mathias Berner
Kemira Fine Chemicals Oy, P.O. Box 330, FIN-00101 Helsinki, Finland
Armandodoriano Bianco
Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali - Universita di Roma "La Sapienza", Piazzale Aldo Moro 5, Roma, Italy
Raimundo Braz-Filho
Setor de Quimica de Produtos Naturais, Universidade, Estadual do Norte l'luminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes Rio de Janeiro, Brazil
Maurizio Bruno
Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy
T. Deyama
Central Research- Laboratories, Yomeishu Seizo Co., Ltd., Minowa-Machi, Nagano 399-4601, Japan
xii Xll Esteban A. Ferro
Departamento de Fitoquimica, Facultad de Ciencias Quimicas, Universidad Nacional de Asuncion, P.O. Box 1055, San Lorenzo, Paraguay
Gerd Gade
Zoology Department, University of Cape Town, ZA-7701 Rondebosch, South Africa
Marina Galvez
Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
Guido Jach
Max-Planck-Institut fur Zuchtungsforschung, Carl von Linne-Weg 10, 50829 Cologne, Germany
Sam Sik Kang
College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul 110-460, South Korea
Hyun Jung Kim
College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea
H. Kobayashi (Late)
Central Research Laboratories, Yomeishu Seizo Co., Ltd., Minowa-Machi, Nagano 399-4601, Japan
Nordin HJ. Lajis
Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selango, Malaysia
M.S. Laya
Institute Venezolano de Investigaciones Cientificas, (IYIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela
Ik-Soo Lee
College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea
Kuo-Hsiung Lee
Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC27599-7360, USA
Li Lin
Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC27599-7360, USA
Mao Lin
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
xiii Xlll Antonella Maggio
Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy
S. Mahboobi
Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany
Heather G. Marco
Zoology Department, University of Cape Town, ZA-7701 Rondebosch, South Africa
Carmen Martin-Cordero
Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
S. Nishibe
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu-Cho, Ishikari-Gun, Hokkaido 061-0293, Japan
Marcia R. Pinto
Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil
Franco Piozzi
Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy
P.S. Poon
Instituto Venezolano de Investigaciones Cientificas, (IVIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela
Alessia Ramunno
Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali - Universita di Roma "La Sapienza", Piazzale Aldo Moro 5, Roma, Italy
M. Carmen Recio
Department de Farmacologia, Facultat de Farmacia, Universitat de Valencia Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain
Jose Luis Rios
Department de Farmacologia, Facultat de Farmacia, Universitat de Valencia Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain
Marcio L. Rodrigues
Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil
Sergio Rosselli
Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy
xiv XIV Thomas J. Schmidt
Institut filr Pharmazeutische Biologie der Heinrich-HeineUniversitlt Diisseldorf, Universitatsstrasse 1, D-40225 Diisseldorf, Germany
A. Sellmer
Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany
Giuseppe Del Signore
Institut fur Organische Chemie, Rheinisch-Westfalische Technische Hochschule, Professor-PMet-Str. 1, 52074 Aachen, Germany
Mariana Sottomayor
Department of Botany of Faculty of Sciences and Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alergre, 823,4150-180 Porto, Portugal
Philip C. Stevenson
Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, UK. and Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey, TW9 3AB, United Kingdom
P. Tu
School of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China
Ivo J. Curcino Vieira
Setor de Quimica de Produtos Naturals, Universidade, Estadual do Norte l'luminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes Rio de Janeiro, Brazil
Pavel F. Vlad
Laboratory of Terpenoid Chemistry, Institute of Chemistry, the Academy of Sciences of Moldova, Academiei Str. 3, Chisinau, MD-2028, the Republic of Moldova
Jochen Winter
Max-Planck-Institut filr Ztlchtungsforschung, Carl von Linne-Weg 10,50829 Cologne, Germany
Yang-Chang Wu
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Chun-Suo Yao
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Bioactive Natural Products
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Vol. 33 33 Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. © 2006 2006 Elsevier B.V. B.V. All All rights rights reserved. reserved. ©
FOCUS ON FLUORESCENT PROTEINS GUIDO JACH AND JOCHEN WINTER
Max-Planck-Institut fir Ziichtungsforschung Carl von Linne-Weg 10, 50829 Cologne, Germany ABSTRACT: A number of fluorescent proteins have been discovered in marine organisms with the green-fluorescent protein (GFP) from Aequorea victoria representing the first member of this family being isolated and well characterized. These polypeptides show marked differences in their spectral properties. Today, blue-, yellow-, cyan- and red-light emitting proteins are known in addition to GFP. These natural products possess the unique ability to autocatalytically form a cyclized />-hydroxybenzylidene-imidazolidinone structure acting as a light emitting chromophore in the presence of the suitable environment provided by Bcan 3D-structure of the proteins. Fluorescence actually represents the biological activity of these protein. In fact, this intrinsic protein property allows for their non-invasive non-destructive detection in living cells, an ability that has caused enormous interest on all areas of molecular biology employing reporter proteins for gene expression and protein localization studies. Today, GFP is widely used and well accepted as valuable reporter protein. However, during the last decade a number of GFP mutants were described showing altered spectral properties and/or improved solubility upon expression in heterologous systems. In addition, numerous other naturally occurring fluorescent proteins were described such as the red-fluorescent protein from Discosoma sp. (DsRED). A comprehensive description of the available fluorescent proteins is given including the spectral properties, amino-acid sequence alignments, comparisons of the secondary and tertiary structures of the proteins. The mechanisms for this self-catalyzed amino-acid modifications leading to the chromophore formation are best characterized for the GFP although some work was carried out on other proteins as well. This review describes the current knowledge about maturation and (possible) oligomerization of the proteins.
FLUORESCENT OVERVIEW
PROTEINS:
AN
HISTORICAL
Numerous marine invertebrates display an extensive palette of visible fluorescence and coloring. In part, the vibrant coloration is due to a growing family of intrinsically fluorescent proteins. Genes encoding fluorescent proteins have been isolated and described for a variety of coelenterates, both hydrozoa such as Aequorea, Obelia,
4
and Phialidium, and anthozoa such as Anemonia, Discosoma and Renilla. Amongst the fluorescent proteins known today the greenfluorescent protein (GFP) from the jellyfish Aequorea victoria not only represents the first member of this protein family being isolated it also is by far the best understood fluorescent protein. Its history dates back to 1962 when it was first observed as a companion protein to Aequorin (a blue-light emitting chemiluminescent Aequorea protein), exhibiting a bright, greenish fluorescence when excited with ultraviolet light [1]. Soon after that the first emission spectrum of GFP was published by the same group [2]. Interestingly, the light emission of GFP peaked at 508 run a value that is rather close to the green bioluminescence peak of living Aequorea tissue. In addition, the excitation spectrum of GFP showed a (secondary) peak around 470 nm, which overlaps with the blue chemiluminescence peak produced by Aequorin. Based on these data it was assumed that the green glow of intact Aequorea tissues results from the conversion of the blue light emission of Aequorin into green light by GFP. Later on similar color shifts were found in the related coelenterates Obelia (a hydroid) and Renilla (a sea pansy) and it was suggested that the in-vivo excitation mechanism for coelenterate GFPs is based on radiationless energy transfer [3]. The first prove for this assumption was provided by Morise and coworkers in 1974 [4]. They managed to purify and crystallize Aequorea GFP, and to demonstrate the efficient luminescence energy transfer between co-adsorbed Aequorin and GFP. In addition, the absorbance spectrum and fluorescence quantum yield was measured. The next important achievement was the determination of the chemical structure of the GFP chromophore in 1979 [5]. Protein fragments obtained by proteolytic digestion were screened for peptides retaining visible absorbance. Analysis of these chromopeptides finally lead to the (correct) proposal that the chromophore is a 4-(p-hydroxy-benzylidene)imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring. Final prove for the chemical structure of the chromophore was provided by Cody et al. [6].
5
Until 1999 Renilla GFP was the only biochemically well characterized fluorescent protein besides the Aequorea GFP. It proved to have a much higher extinction coefficient, resistance to pH-induced conformational changes and denaturation, and tendency to dimerize compared to Aequorea GFP [7]. However, chromophores from Aequorea and Renilla GFPs was shown to be chemically identical [7-9], indicating that the fluorescence properties of the proteins are depending on both, the chromophore and the corresponding environment provided by the amino-acid backbone. The successful cloning of cDNA encoding the Aequorea GFP by Prasher et al. in 1992 and the subsequent demonstration in 1994 that expression of this cDNA in other organisms is sufficient to produce green fluorescence probably are the most important milestones in the history of fluorescent proteins [10-12]. This not only demonstrated that the GFP gene contains all the information necessary for the posttranslational synthesis of the chromophore, and that no jellyfish-specific proteins enzymes are needed. It also caused enormous attention and interest amongst scientists because they instantly noticed the usefulness of GFP with its unique properties to act as a reporter allowing non-invasive and nondestructive detection of gene expression. This ability is a major advantage over commonly used reporter genes such as NPTII, CAT (detection by autoradiography), LUC (detection of light emitted from converted substrate) and GUS (detection of fluorescent or non-fluorescent dyes released from substrate). However, it rapidly turned out that expression of wildtype GFP often results in poor fluorescence yields and that the system is rather insensitive. Further investigations proved that thermosensitivity of GFP protein maturation is one of the major problems leading to the accumulation of improperly folded nonfluorescent, insoluble protein. [13] Several research groups addressed this problem and tried to identify GFP mutants possessing improved properties such as improved light emission properties (higher quantum yield and/or a extinction coefficient) and/or solubility, which both should increase the amount of detectable fluorescence considerably. In. addition, research also focused on the identification of GFP mutants with altered spectral properties (e.g. altered emission peak wavelength) in order to create
6
tools for simultaneous expression monitoring of separate genes or to obtain suitable donor - acceptor pair to investigate FRET (fluorescence resonance energy transfer). In fact, quite a number of different GFP isoforms with an enhanced or altered fluorescence phenotypes were created using various approaches to mutagenize the wildtype GFP coding region [14-21]. Currently, GFP mutants emitting blue, cyan and yellow light are available (BFP, CFP and YFP, respectively). In 1996 the three dimensional structures of crystallized wildtypeGFP and a GFP mutant carrying the chromophore mutation S65T were published almost simultaneously by two independent research groups [22, 23]. Until 1999 the Aequorea GFP remained the only cloned gene encoding a fluorescent protein. All the different GFP isoforms developed and used in molecular biology are derivatives of the GFP 10 cDNA cloned by Prasher and co-workers. Attempts to clone the gene for Renilla-GFV, which is supposed to be several times brighter than the Aequorea-GYV, were not successful. In 1999 Matz et al. reported the cloning of six novel genes from anthozoan species using a PCR approach with degenerate primers derived from conserved amino-acid sequences of known GFPs [24]. Alignments of the deduced amino-acid sequences of these GFP-like proteins with Aequorea GFP showed that the novel proteins share only a low or moderate degree of homology with the Aequorea GFP. The spectral properties of some of these proteins also were markedly different from GFP, with the greatest differences being found between GFP and a protein from reef corals {Discosoma sp.) emitting red light with a peak at 583nm (dsFP583). This protein soon was made commercially available under the name DsRED. About one year after its first description the three dimensional structure of DsRED and chemical structure of the its chromophore was described by two independent groups [25, 26]. The structural data and other experimental evidences proved that the DsRED protein is an obligate tetramer and that the chromophore is GFP-like but possesses an extended n-electron system due to an additional oxidation step [27, 28]. Due to its unique red-fluorescence the DsRED protein gained strong interest amongst researchers, but it soon turned out that the usability of this protein as a reporter is limited by several factors: its
7
tetramerization, its slow maturation rate and its tendency to form aggregates [28]. In order to overcome these limitations several research teams set out to improve the protein properties by means of molecular evolution. In fact, these approaches not only gave rise mutant proteins with faster maturation rates, lowered aggregation and abolished oligomerization, but also to spectral variants emitting far-red light [29-33]. The usability of these DsRED mutants as reporter proteins remains to be seen. hi 2002 Labas and co-workers investigated numerous Anthozoan species and were able to clone 11 new fluorescent proteins with colors ranging from green to red [34]. An additional protein (eqFP611) emitting far-red light was isolated and cloned from Entacmaea quadricolor another anthozoan species [35]. Today 28 members of the family of fluorescent proteins are cloned. Currently research in this field is very active and it is expected that the number of cloned genes encoding fluorescent proteins will grow even further in the near future. NOMENCLATURE During the last two to three years a huge number of new fluorescent proteins were isolated from various organisms and it is now obvious that a unified nomenclature is ultimately needed to distinguish between these proteins and provide them with unique names, hi addition, a unified nomenclature will help to overcome problems arising from the fact, that commonly used abbreviations like BFP, CFP and YFP for blue, cyan and yellow fluorescent proteins are also used in the literature to name completely unrelated proteins such as an E. coli outer membrane lipoprotein related to bundle forming pili (BFPb), a virulence factor (BFP), a cercosporinfacilitator-protein (CFP), culture filtrate proteins (CFPs) from M. tuberculosis , a fluorescent protein from Vibrio fischeri (YFP) [3640], to name only a few examples. Even more confusing is the common practice to provide commercially available FPs with new trivial names resulting in a situation where certain FPs appear under three different names in literature. In 1992, when the first cDNAs encoding the green fluorescent protein (GFP) form Aequorea victoria were cloned names were created by simply adding numbers (probably the number of the
8
clone investigated) to the name of the gene resulting in GFP1, GFP3 and GFP10 [10]. Subsequently, researchers have only worked on the GFP10 cDNA, which therefore represents the ancestor of all Aequorea -GFP known today and is referred to as wildtype - GFP (andnotGFPIO). With green fluorescent proteins becoming available from other organisms scientists started to add the initial letters of the latin names of the organisms as a prefix to the name of the gene. However, this system is not used consequently. Hence, in the literature the wildtype - GFP is also known as AvGFP, but even nowadays in the majority of publications only the term GFP is used for the GFP 1-gene form Aequorea victoria. Furthermore, the system possesses some severe limitations. Since 1994 quite a number of isoforms of the GFP1 protein were created by directed and random mutagenesis in order to improve protein properties. Due to the lack of a defined nomenclature arbitrary names were given to these genes and proteins such as mGFP4, mGFP5, rsGFP4 and smGFP(for modified-GFP, redshifted-GFP and solubility-modified-GFP [13, 41, 42]. Because it is not obvious that these genes are derivatives of the Aequorea victoria GFP1 gene they might be confused with genes from a different organisms named using the above-mentioned system. In addition, problems arise in cases where two organisms share the same initial letters. More recently fluorescent proteins with different colors became available and researchers started to build names for fluorescent proteins and their corresponding genes by using the abbreviation FP (for fluorescent jyotein in contrast to the non-fluorescent colored chromoproteins (CP)) followed by a number indicating the peak wavelength of light-emission [24,43,44]. This nomenclature is also limited since two different fluorescent proteins from different (or even the same) organism can share the same emission peak wavelength resulting in the same name for both of them. Adding the abbreviated latin-name of the source organism as a prefix would help to distinguish between the two proteins, but only if they are originating from different organisms and/or organisms with different names (= abbreviations). To overcome the above-mentioned problems and limitations a novel nomenclature for fluorescent proteins and chromoproteins
9
was proposed in a recent paper [34]. According to this nomenclature the name for a fluorescent protein and its corresponding gene is build by combining a four-letter leader composed of the first letter of the genus name and three initial letters of the species name, followed by definition of color type: BFP, blue, CFP, cyan, GFP, green; RFP, red; YFP, yellow; and CP, chromoprotein (nonfluorescent). Thus a fictitious green fluorescent protein from the fictitious organism "Propose nomenclature " would get the name "pnomGFP". For undefined species the four initial letters of the genus name serve as leader. In the case of multiple unidentified species of the same genus, a number is added to the leader (resulting in "prop 1 GFP", "prop2GFP" etc. for the mentioned fictitious example) In the case of several proteins of the same color type found in the same species, the number is added to the color definition (such as in "pnomGFP 1" and "pnomGFP2"). For A. victoria GFP and drFP583 from Discosoma sp., widely accepted common names, GFP and DsRed, are kept. PROPERTIES OF FLUORESCENT PROTEINS Basks and definitions In this review the term fluorescent protein refers to proteins being able to autocatalytically form a chromophore, thus possessing the intrinsic property to emit light without the need for any substrate, prosthetic group or cofactor. It is important to notice that numerous other light-emitting systems were found in nature. Besides the various classes of marine organisms mentioned above several other bioluminescent species also have emission-shifting accessory proteins, but so far the chromophores all seem to be external cofactors such as lumazines or flavins [40, 45]. Likewise phycobiliproteins and peridininchlorophyll-a protein, which are highly fluorescent and attractively long-wavelength accessory pigments in photosynthesis, use tetrapyrrole cofactors as their pigments [46, 47]. In the literature of fluorescent proteins the terms fluorescence and brightness are often used as synonyms to describe the detected light emission on protein or cell level. Since the usage of these terms is not consistent literature sometimes is misleading especially when it
10
comes to the companion of different GFPs in terms of their "performance" as reporter proteins. In this review the following terms and definitions are used throughout: •
(molar) Fluorescence = (molar) extinction coefficient * quantum yield
•
Apparent fluorescence (brightness) = (molar) extinction coefficient * quantum yield * protein-amount (= fluorescence * protein-amount)
•
Specific fluorescence = (molar) extinction coefficient * quantum yield / amount of protein (= fluorescence/amount of protein)
Protein and chromophore maturation All GFP-like proteins exhibit a p-can motif, formed by 11 ^-sheets, Fig.(l). Several short a-helical segments connect these strands, while one central helix contains the imidazolidinone chromophore. The chromophore is completely encapsulated in this cylinder, thus physicochemically very stable [48]. The a-helical caps at the top and bottom of the p%can support chromophore protection. The neighborhood of the fluorophore contains a number of charged residues and four water molecules to establish hydrogen bonds [49]. The first step in maturation of GFP is the correct folding into the native conformation, with the chromophore being non-fluorescent. Once folding is complete, the tripeptide chromophore motif is deeply buried in the central helix. This 3D-structure presumably promotes maturation of the fluorophore. The fluorescent chromophore is sequentially formed autocatalytically by two distinct chemical processes [50]. Cyclization proceeds through a nucleophilic attack of the amide group of Gly 67 being in close proximity to the carbonyl residue of Ser65, thus leading to a fivemembered imidazolinone ring intermediate. The secondary structure seems to play an important role in the maturation of the chromophore by facilitating attack of the poorly nucleophilic aniido nitrogen of Gly67 on the poorly electrophilic peptidic carbonyl
11
group of Ser65 [48]. Interaction of Arg96 with the carbonyl oxygen of the fluorophore is supposed to activate ring closure and the elimination of a water molecule. In the last step oxidation of the hydroxybenzyl side chain of Tyr66 by molecular oxygen produces the active fluorophore /?-hydroxybenzylideneimidazolinone [49], Fig. (2). The -C=N- double bond of the imidazolidinone moiety is very likely to support the formation of a -C=C- double bond between C2 and C3 of Tyr66 by spontaneous dehydrogenation in the presence of molecular oxygen [48]. This so formed conjugated 71electron resonance system accounts for the fluorescent characteristics [48]. Kinetic studies on chromophore formation suggested a fast protein folding and cyclization process, while oxidation being rate limiting. This model based on elegant experiments comparing the rate of renaturation of
GFP Fig. (1). The B-can-structure of fluorescent proteins
DsRED
12
reduced and non-reduced GFP. Fluorescence develops much faster under non-reduced conditions with the chromophore structure already been formed [50]. The red fluorescent protein (DsRed) from a Discosoma coral forms a GFP-like chromophore structure but with an extended conjugated 71-electron system, thus leading to a red-shifted spectrum. The fluorophore-containing tripeptide consists of amino acids Gln66-Tyr67-Gly68, Fig. (3). Interestingly, DsRED chromophore maturation proceeds via a green-light emitting intermediate state. Therefore, a mechanism similar to GFP was suggested for the first steps of chromophore maturation, with the imidazolinone moiety being derived from Gln66 and Gly68. The final dehydrogenation step then introduces an additional double bond thus extending the 7C-electron system of the chromophore. Two extraordinary features support such extended rc-system: 1. The Ca of Gln66 is observed to be sp2 hybridized, thus creating a double bond with the peptide N-atom of Gln66. The Ca at the identical amino acid residue in GFP exhibits a normal tetrahedral configuration. 2. Phe65 is connected with Gln66 by a unique cis peptide bond which positions the sp2 Ca of Gln66 in the same plane as the chromophore. The analogous peptide bond in GFP is trans configurated [25]. Dehydrogenation of Gln66 requires molecular oxygen, while possible H2O2, presumably generated by the first oxidation step, is ineffective in forming the double bond [27]. Spectral tuning (Effects of mutations)
With regard to spectral tuning of the chromophore by its protein environment, two classes of mechanisms seem to operate in FPs. First, covalent modification of the chromophore through extension of the system of conjugated electrons or through introduction of charge can affect spectra by fundamentally altering the chromophore resonance properties. For example, deprotonation of the phenolic oxygen of the GFP chromophore causes a dramatic red-shifting of chromophore absorbance nearly 100 nm), and is the mechanism of the fluorescein-like 489 nm absorbance peak of GFP. Second, manipulation of electrostatic interactions between the chromophore and its surrounding protein environment
13
folding lOmin)
cyclization
~N
V
dehydration
oxidation (t 1/2 ~20-83min)
H2O2?
Fig. (2). Proposed mechanism for the GFP chromophore formation
t,/2~3min
14 14
folding cyclization dehydration oxidation
dehydration
GREEN
RED Fig. (3). Proposed mechanism for the DsRED chromophore formation
changes the spectra due to selective interaction with photoexcited or ground states. In GFP substitutions of the second position of the chromophore (Tyr66) directly affect spectral properties of the protein, since the aromatic 7i-electron ring system of the hydroxybenzylidene moiety has an important influence on the fluorescence. Replacement of Tyr66 by Phe (Y66F), which lacks an electron donor group on its aromatic side chain, produces blue-shifted excitation and emission spectra (excitation: 358nm, emission: 442nm). In the mutant Y66H (today known as BFP, see below) the imidazole feature of His with
15 15
its stronger ability to withdraw electrons shows a hyperchromatic effect (excitation: 382nm, emission: 448nm), Fig. (4). Presence of the indole side chain of tryptophane in the mutant Y66W (CFP, see below) shifts the spectrum to 433nm and 457nm for excitation and emission, respectively, Fig. (4). The most dramatic effect on fluorescence is derived from the charged phenolate of Tyr66. Up to now pointmutations substituting amino-acid residues of the chromophore leading to altered spectral properties have only be described for Aequorea GFP. However, mutation located outside the chromophore can also change the spectra of fluorescent protein, indicating that the chromophore environment provided by the fi-can structure of the protein is of equal importance, Fig. (5). It is obvious that mutations replacing Ser65 of wtGFP are not directly involved in the conjugated 7t-electron system of the fluorophore. Nevertheless, introduction with aliphatic residues such threonine, alanine, or glycine at this position leads to mutant proteins with red-shifted excitation spectrum and higher molar extinction. These substitutions are thought to prevent Glu222 from deprotonation due to a change in the hydrogen-bonding network around the chromophore. This uncharged Glu leads to a complete ionization of Tyr66 with a phenolate moiety [49]. In the T203Y mutant of GFP, stacking the it-electron system of the tyrosine side chain on the rc-electron system of the chromophore causes red-shifting due to relative stabilization of the photoexcited state to produce yellow fluorescent protein Fig. (4). Not only for GFP, but also for the red fluorescent protein (DsRED) non-chromophore mutations were described altering its spectral properties. The underlying mechanisms, however, are less well understood. Replacing Lys83 by Arg, Glu, Asn, Pro, Phe, Trp (W) and Met results in proteins emitting green light (peaking at 499nm) instead or in addition to the red emission peak at 582nm [28]. Amongst these exchanges the ration of red to green light emission varies considerably. In the mutant K83R the red emission peak is completely abolished. Obviously the second dehydrogenation step required to form the complete DsRED chromophore either is blocked or very inefficient in these proteins. Thus the chromophore formation stops after the first maturation step resulting in a green-light emitting GFP-like chromophore [28].
16 16
GFP
O
GFP-Y66H (BFP)
HN
11
=\
O
V - NH HO —f
N j»
O
GFP-Y66W (CFP)
GFP-S65G/T203Y (YFP)
DsRED NH,
x.
Fig. (4). Known chromophore structures of fluorescent proteins
17
v
T203
S205
E222
Fig. (5). GFP-chromophore and its environment Environment of the GFP ehromophore according to [22] (modified). Side-chains are marked with the one-letter code for the amino-acid and the residue number, whereas groups of the main chain are labeled with the residue number alone (in italics). Hydrogen bonds are shown as dotted lines.
Introduction of the mutations K70R, K70M,Y120H and S197T into the wildtype DsRED protein shows similar results. The "fluorescent timer" is another DsRED mutant with the unique ability to change its light emission peaks from green to red over time. This property results from the pointmutations VI05A and S197T present in "fluorescent timer" [32]. Green fluorescent proteins The proteins of this class and especially its first member, the Aequorea GFP are by far the best understood fluorescent proteins,
18
often serving as model to understand or explain the properties of other fluorescent proteins. Therefore these proteins were chosen the first to be discussed in this review. For a long time the GFPs from Aequorea victoria and Renilla reniformis were the only well characterized proteins, with Aequorea GFP gaining more importance for molecular biology due to the availability of the corresponding cDNA. Meanwhile also genes encoding Renilla GFP became available. Recently a series of other naturally occuring GFP proteins were isolated and spectroscopically analyzed [34]. Table 1 provides an overview over these proteins. It is important to note that in the original publication three additional GFPs (amajGFP, dstrGFP and clavGFP) were described. With emission maxima around 486nm, however, their spectral properties are much more related to those of the cyan fluorescent proteins (CFPs). Consequently, in this review these proteins will be referred to as amajCFP, dstrCFP and clavCFP and will be discussed in the corresponding chapter. Over the last decade quite a number of different recombinant isoforms of Aequorea GFP with improved fluorescence and folding properties were generated and described in literature [14-21]. Commonly used GFP isoform are listed in table 2 together with their spectral properties and the described improvements. Unfortunately, a closer look reveals that some of these published data are inconsistent, best visible upon comparison of the experimental data describing the relative fluorescence of the proteins between each other (on protein and cellular level) as well as with the data describing their extinction co-efficients and quantum yields. When reading the original publications, it becomes obvious that quite different experimental setups were used by the different researchers to measure protein concentrations and fluorescent properties of the recombinant GFPs, some of which appear to be errorprone. This might explain the observed inconsistencies. Unfortunately, a comprehensive, comparative study investigating these proteins under similar experimental conditions is lacking.
19 Spectra
For the majority of GFPs knowledge about the mechanisms and processes responsible for the fluorescence of the proteins is lacking. However, in this respect the Aequorea GFP and its derivatives were investigated intensively and certainly can serve as model for the other proteins [48, 49]. Depending on the chromophore-structure three different types of spectra can be distinguished for wildtype and mutant Aequorea GFP, Fig. (7). Based on these findings Tsien defined three classes of Aequorea GFPs [51]. Class-1 proteins carry the wildtype chromophore and display the typical type-1-spectra. These spectra are complex in that there is are two excitation peaks (usual a major and a minor one) and a single emission peak. In class-2 proteins the chromophore contains a phenolate anion and the excitation-spectra is "red-shifted": the major excitation peak at 399nm disappeared and the amplitude of former minor peak is increased several fold resulting in a type-2- spectrum. Class-3 proteins contain chromophores with neutral phenol. In the resulting spectra (type-3) the minor excitation peak disappeared leaving the major peak at 399nm alone. These proteins still emit green light. Labas and co-workers analyzed the spectral properties of various GFPs isolated from non-bioluminescent anthozoan species with emission peaks between 499 and 516 nm [34]. Interestingly, all anthozoan GFPs displayed spectral properties similar to the type-1 and -2 spectra of Aequorea GFP, Fig. (7). Apparently, these types of spectra can be used in general as a simplified system to group the proteins. However, whether or not the chromophore structures of the type-1 and -2 anthozoan GFPs are related to the corresponding chromophores of Aequorea GPF remains to be determined. As already mentioned wildtype Aequorea GFP possesses a class1 chromophore resulting in a complex type-1-excitation/emission spectrum with a major excitation peak at 395 nm and a three times smaller minor excitation peak at 475 nm. Interestingly, the wavelength of emission peak varies slightly with the excitation wavelength used: excitation at 395 nm results in a single emission peak at 508 nm, whereas excitation at 475 nm gives maximum light emission at 503 nm [52]. In addition, it was found that at alkaline pH values (pH 10-11) the amplitude of the
498 500 500
2 2 2
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * Recombinant isoforms of Aequorea GFP are described in table 2.
506 497
2
-
Scolymia cubensis
scubGFPl
518
508 2
-
Ricordea florida
516 506
2
rfloGFP
508
494 2
-
Dendronephtya sp. Montastraea cavernosa
dendGFP
512
503
2
-
Discosoma sp.3
dis3GFP
mcavGFP
499 (403)480
2
-
nd
nd
506
506
506
500
Ammonia sulcata
nd
nd
nd
nd
496
510
500
2
2
508
405(481)
1
508 496
395(471) 399 (482)
1 1
Peak wai elength's (nm) Em. Ex.
asulGFP (asFP499)
K5E, K10E, N66M
Zoanthus sp.
zoanGFP (zFP506) (zsGreenl)
zoanGFP-0 (ZFP506-N66M-NA)
-
Renilla reniformis
rrenGFP2 (hrGFP)
N66M
-
zoanGFP-I (ZFP506-N66M)
-
Renilla muelleri
-
Ptilosarcus sp.
ptilGFP Renilla reniformis
-
Heteractis crispa
hcriGFP
rmueGFP
-
Aequorea victoria Condylactis gigantea
Type of Spectra
Mutation
GFP*
Organism
cgigGFP
Protein
Cloned green fluorescent proteins
rrenGFPl
Table l.
nd
nd
nd
nd
nd
nd
nd
nd
35600
nd
nd
nd
nd
nd
-
nd
-
-
nd
-
-
1,8
1,8
1
-
nd
nd
nd
nd
nd
nd
0,63
nd
-
nd nd
-
-
-
-
rel. spec. FL
nd
nd
nd
0,80
27600 nd
QY
EC
[34]
[34]
[34]
[34]
[34]
[43]
[55]
[55]
[24]
[54]
[53]
[53]
[34]
[34]
[34]
[10]
Ref
20
21 Table 2. Protein
Properti es of ccimmon isoform s ofAe quorea (IVY Mutation
GFP(gfplO)
Pe ik wavele ngth's (nrn) Em. Ex. 395 (471)
EC
508
25000 30000 25000 30000
GfplO.l
Q80R
395 (471)
508
S65C
S65C
479
507
QY
EC*C
rel.
rel. spec. FL** Prot/Cell
Ref.
0,79
1975023700
1
1/1
[10]
0,79
1975023700
1
1 /I
[11]
6/nd
[17]
0,64
33280 37120
1,6
6/nd
[17]
S65T
S65T
489
511
52000 58000
GFPmutl (EGFP)
F64L S65T
488
508
56000
0,60
33600
1,5
35/39
[18]
GFPmut2
S65A V68L S72A
484
508
n.d.
n.d.
-
-
19/85
[18]
GFPmut3
S65G S72A
504
512
n.d.
n.d.
-
-
21 /77
[18]
Cycle3GFP (smGFP)
F99S M153T VI63 A
398 (480)
508
30000
0,79
23700
1,1
nd/42
[14]
mGFP5
VI63 A I167T S175G
395 473
510
n.d.
n.d.
-
-
nd/20
[13]
mGFP5(S65T)
S65T VI63 A I167T S175G
475
510
n.d.
n.d.
-
-
nd/33
[13]
GFPS65T/S147P
S65T S147P
496
512
n.d.
n.d.
-
-
(6*S65T)
[15]
smRS-GFP
S65T F99S VI63 A I167T
n.d.
n.d.
n.d.
n.d.
-
-
1,68 /nd
[42]
Emerald
S65T S72A N149K M153T I167T
487
509
57500
0,68
39100
1,8
nd/ (8*S65T; 5*EGFP)
[51]
18/
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * for bacteria grown at 37 °C, ** as measured for protein-samples (Prot.) or whole cells (cell)
475-nm excitation peak increases at the expense of the 395-nm peak [9]. It was concluded that purified GFP protein samples are actually protein populations consisting of molecules containing either
22
deprotonated/ anionic chromophores giving rise to the 475-nm peak or protonated/ neutral chromophores responsible for the 395-nm peak [19, 52]. Since phenols almost always become much more acidic in their excited states it has to expected that the protonated/neutral chromophore will be deprotonated in the excited state. This light-induced ionization of the chromophore to the anion was proven experimentally [56] and explains why excitation of the neutral chromophores gives emission at greater than 500 nm, similar to but not quite identical to the direct excitation of anionic chromophores. Class-2 GFPs with phenolate anions in the chromophore are commonly used as reporters in molecular biology because they combine high brightness with simple excitation and emission spectra peaking at wavelengths very similar to fluorescein, a popular small-molecule fluorophore. The ionization of the phenol group of the chromophore is caused by a replacement of Ser65 by Thr, Gly, Ala, Cys, or Leu, with S65T being the most commonly used mutation [13, 17, 18, 41]. The GFP mutant RSGFP4 carrying the triple mutation F64M, S65G, Q69L, shares the same fluorescence phenotype. In both S65T and RSGFP4, the wildtype 395-nm excitation peak is suppressed due to the neutral phenol, whereas the 470nm peak is enhanced five- to sixfold in amplitude and shifted to 489^90 nm due to the anion [17, 41, 57]. The probable mechanism by which replacement of Ser65 promotes chromophore ionization [58, 59] is that only Ser65 can donate a hydrogen bond to the buried side chain of Glu222 to allow ionization of that carboxylate, which is within 3.7 °A of the chromophore. Gly, Ala, and Leu cannot donate hydrogen bonds, and Thr and Cys are too large to adopt the correct conformation in the crowded interior of the protein. Such residues at position 65 force the carboxyl of Glu222 to remain neutral. The other polar groups solvating the chromophore are then sufficient to promote its ionization to an anion, whereas if Glu222 is an anion, electrostatic repulsion forbids the chromophore from becoming an anion as well. The chromophore of class-3 proteins contains a neutral phenol group due to repression of chromophore ionization caused by the mutation Thr203 to He [52, 60]. Presumably a chromophore anion
DTLVNRIELK--'GIDFKEDGNILGHK VQCF SRYPDHMKQHDFFKSAMPEG-YVQE- RTIFFKDDGNYKTRAEVKFEG Q. . .K. . . .G - . 1 . .--.M I. . .A. . .E. . .MN NRAYTG..EEI--S.Y.LQSF...-FTY.- . N. RYQ. G. TAIVKSDISL. D GKFIVNVDF. - •AK. LRRM. PVMQQD NRT.TK..EDI—S...IQSF.A.-F.Y.- ..LRYE.G.LVEI.SDINLIE EMF.Y.V.Y.-- .RN.PN..PVMKKT NRT.TK..NDI--S.Y.IQSF.A.-FMY.- ..LRYE.G.LVEI.SDINLIE .KF.Y.V.Y.-- .SN.PD..PVMQKT NRT.TK. . .DI--A.Y.VQSF.A. -FFY. .NLR.E.GAIVDI.SDISL.D .KFHYKV.YR-- . NG. PSN. PVMQKA IKV.AK..KEI--P....QSL.G.-FSW.- . VSTYE . G. VLSATQ. TSLQ. .CIICKVKVL--. TN. PAN. PVMQK. NKV.AK..—KDHP....QSL...-FTW.- .VSNYE.G.VLTVKQ.TSL.. .CIICK.KAH-- . TN. PA. . PVMQKR NKV.TD...DI--P....QSLSD.-FTWR- .VS*Y**G.VLTVTQDTSLK. .CIICN.KVH-- .TN.P.N.PVMQN. NRV.AK..EDI—A.Y..QTF...-.FW.- . SMTYE. Q. ICIATNDITMMEGVD. CFAYK. RFD- -.VN.PAN.PVMQR. NRA.VN. .KDIPDIFKQTCSG.D.-GFSWQ ..MTYE.G.VCTASNHISVD. ..FYYV.RFN-- . EN. PPN. PVMQKR NRV.TE..ADI--T.Y..QSF...-.SW.- ..MTYE.K.ICTI.SDISL.. .CFFQN.RFN-- .MN.PPN.PVMQK. NRA.TE. .TEIADYFKQSFEFG..-FSW.- .SFT.E.GAICVATNDITMV. GEFQYD.RFD-- .LN. * *. .PVMQK. NRA.TE..KEISDYFKQSFEFG..-FTW.- .SFT.E.GAICVATNDI.MV. .EFQYN.RFD-- .VN.P.. . * *MQK. NRA.VN..EDIPDIFKQTCSG.N.G.SWQ- . .MTYE.G.VCTATSNISW. . .FNYD.HFM—.AN.PL. . PVMQKR NRV.TE..QDI--V.Y..NSC. A. - . TWD-.SFL.E.GAVCICN.DITVSV EENCMYH.S.FY.VN.PA..PVM-K.
GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP
Fig. (6). Alignment of the amino-acid sequences of cloned naturally occurring GFPs
M—SKGEELFTGWPIL VE LDGDVNGHKFSVSGEGEGDATYGKLTLKF-ICT-TGK-:LPVPWPTLVT-T-FSYG .-I. .V. 1. H R D. . . .El ..-...-.-LG. . .--DLAKLGLKE.M.TK IN .E.L.GD.A..ME.V...NILE.TQEV.I-SV.-K.A-:PLPFAFDI.SVA-.... .V-..QILKN..LQE.MSFK.N .E.V..N.V.TME.C.K.NILF.NQLVQI-RV.-K.A- PLPFAFDILSPA-.Q.. .--..QILKN.CLQEVMSYK.N .E.I..N.V.TME.C.K.NILF.NQLVQI-RV.-K.A- PLPFAFDI.SPA-.Q.. PLPFAFDI.SIA-.Q.. .--NRNVLKN. .LKE.M SAKASVE . I. .N.V. .ME . F .K.NVLF .NQLMQI-RV. -K.GPLPFAFDILSHA-.Q.. . YPSIKETMRVQ LS ME.S. .Y.A.KCT.K. . . KPYE .TQS .NI-TI. -E .GPLPFAFDILSHA-.R.. . CSYIKETMQSK .Y ME.K. .D.N.KCTA. .K . EPYK. SQS . TI-TV. -E .GPLPFAFDILSHA-.Q.. . YPWIKETMRSK .Y ME. . . .N.A.KCTAV. . .KPYK.SQD.TI-TV. -E.GPLPFAYDIL.-.V.D.. ME.A V. E . D.K. KPFD.TQ.MDL-TVI-E . A.--TSVAQEKGVIK.DMKMKLR .--.ALK. EMK.K LK MV.V. . .QS.QID. . . K . KPYE . SQK. T L - E W - E PLLFSYDIL.-.I.Q.. . G..FSYDI.T.-A-LH.. . N.IKEDMRVK .H ME.N. . . .A.VIE. . .K.RPYE.TQ. .NL-TVK-E.AP .—QRAGMKVKEHMK. K LR MG.T. . .KH. A.N. T . D. YPYQ. .QI. .L-.VEGSEP-..FAFDI.SA-A-.Q.. ..FAFDI.SA-A-.Q.. . QSAGKKNWKDFMK . T LR M. .A. . . KP . A. N. T . D .NPYG . IQS . .L-TVD-GN.P LT ME.V. . . LP . KIR. D.K.KPYQ . SQE . T L T W K - G..FSYDI.T.-M-.Q.. . P.--.ALK. EMK.N .—AQSKHGL . KEMTMK YR M E . C D . . . .VIT. . . I. YPFK . . Q A I N L C W E - ..FAEDI.SA-A-.N.. G . P-
GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl SCubGFP2 dis3GFP zoanGFP
23
Alignment of the amino-acid sequences of cloned naturally occurring GFPs
VLLEFVTA-AGITH--GMDELYK-* .F. . .FS.-C.H. .--. QHETAIA.-HSTIK--KIEGSLP-. TAIAQL.S-L.KPL—.SLHEWV-. TAIAQM.S-I.KPL--.SLHEWV-. TAIAQL.T-I.KPL--.SLHEWV-. EQH. S . V. -SYSQV--PSKLGHN- . EQH.N.R.-S YFNDSG . - . KQH.Y.V.-SYSKV--PSKIGRQ-. .K.HEHAE-.--R.--.LSRKA.-. LSEDA.AH-NSPLE--KKSQAKA-. ARYSPLPK-S.LVEVQ.KAIMTA-. K.R.HAK.RSSLSP--TSAKER.A. K.Q.-YAK-.RSGL—HLP..Q.-. E.T.VAE.-RYSSL--EKIGKS.A. NQKWHL.E-HA.AS--.--SALP-.
GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP
Fig. (6). (cont.)
LEYNYNSHNVYIMADKQKNG-IKVNFKIRHNIEDGSVQLADHYQQN-TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHM . . . .F P..AN..-L G.G T.-V.L I.I L.T.I...R..T.... IVGMQP.YESMYTNVTSVI.-ECIIAFKLQTGKHFTYHMRTV.KSK-K.VETM.LYHFIQ. R. VKTNVDTASGYW ITGLQP . FE . VY. N. GVLV. -QVILVYRLNSGKFY. C H M R T L M K S K - G W K . F . EYHFIQ. R. --EKTYVE . GGFVEQ. E ILGIEP . FEAMY. NNGVLV. -EVILVYKLNSGKYY. C H M K T L M K S K - G W K E F . SYHFIQ. R. --EKTYVE. GGFVEQ. E ILGMEP.FE.VY.NSGVLV.-EVDLVYKLESGNYY.CHMKTF.RSK-GGVKEF.EYHFIH.R.--EKTYVEEGSFVEQ.E G.-LLLRDTPALMLA. . --GHLSCFMET- . YKSKKE .K. . EL .FHHLRMEKLNISDDWK-TV TCGWEP.TETV.PR. TNGWEP. -TETVIPRGGGIL-MRDVPALKLLGNK.HLLCVMETTYK-SKKKGE.AKPHFH.LRMEKDSV. DDEKTI TDGWEP . STETVI--P . DG. - . VAARSPALRLR. KGHLICHMETTY-K. N K E — . K. . EL. FHHLRMEKLSVSDDGK-TI TLKWEP.TEIMYAR.---GV-L.GDVNMALLL.G.GHYRC.--FKT-.YKAKKV.R...Y.FV--DHRIEIVSHD.DYNK TVKWEP.--TE..FER-DGL-LRGDIAMSLLLKG.GHYRC.FKTIY-..—KRK.NM.GY.FVDHCIEIQ.HDKDYNMAV TLKWEP. TEKLHVR. GLLV. N. NMALLLEGGGHYLCDFKTTYKAKK-WQLPDYHFVDHRIEILSNDSDYNKVKLYEHGV TVKWEP . TEIMY. .NGV-L . GEVNMALLL* . K. HYRC . LKTTY-KAKNNV. -HP. GY. .VDHCIEI LE.RK. .V . GGV-L. GEVNMALLLK. K.HYRC . FKTTYKAKNPVP .TA. . *Y. .VDHCIEIT E.N. . YV TVKWEP.TEIMRV TMKWEP.—TE..FER-DGM-LRGDIAMSLLLKG.GHYRC.FETIY-K.--NKV.KM..Y.FVDHCIEIT-SQQDYYNW MTD.WEPSCEK.IPVPKQGI-L.GDVSMYLLLK..-GR.RCQFDTV-YKAKSV.RKM..W.FI--.HK.TRE—DRS.AK
GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP
24
25 wavelength (nm) 350
400
450
500
550
600 650 I' ' '
1
• • • I i
1,0-
••• Excitation ~~ Emission
0,8type-1-GFP
0,6 0,40,2 0,0 1,08
0,8
•£
0,4-
I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' type-2-GFP
0,2 0,0
' ' • • I
1,00,8 0,6 0,4 0,2 0,0
Fig. (7). Reference spectra for type 1,2 and 3 - GFPs
type-3-GFP
26
cannot be adequately solvated once the hydroxyl-group of Thr203 is gone, so the chromophore is neutral in almost all the ground-state molecules. However, the emission is still at 511 nm because the excited state remains acidic enough to eject a proton. Factors affecting protein folding and maturation
When expressed at temperature ranging from 20 to 23 °C protein maturation of wildtype GFP is quite efficient, whereas at higher temperatures accumulation of improperly folded non-fluorescent, insoluble protein increases dramatically [13]. This effect was called thermosensitivity. Furthermore it was found that only the folding process of the protein is sensitive to higher temperatures. Once matured GFP is stable, soluble and fluorescent at temperatures up to 65 °C. GFP isoforms only containing chromophore mutations such as S65T, S65C etc. show a similar behavior, indicating that these amino-acid exchanges do alter the spectral properties of the proteins but do not significantly alter the initial protein folding required to form the typical B-can-structure (Jach, unpublished). Nevertheless, chromophore mutations do lead to faster maturation of the chromophore itself (about 4 times), which is supposed to happen once the initial folding is finished [52]. Since Aequorea will probably never encounter warm water the thermosensitivity of GFP most likely is of no consequence under natural conditions. Some researchers used the poor maturation efficiency of GFP at higher temperatures in pulse-chase experiments following the fate of fluorescent protein matured at low temperatures after restoration of normal warmth and simultaneous suppression of new fluorescence [61, 62]. So far nothing is known about the occurrence of thermosensitivity effects in case of GFPs from other marine organisms, table 1. However, these proteins have only recently been described and knowledge about their biochemical properties is rather limited. This point will certainly be addressed in future research on these proteins. The demonstration that the expression of GFP cDNA is sufficient to produce green fluorescence in heterologous organisms has caused enormous interest in the scientific community to use the protein as a reporter for non-destructive monitoring of gene expression. However, it quickly turned out that thermosensitivity of
27
GFP protein maturation clearly limits the sensitivity of this system. This problem was addressed in several independent approaches employing various protocols such as DNA shuffling and errorprone-PCR to create and screen randomly mutagenized GFP proteins for improved properties. These approaches gave rise to the GFP isoforms cycle3GFP, GFPmutl (=EGFP), GFPmut2, GFPmut3, mGFP5, and GFP-S65T/S147P [13-15, 18]. The individual point-mutations present in these GFP derivatives are given in table 2. It appears that in all cases two to three amino-acid exchanges are required to improve the protein folding behavior. Interestingly, some point-mutations (M153T and especially VI63 A) were found in independent experimental approaches by several groups, indicating that they may play a major role in GFP folding [63]. As a matter of fact folding mutations do not increase the intrinsic light emission properties of GFP molecules given by the product of extinction coefficient and fluorescence quantum yield. They rather increase the percentage of properly matured molecules under adverse conditions, such as elevated temperatures (e.g. 37°C) and protein concentrations that promote aggregation [64-66]. However, under conditions not severely hampering the GFP folding (e.g. low temperatures) the presence of known folding mutations (as single mutations or in various combinations) has little or no beneficial effect due to the obvious fact that folding efficiency cannot exceed 100%. Additional research proved that the combinatorial use of folding and chromophore mutations is possible leading to protein isoforms with markedly improved apparent fluorescence as for example in the variants mGFP5(S65T), smRS-GFP and Emerald (see table 2) [51]. The beneficial effects of both sets of mutations and their apparent additive effect, suggests that they may play separate roles in the folding or maturation process. Knowledge about the recently described Anthozoan GFPs is still rather limited and chromophore or folding mutations have not yet been described for the these proteins. Factors affecting the mature proteins Photobleaching and -isomerization
Irradiation of FPs causes light emission according to the spectra of the individual protein, but can also induce two distinct changes of
28
the spectral properties itself referred to as photobleaching and photoisomerization [19]. The term photobleaching describes the (general) loss of fluorescence light emission due to exposure of the sample to light of the excitation wavelength for a prolonged time and/or at high intensities. In this respect fluorescent proteins behave just like any other fluorescent dye (the only difference probably is the energy input needed to achieve bleaching). In contrast, photoisomerization describes the light-induced transition between two different forms of a molecular structure (as for example the switch of double-bond conformation from trans to cis, e.g. all-trans retinal to 11-cis retinal). In wildtype-GFP photoisomerization progressively descreases the excitation/absorption peak and 395nm and increases the secondary peak at 475nm thus causing a shift of the spectra rather than a decline of the peaks (as caused by photobleaching) [19]. Clearly, bleaching sets the ultimate limit on the amount of light emission obtainable from a fluorescent protein. Maximum fluorescence can only be achieved by minimizing photobleaching via limiting one or both of the following: (1) the time of exposure to, or (2) the intensity of the exciting light [51]. However, either of these strategies may compromise the quality of the results or limit the types of analyses that can be performed because the signal to noise ratio is unavoidably decreased. Furthermore, kinetics-based assays performed over an extended period of time may not be possible. Fluorescent proteins with improved resistance towards photobleaching could help to overcome these kinds of problems. In fact, all "second generation" protein isoforms derived from Aequorea GFP (cycle3GFP, GFPmutl (EGFP), mGFP5, Emerald etc., see table 2) are known to be less susceptible to photobleaching. Renilla GFP (rrenGFPl) is also known to be relatively resistant towards photobleaching, whereas the remainder of proteins has not yet been investigated with respect to this property. Photoisomerization of wildtype-GFP can be explained as a lightinduced shift from the neutral chromophore to its anionic form [49, 56, 67] caused by the occasional loss of protons during the reversible proton transfers happening during the light absorption/emission cycles. Probably the reversible proton transfer occurs via the hydrogen bonds of a buried water and Ser205 to Glu222 [59, 64], while the phenolate oxyanion is solvated and
29
stabilized by the rotated side chain of Thr203. In the crystal structure of monomeric wildtype GFP, Thr203 exists in two conformations: approximately 85% with the hydroxyl-group facing away from the phenol oxygen, and 15% with the hydroxyl rotated toward it [59]. This proportion agrees well with the spectroscopic estimate for the ratio of neutral to anionic chromophores at equilibrium [56]. Photo-isomerization is of importance for GFPs showing complex type-1 spectra such as GFP, cgigGFP and hcriGFP. However, for the latter two proteins data concerning their photoisomerization have not yet been published. GFPs with type-2 spectra can be regarded at permanently and stably isomerized proteins no longer suffering from this phenomenon. Oligomerization and Aggregation
The Aequorea GFP possesses a weak tendency to form homodimers. This dimerization usually occurs only at high protein concentrations and is most prominent for the wildtype GFP protein. Interestingly, depending on the conditions wildtype GFP can be crystallized either as a monomer or a dimer indicating that oligomerization of GFP is not obligatory [23, 59]. Yang and coworkers determined the three-dimensional structure of dimeric wildtype GFP and found that the protein interface is formed by the hydrophobic residues Ala206, Leu221, and Phe223 as well as the hydrophilic residues Tyr39, Glul42, Asnl44, Serl47, Asnl49, Tyrl51, Argl68, Asnl70, Glul72, Tyr200, Ser202, Gln204, and Ser208. Although there is hardly any report about artifacts or dysfunctions caused by GFP dimerization [68] this tendency to dimerize can be further reduced or eliminated by introducing mutation such as Phe223Arg, Leu221Lys or Ala206Lys, thus replacing hydrophobic residues of the protein-protein interface by charged amino-acids. For rrenGFPl, rrenGFP2 (hrGFP) and asulGFP it is known that they form stable dimers [69]; Jach, unpublished). The high stability of these dimers is reflected by the fact that these proteins only dissociate under denaturing conditions. The zoanGFP (zsGreenl) appears to form dimers and/or tetramers [55], whereas for the other GFPs (see table 1) these data have not been published yet. Mature Aequorea GFP as well as its recombinant derivatives are known to be highly soluble [51]. Formation of protein aggregates has only been seen for the wildtype Aequorea GFP [19]. In GFP
30
isoforms carrying folding/solubility mutations this aggregation tendency is abolished. Not very much is known about the recently described GFPs from Anthozoan species, but it was stated that almost all of them form protein aggregates (at least in vitro). For a number of anthozoan FPs it has been demonstrated that the Nterminal stretch of amino-acids plays a major role in this respect and that substitution of amino-acid residues responsible for the high positive net-charge of the N-terminus by negatively charged residues results in non-aggregating protein isoforms [55]. For zoanGFP such a mutant was generated by introducing the exchanges LysSGlu and LyslOGlu (for more details see the section about red fluorescent proteins). pH and salt
It was found that the excitation and emission spectra of green fluorescent protein (GFP) and its mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells [51]. Typically, GFP and its mutant derivatives are fully fluorescent at pH values ranging from about pH 6.5 to 10. Above pHIO fluorescence strongly declines. At pH values of 4 and below GFP is non-fluorescent, Fig. (8). Renilla GFP (rrenGFP2) shows a quite similar behavior. 100 rrenGFP2 (taGFP)
mGFPS
asulGFP (FP499)
Fig. (8). pH-optima of some green fluorescent proteins
31
In contrast, the pH optimum of asulGFP is shifted towards more acidic conditions and ranges from pH 5 and 8, Fig. (8). Even under relatively strong acidic conditions (pH 3) this particular proteins shows 50% fluorescence (Jach, unpublished). For EGFP and GFP-S65T detailed pH titrations ranging from pH 5 to 8 indicated 10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (EGFP), 5.9 (GFP-S65T) and apparent Hill coefficients of 1 [70]. Under these conditions the fluorescence spectral shape, lifetime, and circular dichroic spectra were found to be pH independent, whereas at pH values below 5, the fluorescence response was slowed and not completely reversible. Due to this it was concluded that GFP pH sensitivity involves simple protonation events at a pH of 5 and above, but both protonation and conformational changes at lower pH values. The pH sensitivity of GFP in living cells was found to be similar to that of purified protein. Furthermore fluorescence responded very rapidly to a pH changes thereby demonstrating the usefulness of GFP as a non-invasive intracellular pH indictor [70, 71]. Salt concentrations, especially inorganic anions (chloride), can vary widely without impacting the fluorescence of GFP and its mutant isoforms. Further work is required to characterize the other members of the green fluorescent protein family (e.g. anthozoan GFPs) with respect to pH and salt dependence of fluorescence. Temperature
Once matured GFP remains fully fluorescent up to 65°C [51]. At temperatures higher than 65 °C the light emission declines (slowly) probably due to unfolding/denaturing of the protein, so that the chromophore is no longer completely shielded by the surrounding fl-can. At 78°C the fluorescence loss of GFP is 50 % [9]. As yet, for the majority of other GFP proteins temperature stability has not been investigated. Blue fluorescent proteins All blue fluorescent proteins known today are derivatives of Aequorea GFP. The first member of these class of proteins was obtained by intentional introduction of a single point mutation
32 32
replacing the second amino-acid of the chromophore (Tyr66) by Histidine [63]. Since the gained protein displayed rather poor fluorescence compared to GFP some work was carried out to identify improved randomly mutagenized isoforms of this very first BFP, but with limited success only. Even the best performing BFP still suffers from a relatively low fluorescence quantum yield and relatively easy bleaching [72, 73]. A list of the most commonly used BFPs in given in table 3. Not very surprisingly, BFPs were found to benefit considerably from the presence of folding mutations originally described for GFP [42]. Crystal structures for several BFPs have been solved [64, 74]. The BFPs should not be confused with other blue-light emitting chemiluminescent proteins (e.g. spent aequorin and lumazinecontaining proteins from Photobacterium phosphoreum) sharing the same acronym [45]. Table 3. Name
GFP derived BFP and its isoforms Mutation
Pe ak wavelc ngth's (nin) Ex. Em.
EC
QY
EC*(
rel. spec. FL
Ref.
abs.
rel.
Prot. / Cell
0,24
5040
1
Nd/1,0
[52]
22300
0,30
6690
1,3
Nd / 2,9
[63]
440 (447)
26300 (31000)
0,17 (0,26)
4471 (8060)
0,9 (1,6)
Nd/5,6
[65]
448
n.d.
n.d.
-
-
0,15*/nd
[42]
BFP (GFP-Y66H)
Y66H
384
448
21000
P4-3
Y66H Y145F
382
446
EBFP
F64L Y66H Y145F
380 (383)
smBFP
Y66H F99S M153T VI63 A
385
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * compared to the fluorescence of cycle3-GFP (=smGFP, Davis 1998)
Spectra
As mentioned above BFPs are mutant isoforms of the Aequorea GFP carrying a histidine residue at position 66. In consequence, an imidazole is placed in the chromophore [52] causing a shift of the excitation and emission peaks towards shorter wavelengths. Further work led to the isolation of improved BFP isoforms with slightly higher molar extinction coefficient and quantum yield (increasing
33 33
total light-emission by about 30 %) by introducing the pointmutation Tyrl45Phe in addition to Tyr66His [63]. All BFPs share rather simple excitation and emission spectra consisting of a single excitation and emission peaks at 383nm and 447 nm, respectively, Fig. (9). Factors affecting protein folding and maturation
The first described BFP (GFP-Y66H, see table 3) also suffers from the thermosensitivity effect described for GFP. In fact, folding of this protein appears to be even more sensitive to elevated temperatures, probably due to the altered steric properties of the mutated chromo- phore. In contrast, the latest recombinant protein wavelength (nm) 350 1,08 c a u £
400
450
500
550 -f-
600
650
••• Excitation — Emission
0,8-
BFP
0,6-
O
_:
0,4
0,20,0 Fig. (9). Typical excitation and emission spectra of BFPs
isoforms (EBFP, smBFP) hardly show any thermosensitivity (Jach, unpublished observation). In the variant P4-3 introduction of the mutation Tyrl45Phe leads to a 30% increase of fluorescence and a three-fold increase of apparent fluorescence, indicating that the major effect of the pointmutation is on protein folding [63]. Later it was shown that addition of the point-mutation Phe64Leu, which was already known to improve GFP folding, further improves the folding of BFP. The resulting clone is commercially available as EBFP. Davis and Viestra combined the Tyr66His chromophore mutation with the folding mutations originally found in cycle3-GFP (Phe99Ser,
34
Metl53Thr and Vail63Ala) giving rise to smBFP [42]. Although neither the extinction coefficient, nor the quantum yield or the relative apparent fluorescence with respect to the original BFP were determined for this protein it is likely that the presence of these mutation will improve the protein folding considerably resulting in increased apparent fluorescence. However, comparative experimental data are missing, so it is impossible to judge the relative "performance" of EBFP and smBFP. Factors affecting the mature protein Photobleaching and -isomerization
All BFPs currently known are very sensitive to prolonged and/or intense excitation light and bleach very fast. For BFP and EBFP the rate of bleaching was estimated to be 40 - 60 times higher with respect to EGFP [75]. Data about the photobleaching of smBFP are lacking, but it is likely that this protein will show similar results, since the only difference to BFP lies in the addition of folding mutations (Phe99Ser, Metl53Thr, Vail63Ala) not altering the spectral properties of the protein. Clearly, the usability of the blue-fluorescent proteins as reporters is limited by their high bleaching rates. Whether further improvement of the properties of theses proteins by means of molecular evolution is possible remains to be seen. Oligomerization and Aggregation
Due to their extremely close relationship to GFP it is highly likely that these proteins share the weak dimerization tendency with its ancestor Aequorea GFP [19]. Although experimental evidence is lacking it has to be assumed that the introduction of mutations blocking the dimerization of GFP (Phe223Arg, Leu221Lys or Ala206Lys) will also be beneficial for GFP-derived BFPs. The known BFPs are highly soluble and do not show significant protein aggregation. pH, salt and temperature
In the published literature there are hardly any data about the impact of these parameters on fluorescence and protein stability of BFPs. The pKi values for the BFP-chromophore were found to be quite similar to those of the achestral GFP-chromophore, although steepness of the slope of the curve describing the pH-dependence of BFP fluorescence is somewhat lower, Fig. (10).
35 35
In term of temperature stability BFPs showed a behavior comparable to that of GFP (Jach, unpublished). No significant losses of fluorescence were seen for mature protein heated to about 65°C. 100%
20%
Fig. (10). pH-dependence of the fluorescence of blue fluorescent proteins
Cyan fluorescent proteins The first described member of the cyan fluorescent proteins (CFPs) resulted from a rationally designed chromophore mutation of Aequorea GFP. Heim and co-worker replaced Tyr66 with Tip and found the peak wavelength for excitation and emission of this GFP derivative (GFP-Y66W) to be shifted to 436 and 476 nm, respectively [52]. Because of this blue-green/cyan light emission the protein was called cyan fluorescent protein or CFP. Since the fluorescence of this protein was several-fold lower compared to GFP researcher attempted to further improve protein properties by random mutagenesis and succeeded to generate improved CFP isoforms. However, only minor improvements were achieved and the best performing GFP-derived CFP known today, ECFP, still is rather dim (see table 4). Recently, a small set of additional cyan fluorescent proteins were described: amajCFP, clavCFP and dstrCFP [34], Table 5, Fig. (11). Unfortunately, besides their spectra not very much is known about these proteins and, in particular, quantitative data allowing to compare the strength of their light emission to ECFP are lacking.
36 36
Nevertheless, one of these proteins has already been made available commercially under the name amCyanl (Clontech). (Note that the latter three proteins were originally classified and published by Labas et al. as the green fluorescent proteins amajGFP, clavGFP and dstrGFP. As mentioned in the chapter about GFPs the renaming and re-classification of these proteins is justified by the fact that their spectral properties are much more related to those of the cyan fluorescent proteins). Spectra
Due to the substitution of Tyr66 by Tip the chromophores of the GFP derived CFPs contain an indole instead of a phenol or phenolate [52]. In consequence, the spectra of these proteins show excitation peaks at 434nm and emission peaks at 476nm to 486nm, which is intermediate between those of GFPs with neutral phenol and anionic phenolate chromophores, Fig. (12). In fact, these proteins possess double-humped rather than conventional single excitation and emission peaks. The origin of the doubled Table 4. Clone
GFP-derived cyan fluorescent proteins (CFPs) Mutation
Pe ak wavelf ngths (mn) Ex. Em.
EC
QY
EC* QY
rel. spec. FL
Ref.
rel.
Prot. / Cell
-
-
n.d. / n.d.
[52]
0,42
10038
1
n.d. / 1
[63]
32500
0,40
13000
1,3
n.d./1,3
[63]
21200
0,39
8268
0,8
n.d./l,6
[63]
CFP (GFP-Y66W)
Y66W
436
485
nd
nd
W7
Y66W N146I M153T VI63 A
434 (452)
476 (505)
23900
WIB(ECFP)
F64L S65T Y66W N146I M153T VI63 A
434 (452)
476 (505)
W1C
S65A Y66W S72A N146I M153T VI63 A
435
495
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined
37 Naturally occurring CFPs and mutant isoforms
Table 5. Protein
Organism
amajCFP (amFP486) (amCyanl)
Anetnonia mqjano
amajCFP-II (amFP486-K68M)
Anemonia majano
amajCFP-lH (amFP486-K68M-NA)
Fe iik waveU njjths (mn) Ex. Em.
EC
QY
rel, spec. FL
Ret
458
486
n.d.
n.d.
1
[24]
K68M
458
486
n.d.
n.d.
1,5
[55]
Anemtmia majano
K6E K68M
4S8
486
n.d.
n.d.
1,5
[55]
dslrCFP (dsFP483)
Dacosoma striata
-
456
484
n.d.
n.d.
n.d.
[24]
clavCFP (CFP484)
Clavularia sp.
-
443
483
n.d.
n.d.
n.d.
[24]
Mutation
Ex: excitation, Em; emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined
emission peaks must be vibrational levels or other quantum states that equilibrate within the lifetime of the excited state, because their shapes and relative amplitudes are the same regardless of the excitation wavelength [51]. Clearly, the amount of light emitted by these molecules is drastically reduced only reaching levels of 3050% compared to their achestor GFP. In contrast to the GFP derived CFPs the naturally occurring anthozoan CFPs (amajCFP, dstrCFP and clavCFP) show simple spectra with single excitation and emission peaks [34], Fig. (12). The excitation maxima found clearly differ from the GFP-derived CFPs. They are shifted about 10-24nm towards longer wavelength (see table 5), whereas emission maxima of these proteins are in the range of 483nm to 486nm, which is quite comparable to the other CFPs. Interestingly, according to their deduced amino-acid sequences anthozoan CFPs possess GFP-like chromophores
38 38 ECFP amajCFP dstrCFP clavCFP
MGKGEELFTGWP .ALSNKFIGDDMK .SCSKSVIKEEML . KCKFVFCLSFLVLAITNANIFLRNEADLEEKTLRIPKALTTMGVIKPDM
ECFP amajCFP dstrCFP ClavCFP
-ILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GK-LPVPWP -MTYHM. .C....Y.T.K.. .N.KPYE.TQ. ST.KV.MANG. P-. AFSFD -.DLH.E.TF...Y.EIK.K.K.QPNE.TN.VTLEV.K .GP..FG.H K.KLKME.N. . . .A.VIE KPYD.TH. .NLEVKE .AP. .FSYD
ECFP amajCFP dstrCFP ClavCFP
TLWTLTWGVQCFSRYPDHMKQHDFFKSAMPEGWQERTIFFKDDGNYKT I.S.VFKY.NR. .TA. .TS.—P.Y. .Q.F.D.MSY. . .FTYE.G.VATA I.CPQFQY.NKA.VHH. . — N I . .YL.LSF. . . .TW. .SMH.E.G.LCCI SW. . .MT.E.K.IV.V I.SNAFQY.NRALTK. . .—DIA.Y. -QSF
ECFP amajCFP ds trCFP C lavCFP
RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQK SW. ISLK.NCFEHKSTFH.VN.PA. . PVMAK.TTGWDP. FEKMTVC.GIL TNDISLT . NCFYYD. KFT. LN. PPN. P W Q K . TTGWEP. TERLYPR . KSDISM. E . SFIYE . RFD . MN. PPN . PVMQK. TLKWEP. TEIMYVR. GVL
ECFP amajCFP dstrCFP ClavCFP
NGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALS KTKK. .TM.P. . W E H R I . — K. DVTA. LMLQGGGNYRC. FHTS . - . VLIGDIHHALTVE. GGHY. CDIKTVYRAKKAALKM. GY. . VD. KLVIW V. D.S.SLLLEGGGHYRCDFKSIYKAKKV.K. . .Y. FVDHRI
ECFP ama j CFP dstrCFP clavCFP
KDPNEKRDHMVLLEFVTAAGITHGMDELYK RTDLD. GGNS . Q. TEHAV. H. . --SWPF* NNDK.FMKVEEHEIA.ARHHPFYEPKKDK* EIL.HDK.YNKVTLYEN.VARYSLLPSQA*
Fig. (11). Alignment of the amino-acid sequences of cloned CFPs
and do not contain an indole, Fig. (11). This emphasizes the importance of the interaction between the chromophore and its environment for the spectral properties of the individual protein. Unfortunately, quantitative data excitation coefficients and the quantum yields of these proteins are lacking, so their brightness can not be compared to the other CFPs. For amajCFP a mutant displaying a 1,5-fold increase in brightness upon expression in E. coli was described and found to contain the point-mutation Lys68Met [55]. However, whether or not this mutation affects the
39 wavelength (nm)
350 1
1,0-
8 c
0,8-
| a
0,6-
400 ' ' ' I•' ECFP
450
500 1
600
550 ' ' I '
650
••• Excitation — Emission
0,20,0
-"-+•
1,0
8
0.8
|
0,6
I
amajCFP (amCyani)
0,2 0,0
Fig. (12). Typical spectra of cyan fluorescent proteins
light emission of the protein remains unclear. It might as well be that the increased brightness results from improved folding behavior of the protein. Factors affecting protein folding and maturation
Inspired by the successful optimization of GFP using molecular evolution techniques similar strategies were applied to the GFP derived CFP (GFP-Y66W) resulting several improved protein isoforms. Interestingly, although generated independently these proteins proved to contain two beneficial point-mutations previously described for improved GFP isoforms, Metl53Thr and
40
Vail63Ala, helping them to overcome thermosensitivity by positively influencing the protein folding. Hence these mutations are expected to exert the same effects on the CFP proteins. Other helpful pointmutations found in these proteins include Phe64Leu, Ser65Thr, Ser72Ala and Asnl46Ile [51]. Knowledge about the behavior of the anthozoan CFPs is rather limited. As already mentioned (see above) the amajCFP-II protein (harboring the mutation Lys68Met) was found to show improved brightness [55], which might either be attributed to higher light emission (spectral mutation) or improved protein maturation (folding mutation). More work is required to clarify this point. Factors affecting the mature protein Photobleaching and -isomerization
The cyan-fluorescent proteins were found to be almost as photostable as GFP indicated by the fact that the rate-constant for bleaching only was about 30% higher than the rate of EGFP [75]. In practical terms this slight difference is not limiting the usability of GFP derived CFPs as reporter proteins in molecular biology. Photoisomerization has not been reported for GFP derived CFPs. Data about the photobleaching and -isomerization of the recently described amajCFP and derived isoforms are not yet available in published literature. Oligomerization and Aggregation
All CFPs being descendants of Aequorea GFP are expected to share the (rather weak) dimerization tendency with its achestor. Due to the close relationship between GFP and CFP introduction of mutations blocking the dimerization of GFP (Phe223Arg, Leu221Lys or Ala206Lys) should also be beneficial for CFPs. However there is no further evidence for this assumption and its truth remains to be proven. The anthozoan protein amajCFP as well as its mutant amajCFPII (see table 5) form tetramers as judged by semi-native gelelectrophoresis [55]. For the anthozoan CFPs protein oligomerization and aggregation needs to be investigated. When it comes to protein aggregation the known GFP derived CFPs should behave just like Aequorea GFP with the mature proteins being highly soluble not giving rise to significant protein
41 41
aggregation. In contrast, amajCFP forms protein aggregates and the other anthozoan CFPs are likely to behave in the same manner. Introduction of a negatively charged residue (Lys6Glu) into the amajCFP protein resulted in the non-aggregating protein version (amajCFP-III), indicating that the high positive net-charge at Nterminus of the protein is responsible for protein aggregation as already described for other anthozoan FPs [55]. pH, salt and Temperature
For the GFP derived CFPs, amajCFP and its mutant isoforms as well as dstrCFP and clavCFP no data were found in published literature describing the pH-dependence, salt-sensitivity and temperature-stability of these proteins. As close relatives of GFP the mature GFP-derived CFPs are supposed to be able to tolerate relatively high temperature (up to 65 °C) before denaturing starts. However, this can only be speculated since experimental data are missing. Yellow fluorescent proteins Together with the protein classes BFP and CFP, also the first yellow fluorescent proteins (YFPs) were originally derived form Aequorea GFP by introduction of amino-acid exchanges at positions 65 (the first amino-acid of the chromophore) and 203 [51]. In fact, these mutants were rationally designed based on crystal structure of GFP in order to create a fluorescent protein sufficiently different in its fluorescence properties to allow for double-labeling experiments, FRET and other experimental applications. Very recently the first naturally occurring yellow fluorescent protein (zoanYFP) was cloned and spectroscopically analyzed [34], Fig. (13). However, further work is required to characterize this protein in more detail. In this review we refer to the GFP derived and naturally occurring YFPs and their isoforms as given in tables 6 and 7. Today GFP derived YFPs are widely used as reporters in molecular biology [76].
42 42 Spectra
As mentioned above the point-mutations at positions 65 and 203 were introduced in order to gain additional interaction of 7Uelectrons and to enhance polarizability around the chromophore, thus reducing excited state energy and increasing both the excitation and emission wavelengths [51]. In the GFP derived YFPs residue 65 is Gly (or Thr) instead of Ser to promote ionization of the chromophore, whereas Thr203 is replaced by an aromatic residues (His, Trp, Phe, and Tyr). In consequence, the aromatic ring of the amino-acid at position 203 is stacked next to the phenolate anion of the chromophore resulting in excitation and emission wavelengths increased by up to 20 nm (30), Fig. (14). When mutation Gln69 to Lys (Q69K) is added in addition to the mentioned pointmutation of positions 65 and 203 the maximum light emission is shifted by 1-2 nm, resulting in an emission peak around 529 nm [51]. The threedimensional structure of zoanYFP and the chemical nature of its chromophore remains to be determined. Consequently, knowledge about the mechanisms underlying the fluorescence properties, Fig. (14), of this protein is lacking. Factors affecting protein folding and maturation
For YFPs derived from the Aequorea GFP (table 6) it has to be expected that they also do suffer from the thermosensitivity effect described for GFP. Indirect evidence is provided by the fact that the point-mutations described to improve the folding of GFP are also helpful for the GFP derived YFPs (see below). However, no direct evidence is present in published literature.
43 Table 6. isoforms Name
GFP-derived yellow fluorescent protein (YFP) and its Mut.
Pe ik wavele ngths (ntn) Em. Ex.
EC
QY
EC* QY
rel. spec. FL
Ref.
rel.
Prot. / Cell
0,70 45850
1
n.d./1
[51]
48500
0,78 37830
0,8
n.d./ 2
[51]
529
62000
0,71 44020
0,9
n.d./ 8,3
[51]
514
527
83400
0,61
50874
1,1
n.d./9,7
[51]
S65G S72A K79R T203Y
514
527
94500
0,60 56700
1,2
n.d./16,7
[51]
Citrine
S65G V68L Q69M S72A T2O3Y
514
524
77000
0,76 58520
1,3
n.d.
[77]
EYFP-F46L
F46L S65G V68L S72A T203Y
515
528
78700
0,61
48007
1
n.d./ 194
[78]
SEYFP
S65G V68L S72A M153T VI63 A S175G T203Y
515
528
101000
0,56
56560
1,2
n.d./29,1
[78]
Venus (SEYFP-F46L)
F46L S65G V68L S72A M153T VI63 A S175G T203Y
515
528
92200
0,57
52554
U
n.d./291
[78]
abs.
GFPS65G/S72A/T203F
S65G S72A T203F
512
522
65500
GFPS65G/S72A/T203H
S65G S72A T203Y
508
518
10C-Q69K.
S65G V68L Q69K S72A T203Y
516
lOC(EYFP)
S65G V68L S72A T203Y
Topaz
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined
44 Table 7.
Naturally occurring YFPs and mutant isoforms EC
QY
rel. spec. FL
Ref.
538
nd
nd
nd
[24]
(494) 528
538
nd
nd
nd
[55]
(494) 528
538
nd
nd
nd
[55]
Name
Organism
Mutation
zoanYFP (zFP538) (zsYellow)
Zoanthus sp.
-
(494) 528
zoanYFP-II (ZFP538-M129V)
Zoanthus sp.
Ml 29V
zoanYFP-III (zFP538-M129V-NA
Zoanthus sp.
K5E K9T Ml 29 V
Pe ak wavelt :ngths (n <«) Em. Ex.
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined
EYFP zoanYFP
MGKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT . AHSKHGLKEEMTMKYHME. C VIT. . . I. YPFK. . Q. INLCVIE
EYF P zoanYFP
GK - LPVPWPTLVTTFGYGLQC FARYPDHMKQHDFFKSAMPEGYVQERTIF .GP..FSEDI.SAG.K..DRI.TE..--QDIV.Y..NSC.A..TWG.SFL
EYFP zoanYFP
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYN . E. - -GAVCICN. DITVSVKE. C. YH. S . FNGMNFPAD. PVMK. MTT. WE
EYFP zoanYFP
SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP ASCEK..PVPKQGIL.GDVSMYLLLK.. GRYRC.FD.VYKAKS.PSK
EYFP zoanYFP
DNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK* MPEWHFI. H.LLR.D.SDAKNQKWQLTEHAIAFPSA.A-*
Fig. (13). Alignment of the amino-acid sequences of cloned YFPs
The recently described zoanYFP represents the only other YFP currently known. As yet there are no data available about its folding behavior under different temperature conditions. The first described amino-acid exchanges improving the protein folding of GFP derived YFP leading to increased amounts of soluble, fluorescent protein were Val68Leu/Gln69Lys or Val68Leu alone. The latter is also widely known as enhanced-YFP (EYFP). Introduction of these mutations improved the apparent fluorescence about nine-fold compared to the initial YFP clones (see table 6). A sixteen times higher apparent fluorescence was described for the YFP isoform Topaz, which carries the point-mutation Lys79Arg [51]. Very recently it was demonstrated that the
45 wavelength (nm) 350 1,0-
400 ' " I "
450
500
550
600
650
••• Excitation — Emission
EYFP
0,80,60,40,2-
0,0 1,0-
1
' ' ' I
zoanYFP
0,80,60,40,20,0
Fig. (14). Exitation and emission spectra of YFPs
introduction of the point-mutation Phe46Leu greatly improves the protein-folding of EYFP [78]. Further increases in the level of soluble and fluorescent protein were achieved by adding the folding mutations Metl53Thr, Vall63Ala and Serl75Gly, which were already described to improve the folding behavior of GFP (see above). The app. fluorescence (brightness) of best performing clone named Venus (SEYFP-F46L) was described to be 30 times higher than that of EYFP (corresponding to a calculated 291-fold increase with respect to the very first YFP described) [78]. For zoanYFP a first mutant protein (zoanYFP-II) was described showing a more complete protein folding (table 7). This protein carries only a single point mutation (Metl29Val) [55].
46 46 Factors affecting the mature protein Photobleaching and -isomerization
As a member of the GFP-derived yellow fluorescent proteins EYFP was investigated for its susceptibility to photobleaching and was found to possess a bleaching-rate which is about 3 times higher then that of EGFP [75]. The increased bleaching rates of GFP-derived YFPs such as YFP and EYFP are believed result from the changes in internal hydrogen bonding and steric packing due to the stacking of Tyr203 and the chromophore [51]. The susceptibility of zoanYFP towards photo-bleaching remains to be determined. Although the differences in photostability compared to GFP are not as dramatic as for the BFPs (40-60 fold, see above), they are significant and limit their usability of as reporter proteins. In consequence, molecular evolution approaches were carried out to create improved proteins resulting in derivatives Citrine and Venus (see table 6), which, in fact, proved to possess higher photostability. Citrine shows a twofold increase in photostability relative to YFPVal68Leu/Gln69Lys, whereas Venus is the brightest and fastest maturing (with reference to the development of fluorescence) YFP so far. Attempts to combine the properties of Citrine and Venus in one proteins were not successful [76]. As yet no data about the photobleaching and -isomerization of the recently described zoanYFP and its derivatives are available in published literature. Oligomerization and Aggregation
For the GFP-derived YFPs it is, again, likely that they share the weak dimerization tendency of Aequorea GFP. As is true for the BFPs and CFP the introduction of mutations blocking the dimerization of GFP (F223R, L221K or A206K) should also be beneficial for YFPs. Nevertheless experimental data are lacking. Analysis of zoanYFP-II (see table 7) proved the tetramerization of this protein. In contrast to GFP-derived YFPs recombinant zoanYFP, as almost any other anthozoan FP, showed substantial aggregation. A non-aggregating zoanYFP isoform was successfully generated by introducing the point-mutations Lys5Glu and Lys9Thr thus lowering the N-terminal positive net-charge [55]. This appears to be
47
a more general phenomenon since similar results were obtained for other anthozoan FPs as well (see GFP, YFP and RFP sections). pH, salt and temperature
GFP-derived YFPs are not only suffering from photobleaching they are also notorious for their sensitivity to pH and anions, especially chloride [76]. However, the already mentioned improved isoforms Citrine and Venus not only show higher photostability (see above), they also show reduced sensitivity to pH changes and are resistant to chloride. Citrine remains 50% fluorescent at pH 5.7, the lowest pH that has been reported so far for a GFP-derived YFP. As yet, no data about the pH-dependence and salt sensitivity of zoanYFP were published. According to the available literature neither the GFP derived YFPs nor the zoanYFP were analyzed with the respect to the temperature stability of the mature proteins. However, GFP derived YFPs will probably behave similar to GFP, which is stable up to 65 °C. For zoanYFP this topic needs to be addressed in future experiments. Red fluorescent proteins The history of red fluorescent proteins (RFPs) dates back to 1999 when the first member of this group of proteins, DsRED (drFP583), was first described as one of six novel fluorescent proteins isolated from nonbioluminescent Anthozoa species [24]. At that time the ability to emit red light was rather unique and due to that the DsRED protein gained much interest amongst scientists looking for alternative reporter proteins to GFP and its various derivatives [79, 80]. It quickly became obvious that the DsRED protein suffers from some severe limitations (e.g. slow maturation rate) and several independent research groups made efforts to generate DsRED mutants with improved properties [29-31, 55]. A list of the currently available DsRED mutants is given in table 9. During the last couple of years a number of other RFPs was isolated from different sources [34], Fig. (15). Important properties of these naturally occurring proteins are listed in table 8. Nevertheless, the DsRED still represents the best understood RFP known today.
595
(522)572
n.d. n.d.
F4L, K12R, F35L, T68A, F84L, A143S, K163E, M202L F4L, K6T, K7E, K12R, F35L, T68A, F84L, A143S, K163E, M202L
asulRFP-III (M35-5)
asuKFP-IV (M35-5-K617K7E)
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * Recombinant DsRED isoforms are described in table 9.
573
T68A, A143S
asulRFP-II (asFPS95-T68A/A143S)
Anemonia sulcata
611
asulRFP(asFP595)
618
588 559
-
-
Heteractis crispa Entacmea quadrieohr
rfloRFP
hcriRFP(HcREDl)
Ricordeaflorida
meavRFP
equaRFP(eqFP6U)
n.d. n.d.
(520)580 (517)574
508 (572) 506 (566)
-
Montastraea cavernosa
552
n.d.
n.d.
595
576
593
-
Zoanlhus sp.2
573
-
Discosoma sp.2
zoan2RFP
n.d.
n.d.
n.d.
n.d.
78000
n.d.
n.d.
n.d.
5200057000
dis2RFP(dsFP593)
583
558
-
Discosomasp.l
Em.
Ex.
DsRED * (drFP583)
EC
Mutation
Organism
Peakwa velengths (nm)
Cloned red fluorescent proteins (RFPs) and recombinant isoforms Name
Tables.
n.d.
n.d.
0,05
0,001
0,45
n.d.
n.d.
n.d.
n.d.
n.d.
0,68
QY
5
5
1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
rel. spec. FL
[55]
[55]
[55]
[44]
[35]
[82]
[34]
[34]
[34]
[81]
[24]
Ref
48
584
607
after 24-32 h maturation (deduced from graphs provided in [31 ])
n.d. / n.d.
***
0,30
values were calculated using 54.4 as average of the exctinction coefficent (EC) of wildtype DsRED
11000
[30]
[31]
[31]
[31]
[55]
[24]
Ref.
[29]
n.d. / 7-20. [32]
n.d. / n.d.
n.d./3***
n.d. 11***
n.d. / n.d.
RedStar is derived from a synthetic gene, yRFP, codon-optimized for yeast expression. This gene carries an additional glycine residue at the NTerminus (position 2). Hence the position numbers of the given mutations are shifted by 1 with respect to wildtype DsRED.
0,25
n.d.
n.d.
0,36
0,79
0,34
n.d./ 2
1 /I
1* 0,65
Prot. / Cell
rel.
rel. spec. FL
**
44,0
n.d.
n.d.
13332
29205
12642
24090
37060*
abs.
EC* QY
*
Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined
mRFPl
R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71 A, K83L, Cl 17E, F124L, I125R, V127T, L150M, R153E, V156A, H162K, K163M, A164R, L174D, V175A, F177V, S179T, 1180T, V195T, Y192A, Y194K, S1971, T217A, H222S, L223T, F224G, L225A
n.d.
n.d.
V105A, S197A
Fluorescent Timer n.d.
n.d.
n.d.
n.d.
G2S, R18K, V97I, F125L, M183K, P187Q, T2031
RedStar •* 499/582
n.d.
30,3
555
0,59 0,44
49,5
587 586
560
R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P, T217A
R2A, K5E, N6D, T21S, H41T, N42Q, V44A, Cl 17S, T217A
DsRED.Tl
DsRED.T4
0,42
30,1
586
554
R2A, K5E, K9T, V105A, I161T, S197A
DsRED2
DsRED.T3
0,55
43,8
587
561
-
0,68
52-57
583
QY
Em.
EC
Ex.
Peak wa^'elength's (n m)
558
Mutation
Properties of recombinant DsRED isoforms
DsRED
Name
Table 9.
49
50 50 DsRED mSSKNVIKEFMRFKOTMEGTVNGHEFEIEGEGEGRPYEGHNTVKLIWTKGGPLPFAWDI .SC K CS. .M. zoan2RFP .AH. .HGLTDD.TMHF. C D . .K.V. . .N.N.N.FK.KQFIN.C.IE. . ...SE.. . N.V.V K TQSMD.T.KE.A. ....Y.. . S. . .SV.KI.L. S .N. L. . .N. .MM.V S...YQ.KCT...D.N..M.TQ.MHI..VE.... ...F.. Y.KC. . . .D.N.FA.TQSMRIH. .E.A....F.. VSGLL. .S. .I.MY . . SAL. .E.KI*LTI
dis2RFP mcavRFP equaRFP HcRED rfloRFP
LSPQFQYGSKVYVKHPADIPDYKKLSFPE GFKWERVMDG— DsRED CFIYKVKFIGVNFDGD dis2RFP — ....E zoan2RFP . -AA.D. .NRLFTEY.EG.V. .F.N.C.A . YT.H.SFVREN. IYHEST.Y. ..VRV , mcavRFP MTTV.H. .NR.FA.Y.KH. . . .F.QM. . . EYS. . .S.GD—. .FN. .R.D. .GDG . , equaRFP .ATS.M. . . .TFI. .TKG. . .FF.Q. . . . . .T. . . .T. . — .LV.HA.VT HcRED .A.CCE. . .RTF.H.T.E. . .FF.Q . .T. . .TTGN--.L VH.T. .GNG rfloRFP .TTIVH. .NRAF.NY.K. . . . IF.QTCSGPGA.YS.Q.T.GD—T.N.DIH.M.AD.GDG DG—CFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGV— DsRED LKGEIHKSTERLYP dis2RFP . . . .E RR.R S — . . .D. .M.S zoan2RFP VREN. IYHEST. Y A K.M.TN. . P.C.KII. INSQKI. . .DVSM.C.KII. mcavRFP G D — . .FN. .R.D PN LK. .P. . .KM.V — .T.D.NM. . .KM.V equaRFP N.A K. . .PN. .M. . .A. .G—.R.YSQMN. .M. . . HcRED . . — .LV.HA.VT GN--.L VH.T. . .A KN.SG. . .P. . . W . .EN. . —.C.RNVM. . . W . . rfloRFP GD—T.N.DIH.M.AD. .US R.VK. .P. . . IMFQC . .L—.R.DVAM. . . IMFQ DsRED STERLYPRDGV—LKGEIHKALKLKDGGHYLVEFKSIYMAKK-P-VQL PGYYYVDS- dis2RFP .S — . . .D. .M. .R.EG V..- zoan2RFP . S. . . .C.KII. INSQKI. . .DVSMY.L R.RCQ.DT. .K. .TE.-KEM .DWHFIQH- mcavRFP . . .KM.V. . . . — .T.D.NM. .L.EG RCD.RTT.R. . .-KG.K. .D.HFE.H- equaRFP N. .M. . .A. .G—.R.YSQM. .NVDG. .YLSCS.ETT.RS. .-T-.ENFKM. .FHF. .HM HcRED . . . W . .EN. . — .C.RNVM. . .VG.-R.LICHHYTS.RS. .-A-.RALTM. .FHFT.IM rfloRFP . . .IMFQC. .L—.R.DVAMS.L..G RCD. .T. .KP. .-N-.KM . . .HF. .HDsRED dis2RFP zoan2RFP mcavRFP equaRFP HcRED rfloRFP
-PGYYYVDSKLDITSHNEDYTIVEQYERTEGRHHLF L * M V K.Q. . . .P. IKPLQ . -.DWHFIQH. .NREDRSDAKNQKW.LIEHAIASRSA .P . -.D.HFE.HSIE.LR.DKE. .E.KL. .HA.AHSG.P RVAK . M. .FHF. .HR.EHLEESDKEMF.V.H.HAVAKFCDLPSKLGR. . M. .FHFT.IR.QMLRKKK.-EYF.L. .ASVA.YSDL PEKAN . -. . .HF. .HCIE. . .QQD. .NV. .h . .-GAVA.YSP .QKPCQAKA.
Fig. (15). Alignment of the amino-acid sequences of cloned RFPs
Spectra
Typical spectra for the known red and far-red fluorescent proteins are given in Fig. (16). hi published literature the values given for the molar extinction coefficient and the quantum yield of DsRED vary considerably. Values published for the extinction coefficient include 22.500, 52.000, 57.000 and 72.500 M-'cnr 1 . In our own work we
51
determined a value of 56.500 M^enr 1 (Jach, unpublished). Although the reasons for these differences have not been investigated yet it might well be that they are due to variations in temperature and concentration during the critical maturation period. The fraction of aged protein that is red, green, or even nonabsorbing may be exquisitely sensitive to the conditions of the maturation [83]. With the exception of one particularly low estimate of 0.23 [24], literature values for the quantum yield of DsRed have been relatively consistent with values of 0.68 and 0.7 determined by several independent groups [28,31,84]. Currently, nothing is known about the extinction coefficients and fluorescence quantum yield values of the other described RFPs (see table 8). These data remain to be determined. Factors affecting protein folding and maturation
Unlike Aequorea GFP and its derivatives the folding/maturation of DsRED does not suffer from thermosensitivity (Jach, unpublished results). However, while working with this protein it quickly turned out that the maturation-rate of this protein is extremely slow. At room-temperature the wildtype DsRED needs 24-36 hours to gain maximum fluorescence [28]. For HcRED maturation rates comparable to GFP have been published. For the other naturally occurring RFPs (see table 8) no such data are available yet. Under appropriate conditions it is possible to produce and isolate almost completely immature DsRED from bacterial expression systems. In contrast to flow cytometry and other whole cell approaches this allows for direct measurement of the maturation rate of the protein without ongoing protein synthesis. Interestingly, it was found that the maturation of DsRED is temperature dependent with the maturation rates increasing with temperatures, Fig. (17). At 37 °C maturation is already about threefold faster and at 60 °C the wildtype matures within ~1 hour. This indicates that maturation of the wildtype protein is ineffective, but not principally limited to low rates. Experiments investigating cell free maturation of immature DsRED under pH conditions ranging from mild acid (pH 5.1) to
52
mild alkaline (pH 8.8) proved that the protein requires pH
wavelength (nm) 350
400
450
500
550
600
650
I '' 'iI iii i I
1.0-
Fig. (16). Typical excitation and emission spectra of RFPs
values of 6.1 or higher to be able to mature and become a fully fluorescent protein, Fig. (18). At pH 5.1 this process is already blocked completely. Interestingly, matured DsRED is fully fluorescent even at more acidic pH values (pH 4.8; at pH 4.3: 50%
53 53
fluorescence). The protein behaves in this way because either the correct folding of the apoprotein required for the autocatalytic formation of the chromophore is hampered, or the apoprotein folds correctly but the chromophore formation itself is blocked. Further work is required to clarify this point. Clearly the slow maturation of DsRED limits its usability as a reporter in molecular biology, especially for approaches such as transient gene expression studies in organisms growing at relatively low temperatures (e.g. plants). To overcome this limitation random mutagenesis and molecular evolution was successfully used to create faster maturing DsRED mutants. In fact, the maturation of the resulting proteins DsRED2, DsRED.Tl, DsRED.T3 and DsRED.T4 (see table 9) was 2 - 1 5 fold faster with respect to wildtype DsRED [31, 55]. In contrast to the approaches to perform molecular evolution of GFP, which lead to the identification of the same (folding) mutations in several independent experiments, each type of fast maturing DsRED isoform
24
Fig. (17). Temperature dependence of DsRED maturation
54 100 i
80
• D • • O
pH5,1 pH6,1 pH7,1 pH7,8 pH8,8
60
40
20
20
30
40
50
60
70
Fig. (18). pH-dependence of DsRED maturation
(DsRED2, DsRED.Tl-4, RedStar) contains its own set of mutations [30, 31, 55]. There is no overlap and the improvement can not be assigned to a single mutation. In addition to the three (DsRED2) or six (DsRED.Tx) aminoacid exchanges required for faster maturation these proteins were engineered at the N-terminus to reduce protein aggregation (see next section), thus bringing the total number of pointmutations present in these proteins up to 6 - 9 [31, 55]. Given the huge number of pointmutations required to improve the properties of the protein, it has to be assumed that the DsRED conformation, unlike GFP, hardly tolerates any changes. This is supported by the fact, that the majority of these mutations only is beneficial in the presence of the other point-mutations (Jach, personal communication). Factors affecting the mature protein Photobleaching and -isomerization
As already mentioned the red-fluorescent protein drFP583 is commercially available under the name DsRED. According to the
55
distributor the protein is not very susceptible to photobleaching and its photostability can be compared to EGFP (Clontech). Using microscope and fluorimeter measurements Baird and co-workers proved the photostability of DsRED to be 3.9-fold and 65-fold higher compared to EGFP and EYFP, respectively [28]. However, under different experimental conditions the values for the photobleaching quantum yield of DsRED were found be 12-fold higher, indicating a lower degree of photostability for the DsRED protein. In these experiments stability of DsRED was only 2.7-fold higher then that of the yellow fluorescent protein Citrine [85]. In our own work fluorescence of DsRED and its derivatives DsRED2, DsRED.T3 and DsRED.T4 proved to be stable even throughout prolonged fluorescence microscopy or measurements of spectral properties. Under similar conditions equaRFPl (eqFP611) bleached rapidly (Jach, unpublished observations), which is in contrast to pub-lished data indicating that this protein should as stable as DsRED [35]. For the other red-fluorescent proteins known today (see table 8) no such data are available. Oligomerization and Aggregation
DsRED has been reported to be an obligate tetramer and the more recently described isoforms with improved maturation rates (DsRED2, DsRED.Tl - .T4) or altered spectral properties (e.g. fluorescent timer) show the same behavior. Preliminary data suggest that hcriRFP (HcRED) forms stable dimers [86], whereas equaRFP (eqFP611) appears to be a monomer [35]. For RedStar [30], another fast maturing DsRED derivative, and the recently published proteins mcavRFP, rfloRFP, zoan2RFP, and dis2RFP [34] oligomerization has not been analysed. Oligomerization of RFPs is not very much of a problem as long as the protein is expressed as non-fusion polypeptides (e.g. as reporter for promoter studies). In protein fusions, however, this can be detrimental, especially if the fusion-partner itself tends of form oligomers. In this case, gross aggregation and precipitation of the fusion protein is highly likely. Recently a monomeric form of the DsRED protein named mRFPl was described [29]. However, this derivative of the DsRED is heavily mutated and carries 33 amino-acid exchanges. Furthermore, the spectral properties differ significantly from those
56
of DsRED: the mRFPl protein emits far-red light (peaking at 609nm), which is about 25nm apart from for DsRED emission peak. In consequence, mRFPl should be regarded as a completely different protein rather than a DsRED mutant. In addition to oligomerization wildtype DsRED and its derivatives (except mRFPl) as well as the anthozoan proteins show a strong tendency to form aggregates upon expression in heterologous expression in bacterial and eucaryotic cells, but without any loss of fluorescence, indicating that the aggregates contain properly folded native protein molecules. This protein aggregation is responsible for the blurring of the microscopic picture, limiting the perceived optical resolution and rendering nuclei and nucleoli invisible [55]. The commercially available HcRED, however, was published to be highly soluble not showing detectable aggregation (Clontech). The actual reasons for the protein aggregation are not quite clear. In case of DsRED aggregation of the protein due to "sticky" hydrophobic surface areas appear unlikely, since the DsRED surface lacks extended hydrophobic surface regions. It was speculated that the positively charged N-terminal region of the protein can interact with the mostly negatively charged surface of other DsRED molecules, thus forming up to four salt bridges between adjacent DsRED tetramers finally leading to the creation of stable net-like structures [55]. In fact, it has been demonstrated that reduction of the positive net-charge of the N-terminal amino-acid sequence greatly reduces or abolishes aggregation of these proteins [55]. pH, salt and temperature
Unlike the other GFP-like proteins mature DsRED and its isoform shows only little pH-dependence displaying maximum fluorescence over a very broad range of pH values ranging from about 5 to 10, Fig. (19). The same holds true for equaRFPl (eqFP611) [35]. The pH-dependence of the other RFPs remains to be determined. Mature wildtype DsRED protein and its recombinant derivatives DsRED2, DsRED.Tl, DsRED.T3 DsRED.T4 proved to be stable (soluble and fluorescent) at temperatures up to 70-75 °C, Fig. (19). For RedStar, mRFPl, "Fluorescent timer", mcavRFP, HcRED and equaRFPl (eqFP611) such data currently are not available.
57
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Fig. (19). pH-dependence and stability of DsRED fluorescence
BIOLOGICAL FUNCTION OF FLUORESCENT PROTEINS Clearly, the biological activity of fluorescent proteins is determined by their intrinsic ability to emit light after irradiation with a suitable excitation wavelength. However, despite the extensive use of these proteins as reporters in molecular biology during the last decade, the biological functions of these proteins in their respective host organisms still are not very well known.
58
For the GFPs from Aequorea victoria and Renilla reniformis it has been shown that they transmute blue chemiluminescence from distinct primary photoproteins, such as the Aequorin in A. victoria, into green fluorescence causing the bioluminescent phenotype of these organisms [51]. The biological significance for the color-shift might be explained by the fact that green light penetrates farther into the sea water than blue and therefore is more efficient for triggering the visual responses in observing animals [87]. GFPs probably not only serve to shift the output color, since this change might as well be achieved by mutating the primary photoprotein. Boosting the overall quantum efficiency might be another function of GFP is this system. The efficiencies of primary photoproteins in terms of chemiluminescence are relatively low, but the organisms can produce more light for a given energy cost, if the excited state energy of this primary protein is efficiently transferred to a GFP that, in turn, emits light with high efficiency [19]. However, it is still not quite clear why these organisms evolved fluorescent proteins and bioluminescence. Currently about 30 different cloned GFP-like fluorescent proteins are listed in the databases and, interestingly enough, only 6 of these proteins are originating from bioluminescent organisms. All other proteins were found in non-bioluminescent, but colored and fluorescent anthozoan species such as reef corals. Moreover, several GFP-like proteins were described possessing very intense absorption abilities, but lacking fluorescence. Together with the mentioned fluorescent proteins these colored proteins (chromoproteins, CPs) might exert their biological function by serving as pigments for the non-bioluminescent host organisms determining their natural coloration [44, 88]. Furthermore, it was proposed coral coloration and fluorescence evolved to provide photoprotection for endosymbiotic algae [89]. However, these protein might exert other yet unknown functions and it is still not clear whether the diversity of colors found in these organisms corresponds to the diversity of functions for which the GFP-like proteins are responsible, or is just the outcome of random variations that occurred under relaxed environmental restraints [90]. It was suggested that at least some of the different colors must have specific functions and are subjected to natural selection [79].
59 59
Clearly, further phylogenetic, physiological and ecological research is required to gain more insights into the biological function and significance of fluorescent proteins
USAGE OF FLUORESCENT PROTEINS During the last decade numerous applications were developed employing fluorescent proteins . This includes passive applications such as the use of fluorescent proteins as fluorescence tags in fusion proteins to monitor the appearance, degradation, location or translocation of appropriate partner proteins, as well as more active applications measuring biochemical parameters such as metabolite concentrations, enzyme activity, or protein-protein interactions by their effects on the fluorescence properties of appropriately designed derivatives of fluorescent proteins (biochemical sensors/indicators) [76]. The most prominent passive application of fluorescent proteins is their use as spatial and/or temporal markers allowing for noninvasive and non-destructive detection [76, 79, 91, 92]. In approaches to just measure promoter activity and expression patterns transcriptional fusions between the promoter under investigation and a fluorescent protein gene are used. More advance approaches such as protein trap strategies, localization of gene activity and transcripts, are based on the construction of suitable fusion proteins consisting of the fluorescent protein and another protein domain mediating organelle-specific expression, membranetransport, RNA-binding, cytoskeleton-binding etc [76, 93]. The recent development of a "fluorescent timer" version of DsRED that changes its light emission from green to red over a fixed period of time (about 24 hours) appears to be an interesting new reporter for temporal analysis of gene expression, which is reflected in the ratio of green to red fluorescence [32, 94]. Hundreds of successful examples of these approaches have already been published. However, there are a few disadvantages in the use of fluorescent proteins a reporters when compared to enzymatic reporter systems. The main disadvantage is the absence of signal amplification. Whereas fluorescent proteins are limited to a single chromophore for each protein, a single copy of 6galactosidase, luciferase or B-lactamase will catalyze the turnover of
60
multiple substrate molecules, allowing for much lower levels of gene expression to be detected. GFP and DsRED proved to be highly stable and protease resistance and therefore can accumulate to high levels in the target cells or tissues. This properties actually represents another limitation causing problems in cases such as oscillating gene expression patterns or leaky promoters, because there is no way to distinguish between the newly synthesized and the already accumulated protein. To overcome this limitation a destabilized GFP was constructed by fusing it to destabilizing protein domains such as the mouse ornithine decarboxylase domain [95]. In fact, in this construct the half-life was lowered to about 2 hours. It is important to note that by using these type constructs a higher level of transcription and translation are required to produce a given level of fluorescence. The use of fluorescent proteins as biochemical sensors allowing measurement of metabolite concentrations, enzyme activity or protein-protein interaction is a more active application for these type of proteins [76]. Such indicators can be further divided into molecules with single ehromophores versus composites in which the light emission dependents on the energy transfer between two ehromophores. Fluorescent proteins can be used as mono-chromophore sensors by exploiting the pH and halide-sensitivity of these proteins [70,9699]. In general, in FPs moderate acidification leads to reversible quenching of fluorescence. This pH sensitivity varies between different mutants and can be used to measure the ambient pH. Successful engineering of fluorescent proteins to render them sensitive to other parameters such and Zn2+, redox potentials or Ca2+ has also been reported [76,100]. The second class of indicators benefits from a quantummechanical phenomenon called fluorescence resonance energy transfer (FRET) that occurs when ehromophores of two separate fluorescent proteins are less than 8 nm apart [76]. These proteins can either be linked to each in a fusionprotein (intramolecular FRET) or can be separate entities (intermolecular FRET). The emission spectrum of the donor fiuorophore should overlap the excitation spectrum of the acceptor fluorophore, but both excitation spectra should be well enough separated to allow independent excitation. When the ratio of donor and the acceptor molecules is
61 61
fixed (e.g. in a fusion of fluorescent proteins), then FRET can be easily readout as the ratio of acceptor to donor fluorescence. Early applications used BFP as the donor and GFP as the acceptor, but the dimness and ability of BFP to be bleached soon led to its replacement by CFP, whereupon GFP had to be replaced by YFP to maintain spectral separation. Based on this principle indicators were cloned and successfully used to measure protease activity (by physically separating previously linked FRET partners) and changes in calcium (by linking the FRET partners via a calmodulin-domain, which changes its conformation and thereby the distances between the two chromophores upon calcium binding) [76]. Intermolecular FRET can be used to detect protein-protein interactions such as homo- or hetero-dimerization and proteindissociation in real-time [80, 101, 102]. However, intermolecular FRET can suffer from false negatives when the donor and acceptor fluorescent proteins are: perturbing the proteins to which they are fused; in close proximity but orientated unfortunately with respect to each other; too far away from each other even when their fusion partners are interacting. In general, false positives could result from the weak dimerization tendency of fluorescent proteins for each other, but so far there is only a single example for this [76]. Furthermore, this problem can be overcome by using FRET partners containing monomerizing mutations (see above). Limiting factors and problems The sensitivity of any reporter system based on fluorescent proteins is determined by numerous factors such as the total amount of fluorescent protein produced in the system, the efficiency of protein maturation/chromophore formation, the individual properties of the fluorescent protein in use, the organism and tissue in which the fluorescent protein is expressed and, last but not least, the available technical equipment [51]. Each of these factors is again influenced by several parameters. The total amount of a protein expressed in an organism or tissue is determined by the rate of protein synthesis vs. degradation. In general, protein synthesis depends on the amount of mRNA produced (promoter-strength and temporal/spatial expression pattern) and its usability for the protein synthesis machinery. The
62
latter is determined by the stability (half-life) of the mRNA, its splicing-efficiency, its codon-usage, the sequence of the translational start site and the presence of translational enhancer sequences [21, 103, 104]. In addition, the protein expression is affected by the copy number of the gene as well as the presence of transcriptional enhancer sequences boosting the promoter strength. The efficiency of protein/chromophore maturation is an intrinsic property of each fluorescent protein or mutant thereof. With respect to this, time, temperature, oxygen-availability and the intrinsic rates of cyclization/oxidation during chromophore formation play important roles [51]. As outlined in this review the latter is strictly dependent on the specific interaction between the chromophore residues and the environmental amino-acid side-chains provided by the fl-can protein backbone. Availability of chaperonins can be helpful [105] but is not required. In case of fusions between host proteins and a fluorescent protein hindrance of the protein folding thus preventing proper maturation can not be excluded. This can only by tested empirically. The usability of a certain fluorescent protein as a reporter protein is mainly dependent on its extinction coefficient and fluorescence quantum yield excitation (determining the amount of light emitted per molecule) and its excitation and emission wavelength, which marks the ability of the protein of overcome/avoid possible autofluorescent background signals (see below). Amongst the factors limiting the usability of a fluorescent protein are oligomerization and susceptibility to photoisomerization /-bleaching [19]. The organism and tissue in which the fluorescent protein is expressed plays an important role because cells might contain autofluorescent material emitting light at the preferred wavelength. This problem is very pronounced in plant molecular biology, because blue-light excitation of GFP also excites chlorophyll resulting in a very strong red fluorescence often covering the green fluorescence of GFP. Furthermore, not all organisms must necessarily tolerate the presence of a fluorescent protein. Although in the vast majority of experiments no problems were reported the proper subcellular localization of the protein might play an important role [103]. With respect to delectability of fluorescent proteins the technical equipment used is certainly of great importance. Not only the quality of excitation and emission filters and dichroic mirrors are
63 63
crucial, but also the choice of the filter set for the chosen experimental setup (e.g. band-pass vs. long-pass filters). In fact, background problems due to auto-fluorescence can often be overcome quite easily by choosing suitable (band-pass) filter sets. Finally, when it comes to imaging and quantification sensitivity, noise, and dark current of the CCD-chip or photodetector should be taken into account. ABBREVIATIONS BFP CAT CFP FRET GFP GUS LUC NPTII PCR RFP YFP
= Blue fluorescent protein = Chloramphenicol acetyl transferase = Cyan fluorescent protein = Fluorescence resonance energy transfer = Green fluorescent protein = fi-glucuronidase = Luciferase = Neomycin phospho transferase II = Polymerase chain reaction = Red fluorescent protein = Yellow fluorescent protein
ACKNOWLEDGEMENTS The authors would like to thank the Max-Planck-Institut for plant breeding research (MPIZ) for providing the fruitful and motivating environment for our work and, in particular, Dr. George Coupland (Head of Dept. Plant Developmental Biology) for his support and Ms. Sabine Frings for her excellent technical assistance.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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STRUCTURE, FUNCTION AND MODE OF ACTION OF SELECT ARTHROPOD NEUROPEPTIDES GERD GADE and HEATHER G. MARCO Zoology Department, University of Cape Town, ZA-7701 Rondebosch, South Africa Dedicated to our parents who have always supported our scientific curiosity whole-heartedly and unconditionally. ABSTRACT: This overview summarizes important features of the majority of neuropeptide families that occur in two species-rich and widely-radiated arthropod taxa, the crustaceans and the insects. The neuropeptides may act as true neurohormones, which are released into the circulation, or as local neurotransmitter and/or neuromodulator. By comparing the primary structures of members of peptide families, the biosynthesis (including preprohormone structure and the peptidergic control of release), the structures of the receptors and transduction of the message via second messenger systems, the inactivation and the multiple functions of selected neuropeptides, we want to draw the reader's attention to the following main conclusions: 1) neuropeptides are important physiological regulators in arthropods; 2) neuropeptides can be structurally and functionally highly conserved in major arthropod groups (for example, proctolin, crustacean cardioactive peptide), or where peptide isoforms exist, there may be different scenarios: - (i) in the case of identical isoforms in insects and crustaceans, the peptide function may have changed in the two taxa (for example, red pigment-concentrating hormone affects pigmentation in crustaceans but mobilizes lipids in insects), - (ii) the isoforms are different in the two taxa and may have the same effect (for example, the ion-transporting peptide in insects and the crustacean hyperglycaemic hormone in crustaceans both play a role in osmoregulation), - (iii) the structurally different isoforms have different functions in the two taxa (for example, pigmentdispersing hormone affects pigmentation in crustaceans, whereas pigment-dispersing factor affects circadian rhythmicity in insects.
INTRODUCTION The phylum Arthropoda contains a number of subphyla, such as Chelicerata (scorpions, spiders and mites), Myriapoda (centipedes and millipedes), Crustacea (crabs, shrimps and woodlice) and Hexapoda (springtails, bristletails and insects).' The most conspicuous and wellknown members of the Arthropoda are, undoubtedly, the crustaceans and
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the insects. Even laymen recognize them and are fascinated by certain morphological features, behaviours and physiological events that are associated with these arthropods. For the specialist the debate is still ongoing as to whether crustaceans and insects are phylogenetically related in a sister group relationship; more and more evidence has recently accumulated from molecular data, ultrastructure of brain and eye, and from neurogenesis to support such a relationship [1-4]. Crustaceans and insects have diversified enormously, and scientific research has, thus, only been conducted on a small number of extant species. The crustaceans are divided into up to 6 classes [5] of which the Malacostraca is the largest and the one that contains the better known crustacean species that occur in the sea, freshwater and on land. Apart from the isopods, research on neuropeptides have mainly been conducted in decapod crustaceans, therefore, this review will be limited to the Decapoda, which contains the most familiar and widespread crustaceans such as shrimps, prawns, crabs, crayfish, spiny lobsters and lobsters and excludes the isopods and amphipods. The decapod crustaceans are a rich source of proteins and famous for their exceptional taste that has made them such a sought-after culinary delicacy. Decapod crustaceans, therefore, form the hub of commercially lucrative seafood industries the world-over. They are, however, also of ecological significance, chiefly dominating the marine and freshwater habitats with only a few terrestrial species. Insects, on the other hand, are the most successful animal group on land and, although insects form a substantial part of the diet of many human tribes, they are far more renowned for (a) their sheer abundance (at least 1 million species are described and a further estimated 3 - 5 million species have not been detected and/or described yet), (b) their striking beauty and (c) their beneficial aspects (e.g. pollination), as well as (d) their potential for disaster to mankind (e.g. crop damage, vector for the transmission of diseases). Insects and crustaceans are also well-known to the general public because of the spectacular changes that they undergo during their life-cycle, events such as metamorphosis and ecdysis; and who has never wondered about how these animals are able to adapt to environments with different salinities, or how they can change colour to camouflage themselves, or how they can use metabolites so efficiently during longdistance migrations or fast swimming? All these events, and more, are under the control of small, bioactive peptides. Because these regulatory peptides are synthesized in modified neurons, they are called neuropeptides.
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In this article we will review the existing literature on a selection of neuropeptides to find out whether the phylogenetically related decapod crustaceans and insects synthesize peptides of the same or very similar primary structures and if so, whether the neuropeptides are employed for the same function and whether this function is exerted by an identical pathway for their mode of action. Although such comparisons may shed some light on evolutionary aspects, by no means is this account meant to treat neuropeptides in a phylogenetic framework.
1. NEUROSECRETION Neuropeptides are peptidergic chemical messengers that are synthesized in specialized neurons and are released into the general circulation, which in insects and crustaceans is called haemolymph, to reach their target organ(s). Most neuropeptides are, in fact, hormones which control a number of physiological processes, hence, the neuroendocrine system represents a form of communication between cells, tissues and organs, other than the classical nervous and endocrine systems. Nervous control mechanisms act rapidly through synapses, releasing neurotransmitters into the synaptic cleft and generating action potentials of short duration; the classical endocrine (hormonal) control is slower acting but of longer duration since the hormones are released into circulation often a long distance away from the target organ and it takes some time before the hormones are degraded. Some neuropeptides do, however, also act as neurotransmitters and neuromodulators as evidenced by their distribution: the neuropeptide-synthesizing neurons may innervate a target organ directly, in addition to projecting to a neurohaemal organ, e.g. proctolinproducing neurons project into the pericardial organs (PO) of crustaceans, as well as to skeletal muscles, and proctolin has a positive modulating effect on neurons of the stomatogastric ganglion [6]. 1.1. Neuroendocrine complexes in insects Most of the endocrine processes in insects are controlled by neuropeptides. The neuroendocrine system in insects is comprised of neuropeptide-synthesizing neurons located in the cerebral ganglia (brain),
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specifically, in the pars intercerebralis and the median and lateral parts of the protocerebrum, as well as neurons in the retrocerebral corpora cardiaca (CC) and ventral nerve cord ganglia (see Fig. 1A). The neuropeptide producing cells are usually arranged symmetrically, and their axons end in so-called neurohaemal organs that serve as the storage and release site of the neuropeptide hormones. The chief neurohaemal organs of insects are (a) the paired retrocerebral CCs, that store neuropeptides from the brain, the sub-oesophageal ganglion and the CC itself and release them into the aorta and, (b) the ventral perisympathetic organs that store neuropeptides made by neurons in the thoracic and abdominal ganglia (see Fig. 1) [7]. A
B
Medial neurosecretory cells
Sinus gland
Lateral neurosecretory cells Brain -
Head
X organ
Corpus cardiacum Corpus allatum
ephalohorax
Gut
Perisympathetic organ
Abdomen
Abdomen
Figure 1. Schematic diagrams of the neuroendocrine system in (A) insects and (B) crustaceans showing the chief location of neurosecretory cells and their neurohaemal release sites. (A) is modified after [10]; fB) is modified after r 1121.
1.2. Neuroendocrine complexes in decapod crustaceans In crustaceans, too, many physiological processes are under neurohormonal control, and in the early years of investigation, these processes were pin-pointed by experimental biological studies that
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involved ablation or implantation of specific tissues/organs, injection of crude extracts of tissues/organs and observations at macroscopic and microscopic levels [8]. In this way it came to light that eyestalks of decapod crustaceans house an important endocrine centre that exerted control over glucose metabolism, moulting, reproduction and epithelial pigmentation. Histology with light microscopy revealed a neuroendocrine complex in the eyestalk that is made up of the X-organ (XO), a cluster of large neurons in the medulla terminalis which is the site of neuropeptide synthesis, and the sinus gland (SG), a neurohaemal organ which is located between the medulla externa and the medulla interna and is formed by the collective axonal endings of the XO neurons, and which serve as the site for storage and release of the neuropeptides into circulation (Fig. IB). Other sources of neuropeptides in decapod crustaceans have been identified [9]: (a) the pericardial organs (POs), neurohaemal organs that lie in the venous cavity surrounding the crustacean heart; the axons ending in the POs arise from thoracic (including the suboesophageal) ganglia and from intrinsic somata; (b) the post-commissural organs, neurohaemal organs with intrinsic neurons and axons arising from the commisure that passes posterior to the oesophagus, connecting the circumoesophageal connectives; not much is really known about this system, and (c) various components of the central nervous system (CNS), such as cerebral (brain), sub-oesophageal, thoracic and abdominal ganglia (Fig. IB). 2. METHODS USED IN NEUROPEPTIDE RESEARCH A suite of methods has been successfully applied over the past few decades in the research on arthropod neuropeptides: the relevant methods are mentioned only briefly here with some examples relating to crustacean neuropeptides; the reader is referred to [10] for a more detailed description of the general methods employed in the isolation and characterization of insect neuropeptides. 2.1. Biological assays Biological assays are employed to (a) determine the physiological relevance of a neuropeptide and (b) to monitor or identify the
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neuropeptide of interest during the procedure of isolation from an extract of biological material. The functional characterization and identification of neuropeptides includes studies at: (i) the gross in vivo level, e.g. which functions are affected by experimental intervention, such as ablation of crustacean eyestalks or re-implantation of sinus glands; (ii) the more-refined in vivo level, e.g. injections of assumed physiological doses of a chemically pure neuropeptide and monitoring specific parameters, such as measuring changes in glucose concentration after injection of crustacean hyperglycaemic hormone; (iii) the indirect in vitro approach, e.g. monitoring the effect that a neuropeptide has on a specific organ where this organ plays an integral role in another process, such as the Y-organ bioassay in which the effect of moult-inhibiting hormone on moulting is studied by measuring the output of ecdysteroids; (iv) the direct in vitro approach, e.g. monitoring the direct effect a neuropeptide may have on an explanted organ, such as cardioacceleratory peptide on heartbeat or the effect of myokinins on isolated hindgut. Other physiological studies with the isolated neuropeptides include dose-responses, time-course, mode of action, peptide titres and effects during stress and different stages of development or reproduction; crossactivity studies at intraspecies, as well as interspecies levels. Before the advent of molecular biological techniques, more often than not, a neuropeptide was first functionally identified by means of biological assays and subsequently sequenced than the other way around, i.e. very few peptides were sequenced without some knowledge of its function. 2.2. Elucidation of neuropeptide structure Once it was established that certain organs/tissues were producing a neuroendocrine factor and the actions of such factors were roughly defined by the experimental biological approach (biological assays), studies focused on the structural elucidation of the neuroendocrine substances. First, the chemical nature of the neurohormones was demonstrated as peptidergic (because they could be inactivated by proteolytic enzymes) [8] and then several other methods were employed to characterize the neuropeptides, e.g. size estimation by using gel chromatography, determining the iso-electric point and investigating whether the neuropeptide is heat-resistant or labile. It lasted several
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decades after its initial functional characterization before the first neuropeptide hormone from invertebrates, the red pigment-concentrating hormone (RPCH), was chemically isolated and its primary structure determined [11]. These early methods, however, required the use of gram amounts of starting material (e.g. whole eyestalks) plus chemically complicated procedures to isolate the active neuropeptide. Amino acid sequencing by Edman degradation in the 1970s was, of course, an important breakthrough in the field of peptide chemistry. Improved methods of microanalytic peptide chemistry, such as isolation of peptides by high pressure liquid chromatography (HPLC), peptide synthesis, reliable mass spectrometry for determining molecular weights and advanced functions of mass spectrometry for peptide sequencing, as well as the use of defined peptidases all contributed to the structural identification of neuropeptide hormones from different insects and crustaceans. 2.3. Localization and quantification of neuropeptides Information on the primary structures enabled comparative endocrinologists to arrange peptides into structurally homologous peptide families; one could also generate very specific antisera for localization of the neuropeptides in cells, tissues and organs with the aid of immunocytochemistry, and for peptide quantification (titre determinations) by means of immunoassays (radioimmunoassay; RIA and enzyme immunoassays; EIA). Polyclonal antisera, raised against conserved structural units of a known peptide, can also be employed to monitor and identify similar peptides during the isolation and purification procedures of the latter. For example, antisera raised against the crustacean hyperglycaemic hormone (cHH) of the edible crab, Cancer pagurus, and the American lobster, Homarus americanus, were successfully used in an enzyme-linked immunoassay (ELISA) to identify cHH molecules in an extract of sinus glands from the South African spiny lobster, Jasus lalandii [12].
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2.4. Molecular biological techniques Conventionally, invertebrate neuropeptides were isolated, chemically characterized and accompanied by some functional analyses. Since the movement towards molecular biological methods in the late 1980s, however, information of DNA encoding peptides with structural homology to known neuropeptide hormone families have "flooded" the scene, and in most of these cases, physiological functions of the encoded peptide could only be guessed at since the peptide itself was not chemically isolated. The chief advantage of using these methods is that relatively small amounts of starting material are required to (a) provide information on the preprohormone structures, (b) allow sensitive expression studies of the specific mRNA by in situ hybridization, Northern blot analyses or polymerase chain reaction (PCR), and (c) there is the potential for recombinant production of neuropeptides for use in physiological studies. The latter can be beneficial for the synthesis of larger and more complicated neuropeptide structures, such as the cHH family peptides which have proven a big challenge for traditional chemical synthesis [13, 14]. The use of improved molecular biology techniques and the world-wide impetus of such studies, also resulted in the sequencing of the complete genome of selected organisms, including the insects, Drosophila melanogaster and Anopheles gambiae. This served as a launch pad for what is now known as "database mining" where putative G proteincoupled receptors, for example, can be identified and then cloned. Although this has provided lots of structural information on receptors for insect neuropeptides, the ligands for these receptors are sometimes not known, hence the term "orphan receptor" has been coined. Prior to these technical advances, the primary structure of only a few receptors for invertebrate neuropeptides was known, and receptors were identified in fractionated cell membranes by specific binding of radiolabeled ligands. Receptor-ligand interaction was also investigated indirectly by performing functional bioassays with analogues of a particular ligand (e.g. the lipid-mobilizing assays with the migratory locust to gather information about the receptor for the insect adipokinetic hormone, AKH) [10]. Several assays are now commonly used to study putative receptors of neuropeptides and to "de-orphanize" the receptors: the receptor is expressed in mammalian cell lines or in amphibian oocytes and is then
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exposed to different peptide ligands; binding between the ligand and the receptor is then monitored in various ways, e.g. by bioluminescence or an electrophysiological signal. With the bioluminescence assay, cells are transfected to express (i) the G protein-coupled receptor of interest, (ii) a promiscuous G protein (so-called because it can activate most G proteincoupled receptors by the phospholipase C pathway) that effects an increase of intracellular Ca2+ when stimulated, and (iii) a Ca2+-sensitive photoprotein, apoaequorin. Aequorin is a bioluminescence complex that was originally isolated from a jellyfish; this complex is made up of three components, viz. (i) apoaequorin which has Ca2+-binding sites and changes conformation to an oxygenase when these sites are occupied, (ii) a luminophore cofactor called coelenterazine, and (iii) molecular oxygen. The molecular oxygen oxidizes coelenterazine and the emission of a blue light is one of the products formed [15]. Thus, in the bioassay when the agonist binds to the G protein-coupled receptor, the G protein is stimulated and results in an intracellular increase of Ca2+; the latter combines with apoaequorin and the protein undergoes a conformational change into an oxygenase, coelenterazine is oxidized and a blue light is emitted and measured [15]. The electrophysiologically based receptor bioassay relies on measuring a change in the membrane potential of cells when a ligand binds to the receptor of interest. One way to do this is by co-injecting the receptor cRNA with murine GIRK (G protein-gated inwardly rectifying potassium channel) cRNA into defolliculated oocytes from the frog Xenopus laevis. The electrophysiological events in the oocyte are recorded by whole cell voltage clamping in a high potassium bathing buffer [16]. When an agonist binds to the receptor, an inward potassium current is induced which is then recorded [16]. To date, the complete sequence information of a crustacean genome has not been published; consequently, there is a wide gap in the available data on receptors for crustacean neuropeptides. 3. INSECT AND CRUSTACEAN NEUROPEPTIDES It is by no means surprising that structurally homologous neuropeptides have been identified in crustaceans and insects - they share, after all, a common ancestry. The functions of some of these neuropeptides have not been conserved in these arthropod groups and we discuss here a selection of neuropeptide families common to both. For each peptide family, we
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will describe the characteristic structural features of the peptide members, when it was first isolated and elucidated in insects and crustaceans, functional activities, localization of the peptide and its synthesis, including available information on the gene and preprohormone, as well as known peptidergic factors regulating the release of the neuropeptide. Information on receptors and their interaction with specific neuropeptide ligands, as well as the peptide mode of action will also be discussed where possible. This review will not provide details, except in a few cases, of the various isolation techniques, nor of the many biological assays used in the characterization of neuropeptides and/or their receptors, since most of the commonly used methods have been elaborated on in previous reviews on neuropeptides [10] or are briefly described in the Introduction above (Section 2). In the various tables provided in the next sections, we will only give structures of those insect peptides that are not shown in our previous overview [10]; for crustaceans, however, we will give all published structures because they are not as readily available in earlier reviews.
3.1. Colour change versus metabolite mobilization: the AKH/RPCH and PDH/PDF families of neuropeptides Already as early as the 1920s to 1940s it was demonstrated that extracts from certain glands of crustaceans and insects caused "blanching" in shrimps [17-21], i.e. the shrimp changes from a dark to a light colour. In crustaceans, colour change is effected by the movement of pigment granules in the integumental cells (chromatophores): when the granules are concentrated in the cytoplasm, blanching results; when the granules are dispersed, a darkening effect is obtained. Later, it was discovered that pigment movement in another cell type of crustaceans was also under hormonal control: the compound eye of arthropods consists of a number of ommatidia which is the smallest functional unit in the process of visual perception. Whereas the movement of the screening pigments in the reticular (photoreceptor) cells is mainly achieved by direct action of light, pigment movement in non-reticular ommatidial cells (either reflecting pigment cells and/or distal pigment cells) is controlled by neurohormones [22,23].
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3.1.1. The AKH/RPCH family After a number of attempts of purification and separation of the peptides that affect pigments in chromatophores of various crustacean species [24], it took until 1968 [25] to achieve a complete purification of the material from the eyestalks of Pandalus borealis. The decisive step in the purification scheme was the elution behaviour of the compound in water/butanol mixtures on Sephadex LH-20 columns. The complete structure elucidation was made possible by: (a) quantitative ammo acid analysis and measurements of ultraviolet absorption (both methods together establishing the equimolar amount of 8 amino acids), (b) the use of the newly developed high-resolution mass spectrometer which determined the N-terminus as pGlu-Leu-Asn, (c) the successful proteolytic cleavage of the molecule into two fragments of 3 residues (the already known N-terminus) and 5 amino acid residues, and (d) the sequencing of the latter by Edman-dansyl sequencing to reveal the Cterminal sequence Phe-Ser-Pro-Gly-Trp amide [11, 26]. Thus, in 1972 the structure of the first invertebrate neuropeptide, now known as PanboRPCH because of its concentrating effect on red pigment granules in crustaceans, was completely elucidated (see Table 1). Parallel to these developments, scientists who were interested in carbohydrate and lipid metabolism in insects were looking for substances that have similar actions to those of the well-known metabolic hormones of vertebrates, glucagon and insulin. The first report on the existence of a glucagon-like factor in insects came from Steele [27]. Extracts of CC from the American cockroach, Periplaneta americana, elevated the concentration of trehalose, a disaccharide, which is the main blood (haemolymph) sugar of insects. A few years later a different effect of extracts from the CC was reported in the desert locust, Schistocerca gregaria [28], and the migratory locust, Locusta migratoria [29]. Here, the concentration of lipids in the haemolymph was increased upon injection of CC extract, thus, a hyperlipaemie or adipokinetic effect was measured. After a number of purification attempts, a decapeptide, which is now called locust adipokinetic hormone I (Locmi-AKH-I) was isolated from 3000 corpora cardiaca by size-exclusion chromatography on controlled-pore glass and thin layer chromatography on silica gel [30]. Structure elucidation was achieved by a combination of enzymatic
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cleavage and mass speetrometry resulting in the primary sequence of a decapeptide [30]. Table 1. Primary structures of members of the AKH/RPCH family of peptides. Peptide name, code name:
Species
Crustaceans red pigment-concentrating hormone Panbo-RPCH Pandalus borealis and various other crustaceans
Sequence
Reference
pELNFSPGW-NH2
[11.31]
Insects adipokinetic hormone Locmi-AKH-I
Locusta migratoria
pELNFTPNWGT-NH2
[30]
Loemi-AKH-H Locmi-AKH-ffl Panbo-RPCH Eiysi-AKH Pyrap-AKH Tenar-HrTH
L. migratoria L. migratoria Nezara viridula Erythemis slmpllcicollis Pyrrhocoris apterus Tenthredo arcuata
pEQLNFSAGW-NHj pELNFTPWW-NH2 pELNFSPGW-NHa pELNFTPSW-NH2 pELNFTPNW-NH2 pELNFSTGWGG-NH2
[232,233] [35] [32] [234] [235] [236]
A complete list of sequences up to 1997 is available for insect members of this family [10,37].
The primary structures of Locmi-AKH-I and Panbo-RPCH are strikingly similar (Table 1). Both peptides are cross-active in the reciprocal system and the structural similarity also explains why crude extracts of insect CCs (which contain one or the other form of "AKH") cause blanching in shrimps (see above). The structural similarity was also the basis for classifying these peptides as members of a peptide family, viz. the AKH/RPCH family of peptides. Thus, the peptide family includes structurally related peptides which have diverse functions in the two main arthropod taxa. New developments in the analysis of small peptides have greatly advanced this field. For example, improvements in the techniques of isolation and sequencing have occurred along with the introduction of modern mass spectrometric methods for accurate mass determination and sequencing tasks as first achieved with the fast atom bombardment (FAB) mode, and later with matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass speetrometry. These new techniques, combined with the large amount of peptidic material typically stored in the CC, made it relatively easy to determine the primary structure of almost 40 different natural analogues in about 100 insect species (see Table 1 for some examples). Far fewer decapod crustaceans have been investigated but, interestingly, the structure of RPCH is highlyconserved among crustaceans. In all species from which it has been
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sequenced or characterized by chromatographic behaviour and amino acid composition data, only RPCH can be found of the AKH/RPCH family peptides in crustaceans [see 31]. RPCH is, however, also synthesized in an insect species, the bug Nezara viridula (belonging to the large taxon of Hemiptera), where it mobilizes lipids [32]. In insects, representative peptides of the AKH/RPCH family have been found in most orders [33, 34]. In a number of taxa, gene duplication has taken place and two or even three AKH peptides are found in one species [10, 35-37]. In contrast to crustaceans (only RPCH present), insects show a high degree of variability in isoforms of AKH peptides. Common characteristics of the family are: a chain length of 8 to 10 amino acids; the N-terminus blocked by pyroglutamic acid (pGlu); the C-terminus blocked by a carboxyamide; amino acids at positions 8 and 9 (when present) are Tip and Gly; most of the peptides are uncharged, but there are a few that have an aspartic acid at position 7; there are at least two aromatic acids present, at position 4 mostly Phe (but sometimes Tyr) and at position 8 (Tip), and a few peptides have a third aromatic acid either at position 2 (Tyr or Phe) or at position 7 (Tip). In addition to the post-translational modifications at the terminals, the hypertrehalosaemic hormone (HrTH)-I of the stick insect, Carausius morosus (Carmo-HrTH-I) is glycosylated [38]. The site of glycosylation is not the usual Ser/Thr (O-glycosylation) or Asn (N-glycosylation) but Tip. As in human ribonuclease [39], the hexose in Carmo-HrTH-I is very likely C-glycosidically linked to the C-2 atom of the indole ring of Tip. In some insects, notably Lepidoptera (e.g. the butterfly Vanessa cardui) the AKH is not completely processed from the prohormone and, thus, occur in a C-terminally extended form (..-Gly-Gly-Lys) together with the fully processed peptide Manse-AKH [40]. Unfortunately, no rigorous tests have been conducted as to whether this compound is biologically active or whether the measured biological activity is the result of a breakdown product. Another, as yet unidentified, modification apparently occurs in a hypertrehalosaemic neuropeptide of cicadas: in a number of species two peaks are always found when using HPLC separation methodology but the material of both peak fractions have the same mass and amino acid sequence [10, 37]. When the synthetic peptide was synthesized according to the sequence information, its retention time on HPLC coincided only with one of the two peaks derived from natural material. The detailed pathways of the biosynthesis of the two adipokinetic hormones from S. gregaria, including the characterization of the
82
preprohormones, have been elucidated by direct protein chemical methodologies, as well as molecular cloning [41, 42]. Similar studies have been executed for AKH family members in other insect species and also for RPCH of crustaceans. There is a distinct mRNA encoding for each AKH precursor (up to 3 in L migratoria) [43] which is translated into the discrete precursor, the prepro-AKHs. The organization of the precursor is basically always the same for all AKH and RPCH peptides: a signal peptide is followed by the respective AKH sequence, followed by the Gly residue for amidation of the AKH, the dibasic processing site, and C-termmally, a "tail peptide" or "precursor-related peptide". The latter is very long (more than 70 ammo acids) in the crustaceans compared to the 28 to 46 residues in insects. There are almost no structural similarities between signal peptides and "tail peptides" of insects and crustaceans. No biological function is known for any of the "tail peptides". Studies on the biosynthesis of the AKHs from the desert locust have revealed a unique strategy: after cleaving off the signal peptide, the two independently translated monomers of the pro-Locmi-AKH-I (as an example), consisting of the sequence for Locmi-AKH-I and the Cys-containing "tail peptide", respectively, are oxidized to a unique precursor dimer forming a disulfide bond. Thereafter, the precursor is processed to the following products: two monomeric molecules of Locmi-AKH-I extended by Gly-Lys-Arg and one dimeric molecule of the precursor-related peptide. The extended Locmi-AKH is subsequently cleaved by a carboxypeptidase H-like enzyme, which removes first the Arg and thereafter the Lys residue. A peptidylglycine-a-amidating monooxygenase then produces, from this Gly-extended form, the amidated Locmi-AKH. For RPCH and also other insect AKHs it is not known whether dimers are formed. The processes described above take place in the intrinsic neurosecretory cells of the CC of insects. The localization of AKH peptides in the neurosecretory cells of the CC has been shown numerous times by immunocytochemistry, and in situ hybridization experiments have demonstrated that the signals for the mRNA of all three AKH preprohormones of the migratory locust are co-localized in the neurosecretory cells of the CC [43]. RPCH immunoreaetive cells are found in neurons of the XO but also in other neurons, including those projecting into the neuropil of the stomatogastric ganglion of the crab, Cancer borealis and the spiny lobster, Panulirus interruptus or neurons of the abdominal ganglion of Pacifastacus leniusculm [see 6].
83
Peptidergic releasing factors for AKHs of the migratory locust have been reported. In vitro studies identified the locust tachykinin [44] (see Section 3.4.6) and the crustacean cardioactive peptide [45, 46] which also occurs in insects (see Section 3.4.2) to promote the release of all three AKHs. The peptides FLRFamide from the desert locust and the peptide FMRFamide (see Section 3.4.4) were shown to be inhibitors of AKH release in the migratory locust in vitro [47]. There are no convincing reports on peptidergic releasing factors for RPCH in crustaceans and the reader is referred to [24] for information on the possible action of aminergic control factors. Release of AKH in locusts was also shown upon flight and quantitated by using a biological assay [48]. Only recently, sufficiently sensitive methods were published to determine reliable AKH titres by a RIA [49], and an ELISA [50]. In both immunoassays the major hurdle to overcome to obtain consistent measurements were the processing of the haemolymph samples. These are cumbersome and time-consuming and prevent the methods from being used on a routine basis. With the RIA, the release of Locmi-AKH-I and Schgr-AKH-II of the desert locust was shown to increase 15- and 6-fold upon 5 min of flight [49]. Since the maximal released levels are between 3 and 1 pmol for the two AKHs, it is clear that only a small fraction of the stored AKH material is released when locusts fly for 30 min. No titre determinations of RPCH have been executed, although there may be an RPCH analogue that could be useful for successfully developing a RIA method: when the Phe residue at position 4 in RPCH is replaced by Tyr this can be iodinated with 125I; a Tyr4 RPCH compound is even 4-fold more active than RPCH itself [51]. Once released into the haemolymph, the peptides are prone to proteolytic breakdown. Whereas no data are available on RPCH, there are quite a number of publications that deal with the fate of various AKHs. Only a few studies are reliable, those in which the AKH peptide concentration was used at physiological concentrations of about 1-3 pmol. In migratory locusts, for example, the three AKHs are not associated with carrier proteins during transport in the haemolymph, they have short half-lives which are not only different for the three peptides but also different for each peptide during a flight period [52]. One of the peptidases involved in the process of AKH breakdown is an enzyme with actions and physical and kinetic properties closely resembling those of the mammalian endopeptidase24.11 [10].
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Structure-activity studies are an indirect method to investigate a possible interaction of AKH peptides with their receptor proteins. For various members of the AKH family a vast body of literature has accumulated, employing bioassays such as lipid mobilization in locusts and the tobacco hornmoth Manduca sexta, carbohydrate mobilization in various cockroach species, the activation of phosphorylase in the larvae of M. sexta, or the inhibition of fatty acid synthesis in the fat body of locusts [10, 33, 37, 53]. Contrary to most other invertebrate neuropeptides, the insect members of the AKH family do not have a "core sequence" which is essential for potency, however, the N-terminal pGlu residue and the Cterminal amide are important, as well as the aromatics at position 4 and 8. To achieve full efficacy, all amino acids are apparently important. There is also no superagonist found and also no inhibitor. Two noteworthy sets of structure-function studies on RPCH have been performed. The chemical approach using synthetically modified analogues of RPCH and testing their effect on pigment movement in the shrimp, Palaemon (=Leander) adspersus revealed, surprisingly, that the blocked N-and C-termini are not essential for biological activity, in contrast to the situation in insects. Moreover, the Tip residue is very important for interaction with the receptor; replacement of the Phe residue at position 4 by Tyr results in an agonist with 4-fold higher activity [26]. A second approach made use of some of the then-existing natural analogues of RPCH, namely the insect AKH members, and measured their effect on erythrophores of the crayfish Cambarellus shufeldtii [54]. The native Panbo-RPCH was always the most potent compound, and the insect Schgr-AKH-II, which differs from Panbo-RPCH only in the Thr at position 6, had about 80 % of that potency; all other compounds were markedly less active. The first report on an AKH receptor protein in insects mentioned specific binding studies with tritiated Manse-AKH on membrane fractions purified from the fat body of adult M. sexta [55]. A competitive receptorbinding assay was developed and the analogues tested, revealing again that almost each amino acid of the AKH molecule is equally involved in interaction with the receptor [56]. In crustaceans to date, it has only been shown that membrane proteins from brain tissue, thoracic ganglia and the abdominal nerve cord are apparently able to bind RPCH [57], but no receptor has been isolated. This will, however, be feasible now, because the latest studies on insect AKH receptors have reported on molecular biological methods which may also be applicable to finding the RPCH
85
receptor. AKH receptors have been cloned from the fruit fly, D. melanogaster and the silkworm, Bombyx mori [58, 59]. These G proteincoupled receptors have 7 membrane-spanning domains and are structurally related to receptors of the vertebrate gonadotropin-releasing hormone. Following functional expression of the D. melanogaster AKH receptor in Chinese hamster ovary (CHO) cells [58] or in frog oocytes [59], the receptor responded to the AKH peptide of D. melanogaster with the lowest EC50 value of all AKH peptides tested. As a caveat, however, one has to stress that these tests are not testing for true receptor binding but are heterologous "functional" bioassays and often use a promiscuous G protein, which does not allow anyone to test whether the ligand really signals through Ca +. Studies concerning the cellular signalling pathways, thus the mode of action of AKH/RPCHs, have been numerous in insects but only few in crustaceans. If we consider the classical action of insect AKHs, namely to elicit an increase of carbohydrates, lipids or proline in the haemolymph, the following signalling pathways are known [37, 60-62]: to achieve the efflux of trehalose from the fat body cells, glycogen phosphorylase has to be activated and for this, AKH activates a Gq protein after binding to the receptor and this activation leads to the stimulation of phospholipase C (PLC) and the production of the second messengers inositol trisphosphate (IP3) and diacylglycerol; IP3 is responsible for Ca2+ mobilization from internal stores; external Ca2+ plays an important role as well. In the migratory locust, stimulation of adenylate cyclase (AC) and therefore the second messenger, cyclic AMP, is additionally involved in the activation of glycogen phosphorylase. On the other hand, those AKHs that are finally responsible to increase the concentrations of diacylglycerols or the amino acid proline in the haemolymph, signal via a Gs protein and a subsequently stimulated AC produces cyclic AMP. Again, extracellular and intracellular Ca2+ (from IP3 insensitive stores) is necessary to stimulate lipase. Pigment aggregation in the shrimp Macrobrachium potiuna is thought to proceed via a similar route involving PLC, IP3 and Ca2+ as described above, however, the evidence for this is weak and mostly indirect [63]. That insect AKHs are truly multifunctional and have pleiotropic tasks will be evident from the following list that summarizes the various effects observed besides the control of mobilization of metabolites [10, 37, 53]: 1. stimulation of lipid oxidation by flight muscles
86
2. increase of the lipid-carrying capacity of lipoprotein carriers in the haemolymph 3. inhibition of protein synthesis; maybe even a specific effect on vitellogenin and, thus, on reproductive processes 4. inhibition of synthesis of RNA 5. inhibition of synthesis of fatty acids 6. stimulation of heart beat and certain other muscles, and locomotory activity in general 7. aiding the immune response in the migratory locust The list of functions corroborated for RPCH is smaller [31]: 1. stimulation of the release of methyl farnesoate from mandibular organs (MOs) 2. modulation of the rhythms of certain parts of the crustacean stomatogastric and swimmeret system 3. modulation of the crustacean photoreceptor cells. RPCH is not known to have any true metabolic effect in crustaceans, however, RPCH can (of course) elicit most of the actions of the insect AKHs in the appropriate insect recipient. Likewise, insect AKHs are potent to concentrate pigments in the appropriate crustacean species but to date, no report has shown that AKH can effect a colour change in insects. 3.1.2. The PDH/PDF family Early studies had shown that there are substances in the eyestalks of decapod crustaceans that have an influence on retinal pigments (see above). In 1971 a peptide that caused the dispersion of distal retinal pigment was purified from eyestalk extracts of P. borealis mainly by chromatography on CM-Sephadex [64]; the biologically active fraction was sequenced by Edman-dansyl sequencing and the compound turned out to be an octadecapeptide with an amidated C-terminus but with a free N-terminus [65] (see Table 2). Due to its function, the peptide was originally called light adapting distal retinal pigment hormone (DRPH). It is now better known as pigment-dispersing hormone (thus, Panbo-ccPDH), because it also translocates the pigments in the chromatophores centrifugally [66]. About a decade later, a second PDH was chemically identified from the eyestalks of the fiddler crab, Uca pugilator, a so-
87
called P-PDH which differs from a-PDH in six positions (see Table 2) [67]. To date, PDHs from 14 decapod crustacean species are known; there are some modifications of a- and P-PDHs occurring. It was later discovered that extracts from heads of insects were able to elicit a dispersion of pigments in the epidermis of eyestalk-less fiddler crabs; this bioassay was employed to isolate the active principle from the grasshopper, Romalea microptera - a modified P-PDH - via a number of complex chromatographic steps including partition-, gel filtration- and ion-exchange chromatography [68]. Since then, the structures of pigmentdispersing factors (PDFs) have been completely elucidated from cricket, stick insect and cockroach; and orthologues have been identified in the genomic cDNA databases of D. melanogaster [69] and the malaria mosquito, A. gambiae [70]. There are many conserved structural features in the PDH/PDF family; namely the chain length (18 ammo acid residues), the N-terminal Asn and C-terminal amidated Ala residues, the residues Ser, He, Asn, Ser and Leu at positions 2, 5, 6, 7 and 9; moreover, the substitutions occurring at positions 4, 8, 10, 12, 15 and 17 can all be explained by point mutations [31]. Interestingly, like AKHs in insects, PDHs in crustaceans have undergone gene duplication and up to three forms may be found in one species, for example two a-PDHs and one P~ PDH, are found in Pandalus jordani [31] (Table 2). A phylogenetic tree for PDH/PDF peptides that is constructed with the sequence data, suggests that P-PDH may be an "ancient molecule from which the PDHs and PDFs evolved as a highly conserved family of neuropeptides" [31]. There is nothing known about the actual biosynthesis of these peptides. Molecular biological studies, however, have identified the preprohormone sequence from some PDHs and PDFs [31]. The general organization is strikingly different to that of the AKH/RPCH precursor: the signal peptide is followed immediately by a precursor-related peptide of unknown function and variable sequence and length, a dibasic or tribasic cleavage site and the PDH/PDF octadecapeptide with a C-terminal Gly for amidation and a monobasic or dibasic cleavage site at the end of the open reading frame (ORF). Localization of cells producing PDHs/PDFs has been achieved by immunoeytochemistry. Mapping of PDH-immunoreactivity in a few crustacean species revealed not only association with neurosecretory cells but also with intemeurons suggesting an additional role of PDH as neurotransmitter or in neuromodulation [71]. In several insects, including
88 88
D. melanogaster, PDF is localized in a few specialized neurons which have their cell bodies in the optic lobe and some processes in the accessory medulla of the optic lobe, an area which is known to house the neurons of master circadian pacemakers responsible for daily locomotory rhythms [72, 73]. It is from these and a number of other observations that PDFs are thought to be the communicator of these pacemaker cells to spread the message of the biological clock, thus acting as peptidergic neurotransmitters [74]. The PDFs do not affect pigment movement in insects. Table 2. Primary structures of members of the PDH/PDF family of peptides. Species Sequence Peptide name, code name Crustaceans a-pigment-dispersing hormone Panbo-PDH Pandalus borealis, Pandalusjordani Panjo-PDH-III P.jordani Macro-PDH Macrobrachium rosenbergii [5-pigment-dispersing hormone Ucapu-PDH Uca pugilator, Callinectes sapidus, Cancer magister, Carcinus maenas, Pacifastacus leniusculus Procl-PDH Procambarus clarkii, Orconectes immunis, Orconectes limosus Penaz-PDH Penaeus aztecus, Penaeus vannamei Panjo-PDH-I P.jordani Calsa-PDH-II C. sapidus Penva-PDH-III P. vannamei Penja-PDH-I Penja-PDH-II
Penaeus japonicus P. japonicus
Insects Pigment-dispersing factor Drome-PDF Drosophila melanogaster
Reference
NSGMINSILGIPRVMTEA-NH2 NSGMINSILGIPKVMADA-NH2 NSGMINSILGIPKVMAEA-NH2
[31,65] [31] [31]
NSELINSILGLPKVMNDA-NH2
[31]
NSELINSILGLPKVMNEA-NH2
[31]
NSELINSLLGIPKVMNDA-NH2 NSELINSLLGLPKVMTDA-NH2 NSELINSLLGISALMNEA-NH2 NSELINSLLGLPKVMNDA-NH2
[31] [31] [31] [31]
NSELINSLLGIPKVMTDA-NH2 NSELINSLLGLPKFMIDA-NH2
[31]
NSELINSLLSLPKNMNDA-NH2
[31]
[31]
Only one PDF structure is included; a complete list is available [10, 31].
A diurnal and circadian pattern of staining in terminals of specific PDF-expressing neurons in D. melanogaster was found that was consistent with a hypothesis of daily release of PDF [75]. Whether peptidergic compounds cause release of PDHs/PDFs is not known, but the involvement of aminergic compounds has been implicated [24]. A quantitative ELISA with an antiserum against the R. microptera PDF has been developed. It was used to determine that the optic ganglion of this grasshopper is the richest source of PDF, but titres of PDHs/PDFs in the haemolymph of any species have not been quantified [31]. It is well-
89
known that the two Met residues at positions 4 and 15 in certain PDHs are prone to oxidation and that the Met-oxidized PDH loses a great deal of its activity, but no systematic study has been published on the inactivation or the half-life of any member of this peptide family [31]. A G proteincoupled receptor is suggested for PDFs, but such a receptor has not yet been identified. There are, however, a few facts known about the interaction of the PDH ligand with its putative receptor from structureactivity studies [71]: a chain length of 13 amino acids (loss of the first 5 amino acids from the N-terminus) is the minimum structure to elicit pigment dispersion, albeit very weakly active. Progressively extending the molecule towards the N-terminus restores more and more activity. Replacing the Arg residue at position 13 with an arginine diphenylglyoxal derivative results in an agonist that is 14-fold more active than the parent molecule; it is suggested that this may be due to increased resistance to/protection against proteolytic cleavage, but this is not proven experimentally. Only a few studies have been conducted on the signalling of PDH in crustaceans. It is suggested that pigment dispersion is achieved by the ligand binding to a Gs protein-coupled receptor, resulting in the activation of AC and the increase of the intracellular concentration of cyclic AMP which in turn activates a cyclic AMP-dependent protein kinase (PKA) [63]. This PKA has been shown to phosphorylate a protein; such a signalling mechanism is also known to elicit pigment dispersion in vertebrate cells [63]. The main functions of PDHs in crustaceans are [see 31]: 1. inducing pigment-dispersion in all kinds of chromatophores, but often with a different potency for the various types of chromatophores, and, if more than one form of PDH is present in one particular species, they may have different potencies on the same type of chromatophores. 2. eliciting light adaptive movement of screening pigment in distal eye pigment cells, perhaps also in the reflecting pigments of some species. A vast body of information, including molecular and genetic studies, has accumulated on the role of PDFs mainly in D. melanogaster, the house fly, Musca domestica and the Madeira cockroach, Leucophaea maderae. All these activities have to do with rhythmic behaviours and biological clocks and it is suggested that PDFs act directly on brain or optic lobe neurons as both input and output factors for the circadian clock
90
controlling daily locomotion [31, 72, 74, 76]. However, there are also reports that PDF immunoreactive neurosecretory cells are found in locust abdominal ganglia which have neurohaemal release sites and, thus, the synthesized material could act as a neurohormone in the haemolymph [73]. 3.2. The cHH peptide family For many years, scientists were aware that peptide factors from the eyestalks of decapod crustaceans regulated glucose metabolism, moulting and female reproduction. It was only much later when these peptides were isolated from the XO-SG complex and structurally elucidated, that it became apparent that the peptides controlling these diverse processes were structurally homologous to each other and, hence, warranted inclusion as members of one peptide family. 3.2.1. Crustacean hyperglycaemic hormone The first member of the cHH peptide family to be functionally and structurally characterized from crustaceans was the crustacean hyperglycaemic hormone (cHH) of the shore crab, Carcinus maenas [77]; cHH got its name from the first function ascribed to this neuropeptide, viz. when injected into the haemolymph of a crustacean, there is a consequential and significant rise in the concentration of glucose in the haemolymph that peaks around 2 hours after injection. This is a relatively straight-forward biological assay to perform; cHH does, however, also have other functions which will be outlined below. The crab cHH has 72 amino acid residues, is blocked at the N-terminus by a pGlu residue and by an amide at the C-terminus, and contains six cysteines that form three intramolecular disulflde bridges [77]. The amino acid sequence of other peptides with cHH activity was later published from a number of other decapod crustacean species (see Table 3). In the American lobster, (Infraorder: Astacidae), 4 cHHs were isolated which arise from 2 different genes and subsequent post-translational modification. This entails isomerization of the Phe residue to yield peptides with L- or D-Phe3 residues. The D-Phe3-cHH displays a higher potency and an extended hyperglycaemic effect [78]; this isomerization of
91
cHH also occurs in other other astacideans, such as crayfish. In crabs (Infraorder: Brachyura), two cHHs are always isolated from the sinus glands in different concentrations; they are the products of one gene and subsequent post-translational modification, viz. the formation of a pGlu at the N-terminus; the cHH which is present in minor quantities, however, has the non-cyclized glutamine (Gin1) residue yet both cHH isoforms have the same potency in a hyperglycaemic bioassay [79]. The dynamics of the N-terminally blocked cHH peptide versus the non-blocked cHH of C. maenas was investigated, both peptides were cleared from the circulation at equivalent rates and no distinct differences in degradation and activity could be found between the two [80]. Thus, it seems as if the cHHs are not less stable or more vulnerable to exopeptidase degradation by having a free N-terminus. Such an unblocked cHH is, indeed, the norm in some other decapod crustaceans (see Table 3), e.g. prawns (Infraorder: Penaeidae) and spiny lobster (Infraorder: Palinura) where the multiple forms of cHHs are all unblocked at the N-terminus although the Cterminus is amidated; these cHHs are products of different genes. Table 3 shows that there are distinct similarities in peptide structure between species of the same infraorder. This may possibly explain the observed results of early heterologous hyperglycaemic bioassays in which cHH elicited a hyperglycaemic response in a phylogenetic group-specific manner [81]. Functional activity of cHH (and perhaps also its observed group-specificity) may be conferred by the C-terminal sequence of the cHH peptide; indirect evidence for this with respect to biological function came from conspecific bioassays with two C-terminally truncated cHHs of Jasus lalandii: such truncated cHH peptides did not elicit hyperglycaemia [82]. Support for this hypothesis was recently provided with conspecific bioassays in which recombinant cHH from the Kuruma prawn Penaeus japonicus was used: the non-amidated cHH peptide showed low hyperglycaemic activity compared to that induced by the amidated recombinant cHH [83]. The secondary structure of the amidated recombinant cHH (as determined from circular dichroism spectra) also differed to that of the non-amidated cHH, indicating that the C-terminal amide may be significant in the folding of the molecule [83]. The elevation of glucose in the haemolymph after injection of cHH into crustaceans results from the hormone acting on its target tissues to mobilize glycogen stores. Significant binding of radiolabelled C. maenas cHH to crude membrane preparations from hepatopancreas (midgut gland), heart muscle and epidermis have been observed [84]. Receptors
92
for cHH and their binding affinity for homologous and heterologous cHHs were also studied using classical binding assays with plasma membranes purified from the hepatopancreas of C. maenas and the crayfish Orconectes limosus; species specificity was demonstrated in this study, suggesting that the previously observed group specificity of cHH with regards to biological activity, reflected co-evolution of both the hormone and its receptor [85]. One should caution, however, that only one species from the two infraorders were tested in the heterologous displacement. Eyestalk ablation (i.e. removal of cHH) results in hypoglycaemia (low concentration of glucose in the haemolymph). This hypoglycaemia is accompanied by an increase in glycogen content in different tissues (e.g. hepatopancreas, muscle, epidermis), an inactivation of phosphorylase and an activation of glycogen synthetase [86]. Injection of purified cHH into O. limosus caused an increase in cyclic nucleotide levels and inactivated glycogen synthetase in hepatopancreas [87] and in muscle [88]. Although both cyclic nucleotides are also involved in the mode of action of cHH, cyclic GMP and not cyclic AMP appears to be the relevant second messenger of cHH in O. limosus [86]. It has been reported that synthesis of cHH is not restricted to the XO in the eyestalks of crustaceans, but is also shown by immunocytochemistry to be localized to the suboesophageal ganglion and thoracic second roots in H. americanus [89], in the POs of C. maenas [90] and is transiently expressed in gut paraneurons of C. maenas where it is involved with water uptake to facilitate ecdysis [91]. Interestingly, the PO-cHH of C. maenas has a free C-terminus, its first 40 amino acid residues are identical to the SG-cHH and, not surprisingly, it displays no functional activity in the cHH bioassay [90].
Species
P.japonicus P.japonicus P.japonicus P.japonicus
PenjacHH-II
Penja cHH-ffl
Penja cHH-V
Penja cHH-VI
Penmo cHH-IV
Penmo cHH-III
Penmo cHH-tf
Penmo cHH-I
3
Penaeusjaponicus
Penja cHH-I
3
Bythograea thermidron
BytthcHH*
P. monodon
P. monodon
P. monodon
Penaeus monodon
Cancer paguna
Canpa cHH-tf
3
Carcinus maenas
Procambarus bouvieri, Procambarus clarkii, Orconectes limasus
CarmacHH-II2
OrclicHH
1
Probo cHH1
Crustaceans Crustacean hyperglycaemic hormone Homam cHH-A1 Homarus americams (=HomamMrH) 1 Homam cHH-B H. americanus
Peptide name, code name
ANFDPSCAGVYNRELLGRLSRLCDDCYNVFREPKVATECRNNCFYNPVFVQCLEYLI PADLHEEYQAHVQTV-NHj SLFDPACTGIYDRQLLGKLGRLCDDCYNVFREPKVATGCRSNCYYNLIFLDCLEYLI PSHLQEEHMEALQTV-NH2
ANFDPSCAGVYNRELLGRLSRLCDDCYNVFREPKVATECRSNCFYNPVFVQCLEYLI
pEVFDOACKGVYDRNLFKKLDRVCEDCYNLYRKPFVATTCREHCYSNWVFRQCLDDLL LSDVIDEYVSNVQMV-NH2 pEVFDQACKGVYDRNLFKKLNRVCEDCYNLYRKPFVIVTCRENCYSNRVFRQCLDDLL MIDVIDEYVSNVQMV-NH2 pEVFDQACKGIYDRRIFKKLDRVCEDCYNLYRKPYVATTCRQNCYANSVFRQCLDDLL LIDWDEYISGVQTV-NH2 pEVFDQACKGI YDR&I FKKLDRVCEDCYNLYRKPY¥aTTCRQNCYJ«JSVFRQCLDDLL LIDVLDEYISGVQTV-NH2 pEIYDTSCKGVYDR&LFNDLEHVCDDCYNLYRTSYVASACRSNCYSNLVFRQCMDDLL MMDEFDQYARKVQMV-NH2 pEIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNWFRQCMEELL MMDEFDKYARAVQMV-NH2 pEIYDRSCKGLYDRRLFSDLDHVCDDCYNLYRNSRVANACRENCYSNLVFRQCMEDLL LMDQFDKYARAVQTV-NH2 SLFDPSCTGVFDRQLLRRLGRVCDDCFNVFREPNVATECRSNCYNNPVFRQCMAYW PAHLHNEHREAVQMV-NHs SLFDPSCTGVFDRQLLRKLGRVCDDCFNVFREPNVAMECRSNCYNNPVFRQCMEYLL PAHLHDEYRLAVQMV-NH2 SLFDPACTGIYDRQLLRKLGRLCDDCYNVFREPKVATGCRSNCYHNLIFLDCLEYLI PSHLQEEHMAAMQTV-NHz LVFDPSCAGVYDRVLLGKLNRLCDDCYDVFREPDVATECRSNCFYNLAFVQGLEYLM PPSLHEEYQANVQMV-NH2 LVFDPSCAGVYDRVLLGKLNRLCDDCYNVFREPNVATECRSNCFYNLAFVQGLEYLL PPSLHEEYQANVQM¥-NH2 SLFDPSCTGVFDRQLLRRLSRVCDDCFNVFREPNVATECRSNCYNNEVFRQCMEYLL
Sequence
Table 3, Primary structures of members belonging to the crustacean hyperglycaemic hormone family.
[243]
[243]
[243]
[243]
[242]
[242]
[242]
[242]
[242]
[241]
[79]
[77]
[240]
[238,239]
[237]
[102,237]
Reference
93
Penaeus schmidtii
Pensc cHH
J. lalandii
Jasla cHH-II
Moult-inhibiting hormone
Penva MIH*
Penmo MIH-II*
Penmo MIH-I*
PenjaMIH
Canma MIH*
Chafe MIH*
Calsa MIH*
Canpa MIH
Carma MIH
Probo MIH
Procl MIH
Penaeus vannamei
P. monodon
Penaeus monodon
Penaeus japonicus
Cancer magister
Charybdisferiatus
Callinectes sapidus
Cancer pagurus
Carcinus maenas
Procambarus bouvieri,
Procambarus. clarkii
Macrobrachium rosenbergii Jasus lalandii
Macro cHH
Jasla cHH-I
Metapenaeus ensis
Meten cHH-B*
Meten cHH-A*
P. monodon
Penmo cHH-V3
RYVFEECPGVMGNRAVHGKVTRVCEDCYNVFRDTDVLAGCRKGCFSSEMFKLCLLAM ERVEEFPDFKRWIGILNA-NH2 pEVFDQACKGIYDRAIFKKLELVCDDCYNLYRKPKVATTCRENCYANSVFRQCLDDLL LINVVDEYISGVQIV-NH2 RVINDECPNLIGNRDLYKKVEWICEDCSNIFRKTGMASLCRRNCFFNEDFLWCVHAT ERSEELRDLEEWVGILGAGRD RVINDDCPNLIGNRDLYKKVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYAT ERTEEMSQLRQWVGILGAGRE RVINDDCPNLIGNRDLYKKVEWICDDCANIYRSTGMASLCRKDCFFNEDFLWCVRAT ERS S DLAQLKQWVTILGAGRI RVINDDCPNLMGNRDLYKKVEWICDDCANIYRITGMASLCRKDCFFNFDFLWCVRAT FRS FDMTQLKQWVRILGAGRI RVINDDCPNLIGNRDLYKRVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYAT ERTEEMSQLRQWVGILGAGRE SFIDNTCRGVMGNRDYNKKVVRVCEDCTNIFRLPGLDGMCRNRCFYNEWFLICLKAN REDEIEKFRVWISILNAGQ SLTDGTCRGRMGNREIYKKVDRVCEDCANIFRLPGLEGLCRDRCFYNEWFLLCLKAA NREDEIENFRVWVSILNA SLTEGTCRGRMGNREIYKKVDRVCEDCANIFRLPGLEGLCRDRCFYNEWFLLCLKAA NREDEIENFRVWISILNA DTFDHSCKGIYDRELFRKLDRVCEDCYNVFREPKVATECKSNCFVNKRFNVCVADLR HDVSRFLKMANSALS
ANFDPSCAGVYDRELLGGLSRLCDDCYNVFREPKVATECRSNCFYNSVFVQCLEYLI PADLHEEYQAHVQTV-NH2 ANFDPSCTGVYDRELLGRLSRLCDDCYNVFREPKVATECRSNCFYNPVFVQCLEYLI PADLHEEYQAHVGTV-NH2 SLFDPSCSGVFDRELLGRLNRVCDDCYNVFRDPKVAMECKSNCFLNPAFIQCLEYLL PEDLHEEYQSHVQVV-NH2 SLFDPSCTGVKDRELLGRLNRVCDDCYNVFREPKVATECRSHCFLNPAFIQCLEYII FEVLHEEYQANVQLV-NH2 AILDQSCKGIFDRELFKKLDRVCDDCYNLYRKPYVAIDCRRGCYQNLVFRQCIQDLQ LMDDLDEYANAVQTV-NH2 AVFDQSCKGVYDRSLFSKLDRVCDDCYNLYRKHYVATGCRRNCYGNLVFRQCLDDLM LVDVVDEYVASVQMV-NH2 AVFDQSCKGVYDRSLFKKLDVVCDDCYNLYRKPYVATGCRENCYSNLVFRQCLDDLM LVDVVDEYVSTVQMV-NH2
[252]
[251]
[251]
[104]
[250]
[249]
[248]
[100]
[99]
[103]
[101]
[82]
[12]
[247]
[246]
[245]
[244]
[243]
94
M. ensis Macrobrachium rosenbergii M. rosenbergii Jasus lalandii
Meten MIH-B*
Macro MIH-A*
Jasla MIH4
Procambarus bouvieri
Nephrops norvegicus
Libinia emarginata
Libem MOIH*
Drosophila melanogaster
SFFDIQCKGVYDKSIFARLDRICEDCYNLFREPQLHSLCRSDCFKSPYFKGCLQALL LIDEEEKFNQMVEIL-NH2 SFFTLECKGVFDAAIFARLDRICDDCFNLFREPQLYTLCRAECFTTPYFKGCMESLY LYDEKEQIDQMIDFV-NH2 SNFFDLECKGIFNKTMFFRLDRICEDCYQLFRETSIHRLCKQECFGSPFFNACIEAL QLHEEMDKYNEWRDTL-NH2
RRINNDCQNFIGNRAMYEKVDWICKDCANIFRKDGLLNNCRSNCFYNTEFLWCIDAT ENTRNKEQLEQWAAILGAGWN RRINNDCQNFIGNRAMYEKVDWICKDCANIFRQDGLLNNCRSNCFYNTEFLWCIDAT ENTRNKEQLEQWAAILGAGWN QIFDPSCKGLYDRGLFSDLEHVCKDCYNLYRNPQVTSACRVNCYSNRVFRQCMEDLL LMEDFDKYARAIQTV-NH2
ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRNNDVGVMCKKDCFHTMWFLWCV YATERHGEIDQFRKWVSILR ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRINDVGVKCKKDCFHNMDFLWCV YATERHGEIDQFRKWISILRAGRK pEVFDQACKGIYDRAIFKKLELV****YN******VATTCRENCYAN
SYIENTCRGVMGNRDIYKKVVRVCEDCTNIFRLPGLDGMCRDRCFNNEWFLVCLKAA NRDDELDKFKVWISILNPGL FSIDYTCTGAMGNRDIYNKVSRVCDDCANIYRLPGLDGMCRNRCFNNFWFMICLRAA KREDEIDKFRVWISILNPGGAW RYLDDECPGVMGNRDLYEKVVRVCDDCSNIFRMNDMGTRCRKDCFYNVDFLWCVYAT ERHGDVDQLNRWMSILRAGRK RFLDDECRGVMGNRDLYEYVVRICDDCENLFRKSNVGSRCKKNCFYNEDFMWCVRAT ERTDELEHLNRAMSIIRVGRK RFTFD-CPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCL QATMREHETPRFDIWRSILKA-NH2
[131]
[130]
[126, 128]
[122]
[256]
[256]
[255]
[116]
[254]
[105]
[107]
[107]
[106]
[253]
Cys residues are indictaed in BOLD text. 'Present as L-Phe3 and D-Phe3 isoforms. 2Sequence of cHH-I differs only by Gin1 residue. 3Sequenced by mass spectrometry. 4 A gap has been introduced to align the Cys residues. 5Partially sequenced; unclear residues are shown as*. "Sequence deduced either in part or completely from the nucleotide sequence.
Drome ITP*
Bommo ITP*
Schgr ITP*
Ion-transporting peptide Schistocerca gregaria, Locusta migratoria Bombyx mori
Cancer pagurus
CanpaMOIH-2
Insects
Cancer pagurus
Canpa MOIH-1
Mandibular organ-inhibiting hormone
Probo VIH
5
Nepno VIH*
Vitellogenesis-inhibiting hormone Homarus americanus Homam VIH1
Macro MIH-B*
Metepenaeus ensis
Meten MIH-A*
95
96
3.2.2. Moult-inhibiting hormone The second group of peptides that belong to the cHH peptide family are the moult-inhibiting hormones (MIH), so-called because it increases the interval between subsequent moults by exerting an inhibitory action on the Y-organ (YO), a cephalothoracic gland in which the moultpromoting steroid hormones (ecdysteroids) are synthesized [8]. The accepted, simplified paradigm for this is as follows: like all arthropods, crustaceans have a chitinous exoskeleton that must be shed and replaced by a larger exoskeleton in order for the animal to increase in size but, unlike insects, the moult cycle of crustaceans is under negative control and, only when the repressive hold of MIH on the YO is lifted, can the YO produce sufficient titre of ecdysteroids that leads to ecdysis. Confirmation of this paradigm has been provided in numerous cases where eyestalk ablations (thus, removal of the source of MIH) accelerated the moult cycle and enhanced the synthesis of ecdysteroids by the YOs [92] and by quantitative, moult-specific changes in MIH gene expression, as determined by Northern blots with eyestalk neural ganglia from the blue crab Callinectes sapidus at different stages of the moult cycle [93]. The latter result is in contrast to a similar study carried out with MIH mRNA from P. japonicus where no moult stage-specific fluctuation in MIH expression was observed [94]. Both studies have revealed, however, that MIH is expressed throughout the moult cycle. To date, however, the titre of MIH has not been determined in the haemolymph of any crustacean during all stages of the moult cycle, despite having MIHspecific antisera available; a chief reason for this is that the hormone is released in a pulsatile manner in minute quantities and has a short half-life of 5-10 min [95]. On the other hand, the titre of circulating ecdysteroids has been established in several decapod crustaceans (see for example [96]). On the basis of ecdysteroid measurements, a reliable in vitro bioassay was developed by Soumoff and O'Connor [97] to measure the reduction of ecdysteroid synthesis in YOs after exposure to crude extracts of SGs (or MIH). This assay was successfully used to characterize the first MIH to be sequenced [98]; the primary structure of this neuropeptide (see Table 3), isolated from SGs of C. maenas [99] was, surprisingly, similar to the neuropeptides involved in glucose metabolism, i.e. the cHHs. The structure of MIH isolated from another brachyuran crab, viz. the edible crab Cancer pagurus [100] showed that the sequences are very
97
highly related (around 80 % structural identity): both consist of 78 residues with a free N- and C-terminus and 6 cysteine residues in conserved positions that form 3 intrachain disulfide bridges (see Table 3). On the other hand, there is only around 30 % sequence identity between the brachyuran MIH and cHH peptides. In astacurans, a structurally distinctive MIH has, thusfar, only been reported in the crayfish Procambarus clarkii [101]; a peptide with MIH activity in H. americanus [102] and the Mexican crayfish Procambarus bouvieri [103] has also been reported on but, structurally, these neuropeptides are more characteristic of cHHs than MIHs (see Table 3). Peptides with MIH activity and which are structurally similar to the brachyuran MIHs have also been isolated and functionally characterized from sinus glands of P. japonicus [104] and J. lalandii [105]. Many more putative MIH sequences have been deduced from cDNA sequences of a variety of decapod crustaceans, notably brachyuran crabs and prawns (see Table 3). Although these deduced sequences show homology to MIH peptides, there is, in most cases, no additional evidence that they are indeed functional MIHs. One exception is the study in which the expression of a putative MIH of C. sapidus was investigated by Northern blots during the moult cycle [93]. In another study that tried to functionally characterize 2 putative MIH clones from the prawn Metapenaeus ensis, recombinant MIHs were produced in Escherichia coli and shown to increase the interval between subsequent moults when injected into M. ensis specimens [106]. There are other reports of possibly more than one MIH isoform being present in crustaceans: 2 MIH-like sequences have been deduced from cDNAs of the giant freshwater prawn Macrobrachium rosenbergii [107] and from the giant tiger prawn Penaeus monodon (see Table 3); it should be noted, however, that these putative MIHs have not yet been functionally characterized. There are several reports that cHH can inhibit the ecdysteroid synthesis by YO in vitro but that MIH cannot act as a cHH by increasing glucose concentration (see for example [108]). It was probably this multifunctionality of cHH that led to it being mistaken for MIH in certain instances. Why should cHH duplicate the role of another hormone during the moult cycle? The definitive answer to this question is still not known but specific receptors for cHH (along with MIH-specific receptors) have been demonstrated, by classical membrane binding studies, to be present on the YOs of intermoult brachyuran crabs [84]. Unlike cHH, radiolabelled MIH binds only to membrane preparations of YOs and not
98
to epidermal, heart muscle and hepatopancreas preparations [84]. Not surprisingly, MIH peptides from 2 other crab species could bind effectively to the C. maenas MIH receptor, which suggests that there is a high degree of conservation in the binding domains of the crab MIHs [84]. What is surprising, though, is that neither the density nor the affinity of the MIH and cHH receptors on the YO change significantly during the moult cycle of the crab [95], yet MIH and cHH have very little inhibitory effect in vitro on YOs at certain stages of the moult cycle (see for example [96]). This seems to suggest that the degree of YO inhibition may be independent of hormone titre and receptor expression, and further, that the control of signal transduction must lie downstream of the MIH receptor, thus the accepted paradigm of moult control is certainly oversimplified and possibly inaccurate [95]. The binding of MIH to its receptor on the YO cell membrane activates a signalling pathway that results in the inhibition of: (a) the cellular uptake of the ecdysteroid precursor (cholesterol), (b) protein synthesis and (c) the expression of steroidogenic enzymes [109]. Crustaceans are unique with respect to the negative control of ecdysteroid synthesis: after receptor-ligand interaction, there is an increase in cyclic nucleotide levels and this shuts down the YO cell's activity [110]. Although both cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) are present in YOs of O. limosus, only the activity of PKG is decreased by the removal of eyestalks, indicating that cyclic GMP is the second messenger of MIH [109]. Steroidogenesis is clearly enhanced by intracellular Ca2+ which arises from an influx from the extracellular space (via channels) and from the release of intracellular stores via IP3 [109]. Numerous investigations have shown that MIH functions independently of Ca2+ concentration, although its action can be eliminated by a high concentration of Ca2+ [110]. Few studies have looked at the expression of MIH apart from the classical XO-SG distribution, and the results are not all in agreement. Northern blots, PCRs and immunocytochemistry with antisera raised to recombinant MIH-B of M. ensis showed that MIH-B is expressed in the eyestalk (XO) and ventral nerve cord [106], whereas PCRs and Northern blots showed expression of a putative MIH only in the eyestalks of M. rosenbergii [107]. In C. pagurus, MIH-like transcripts were detected by Northern blotting only in XOs, whereas the more-sensitive nested PCR approach revealed that MIH could be amplified from optic nerve and the
99
ventral nerve cord [111]. Whether this non-XO production of MIH has any physiological role is still not clear. 3.2.3. Vitellogenesis-inhibiting hormone A third member of the cHH peptide family is also synthesized by neurons of the crustacean XO and has been named the vitellogenesisinhibiting hormone (VIH) or the gonad-inhibiting hormone (GIH) because of its negative influence on reproduction; this inhibitory peptide does not act in a species- or group-specific manner (for review see [112]. The identification of VIHs has been slow owing to a lack of reliable and fast bioassays [92]: in vivo assays in which ovarian growth or oocyte diameter is measured after injection of VIH, is only feasible with crustaceans that have short vitellogenic cycles, hence, in vitro assays which measure the endocytotic uptake of gold-labelled vitellogenin, or measures the endogenous synthesis of ovarian proteins by means of the incorporation of radiolabelled amino acids have been favoured. Heterologous bioassays with shrimps and prawns are most often used because these crustaceans have short and numerous reproductive cycles per year, the approximate vitellogenic stage of the ovary can be visually assessed through the opaque exoskeleton of the living animal and the reproductive/vitellogenic cycle is well documented (see for example [113]). Although the heterologous peptides have a significant effect at physiological doses, a homologous bioassay would provide unequivocal data/evidence as to whether the peptide is a true VIH in its own system. All VIHs sequenced to date from decapod crustaceans have been functionally characterized in heterologous assay systems: the first was isolated from H. americanus and tested in an in vitro assay with shrimps [114]. The VIH peptide consists of 78 amino acid residues, a free Nterminus but an amidated C-terminus and 6 Cys residues with 3 disulfide bridges (Table 3). It is around 53 % identical to MIH molecules and 25 % identical to cHHs [92]. The only other functionally characterized decapod VIH is a partially sequenced peptide that was isolated from P. bouvieri; this peptide inhibited the growth of prawn oocytes in vitro [115] but this activity was attained with an unphysiologically high peptide dosage and further, its structure resembles a cHH rather than the lobster VIH (Table 3). Recently, a cDNA sequence from the eyestalks of the Norway lobster Nephrops norvegicus was published [116]; the cDNA encodes a peptide
100
that is 96 % identical to the preproVIH of//, americanus [117]. The N. norvegicus VIH must, however, still be characterized to confirm its biological significance. To date, a characteristic VIH peptide has not been found in any brachyuran crab, spiny lobster, shrimp or prawn [see 113]. VIH activity, as gleaned from heterologous in vitro assays, is not associated with a unique peptide from the XO-SG complex of the spiny lobster J. lalandii [113] and the prawn P. japonicus [118], but was elicited by previously characterized cHHs, whereas MIH had only a neglible effect in both studies. In contrast, the lobster VIH displayed no hyperglycaemic activity and the lobster cHH did not have a VIH effect [119], although the lobster cHH clearly has MIH activity [102]. There is, unfortunately no report that a brachyuran MIH has been tested in a VIH assay. Interestingly, immunocytochemistry and in situ hybridization studies show that VIH is localized not only in female crustaceans but also in males and larvae [92]; the biological significance of this is still unknown but this distribution of VIH is a strong argument for changing the name of the peptide since its function may not be limited to the inhibition of vitellogenesis in female crustaceans.
3.2.4. Mandibular organ-inhibiting hormone The most recent member of the large cHH peptide family in crustaceans was functionally characterized as a mandibular organinhibiting hormone (MOIH) in brachyuran crabs [120]. The MO, which is located at the base of the tendon associated with the adductor muscle of the mandible, synthesizes methyl farnesoate (MF), a precursor of the juvenile hormone (JH) that occurs in insects, viz. JH III [121]. Earlier, observations were made that the removal of eyestalks leads to hypertrophy of the MO and more recently, it was shown that the circulating MF titres are increased after eyestalk ablation in three different crustacean species [121]. Thus, it was not surprising that the MOIH peptide was isolated from the SG of crustaceans. The biological assay used in identifying a MOIH peptide is based on the quantitative inhibition of MF synthesis by MOs in vitro, following incubations with the peptide. In this way, two MOIHs were identified in C. pagurus, isolated from SGs by HPLC and N-terminally sequenced [120]. These MOIHs are 78 residues long, have free N- and C-termini, three intrachain disulfide bridges and differ by only one amino acid substitution (Table 3).
101 101
Although there is 59 % sequence identity between C. paguras MIH and MOIH, the MIH is not active as an MOIH and the MOIH displays only limited activity in the MIH bioassay; furthermore, the cHH of this crab did not affect the MO in an in vitro MOIH assay [120], hi contrast, the 2 cHHs of C. maenas were equipotent at inhibiting the MOs in vitro (inhibition of around 83 %), whereas the MIH effected only 12.5 % inhibition [108]. hi another conspecific in vitro bioassay, the MIH and the 2 cHH peptides of O. limosus effected 80-89 % inhibition of MF synthesis [108], Peptides with MOIH activity have also been identified in the spider crab Libinia emarginata (Table 3) with an in vitro bioassay using dissociated MO cells; these peptides, however, also demonstrated cHH activity and are structurally more similar to the cHH than to the MOIHs of C. pagurus [122]. Criticism has been levelled against this study on the basis of a relatively high ED50 value for the inhibition of MF synthesis in vitro [121]. An in vivo bioassay was also established to measure MF titres in haemolymph samples of H. americanus and C. pagurus following the injection of putative MOIHs. Intriguingly, such bioassays identified active peptide fractions that are distinct from the previously identified and sequenced MOIHs of C. pagurus [121]. The peptide sequences of these unique MOIHs have not been published yet but the results indicate that there may be two groups of MOIHs acting on crustaceans: those having a direct effect on the MO (e.g. the MOIHs identified in in vitro assays), and those acting indirectly on the MO to elevate MF titres in the haemolymph. Should both groups of peptides be considered as physiologically relevant regulators of the MO when only one group demonstrates an effect in vivol Like JH III in insects, MF seems to play a role in enhancing crustacean reproductive maturity, as well as maintaining the juvenile morphology [123]. The synthesis of JH III in insects is also regulated by neuropeptides, one group of regulators (the allatostatins) is, in fact, similar in structure to neuropeptides that have been isolated from crustaceans (see Section 3.3) but there is no structural resemblance between the allatostatins and the MOIHs and there is no report on the role of allatostatins in regulating the crustacean MOs. Investigations into the mode of action of MOIH (or SG extracts) on the MO have been carried out in a few crustaceans only and not much is known. Cyclic nucleotides are involved in signal transduction - cyclic GMP is apparently important in H. americanus, whereas cyclic AMP is essential in C, pagurus [see 121].
102 102
3.2.5. Ion-transporting peptide Peptides that are structurally related to the cHH family of peptides are not restricted to crustaceans, although for a long time this was thought to be the case. In several insect species a peptide with structural features characteristic of the cHH has been isolated or deduced from cDNA sequences. This peptide is named "ion-transporting peptide" (ITP) because it stimulates the reabsorption of ions and water from primary urine in the hindgut of locusts [124], thus, it plays a role in osmoregulation. ITP was first isolated from CCs of S. gregaria and partially sequenced (amino acids 1-33); in a conspecific in vitro bioassay 1 -5 nM ITP stimulated reabsorption of Cl", Na+, K+ and fluid in isolated ilea [124, 125]. The complete peptide sequence of ITP from S. gregaria was later deduced from cDNA from locust brains; the 72 amino acid peptide (Table 3) has a free N-terminus, amidated C-terminus and the characteristic 6 Cys residues with about 40 % overall identity to cHHs from decapod crustaceans [126]. The in vitro bioassay used to identify ITPs, measured the active transport of Cl" from the lumen side of ileum preparations (mounted as a flat sheet), this transport is reflected by the positive change in short circuit current (Isc) across the ileum membrane [127]; compounds to be tested in this assay were always applied to the haemocoel side of the ileum preparation. In similar bioassays, crude extracts of CCs from a variety of orthopteran insects (migratory locust, crickets, cockroaches, stick insect) stimulated Cl" transport across the ileum of S. gregaria, whereas CC extracts from a lepidopteran and a dipteran showed no ITP activity in this heterologous assay, regardless of CC dosage applied [128]. This is most likely due to group-specific structural features of the peptide or its receptor, since peptides that are structurally homologous to the locust ITP are present in Lepidoptera and Diptera and further, cHH-family peptides from crustaceans have also not shown ITP activity in the locust ileal bioassay [129]. Putative ITP sequences (Table 3) have been deduced from cDNA of L. migratoria [128] and B. mori [130], as well as postulated from the genome of D. melanogaster [131]. In addition to these putative ITP sequences, an ITPlike peptide was identified, cloned and sequenced from a locust ileal mRNA library [126]. The ITP-like cDNA sequence was identical to that of the brain ITP cDNA except for an insert of 121 bp that preceded the
103 103
ITP C-terminus, and the predicted absence of a terminal amidation; that means, only the first 40 amino acid residues of ITP and the ITP-like peptide are identical [126]. This partial identity is reminiscent of the situation with C. maenas SG-cHH and PO-cHH and suggests also that the peptides arise from alternative splicing of a single gene (see Section 3.2.1). Like the crab PO-cHH [90], the non-amidated ITP-like peptide of the desert locust does not display the classic function in the original bioassay: instead of stimulating the ion/fluid transport in the ileal bioassay, recombinant ITP-like peptide inhibited the stimulation brought on by synthetic ITP [126]. Structure-function studies were conducted with recombinant ITPs that were suitably modified by site-directed mutagenesis. These studies are discussed in a recent review [129]. Briefly, the C-terminus is important for biological activity and Cterminally truncated ITPs, or the absence of the amide abolishes stimulation of ileal Isc; the 3 disulfide bridges are also important for activity. Interestingly, single deletions indicated that He 1 is important for receptor binding: when this residue is deleted in ITP-like peptides, the antagonistic effect to ITP is completely abolished. Hence, the observed inhibitory effect of ITP-like peptide on ileum transport appears to simply be a consequence of competitive receptor binding, with ITP-like peptide being able to bind to the ITP receptor but not being able to activate the receptor due to its changed C-terminus [129]. Factors from the eyestalks of H. americanus, specifically cHH, have been implicated in playing a role in osmoregulation too [132]. The involvement of cHH in osmoregulation was confirmed in another decapod crustacean, viz in the crayfish Astacus leptodactylus, where the injection of cHH into eyestalk-ablated specimens caused a significant increase in the haemolymph osmolality and Na+ concentration, whereas the concentration of Cl" remained unchanged [133]. 3.2.6. Sub-grouping of the cHH peptide family Members of this extensive and functionally diverse family of peptides that occur in arthropods show strong structural homology to each other, yet they can be sub-grouped on the basis of primary structure, as well as on preprohormone structure. Computer analyses, based on structural motifs of the mature peptides and their preprohormones, were carried out [134]. Thirty-two sequences were included in the analyses which showed that
104
the cHH peptide family subdivides into two groups, Group I contains the cHHs (including the MOIH from the spider crab, the MIH of P. bouvieri and the insect ITPs), while Group II contains the lobster VIH, MIHs and the remaining MOIHs. The preprohormone structure of Group I peptides is characterized by a signal peptide, a so-called cHH precursor-related peptide (CPRP), followed by a dibasic cleavage site and the sequence for the mature cHH (or ITP) sequence with an amidation signal at the Cterminus (see for example [135]). Preprohormones of Group II peptides are arranged into a signal peptide and the sequence for the mature VIH, MIH or MOIH peptide [134]. Many questions are still unanswered about the cHH peptide family: (a) there is clearly structural homology amongst the CPRPs [see 136] but what is the role of the CPRPs, if any? (b) how does the cHH effect its multi-functionality, while the VIH, MIH and MOIH seem to be more limited in functional activity, and is there any significance in this? (c) why are certain peptide functions not group- or species-specific and others are, and what does this imply about receptorligand interactions?
3.3. The allatostatin superfamily Two groups of hormones regulate development and reproduction in insects, namely the ecdysteroids and the JHs. In crustaceans too, ecdysteroids are involved in the hormonal control of growth, and a chemical compound, which is similar to the JHs of insects, is present and thought to play a role in crustacean reproduction and development. The insect JH is a species-specific acyclic sesquiterpenoid epoxide, which is synthesized in a pair of retrocerebral epithelial organs called the corpora allata (CA; see Fig. 1). In decapod crustaceans, MF is the unepoxidated form of the insect JH III and it is synthesized and secreted from the MOs (see Section 3.2.4). In insects and in crustaceans, the synthesis of both ecdysteroids and JH/MF are subject to control by neuropeptides. Although ecdysteroids are considered to be "growth promoting hormones" in both insects and crustaceans, these steroid hormones are differently regulated in the two taxa. In insects, the synthesis and release of ecdysteroids from the prothoracic gland is positively regulated by the prothoracicotropic hormone (PTTH) which is synthesized in neurosecretory cells of the brain and released from the CA [37]. PTTH was first completely identified in B.
105
mori as a 109 amino acid long polypeptide with an N-glycosylation site and 7 Cys residues which form disulfide bridges [10]. hi crustaceans, on the other hand, the synthesis of ecdysteroids in the YO is inhibited by MIH (see Section 3.2.2). There is no structural resemblance at all between PTTH and MIH. JH in insects maintains larval and nymphal characteristics during development and suppresses metamorphosis into the adult form; similarly, MF in crustaceans retards metamorphosis and larval development [123]. Two types of neuropeptides control the production of JH in the CAs of insects in vitro: (a) allatotropins, which stimulate the biosynthesis of JH and have only been found in insects to date, and (b) allatostatins (ASTs) which, inter alia, inhibit the biosynthesis of JH but which also have numerous other effects (mainly myoinhibitory) [137]. Neuropeptides that are structurally similar to the ASTs are also present in crustaceans and, therefore, we will review here what is known from the ASTs of insects and crustaceans. hi insects, one distinguishes between ASTs of three major structural forms [53, 137]: the "cockroach" ASTs or A-type ASTs which are characterized by the C-terminal pentapeptide sequence Y/FXFGLamide, the "cricket" ASTs or B-type ASTs which are generally characterized by Trp residues at position 2 and 9, and the "moth" ASTs or C-type ASTs which are only found in a few species to date (various moths and in D. melanogaster) and are 15 amino acid residues long with a pGlu residue at the N-terminus, a free C-terminus and two Cys residues forming an intramolecular disulfide bridge. Thusfar, only peptides of the "cockroach" AST-type have been found in crustaceans [138-140], and, hence, we will limit our inspection only to this group of ASTs which is common to insects and crustaceans. To date, more than 60 members of the A-type ASTs are structurally known from insects and about 20 species have been analyzed. Although only three species of decapod crustaceans were investigated, more than 60 isoforms are known in these crustaceans (see Table 4); interestingly, none is identical to the ASTs identified so far in insects [139]. Thusfar, in each insect and crustacean species investigated to date, multiple isoforms of the A-type Asts have been isolated or demonstrated to be present (see Table 4). Whereas all members of the A-type Ast family contain the relatively conserved pentapeptide core C-terminal of Y/FXFGLamide (for Leu there can be conservative changes such as He or Val but also Met in the blowfly, Calliphora vomitoria), the N-terminal part of the peptide can
106
be of variable length and may be blocked at the N-terminus. Some ASTs are only pentapeptides, while the longest ASTs have 30 residues (Table 4). Table 4. Primary structures of members of the allatostatiD family of peptides. Peptide name, code name Crustaceans Carma-AST 1 Carma-AST 2 Carma-AST 3 Carma-AST 4 Carma-AST 5 Carma-AST 6 Carma-AST 7 Carma-AST 8 Carma-AST 9 Carma-AST 10 Carma-AST 11 Carma-AST 12 Carma-AST 13 Carma-AST 14 Carma-AST 15 Carma-AST 16 Carma-AST 17 Carma-AST 18 Carma-AST 19 Carma-AST 20 Penmo-AST 1 Penmo-AST 2 Penmo-AST 3 Penmo-AST 4 Penmo-AST 5 Penmo-AST 7 Penmo-AST 8 Penmo-AST 9 Penmo-AST 10 Penmo-AST 11 Penmo-AST 12 Penmo-AST 14 Penmo-AST 15 Penmo-AST 16 Penmo-AST 17 Penmo-AST 18 Penmo-AST 19 Penmo-AST 20 Penmo-AST 21 Penmo-AST 22 Penmo-AST 23 Penmo-AST 24 Penmo-AST 25 Penmo-AST 26 Penmo-AST 27 Penmo-AST 28
Species
Sequence
Carcinus maenas, Penaeus monodon YAFGL-NH2 EAYAFGL-NH 2 C. maenas EPYAFGL-NHa C. maenas DPYAFGL-NH2 C. maenas NPYAFGL-NH 2 C. maenas SPYAFGL-HH 2 C. maenas ASPYAFGL-NH 2 C. maenas C. maenas, Orconectes limosus, P. monodon AGPYAFGL-NH2 GGPYAFGL-NH 2 C. maenas APQPYAFGL-NH 2 C. maenas ATGQYAFGL-NH 2 C. maenas PDMYAFGL-NH2 C. maenas EYDDMYTEKRPKVYAFGL-NH2 C. maenas YSFGL-NH2 C. maenas, P, monodon AGPYSFGL-NH 2 C. maenas GGPYSYGL-NHa C. maenas SGQYSFGL-NH 2 C. maenas SDMYSFGL-NH 2 C. maenas APTDMYSFGL-NH2 C. maenas C. maenas
P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon P. monodon
GYEDEDEDRPFYALGLGKRPRTYSFGL-NH2 MJEDEDAASLF&FGL-NH 2 PDAEESHKRDRLYAFGL-NH 2 DRLYAFGL-NHa TGGPYAFGL-NH 2 SAGPYAFGL-NH 2 SGHYAFGL-NH 2 ANQYAFGL-NH2 AGQYAFGL-NH2 TPSYAFGL-NH 2 PQRDYAFGL-NH 2 SDYAFGL-NH 2 ANQYTFGL-NH 2 ASQYTFGL-NH 2 SQYTFGL-NH 2 YTFGL-NH 2 SGHYNFGL-NHj GHYNFGL-NH 2 AGPYEFGL-NH 2 GGPYEFGL-NH2 AAPYEFGL-NH 2 GPYEFGL-NH2 SPYEFGL-NH 2 NPYEFGL-NH 2 NEVPDPETERNSYDFGL-NH 2 EVPDPETERNSYDFGL-NH 2 PETERNSYDFGL-NH 2
Reference
[138,139] [138] [138] [138] [138] [138] [138] [138-140] [138] [138] [138] [138] [138] [138, 139] [138] [138] [138] [138] [138] [138] [139] [139] [139] [139] [B9] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139]
107 Penmo-AST 29 Penmo-AST 30 Penmo-AST 31 Penmo-AST 32 Penmo-AST 33 Penmo-AST 34 Penmo-AST 36 Penmo-AST 37 Penmo-AST 38 Penmo-AST 39 Penmo-AST 40
P, P. P. P. P. P. P. P. P. P. P.
monodon monodon monodon monodon monodon monodon monodon monodon monodon monodon monodon
Orcli-AST 1 Orcli-AST 2
Orconectes limosus O. limosus
NSYDFGL-NH 2 YDFGL-NHa AGHYSFGL-NH 2 DRTYSFGL-NH 2 PSAYSFGL-NH2 pENMYSFGL-NH 2 DARGALDLDQSPAYASDLGKRIGSAYSFGL-NH 2 TARGALDLDQSPAYASDLGKRIGSAYSFGL-NH 2 SVAYGFGL-NH 2 TVAYGFGL-NH 2 (X)GIYGFGL-NH 2
[139] [139] [139] [139] [139] [139] [139] [139] [139] [139] [139]
SAGPYAFGL-NH 2 PRVYGFGL-NH 2
[140] [140]
SRPFGFGL-NH2
[142]
AGMYSFGL-NH2 GEGRMYSFGL-NH 2 PNYERMAGSRFNFGL-NH2 GPDHRFAFGL-NH 2 SLHYGFGI-NH 2 PYSFGL-NH 2 VPMYDFGI-NH 2
[142] [142] [142] [142] [142] [142] [142]
GRQYSFGL-NH a ADGRTYAFGL-NHz IPMYDFGL-NH2 TSSLYSFGL-NHs
[257] [257] [257] [257]
TTRPQPFNFGL-NH 2
[258]
Insects Grybi-AST-A6
Gryllus bimaculatus
Grybi-AST-A7 Grybi-AST-A9 Giybi-AST-AlO Gtybi-AST-All Grybi-AST-A12 Giybi-AST-A13 Grybi-AST-A14
G. G. G. G. G, G. G,
Carmo-AST-Al Carmo-AST-A2 Carmo-AST-A4 Carmo-AST-A5
Carausius morosus C. morosus C, morosus C. morosus
Drome-AST 4
D. melanogaster
bimaculatus bimaculatus bimaculatus bimaculatus bimaculatus bimaculatus bimaculatus
A complete list of sequences up to 1999 is available for insect members of this family [10,149],
The AST precursor, known only from insects to date, is characterized by a long leader peptide which consists of a signal peptide and another peptide of no known function, then follow clusters of ASTs interspaced by "acidic spacers", i.e. peptides with a high number of Asp and Glu residues and a resultant low isoelectric point [141], and a short tail peptide of unknown function [142]. When one considers the array of insect ASTs from an evolutionary view, it appears that the evolutionary more ancient insects, such as cockroaches and crickets, have a larger number of AST isoforms encoded on the precursor than more recent insect taxa, and it is speculated that this may be linked to functional diversity of the peptides: the ASTs of "older" insects have true allatostatic action in addition to other functions (see below), and those of the evolutionary "younger" insects have lost the allatostatic function [142]. For some insect species it is known that virtually all the encoded AST peptides of the preprohormone are, indeed, expressed. This was done exemplarily for the American cockroach by the employment of sensitive methods of mass spectrometry [143] showing additionally that a
108 108
particular internal dibasic cleavage site that is present in a peptide called Peram-AST-2 in the preproallatostatin is used to produce a small octapeptide. Such an octapeptide is quite ubiquitous and occurs not only in cockroaches, but locusts, moths and blowflies suggesting a possibly similar mechanism of prohormone processing. Is there a biological need or relevance for so many structurally similar peptides, such as the allatostatins, in one organism? In C. vomitoria different AST peptides have myoinhibitory actions on different segments of the gut, thus, different ASTs may have different targets within the same animal, and there is evidence of the insect ASTs having different potencies of action on one target [144]. Although direct radioligand-binding assays were successfully used to partially characterize an insect AST receptor [145], all the AST receptor sequences known to date are derived from molecular biological approaches. A G protein-coupled receptor was cloned from D. melanogaster and expressed in frog oocytes for a functional test; the results demonstrated that an A-type AST is the ligand for this receptor [16]. Structurally, the transmembrane regions of this AST receptor are related to the mammalian somatostatin/galanin/opioid receptors; in fact, most closely related to the galanin receptors although the peptides, galanin and AST, have little structural commonality. After the first AST receptor was cloned, similar receptors were cloned from P. americana, C. morosus, B. mori and a second one from D. melanogaster [137]. In a functional electrophysiological test, the characteristic C-terminal pentapeptide core sequence of A-type ASTs is able to mediate activation of the D. melanogaster [146] and P. americana receptor (G. Gade, H. Marco, R. Weaver and D. Richter, unpublished results). The latter study also showed that all 14 A-type ASTs from P. americana are potent in the electrophysiological functional assay, thus, it appears that there is no need for each peptide binding to a specific receptor protein. This contrasts with a report on a putative AST receptor from the cockroach Diploptera punctata, where a competitive binding assay showed that some of the ASTs have a higher affinity to the receptor [145]; the receptor has not yet been sequenced. Although present in a large number of insect species, the A-type ASTs apparently display an allatostatic effect (inhibiting the biosynthesis of JH in vitro) only in cockroaches and crickets. In other insect species, the conspecific AST peptides do not have an inhibitory effect on the CA in vitro [137, 147]. Moreover, immunocytochemical methods have identified numerous cells in a wide variety of insects that
109
react positively to A-AST antisera. Thus, not only cells in the brain but in most parts of the CNS, as well as in nerves innervating visceral muscles, midgut cells and haemocytes are immunopositive, and this distribution suggests, of course, different and multiple functions, inter alia also functions of A-ASTs as neurotransmitters and/or neuromodulators [137, 148]. To date, the following effects have been demonstrated by conspecific A-type ASTs in insects [137,149]: 1. Myoinhibitory actions of ASTs were shown in a number of insect species, for example, both spontaneous and proctolin-induced contractions of the Mndgut of D. punctata were inhibited; as were the peristaltic contractions of the ileum of C. vomitoria, the spontaneous contraction of the muscles of the oviduct of S. gregaria, the contraction of muscles from oesophagus and crop of the codling moth, Cydia pomonella, and the contraction of heart muscle in the German cockroach, Blattella germanica. 2. In B. germanica an endogenous AST inhibited the production of vitellogenin in vitro. 3. In D. punctata an endogenous AST also had a stimulating action: using ligatured midgets in vitro it was found that this AST stimulated the activity of the carbohydrases, amylase and invertase. Despite the similarity of the crustacean MF, in structure and function, to the insect JH, the synthesis of MF in the MO is probably not regulated by the numerous allatostatin peptides identified in crustaceans. Instead, the synthesis of MF is inhibited by MOIH (see Section 3.2.4). It should be noted, however, that the crustacean ASTs have not yet been fully tested for a role in the regulation of MO activity, so they could perhaps act on the MO together with the MOIHs. The crustacean AST peptides show a widespread distribution in immunocytochemical investigations with immunoreactive neurons localized in the brain, ventral nerve cord and commissural organs [139], the stomatogastric nervous system, entire CNS and the POs [140] and with immunoreactive fibres projecting into the walking legs, eyestalks (medulla extema, medulla interna and medulla terminalis) and into neurohaemal release sites, such as the suboesophageal ganglia, POs and the thoracic ganglia [140]. Such a distribution has been interpreted as clues that the crustacean ASTs play a role in neurotransmission or neuromodulation. In conspecific bioassays, the application of ASTs to hindgut preparations of crayfish in vitro resulted in a decrease in amplitude and
110
frequency of myogenic contractions; electrophysiological measurements also revealed a decrease in the cycling frequency of the pyloric motor rhythms which is generated by the stomatogastric ganglion. Since no AST immunoreactivity was found associated with the hindgut or its innervation, it is possible that the effect of ASTs on hindgut is not neuromodulatory [140]. A number of different crayfish species have now been shown to contain motor neurons in their stomatogastric nervous system that display AST-like immunoreactivity [150]. 3.4. Muscle activity regulated by various neuropeptide families An overwhelming number of fully characterized neuropeptides, especially in insects, have myotropic activity: one way or the other these peptides regulate the contractile activity of visceral and/or skeletal muscles. One possible reason for this abundance of sequenced neuropeptides that regulate muscle activity may be the successful usage of a very simple bioassay technique [see 151]. 3.4.1. Proctolin The first insect neuropeptide to be isolated and structurally identified from the American cockroach was the pentapeptide proctolin (RYLPT) [152]. It stimulates and modulates the contractions in visceral and skeletal muscles and has a role as co-transmitter but not as a neurohormone. The identical structure has been found in crustaceans (see Table 5): first in the PO of Homarus vulgaris [153] and later confirmed from the PO of C. maenas [154] here it has inotropic effects on the heart, i.e. it enhances the frequency of the spontaneous contractions. In the crab, C. borealis, proctolin has modulatory functions on neural circuits of the stomatogastric system [155]. More information, especially on structureactivity studies, breakdown and localization of proctolin-immunoreactive neurons can be gleaned from [10, 73, 156]. Based on binding studies, a receptor for proctolin was proposed in the locust oviduct [157]. Recently, with the help of the D. melanogaster genomic data base [158] and molecular cloning, a G protein-coupled receptor was identified, which, when expressed in HEK (human embryonic kidney) cells responded very sensitively (EC50: 3 x 10"10 M) to proctolin in a functional bioassay
111 Ill
measuring Ca2+ mobilization [159]. Another group found similar results and reported an EC50 value of 6 x 10"10 M [160]. It was also shown that the recombinant receptor binds proctolin in a competitive radioreceptor assay with high affinity (IC50 of about 4 x 10'9M) [159]. In addition, with the use of an antibody directed against the receptor, the latter authors demonstrated receptor-positive tissues in D. melanogaster which correlate well with those tissues/organs known to respond to proctolin. Interestingly, the genome of A. gambiae apparently does not contain an orthologue gene to the D. melanogaster proctolin receptor [159-161]. Previous studies in A. gambiae also reported an inability to detect proctolin-like immunoreactivity, proctolin-like biological activity, or proctolin responsiveness [159]. 3.4.2. Crustacean cardioactive peptide Another neuropeptide that has the same structure in crustaceans and insects is the cyclic nonapeptide, crustacean cardioactive peptide (CCAP; PFCNAFTGCamide) (Table 5), which was first isolated and sequenced from POs of C. maenas [162] and shown to have a stimulatory influence on the frequency of the heart beat in the shore crab and of the hindgut of O. limosus [163]. Later, the same peptide was isolated and sequenced from a number of insects such as migratory locust, tobacco hornmoth, southern armyworm (Spodoptera eridania), the mealworm Tenebrio molitor [10, 73] and the stick insect C. morosus [164]. Molecular biological and bioinformatic studies have identified or predicted genes which encode the precursor for CCAP in some insects, such as M. sexta [165], D. melanogaster [158, 166] and A. gambiae [70], but not in crustaceans. The CCAP precursor encodes a signal sequence of 20 amino acids followed by a dibasic cleavage site and a single copy of CCAP with a Gly residue (to form post-translationally the amidated peptide) and another two basic amino acid residues for proteolytic cleavage; no other putative peptides are encoded [165]. Localization of CCAP in crustaceans and various insect species has been studied in detail with the aid of immunocytochemistry [73, 167]. The distribution, for example, in neurons of brain, midgut and ventral nerve cord of insects suggests multiple and diverse functions, some of those are surely neuromodulatory (immunopositive interneurons which have processes into brain neuropil) but others are neurohormonal roles
112
(immunopositive processes to corpora cardiaca). It is not known what regulates the release of CCAP in crustaceans and insects, but in insects CCAP itself is a releasing factor for the well-known metabolic AKHs (see Section 3.1.1). Concentrations of CCAP were measured by RIA in haemolymph (10 pmol per liter) and POs (40 pmol per PO) of the shore crab [167]. CCAP is also released in massive amounts into the haemolymph of C. maenas and O. limosus just prior to ecdysis [168]. No detailed studies on inactivation of CCAP in any organism has been conducted. A G protein-coupled receptor for CCAP from D. melanogaster was cloned and functionally expressed in frog oocytes [59]. Although in this study the receptor also recognized the AKH of D. melanogaster to some extent, this can possibly be explained by some technical problems with the measurement as outlined elsewhere (169]. In the latter study CCAP appeared to be a very good candidate ligand for the cloned receptor with an EC50 of 5.4 x 10"10 M when expressed in CHO cells. The mode of action of CCAP is not known, but there are a large number of functions that are attributed to this peptide in crustaceans and insects: in crustaceans, it has an inotropic and a chronotropic (i.e. enhances the amplitude of spontaneous contractions) effect on the heart in the shore crab, a myotropic action on the hindgut of O. limosus, but no cardioactive effect in the American lobster and the Dungeness crab [167]. It has a neuromodulatory role or acts as neurotransmitter on the pattern of motor neurons that activate swimmeret beating in the crayfish P. leniusculus [170], Yet another effect of CCAP is the reduction of the amplitude of the electroretinogram in O. limosus [171]. In M potiuna high concentrations of CCAP were able to disperse the erythrophores; but this may rather be a pharmacological effect [63]. In insects, CCAP stimulates the contractions of heart, hindgut and oviduct in various species, but it is also one of the releasing factors for AKHs in the migratory locust [73, 172] and it is involved in inducing the motor programme that is so important for larval ecdysis and adult eclosion [73, 173]. In fact, a recent publication reports that genetic ablation of the CCAP neurons in D. melanogaster (although not lethal during the larval stages but lethal during pupal ecdysis) disrupts "the timing and organization of ecdysis behaviour [174].
113 Table 5. Primary structures of members of various peptide families with myoactivity. Peptide name, code-name
Species
Sequence
Reference
1. Proctolin Crustaceans Peram-proctolin
Homarus vulgaris and other crustacean species
RYLPT
[153]
Insects Peram-proctolin
Periplaneta americana and other insect species ;s
RYLPT
[152, 156]
PFCNAFTGC-NH 2
[162]
Carausius morosus
PFCNAFTGC-NH 2
[164]
Penaeus vannamei
ASFSPWG-NH 2
[177]
2. Crustacean cardioactive peptide Crustaceans Carma-CCAP Carcinus maenas Insects Carma-CCAP 3. (myo)kinins Crustaceans Penva-K-I Penva-K-II
P. vannamei
DFSAWA-NH2
[177]
Penva-K-IH Penva-K-IV Penva-K-V Penva-K-VI
P. P. P. P.
vannamei vannamei vannamei vannamei
PAFSPWG-NH 2 VAFSPWG-NH 2 pEAFSPWA-NH 2 AFSPWA-NH 2
[259] [259] [259] [259]
P. P. P. P. P. P.
americana americana americana americana americana americana
RPSFNSWG-NH 2 DASFSSWG-NH 2 DPSFNSWG-NH 2 GAQFSSWG-NH2 SPAFNSWG-NH 2 AFSSWG-NH 2
[260] [260] [260] [260] [260] [260]
P. americana
DPAFSSWG-NH 2
[260]
P. americana
GADFYSWG-NH2
[260]
NTVVLGKKQRFHSWG-NH2
[176, 261] [176, 180]
Insects Peram-K-I Peram-K-II Peram-K-III Peram-K-IV Peram-K-V Peram-K-VI (= Locmi-KI) Peram-K-VII (= Leuma-K-VII) Peram-K-VHI (= Leuma-K-VIII) Musdo-K Drome-K.
Musca domestica, Stomoxys calcitrans, Haematobia irritans D. melanogaster, Neobelliera bullata, Anopheles gambiae
NSVVLGKKQRFHSWG-NH2
4. FMRF-NH2-related peptides a) FMRF-NH? Crustaceans Do not have FMRF-NH2s Insects Neobu-FMRF
N. bullata
pEPSQDFMRF-NH2
[189]
SDRNFLRF-NH2 TNRNFLRF-NH2 GYNRSFLRF-NH2
[194] [194] [262]
b) FLRF-NHVmvosuppressins Crustaceans Homam-FLRF-I Homam-FLRF-II Calsa-FLRF
Homarus americanus H. americanus Callinectes sapidus
114 Procl-FLRF-I Procl-FLRF-II (=Macro-FLRF-I) Macro-FLRF-II Macro-FLRF-III Macro-FLRF-IV Macro-FLRF-V Macro-FLRF-VI Macro-FLRF-VII Macro-FLRF-VIII Insects Leuma-MS
Procambarus clarkii P. clarkii, Macrobrachium rosenbergii M. rosenbergii M. rosenbergii M. rosenbergii M. rosenbergii M. rosenbergii M. rosenbergii M. rosenbergii Leucophaea maderae, P. americana, Diploptera put
NRNFLRF-NH2 DRNFLRF-NH2 ADKNFLRF-NH2 NYDKNFLRF-NH2 APALRLRF-NH2 DRTPALRLRF-NH2 DGGRNFLRF-NH2 GYGDRNFLRF-NH2 VSHNNFLRF-NH2
[263] [263, 264] [264] [264] [264] [264] [195] [195] [195]
pEDVDHVFLRF-NH2
[190, 265, 266]
pEFDEY(SO3H)GHMRF-NH2
[197, 198] [197,
c) sulfakinins Crustaceans Penmo-SK-I Penmo-SK-II Penmo-SK-III Insects Peram-SK-I Blocked Peram-SK-I Glu-methylated Peram-SK-I Leuma-SK-II
P. monodon, P. vannamei Penaeus monodon, P. vannamei P. monodon
AGGSGGVGGEY*DDY(SO3H)GHLIRF-NH2
198] VGGEYDDY(SO3H)GHLRF-NH2
L. maderae, P. americana P. americana P. americana L. maderae, P. americana
EQFDDY (SO3H) GHMRF-NH2
[197] [267]
pEQFDDY(SO3H)GHMRF-NH2
[199]
(CH3) EQFDDY (SO3H) GHMRF-NH2 pESDDY (SO3H) GHMRF-NH2
[199] [268,
199] unblocked Leuma-SK-II
P. americana
QSDDY(SO3H)GHMRF-NH2
[199]
RARPRF-NH2 YSQVSRPRF-NH2 YAIAGRPRF-NH2 YSLRARPRF-NH2
[208] [208] [208] [208]
SNSRPPRKNDVNTMADAYKFLQDLDTYYGDR ARVRF-NH2 £SFTDARPQDDPTSVAEAIRLLQELETKHAQHA RPRPF-NH 2
[213]
d) NPFs Crustaceans Onlv short NPFs Penmo-PYF-I Penmo-PYF-II Penmo-PYF-III Penmo-PYF-IV
Penaeus monodon P. monodon P. monodon P. monodon
Insects Long NPFs Drome-NPF
D. melanogasler
Aedae-NPF
Aedes aegypti
Short NPFs Drome-NPF-I Drome-NPF-II Drome-NPF-III Drome-NPF-IV
D. D. D. D.
e) pvrokinins Crustaceans Penva-PK-I Penva-PK-II
P. vannamei P. vannamei
melanogasler melanogaster melanogaster melanogaster
[214]
AQRSPSLRLRF-NH2 WFGDVNQKPIRSPSLRLRPF-NH2 PQRLRW-NH2 PMRLRW-NH2
[166] [166] [166] [166]
DFAFSPRL-NH2 ADFAFNPRL-NHz
[219] [219]
115 Insects Peram-PK-I Peram-PK-II Peram-PK-III Peram-PK-IV Peram-PK-V Peram-PK-VI Perfu-PK-IV Carmo-PK Drome-PK-I Drome-PK-II
P. americana P. americana P. americana P. americana P. americana P. americana Periplaneta fuliginosa Carausius morosus D. melanogaster D. melanogaster
HTAGFIPRL-NH2 SPPFAPRL-NH 2 LVPFRPRL-NH 2 DHLPHDVYSPRL-NH 2 GGGGSGETSGMWFGPRL-NH2 SESEVPGMWFGPRL-NH 2 DHLSHDVYSPRL-NH 2 DEGGTQYTPRL-NH 2 TGPSASSGLWFGPRL-NH2 SVPFKPRL-NH 2
[269] [269] [270] [270] [270] [271] [271] [164] [272] [220, 221]
APSGFLGMR-NH 2 SGFLGMR-NH 2
[226] [226, 177]
APSGFLGVR-NH 2 APEESPKRAPSGFLGVR-NH 2 NGERAPGSKKAPSGFLGTR-NH 2 APSGFMGMR-NH2 APAMGFQGVR-NH2 APAAGFFGMR-NH2 VPASGFFGMR-NH 2 GPSMGFHGMR-NH2 APSMGFQGMR-NH2
[273] [273] [273] [273] [273] [274] [274] [274] [274]
f) tachvkinins Crustaceans Canbo-TRP-Ia CanboTRP-Ib
Cancer borealis C. borealis, P. vannamei
Insects Leuma-TRP-I Leuma- TRP-II Leuma- TRP-III Leuma- TRP-IV Leuma- TRP-V Leuma- TRP-VI Leuma- TRP-VII Leuma- TRP-VIII Leuma- TRP-IX
L. L. L. L. L. L. L. L L.
maderae maderae maderae maderae maderae maderae maderae maderae maderae
A complete list of sequences for insect peptides up to 1997 is available [10, 37]. Insect tachykinin-related peptides are given in [73]. *also sulfated in P. vannamei [198],
3.4.3. The (Myo)kinin family The (myo) kinin family of peptides is especially a very interesting one, because its members have two very different functional activities in insects. The first members of this family were originally isolated on the basis of stimulation of the cockroach hindgut in vitro from extracts of whole heads of L. maderae [151]. This species contains 8 isoforms of kinins. Shortly after their first isolation, it was reported that these kinins also stimulate diuretic activity of isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti [175]. This function of kinins has now been shown regularly in conspecific diuretic assays with a number of other insect species. To date, the structure of kinins (see Table 5) have been fully elucidated in insects from the American cockroach (8 isoforms), the house cricket, Acheta domesticus (5 isoforms), the migratory locust (1 isoform), the mosquitoes, Culex salinarius and A.
116 116
aegypti (each 3 forms), housefly (M domestica), stablefly (Stomoxys calcitrans), hornfly (Haematobia irritans), flesh fly (Neobelliera bullata), D. melanogaster (each one form) and corn ear moth, Heliothis zea (3 forms) [129, 176]. In crustaceans, 6 kinin isoforms (Table 5) are known from the prawn Penaeus vannamei [177, 178]. Structurally, the kinin family is characterized by the C-terminal pentapeptide sequence PheXaa -Xaa -Trp-Gly amide; in a few crustacean kinins the ultimate Cterminal amino acid is however Ala (see Table 5). A preprohormone for kinins is reported from A. aegypti [179]. The organization is: a putative signal peptide (18 amino acids) followed by a 210 amino acid long prokinin which encodes one copy each of the 3 kinins of A. aegypti. The kinin precursor is also deduced from the genome of D. melanogaster [180], but no information has been gathered for any crustacean species. Localization of kinin-containing neurons has been achieved in many insect species by immunocytochemistry. Immunoreactivity with kinin antibodies occur in neurosecretory cells, in interneurons and in wellknown release sites but apparently not in neurons that directly innervate the hindgut and/or Malpighian tubules [10, 73]. Thus, it is suggested that kinins act as true neurohormones. RIAs have been developed and titre determinations executed but overestimation of kinin titres in non-purified haemolymph is possible; especially because the antisera are not well characterized and it is known that inactive breakdown products of kinins are recognized as well [see 129]. Degradation of kinins is well studied in the corn ear moth H. zea [181]: an enzyme that is similar to the vertebrate metalloprotease neprilysin is bound to membranes of the Malpighian tubules of the moth and cleaves an endogenous kinin primarily between Pro5 and Trp6, hence, destroying the C-terminal pentapeptide that is so important for biological activity. Structure-function studies had previously shown, when testing kinin analogues for their diuretic effects on cricket tubules in vitro, that this C-terminal pentapeptide is the minimum sequence required to elicit the same activity and potency as the parent molecule [10, 129]. Further studies revealed that the active conformation of the core sequence is a type VI P-turn where the aromatic side chains of the invariant Phe and Trp residues of the pentapeptide are very likely interacting with the receptor. The latter is G protein-coupled, has been cloned from D. melanogaster and expressed in heterologous cell lines and shown to interact well with the endogenous kinin of D. melanogaster [182]. The receptor is localized, as shown by
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immunocytochemistry, in the secondary stellate cells of D. melanogaster, where these cells are the target for the kinin. Studies on its mode of action have demonstrated that signalling is dependent on activation of PLC, IP3 production and increase of intracellular Ca +, resulting in an activated Cl~ shunt which may be located transcellular through apical and basal Cl" channels [129, 183]. Apart from their role as true neurohormones in insects, the insect kinins may also fulfil neuromodulatory roles on specific neuronal circuits, as deduced from immunocytochemical studies with various insect species [see 73]. In crustaceans, the kinins were isolated first on the basis of a heterologous bioassay, namely to stimulate the contraction of the hindgut of the Madeira cockroach but the crustacean kinins were also active in another insect assay, i.e. they stimulated fluid secretion in the Malpighian tubules of A. domesticus [177]. Later, the 6 kinins from the prawn (Table 5) were tested in another heterologous bioassay, but at least it was on crustacean tissues: all prawn kinins were able to stimulate the basal tonus of the isolated oviduct of A. leptodactylus and also enhanced the frequency and amplitude of the spontaneous contractions of the isolated hindgut of this crayfish [184]. Conspecific assays on the hindgut of the prawn were only successful when the endogenous peptide Penva-K-1 was used at a concentration of 10"6 M [178]. 3.4.4. The FMRFamide-related peptide superfamily There is a very diverse superfamily of neuropeptides present in most invertebrates that are called FMRFamide-related peptides (FaRPs); the name is based on the first member of this group isolated from ganglia of a clam (mollusc) and identified as Phe-Met-Arg-Phe amide (FMRFamide); this peptide had a strong cardioexcitatory action [185]. A number of important families are grouped into this superfamily: the FMRFamides and extended FMRFamides, myosuppressins (FLRFamides), sulfakinins (HMRFamides) and, neuropeptides F and Y (NPFs and NPYs). We will not attempt to review all these peptides in detail but will make only a few comments and dwell a bit deeper into the sulfakinins, NPFs and NPYs as far as resemblance between insect and crustacean peptides is concerned.
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3.4.4.1. The FMRFamides FMRFamides have been found in a variety of insects and detailed accounts of their distribution, structure, preprohormone organization and function have been given in previous reviews [10, 73, 186, 187]; Table 5 shows an example of a FMRFamide. Recently, a G protein-coupled receptor was cloned from D. melanogaster which, when stably expressed in CHO cells, reacted with high affinity (EC50: 9 x 10~10 M) to one of the 8 endogenous FMRFamides and with lower affinity to some others, suggesting that more FMRFamide receptors must exist in D. melanogaster [188]. The same receptor was also cloned by another research group [189] and their experiments did not show this differential affinity of the receptor for the endogenous peptides. Thus, there is a disagreement in the results of these two research groups with respect to the rank order potencies of the different ligands. 3.4.4.2. The FLRFamides A close structural relative of the FMRFamides are the FLRFamides. Its first candidate in insects was characterized as a myosuppressin from L. maderae where it inhibits the spontaneous contraction of the hindgut [151]. Thereafter, a number of FLRFamides were isolated from various insects (see Table 5 for post-1997 sequences) [73, 187]. The cockroach D. punctata contains the same myosuppressin as L. maderae in its brain. The cDNA encoding the precursor for that peptide was isolated and shown to have a single copy of the myosuppressin at its C-terminus and, additionally, the necessary sites for cleavage and amidation to generate the mature peptide [190]. A single-copy gene for a myosuppressin is also found in the genome of D. melanogaster [191]. Recently, two G proteincoupled receptors were identified in D. melanogaster, by cloning their genes and expressing the cDNA in CHO cells, these receptors were shown to be activated by low concentrations of the endogenous fly's myosuppressin (EC50: 4 x 10"8 M) [192]. These results were confirmed and expanded on in a more substantive study that made use of a fluorescence translocation assay to identify ligands of G protein-coupled receptors (Johnson, E.C.; Bohn, L.M.; Barak, L.S.; Birse, R.T.; Nassel,
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D.R.; Caron, M.G.; Taghert, P.H.; personal communication). This assay is based on the rationale that most G protein-coupled receptors have a common mechanism to terminate the hormonal signal after receptor activation. In vertebrates, such receptors are desensitized by the action of G protein-coupled receptor kinases and |3arrestins. A previous report showed the desensitization of known and diverse mammalian G proteincoupled receptors with chimeras of parrestin2 tagged with green fluorescent protein (Parr2-GFP) [193]. In such an assay, unstimulated cells which expressed the receptor showed a diffuse distribution of GFP fluorescence in the cytoplasm and when these cells were exposed to the appropriate ligand (which binds the receptor), most of the fluorescence (i.e. Parr2-GFP) then translocated to the cell membrane. Using this Parr2GFP translocation assay, it was shown that 1 x 10"6 M Dromemyosuppressin triggered the translocation of Parr2-GFP to the cell membrane of HEK cells that expressed receptor cDNA of the D. melanogaster gene CG 13803, as well as in cells expressing the paralogous receptor gene CG 8985 (Johnson, E.C.; Bonn, L.M.; Barak, L.S.; Birse, R.T.; Nassel, D.R.; Caron, M.G.; Taghert, P.H.; personal communication). In accordance with its inhibitory physiological action, the activation of CG 13803 and CG 8985 receptors by Dromemyosuppressin, in the presence of forskolin, caused a decrease in the levels of cyclic AMP, hinting that the receptor couples via inhibitory G proteins (Johnson, E.C.; Bohn, L.M.; Barak, L.S.; Birse, R.T.; Nassel, D.R.; Caron, M.G.; Taghert, P.H.; personal communication). In crustaceans true FMRFamides are not known, but only FLRFamides (Table 5). They were first isolated from the PO of//, americanus [194] and later found in crabs, crayfish and a prawn [195]. Their functional role is most likely neurohormonal, i.e. probably cardioexcitatory and influencing contractions of visceral muscles, as well as neuromodulatory, i.e. modulating motor patterns in the stomatogastric ganglion [150]. 3.4.4.3. The sulfakinin family The first sulfakinins were again isolated from whole heads of L. maderae and its sequence elucidation suggested that this family is structurally related to the mammalian peptide family comprising gastrin and cholecystokinin [151]. Its biological activity was measured by stimulation of contractions of the cockroach hindgut. Later, members of this family
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were found in a number of insects, such as the American cockroach, the migratory locust, and various dipteran species, including D. melanogaster [37, 73] (see Table 5), In crustaceans, sulfakinins have been characterized only in the two prawn species P. vannamei [196] and P. monodon [197]. The characteristics of this family in insects and crustaceans are a Cterminal YGHMRFamide and a sulfated Tyr residue (Table 5). In the prawn P. vannamei, one of the two sulfakinins is even sulfated at both Tyr residues [198]. Non-sulfated forms apparently also occur in insects, hi the American cockroach, other post-translational modifications have taken place, namely a pGlu residue at the N-terminus or even Omethylation of the N-terminal glutamic acid [199] (see Table 5). The precursor of sulfakinins has been analyzed in D. melanogaster. it contains three putative peptides of which two are clearly sulfakinins [200]. A G protein-coupled receptor has been cloned from D. melanogaster; when it was functionally expressed in CHO cells, the endogenous Dromesulfakinin was the most active ligand [201]. Imrnunocytochemical studies revealed that sulfakinins are predominantly located in the CNS and are only found at neurosecretory release sites of a few insects; sulfakinins, apparently, act as central neuromodulators and do not have a neurohormonal role [73]. Structure-activity studies demonstrated that non-sulfated analogues were inactive and that the C-terminal hexapeptide is the smallest functioning fragment (possessing about 10 % of the myotropic activity of the parent molecule) but full potency and efficacy is achieved by the C-terminal octapeptide [see 10]. Whereas the C-terminal peptide is HMRFamide for the sulfakinins, it is WMDFamide for gastrin and cholecystokinin which are also both sulfated. Although the vertebrate peptides as such are not active in the cockroach hindgut assay, replacement of the Asp residue with an Arg residue transformed them into active analogues in this bioassay [202]. The functional tasks of insect sulfakinins, besides stimulation of the contractions of the hindgut, are stimulation of heart beat in cockroaches, induction of the secretion of aamylase in the midgut of a beetle and reduction of the amount of food eaten by locusts and cockroaches upon injection of sulfakinin [53, 73], Similar actions (induction of satiety, secretion and muscle contractions) are known from the mammalian counterpart, cholecystokinin [203]. The crustacean sulfakinins have only been proven to have an effect on the cockroach hindgut; they were not active at all on hindgut or oviduct of the crayfish [198].
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3.4.4.4. The NPY superfamily, including the insect NPF family The vertebrate NPY family contains such prominent members as pancreatic polypeptide (PP), characterized initially from chicken where PP is synthesized and released from the endocrine cells of the pancreatic islets [204], neuropeptide Y (NPY) found in porcine brain [205] and peptide YY (PYY) isolated from porcine intestine [206]. Such peptides are characterized by a chain length of 36 amino acids and Tyr-amide at the C-terminus. Whereas NPY is exclusively expressed in neurons of mammals and is known to control processes such as stimulation of food intake, vasoconstriction, sexual behaviour and circadian rhythm, PP and PP Y are synthesized in endocrine cells of the gut and are inhibitors of gut motility and of the secretion of exocrine products from the pancreas. In insects, members of the NPY superfamily have been identified, but here they are called members of the neuropeptide F (NPF) family because their C-terminus is characterized by a Phe-amide (Table 5). In fact, NPFs have been elucidated before in a number of other invertebrate taxa such as Cestoda (tapeworms) and Mollusca [see 73]. According to chain length, insects possess long NPF-like peptides and short ones (see Table 5 for some examples). This distinction appears to be justified not only because of the different chain length but also because in D. melanogaster, different genes have been identified to code for short and long NPF-like peptides; similarly, the two types seem to have different receptors [73]. Fully characterized short NPF-like peptide structures are known from the insects A. aegypti, Leptinotarsa decemlineata, P. americana [207], H. zea, S. gregaria and are predicted for D. melanogaster and A. gambiae [70,73,158]. The C-termini of these peptides are either PXLR/KL/TRFamide or RPRFamide. In the prawn, P. monodon, four short NPF-like peptides have been isolated from the eyestalks and the primary sequence determined (Table 5); the one hexapeptide and three nonapeptides share the four C-terminal amino acids RPRFamide [208]. For the so-called A. aegypti "head peptide" the encoding gene has been cloned and the preprohormone is characterized by coding for a signal peptide, three identical "head peptides" and a 38-mer at the carboxy terminus that does not resemble any known insect neuropeptide [209]. A receptor that can be activated by the D. melanogaster short NPFs was recently identified [210, 211]. Expression of the receptor in CHO cells [210] or X. laevis oocytes [211] results in some different data for the
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potencies of the endogenous D. melanogaster NPFs; whereas they were equipotent in CHO cells, the electrophysiological results with the oocytes point to a higher potency for Drome-NPF 1. The receptor is expressed in brain, gut, Malpighian tubules, fat body of D. melanogaster larvae, as well as in ovaries of adult females [210]. A variety of functional activities have been attributed to short NPFs of insects, ranging from activation of muscle activity and inhibition of host seeking behaviour (A aegypti "head peptide") to stimulation of ovarian growth and an increase of vitellogenin titres in the haemolymph, thus myotropic and gonadotropic effects are suggested [73,209]. It is not known, however, whether such gonadotropic effects are direct or indirect, i.e. via stimulation on the ecdysone or juvenile hormone systems. In D. melanogaster the endogenous NPF is reported to control foraging and social behaviour [212]. In brief, the level of NPF RNA was high in the CNS tissues of larvae attracted to food, gene expression is, however, turned off in those larvae that showed aversion to feeding. When the feeding response was tested in transgenic larvae, in which the NPF neurons were ablated, it resulted in a premature insensitivity to the feeding stimulus. Moreover, developmental downregulation of NPF also suppresses cooperative burrowing behaviours which are normally displayed by older larvae; thus, the NPF system is necessary for social interaction as well [212], Immunocytochemical studies on the distribution of these peptides have not been very helpful and are ambiguous, largely because no specific antisera have been developed (the antisera would most likely cross-react with other members of the FaRFamide superfamily). Long NPFs have only been isolated in insects but not in crustaceans (see Table 5). The insect forms are known from D. melanogaster [213] and A. aegypti [214] and an orthologue has been found in the genome of A. gambiae [70]. The prepropeptide is organized into signal peptide and the 36 amino acid long NPF peptide and this organization is common to all members of the NPF superfamily of this length [213]. The long NPFs are found in brain neurosecretory cells and midgut endocrine cells as shown by imrnunocytochemistry and Northern blots [214]. Some years ago, a putative receptor that was structurally similar to the vertebrate NPY receptors had been cloned from D. melanogaster, first called PR4 and later re-named NepYr [215, 216]; recent data has questioned its role as a true receptor for the long NPF of D. melanogaster [217]. hi the latter study a novel receptor was cloned, called DmNPFRl, which was stably expressed in CHO-K1 cells along with the NepYr. Only DmNPFRl-
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expressing cells showed high affinity binding of the endogenous NPF in a radioreceptor assay and inhibition of forskolin-stimulated activity of AC; NepYr-transfected cells were negative in both assays [217]. The receptor is localized in the CNS and midgut of larvae of D. melanogaster. Genome analysis of A. gambiae has identified an orthologue of the DmNPFRl gene [161]. As to the function of long NPFs in insects, there is no conclusive evidence but it is speculated, mainly on the basis of its distribution, that it plays a role in regulation of feeding behaviour and digestion, thus acting very likely in a neuromodulatory capacity and as true hormone [213]. 3.4.5. The pyrokinin family and structurally related peptides Peptides of this family have received their name from the first pyrokinin (PK; isolated from L. maderae in which it was demonstrated to have a stimulatory action on the hindgut), which is characterized by a pGlu residue at the N-terminus and the pentapeptide F/YXPRLamide at the Cterminus [10, 73]. In other insects, peptides with an identical C-terminus to that of the PKs have been found and have been named myotropins (MTs), pheromone biosynthesis-activating neuropeptides (PBANs), diapause hormones (DHs), puparium acceleration factor, and melanization and reddish colouration hormones (MRCHs), according to the biological effects these peptides have been attributed to [10, 73]. PKs sensu strictu have been identified in cockroaches, locusts, and D. melanogaster [10, 73]. In P. americana, for example, six PKs are present (Table 5): whereas the isoforms 1 to 4 and 6 are produced in the neurosecretory cells of the suboesophageal ganglion and tritocerebrum, isoform 5 (and much less so isoform 6) is the main candidate present in the abdominal neurosecretory cells of the perisympathetic organs [73, 218]. In crustaceans, two PKs have been identified from P vannamei (Table 5) [219]. In D. melanogaster there are different genes encoding putative peptides of this family. One is called capability and encodes, in addition to two cardioacceleratory peptides with the C-terminal FPRXamide (cap 2b-1 and cap 2b-2), a third peptide (cap 2b-3) with the C-terminal FXPRL characteristic of the PK family [191]. The second gene is called hugin and encodes two peptides, one of which belongs to the PK family (Drome-PK-2); the other peptide called hug y, is rather related to the family of ecdysis-triggering hormones (ETHs) which lack
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the characteristic Phe residue in the C-terminus of PKs [220, 221]. Genes and precursors of PBANs/DHs have also been cloned from various Lepidoptera [73]. A G protein-coupled receptor was cloned and expressed in frog oocytes and shown to be most sensitive to Drome-PK-2 (and hug y) and had moderate sensitivity to cap 2b-3 and ETH [59]. Not surprisingly, considering the variety of peptides belonging to the PK family, there is also an array of functions known: the "true" PKs/MTs are myostimulatory in action on foregut, hindgut, oviduct and hyperneural muscle of locusts and/or cockroaches [73, 218], whereas the two crustacean PKs are potent stimulators of the hindgut of A. leptodactylus but are not active on the oviduct of this crayfish [184]. The other members of this peptide family are well known for their ability to stimulate the bioysnthesis of sex pheromones in a number of Lepidoptera (PBANs), to regulate melanization in larvae of some moths (MRCHs), to induce diapause in silkworm eggs (DHs), and to accelerate the formation of a puparium in flies [10, 73]. Extensive structure-activity studies have been performed, especially on PBAN. This peptide is also best analyzed with respect to: (a) analogues that display antagonistic effects, (b) pseudopeptides which have better penetration of the insect cuticle, and/or (c) resistance to peptidases [222, 223]. Such studies have been conducted in the context of pest control. The interested reader is referred to a recent overview which discusses some ideas centred around the possibility of using insect neuropeptides as agents to control pest insect species [53]. 3.4.6. Tachykinin-related peptides The best-known member of the vertebrate neuropeptide tachykinin (TK) family is the undecapeptide, substance P (RPKPQQFFGLMamide), which has diverse actions as excitatory neurotransmitter and also as modulator of various functions, including sensory processing, control of movement, gastric mobility, vasodilation and salination [10, 224]. The migratory locust was the first insect in which neuropeptides were found which had limited structural homology to vertebrate TKs; they are characterized by the C-terminal pentapeptide FX'GX2Ramide (see Table 5) instead of the FXGLMamide of vertebrate TKs [225]. To date, such peptides have been isolated and up to 9 isoforms per species sequenced from the migratory locust, the Madeira cockroach (Table 5), the blowfly C. vomitoria, the mosquito C. salinarius and the stable fly S. calcitrans
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[224]. In D. melanogaster, 6 TK-related peptides have been identified by molecular biological methods, including a slightly modified form [73] and in A. gambiae a gene has been identified which is predicted to encode TK-related peptides [70]. hi crustaceans, 2 TK-related peptides were found in C. horealis [226], one of which is also present in P. vannamei (Table 5) [177]. The precursor gene is only known from D. melanogaster. It consists of 4 exons, but only exon 2 and 3 encode multiple isoforms of TK-related peptides, viz. 4 isoforms on exon 2 and a further 2 forms on exon 3. Additionally, other peptides are encoded on this precursor but they are not related to known insect neuropeptide families, and no function is known either [227]. The first receptor (belonging to the G protein-coupled type) in insects that have some sequence similarities (in the transmembrane region) with mammalian TK receptors were cloned as early as the beginning of the 1990s from D. melanogaster [228, 229]. Whereas the earlier study used the mammalian substance P to activate the receptor, the later study tested, heterologously, the TK-related peptide II from L. migratoria when the receptor cDNA was stably expressed in mouse NIH-3T3 cell lines. Recently, expressing the cDNA of the 2 putative receptors for TKs from D. melanogaster and parr2-GFP cDNA in HEK-293 cells, it was demonstrated that both these paralogous receptors translocate the parr2-GFP to the cell membrane upon exposure to the putative Drome-TK-I [Johnson, E.C.; Bohn, L.M.; Barak, L.S.; Birse, R.T.; Nassel, D.R.; Caron, M.G.; Taghert, P.H.; personal communication]. Later, a similar receptor was cloned from 5. calcitrans [230] and, when expressed in a stable D. melanogaster Schneider 2 cell line, a number of insect TK-related peptides, including the conspecific one from the stable fly, were found to be potent agonists [231]. Moreover, structure-activity studies using the functional system described above, suggested that (1) the conserved residues Phe and Arg at the C-terminus are essential for receptor interaction, (2) the C-terminal pentapeptide FTGMRamide is the active core region of these peptides which display a large percentage of the possible maximal activity and (3) in accordance with its C-terminal amino acid, Arg-amide in insect TK-related peptides and Met-amide in mammalian TK, the respective insect and mammalian receptors show increased activity with their "own" type of C-terminal peptide [231]. In insects, TK-related peptides are mainly localized in brain and gut tissue. Their major actions are to stimulate title contractility of muscles in the cockroach hindgut and the locust oviduct, but it has also been shown
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in some insects that TK-related peptides stimulate muscles of fore- and midgut, the heart and even of Malpighian tubules; they also have a myomodulatory action on the extensor tibiae muscle of the migratory locust, are apparently involved in the control of release of adipokinetic hormone from the neurosecretory cells in the CC of this insect, and have been attributed various neuromodulatory roles in photoreceptors of the crayfish P. leniusculus and on motor patterns of the stomatogastrie ganglion of the crab C. borealis [73]. CONCLUSIONS: The analysis of the structures, function and modes of action of major neuropeptide families of insects and crustaceans has revealed the following: 1. a few peptides, such as proctolin and CCAP belong to a "one member family", they are ubiquitously distributed in invertebrates and occur in both taxa under review here; their function has apparently not changed during the course of evolution. 2. some peptides belong to families that are, to date at least, only known with certainty to occur in arthropods. These are the AKH/RPCH (in short, AKH) family and the eHH/MH/VIH/MOIH/ITP (in short, cHH) family. In both families, the members found in the crustaceans primarily have different functions to those found in insects. The differences between the two peptide families are clear: (a) most variant members of the AKH family have been sequenced from insects, whereas most members of the cHH family occur in crustaceans. (b) In the AKH family, only one member is found in crustaceans and this member is also present in at least one insect species. However, whereas it functions as a chromatophorotropin in crustaceans, it controls metabolism in the insect. The receptors in both groups are rather specific but will still react to other peptide members of the family: the crustacean RPCH and the insect AKHs cross-react biologically in the other's system. (c) In the cHH family there are (as yet) no identical members in any insect and crustacean, and the particular members do not cross-react in the other's system, i.e. ITP will not increase the glucose concentration in the circulation of a crustacean and the
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crustacean cHHs are not potent in affecting the transepithelial potential of a locust's hindgut. Some families have a number of members but are not restricted to arthropods, for example, the FaRPs which occur in most major taxa, including vertebrates. Their myoactive effect also appears to be ubiquitous. Although it is not always clear why numerous isoforms of a peptide family do exist in organisms (with respect to physiological relevance), it is interesting to see just how these multiple forms are produced in insects and crustaceans: (a) by distinct genes that may (or may not) also code for an unrelated peptide, e.g. the 2 cHH isoforms of the lobster, the 3 AKHs of the locust and the 2 MOIHs of the crab (b) by a single gene, e.g. all 14 allatostatins of the cockroach are encoded by one gene (c) by alternative splicing sites on one gene, e.g. ITP and ITP-like peptides in insects, and the PO-cHHs in crabs. The complete subject area of ligand/receptor co-evolution cannot really be answered and fully exploited since there is no detailed sequence information on receptors from crustaceans. The chief reason for this is because the complete genome of a crustacean has not been sequenced yet. Perhaps major progress in this area will be achieved when industry is convinced of the profitability of transgenic lobsters! ABBREVIATIONS PO CC XO SG CNS RPCH HPLC RIA EIA cHH ELISA PCR
= pericardial organ = corpora cardiaca = X-organ = sinus gland = central nervous system = red pigment-concentrating hormone = high pressure liquid chromatography = radioimmunoassay = enzyme immunoassay = crustacean hyperglycaemic hormone = enzyme-linked immunoassay = polymerase chain reaction
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AKH GIRK FAB MALDI ESI HrTH pGlu CHO PLC IP3 AC MO DRPH PDH PDF ORF PKA MIH YO PKG VIH GIH MOIH MF JH ITP Isc CA PTTH AST HEK cells CCAP FaRP NPF NPY Parr2-GFP PP PYY PK
= adipokinetic hormone = G protein-gated inwardly rectifying potassium channel = fast atom bombardment = matrix-assisted laser desorption/ionization = electrospray ionization = hypertrehalosaemic hormone = pyroglutamic acid = Chinese hamster ovary = phospholipase C = inositol trisphosphate = adenylate cyclase = mandibular organ = light-adapting distal retinal pigment hormone = pigment-dispersing hormone = pigment-dispersing factor = open reading frame = cyclic AMP-dependent protein kinase = moult-inhibiting hormone = Y-organ = cyclic GMP-dependent protein kinase = vitellogenesis-inhibiting hormone = gonad-inhibiting hormone = mandibular organ-inhibiting hormone = methyl farnesoate = juvenile hormone = ion-transporting peptide = short circuit current = corpora allata = prothoracicotropic hormone = allatostatin = human embryonic kidney cells = crustacean cardioactive peptide = FMRFamide-related peptide = neuropeptide F = neuropeptide Y = Parrestin2-green fluorescent protein = pancreatic polypeptide = peptide YY = pyrokinin
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MT PBAN DH MRCH ETH TK
= myotropin = pheromone biosynthesis-activating neuropeptide = diapause hormone = melanization and reddish colouration hormone = ecdysis-triggering hormone = tachykinin
ACKNOWLEDGEMENTS The authors thank Dr. L. Auerswald (University of Cape Town) for his help with the preparation of the manuscript and figures, Prof. P. Taghert (Washington University School of Medicine, St. Louis, USA) for critically reading some sections on myotropic peptides and The National Research Foundation (Pretoria, South Africa; grant number 205 3396) and the University of Cape Town (staff award to G.G.) for financial support.
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NATURAL PRODUCTS AS MODULATORS OF APOPTOSIS AND THEIR ROLE IN INFLAMMATION JOSE LUIS RIOS, M. CARMEN RECIO Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia. Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain ABSTRACT: Modifications in apoptosis, a programmed form of cell death that participates in a wide variety of physiological processes, can contribute to many diseases, principally those associated with cell accumulation, such as inflammation. One hallmark of inflammation is the infiltration of leukocytes, which are programmed to undergo apoptosis at inflamed tissues. Resolution of inflammation thus involves the elimination of excess inflammatory cells by physiological cell death and the subsequent removal of apoptotic cells by phagocytes. Controlling this process may therefore be useful in the treatment of pathologies, which include an inflammatory component. Interestingly, while in some cases apoptosis is the mechanism that actually produces the resolution of inflammation, in other pathologies it is the cause of inflammation rather than its solution. This is the case of eczematous dermatitis. Depending on the type of inflammatory disease, then, possible treatment may include either augmenting or blocking the apoptotic process. Different anti-inflammatory agents, e.g. teophylline or the glucocorticoid family of compounds, have already been shown to modify cell apoptosis. In this chapter, we review the possible new anti-inflammatory agents isolated from plants with proapoptotic or antiapoptotic properties, with special emphasis on alkaloids and phenolics as the most relevant phytochemical groups in the resolution of inflammation to date.
INTRODUCTION Apoptosis is a physiological process critical for the development, homeostasis, and normal functions of the immune system. It is a process that involves the removal of cells from tissues in a deliberate and systematic manner, without an inflammatory reaction. It can thus be considered a process that is opposite, but complementary to that of cell proliferation, otherwise known as mitosis [1]. In contrast with apoptosis, necrosis is a type of cell death, which typically occurs in numerous cells of a given tissue in response to physiological trauma such as acute anoxia or extreme injury. This process involves loss of membrane integrity and uncontrolled release of cellular contents and often triggers an inflammatory response [2]. While
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necrotic cells die in an indiscriminate fashion and generate serious health problems, in apoptosis, cells play an active role in their own demise. For this reason, apoptosis can play a critical role in many pathological conditions. It is well known that a balance between cell death and cell proliferation is essential for maintaining a cellular homeostatic state. Deviation from this cellular balance can lead to human disease; thus, for example, cell accumulation due to insufficient apoptosis can contribute not only to cancer, but also to inflammations such as rheumatoid arthritis, as well as to autoimmune diseases such as lupus and lupus-like syndromes. On the other hand, excessive apoptosis may play an important role in neurodegeneration, acquired immunodeficiency syndrome (AIDS), osteoporosis, and heart failure. A fuller understanding of this complex physiological process of cell death would therefore be of great benefit in maintaining human health [3,4]. Characteristics of apoptosis The complete apoptosis of a cell is a rapid process defined by a distinct set of morphological and biochemical characteristics, which are observed exclusively with this mode of, cell death. These features include cell shrinkage and loss of the cell-cell contact, condensation of nuclear chromatin followed by internucleosomal DNA fragmentation (when examined by means of agarose gel electrophoresis, these multiple fragments form a ladder pattern that is characteristic for most cells undergoing apoptosis), and the formation of apoptotic bodies. In order to facilitate phagocytosis by macrophages, the membranes of apoptotic cells undergo several changes, including the translocation of the phosphatidylserine molecules from the inner leaflet of the plasma membrane to the outer surface. This phosphatidyl exposure serves as a stimulus to macrophages to remove apoptotic cells from tissues in a clean and tidy fashion, thereby avoiding many of the problems associated with necrotic cell death [5]. This efficient engulfment of the apoptotic cell by its neighbors is likely what makes this type of cell death appear to be "silent." At the center of the apoptosis machinery is a family of intracellular proteases, known as caspases (cysteine aspartyl-specific proteases) that are responsible, either directly or indirectly, for the morphological and biochemical events involved in the process Table 1 [6,7]. Recent work has provided evidence that mitochondria also play an important role in
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the regulation of apoptosis by acting as reservoirs for pro-apoptotic proteins such as cytochrome c, Smac/DIABLO (second mitochondriaderived activator of caspases/direct 1AP binding protein), AIF (apoptosis inducing factor), and procaspase-2, -3, -8, -9 (for more information, see review [8]). Table 1. The Caspase Family Name (Previous name)
Function
Caspase 1 (ICE)
Cytokine processing
Caspase 2 (ICH-1)
Initiator
Caspase 3 (Apopain, CPP32, Yama)
Effector
Caspase 4 (ICH-2, TX, ICE-rel II)
Cytokine processing
Caspase 5 (ICE-rel III) Caspase 6 (Mch-2) Caspase 7 (Mch3, ICE-LAP3, CMH-1) Caspase (MACH, FLICE, Mch5)
Cytokine processing Effector Effector
Initiator
Caspase 9 (Mch6, ICE-LAP6)
Initiator
Caspase 10 (Mch4, FLICE-2) Caspase 11 (ICH-3)
Initiator Cytokine processing
Substrate specificity Comments Pro-caspase Others Pro-IL-lp It is involved in TNF1,3,4 Pro-IL18 mediated apoptosis, apoptosis induced by the IFN-y destruction of the extracellular matrix. ? ? It is related to the C. elegans death protein CED3. Its activation occurs early in the apoptotic process. Activation of caspase-3 1,6,7,9 PARP occurs rapidly. PKC-8 DNA-PK Staurosporine is one of the most potent chemical activators PARP 1,3,4 It appears to exist as a single molecular weight species somewhat smaller than caspase-2. ? 5 PARP 3,7 It is involved in the final apoptotic execution phase. 7 PARP It is processed by caspase10, granzyme B and caspase-9. 3,4,7,9,13, 14 ? It is directly involved in apoptosis mediated through Fas and TNF receptors. 9 7 It is involved in the early activation cascade that occurs following an apoptotic signal. ? Similar to caspase-8. 1,3,7, 14 7
1,3
Caspase 12
Cytokine processing
9
?
Caspase 13
Cytokine processing
7
?
Caspase 14
Cytokine processing
?
9
Pro-inflammatory caspase involved in the caspase activation cascade. Apoptotic protease localized in the endoplasmic reticulum. Also known as the evolutionarily-related IL-ip converting enzyme (ERICE). -
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THE MECHANISMS OF APOPTOSIS The mechanisms that regulate apoptosis are not yet fully understood, but it is probable that alterations in the environmental conditions of the cell can trigger, accelerate, or slow down the process. The best evidence for which specific molecules are essential for apoptosis comes from research on the nematode Caenorhabditis elegans [9]. This work has demonstrated that in the system found in this species, the apoptosis-inducing genes are Ced-3 and Ced-4. In mammals, caspases fill the role of Cd-3 while the mammalian homologue of Ced-4 is the apoptosis protease activating factor-1 (Apaf-1). This latter protein is actually involved in activating executioners of programmed cell death following cytochrome c release from the mitochondria of mammalian cells. An additional regulatory pathway for this process is the system involved in the production of ceramide, which not only mediates apoptotic signals in some cells, but can also modulate apoptosis itself. Ced-9 acts as an antagonist toward the activities of Ced-3 and Ced-4, thereby protecting the surviving cells from any accidental death program activation. The human homologue of Ced-9 is a large family of compounds dubbed the Bcl-2 family (B-cell leukemia oncogen-2), whose anti-apoptotic members act by preventing caspase activation [10]. Caspase activation mechanisms The first caspase to be identified, interleukin-ip (IL-ip) converting enzyme (ICE, caspase-1), was discovered through its involvement in the processing of proIL-ip to the pro-inflammatory IL-ip molecule. Although caspase-1 plays no obvious role in cell death, it is of interest as the first identified member of a large family of proteases whose members have been shown to have distinct roles in both inflammation and apoptosis [11,12]. The caspase gene family contains 14 mammalian members; of these, 11 human enzymes have been identified (Table 1). This gene family is composed of two major subfamilies: ICE (caspase-1; inflammation group) and ICH (ICE and Ced-3 homologue; apoptosis group) [13]. All the caspases share similarities in their amino acid sequence, structure, and substrate specificity. They are expressed as proenzymes or zymogens
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and are triggered into action as a result of their proteolytic processing at conserved aspartic acid residues. Although cell death can occur through caspase-independent, non-apoptotic mechanisms, the morphological characteristics that define the process of apoptosis depend on caspases [14]. Apoptosis can be induced in mammalian cells by a number of mechanisms, but only two pathways, both for activating caspases, have been elucidated in detail [13]. The death receptor or "extrinsic" pathway can be induced by members of the tumor necrosis factor (TNF) family of cytokine receptors such as TNFR1 (TNF receptor-1) and Fas. The TNF family of receptors use caspase activation as a signaling mechanism, connecting ligand binding at the cell surface to the induction of apoptosis [15]. The "intrinsic" pathway for caspase activation in cells involves the participation of the mitochondria, which release caspase-activating proteins such as cytochrome c into the cytosol [16]. These mechanisms are commonly viewed as separate pathways and are capable of functioning independently, but cross-talk can occur between them at multiple levels, depending on the apoptosis-modulating proteins expressed. Numerous experimental results have demonstrated that caspase-8 is the main caspase in the TNF family death receptor pathway (Fig. 1) whereas caspase-9 is the principal caspase of the mitochondrial pathway (Fig. 2) [17,18]. However, not all caspases are directly involved in apoptosis. The primary role of some caspases, including caspase-1, is the activation of pro-inflammatory cytokines. The inhibition of these proteases could thus be useful in controlling numerous diseases. The peptide drug VX-740, for instance, is a selective and reversible caspase-1 inhibitor that is intended to treat arthritis and other inflammatory diseases [19]. All the protease systems are under negative control by endogenous inhibitory proteins called inhibitor of apoptosis proteins (IAPs) [reviewed in 20 and 21]. Some IAPs are overexpressed in cancers, and are associated with resistance to apoptosis [22]. Antisense experiments have in fact established that certain IAPs (e.g. survivin) could be considered as potential drug targets for cancer since interference with IAP function leads to caspase-induced apoptosis of cancer cells. In addition, there is increasing interest in the use of IAPs in gene-therapy strategies to reduce neuronal cell loss following stroke or other brain injuries.
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FasL
Receptor
0
Membrane
FADD
L
™.p
Procaspase 8
Nucleus
DNA
Fig. (1). Fas signaling pathway and caspase cascade
147
Stimuli
Membrane
Bad
Proapoptotic Bcl-2 family proteins
Bid
Antiapoptotk Bcl-2 family proteins
Cvtochromc c Procaspase 9 ApaM
LI
\> & £ Apoptosome
Caspase 9
Effector caspase
Fig. (2). Mechanism of apoptosis
U
dATP
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Bcl-2 family proteins Bcl-2 was first discovered as a proto-oncogene in follicular B-cell lymphoma. It has since been identified as a mammalian homologue to the apoptosis repressor Ced-9. The mitochondrial-dependent pathway in the human Bel-family includes both pro-apoptotic (Bax, Bak, and Bok) and anti-apoptotic proteins (Bcl-2, BCI-XL, Bcl-w, etc.) [10]. Although the precise mechanisms of the mammalian Bcl-2 family of proteins are still a matter of debate, it has been established that their main function is to regulate the release of cytochrome c and other proteins from the mitochondria [24]. The relative ratios of anti- and pro-apoptotic Bcl-2-family proteins dictate the ultimate sensitivity to or resistance of cells to various apoptotic stimuli, including hypoxia, radiation, anticancer drugs, oxidants, Ca2+ overload, ceramide, and growth-factor/neurotrophin deprivation [25]. During apoptosis, the pro-apoptotic Bcl-2 family members are activated through several mechanisms such as dephosphorylation or proteolytic cleavage brought about by caspases. They are then translocated to the mitochondria. The translocation of Bax, Bid, or Bad can induce the release of the proteins contained in the intermembrane space, including cytochrome c, which, only after subsequent coupling with a heme group, functions to induce caspase activation [25,26]. The anti-apoptotic proteins Bcl-2 and BCI-XL prevent cytochrome c release, thus preserving cell survival. Cytochrome c release is thus an early event during apoptosis, occurring hours before phosphatidylserine exposure and loss of plasma membrane integrity. As mentioned above, it is only after cytochrome c release that caspases areactivated and the cell undergoes apoptosis. The actual apoptotic process occurs through the formation of an "apoptosome" (comprised of cytochrome c, apoptosis protease activating factor 1 (Apaf-1) and procaspase-9). This apoptosome then recruits procaspase-3, which is cleaved and activated by the active caspase-9 and is subsequently released to mediate apoptosis (Fig. 2) [27]. In some cellular systems, cytochrome c is necessary, but not sufficient, for cell death. In these systems, a second mitochondrial activation of caspases is promoted by a protein with the dual name of Smac/DIABLO [28,29], which is released from the mitochondria with
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cytochrome c in a coordinated fashion during apoptosis. While cytochrome c activates Apaf-1, Smac/DIABLO relieves the inhibition of caspases by binding to the IAPs, thereby allowing caspase-9 to activate caspase-3 [30]. Alterations in the amounts of Bcl-2 proteins have been associated with diseases in which too much or too little cell death occurs (this is referred to as cell loss and cell accumulation, respectively). These diseases include cancer, autoimmune disorders such as lupus, immunodeficiency associated with human immunodeficiency virus (HIV) infection, and ischemia-reperfusion injury during stroke and myocardial infarction, among others [31]. Death ligands and death receptors The "death receptors" of the TNFR family include TNFR1, Fas (CD95), DR3/WSL, and the TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) receptors. When these receptors are bound by a ligand (TNF, Fasligand (FasL), etc.), apoptosis may occur [25]. Fas is a glycosylated cell surface molecule that can also be found in soluble form. It is expressed on several different cell types (mainly in macrophages, activated T and B lymphocytes, and in organs such as the thymus, liver, spleen, lungs, testes, brain, intestines, heart, and ovaries), and its expression can be augmented both by cytokines (interferon-y (IFN-y) and TNF) and by lymphocyte activation. In contrast, expression of FasL is more tightly regulated, often being induced only under very specific conditions. Moreover, FasL expression is restricted to immune cells including T and B lymphocytes, macrophages and natural killer cells, and to non-immune sites such as the testes, kidneys, lungs, intestines, and eyes [25]. A fuller understanding of the Fas-FasL interaction is necessary to comprehend better the signaling pathway involved in death receptorinduced apoptosis. Inappropriate expression of Fas and FasL on lymphocytes and other immune cells has previously been documented in patients with HIV infection; it has also been implicated in the loss of lymphocytes that characterizes this immunodeficiency syndrome [32]. Conversely, hereditary mutations in the death domain of the Fas gene are known to cause an autoimmune lymphoproliferative syndrome in humans [33].
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Researchers have discovered that Fas-mediated apoptosis is blocked by a molecule that has been assigned various names, but is usually referred to as c-FLIP (cellular FLICE-inhibitory protein) (other names include FLAME, I-FLICE, Casper, etc.) [34,35]. The expression of c-FLIP is down-regulated by IL-2 [36,37], a fact which explains why IL-2 can sensitize activated T-cells to Fas. Ligation of a death receptor does not necessarily lead to caspase-8 activation and death. TNFR1 can also activate nuclear factor-KB ( N F - K B ) in cells that are resistant to TNFR1-mediated apoptosis. Inhibition of this transcription factor can thus sensitize the cells to this particular form of cell death [38]. In summary, these are only some of the major molecular pathways for the induction and control of caspase activation and apoptosis following a variety of stimuli for cell death. It should be noted, however, that apoptosis induced by ultraviolet (UV) light and other stimuli has been shown to occur in animals lacking caspase-9 [39], thus suggesting at least one alternative to the apoptosome. APOPTOSIS AND INFLAMMATION Defects in the physiological pathways of apoptosis have been shown to play a role in many diseases (Table 2). Consequently, there has a been a great surge of interest in devising therapeutic strategies for modulating the key molecules that make life-or-death decisions in cells. In addition to targeting the core components of the cell-death machinery (caspases, IAPs, and Bcl-2 family proteins) directly, opportunities exist to affect apoptosis indirectly by modulating the input into cell-death pathways through protein kinases, protein phosphatases, and transcription factors, as well as by affecting the cell-surface receptors for cytokines, neurotrophins, cardiotrophins, and growth factors [19]. For example, the inhibitor of the N F - K B (IKB) kinase (IKK) complex has emerged as a potential target for promoting the apoptosis of cancer cells. N F - K B has been found to suppress cell death in a variety of contexts, and has also been seen to induce the transcription of several anti-apoptotic genes, including members of the Bcl-2 family and some IAPs. Moreover, N F KB hyperactivity has been observed in several types of cancer. Certain non-steroidal anti-inflammatory (NSAIDs) drugs, as well as synthetic triterpenoids and other compounds have been reported to inhibit the
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catalytic activity of IKK. These agents should be examined not only for their ability to sensitize tumors to apoptosis, but also for their potential in treating inflammatory diseases in which N F - K B has been implicated [19, 40].
Table 2.
Diseases associated with defective apoptosis regulation
Insufficient apoptosis
Cancer, autoimmunity, restenosis, persistent infections, atherosclerosis, metabolic disorders.
Excessive apoptosis
Neurodegenerative disorders (Alzheimers's disease, Parkinson's disease), hematological disorders, autoimmune disorders (graft versus host disease, type I diabetes, rheumatoid arthritis), ischemia, heart failure, inflammation, osteoarthritis, human immunodeficiency virus, bacterial infections, allograft rejection, trauma.
There are several areas in which the modulation of apoptotic death could advance medical treatment of disease in the future: 1) Control of malignant diseases, 2) Delay of premature senescence/neurodegenerative disorders, 3) Treatment of transplant rejections, 4) Regulation of tissue regeneration/repair, and 5) Regulation of inflammatory diseases through the induction of apoptosis and phagocytosis, which would aid in suppressing and/or resolving the inflammatory response [41,42]. The rest of this section will deal with how new findings concerning the apoptotic process may influence the treatment of inflammatory diseases. To date, one of the main methods of controlling inflammations has been the use of glucocorticoids, which have proven highly effective in attenuating the inflammatory response. These hormones are able to induce apoptosis in the monocytes, macrophages, and T lymphocytes involved in the inflammation reaction. However, in fibroblasts and glandular cells such as hepatocytes and ovarian follicular cells, glucocorticoids actually protect against apoptosis induced by cytokines, cyclic adenosine monophosphate (cAMP), tumor suppressors and death genes. This phenomenon is probably due to composite regulatory crosstalk among multiple nuclear coactivators or corepressors, which mediate the transcription regulation of the genes by interacting with the glucocorticoid receptor [43]. Another line of research in this area includes the study of the apoptotic processes of other cells, which play major roles in the immune system. Recent work has thus focused on the apoptosis of a key component of
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this system, namely the polymorphonuclear (PMN) leukocytes known as neutrophils. Aged neutrophils have been found to undergo spontaneous apoptosis in the absence of cytokines or other proinflammatory agents prior to their removal by macrophages. In acute inflammation, however, the number of neutrophils within tissues may reach excessive levels due to recruitment from circulation and also because their constitutive apoptotic pathway is delayed by the action of local inflammatory mediators [44]. Because the potential for inflammatory neutrophil tissue damage via the release of toxic reactive oxygen species and granule enzymes such as proteases is very high, death by apoptosis and safe removal by phagocytic cells helps limit tissue damage during the resolution of inflammation. It has been demonstrated that neutrophil apoptosis can be promoted by a wide variety of agents, including granulocyte-macrophage colony stimulating factor (GM-CSF), P2 integrins, and TNF-a. In the regulation and execution of neutrophil apoptosis, members of the Bcl-2 family and caspases (caspases-1, -3, -4 and -8) are also involved. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases (MAPK), also play critical roles. These and other aspects related to the regulation of neutrophil apoptosis have recently been reviewed [45]; from the research to date, it is clear that promoting the safe clearance of apoptotic cells may be a new avenue for therapy in inflammatory responses which persist rather than resolve [46]. Controlling apoptotic processes may also be useful in devising new treatments for numerous inflammations of the skin. One of the principal cell groups found in the skin is that of the keratinocytes, the epithelial cells which comprise the epidermis of the skin and the epithelium of the oral mucous membranes. The main function of these cells is to provide an intact epithelial covering for the body to serve as an impermeable barrier. While keratinocyte stem cells are found in the deepest tips of the dermal papillae, proliferating keratinocytes are found in the basal and the immediately suprabasal epidermis. There are other cellular elements in the epidermis, which influence epidermal keratinocyte function, notably melanocytes. These cells produce melanin, a complex heteropolymer which protects against the toxic effects of light. Once synthesized, melanin is transferred to keratinocytes to form supranuclear shields against ultraviolet radiation effects. In addition, keratinocyte function is influenced by the cytokine-releasing epidermal Langerhans cells. These
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are specialized dendritic antigen-presenting cells that reside in the upper epidermis, but which emigrate to regional lymphatic when presented with exogenous antigens. In many inflammatory skin diseases, recirculating lymphocytes can enter the epidermis in response to cytokine and chemoMne release. Keratinocyte apoptosis has been observed not only during a number of biological processes, such as epidermal differentiation, but also in certain skin diseases in which the epidermis is the target of immunological cytotoxicity [47,48]. The skin, and especially the epidermis, is constantly at risk for induction of cytotoxicity by ultraviolet radiation, oxidant stress, cytokines, chemokines, and neuropeptides, as well as cytotoxic lymphocytes and macrophages. Modulation of the members of the Bcl-2 family is thus a potential mechanism for the defense of the epidermis against apoptosis. Since immunohistochemical analyses have demonstrated that Bcl-2 proteins are localized in the basal keratinocyte layer, it has been proposed that these proteins may be a major inhibitor of apoptosis in keratinocytes. Indeed, transgenic mice which overexpress Bcl-2 in the epidermis showed decreased susceptibility to apoptosis induced by ultraviolet radiation or 12-0-tetradecanoylphorbol 13-acetate (TPA) [49]. However, despite the intrinsic defenses of the basal layer of the epidermis against the induction of apoptosis, keratinocyte apoptosis is largely involved in a number of immunological skin diseases, including lichen planus, graft versus host disease, photosensitive lupus, erythema multiforme, and other forms of dermatitis. By the same token, it has been demonstrated that keratinocyte apoptosis represents the major mechanism in the pathogenesis of atopic dermatitis as well as other eczematous disorders. The process begins as T cells infiltrate the skin and upregulate the Fas receptor on the keratinocytes, thus rendering them susceptible to apoptosis by IFN-y. Apoptosis is simultaneously induced by the FasL expressed on the surface of the T cells themselves [50]. Chondrocytes, the only cell type found in normal mature articular cartilage, are responsible for the maintenance and repair of this tissue, which happens to be the site of pathologic matrix remodeling and degradation in arthritis [51]. Indeed, chondrocyte apoptosis is a feature of osteoarthritic cartilage and is closely associated with extracellular matrix degradation. Fibrillated cartilage from osteoarthritic joints has been found to contain apoptotic cells in both the superficial and mid zones. In
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contrast, very low numbers of apoptotic cells are detected in normal cartilage. This is due to the fact that cartilage does not contain mononuclear phagocytes; therefore, apoptotic bodies are more likely to exert pathogenic effects on this tissue. Chondrocyte apoptosis in osteoarthritis may be the consequence of aberrant hypertrophic chondrocyte differentiation, but it may also be induced by extracellular stimuli such as FasL and other cytokines. In addition, nitric oxide (NO) production may lead to chondrocyte apoptosis. Various factors thus contribute to the pathogenesis of cartilage degradation. Inhibitors of NO synthesis and chondrocyte apoptosis may therefore be of therapeutic value after cartilage injury and in osteoarthritis [52]. In contrast to osteoarthritis, rheumatoid arthritis is a chronic inflammatory synovitis dominated by the presence of macrophages, lymphocytes, and synovial fibroblasts and which ultimately leads to the destruction of bone and cartilage. The effectiveness of therapies that are directed against TNF-a and IL-1 lends credence to the notion that macrophages are a crucial target for therapeutic intervention in this disease [53]. Because inadequate or insufficient apoptosis appears to play a significant role in the increase in cellularity of rheumatoid synovial tissue, lack of apoptosis is seen as contributing to the perpetuation of the disease. The mechanisms that prevent apoptosis of inflammatory cells in rheumatoid arthritis have recently been reviewed by Pope [54], whose findings support the idea that interventions aimed at enhancing apoptosis in the synovium are potentially effective forms of treatment in patients suffering from this disease. ANTI-INFLAMMATORY NATURAL PRODUCTS As mentioned above, the resolution of inflammation involves clearing away the excess of inflammatory cells by apoptosis and the subsequent recognition and removal of apoptotic cells by phagocytes. One hallmark of inflammation, especially in the skin, is macrophage infiltration, which quite often can mediate chronic inflammations such as psoriasis, atopic dermatitis, and chronic contact dermatitis [55]. In both atopic dermatitis and allergic contact dermatitis, a disruption of the epidermal barrier occurs, leading to spongiosis, which in turn can rupture the intercellular attachments of the keratinocytes, thus generating vesicles. Secretion of IFN-y by T lymphocytes then promotes Fas upregulation in
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keratinocytes, which undergo apoptosis as part of the normal inflammatory reaction. In the case of keratinocytes, however, the inflammatory infiltrate is the cause rather than the consequence of their apoptosis [56]. Finally, apoptosis is implicated in the resolution of T cellmediated cutaneous inflammation, such as that associated with delayedtype hypersensitivity (DTH) reactions [57]. There are thus several different mechanisms by which apoptosis can be involved in the inflammatory response. The fact that the apoptosis of inflammatory cells can be regulated pharmacologically makes this phenomenon extremely interesting, as it can therefore be exploited to develop new drug therapies [44]. In fact, as discussed above, the effect of many anti-inflammatory drugs, such as glucocorticoids, NSAIDs, and anti-oxidant agents, is based on the role these drugs play in this particular physiological process. Glucocorticoids and their implication in anti-inflammatory effects Glucocorticoids, which play a major role in attenuating the inflammatory response, do so via a mechanism in which a specific receptor is implicated. As mentioned above, the anti-inflammatory action of glucocorticoids is exerted by two complementary, but opposite pharmacological effects: a) by an apoptotic mechanism that induces the death of the inflammation-provoking cells such as monocytes, macrophages, and T lymphocytes; and b) by an anti-apoptotic mechanism that protects the resident cells of the inflamed tissue by arresting the apoptotic signals evoked by cytokines, cAMP, tumor suppressors, and death genes on glandular cells and fibroblasts. This latter mechanism involves the modulation of several survival genes such as Bcl-2, BCI-XL, and N F - K B in a cell-specific manner [43,58]. Modification of transcription signals and apoptotic effects
The glucocorticoids have been found not only to increase apoptosis, but also to modulate the expression of apoptosis-related markers in both unstimulated and IL-2-stimulated T lymphocytes. In one study, this class of drugs induced apoptosis while reducing Bcl-2, Fas, and CD25 expression. Only negligible effects were detected on Bax expression, a fact which points towards a potential mechanism by which some corticoids exert their anti-inflammatory effects [59].
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In another study on the effects of glucocorticoids on proliferation and apoptosis, as well as on the activity and expression of N F - K B in intestinal epithelial cells, glucocorticoids were seen to modulate the repair mechanisms of intestinal epithelial cells in vitro while profoundly modulating the inflammatory regulator N F - K B , a known regulator of apoptosis and inflammation [60]. On the other hand, glucocorticoids decreased the expression of cyclooxygenase-2 (COX-2). This is of interest because upregulated COX-2 expression plays a relevant role in pathological processes characterized by increased local prostaglandin (PG) production and consequently in the process of inflammation itself [61]. Role of cells in inflammation and their modification by apoptosis
While the role of neutrophils in asthma remains relatively obscure, eosinophils have been shown to play a major part in the onset and maintenance of the bronchial inflammation and tissue injury in this condition [62]. Like other leukocytes, eosinophils present in excessive numbers in inflamed tissues are removed by apoptosis, which allows for the elimination of dangerous cells [63,64,65]. Conversely, a defect in eosinophil apoptosis leads to the development and persistence of allergic airway inflammation in asthma. It is also thought that a defect in apoptosis might contribute to the chronic tissue eosinophilia associated with the malady. It has been determined that this delay of eosinophil apoptosis in asthma occurs in part due to the production of GM-CSF. Traditional treatments for asthma are based almost exclusively on the use of inhaled glucocorticoids, which totally reverse the delayed eosinophil apoptosis in this condition [66]. It is clear that a better understanding of the mechanisms underlying eosinophil apoptosis would help delimit the molecular events involved in eosinophil accumulation in the blood and tissues, which in turn would uncover potential new targets for the treatment of allergic diseases in general, and asthma in particular [67]. One line of research that has proven of interest is that concerning the role of caspases in eosinophil apoptosis. Thus, although it has been established that treatment with dexamethasone induces eosinophil apoptosis via a mechanism mediated through the glucocorticoid receptor, as evidenced by the fact that the effect was nullified by the glucocorticoid receptor antagonist mifepristone [68], it has also been shown that this dexamethasone-induced apoptosis and activation of c-jun
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NH2-terminal kinase (JNK) and p38 MAPK activity in eosinophils is regulated by caspases. Interestingly, the caspases involved are not the common apoptosis-related caspase-3 or -8, as is the case in other cells. The elucidation of the role of caspases in eosinophil apoptosis may thus facilitate the development of more specific and effective treatments for this type of allergic inflammation [69]. Neutrophils, for their part, have been implicated in mediating the tissue damage associated with chronic inflammatory diseases. Glucocorticoids have been found to exert significant inhibitory effects on both neutrophil activation and neutrophil functions such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis [70]. Since phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis all promote the resolution of inflammation, this hitherto unrecognized ability of glucocorticoids to potentiate the nonphlogistic clearance of apoptotic leukocytes by phagocytes has potential implications for therapies aimed at promoting the resolution of inflammatory diseases [71]. Basophils comprise another group of cells actively involved in allergic inflammations. The apoptogenic effects of glucocorticoids on these cells might have implications for the mechanism of action of these drugs in allergic inflammation [72]. Mast cells, positioned in the asthmatic airways, also play a major role in the maintenance of the condition. During the active disease, these cells are primed to secrete preformed and newly generated inflammatory mediators, neutral proteases, cysteinyl leukotrienes, cytokines, and chemokines [73]. The local delivery of glucocorticoids to the affected tissues has been found to reduce significantly the number of mast cells present. In one study in which glucocorticoids were directly applied to mouse dermis, the number of mast cells decreased. No direct effect of the glucocorticoids on the mast cells themselves was observed; thus, the decrease was probably the result of increased apoptosis [74]. T-cells are involved in various inflammatory pathologies such as asthma, multiple sclerosis, and human idiopathic polymyositis. The modification of these cells may thus be an important mechanism to help avoid the damage caused by these afflictions. In the natural disease course of multiple sclerosis, for example, apoptosis contributes to the elimination of T-cells from the inflamed central nervous system. Using corticoids to induce apoptosis could contribute to the down-regulation of
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T-cell activity, thereby terminating the inflammation of the central nervous system [75]. Moreover, corticoids may reduce allergen specific T-cells through apoptosis, which is one of the mechanisms of effectiveness of corticoids in asthmatics [76], as well as induce apoptosis of endomysial T-cells in human idiopathic polymyositis, thus modifying the inflammation [77]. Interestingly, the expression of Bcl-2 may be an important factor in protecting the lymphocytes in inflamed synovia! membranes from glucocorticoid-induced apoptosis [78]. Inflammatory cytokines and apoptosis
Phagocytosis of apoptotic cells by macrophages leads to the production of anti-inflammatory cytokines as a way of preventing inflammation. Kurosaka et al. [79] have demonstrated that human serum potentiates the production of two anti-inflammatory cytokines, IL-10 and TGF-p, by both TPA-treated THP-1 cells and human monocyte-derived macrophages. This enhanced response to the presence of apoptotic cells also results in the suppression of the production of the pro-inflammatory cytokine IL-8. In addition, human IgG and FcyRI appear to be critical in triggering the production of anti-inflammatory cytokines by macrophages in response to apoptotic cells. Glucocorticoids repress the expression and release of numerous cytokines in macrophages, thymocytes, and CD4+ splenocytes; in addition, a protein-protein interaction with transcription factors such as N F - K B is involved in their anti-inflammatory activity. The only defect of immune suppression brought about by this class of drugs detected so far concerns the induced apoptosis of thymocytes and T lymphocytes [80]. Non steroidal anti-inflammatory drugs (NSAIDs) The anti-inflammatory mechanism of the NSAIDs is principally related to any or all of the following three factors: the inhibition of different enzymes implicated in the aracMdonic acid metabolism, the modification of the effects of relevant mediators, and the inhibition of the tissue damage produced by free radicals. It is thought that the phenomenon of overexpression of the enzymes implicated in inflammation may be avoided by the effect of apoptotic mechanisms on inflammatory cells. The role of eicosanoids and NSAIDs, with either a positive or negative
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effect, thus provides exciting possibilities for new anti-inflammatory agents. Arachidonic acid metabolites and apoptosis
The arachidonate cascade includes the COX pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. While the exact mechanism remains unknown, it has been shown that eicosanoids play a dual role in regulating both cell survival and apoptosis in various types of cells [81]. One recent study, for example, demonstrated that arachidonic acid metabolites are involved in the regulation of apoptosis in human polymorphonuclear neutrophil granulocytes [82]. NSAIDs such as the selective COX-2 and LOX inhibitors were initially developed to suppress inflammation and pain by inhibiting the production of PGE2 and its metabolites. Since then, however, the arachidonic acid metabolites, particularly 12-hydroxy-6,8,11,14eicosatetraenoic acid (12-HETE), 5-HETE, and PGE2, have been found to play a pivotal role in prostate cancer. In a recent study with human prostate cancer cell lines, for instance, the addition of a 5-LOX inhibitor induced apoptosis and decreased cell life duration. In contrast, 5-HETE prevented cell death and led to the overexpression of COX-2 as well as a clear increase in PGE2 production. Inhibiting COX-2 may thus be a possible key to the treatment of prostate cancer since selective inhibitors of COX-2 actually reduce PGE2 production in this cancer, and this, in turn, leads to cell apoptosis [83]. Moreover, while overexpression of 12LOX and 15-LOX-1 in prostate cancer cells stimulates prostate tumor angiogenesis and growth, the expression of 15-LOX-2 is reduced during the initiation and progression of prostate tumors. It has been found, however, that 15(5)-HETE, the product of 15-LOX-2, inhibits proliferation and causes apoptosis in human prostate cancer cells. This fact suggests an inhibitory role for 15-LOX-2 in the progression of the prostate tumor [84]. The relationship between 5-LOX activity and apoptosis has been reported in several different studies. Anderson et al. [85] studied the effect of the compound MK886, which at nM concentrations is a selective in vivo inhibitor of 5-LOX, while at uM concentrations it inhibits the proliferation of monoblastoid cells by means of an apoptotic mechanism. In human pancreatic cancer tissues, which show a marked
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expression of 5-LOX and the leucotriene B4 (LTB4) receptor, it has been demonstrated that the inhibition of 5-LOX not only blocks the proliferation of cancerous cells, but also that it induces their apoptosis [82]. Moreover, the 5-LOX branch of the arachidonate cascade is also responsible for membrane peroxidation, oxidative stress, and apoptosis of peripheral blood mononuclear cells; thus, the administration of substances such as vitamin E, which inhibits 5-LOX activity, may be helpful in controlling oxidative stress-related diseases [86]. Lipoxins (LXs) are LOX-derived eicosanoids generated during inflammation that inhibit chemotaxis and adhesion of PMN neutrophils. They also function as putative braking signals for PMN neutrophilmediated tissue injury. Lipoxin A4 (LXA4), for example, promotes the phagocytosis of apoptotic PMN neutrophils by monocyte-derived macrophages, thus acting as an endogenous stimulus for PMN neutrophil clearance during inflammation [87]. Moreover, LXA4 has been shown to inhibit TNF-a-stimulated neutrophil adherence to epithelial monolayers at nM concentrations. This is noteworthy as TNF-a not only induces disruption of mucosa architecture, but also enhances colonocyte apoptosis via a caspase-3-independent mechanism [88]. Nuclear factor-KB (NF-KB) and apoptosis
comprises a family of inducible transcription factors that serve as relevant mediators of the inflammatory response. This factor is also involved in protecting cells from undergoing apoptosis in response to DNA damage or treatment with cytokine [89]. Normally, N F - K B is kept inactive by a cytoplasmic inhibitor of KB (IKB) proteins, which are phosphorylated by a cellular kinase complex known as IKK, made up of two kinases, IKK-a and IKK-p. The phosphorylation of IKB by these kinases leads to the degradation of the proteins and to the translocation of N F - K B to the nucleus. Once in the nucleus, N F - K B activates gene expression of cells exposed to growth factors and cytokines [90,91]. Activation of the N F - K B pathway is thus involved in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and asthma [92], while an altered N F - K B regulation is in part responsible for the inflammatory response of other pathologies such as Alzheimer's disease [93]. Taking this into consideration, then, it is not surprising that the mechanisms of actuation of various pharmacological agents are based on
NF-KB
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how these anti-inflammatory drugs modulate N F - K B effects. In the case of glucocorticoids, the repression of the entire N F - K B pathway is implicated in their mechanism of action, while different NS AIDs inhibit this pathway at various stages, including that of IKK inhibition [89]. Despite evidence that N F - K B is generally an antiapoptotic factor, there are cases in which N F - K B acts as a proapoptotic [94]. For this reason, the modification of N F - K B could be an interesting target for different antiinflammatory drugs. Inhibition of N F - K B activation by NSAIDs has been described in many cells. These drugs, however, failed to impair IKB kinase activity, the processing of N F - K B , or the expression of N F - K B dependent genes such as iNOS in hepatic cells. Moreover, selective COX-2 inhibitors did not promote apoptosis in hepatocytes under inflammatory conditions, a fact which suggests that PGs are not required to maintain cell viability [95]. Nitric oxide (NO) and apoptosis
NO, derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in the regulation of relevant physiological and pathophysiological functions. The mechanisms by which NO exerts its effects include activation of guanylate cyclase, formation of peroxynitrite, apoptosis, and COX regulation [96]. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression [97]. NO is generated under inflammatory conditions and may serve as a cytotoxic molecule to produce cell death along either an apoptotic or a necrotic pathway. NO formation is established to initiate apoptosis, characterized by upregulation of the tumor suppressor p53, changes in the expression of pro- and anti-apoptotic Bcl-2 family members, activation of caspases, and DNA fragmentation. Prestimulation of macrophages with cytokines or low-level NO has been shown to activate the transcription factor N F - K B and to promote immediate early gene expression of COX-2 [98]. In a recent study, the NO radical quenching activity of NSAIDs and steroidal drugs demonstrated that NSAIDs directly scavenged generated NO and prevented the reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of iNOS. In contrast, corticoids, which had no scavenging effects in vitro, showed
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almost no protective effects. These data suggest that the protective effects against apoptosis of the NSAIDs studied might be due mainly to their direct NO radical scavenging activities in neuronal cells [99]. Antioxidant compounds and apoptosis
While apoptosis is related to the production of reactive oxygen intermediates, the modulation of apoptosis by antioxidants correlates to the modification of cell proliferation. There is evidence that oxidative stress acts as a major determinant of apoptotic cell death [100]. For this reason, the ability to differentiate and modulate apoptosis and necrosis by antioxidants opens up a wide range of possibilities in anti-inflammatory therapies [101]. It has been well established that fatty acid metabolites of LOX and cytochrome P450 are implicated in essential aspects of cellular signaling, including the induction of apoptosis. The enzymatic and non-enzymatic products of polyunsaturated fatty acids thus control cell growth and apoptosis, and the spontaneous oxidation of polyunsaturated fatty acids gives rise to reactive aldehydes and other products of lipid peroxidation that are potentially cytotoxic and which may also signal apoptosis [102]. MODULATION OF APOPTOSIS BY NATURAL PRODUCTS Anti-inflammatory properties have been attributed to many natural products, some of which are antioxidants while others inhibit the arachidonic acid metabolism. Still others act through a mechanism related to the modification of transcription signals. The apoptotic effects of natural products have received little attention to date, although more in-depth studies of these effects would help determine the precise mechanisms through which these compounds act as anti-inflammatory agents. The majority of studies in this area have focused on the apoptotic effects of phytochemicals in cancer research with the goal of identifying anti-cancer agents, but much of this research could also be applied to the study of the anti-inflammatory properties of natural products. Such is the case of alkaloids or phenolics such as flavonoids, both of which are widely studied in phytochemical and pharmacological research. For this review, we have selected a few representative natural products, many of which have previously been described as anti-
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inflammatory agents. We have, however, also included several compounds whose mechanisms and known properties may be related to the inflammatory process. We thus classify this review into two main groups, namely apoptosis-inducing compounds and those compounds with anti-apoptotic effects. Natural products as activators of apoptosis and their implications in inflammatory diseases Alkaloids
Alkaloids comprise the most relevant group of phytochemical compounds with pharmacological and biological activities. Indeed, a wide range of alkaloids has been described as being analgesics, antiinflammatories, or anticancer agents, with various mechanisms of action depending on their pharmacological effects. As for their role in the resolution of the inflammatory process, recent data have implicated the apoptosis of specific cells, a fact which makes the investigation of apoptosis an interesting new approach to studying different compounds, both known and new. The xanthine theophylline has been used for several decades in the treatment of asthma. This compound produces different effects at the cellular level, including phosphodiesterase isoenzyme inhibition, adenosine antagonism, catecholamine secretion enhancement, and the modulation of calcium fluxes. Recently, theophylline was found to have both immunomodulatory and anti-inflammatory properties; therefore, interest in its use in patients with asthma has been renewed [103]. Recent studies have thus discovered that at low doses, theophylline is able to decrease airway inflammation, accelerate eosinophil apoptosis, and decrease recruitment of lymphocytes and neutrophils to the lungs. Although it is classified as a phosphodiesterase inhibitor, its exact therapeutic mechanism of action remains undetermined [104]. Of the new mechanisms that have been included in the potential mode of action of theophylline, one is the apoptosis of inflammatory cells. In eosinophils and lymphocytes, for example, this effect is due to the compound's ability to inhibit phosphodiesterase, which leads to an even more pronounced increase in intracellular cAMP levels than that which occurs when adenylate cyclase, the enzyme that synthesizes cAMP, is activated. This inhibition and the resulting cAMP level increase thus lead to
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apoptosis in the aforementioned cells. Since inducing apoptosis is generally beneficial in allergic inflammations, the use theophylline in combination with corticoids may also be appropriate to induce apoptosis in eosinophils and lymphocytes [105]. Inflammatory cytokines such as GM-CSF and IL-5 are upregulated in bronchial asthma, and because they inhibit granulocyte apoptosis, they cause neutrophil and eosinophil infiltration into the airways. The administration of theophylline counteracts this affect by accelerating granulocyte apoptosis, which is important not only in combating the inflammation, but also in controlling granulocyte longevity regardless of the elevation of intracellular cAMP levels. Studies have shown that after theophylline administration, the percentages of GM-CSF-induced delayed apoptosis increased in both neutrophils and eosinophils, and that the percentage of IL-5-induced delayed eosinophil apoptosis was also higher. Moreover, cAMP-increasing agents inhibited granulocyte apoptosis both in the control and in anti-Fas antibody-treated cells, with the expression of Bcl-2 protein also decreasing after incubation of eosinophils with theophylline [106]. Additionally, theophylline was shown to induce apoptosis of leukemia cells in humans [107].
Sanguinarine, isolated from the root of Sanguinaria canadensis, possesses both anti-inflammatory and antioxidant properties. In addition, this alkaloid has displayed anti-proliferative and apoptotic effects against human epidermoid carcinoma cells and normal human epidermal keratinocytes. While treatment with sanguinarine has been shown to decrease the viability of both kinds of cells, this loss of viability occurred at lower doses of the compound and was much more pronounced in the carcinoma cells than in the normal keratinocytes [108].
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In a study on the mechanisms of apoptosis induced by Chinese and Western anti-rheumatic drugs in human T-cells, Lai et al. [109] demonstrated that Tripterygium wilfordii and its alkaloid tetrandrine can cause T-cell death. It is noteworthy that the apoptotic effect of tetrandrine was selective toward especially activated T-cells. Although the cytotoxicity of this compound was mediated through apoptotic mechanisms, Fas/FasL interaction was not required. Moreover, tetrandrine-induced T-cell DNA damage required caspase-3 activity. The induction of apoptosis brought about by tetrandrine was much faster than that caused by treatment with glucocorticoids, and did not require de novo protein synthesis. These results suggest that the anti-inflammatory and irnmunosuppressive properties of tetrandrine are mediated by novel mechanisms that have yet to be determined [40]. Nevertheless, cepharanthine, an alkaloid analogous to tetrandrine, has been shown to restore the aberrant in vitro morphogenesis of apoptotic cells treated with both TNFoc and plasmin. This alkaloid suppressed the TNFa-stimulated N F - K B activity by partially preventing the degradation of IKBO: protein in NS-SV-AC cells. In addition, cells which were pretreated with cepharanthine and which then received subsequent treatment with both TNFa and cepharanthine exhibited suppressed production of matrix metalloproteinase 9 [111]. Other alkaloids with potential interest as proapoptotic agents are acutiaporberine from Thalictrum acutifolium, which induces apoptosis by down-regulating the Bcl-2 gene while simultaneously up-regulating the Bax and c-myc genes [112]; acrocynine, a cytotoxic alkaloid isolated from Achronychia baueri used to synthesize active proapoptotic agents [113]; sinococuline from Stephania sutchuenensis [114]; solamargine from Solarium incanum [115]; and cryptolepine and neocryptolepine, both isolated from Cryptolepis sanguinolenta [116].
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OCH 3
OCH,
OCH 3 Cepharanthine
Tetrandrine
CH3O
OCH3 OCH3 OCH,
CH3O
OCH3
OCH3 Acutiaporberine
Phenolics
Phenolics and their functional derivatives are widely found throughout the plant kingdom. One defining characteristic of these compounds is that their aromatic ring usually contains at least one hydroxyl substituent. In a broad sense, phenolics, which are classified according to their structural skeleton, are basically derivatives from simple phenols and phenolic acids, phenylpropanoids including coumarins and lignans, flavonoids and related compounds, and stilbenes. Some of these compounds which show anti-inflammatory activity are reviewed in this section.
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Tiram'-resveratrol, a natural product obtained from grapes and grape products such as red wine, has recently been shown to have antiinflammatory properties. Indeed, resveratrol is a potent inhibitor of both N F - K B activation and NF-KB-dependent gene expression due to its ability to inhibit IKK activity. By this mechanism, resveratrol blocks the expression of genes that promote inflammation and protect against apoptosis [117]. Moreover, resveratrol induces apoptosis through activation of p53 activity in those cells that express wild-type p53 protein, but not in p53-deficient cells. This mechanism may thus be responsible for resveratrol's proven anti-carcinogenic activity [118].
/raws-Resveratrol
Capsaicin is a natural compound that has been described as both antigenotoxic and anti-carcinogenic. In addition, it is surmised to have a potential chemopreventive activity [119]. The compound's antiinflammatory properties have been demonstrated in different in vivo pharmacological tests, which have shown that it inhibits, among others, carrageenan-induced inflammation in rats and croton oil-induced mouse ear edema. These effects are associated with its interference of phospholipase A2 (PLA2), the enzyme that produces arachidonic acid from the membrane phospholipids. Moreover, the proapoptotic effects of capsaicin are widely documented in the literature [120]. Although the exact mechanism responsible for the proapoptotic effects of capsaicin remains unknown, several different mechanisms have been proposed, including the inhibition of plasma membrane nicotinamide adenine dinucleotide reduced (NADH) oxidase activity [121], regulation by Bcl-2 and calcineurin [122], and the overexpression of the p53 tumor suppressor gene and/or c-myc proto-oncogene [123]. Since tumor promotion is related to inflammation, the anti-inflammatory and antitumoral effects of capsaicin are most likely directly related to each other and are thus both of interest. In addition, the activation of N F - K B by
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external signals provoked the migration of this factor to the nucleus, where it binds to a specific segment of DNA. This triggers the expression of a variety of rapid-response genes involved in important physiopathological reactions, including inflammation [120]. Vanilloids such as capsaicin are recognized at the cell surface by vanilloid receptor type 1 (VR1), which mediates the effects of capsaicin in VR1-expressing cells. There are, however, pathways which are not mediated by VR1 through which vanilloids are able to induce apoptosis. [124]. Macho et al. [125], for example, demonstrated that capsaicin induces apoptosis in transformed cells and produces a rapid increase of reactive oxygen species (ROS). Interestingly, this latter effect is not a consequence of calcium signaling; thus, the apoptotic pathway may be separated from that which mobilizes calcium. Moreover, the authors evoke the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation.
CH 3 O
Capsaicin
Curcumin, the major component of the spice turmeric {Curcuma longa), exhibits anti-inflammatory and antioxidant activities, and inhibits both the generation of ROS and the JNK pathway. This compound reduces or inhibits not only the effects of PLA2 and phospholipase Cyl (PLCyl), which are involved in arachidonic acid release; but also the protein kinase C (PKC) activity induced by treatment with TPA. It has also been shown to reduce the inhibition of tyrosine protein kinase activity and to inhibit oxidative DNA damage both in the epidermis of mice as well as in cultured fibroblast cells of mice. The compound also inhibits the generation of ROS, including superoxide and hydrogen peroxide in peritoneal macrophages. Finally, it has been shown to inhibit the expression of proto-oncogenes such as c-fos, c-jun and c-myc [126,127,120]. Different researchers see a relationship between these effects and the ability to induce apoptosis. Thus, Kuo et al. [128] demonstrated that while protein synthesis inhibitors did not affect the apoptosis-inducing activity of curcumin, antioxidant agents prevented it,
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suggesting that curcumin-induced cell apoptosis is mediated by ROS, a proposal that gives Bcl-2 an important role in the early stages of curcumin-triggered apoptotic cell death. Recently, Piwocka et al. [129] demonstrated that curcumin induces caspase-3-independent apoptosis in human multidrug-resistant cells. In addition, Deeb et al. [130] demonstrated that a mixture of curcumin and TNF-related apoptosisinducing ligand (TRAIL), a member of the TNF family of cell deathinducing ligands, induced the cleavage of procaspase-3, procaspase-8, and procaspase-9, as well as the truncation of Bid and the release of cytochrome c from the mitochondria. These effects indicate that, at least in prostatic cancer cells, treatment with the aforementioned mixture triggers both the extrinsic and intrinsic pathways of apoptosis, thus defining a potential use of curcumin to sensitize cancer cells via TRAILmediated immunotherapy. In another study, however, experimental dietary supplementation of curcumin was accompanied by decreases not only in the activation of apoptosis by cyclophosphamide, but also in that of JNK. These results thus demonstrate that curcumin can inhibit chemotherapy-induced apoptosis through inhibition of ROS generation and blockade of JNK function, suggesting a possible negative effect on breast cancer patients undergoing chemotherapy [131].
Curcumin
Studies of the methanolic extract of Alpinia oxyphylla have shown that this extract suppresses the promotion of skin tumors in mice and that it induces apoptosis in cultured human promyelocytic leukemia cells. In addition, two phenolic diarylheptanoids isolated from the active extract, yakuchinone A and yakuchinone B, were found to ameliorate tumor promotion as well as inhibit both TPA-induced epidermal ornithine decarboxylase (ODC) activity and ODC RNA expression. Moreover, yakuchinones A and B reduced production of the TNF-a in the TPAstimulated skin of mice. Furthermore, both compounds inhibited the TPA-induced expression of COX-2 at both transcriptional and
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translational levels. Doubtless, the anti-inflammatory properties attributed to these compounds are related to these inhibitory effects [132].
CH 3 O
Yakuchinone A
o CH3O
Yakuchinone B
[6]-Gingerol, which is the major pungent ingredient of the ginger rhizome (Zingiber ojjicinalis), has also been shown to exhibit strong antiinflammatory activity while [6]-paradol, a closely related compound from the same species, possesses chemopreventive potential. Both compounds showed similar effects when studied as potential inducers of apoptotic cell death [133]. H
OH
CH3O
CH3O
CH3
[6]-Paradol
Various phenolics have been found to induce apoptosis in human cancer cells, with some of them serving as potential anti-inflammatory
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agents as well. It is thought that the mechanism of action in both cases is related. Of the compounds studied, honokiol, from Magnolia officinalis, induced apoptotic cell death in different types of human cancer cells using a three-pronged attack: modulation of Bcl-XL and Bad proteins, induction of the release of mitochondrial cytochrome c, and activation of caspase-3 [134]. Another compound, humulone, isolated from hops extract (Humulus lupulus), induced apoptosis in the promyelocytic leukemia cell line HL-60 by means of a mechanism which implicated the compound's antioxidative activity [135].
OH
OH O Honokiol
O
Humulone
Flavonoids are natural products with a wide range of pharmacological effects, including anti-inflammatory and antioxidative properties [89]. Some of them, e.g. naringin, naringenin, quercetin, and myricetin, inhibit the enhanced expression of iNOS through down-regulation of N F - K B binding activity [89,136]. Because N F - K B is involved in both inflammatory diseases and apoptosis, the modulation of N F - K B activity may be a suitable target for altering the inflammatory process. The LOX inhibitors nordihydroguaiaretic acid (NDGA) and baicalein were found to induce apoptosis and inhibit proliferation of different breast cancer cells in vitro. In contrast, the LOX products 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of the same cell lines. Blocking both 5-LOX and 12-LOX pathways led to several different effects, including apoptosis in breast cancer cells. This effect occurred through cytochrome c release and caspase-9 activation, as well poly-(ADP-ribose) polymerase (PARP) cleavage. Blocking these pathways also reduced the levels of the anti-apoptotic proteins Bcl-2 and Mcl-1 while increasing the levels of the pro-apoptotic protein Bax [137]. The 12-LOX inhibitors might be of interest for the treatment of
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Alzheimer's disease, first because of the chronic inflammation which occurs in this illness, and second because the neurodegeneration associated with Alzheimer's is related to the accumulation of amyloid p peptide. Thus, studies with the 12-LOX inhibitor baicalein found that this compound attenuated both neuronal apoptosis as well as the c-jun protein over-expression induced by amyloid P peptide (25-35) on rat cortical cells. In contrast, neither the broad spectrum LOX inhibitor NDGA nor the 5-LOX inhibitor caffeic acid exhibited cell protecting effects [138]. As for baicalein's mechanism of action, Chang et al. [139] demonstrated that the apoptotic effect of this compound on human Hep G2 cells was induced by mitochondrial dysfunction and Bcl-2 regulation. Quercetin and its 3-rhamnoglucoside rutin were studied as possible modifiers of tumor formation, but neither showed any effect in this respect [140]. In contrast, the aglycone quercetin was found to induce apoptosis in HeLa cells by means of a mechanism which reduces the level of expression of Hsp27 and Hsp72 [141]. Moreover, the same authors [142] demonstrated that quercetin can induce apoptosis and necrosis in vitro in the kidney cells of monkeys, although the percentage of affected cells was very low. On the other hand, quercetin, apigenin, myricetin, and kaempferol are all able to induce apoptosis in human leukemia cells. When administered separately, each of these compounds caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of PARP. They also induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing [143]. The authors correlate the apoptogenic potency of these compounds with the presence of hydroxyls in ring B, along with the absence of a 3-hydroxyl in ring C. OH
H< OH
O Baicalein
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Tangeretin is a citrus flavone that inhibits the release of both ROS by human neutrophils and histamine by human basophils. In addition, it inhibits the proliferation of malignant tumor cells in vitro. Hirano et al. [144] demonstrated that the compound's anticancer activity is mediated in part through induction of apoptosis, but that this occurs without affecting the immune cells. OCH 3 H3C0.
Studies with tectorigenin and its 7-glucoside, tectoridin, both isolated from the Korean plant Belamcanda chinensis, long used as an antiinflammatory, found that these compounds suppressed PGE2 production by rat peritoneal macrophages that had been stimulated by either TPA or thapsigargin. This effect arose from the inhibition of COX-2 induction in the inflammatory cells; interestingly, the compounds do not directly affect the activity of either COX-1 or COX-2 [145]. Among other effects, tectorigenin has been shown to induce the transformation of human promyelocytic leukemia cells into granulocytes and monocytes/ macrophages, thus causing apoptotic changes of DNA in cells. Tectorigenin also inhibits autophosphorylation of epidermal growth factor (EGF) receptors by EGF itself, and decreases the expression of Bcl-2 protein [146]. The study of this compound with other related isoflavones demonstrated that the cytotoxic properties of tectorigenin depend on the isoflavone configuration and the presence of the 5hydroxyl group. Genistein, or 6-demethoxy tectorigenin, was shown to exert inhibitory effects on IL-3, IL-5, IL-6, and GM-CSF bioactivities, a fact which sheds light on its possible mechanism of action as an anti-inflammatory and immunosuppressive agent [147]. Moreover, genistein induces apoptosis in prostate cancer cells [148] and in non-small-cell lung cancer cell lines
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[149]. Studies of this compound demonstrated that genistein causes a typical DNA laddering, which is a hallmark of apoptosis. HO.
Finally, Kuntz et al. [150] have established that the capability of flavonoids for inhibiting growth and inducing apoptosis cannot be predicted on the basis of their chemical structures or substituents. A great number of pharmacological effects for licorice {Glycyrrhiza glabrd) have been documented, including its anti-inflammatory properties. Glycyrrhizic acid has been found to inhibit both LOX and COX; it also inhibits PKC and downregulates the EGF receptor. Although research on other phenolics has shed light on their various applications, it is the polyphenols that are thought to be mainly responsible for inducing apoptosis in cancer cells [151]. For instance, the major polyphenol isolated from green tea {Camellia sinensis), epigallocatechin gallate, has been found to exhibit multiple biological effects, including antioxidative, anti-inflammatory, and antiproliferative properties. The pharmacological effects of this compound are myriad and include reduction of the inhibitory effect of peroxinitrite on COX, attenuation of NO generation, blocking of iNOS expression and activity, inhibition of N F - K B binding activity [152], and inhibition of DNA synthesis [153]. This compound also protects against cancer by causing cell cycle arrest. Finally, it has been found to induce apoptosis in different cell lines by forming internucleosomal DNA fragments [154,155,156]. With respect to the activity of tannins, Momose et al. [157] have demonstrated that gallic acid exhibits higher activity than the tannins derived from it. Gallic acid was found to induce apoptosis in different cell lines; this induced cell death was mediated by ROS such as hydrogen peroxide and superoxide anion, as well as by Ca2+. However, the induction of apoptosis depends on the acid's distinctive structural features rather than on its antioxidative activity [158]. In fact, the
175
apoptotic effect of gallic acid seems to require the presence of Ca2+ since the depletion of this ion from the culture medium reduced the acid's apoptosis-inducing activity. In contrast, lack of Ca2+ did not affect the activity of either tannic or caffeic acid [157].
OH
Epigallocatechin gallate
Caffeic acid phenethyl ester, isolated from the propolis found in bee hives, has also been shown to induce apoptosis in different cell lines, probably by modulating the redox state of cells. This ester was studied under different experimental conditions and in the presence of various agents, including Bcl-2, which protects cells from oxidative stress. Indeed, this agent had a protective effect against the apoptosis induced by caffeic acid phenethyl ester [159,160]. OH
In a study on different anthraquinones from Rheum palmatum, Chen et al. [161] found that emodin, but not physion or chrysophanol, induced apoptosis in HL-60 cells though activation of the caspase 3 cascade. The
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authors further demonstrated that this effect was independent of ROS production, and they hypothesized that the presence of a hydroxy substituent on C-6 was essential for the apoptotic activity of the compound. Terpenoids
A great number of triterpenes have been studied as anti-inflammatory agents, both in vivo and in vitro. The mechanism of action has been found to depend on the type of terpene as well as its structural group [162,163,164]. In Hata et a/.'s [165] study of the possible pro-apoptotic effect of a number of triterpenes on a melanoma cell line in mice, many of the compounds tested showed anti-inflammatory activity. These tests, together with similar studies on the apoptotic effects of the same compounds, indicate that the ability to induce apoptosis in proinflammatory cells is strongly correlated to anti-inflammatory activity. Of the 21 triterpenes assayed, betulinic acid and its methyl ester, along with lup-28-al-20(29)ene-2p-ol and lup-28-al-20(29)-en-3-one all inhibited cell proliferation by inducing apoptosis. The authors suggest that the carbonyl group at C-27 may be essential for the apoptotic effects of these compounds. While previous papers [163,164] had already described the relevance of this carbonyl group for the anti-inflammatory activity of triterpenes, more research on the apoptotic effects on proinflammatory cells is necessary to better understand these compounds.
Ri
Betulinic acid Betulinic acid methyl ester Lup-28-al-20(29)ene-2p-ol Lup-28-al-20(29)-en-3-one
a-H, P-OH a-H, P-OH a-H, p-OH =O
R2 COOH COOCH3 CHO CHO
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Ginsenoside Rg3 from Panax ginseng not only induced classic apoptotic morphology, but it also interfered with the expression of Bcl-2, caspase-3, and apoptosis-related genes in human prostate carcinoma LNCaP cells. By the same token, ginsenoside Rg3 activated the expression of the cyclin-kinase inhibitors p21 and p27, arrested LNCaP cells at Gl phase, and subsequently inhibited cell growth through a caspase-3-mediated apoptosis mechanism [166]. Moreover, ginsenosides Rbl, Rb2, and Re were shown to be metabolized by intestinal bacteria to yield ginsenoside Ml, which in turn inhibited cell proliferation and induced cell death by regulating apoptosis-related proteins [167].
Ginsenoside Rg3
Parthenolide, isolated from Tanacetum parthenium and other species, is a sesquiterpene lactone widely investigated for its anti-inflammatory activity [168,169,170]. Recent in vitro studies have shown that this compound inhibits the N F - K B pathway. A study on the effect of parthenolide in endotoxic shock in rodents showed that treatment with this compound stopped nitrotyrosine formation, PARP synthetase expression, and apoptosis. It also reduced iNOS mRNA content in the tissues studied. All these effects are brought about by the compound's inhibition of N F - K B [171]. In addition, parthenolide mimicked the effects of IKBOC in that it inhibited both N F - K B DNA binding activity as well as Mn-SOD expression, while simultaneously increasing paclitaxel-induced apoptosis of breast cancer cells [90].
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Triptolide from Tripterygium wilfordii was also shown to exhibit antitumoral and anti-inflammatory effects by inhibiting cell proliferation, inducing apoptosis, and inhibiting both N F - K B and AP-1 transeriptional activity. However, since this compound neither inhibited growth nor induced apoptosis in cells with mutant p53, it clearly requires a functional p53 to exhibit any proapoptotic, anti-inflammatory, or antitumoral effect. [172].
Parthenolide
Jolkinolide B from Euphorbia fischeriana was found to induce the apoptosis of human prostate cancer cells, but its exact mechanism has yet to be determined [173].
Jolkinolide B
Zerumbone, from Zingiber zerumbet, was shown to effectively suppress free radical generation, pro-inflammatory protein production, and cancer cell proliferation while additionally inducing the apoptosis of these same cells. An analysis of the relationship between the compound's structure and its activity indicated that the a,p-unsaturated carbonyl group in the sesquiterpene structure is a requisite for the compound's
179
effects since ot-humulene, an analogous compound without the carbonyl, is ineffective [174].
Zerumbone
a-Humulene
Other terpenoids with potential proapoptotic effects are farnesol and geranylgeraniol [175], y-tocotrienol and P-lonone [176], perillyl alcohol [177], limonene [178], and paclitaxel [179], all of which induce apoptotic cell death via a signaling pathway that is independent of G2/M arrest and microtubules [180]. Lectins
In general, lectins are divalent or multivalent carbohydrate-binding proteins with the ability to agglutinate cells. They have been used in biochemistry, immunology, and molecular physiology, but the interest in this phytochemical group has increased greatly since recent studies have established their utility as potential pharmacological agents. One of the potential uses of lectins is as a modifier of cell evolution. Thepen et al. [181], for example, have reported the resolution of cutaneous inflammation after local elimination of macrophages by an apoptotic mechanism. For this study, an immunotoxin composed of an antibody directed against the high-affinity IgG receptor CD64 and the lectin ricinA was synthesized. This immunotoxin was found to induce apoptosis in cultivated macrophages while leaving the low CD64- expressing macrophages unaffected. This activity was corroborated in vivo on transgenic mice expressing human CD64; the cutaneous inflammation was resolved in 24 h, and both the skin temperature and vasodilatation decreased. Apart from their postulated immunostimulatory properties, lectincontaining extracts from mistletoe (Viscum album) have also been found to induce apoptosis [182]. The activity of the extracts depends on the
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manufacturing process, host tree, and time of harvest [183]. Although the lectin content of these extracts is strongly correlated with their apoptosis-inducing properties on cultured lymphocytes, its presence does not totally account for their biological activity; thus, other compounds are probably involved in the modulation of the mistletoe lectin activity [184,185]. In the case of the lectin isolated from Korean mistletoe (V. album coloratum), for example, the activity against tumor cells occurs by means of an apoptotic process that is mediated by Ca2+/Mg2+-dependent endonucleases [186]. Three additional lectins, obtained from Canavalia brasiliensis (ConBr), Dioclea violacea (DVioL), and Dioclea grandiflora (DGL), have all been found to stimulate T-cell activation and apoptosis in vivo, but they also produce important side effects, including inflammation associated with high endothelial venule necrosis [187]. Other natural products
Oh et al. [188] isolated the principle (3i?,6i?)-4-methyl-6-(lmethylethyl)-3-phenylmethylperhydro-l,4-oxazine-2,5-dione from the fruiting bodies of Isaria japonica to study its effects as an apoptosisinducing agent on human leukemia HL-60 cells.
\
^s
T
(3^,6«)-4-Methyl-6-(l-methylethyl)-3-phenylmethylperhydro-l,4-oxazine-2,5-dione
Inhibitors of apoptosis and their implications in inflammatory diseases Alkaloids
The alkaloid boldine has been found to decrease dopamine-induced cell death, including apoptosis, in PC 12 cells through a scavenging action on ROS and inhibition of both melanin formation and thiol oxidation [189].
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CH3O
CH3O OH Boldine
Phenolics
As mentioned above, experimental dietary supplementation of curcumin was accompanied by a decrease in the activation of apoptosis by cyclophosphamide, as well as a decrease in JNK activation. These results demonstrate that curcumin can inhibit chemotherapy-induced apoptosis by inhibiting ROS generation and blocking JNK function, effects which would be counterproductive for breast cancer patients undergoing chemotherapy [131]. Agastinol and agastenol, two lignans from Agastache rugosa, have been found to inhibit etoposide-induced caspase-3 induction in U937 cells. Both compounds have a similar potency range and thus seem to comprise a new type of anti-apoptotic agent [190]. Other natural products
Cat's claw (Uncaria tomentosa and Uncaria guianensis), an herbal medicine from the Amazon, is widely used to treat inflammatory disorders. Both species of cat's claw provide effective antioxidant and anti-inflammatory activities, but Uncaria guianensis is the more potent of the two. Non-alkaloid fractions from both species have been found to decrease lipopolysaccharide (LPS)-induced TNF-a and nitrite production in RAW 264.7 cells, and oral pretreatment for 3 days with Uncaria tomentosa actually prevented TNFa mRNA expression and apoptosis. Interestingly, the pharmacological properties of these species do not seem to be dependent on the presence of oxindole or pentacyclic alkaloids [191].
182
OH
OH OCH 3
CHjO
OGH3
CH3O Agastinol
Agastenol
CONCLUSIONS The study of natural products as potential anti-inflammatory agents is an exciting topic for future research. Many known compounds have been described as anti-inflammatory agents because of their ability to affect the arachidonic acid metabolism and/or the induction of new proinfiammatory agents and proteins. However, a new approach to the investigation of natural products and their part in inflammations has arisen from the recent revisiting of the role of glucocorticoids and theophylline in inflammatory processes. Thus, while glucocorticoids have been found to promote safe clearance of apoptotic cells through phagocytes, theophylline has been shown to induce apoptosis in eosinophils and lymphocytes. Both drugs produce beneficial effects in allergic inflammation, especially in the treatment of asthma if used in combination with corticoids. Moreover the application of apoptotic agents could be highly effective in the resolution of skin diseases such as eczematous dermatitis. Recent research seems to single out phenolics as being those natural products with the most potential for use as anti-inflammatory and proapoptotic agents. Many of these compounds are proven antioxidants that have been found to inhibit the enzymes implicated in the inflammatory process as well. Some of the mechanisms in question may be related to their pro-apoptotic effects and as such may provide ways to potentiate the compound's actual pharmacological effects. Alkaloids and terpenoids may also be of interest, but the research on them to date has focused more on their use as anticancer agents.
183 183
ABBREVIATIONS = adenosine diphosphate = acquired immunodeficiency syndrome = apoptosis inducing factor AIF AP-1 = activator protein-1 Apaf-1 = apoptosis protease activating factor-1 Bcl-2 = B-cell leukemia oncogen-2 cAMP = cyclic adenosine monophosphate = cluster of differentiation CD CD64 = FcyRI (immunoglobulin G receptor I) CD95 = Fas c-FLIP = cellular FLICE-inhibitory protein c-fos = cellular family of genes c-jun = cellular family of genes c-myc = cellular family of genes COX, COX-1, COX-2 = cyclooxygenase, eyclooxygenase-1, -2 DNA = deoxyribonucleic acid = delayed-type hypersensitivity DTH = epidermal growth factor EGF FADD = Fas-associated death domain FasL = Fas ligand FcyRI (CD64) = immunoglobulin G receptor I FLICE = FADD-like interleukin-ip converting enzyme GM-CSF = granucolyte-macrophage colony stimulating factor 5-HETE(12-,15-) = 5-hydroxy-6,8,l 1,14-eicosatetraenoic acid (12-, 15-) = human immunodeficiency virus HIV Hsp, Hsp 27,Hsp 72 = heat shock protein = inhibitor of apoptosis protein LAP = interleukin-ip converting enzyme (caspase-1) ICE = ICE and Ced-3 homologue ICH = C-FLIP, FLAME, Casper I-FLICE = immunoglobulin G IgG IKB = family of inhibitory proteins of NF-KB = inhibitor of the NF-KB kinases, IKB kinase IKK IL,-lp, -2,-3,-5,-6,-8,-10 = interleukin, interleukin-2,-3,-5,-6,-8,-10 INF-y = interferon-y = c-jun NHrterminal kinase JNK = lipoxygenase, lipoxygenase-5, -12, -15 LOX, -5,-12,-15 = lipopolysaccharide LPS = leukotriene, leukotriene B 4 LT, LTB4 LX,LXA4 = lipoxin, lipoxin A4 MAPK = mitogen-activated protein kinase = nordihydroguaiaretic acid NADG NADH = nicotinamide adenine dinucleotide reduced
ADP
AIDS
184 NF-KB
NO NOS, iNOS NSAID ODC PARP PG, PGE2 PKC PLA2 PLC PMN mRNA ROS Smac/DIABLO SOD TGF, TGF-(3 TNF, TNFa TNFR1 TPA TRAIL UV VR,
:
nuclear factor-KB = nitric oxide ; nitric oxide synthase, inducible nitric oxide synthase = non-steroidal anti-inflammatory drug = ornithine decarboxylase = poly-(ADP-ribose) polymerase = prostaglandin, prostaglandin E2 = protein kinase C = phospholipase A2 = phospholipase C = polymorphonuclear = messenger ribonucleic acid = reactive oxygen species = second mitochondria-derived activator of caspases/direct IAP binding protein = superoxide dismutase = tumoral growth factor, tumor growth factor-(3 = tumor necrosis factor, tumor necrosis factor-a = TNF receptor-1 = 12-0-teradecanoylphorbol 13-acetate = TNF-related apoptosis-inducing ligand = ultraviolet light = vanilloid receptor type 1
Cell lines cited in text A431 B16 2F2 Caco-2 C3H 10T1/2 CH27 CREF DU145 GMK H460 HaCaT HeLa Hep3B HepG2 HL-20 HL-60 L5178Y LLC-PK1 LNCaP MCF-7 Molt4B
:
human epidermoid carcinoma cells mouse melanoma derived subclone cells : human colon cancer cells = mouse embryonic fibroblasts = human squamous lung cancer cells = non-tumorigenic rat embryo fibroblasts = human prostate carcinoma cells = monkey kidney cells = non-small lung cancer cells = human carcinoma keratinocytes = human negroid cervix epitheloid carcinoma cells = human hepatocytes = human hepatome cells = human Caucasian promyelocytic leukaemia cells = human promyelocytic leukaemia cells = mouse lymphoma cells = renal tubular cells = androgen-sensitive human prostate cancer cells = human breast adenocarcinoma cells = human lymphoid leukaemia cells :
185 NHEKs NSCLC NS-SV-AC PCa PC 12 PLA-801 PBMCs RAW 264.7 SK-N-SH THP-1
= normal human epidermal keratinocytes = human non-small cell lung cancer cells = normal human salivary gland cells = prostate adenocarcinoma cells = undifferentiated cells = cultured NSCLC cells = human peripheral blood mononuclear cells = mouse monocyte macrophages = human neuroblastoma cells = human monocytic cells
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[127] Christensen, S.B.; Norup, E.; Rasmussen, U.; Madsen, J.0.; Phytochemistry 1984,23,1659-1663. [128] Thastrup, O.; Dawson, A.P.; Scharff, O.; Foder, B.; Cullen, J.P.; Drebak, B.K. Bjerrum, P.J. Christensen, S.B.; Hanley, M.R.; Agents and Actions, 1989, 27, 17-23. [129] Treiman, M.; Caspersen, C; Christensen, S.B.; TiPS, 1998, 19, 131-135. [130] Rasmussen, U. Christensen, S.B.; Sandberg, F.; Ada Pharm. Suec, 1978, 15, 133-140. [131] Ali, H.; Christensen, S.B.; Foreman, J.C.; Pearce, F.L.; Piotrowski, W.; Thastrup, O.; Br. J. Pharmac, 1985, 85, 705-712. [132] Malcolm, K.C.; Fitzpatrick, F.A.; J. Pharmacol. Exp. Ther., 1992, 260, UAA1249. [133] Tsukamoto, A.; Kaneko, Y.; Cell Biol. Internal, 1993, 17, 969-970. [134] Wei, H. Wei, W.; Bredesen, D.E. Perry, D.C.; J. Neurochem., 1998, 70, 23052314. [135] Hakii, EL; Fujiki, H.; Suganuma, M.; Nakayasu, M.; Tahira, T.; Sugimura, T.; Scheuer, P.J.; Christensen, S.B.; J. Cancer Res., 1986, Clin. Oncol. Ill, \11181. [136] Pahl, H.L.; Baeuerle, P.A.; Trends Cell Biol, 1997, 7, 51-55. [137] Pahl, H.L.; Sester, M.; Burgert, H.-G.; Baeuerle, P.A.; J. Cell Biol, 1996, 132, 511-522. [138] Sagara, Y.; Inesi, G.; J. Biol. Chem., 1991, 266, 13503-13506. [139] Inesi, G.; Sagara, Y. Arch. Biochem. Biophys., 1992, 298, 313-317. [140] Inesi, G.; Sagara, Y.; J. Membrane Biol. 1994, 141, 1-6. [141] Sagara, Y.; Wade, J.B.; Inesi, G.; J. Biol. Chem., 1992, 267, 1286-1292 [142] Sagara, Y.; Fernandez-Belda, F.; de Meis, L.; Inesi, G.; J. Biol. Chem., 1992 , 267, 12606-12613. [143] Witcome, M.; Henderson, I.; Lee, A.G.; East, J.M.; Biochem. J., 1992, 283, 525-529. [144] Toyoshima, C ; Nakasako, M.; Nomura, H.; Ogawa, H.; Nature, 2000, 405, 647-655. [145] Christensen, S.B.; Andersen, A.; Poulsen, J.-C.J., Treiman, M.; FEBS Lett., 1993, 335, 345-348. [146] N0rregaard, A.; Vilsen, B.; Andersen, J.P.; J. Biol Chem., 1994, 269, 2658926601. [147] Toyoshima, C ; Nomura, H.; Nature, 2002, 418, 605-611. [148] Holub, M.; Budesinsky, M.; Phytochemistry, 1986, 25, 2015-2026. [149] Christensen, S.B.; Hergenhahn, M.; Roeser, H.; Hecker, E.; Cancer Res. Clin.
391 Oncol 1992,7/5,344-348. [150] Nomp,E.;Smitt,U.W.;Christensen,S.B.;P/aKtoMeJ.,198S, 251-255., [151] Nielsen, S.F.; Thastrup, O.; Pedersen, R.; Olsen, C.E.; Christensen, S.B.; J. Med. Chem., 1995, 38,272-276. [152] Christensen, S.B.; Andersen, A.; Kromann, H.; Treiman, M.; Tombal, B.; Denmeade, S.; Isaacs, J.T.; Bioorg. Med. Chem,, 1999, 7, 1273-1280. [153] Jakobsen, CM.; Denmeade, S.R.; Isaacs, J.T.; Gady, A.; Olsen, C.E.; Christensen, S.B.; J. Med. Chem., 2001, 44, 4696-4703. [154] Egan, M.E.; Glockner-Pagel, J.; Ambrose, C.A.; Cahill, P.A.; Pappoe, L; Balamuth, N; Cho, E.; Canny, S.; Wagner, C.A.;Geibel, J.; Caplan, M.J; Nature Medicine, 2002, 8, 485-492. [155] Delisle, B.P.; Anderson, C.L.; Balijepalli, R.C.; Anson, B.D.; Kamp,TJ.; January, C.T.; J. Bio!. Chem., 2003,278, 35749-35754. [156] Denmeade, S.R.; Jakobsen, CM.; Janssen, S.; Khan, S.R.; Garrett, E.S.; Lilja, H.; Christensen, S.B.; Isaacs, J.T. Journal of the National Cancer Institute 2003, 95, 990-1000. [157] Schmidt, T.J.; Miiller, E.; Fronczek, F.R.; J. Nat. Prod., 2001, 64,411-414. [158] Squires, R.F.; Casida, J.E.; Richardson, M.; Saederup, E. Mol. Pharmacol., 1983,23, 326-336. [159] Kudo, Y.; Oka, J.-I.; Yamada, K. Neurosci. Lett., 1981,25, 83-88. [160] Mateumoto, K.; Fukuda, H. Neurosci. Lett, 1982, 32, 175-179. [161] Matsumoto, K.; Fukuda, H. Brain Res., 1983,270,103-108. [162] Kuriyama, T.; Schmidt, T.J.; Okuyama, E.; Ozoe, Y.; Bioorg. Med. Chem., 2002, 10, 1873-1881. [163] Schmidt, T.J.; Gurrath, M.; Ozoe, Y.; Bioorg. Med. Chem., 2004, 12, 41594167. [164] Hosie, A.M.; Ozoe, Y.; Koike, K.; Ohmoto, T.; Nikaido, T.; Sattelle, D.B. Br. J. Pharmac, 1996,119, 1569-1576. [165] Ozoe, Y.; Akamatsu, M.; Higata, T.; Ikeda, I.; Mochida, K.; Koike, K.; Ohmoto, T.; Nikaido, T. Bioorg. Med. Chem., 1998, 6,481-492. [166] Klunk, W.E.; Kalman, B.L.; Ferrendelli, J.A.; Covey, D.F. Mol. Pharmacol., 1983,23, 511-518. [167] Inoue, M.; Akaike, N. Neurosci. Res., 1988, 5,380-394. [168] Newland, C.F.; Cull-Candy, S. G. J. Physiol, 1992, 447,191-213. [169] Yoon, K.-W.; Covey, D.F.; Rothman, S.M. J. Physiol., 1993, 464,423-439. [170] Zhang, H.-G.; ffiench-Constant, R.H.; Jackson, M.B. J. Physiol., 1994, 479, 65-75. [171] Jarboe, C.H.; Porter, L.A.; Buckler, R.T J. Med. Chem., 1968, / / , 729-731. [172] Ozoe, Y.; Akamatsu, M.; Pest Manag. Sci, 2001, 57,923-931. [173] Akamatsu, M.; Ozoe, Y.; Ueno, T.; Fujita, T.; Mochida, K.; Nakamura, T.; Matsumura, F.; Pestic. Sci. 1997, 49,319-332. [174] Wong, M.G.; Andrews, P.R. Eur. J. Med. Chem., 1989,24,323-334. [175] Calder, J.A.; Wyatt, J.A.; Frenkel, D.A.; Casida, J.E. J. CAMD, 1993, 7, 45-
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33 Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 33 © 2006 2006 Elsevier B.V. All Allrights reserved. ©
393
SYNTHETIC INVESTIGATIONS IN THE FIELD OF DRIMANE SESQUITERPENOIDS PAVEL F. VLAD Laboratory of Terpenoid Chemistry, Institute of Chemistry, the Academy of Sciences of Moldova, Academiei Str. 3, Chisinau, MD-2028, the Republic of Moldova ABSTRACT: In this article, the results of the author's investigations in the field of the synthesis of drimane sesquiterpenoids, a group of terpenic compounds which possess a wide variety of biological activities (antifeedant, antibacterial, antifungal, anticomplemental, cytotoxic, antiallergic, piscicidal, molluscicidal, plant growth regulatory, insecticidal and others) have been reviewed. The original structural- and stereoselective methods for obtaining natural compounds in the optically-active form, and also of important intermediates on the pathway to them, including alcohols, poliols, ketones, lactones and acids, have been elaborated. Most of the compounds obtained are polyfunctional. Much attention has been focussed on the synthesis of drimenol, a drimanic sesquiterpene key compound, which is widely used as a starting substance in syntheses of many biologically active drimanes. Much effort has been devoted to elaborate the syntheses of drim-8-en-7-one and drim-5,8-diene-7-one, compounds possessing a great synthetic potential. Indeed, using those above a large number of natural compounds, such as isodrimenin, 7-oxoisodrimenin, 7-oxo-5,6dehydroisodrimenin, /raras-tetrahydroactinidiolide and intermediates for the syntheses of polygodial and warburganal, compounds with various and high biological activities, were prepared.
INTRODUCTION Drimanes are sesquiterpenes with a carbon skeleton of drimane whose structure and stereochemistry are depicted in formula (1) [1]. The name "drimane" comes from the name of a South American tree Drimys winteri Forst., from the bark of which the alcohol (-)-drimenol (2), the first representative of this group of sesquiterpenoids, has been isolated [2]. The chemistry of drimanes had been particularly intensively developed within the last 15-20 years, when many members of this group of terpenoids were isolated and investigated. It was shown that they are sufficiently widespread in the nature. Drimanes have been isolated from higher plants, fungi, microorganisms, and marine organisms [3-4]. Drimanes have been of interest first of all owing to their high and various biological activities, considered in detail in excellent reviews [3-4]. Among them are
394
substances with diverse activities: antifeedant, antibacterial, antiviral, antifungal, cytotoxic, phytotoxic, anticomplemental, plant growth regulatory, piscicidal, molluscicidal, and others [3,4]. The practical value of many drimanes and their often small content in natural sources stimulated their synthetic preparation [5, 6]. Both total and partial syntheses of drimanes have been described in literature. The former have two drawbacks: they are, as a rule, multistep procedures and consequently the yields of final products are low. In addition, these compounds are obtained in racemic form [6]. As it is known, usually only one of the enantiomers of such compounds possesses biological activity and hence the yield of the desired stereoisomer a priori can not exceed 50%, but actually it is lower. Moreover, in most cases the racemates resolution is not a simple, empiric process. The latter approach to drimane syntheses is based on their structural relationships with higher terpenoids: di-, sesterand triterpenoids. With their cleavage, drimanic compounds can be obtained. Such partial syntheses of drimanes are attractive since the target compounds are obtained in natural, optically-active form and besides most of these syntheses involve a few steps [6, 7].
OH
(5)
(6)
(7)
Most of the known partial syntheses of drimanes were carried out starting with bi- and tricyclic diterpenoids. However, bicyclic labdane diterpenoids mostly are structurally similar to drimanes. Their only
395
difference from drimanes is the presence of an extra isoprene unit in their side chain. Elimination of that unit yields the desired drimanes. Labdane diterpenoids have one more important advantage since most of them, for example, sclareol (3), manool (4), larixol (5), labdanolic acid (6), the mixture of AX3-cis- and trans-neoabienols (7) and others, are easily available and can be obtained in large amounts [8]. In this review, the results of our investigations in the field of the synthesis of a number of optically-active, drimanic compounds from norlabdanic derivatives, the cleavage products of many available labdanoids, and also from a mixture of neoabienols (7) are summarized. The respective literature data are also discussed in the present article. A number of the prepared drimanes are naturally occurring biologically active substances. Others have been used as intermediates in the syntheses of important natural bioactive polyfunctional drimane sesquiterpenoids. Below the syntheses of the representatives of the drimane (1) group are described. 1 Synthesis and Use of (-)-Drimenol (2) Besides the Drimys winteri, drimenol (2) was also found in many other natural sources [3,4]. For example, it has been isolated from some species of the plants of the Warburgia [9] and Porella [10] families, from Polygonum hydropiper [11], some liverworts [12], Ferula ceratophylla [13] and fungi [14]. According to [3], drimenol (2) has the plant growth regulatory activity comparable with that of heteroauxin (indole-3-acetic acid). However, more important is the fact that drimenol (2) has been used as a starting compound for the synthesis of a series of natural biologically active drimanes and nordrimanes. So, two syntheses of the drimane dialdehyde polygodial (8) were carried out starting with the drimenol (2) (Scheme 1). In one of them [15], drimenol (2) was oxidised into drimenal (9), which then transformed into acetal (10). The latter was oxidised into aldehyde (11) with selenium reagents. Treatment of aldehyde (11) with ptoluenesulphonic acid gave the target polygodial (8) (a 30% overall yield), isolated earlier from some plants and marine organisms [3, 4]. In the second synthesis [16], drimenol (2) was acetylated and its acetate (12) was oxidised with selenium reagents into a mixture of
396
CHO (2)
CHO
a) PCC, CH2C12, 75.4%; b) CH(CH 2 OH) 2 , H 1 , 88.5%; c) SeO2 (cat), (p-MeOC 6 H 4 ) 2 SeO, 45%; d) TsOH, acetone, 100%; e) Ac 2 O, Py, 98%; f) K 2 CO 3 , MeOH, 100%; g) (COC1)2, DMSO, 98%.
Scheme 1 hydroxy acetates (13) and (14). Saponification of the latter gave diol (15), which was oxidised by the Swern reagent into polygodial (8) in a high yield. The particular interest of researchers to polygodial can be explained by its various biological activities: antifeedant [17, 18], antibacterial [10, 19], cytotoxic [10, 20], allergenic [10], piscicidal, molluscicidal, anticomplemental, and plant growth regulatory [3, 4]. Polygodial is a powerful analgetic agent (15 times more powerful than aspirin) [10,21]. Some substances (anetol, saphrol, methylevgenol) have the synergistic effect on polygodial (8), intensifying its antifungal activity 32-128 times [22]. It is necessary to note that polygodial has bitter taste [17]. Drimenol (2) has also been used as a starting compound for the synthesis of another drimanic dialdehyde, warburganal (16), which is
397
similar to polygodial (8) in the variety and level of biological activities [23] (Scheme 2). _OAc
CHO
(17)
(16)
a) SeO 2 , dioxane, A, 45%; b) KOH, MeOH, 100%; c) DMSO, (CF 3 CO) 2 O, 64%.
Scheme 2 Drimenyl acetate (12) was oxidised with selenium dioxide into the acetoxy diol (13). This compound was saponified into triol (17) which on oxidation with DMSO and (CF3CO)2O gave the final product (16). Afterwards, the yield at the step of the triol (17) oxidation was improved using the Swern reagent [24]. Warburganal possesses antifungal, antibacterial, antimicrobial, cytotoxic, antiallergic, antifeedant, insecticidal, plant growth regulatory, helicoccidic activities, and a bitter taste [3, 4]. Scheme 3 shows the synthesis of (-)-cinnamodial (ugandensidial) (18), the warburganal-related dialdehyde, from the drimenol (2) [25]. Cinnamodial also has a bitter taste [3], and possesses antimicrobial, antifeedant, piscicidal and antihelmintic activities [3, 4]. Cinnamodial (18) has been simultaneously isolated from Cinnamosma fragrans [26] and from Warburgia ugandensis [27], and then from other plants [4]. Cinnamodial (18) was obtained from drimenol (2) in 12 steps in an overall 10% yield. The diol (22) was synthesised from drimenol (2) according to the method [28], and further synthesis was continued as described in [25] (Scheme 3). Drimenol (2) has also been used as a starting material for the preparation of uvidin C (26), a metabolite of fungus Lactarius uvidus Fries [29]. The eight-step synthesis was carried out according to Scheme 3 and includes oxidation of the diol (22) with m-chloroperbenzoic acid. The overall yield of (26) was 20% [28].
398 OH
OH
'lOH (2)
\z
(19)R=O (20) R=NNHTs
OH
CHO
OH (26)
OAc (18)
a)Ac2O/Py;OsO4,NMO;NBS,CH2Cl2;KOH,MeOH; b) TsNHNH2, BF3 Et2O, QH^; c)BuLi,THF; Py; PCC; D1BAL, THF; e) Ai^O, Py, DMAP; f) SeO2 (cat), (p-MeOCyi^SeO, dioxane; g) K2CO3, MeOH; h) (COCl^, DMSO; i) m-CPBA, CH2C12.
Scheme 3 Swedish chemists [30], starting with drimenol (2), accomplished the synthesis of drim-8-en-7-one (27), thus confirming its structure and determining its stereochemistry (Scheme 4). Drimenyl acetate (12) was epoxidised with m-CPBA and the product was saponified into (28). Its oxidation with the Brown reagent, the base isomerisation of the resulting epoxy aldehyde, and the sodium borohydride reduction of the isomerisation product afforded the diol (29). Its monoacetate (30) was oxidised with the Jones reagent into the keto acetate (31) whose hydrogenolysis gave the target drim-8-en-7-one (27). It should be mentioned that this synthesis has no preparative importance since it involves many steps and at some stages (for example "c") mixtures of products were formed. Furthermore, some intermediates have not been characterised.
399 ,OH
(12) OH (28)
I
(29)R=H (30) R=Ac
,OAc
(31) (27) a) m-CPBA, CHjCl2; KOH, MeOH; b) CrO3, EfcO; KOH, MeOH; NaBH,, MeOH; c) Ac2O, Py; d) Jones reagent; e) Zn, AcOH.
Scheme 4
CHO
CHO
'V,r
OH
(1) 8aH
CHO
OH
(1) 8PH
(38)
Drimenol (2) has also been used in the syntheses of a series of natural nordrimanic compounds: the fragrant hydroxy ketone (32) [31] isolated from tobacco [32]; isonordrimenone (33) isolated also from tobacco [33];
400
and its unnatural isomer (34) [34], polygonal (35), isopolygonal (36) and polygonone (37) [35] isolated from Polygonum hydropiper L [21, 37]. Finally, drimenol (2) was transformed into the saturated 8aH- and 8(3Hdrimanes (1) found in benzenes [37]. From the data mentioned above it follows that drimenol (2) is a valuable synthon for the synthesis of drimanic compounds, including bioactive ones. This fact stimulated the elaboration of the methods for its preparation from available natural compounds, first of all, from labdanoids. One of the first syntheses of natural drimenol (2) was accomplished by Wenkert and Strike [38] from dehydroabietic and podocarpic derivatives of the type (38), obtained from resin acids. Because of its complexity due to a multistep path (ca 25 steps) and also of no preparative importance, these syntheses are not discussed here. Pelletier et al. [39] carried out the drimenol (2) synthesis from the ambreinolide (39), the product obtained from a series of available labdanoids [7, 8] (Scheme 5). Dehydrogenation of the ambreinolide (39) OR HOH
I
(41)R=H
I „ (42)R=Ac
OH
(2)
(43)
a) DDQ, dioxane, A; b) O,, CH2C17, -70°C; Red-Al, C 6 H 6 ; c) Ac 2 O, Py; d) POC13, Py; KOH, MeOH; e) BF 3 Et 2 0, CH2C12.
Scheme 5 with DDQ led to A -dehydroambreinolide (40), whose ozonolysis followed by reduction of the resulting products with Red-Al gave (+)drimane-8a,l 1-diol (41). Its monoacetate (42) was dehydrated with POCI3 into a mixture of unsaturated acetates followed by their saponification to a lz 112
401
mixture of alcohols (2) and (43) separated by column chromatography on silica gel. The total yield of the drimenol (2) in this seven-step synthesis was ca 13%. It is necessary to mention that authors [40] showed that the albicanol (43) isomerises into the drimenol (2) in high yields (93%) on treatment with boron trifluoride etherate.
CO 2 Me
CO,Me
(2)
a) SOC12, Py; b) Na 2 S 2 O 4 , PTC; O3,CH2C12, MeOH; H 2 O 2 , NaOH; c) NaBH,, MeOH; d) (CH 3 ) 2 C(NH 2 )CH 2 OH, H 3 BO 3 ; e) PhSeOH, H 2 O 2 ; f) O 3 , CH2C12, MeOH; CrO 3 , H 2 SO 4 ; g) NaBH,, MeOH; CH 2 N 2 ; h) HMPA, A; i) LiAlH 4 , THF.
Scheme 6 The drimenol (2) was also synthesised from the available labdanoid gispanolone (44) [41] (Scheme 6). The latter was dehydrated into the unsaturated ketone (45), the conjugated double bond of which was reduced, and the resulting product was ozonolysed, giving the keto acid
402
(46). Its reduction with NaBELt led to a mixture of epimeric hydroxy acids (47), which on interaction with 2-amino-2-methylpropanol-l afforded a mixture of 4,5-dihydrooxazols (48). This mixture was dehydrogenated with phenylseleninic acid into a mixture of compounds (49), which was converted into the keto acid (50) by subsequent ozonisation and the Jones oxidation. Reduction of this keto acid with NaBH4 followed by methylation with CH2N2 afforded the hydroxy ester (51). On its heating with HMPA, the drimic acid ester (52) has been obtained. Its reduction with LiAltLt led to the drimenol (2). This synthesis of the drimenol (2) includes 11 steps, the overall yield being 8%. A shorter synthesis of the drimenyl acetate (12) was elaborated by Barrero et al. [42] from the sclareol (3) (Scheme 7). Oxidative cleavage of
(3)
(12)
(42) a) OsO 4 , NaIO 4 , 73%; b) tBuMezSiCl, NaH, 99%; c) 0,, CH2C12, MeOH, -78°C; NaBH 4 , MeOH, 95%; d) SnCL,, CH 2 C1 2 ,25%.
Scheme 7 the sclareol (3) side chain by osmic acid and sodium periodate led to the acetoxy aldehyde (53), whose enol silylation gave a mixture of the acetoxy silyl esters (54). On its ozonolysis, followed by reduction of the ozonolysis products with NaBH4, the 11-monoacetate of drimanediol (42) has been formed. On its interaction with SnCU, the drimenyl acetate (12) was obtained in low yield. The total yield of the desired product in this four-step synthesis was ca 17%. The final stage of this synthesis turned out to be less efficient. Synthesis of the drimenol (2) from the readily available larixol (5) has been recently described in [43] (Scheme 8). The exocyclic double bond of
403
larixol has been selectively epoxidised with oxone. The obtained epoxy diol (55) was then reduced into the triol (56), whose C-6 hydroxy group OH
(59)
OH
(60)
(2)
a) oxone. CH2C12, acetone, H 2 O, NaHCO 3 . 18-crown-6, 81%; b) LiAlH 4 , THF, 94%; c) Ac 2 O, Py, 99%; OsO 4 , NaIO 4 , THF, H 2 O, 84%; d) tBuMe 2 SiCl, NaH, THF, 96%; e) O 3 , CH2C12, MeOH, -78°C, IVfeS, 78%; f) collidine, 200°C, 77%; g) NaBH,, EtOH, 0°C, 89%.
Scheme 8 was selectively acetylated and the resulting compound was cleaved with OsO4-NaIO4 into the diacetoxy aldehyde (57). This compound was converted into the mixture of the silyl enol esters (58), ozonolysis of which led to the diacetoxy aldehyde (59). On subsequent heating of compound (59) eliminated the acetic acid, affording the diene aldehyde (60). Reduction of (60) with NaBELt gave only drimenol (2) as a result of selective 1,4-addition. Selectivity of this method and sufficiently high yields of products at all stages of this synthesis are its main advantages. The overall yield of this eight-step synthesis was 32.5%.
404
We accomplished the drimenol (2) synthesis from the sclareol (3) [44] (Scheme 9). This was the first synthesis of a drimanic sesquiterpenoid
(64) A' (65) A8
(67) A s
8 14
(66)A < > (68)A8(12) a) KMnO 4 , AcOH, H 2 O; KOH, MEOH, A; H 2 SO 4 ; b) CH 3 Li, Et 2 O; c) H 2 O 2 , BF 3 Et 2 O; d) KOH, EtOH, A.
Scheme 9 from a labdanic diterpenoid. This synthesis, which correlated these two groups of terpenic compounds, confirmed the stereochemistry of the drimenol (2). The sclareol (3) was oxidised according to the method [45], and the obtained hydroxy acid (61) was introduced in reaction with methyl, lithium. The reaction product was a mixture of the ditertial diol (62) (18% yield) and of the hydroxy ketone (63) (80%). The latter on interaction with a mixture of concentrated (93.6%) hydrogen peroxide and boron trifluoride etherate afforded a mixture of the unsaturated ketones (64)-(66) and of the isomeric acetoxy drimenes (12), (67) and (68). All these substances were separated and isolated in individual form by column chromatography with SiC^'AgNOs and characterised. On saponification of the acetate (12), drimenol (2) was obtained. Later on in [46] it was established that, instead of the unstable, easily lactonised hydroxy acid (61), in the reaction with methyl lithium it is possible to use the commercially available norambreinolide (69), a common cleavage product of many labdane diterpenoids [7, 8]. In this case the yield of the hydroxy ketone (63) is a little lower (65%), but it is more convenient to carry out
405
this reaction. The total yield of the drimenol (2) in this synthesis is small (6.5%), therefore this method is of no preparative interest. It is necessary to note that later on we elaborated an alternative, shorter route for preparation of the hydroxy ketone (63) from the sclareol (3) [47] (Scheme 10). On the ozonolysis of the sclareol (3) and subsequent treatment of the ozonolysis products with ammonium chloride, the dimeric product (70) is formed [48]. Its further ozonolysis gave the (3-diketone (71).
~CH,
(3)
OAc
'OH
(72)
a) O 3 , MeOH, 5-10°C; NH4C1, 80%; b) O 3 , hexane, -65...-70°C; H 2 O, A, 100%; c) NaOH, EtOH, A; d) H 2 O 2 , CH2CI2, (CF 3 CO) 2 O/NaHCO 3 (l:l), 100%; e) H 2 SO 4 , EtOH, r.t, 56%; f) KOH, MeOH, 98%; g) 1 mmole FSO3H, C 3 H 7 NO 2 , -8O...-85°C, 1 h, 7 1 % ; h) 10 mmoleFSO3H, -8O...-85°C, 5 min, 77%.
Scheme 10 Alkaline cleavage of the latter produced a mixture (1:1) of the hydroxy acid (61) and the hydroxy ketone (63). As a result, this three-step synthesis led to the hydroxy ketone (63) in an overall 37% yield. Furthermore, the hydroxy acid (61) can be converted into (63), as is indicated in Scheme 9.
406
Recently, we have elaborated an alternative method of the drimenol (2) synthesis from the hydroxy ketone (63) of preparative value [49]. In the article [46] it was shown that on oxidation of the hydroxy ketone (63) with trifluoroperacetic acid under certain conditions, the 11-monoacetate of drimane-8a,ll-diol (42) is obtained in the quantitative yield. On treatment of the compound (42) at room temperature with 30% solution of concentrated sulphuric acid in ethanol by using 10 ml of this solution per 1 g of the compound (42), the crystalline drimenol (2) was obtained in 56% yield, which could be purified by recrystallisation from n-hexane [49] (Scheme 10). In such a way, at stage e) selective dehydration and transesterification of the hydroxy acetate (42) took place. Finally, we elaborated a highly efficient, structure- and chemoselective, stereospecific one-step method for the synthesis of the racemic drimenol (2) and hydroxy acetate (42) by the superacidic, low temperature cyclisation of E,E-farnesol (72) and its acetate (73), respectively [50] (Scheme 10). 2 Synthesis and Use of Drimane-8a,ll-diol and Its 11-Monoacetate Drimane-8a,ll-diol (41) and its 11-monoacetate (42) are suitable starting compounds for the synthesis of a series of drimanes and not only of them. Only the diol (41) was found in natural sources and was isolated from tobacco [51] and from a special gland of African elephant [52]. Data about the synthesis of these compounds from the ambreinolide [39] and of the hydroxy acetate (42) from the sclareol (3) have been already reported [42]. Barrero et al. [42] showed also that if the reduction of the ozonolysis product of the mixture of esters (54) is done with L1AIH4 instead of NaBH4, the diol (41) is obtained in a 95% yield (Scheme 11). Ohloff and Giersch [53] accomplished the synthesis of the drimanediol (41) from the norambreinolide (69). The latter was reduced into the semiacetal (74), whose acetate (75) on pyrolysis gave the dihydrofuran (76). Its ozonolysis and subsequent reduction of the ozonolysis products with NaBH4 afforded the diol (41). Unfortunately, the yields of the products in [53] are not given. As it was described above, the hydroxy acetate (42) was transformed into the drimenol (2) [39]. It should be mentioned that the reverse conversion of the drimenol (2) into the diol (41) [54] also takes place. For this purpose, the drimenyl acetate (12) was
407
epoxidised into the epoxy acetate (77) which was further reduced to the diol (41) (Scheme 11).
(54)
a) O 3 , MeOH, CH2C12, -78°C; LiAlH,, THF, 95%; b) iBujAlH, toluene; c) Ac 2 O, Py; d) 350°C; e) O,, EtOH; NaBH 4 ; f) m-CPBA, CH2C12; g) LiAlH,, THF.
Scheme 11 Earlier it was indicated (Schemes 5, 7, 10) that the hydroxy acetate (42) was transformed into the drimenol (2) [49], its acetate (12) [42] or a mixture of the drimenol (2) and the albicanol (43) [39]. Authors [55] accomplished the targeted synthesis of the albicanol (43) and its acetate (68) starting with the hydroxy acetate (42) (Scheme 12). The hydroxy acetate (42) was dehydrated with thionyl chloride into the mixture of acetates (12), (67) and (68), which was subjected to oxidation with mCPBA. On that treatment the acetates (12) and (67) are selectively oxidised, but the acetate (68) remained unreacted. The latter was than isolated by chromatography and saponified into the albicanol (43). The overall yield of the albicanyl acetate (68) and the albicanol (43) was 61% (at the saponification step the yield of (43) was quantitative). Barrero et al. [42] synthesised a mixture (1:1) of the albicanyl acetate (68) and its isomer (67) from the hydroxy acetate (42) by its successive acetylation into the diacetate (79) and pyrolysis of the latter (Scheme 12). It should be mentioned that the albicanyl acetate (68) is a biologically active substance, being a powerful antifeedant for fish [56]. The albicanol (43) itself is inactive, but it is easily acetylated with acetic anhydride in
408 ,OAc
OAc
,-OAc
(67)+(68)
(41)
'"OH
a) SOC12, DMAP, Py; b) m-CPBA, CH2C12, NaHCO 3 ; c) KOH, MeOH; d) Ac 2 O, Et 3 N, DMAP, THF, A, 92%; e) collidme, A,; f) TsCl, DMAP. Py; g) Nal, Zn, (CH 2 OMe) 2 , A, 59%.
Scheme 12 pyridine to give the active compound (68) [57]. The albicanol (43) was isolated from liverworts [12, 58], where it was found for the first time, and also from marine molluscs [56], where it is present together with its acetate (68), the predominant metabolite. It is necessary also to note that the diol (41) has been used as a starting compound for the synthesis of drim-9(ll)-en-8a-ol (80), one of the two C-8 epimeric metabolites of the fungus Aspergillus oryzae [59], used in Japan in baking and in manufacturing of some beverages (sake and others). On tosylation, the diol (41) afforded the monotosylate (81) which was transformed to the unsaturated alcohol (80) on treatment with Nal and Zn [60] (Scheme 12).
409
We succeeded in the elaboration of several syntheses of the drimanediol (41). One of them [49] was already discussed above. On peracidic oxidation, the hydroxy ketone (63) led to 11-monoacetate of drimane8a,ll-diol (42), which on alkaline saponification gave the diol (41) in almost quantitative yields (Scheme 10). In two syntheses of the diol (41), the sclareol (3) has been used as a starting compound. The sclareol oxidation product, the sclareol oxide (82) [61], on bromination in methanol afforded the dibromomethoxy derivative (83) which on interaction with potassium hydroxide eliminated hydrogen bromide, giving the unsaturated oxide (84). Its successive ozonolysis and reduction of the ozonolysis products with LiAlELi led to drimane-8a,lldiol (41). The overall yield of the diol (41) from the sclareol (3) was ca 32% [62] (Scheme 13).
(83)
(84)
a) KMnO 4 , acetone, 80%; b) 2 mol Br2, MeOH, 76%; c) KOH, toluene, PEG-600, A, 76%; d) O 3 , CH2C12> 65...-70°C; LiAlH,, EtjO, 70%.
Scheme 13 In the other synthesis of the diol (41) from the sclareol (3) [63], the latter was ozonised according to [64]. Under these conditions, a mixture of C-12 epimeric bisnorlabdanic oxidoketones (85) has been formed in a high yield. Its oxidation with monoperphtalic acid afforded a mixture of acetoxy oxides (75), from which acetic acid has been eliminated to give the unsaturated oxide (76). The latter was ozonised and the ozonolysis products were reduced with NaBtLj into the diol (41). This four-step synthesis led to the diol (41) in an overall 44% yield (Scheme 14).
410
However, the preferable method for the diol (41) synthesis is its preparation from a mixture of the isomeric neoabienols (7) [65], which has been isolated from the highly boiling fraction on distillation of oleoresin of silver-firs, for example, Siberian silver-fir [66]. On heating of the oleoresin, the cis-abienol (86), presented in it, suffers the sigmatropic shift giving a mixture of stereoisomeric neoabienols (7) [67]. On the exhaustive ozonisation of this mixture of neoabienols in methanol, followed by reduction of the resulting peroxidic products with NaBH4, drimane-8ot,l 1-diol (41) was obtained in a high yield (74%) (Scheme 14). H_
V-n
0Ac
(3)
"'OH
a) Oj, NaIO4, MeOH, H2O, -78°C, 91%; b) MPPA, Et2O, 90%; c) DMSO, NaHCO3, 150°C, 80%; d) O3, MeOH, -70°C; NaBH<, 67%; e) A; f) O3, MeOH, -75°C, NaBHt, 74%.
Scheme 14
3 Synthesis of Drim-9(ll)-en-8a-ol As was described above (see page 16), K. Wada et al. [59] have isolated the epimeric alcohols (87) and (88) from the culture filtrate of fungus Aspergillus oryzae and have established their structures and relative configurations, but their absolute configurations remained unknown. Later on, Brazilian chemists [68] accomplished the synthesis of alcohols having the structures (80) and (89) from the manool (4). The latter was epoxidised selectively at exocyclic double bond, and the mixture of epoxides (90) and
411
(91) was reduced into the mixture of sclareol (3) and 8-episclareol (92). This mixture was oxidised with KMnC>4 to a mixture of C-8 epimeric 14,15-bisnorlabdan-8-ol-13-ones (93) and (94). The latter mixture was subjected to Norrish II photolytic cleavage to afford compounds (80) and (89) (Scheme 15). On the basis of polarimetric data, authors [68] concluded that the alcohols, isolated from the fungi, belong to the series of ent-drimanes. The results of the work [60] (Scheme 12) are also in conformity with this conclusion. OH
(87)R,=OH,R2=CH3 (88)R,=CH3,R2=OH
(3) R,=CH3, R2=OH (92) R!=OH, R2=CH3
(90) 8R (91) 8S
"'OH i_H (93) RrCH 3 , R2=OH (94)R!=OH,R2=CH3
(80)
a) rn-CPBA, CH2C12, NaHCO.,, 78%; b) LiAlH,, THF, 75%; c) KMnO4, MgSO4, acetone, 100%; d) hv, petroleum ether, Ar.
Scheme 15 Afterwards, G. Dominguez et al. [69] accomplished the synthesis of alcohols (80) and (89) from the methyl ether of the natural quinone royleanone (95) (Scheme 16). They investigated three different approaches for compounds (80) and (89) preparation from the quinone (95). It was found that the following ones were optimal. The quinone (95) was ozonised, and the ozonolysis products were oxidised with periodic
412 OH
OMe
(80) + (89)
(101)
(102)
acid and methylated. The obtained keto diesther (96) was reduced to a mixture of C-ll epimeric triols (97), which were transformed to acetonides (98). The free hydroxylic group of (98) was reduced via the sulphoester, and the mixture of compounds (99) was obtained. Hydrolysis of the acetonide group of (99) gave the diols mixture (100), whose glycolic group was cleaved by oxidation and the resulting product was then reduced to the alcohol (101). The latter was transformed to the selenophtalimide (102) which on oxidation with H2O2 afforded the corresponding selenoxide spontaneously rearranged to give a mixture of the alcohols (80) and (89). a) O3, CH2C12, -80°C; H5IOfo EtOH, H2O; CH2N2; b) LiAlH,, THF; c) acetone, CuSO4; d) PySO3, THF; LiAlH,; e) AcOH, H2O; f) Pb(OAc)4. O f t , MeOH; LiAlH,, Et2O; g) N-(phenylseleno) phtalimide; h) H2O2, CH2C12.
Scheme 16 Although manool (4) and royleanone (95) belong to the same stereochemical series, the results of polarimetric measuring, carried out in [69], turned out to be opposite to what Brazilian chemists concluded [68]. Authors [69] drew the conclusion that the natural drimenols, isolated by authors [59], belong to the normal drimane row.
413
To clarify this confusing situation, we [70] carried out the synthesis of the alcohol (80) from sclareol (3) which had been earlier used by authors [68] as one of the intermediates in their synthesis of the same substance (80) (Scheme 17). Taking into consideration that the hydroxy ketone (90) is unstable and is easily transformed to sclareoloxide (82) [8], sclareol (3) was first acetylated according to the method [71] to the diacetate (103), which was cleaved to the acetoxy ketone (104) by ozonolysis. Norrish II photolytic cleavage of this ketone yielded the acetate (105). Under these conditions a part of the starting material remained unreacted. The acetate (105) is an extremely unstable substance. As a result, it decomposes on chromatographic purification to give the hydrocarbon (106). Apparently, this was the reason why authors [72] obtained only the hydrocarbon (106) on photolysis of the acetoxy ketone (104).
(3)
(105)R=Ac (106) (80) R=H a) AcCl, DMA, 93%; b) O3, MeOH, CH2C12> -60T; Cu(OAc)2H2O, MeOH, toluene, 70°C, 52%; c) hv, hexane.99%; d) SiO2, r.t, 100%; e) KOH,EtOH,A.
Scheme 17 Taking into account this fact we saponified the crude photolysis product and obtained the alcohol (80) which is more stable and can be purified by chromatography. Positive optical rotation of the alcohol (80) obtained by us demonstrated that metabolites of Aspergillus oryzae [59] belong to entdrimane series, and their absolute configurations should be depicted by formulas (87) and (88). The overall yield of the alcohol (80) synthesised from sclareol (3) in four steps was 45% (taken into consideration the unreacted acetoxy ketone (102) at the photolysis stage).
414
4 Synthesis of Drim-8-en-7-one and Drim-5,8-dien-7-one To date, drim-8-en-7-one (27) has been found only in one natural source, tobacco [30]. Authors [30] accomplished also its first multistep and
•(27)
-co2
(110) (HI) (112) a) H2SO4, MeOH, A, 100%; b) K2Cr2O7, AcOH, A, 41.5%; o) KOH, EtOH, A; HC1, 98%; d) SeO2, dioxane, A, 93%; e) NaOH, EtOH; HC1, 100%; f) 200°C, 80%.
Scheme 18 low-efficient synthesis from drimenol (2) (Scheme 4). Another known synthesis of the racemic ketone (27) [73] also involves many steps and is not considered here. Drim-8-en-7-one (27) has been found among the neutral products of the sclareol (3) oxidation with chromic mixture [74]. We have found that this substance possesses a strong ambergris odour, and may be of interest for perfumery [75] and tobacco industry [76]. The discovery of these important properties stimulated the search for a new, shorter, simpler and more efficient synthesis of the ketone (27) from available starting materials. We could accomplish such a synthesis from commercially available norambreinolide (69) [77]. The latter was converted according to the known procedure [78] to a mixture of esters (107) whose oxidation with K2Cr2O7 in acetic acid led to the keto ester (108). On its saponification, the corresponding keto acid (109) was obtained, which spontaneously decarboxylated to give the drimenone (27) in an overall 40.5% yield. A disadvantage of this method is the low yield of the keto ester (108). All attempts to improve it by variation of chemical oxidants and reaction conditions were unsuccessful [77]. We could obtain
415
an acceptable yield (65%) of the keto ester (108) only on electrooxidation of the mixture of esters (107) [79] (Scheme 18). This method allowed the simplicity of drimenone (27) isolation; the electrooxidation product was saponified and separated into the acidic and neutral parts. The latter consisted of the drimenone (27) which was purified by crystallisation. Thus, this ketone became a relatively available compound. This fact and also the specific chemical structure of drim-8-en-7-one make it attractive as a starting compound for the syntheses of the natural polyfunctional drimanic sesquiterpenoid, including the biologically active ones. Indeed, its functional groups, double bond at C-8-C-9 and the carbonyl group at C7, activate the carbon atoms C-6, C-ll and C-12, providing a certain synthetic niche for the ketone (27). Below are the data concerning the syntheses of poly functional drimanes from drim-8-en-7-one (27). The keto ester (108) is easily dehydrogenated with SeCh to the dienone ester (110). On its saponification, the corresponding acid (111) is obtained. Unlike the keto acid (109), the keto acid (111) is sufficiently stable and decomposes only on heating to give drim-5,8-dien-7-one (112) [79] (Scheme 18). Like the drim-8-en-7-one (27), the ketone (112) is also of interest as a starting compound in the syntheses of polyfunctional drimanes. 4.1 Synthesis of 1 l-Acetoxydrim-8-en-7-one from Drim-8-en-7-one
ll-Acetoxydrim-8-en-7-one (31) is of interest as an intermediate in the syntheses of biologically active drimanes, particularly, of warburganal (16). For the first time, compound (31) was obtained by authors [30] as an intermediate in the synthesis of drim-8-en-7-one (27) from drimenol (2) (Scheme 4). Later on this substance was prepared by Chilean chemists [80] (Scheme 19). On the interaction of drimenyl acetate (12) with selenium oxidants a mixture of compounds (13), (14) and (113) was obtained, in which the latter predominates (65% yield). Oxidation of the hydroxy acetate (113) with pyridinium dichromate and tert-butyl hydroperoxide led also to a mixture of three compounds (114), (115) and (31). The yield of the keto acetate (31) reached only 20% at this stage, and the overall yield of (31) from (12) was 13%. Barrero et al. [81] carried out a more efficient synthesis of the keto acetate (31) from the unsaturated acetate (67), which they obtained earlier
416
(12)
(113)
OH
(16)
a) SeO 2 (cat), (p-MeO-C6H4)2SeO, dioxane, A; b) 2Py Cr2O7> tBuO 2 H, O f t ; c) Na 2 Cr0 4> AcjO, AcOH, AcONa, 70°C, 9 1 % ; d) H 2 O 2 , NaOH, MeOH, 88%; e) N2H4, AcOH, A, 95%; f) Ac 2 O, Py, 92%; g)NaBH4, EtOH, 96%; h) MsCl, Et 3 N, DMAP, THF, A, 8 1 % ; i) SeO 2 , dioxane, A; KOH,MeOH, 7 1 % .
Scheme 19 [42] from sclareol (3) via the 11-monoacetate of drimane-8a,l 1-diol (42). The acetate (67) was converted into the keto acetate (31) by oxidation of sodium chromate in a very high yield (91%) (Scheme 19). The same authors [81] performed the transformation of the keto acetate (31) into biologically active warburganal (16) by two convergent pathways, (Scheme 19). According to one of them, compound (31) was epoxidised, and the epoxy ketone (116) was subjected to a WhartonBollen rearrangement. The resulting diol (117) was acetylated to give the
417
hydroxy acetate (113). According to the second, a shorter route, the keto acetate (31) was reduced to the hydroxy acetate (118), which was isomerised to the hydroxy acetate (113). Its subsequent oxidation with selenium dioxide and saponification led to the triol (17), whose conversion to warburganal (16) was described above. We carried out a three-step synthesis of the keto acetate (31) from drim8-en-7-one (27) in the overall yield of 66.5% [82] (Scheme 20). The ketone (27) was enolacetylated to the diene acetate (119), whose oxidation with MPPA gave the keto alcohol (120), acetylated to the keto acetate (31). It should be noted that the structure of the keto alcohol (120) was determined not only on the basis of spectral data, but also was confirmed by X-ray [83].
(31) O
> f £ ^
^OAc
°
(27) (119) (120) a) CH3C(OAc)=CH2, TsOH, A, 84%; b) MPPA, Et2O, 80%; c) Ac2O, Py, 99%.
Scheme 20 We proposed also an alternative method to pass from the keto acetate (31) to the triol (17) (see below). 4.2 Synthesis of 11,12-Diacetoxydrim-8-en-7-onefrom
Drim-8-en-'/'-one
For the first time, the 1 l,12-diacetoxydrim-8-en-7-one (121) was prepared in the racemic form by Japanese chemists [84] and was then converted in two stages via the epoxide (122) into the racemic triol (17). This triol was further transformed to biologically active (l)-warburganal (16) by the same authors in a very complicated way of eight steps. Afterwards, warburganal (16) was obtained from (17) in one step by the Swern oxidation [24] (Scheme 21). The keto diacetate (121) was obtained in optically active form from royleanone (95) [69]. The ozonisation of the latter, and subsequent treatment of the resulting products by hydrogen peroxide in alkaline medium and then with Pb(OAc)4, gave the dicarboxylic acid (123). This diacid was converted in two steps into drim8-en-11,12-diol (124) followed by the acetylation to the diacetate (125). Chromic anhydride oxidation of diacetate (125) afforded the (+)-keto diacetate (121) (Scheme 21).
418
OH
(16)
(95)
^ (125)R=Ac
*•
(127)
(124)
(128)
Later on the (+)-keto diacetate (121) was synthesised by Nacano et al. a) H 2 O 2 , NaOH, MeOH, H 2 O, 82%; b) N 2 H,H 2 O, 23%; c) (COC1)2, DMSO, 96%; d) O3, CH2C12, -78°C; H 2 O 2 , NaOH, 90%; Pb(OAc) 4 , C f t , MeOH, 80%; e) CH 2N2, 100%; LiAlH,, Et 2 O, 90%; f) Ac 2 O, Py, 100%; g) CrO 3 , AcOH, 69%; h) KMnO 4 , acetone, 60%; i) hv, pentane, 73%; j) hv, O 2 , maso-tetraphenylporphine, CC14, 72%; k) LiAlH4, THF, 100%.
Scheme 21 [85] also by oxidation of the diacetate (125) with CrC>3. However, the diacetate (125) was obtained from manool (4) oxidised to the ketone (126) followed by photolysis to give the diene (127) [86]. Subsequent photooxidation of this diene to the endoperoxide (128), its lithium aluminium hydride reduction to the diol (124) [88] and acetylation led to the diacetate (125) [85] (Scheme 21).
419
We elaborated two pathways for obtaining the keto acetate (121) from drim-8-en-7-one (27) [82] (Scheme 22). According to one of them, the keto acetate (31) was brominated to ll-acetoxy-12-bromodrim-8-en-7-one (129), in which the acetoxy group was substituted for the bromine atom to give the keto acetate (121) in an overall 28% yield from drim-8-en-7-one (27) (5 steps). According to the second pathway, compound (27) was
(31)-
(121) OH (132) l l a OH (133) lip OH a) NBS, CaCO3, CC14, A, 60%; b) AcOK, DMSO, 84%; c) AcOK, DMF, 65%; d) K2CO3, MeOH, 72%; e) KOH, O2, MeOH, 50%; f) SOC12, Py, 78%; NaBHj, EtOH, 94%; g) O2, eosin, hv, tBuOH, 2,6-lutidine, 40%.
Scheme 22 brominated with NBS to afford the dibromoketone (130) in a high yield. Its structure was confirmed by X-ray data [79]. The keto diacetate (121) was then obtained from (130) by substitution of both bromine atoms by acetoxy groups. In this two-step synthesis of the product (121) from the ketone (27) the overall yield was reached to 59%. It is necessary to note that in [69], the keto diacetate (121) has been described as a crystalline substance, whereas in [85] as an oil. We did not succeed in crystallising this compound either. However, on saponification of (121), we obtained the keto diol (131) in the crystalline form. The structures of (121) and (131) were confirmed by spectroscopic and X-ray data [88]. It should be noted that the keto diacetate (121) served as a starting compound for the synthesis of one more natural drimane (+)-fuegin (135) [85], isolated from Drimys winteri Forst [89]. On saponification of
420
compound (121) in the presence of oxygen a mixture of epimeric semiacetals, (132) and (133), was formed, whose further dehydration and subsequent reduction gave hydroxyeuryfuran (134), photooxidated into fuegin (135). 4.3 Synthesis of 7-Oxoisodrimenin and Isodrimenin from Drim-8-en-7-one
7-Oxoisodrimenin (136) has been isolated from Porella cordeana [90], though much earlier it had been synthesised on the Beckmann mixture OH.
OH j
(16)
•i.H
(141)a-epoxi(68%) (142) |3-epoxi(29%)
I
(17)
a) Beckmann mixture; b) CrO 3 , Py, 38%; c) PCC, CH2C12, 92%; d) MnO 2 , CH2C12; e) (CH 2 OH) 2 , TsOHHjO, C6H6, A, 94%; f) LiAlH4, Et 2 O; 10% HC1, 95.5%; g) LiAlH 4 , Et 2 O, 88.5%; h) m-CPBA, CH2C12; i) Et 2 NLi, THF.A, 54%; j ) (COC1)2, DMSO, 70%.
Scheme 23 oxidation of drimenin (137) and isodrimenin (138), isolated for the first time from Drimys winteri [91]. 7-Oxoisodrimenin (136) was also obtained on oxidation of natural isodrimeninol (139) with the Collins reagent [10]. Nacano et al. [85] have prepared 7-oxoisodrimenin (136) on oxidation of the mixture of oxosemiacetals, (132) and (133).
421
We obtained 7-oxoisodrimenin by oxidation of the keto diol (131) with an excess of PCC [92]. On oxidation of (131) with MnO 2 or PCC (2 mol. equivalents), a mixture of the semiacetals (132) and (133) and keto lactone (136) was obtained. One of these semiacetals, (133) was isolated by crystallization, and its structure and stereochemistry were confirmed by Xray data [93]. The mixture of semiacetals (132) and (133) was further oxidised to the keto lactone (136). This lactone has no biological activity itself, but it has been used as an intermediate in the total synthesis of bioactive (±)-warburganal (16) [84]. The ethylene ketal (140) of the keto lactone (136) was converted into the keto diol (131), whose transformation to warburganal (16) was discussed above (Scheme 23). There are many syntheses of isodrimenin (138), both total in the racemic form, and partial in the optically active form from available bi- and tricyclic diterpenoids or other drimanes. These syntheses will not be discussed here since most of them have already been reviewed [6,7]. Isodrimenin (138) possesses antifeedant activity against larva of kolorado beetle [4]. Besides, compound (138) can be transformed to biologically active warburganal (16) via the following series of transformations: (138) -^ (136) - • (140) -> (131) -> (122) - • (17) -» (16). There is also known another transition from isodrimenin (138) to warburganal (16) via the diol (124), the isodrimenin reduction product [91] (Scheme 23). On the m-chloroperbenzoic acid oxidation, the diol (124) gives a mixture of the epoxy diols, (141) and (142). The predominant one (141) (68% yield) reacts with lithium diethyl amide to afford the triol (17), oxidised to warburganal (16) [87, 94]. We prepared isodrimenin (138) from drim-8-en-7-one (27) according to the following sequence of transformations: (27) —> (130) —» (121) —» (131) -» (136) -> (143) - • (138) (schemes 22-24) [92].
422
4.4 Synthesis of5,6-Dehydro-7-oxoisodrimeninfrom Drim-8-en-7-one
5,6-Dehydro-7-oxoisodrimenin (144) was isolated from Porella cordeana [90], though earlier this compound (144) had been synthesised
(136)
OAc
(112)
(145)
\Z
(146) (147)
(144)
a) (CH 2 SH) 2 , SnCl 2 2H 2 O, THF, A, 92%; b) MC1 2 6H 2 O, NaBtt,, DMF, 85%; c) SeO 2 , AcOH, A, 72.5%, d) NBS, CCU, A, 76%; e) AcOK, DMSO, 95%; f) K 2 CO 3 , MeOH, 60%; g) PCC, Me 2 CO, 98%; h) CH 3 C(OAcj=CH 2 , TsOH, A, 78%; i) O 2 , hv, tetraphenylporphine, CC14, 69%.
Scheme 24 by dehydrogenation of 7-oxoisodrimenin (136) with selenium dioxide [91]. However, the keto lactone (144) prepared in such a way is contaminated with selenium, which is difficult to get rid of. We elaborated two pathways for the preparation of compound (144). In one of them, the ketone (112) has been brominated to the dibromide (145), by replecing the bromine atoms with the acetoxy groups. The resulting product (146) has been saponified to the keto diol (147), which was oxidated to the target lactone (144). The overall yield of compound (144) was 42%. It is necessary to note that the dibromide (145) is formed also on bromination of drim-8-en-7-one (27) [79]. According to the second route, the keto lactone (136) has been enolacetylated to the enol acetate (148) photooxidated into the keto lactone (144). The overall yield of (144) from drim-8-en-7-one (27) (six steps) was 22.4%.
423
c (99%)
4.5 Synthesis ofDrim-7fi8/3,9a-triolfrom Drim-8-en-7-one
Polyhydroxylated drimanes are of interest as potentially biologically a)H2O2, NaOH, MeOH, 96%; b) KBH4, MeOH, 100%; c) Ac^O, Py, 97%; d)HClO4, THF; e)KOH,MeOH, 93%; f) (CH3)2CO, CuSO4, HC1O4, 74%; g) SOC12, Py, 79%.
Scheme 25 active substances and as starting compounds in the syntheses of bioactive labdanic diterpenoids (forscolin, manoyloxides). We carried out the synthesis of drim-7|3,8(3,9a-triol (149) starting with drim-8-en-7-one (27) [95]. The latter was epoxidised to the epoxy ketone (150), which was reduced to the epoxy alcohol (151), whose acetate (152) was transformed, on interaction with HCIO4, to a mixture of the triol (149) (41% yield) and its monoacetate (153) (54%). Compound (153) gives the triol (149) on saponification, and vice versa, the triol (149) is quantitatively acetylated to the monoacetate (153). The triol (149) forms the acetonide (154). That conversion proves its absolute configuration at C-8 and C-9, confirmed by spectral data. It is interesting to note that on phosphorus oxychloride treatment the acetoxy diol (153) is transformed into the epoxy acetate (152), but the dehydration products were not formed (Scheme 25).
424
5 Synthesis of (-)(3aS,7aS)-ft"a«s-tetrahydroactinidiolide from Drim-8en-7-one 7?ans-tetrahydroactinidiolide (155) has been isolated from tobacco [96]. This compound is of interest since it can easily be transformed to dihydroactinidiolide (156) [97, 98], an important odorous component of the fragrances of tea [99], tobacco [100], tomato [101], essential oils from Actinidia polygama [102] and Acacia famesiana [103], and also the one of the components of the red ants pheromone [98]. Dihydroactinidiolide (156) was patented as an analeptic for the respiratory depression [104]. We carried out a short and efficient synthesis of transtetrahydroactinidiolide (155) from drim-8-en-7-one (27) [105]. On exhaustive ozonolysis of drim-8-en-7-one (27) and subsequent treatment of resulting products with hydrogen peroxide, the keto acid (157) was obtained in a quantitative yield. The Baeyer-Villiger oxidation of this acid leads directly to (-)(3aS, 7aS)fra«s-tetrahydroactinidiolide (155) (Scheme 26).
(157)
(155)
(156)
a) O 3 , AcOEt; H 2 O 2 , 100%; b) m-CPBA, conc.H 2 SO 4 , CH2C12, 67%; c) LDA, THF; (PheSe) 2 , HMPA, 59%; H 2 O 2 , THF, AcOH, 37% [97].
Scheme 26 Thus, starting with drim-8-en-7-one (27), we succeeded in performing the syntheses of a series of drimanic compounds. 6 Synthesis of (+)-drim-8-en-ll-oic Acid Drim-8-en-l 1-oic acid (158) is a convenient synthon for the preparation of polyfunctional drimanes. This acid has been used as an intermediate in the total synthesis of 1,6,7-tridesoxyforskolin (159), a related compound to forskolin (160), possessing ionotropic, antihypotensive and other activities [106]. Optically active compound (159) was isolated together with forskolin from the plant Coleus forskohlii [107]. The synthesis of the acid (158) was accomplished by us starting with the mixture of esters (107) [108], obtained from norambreinolide (69)
425
[78]. The mixture (107) reacts with methyl lithium to give a mixture of alcohols, (161) and (162), separated by chromatography. Alcohol (162) was dehydrated into the diene (163) cleaved by ozonolysis to a mixture of acids (158) and (164), separated by chromatography. It was found that on acid treatment, the unsaturated alcohol (161) is easily cyclised to the oxide (165), which is formed together with a mixture of hydrocarbons (Scheme 27). The overall yield of the unsaturated acid (158) was 28.8%. OH
_CO-,Me
OH
OAc OH (160) a) CH3Li, Et2O, 90%; b) TsOH, O f t , A, 90%; c) O3, AcOEt, Py, -50°C.
Scheme 27 7 Synthesis of 8a-Acetoxydrimane-ll-oic Acid 8a-Acetoxydrimane-ll-oic acid (166) is a convenient synthon for the preparation of biologically active drimanes [72]. The synthesis of this compound was accomplished by us [109] from commercially available
426
sclarodiol (167) [8]. The latter was exhaustively acetylated to the diacetate (168) followed by selective saponification to give the hydroxy acetate (169) which was then oxidised to the acetoxy aldehyde (53). Its enolacetylation and recrystallisation of the reaction product led to the trans-enol acetate (170), which was ozonised to afford the acetoxy acid (166) in a good yield. The overall yield of (166) from sclarodiol (167) was 16% (Scheme 28). It should be mentioned that authors [72] synthesised the acetoxy aldehyde (53) from labdanolic acid (6) in 5 steps in an overall 50 %
lOAc
(171)R=CH2OH (172) R=CHO
yield. a) Ac2O, Et3N, DMAP, 80%; b) NaHCO3, MeOH, H2O, 58%, c) CrO32Py, CH2C12, 63%; d) O3, AcOH, Py, -70°C, 88%; e) O3, NaBU, 45%; f) (COC1)2, DMSO, 25%; g) NaClO2, 90%.
Scheme 28 They obtained the enol acetate (170) in 92% yield, probably, as a mixture of cis- and tram- isomers. Compound (170) was ozonised, and the resulting product was reduced with NaBELi to the hydroxy acetate (171) which, on the Swern oxidation, afforded the acetoxy aldehyde (172). The
427
latter was then oxidised to the acetoxy acid (166). The overall yield of (166) from labdanolic acid (6) was 4.6% (Scheme 28). Thus, the present review summarises the results of our investigations, concerning the synthesis of a series of drimanic sesquiterpenoids with biological activity or being valuable intermediates on the way to important, biologically active natural compounds. The literature data concerning the presence of these compounds in natural sources, their biological activity and methods of preparation are also discussed. Starting from available labdanoids, sclareol and neoabienols, a large number of drimanic sesquiterpenoids have been prepared. In many of these syntheses, the commercially available norambreinolide served as an intermediate on passing from labdanoids to drimanes. It is well known that norambreinolide is a cleavage product of a large number of labdanoids. In particular, using norambreinolide as a convenient method for preparing the drim-8-en-7-one, a derivative with a high synthetic potential, was worked out. Using this compound, a series of drimanic substances has been prepared. At present, the investigations in the synthesis of drimanic polyfunctional sesquiterpenoids are in progress in our laboratory. ABBREVIATIONS
Ac
AcOH (Ac2O)2O
NBS BuLi m-CPBA DMAP DMA DIBAL DDQ DMF DMSO HMPA NMO MPPA
PTC Py
Acetyl Acetic acid Acetic anhydride N-Bromosuccinimide n-Butyllithium m-Chloroperbenzoic acid 4-Dimethylamino pyridine N,N-Dimethylaniline Diisobutylaluminium hydride 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone N,N-Dimethylformamide Dimethyl sulphoxide Hexamethylphosphoric triamide N-Methylmorpholine-N-oxide Monoperphtalic acid Phase transfer catalyst Pyridine
428
PCC Red-Al Tert t-Bu THF TsOH TSNHNH2 TsCl
= = = = = = = =
Pyridinium chlorochromate Sodium bis(2-methoxy)-aluminium dihydride Tertiary Tertiary butyl Tetrahydrofuran p-Toluenesulphonic acid p-Toluenesulphonic acid hydrazide p-Toluenesulphonyl chloride (tosyl chloride)
ACKNOWLEDGEMENTS The author Kuchkova, preparation contributed
is sincerely grateful to Doctors Aculina N. Aricu, Kaleria I. and Olga C. Iliashenco for skillful assistance in the of the manuscript. Thanks are also due to co-workers who to these investigations, whose names are in references.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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QUASSINOIDS: STRUCTURAL DIVERSITY, BIOLOGICAL ACTIVITY AND SYNTHETIC STUDIES IVO J. CURCINO VIEIRA* AND RAIMUNDO BRAZ-FILHO Setor de Quimica de Prodntos Naturals, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes, Rio de Janeiro, Brazil. (*Email: curcino@uenf. br) ABSTRACT: Quassinoids are bitter constituents of Simaroubaceae and the secondary metabolites characteristic of this family. The generic term quassinoids arises from quassin, the name of the first structurally identified member of this class isolated from specimen Quassia amara. Quassinoids can be divided into distinct groups according to their basic skeletons C!8, C19, C20, C22 and C2s. The chemistry and biogenesis of quassinoids have been reviewed several times. They remain exclusively of Simaroubaceous origin and biogenetically can be regarded as degraded triterpenoids and are almost certainly derived from tetracyclic triterpenes. Several quassinoids have been isolated and structurally elucidated and the majority of them have been biologically tested, including antifeedant, inseticidal, herbicidal, antLparasitic, antimalarial and anticancer activities. The interest in the chemistry of quassinoids has accelerated rapidly with the American National Cancer Institute finding in early 1970s, showing that these compounds display marked antileukemic activity (e.g. bruceantin). Chemical modifications of biologically inactive quassinoids have been performed, attempting to yield active ones, either by esterification or conversion of glycosides to the corresponding aglycones. Many studies on semisynthesis of rings member, intermediates and total synthesis of the molecular backbones or same leads such as bruceantin have been published. This review covers the structural variations, biological activity and some quassinoids synthetics studies.
1. STRUCTURAL DIVERSITY OF QUASSINOIDS /. / Introduction In elapsing the scientific development, the phytochemical has been using growing efforts to search new secondary metabolites of vegetable origin, not only for its economic value for the human society but also for what they represent in aiding the amplification of
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the knowledge on the natura phenomena. The tracks that motivate and guide that type of investigations are based most of the times on the popular use of several plants. With that approach researchers could isolate and identify the quassinoids, micromolecules by proceeding that, what was verified so far, i.e. they are produced exclusively by plants of Simaroubaceae family. There are lot several of those plants, it tending to be of popular use in the amebiasis treatment, disinters, arthritis and intestinal parasites, in the combat the fever and, also as insecticides. Several pharmacological studies led to the conclusion that quassinoids are the main ones responsible for the healing properties of those plants. For such review the collected data are from 1985 up to 2004 focusing on all the isolated and identified quassinoids, as well as, the main biological activities and some synthetic processes. 1.2 General Features of Quassinoids The review containing structural data, biological activities and chemical modifications of quassinoids was published last decade with references up to September 1984 by Polonsky [1]. According to Simao et al. [2] more than two hundred quassinoids were isolated and identified until December 1984.
cm,. Quassinoids are degraded triterpenes. They present five basic skeletons: a) skeleton of 25 carbon atoms (C25), denominated simarolidane skeleton, b) skeleton of 22 carbon atoms (C22),
435
denominated here of picrolemmane skeleton; c) skeleton of 20 carbon atoms (C20), denominated quassotidane skeleton; d) skeleton of 19 carbon atoms (Cw), denominated cedralidane skeleton; and e) skeleton of 18 carbon atoms (Cig), denominated of lauricolactme skeleton [2]. The quassolidane skeleton (C20) is of larger occurrence. Quassinoids are heavily oxygenated lactones (5-lactone in the quassolidane skeleton and yfoctone in the cedrotidane and lauricolactane skeletons, and 8-lactone and y-lactone in the other skeletons), with variable of groups hydroxyl amount, hydroxyl esterified, carbonyt, methoxyl and carbomethoxyl. In the quassolidane skeleton, these oxygenated functions can be found in most of carbon atoms, except carbons C-5, C-9, C-19 and C-29. Carbons C-l, C-2, C-7, C-ll and C-12 almost meet that obligatorily oxygenated, except in the quassinoids of the simarolidane type, once they do not happen oxygenated in C-12 position [1]. Ring D of quassolidane skeleton can present one group hydroxyl in carbon C-l 5, group that is generally esterified with various small fatty acids. In ring B, the carbon C-6 can present group hydroxyl, esterified or not. Ring A introduces one or two double bond in all skeletons. All isolated quassinoids he has only a group methyl in the carbon C-4 so far. Quassinoids O-glycosylated were isolated from species of genera Bntcea and Picrasma. 1.3 Biogenesis of Quassinoids Biogenetically quassinoids can be regarded as degraded triterpenoids and are almost certainly derived from tetracyclic triterpenes. The conversion of the tetracyclic triterpene into quassinoid has been experimentally verified by using labeled mevalonate precursors [3-5] and follows the route originally proposed by Arigoni et al. [6] but extended by further degradation to quassinoids as shown in Scheme 1. Support for this biogenetic picture has also come from the isolation of compounds representing the various stages and structural variations among quassinoids.
436 H
HO
Q5 Skeletons
Ct8 Skeleton
Scheme 1. Biogenesis of Quassinoids The postulated precursor, A7 - euphol (1) is proposed to undergo skeletal rearrangement to the hypothetical 7oc-hydroxy apo compound (2). Ring D is then oxidatively expanded to the 5-lactone (3) [6]. During these transformations one of the methyl groups at C-4 and four terminal carbons at the side chain must be lost. Opening of the 8-lactone (3) and relactonisation to the 7a-hydroxyl would then
437
give the intermediate 5-lactone (4) from which the basic skeletons observed amongst quassinoids can be derived. Thus, the lactonisation through the C-17 hydroxyl leads first to C25 skeleton quassinoid, while lactonisation through the hydroxyl at C-21 and subsequent oxidation of the residual C-17 hydroxyl may result in second C25 skeleton quassinoid. The C22 and C20 skeletons quassinoids may then be formed by cleavage of the C-13 - C-17 bond while the C19 skeleton compounds require the additional loss of the carbon atom C-16. The C18 skeleton results from the C19 skeleton quassinoid by loss of one carbon atom in ring A, presumably due to a benzylic rearrangement resulting in a ring contraction. 1.4 Structures of Quassinoids To follow what has been described, all quassinoids (divided by basic skeletons) were isolated and identified since 1985 to June 2004. 1.4.1 (
Quassinoids
xI
.,o
..,-"
HO,
o \ It J 5
c ^O
O
H
ft
6
o R-
O \\
iX i
A
^o
•o
/
7R=H 8R=C1
Until 1985 only three quassinoids with C18 skeleton had been isolated from Simaroubaceae species, Samaderine A (5) isolated from Samadera indica [7], and laurycolactone A (6) and B (7) isolated from Eurycoma longifolia Jack [8].
438
Then, two decades passed and only three more quassinoids with Ci8 skeleton were isolated from Eurycoma longifolia, eurycolactone B (8), C (9) [9] and D (10) [10], in a total of six quassinoids with Ci8 skeleton, isolated and identified so far. Compound (8) is the first halogenated quassinoid separated from plant sources. 1.4.2 Ci9 Quassinoids o
11. Ri= O; R2= O 12. Ri= OH-a; R2= O 13. 5,6-dehydro; Ri= OH-a; R2= O 14. 3,4-dihydro; Ri= O; R2= O 15. R]= O; R2= OH-a
o
18 17. 5,6-dihydro
Only nine quassinoids possessing the C19 basic skeleton were isolated until 1985: samaderins B (11), C (12), D (13), an 3,4dihydrosamaderin B (14) from the stem and leaves of Samadera indica, cedronin (15) and cedronolin (16) isolated from the fruits of Simaba cedron, eurycomalactone (17) and 5,6dehydroeurycomalactone (18) from Eurycoma longifolia Jack, and shinjulactone B (19) isolated from Ailanthus altissima Swingle [1]. Two decades passed and twenty-three more quassinoids were isolated from Simaroubaceae species in a total of thirty-two quassinoids with C19 skeleton identified so far. Eurycoma longifolia Jack is one of the most well known folk medicines for intermittent fever (malaria) in Southeast Asia [11]. This plant possesses the largest number of quassinoids with C19 skeleton identified so far. 7a-hydroxyeurycomalactone (20) [12], 6ahydroxyeurycomalactone (21) [13], eurycolactone E (22) [10], eurycomalide B (23) [14], and quassinoids (24) and (25) [15] isolated from the E. longifolia Jack and cymosanine (26) [16] isolated from
439
Simaba subcymosa possessing an y-lactone linked to carbons C-12 and C-13 in ring C in basic skeleton. Quassinoid (24) showed the presence of an exomethylene group in ring A. Cymosanine (26) showed an oxymethylene group (CH2-30), involving the formation of a tetrahydrofuran ring linked to carbon C-13. The p-configuration of the methyl group in position C-4 in ring A in cymosanine (26) and quassinoid (24) has been the unedited factor in all quassinoids.
OH
20. Ri= O; R2= OH-a; R3= H2 21. R,= O; R2= O; R3= OH-a 22. R,= OH-a; R2= O; R3= H2 23. 5,6-dehydro; Ri= OH-a; R2= O; R3= H 25. 3.4-dihvdro: Ri= OH-a: R2= O: R3= OH-a
26
The eight new quassinoids C19 type shinjulactone B (19) with a presence of a l,2-seco-l-nor-(5->10)-aZ>eo-picrasan-2,5-olide skeleton have been isolated from Simaroubaceae species. The structures of polyandrol (27) and 15-(9-acetyl-5(<S)polyandrol (28) isolated from Castela polyandra were established by a combination of spectroscopic and X-ray analysis [17,18]. Cedronolactone B (29) and cedronolactone C (30), isolated from wood of Simaba cedron were shown, exhibited in vitro cytotoxicity (IC50 6.5 and 49 u,g/mL) against P-388 lymphocytic leukemia cells [19]. Brucea javanica Merr. is a shrub, which is distributed from Southeast Asia to northern Australia having its seeds been used in the treatment of dysentery, malaria and cancer [20]. Javanicolide A (31) and Yadanziolide D (32) were isolated from seeds of B. javanica Merr., and their structures were elucidated by the analysis of spectral data and chemical evidence [20].
440
Ailanthm malabarica is a large indigenous tree from India and Southeast Asia and is used in the treatment of dyspepsia, dysentery, bronchitis, opthalmia and snake bites [21]. Ailanquassin A (33) was isolated from the wood of Ailanthus malabarica, and its structure was established by a combination of spectroscopic and X-ray analysis [21]. Two new quassinoids (34) and (35) with a presence of a 1,2seco-l-nor-(5—»10)-«&eo-picrasan-2,5-olide skeleton were isolated from wood Ewycoma longifolia Jack [15]. OH
OH ..-•""
R,
o
H ,
Four quassinoids Cw skeleton type with ring D contracted have been isolated from Eurycoma longifolia Jack., longilactone (36) [22], eurycolactone F (37) [10], 6-dehydroxylongilactone (38) [12], and eurycomaoside (39) [23]. The eurycomaoside (39) represents the first entry of the series of quassinoids with C19 skeleton possessing a glycosyl moiety at C-l. The 6-dehydroxylongilactone (38) showed potent activity (IC50 0.66 ng/mL) against P-388 leukemia cells. OH
OH OH 39
441
Quassinoid (40) isolated from the roots from Eurycoma longifolia Jack. [14] showed lactonization rarely-occurred C ring with hydroxyl group at C-ll with the carbonyl group at C-13, and eurycolactone A (41) showed a novel carbon framework with ring A contracted [9].
42
41
Cedronolactone E (42) isolated from Simaba cedron possesses a unique pentacyclic structure [24]. The structural similarity between cedronolactone C (30) may suggest a biogenetic relation between the former and the latter (Scheme 2). The cleavage of hemiketal at C-ll produces the cyclic ketone intermediate (30a), which via Michael addition of the ketone oxygen atom to C-4 from the si face produces OH
O H Michael addition „
30
Scheme 2. Proposed Biogenetic pathway from Cedronolactone C (30) to Cedronolactone E (42)
Ailantinol G (43) a quassinoid isolated from Aikmthus altissima was evaluated for its antitumor effects promotion against Epstein-Barr virus early antigen activation introduced by 12-0tetradecanoylphorbol-13-acetate in Raji cells [25], OH
HO, OH
442
1.4.3 C20 Quassinoids Despite their diversity in structure in quassinoids with C20 skeleton certain general features have been rationalized as follows [1]: i) The number and the positions of the methyl groups are the same of all skeletons; ii) Quassinoids are heavily oxygenated 8-lactones in the C20 skeleton, with the exception of C-5 and C-9 and the methyl groups at C-4 and C-10 oxygenated functions have been found on all the other carbon atoms; iii) Ring A may have the structures (a), (b), (c), (d), and (e); iv) Ring C may possess at position C-8 either a methyl group or a hydroxymethyl group which forms a hemiketal bridge to C-11 or an oxide bridge to C-13; v) Ring D and ring B may have at C-15 and/or C-6 hydroxyl groups which are generally esterified with a small fatty acid e.g. acetic, 2-methylbutiric, isovaleric, senecioic, 2-hydroxy-2methylbutyric, 3,4-dimethyl-4-hydroxyvaleric acids. OH
OH
o
o
H
°
I H (e)
To proceed, they are striped, all quassinoids identified since the year of 1985 up to 2003. The identified quassinoids are divided by Simaroubaceae species. 1.4.3.1 Quassinoids from Ailanthus altissima Swingle Ailanthus altissima Swingle, the tree-of-heaven, is native of China and was introduced in Europe around the end of the 18th century. Ailanthus altissima is used in Chinese traditional medicine as a bitter aromatic drug and in the treatment of colds and gastric diseases [26]. Seven quassinoids were isolated from Ailanthus altissima Swingle, ailantinol A-F (44-49) [26-28], and their structures were elucidated from spectral data. The ailantinol E (47) and F (48) were evaluated for its antitumor promoting effects against Epstein-Barr virus through early antigen activation introduced by 12-0-
443
tetradecanoylphorbol-13-acetate in Raji cells. Quassinoids were found to show potent activity without showing any cytotoxicity [25].
Shinjulactones L (50), M (51), N (52) [29-30], and shinjuglycosides E (53), and F (54) [31] were isolated from the root bark of Ailanthus altissima. OAc
OH HQOl OH
HO. OH
CH;OH
9H
,
HO,
GbO4l
1.4.3.2 Quassinoids from Ailanthus excelsa Chemical examination of Ailanthus excelsa has been carried out by several workers resulting in the isolation of quassinoids, alkaloids and
444
terpenoids [32], Three quassinoids (55), (56) and 3,4dihydro excelsin (57) were isolated from the stem bark of Ailcmthus excelsa, and structural elucidation is based on the analysis of spectroscopic data [32]. Quassinoids (55) and (56) showed the presence an A ring aromatized. OH HO O I
1.4.3.2 Quassinoids from Ailanthus vilmoriniana Six quassinoids, named vilmorinine A-F (57-62) were isolated from the cortex of Ailanthus vilmoriniana. The structures were elucidated by various spectroscopic methods, X-ray analysis, and computational chemical methods. Vilmorinines did not show cytotoxic activity (IC50 > 100 ug/mL) [33-34]. OH
O, , O H V
COzH "OR?
R2 Ri
SapOH 59. POH 60.POH 61. POH 62. pOH
H R4
R2
R3
aH aH aH aH aH
Me PMe H PMe H PMe H PMe H aMe
445
1.4.3.4 Quassinoids from Brucea antidysenterica Milt, Various quassinoids with antileukemic activity have been isolated from the Ethiopian tree Brucea antidysenterica Mill, including the antileukemic compound bruceantin (234) by kupchan and associates [35]. Eight quassinoids named bruceanols A-H (64-71) were isolated from Brucea antidysenterica, and all of these compounds exhibited cytotoxicity against murine lymophocytic leukemia (P-388) [35-38], Bruceanols G (70) and H (71) were evaluated against three cancer cell lines: SK-MEL-5 (melanoma), COLO-205 (colon cancer), and KB (nasopharynx carcinoma). These compounds were only marginally cytotoxic in the melanoma cell line with ED50 values of 4.08 and 6.37 uM, respectively. However, bruceanol G (70) showed activity against the COLO-205 and KB cell lines with ED50 values of 0.44 and 0.55 uM, respectively [38]. Quassinoid glycosides named bruceantinoside (72) and the new bruceanic acids B (73), C (74), and D (75) were isolated from wood Brucea antidysenterica [39-40]. Bruceanic acid D (75) was cytotoxic against P-388 (ED50 0.77 ug/mL).
446 OH
OAc
1.4.3.5 Quassinoidsfrom Bntceajavanica (L.) Merr. Brucea javanica Merr, is a shrub, which is distributed from Southeast Asia to northern Australia having its seeds been used for the treatment of dysentery, malaria and cancer [20]. Seeds ofBrucea javanica are known as "Ya-dan-zi" in Chinese folklore and have been used as a Chinese medicine for cancer, and the main active compounds of the plant has been extensively studied and thirty-four quassinoids have thus far isolated the last two decades. Quassinoids glycosides, yadanziosides A (76), B (77), C (78), D (79), E (80), G (81), H (82) and dehydrobrasatol (83) and dehydrobraceantinol (84) [41], and yadanziosides K (85), M (86), N (87), O (88) [42], and P (89) [43] were isolated from seeds Brucea javanica Merr..
447 447 OH HO.
io
GlcO.
GlcO1
GlcO,
Three quassinoids named yadanziolides A (90), B (91), and C (92), and four quassinoids glycosides named yadanziosides F (93), I (94), J (95), and L (96) were isolated from water-soluble fraction methanol extract of seeds of Brucea javanica Merr., and their structures were determined by spectral and chemical means [44]. Yadanziosides F (93), I (94), J (95), and L (96) were demonstrated to have in vivo antileukemic activity against the murine P-388 lymphocytic leukemia.
448 OH
OH
Bruceosides C (97) isolated from the fruits of B. javanica [45] demonstrated potent cytotoxicities against human epidermoid carcinoma of the nasopharynx (KB) (ED50 < 0.1 u,g/mL), human lung carcinoma (A-549) (ED50 < 0.44 ug/mL), colon carcinoma (HCT-8) (ED50 < 4.51 ug/mL), melanoma (RPMI) (ED50 < 0.1 ug/mL), and CNS carcinoma (TE-671) (ED50 < 0.29 ug/mL), as well as murine lymphocytic leukemia (P-388) (ED50 < 5.11 |J.g/mL) [45]. Bruceosides D (98), E (99), and F (100) show selective cytotoxicity in the leukemia and non-small cell lung, colon, CNS, melanoma, and ovarian cancer cell lines with log GI50 values in the range of-4.14 to -5.72 [46]. HO, GfcO. GfcO
449
Javanicin (101) an unusual quassinoid with seco ring A had its structure and relative configuration established unequivocally by single crystal X-ray analysis [47]. Javanicolides B (102) [20], C (103), and D (104) [48] isolated from the seeds of Brucea javanica showed weak cytotoxicity against P-388 murine leukemia cells with an IC50 values of 8, 10 and 18 Hg/mL, respectively, whereas javanicosides A (105), B (106), C (107), D (108), E (109), and F (110) had no activity [20, 48]. OAc
OH
HO,
I H
H 103
HO_
OH J.R .COjCH,
YY OH f O
HO. OH
HO.. J.R
rr
GkO.
,CO2CH3
GfcO,
107
106 OH
I H
H
1.4.3.6 Quassinoids from Castela peninsularis The structure of a new bitter-tasting quassinoid, named peninsularinone (111) isolated from the roots of Castela peninsularis
450
were determined by NMR spectroscopic and single X-ray analysis [49].
9H
Ill
1.4.3.7 Quassinoids from Castela texana A new quassinoid, ll-0-/ra«s-p-coumaroyl amarolide (112) isolated from Castela texana and the structure was elucidated by spectroscopic analysis. Compound (112) is the first coumaroyl quassinoid derivative to be isolated from nature [50]. Testing in the antimalarial bioassay showed that (112) possessed moderate antimalarial activity without potent cytotoxicity.
o o
112
1.4.3.8 Quassinoids from Castela tortuosa Castela tortuosa Liemb, a medicinal plant known as "chaparro amargo" in Mexico, was administered by the ancient Mexican people to treat liver diseases and is currently used to heal stomach aches and spasmodic pain [51], Quassinoids, castelalin (113) [52] and chaparramarin (114) [51] have been isolated and identified from the bark of C tortuosa.
451
Chaparramarin (113) exhibited moderate insect inhibitory activity against the lepidopteran pest insect, Heliothis virescem (tobacco budworm) £51]. Three quassinoids glycosides, casteloside A (115), casteloside B (116), and casteloside C (117) were also from the bark of Castela tortuosa [53-54]. OH
116. R= OH
1.4.3,9 Quassinoidsfrom Castela polyandra The structures of six new quassinoids, l-e/w-holacanthone (118), 15O-acetyl-glaucarubol (119), 15-O-aeetyl-A4>5-glaucarubol(120), l-epi5-wo-glaucarubolone (121), 1-e/w-glaucarubolone (122), and A4'5glaucarubol (123) all isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis [18].
452
1.4.3.10 Quassinoids from Eurycoma harmandiana Eurycoma harmandiana Pierre is a small Simaroubaceae plant (Thai name: Ian-don) distributed in the border regions between Thailand and Laos. Three new unusual 15a-0H quassinoids named iandonosides A (124), B (125), and iandonone (126) were isolated from the roots of Eurycoma harmandiana [55]. OH
^,OH
126
1.4.3.11 Quassinoids from Eurycoma longifolia Jack. Eurycoma longifolia Jack is one of the most well known folk medicines for intermittent fever (malaria) in Southeast Asia [11]. This plant possesses the largest number quassinoids with C19 skeleton identified so far. Seven novel highly oxygenated quassinoids were isolated from the leaves of Eurycoma longifolia, 13a(21)-epoxyeurycomanone (127), 15-acetyl-13oc(21)-epoxyeurycomanone (128), 12,15-diacetyl13a(21)-epoxyeurycomanone (129), 12-acetyl-13,21dihydroeurycomanone (130), 153-acetyl-14-hydroxyklaineanone (131), 6a-acetoxy-14, 15f3-dihydroxyklaineanone (132), 6a-acetoxy, 15(3-hydroxyklaineanone (133) [12]. The quassinoids (127-133) showed moderate antileukemic activity against P-388 cell lines, with IC50 14.0, 6.6, 7.2, 0.94, 7.8, 12.0, and 15.0 (ig/mL, respectively. The new 12-ep;-ll-dehydroklaineanone (134) isolated from the leaves Eurycoma longifolia showed moderate activity (plant growth inhibitor) against cucumber seedling [56]. Two highly oxygenated quassinoids, (135) and 13(3,18dihydroeurycomanol (136) were also isolated from the roots of Eurycoma longifolia [57-58].
453
Eurycomanol-2-O-P-D-glycopyranoside (137) isolated from the «-butanol extract roots of E. longifolia showed moderate activity against Plasmodium falciparum with IC50 of 1.590±0.169 |ig/mL, less potent than chloroquine (225) and quinine (222) [59]. OAo
OAo
OR2 O.
127. R,=R2= H 128. R,= H; R2= Ac 129.R1=R2=Ao OH OH
OAc 132. R= OH 133. R= H
1.4.3.12 Quassinoids from Hannoa chlorantha Hannoa chlorantha Planch, is a shrub used in Angolese traditional medicine, and the last two decades only a quassinoid was isolated of Hannoa chlorantha, 14-hydroxychaparrinone (138) [60]. HO,
454
1.4.3.13 Quassinoids from Harmoa klaineana Hannoa klaineana Pirre et Engler decoctions are used in African traditional medicine against fever and intestinal diseases [61]. Two quassinoids glycosides 15-0-P-D-glycopyranosyl-21hydroxyglaucarubolone (139) and 15-0-a-D-xyloruranosyl(l-»6)-pD-glycopyranosyl-21-hydroxyglaucarabolne (140) were isolated from the roots of Hannoa klaineana [62].
1.4.3.14 Quassinoids from Picrasma ailanthoides Planchon Quassinoids of the Japanese Picrasma ailanthoides Planchon (= P. quassioides Bennett) have been investigated in detail and more than twenty quassinoids have been obtained until 1984. However, very few reports for quassinoid glycosides have been found [63]. Eight quassinoids glycosides, picrasinoside A (141), B (142), C (143), D (144), E (145), F (146), G (147), H (148), and quassinoids hemiacetals, picrasinol A (149), B (ISO), C (1S1), and D (152) were isolated from Picrasma ailanthoides the last two decades [63-66].
455 OMe
OMe
GlcO,
MeO, OGk
MeO, ""OH
MeO,
I H 151
152
OH
150
1.4.3.15 Quassinoids from Picrasma crenata Picrasma crenata (Veil.) Engler is a Brazilian tree, which is used in traditional medicine to treat Diabetes mellitus, gastric disturbance and hypertension [67]. Three quassinoids quassin type, Pdihydronorneoquassin (153), 16-P-O-methylneoquassin (154), and 16p-0-ethylneoquassin (155) were isolated from Picrasma crenata [6768].
456 OMe
OMe HO,
MeO,
MeO,
MeO, OH
OMe
. i \ H 155
154
153
1.4.3.16 Quassinoids from Picrasma javanica Picrasma javanica is a medium-sized tree found in New Guinea, Southeast Asia and Indonesia. Decoctions of its bark are used in folk medicine as a febrifuge and as substitute for quinine [69]. OMe
OMe HO,, MeO,
MeO,
157. H l«0. H 164. H 166. OH 170. H
H OH H H H
H aOMe H O H aOH H O OH O
159. Me 162. Ac 163. PhCO 167. H 169. Ac
MeO. OMe
OMe
HO, MeO,
MeO, OMe
O MeO,
172. H Ac OH O 177. OH Me H O 179. OH Me H aOH
OMe HO,
175. R!= Me R2= H 176. R,= H R2= OH
MeO,
R2 Me Me Ac OH Me H Me H Me H
457
Twenty-four new picrasane quassinoids have been isolated from the leaves, roots and bark of Picrasma javanica, and they were named of javanicin B (156) [69], E (157), F (158), G (159) [70], H (160), I (161), J (162) [71], K (163), L (164), O (165), R (166), S (167), T (168) [72], N (169), P (170), Q (171) [73], U (172), V (173), W (174), X (175), Y (176) [74], Z (177), dihydrojavanicin Z (178), and hemiacetaljavanicin Z (179) [75]. Quassinoids glycosides has also been isolated of Picrasma javanica, and were denominated of javanicinoside B (180), C (181) [69], D (182), F (183), G (184), H (185) [76], I (186), J (187), K (188), L (189), and A (190) [77].
MeO,
"OGfc
180. Ac 183. Ac 184. Me 185. H
Ac Me Me Ac
OH H H OH
MeO,
187. Me OAo 188. H OAo 189. Me OH 190. H OMe
OH OH OH H
J. 4.3.17 Quassinoids from Quassia amara Quassia amara Wood is still widely used in traditional medicine and some quassinoids and quassinoid glycosides isolated from Quassia have received renewed attention due to their biological activity as potential antitumor agents [78].
458
HO
MeO. OMe
MeO.
= H 194
Six new quassinoids were isolated from Quassia amara Wood, dihydronoraeoquassin (191) [79], ll-a-0-(P-D-glycopyranosyl)-16a-O-methylneoquassin (192), l-a-0-methylquassin (193), 12-cthydroxy-13,18-dehydroparain (194), and 16-a-O-methylneoquassin (195), and 11-acetylparain (196) [78]. 1.4.3.18 Quassinoids from Quassia indica Indaquassin C (197), D (198), and E (199) were isolated from bark of Quassia indica. Their structures were determined by spectroscopic and chemical evidence [80].
OH
1.4.3.19 Quassinoids from Simaba multiflora
459
Two new quassinoids, 13,18-dehydro-6a-senecioyloxychaparrin (200) and 12-dehydro-6o>senecioyloxychaparrin (201), have been isolated from the fruits of Simaba multiflora [81]. HO, HO,.
200
1.4.3.20 Quassinoids from Simaba guianensis Simaba guianensis is a small tree that occurs in the flooded areas of the Amazon basin and is locally known as "cajurana". Its red fruits that ripen during the time of flooding are highly appreciated by fish and its bark is very bitter and is used by native populations against fevers [82], The new antimalarial (IC50 3.9-4.1 ng/mL) quassinoid named gutolactone (202) was isolated from the bark of Simaba guianensis [82]. OH I O .,
1.4.3.21 Quassinoids from Simaba orinocencis on
460
Simaba orinocencis Hunth (= S. multiflora) is a native tree found in the Amazonian riversides and seasonally inundated areas of South America [83]. A new antimalarial quassinoid, namely, orinocinolide (203), was isolated from the root bark of Simaba orinocencis [83]. The orinocinolide (203) was potent against Plasmodium falciparum clones D6 and W2 (IC5o 3.27 and 8.83 ng/mL). Orinocinolide (203) also inhibited growth of human cancer cells SKMEL, KB, BT-549, and SK-OV-3 [83], 1.4.3.22 Quassinoids from Soulamea amara Soulamea amara Lam., is a Simaroubaceae indigenous to Vanuatu (New-Hebrides). The new quassinoid 15-0-benzoylbrucein (204) was isolated from the aerial parts, and the structure has been established from spectral data and by single-crystal X-ray [84].
1.4.3.23 Quassinoids from Soulamea fraxinifolia Soulamea raxinifolia is a small tree New Caledonia that occurs in forest gallery of low altitude. It is characterized by the composed, barefaced leaves.
205
The studied sample comes from the region of Dumbea and the present survey has been led on peels of stems and leaves [85], From
461
the stem bark and leaves, a new quassinoid 6a-acetoxypicrasine B (205) was isolated [85]. 1.4.4 C22 Quassinoids Until 1985, only two quassinoids possessing the C22 basic skeleton were known: sergeolide (206) [86] and 15-deacetylsergeolide (207) [87] isolated from the roots of Picrolemma pseudocoffea. Sergeolide (206) is the first quassinoid to possess a butenolide function [1]. Two decades passed and only a new quassinoid with C22 skeleton was isolated so far. The new quassinoid (208) contained the y-lactone function bonded to ring A, in angular fashion, instead of linear for sergeolide (206) [88-89]. OH
O
o 208
1.4.5 C25 Quassinoids Until 1985, only six quassinoids possessing the C25 basic skeleton were known: simarolide (209) from the bark of Simarouba amara, picrasin A (210) isolated from Picrasma quassioides, soulameolide (211) from Soulamea tomentosa, simarinolide (212) and guanepolide (213) from the root bark of the Simaba cf. orinocencis, and deacetylsimarolide (214) isolated from the fruits of Simaba moretii [1].
462
Since then, seven more C25 quassinoids have been discovered: Odyendane (215) and odyendene (216) from the Odyendea gabonensis [90], klaineanolide A (217) and B (218) from the Hannoa klaineana [61], indaquassin F (219) from the Quassia indica [80], javanicinoside E (220) isolated from the Picrasma javanica [76] and the new epimer from simarolide (221) [91].
MeO,
R,0
H 209. Rj= H; R2= Ac; R3= pH 210. R!= Me; R2= Ac; R3= 0H; 2,3-dehydro 214. R!=R2= H; R = PH 221. Ri= H; R2= Ac; R3= ocH
212
213
2. BIOLOGICAL ACTIVITY OF QUASSINOIDS 2.1 Antimalarial Activity One estimates that throughout the world more than 300 million clinical cases of malaria occur every year, and over 1 million people
463
die of malaria [92], The vast majority of deaths occurs among young children in Africa, especially in remote rural areas with poor access to health services. In malaria-endemic regions and countries this disease is not only a health problem, but it also causes delay in economic development [92]. Malaria is a disease caused by Plasmodium protozoa and transmitted by of mosquito vectors (Anopheles spp.), bites [93] to men, monkeys, rodents, birds, and reptiles [94]. The main agents involved in human malaria fever are four species of Plasmodium protozoa (single-celled parasites): P. fakiparum, P. vivax, P. ovale, and P, malarie. Among these, P. falciparum accounts for the majority of infections, and it is the most lethal one. The control of disease is, among other factors, hampered by the ongoing spread of multidrug-resistant of Plasmodium falciparum [95] and the resistance of vector (Anopheles spp.) to insecticides [96]. Although malaria is a curable disorder if promptly diagnosed and adequately treated, a limited number of drugs for its treatment are available today [97]. In fact, the two most effective drags for malaria, quinine (222) and artemisinin (223), originate from plants; it is probable that other plants still contain undiscovered antimalarial substances. Many researches have focussed on trying to isolate and purify antimalarials from plants. The first studies about the antimalarial activity of Simaroubaceae's extracts date back to 1947 [98]. Cedronin (224) was isolated from Simaha cedron, a species popularly believed in South America to have antimalarial properties. It was examined for in vitro and in vivo antimalarial activities and for cytotoxicity against KB cells. Experimental results showed that cedronin was active against chloroquine-sensitive and resistant strain, with an ICso of 0.25 u,g/mL (0.65 ujnol/mL). It was also found to be active in vivo against P. vinkei with an IC50 of 1.8 mg/Kg (4.7 nM/Kg) in the classic 4-day test. Cedronin (224) show a rather low cytotoxicity against KB cells (IC50- 4 jig/mL, 10.4 uM); however its toxic/therapeutic ratio (10/1.8) remains lower than chloroquine (225) (10/0.5) [99].
464
222
225
223
Hannoa chlorantha and Hannoa klaineana are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and for quassinoids from H. chlorantha were examined in vitro against P. falciparum NF 54 [100]. OH
O H I H Otiglate 226.Ri=R 2 =R 3 =O 227. Rt= O-tiglale; R2= H; R3= OH 22&R,=R 3 =H;R 2 -OH
O
229
The extracts displayed good activities, while the quassinoids were highly active, with IC50 values very below 1 (xg/mL, those chaparrinone (226) and 15-desacetylundulatone (227) being much lower than 0,1 |ig/mL (0.037 and 0.047 M-g/mL, respectively). Chaparrinone (226) is five times more active than 14hydroxychaparrinone (228) against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity [100]. As 14-hydroxychaparrinone (228) has a seven-time higher cytotoxic activity against P-388 cells than chaparrinone (226), the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were also evaluated in vivo in a standard 4-day test. 15-desacetylundulatone (227) was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinnone (226) and 14hydroxychaparrinone (228) were active for at least 4 days.
465
Quassinoids have ED50 values much lower than 50 mg/Kg body weight/day and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone (227) is apparently of crucial importance for its high activity. 6-a-Tigloyloxyglaucarubol (229) was not active at all. Chaparrinone (226) is considered the most interesting of the investigated quassinoids and its in vivo antimalarial potential will be examined further [100]. In recent literature [101] the synthetic compound 3,15-di-Oacetylbruceolide (230), a derivative from bruceoside showed a potent in vitro antimalarial activity against Plasmodium falciparum equivalent to that of chloroquine (225) [102] and also a potent in vivo activity against Plasmodium berghei in mice, with low cytotoxicity against mouse mammary tumor, representing a model in the same host.
OCOCH,
o
o 230
The results obtained showed that 0.46 + 0.06 mg/Kg per day of 3,15-di-O-acetylbruceolide (230) caused 50% suppression of P. berghei in mice, while the ED50 values of chloroquine (225) and artemisinin (223) were 0.2 and 5.6 mg/kg per day [101]. 2.2 Anti-EW Activity In order to combat the Human immunodeficiency virus (HIV), the causative agent of the debilitating disease acquired immune deficiency syndrome (AIDS), colossal amount of money, manpower time and energy have been dedicated to research on compounds which can be developed as therapeutic agents. Research groups have devoted their efforts to hunt for compounds in different plant species, which inhibit HIV replication
466 466
and/or activities of HIV enzymes. A number of terpenoids and their derivatives, such as quassinoids have been reported to be anti-HIV agents [103]. Eighteen quassinoids glycosides and nine known quassinoids isolated from Brucea javanica, Brucea antidysenterica and Ailanthus altissima were tested for inhibitory activity against HIV replication in H9 lymphocytic cells [103]. Bruceoside-B (231), as well as yadanzioside-B (77) and -L (96), showed good EC50 values of 3,5, and 5 uM, respectively. However, their therapeutic indexes (TI) of 1, 0.8, and 2, respectively, indicate that they are toxic as anti-HIV agents. Likewise, quassinoid shinjulactone A (232) had a good EC50 value of ca. 5 uM but was cytotoxic at this concentration. Shinjulactone B (19) and ailantinol A (43) showed no cytotoxicity and were marginally active with EC50 values of 28 and 30 [oM, respectively. The most promising compound was shinjulactone C (233); this compound had a TI of >25 and showed significant anti-HIV activity with an EC50 value of 10.6 uM. Further studies on analogs and related compounds to increase the pharmacological profiles of (233) are in progress [103]. OH
OH
OH
GlcO
2.3 Anticancer Activity
O
HO
234
467 467
Since the isolation and characterization of braceantin (234) by Kupchan and associates over 30 years ago [104], several quassinoids have been tested against several types of tumors [1]. Several quassinoids isolated from Brucea antidysenterica in the series of antitumor agents of the National Cancer Institute, including: Bruceanol-A (64) and Bruceanol-B (65) [35] showed significant (T/C >120%) antileukemic activity in vivo against P-388 lymphocytic leukemia (3-day dosing) at T/C= 130% (0.5 mg/Kg), 129% (1 mg/Kg) and 134% (2 mg/Kg), and 123% (0.5 mg/Kg), respectively. The control, 5-fluorouracil, had T/C= 135% (200 mg/Kg, 1-day dosing). Braceantinoside C (72), Yadanzioside G (81) and Yadanzioside N (87) showed significant (EDso< 4,0 ug/mL) cytotoxicity in vitro against P-388 and L-1210 lymphocytic leukemia tissue culture cells at ED50= 2.12 and 3.50, 1.25 and 2.58, and 4.56 ug/mL, respectively [39]. The control drag, 5-fluorouracil, used in this assay showed EDso= 3.72 and 1.94 ug/mL, respectively. Bruceanol C (66) demonstrated potent cytotoxicity against human KB, A-549 lung carcinoma, and HCT-8 colon tumor as well as murine P-388 lymphocytic leukemia with ED50 values of < 0.04, 0.48, <0.40, and 0.56 ug/mL, respectively [36]. Bmceanols D (67), E (68), and F (69) were tested for in vitro cytotoxicity in five human tumor cell lines (KB, A-549, HCT-8, KPM-7951, TE-671) and a murine lymphocytic leukemia (P-388). The results shown in Table 1 [37]. All the compounds showed significant activity in each tested cell lines. Table 1. Cytotoxicity of Bruceanols D (67), E (68), and F (69) against various tumor cell lines.
67 68 69
KB
A549
0.08 0.55 0.43
0.55 3.75 0.55
Cell Line (EDso, pi/mL) TE-671 HCT-8 P-388 0.09 0.37 0.13
0.09 0.57 0.36
0.08 0.12 0.09
RPMI-7951 0.09 0.11 0.09
Sergeolide (206) and isobrucein B (235) isolated from Cedronia granaiensis showed significant activity against several
468
melanoma, colon, and non-small cell lung cancer with concentrations in range of 9-50 ug/mL [105]. OH OH_
,
,
.OCOCH•3
o
"hoH
H 235
Bruceoside C (97) isolated from Brucea javanica demonstrated potent cytotoxicities against human epidermoid carcinoma of the nasopharynx (KB) (ED50 < 0.1 |ig/mL), human lung carcinoma (A549) (ED50= 0.44 ug/mL), colon carcinoma (HCT-8) (ED5o= 4.51 lig/mL), melanoma (RPMI) (ED50 < 0.1 u,g/mL), and CNS carcinoma (TE-671) (EDso^ 0.29 u.g/mL), as well as murine lymphocytic leukemia (P-388) (ED50= 5.11 ug/mL) [45]. Bruceosides D (98), E (99), and F (100) showed selective cytotoxicity in the leukemia and non-small cell lung, colon, CNS, melanoma, and ovarian cancer cell lines with log GI50 values in the ranger of -4.14 to -5.72 [46]. Several natural products have been investigated for their inhibitory effects on 12-0-tetra-decanoylphorbol-13-acetate (TPA)induced Epstein-Barr virus early antigen (EBV-EA) activation and thus, as potential antitumor promoting agents [106]. Many quassinoids were isolated from Simaroubaceous plant to demonstrated antitumor activity, however, these compounds have not been subjected to a test yet for their antitumor promoting effects. Forty-five quassinoids isolated from the three Simaroubaceous plants, Brucea javanica, Brucea antidysenterica and Picrasma ailanthoides, and fourteen isolated from Ailanthus altissima were tested for Okano et al. [106-108] for their inhibitory activities using a short-term in vitro assay of the EBV-EA activation induced by TPA in Raji cells. The most seven potent compounds include isobrucein-B (235), bruceanol A (64), G (70), D (67), B (65), E (68), and bruceantin (234), which were all isolated from Brucea antidysenterica. All these compounds are aglycones with C=O and OH groups in ring A, a -
469
CHbO- bridge between Cg and Co, and an ester side chain at [106], Sixty percent of these compounds are obtained from Brucea javanica. Eighty-six percent in 60% compounds are glycosides. Like the most potent compounds, these glycosides also have C=O and OH groups in ring A, a -CH2O- bridge between Cg and C13 and an ester side chain at C15, but they are less active. Therefore, these results suggest that glycosylation reduces activity in quassinoid-type compounds [106]. Six quassinoids isolated from Eurycoma longifolia Jack, were subjected to in vitro tests on anti-tumor promoting. The most active compound for inhibition of tumor promoter-induced Epstein-Barr virus activation was 14,15J3-dihydroklaineanone (136, ICso= 5uM) [109]. This inhibitory potential was much higher than quercetin (ICso23 uM) and p-carotene ( I C J I F 30 uM), two common antitumor promoting natural agents [110]. 2.4 Antifeedant and Insecticidal Activity Four semi-synthetic and fourteen quassinoids were tested for their antifeedant and insecticidal activity against 3 rf instar larvae of the diamondback moth {Plutella xylostelld) [111], Results indicate that methoxyl groups at C2 and/or C12 or a methylenedioxy group between Cn and Cn were required for both activities. The very weak activity suggest that the conjugated double bond system from Cn to Cie did not contribute to the antifeedant and insecticidal activities [111]. Compounds showed weak activity in both assays, due to their hemiacetal structure. In general, all compounds showed potent antifeedant and insecticidal activity at concentrations of 16.0-63.7 pg/cm2. These compounds contained the following partial structures: an a,(3-unsaturated carbonyl in ring A, vicinal hydroxy groups at Cn and C12, a methyleneoxy bridge between Cg and C13 and a side chain
atCistlll]. The most potent compounds were further tested at a lower concentration of 1.0-4.0 jig/cm2. These results can be summarized as follows: a) the aglycone, isobrucein-B (235), showed the highest potency in both assays. The antifeedant activity may result in its
470
insecticidal activity; b) The glycosides, bruceoside-C (97), yadanzioside-B (77) and yadanzioside-L (96), did not show insecticidal activity on the first day; however, on the second day, they showed potent activity [111]. Polonsky et al [112] reported the effect of forty-six natural and semi-synthetic quassinoids, on feeding of tobacco budworm {Heliothis virescens). Their activity is compared to that of the well-known antifeedant azadirachtin from Azadirachta indica Juss. Four quassinoids, indaquassin C (197), samaderins C (12), B (11) and A (5), isolated from the seeds and bark of Samadera indica [113], were tested for insect antifeedant against the tobaco cutworm, Spodoptera litura, Indaquassin C (197) was the most effective antifeedant. 2.5 Anti-parasitic Activity Some quassinoids isolated from the leaves of Eurycoma longifolia Jack, were subjected to in vitro antischistosomal activities [109]. Compounds longilactone (36), 11-dehydroklaineanone (134) and 14,15f3-dihydroxyklaineanone (136) showed significant inhibitory effects on adult schistosome movement (IM) and egg-laying (EL) of S. japonicum at 200 ug/mL as compared with those of control experiments using only DMSO (Table 2). At concentration of 20 u,g/mL, all compounds inhibited movement of schistosomes and had slight effects of three quassinoids, being evaluated to be weaker than that of a known drug, praziquantel. Table 2. Antischistosomal activities* of the guassinoids. 200 i-ig/mL
36 134 136 Control l d Control 2d Drug
IM" + + + +
EL° 20 ±16 193 ±34 113 ±146 1273 ±328 726 ±310 0
20 Ug/mL ELC + 133 ±108 + 306 ±306 166 ±165 +
Mb -
2M.g/mL
EL° 933 ±177 853 ± 537 833 ±99
Tasted using S. japonicum in triplicate e}speriments. *IM, inhibition of movement of adult schistogotnes; +, incomplete inhibition; -, no inhibition, C EL, number of eggs laid. ••Control (DMSO-1 and 2): Data in the medium containing 1% DMSO (v/v) in triplicate experiments. 'Drug, Praziquantel (2 ng/mL).
471 471
The nematocidal activity of 38 quassinoids, C19 or C20 was measured by using a species of Diplogastridae (Nematoda) to develop lead parasiticides [114]. From various quassinoids tested, samaderine B (11) displayed the most potent nematocidal activity with a minimum lethal concentration (MCL) of 2.0 x 10"5 M. The nematocidal activities of samaderine B (11) was 15-fold greater than that of albendazole (3.0 x 10"4 M), 10-fold greater than that of thiabendazole (2.0 x 10"4 M) and 7.5-fold greater than that of avermectin (1.5 x 10"4 M). Thus, samaderine B (11) may eventually be used as lead parasiticides. 2.6 Herbicidal Activity
H 236
Extracts of Ailanthus altissima stem bark were evaluated for herbicidal effects under field conditions in two outdoor trials. Previous investigations has shown Ailanthus altissima bark, extracted with methanol, yielded a strongly phytotoxic extract that contained ailanthone (236) as one of the major herbicidal compounds [115-116]. The first field trial investigation taking into account the level of activity and selectivity of the extract Ailanthus altissima bark extract was sprayed post-emergence into 17 species of weeds and crops at rates of 366, 177, 93, 47, 23, and 0 Kg/ha. These application rates provided herbicidal activity equivalent to 4.5, 2.2, 1.1, 0.6, 0.3, and 0.0 Kg of pure ailanthone (236) per hectare, based on the results of an extract and pure ailanthone (236) laboratory bioassay [115-116]. Strong herbicidal effects were observed within several days. Even the lowest rate caused mortality and injury in excess of 50% for nine out of 17 species, and a significant reduction in shoot biomass for 13 species.
472
The second field trial tested the ability of bark extract to control weeds under field conditions with horticultural crops (bush bean, cauliflower, sweet corn, and tomato). Ailanthus altissima bark extract was sprayed post-emergence at rates of 99, 50, 26, 13, and 0 Kg/ha, providing herbicidal activity equivalent to 1.1, 0.6, 0.3, 0.14, and 0.0 Kg of pure ailanthone (236) per hectare. Extract treatment provided partial weed control (greatest reduction in weed biomass was 40%), but also caused serious crop injury. Bush bean was the only crop that showed a significant increase in shoot biomass and fruit yield, compared to the non-weeded control. None of the crops, regardless of application rate, showed a level of shoot biomass or fruit yield comparable to the hand-weeded control. The herbicidal effects of Ailanthus altissima bark extract declined within the first few weeks after application, supporting previous evidence that ailanthone (236) is rapidly degraded under field conditions [115-116]. Seven quassinoids isolated from the leaves of Eurycoma longifolia showed as plant growth inhibitors [117]. Taking into account the tested compounds, the activities of 15Phydroxyklameanone (133) and 14,15f}-dihydroxyklaineanone (136) were significantly higher than the others. In particular, the activity of (136) against root growth is remarkable (Table 3). Table 3. Inhibitory activities of quassinoids 133 and 136 against the growth of cucumber and rice seedling. Cucumber seedling IC 50 (nM) ICso(MM) root growth Shoot growth
133 136
10.5 ±0.5 2.510.5
23.7 ±0.5 22.7 ±0.5
Rice seedling ICso(nM) ICso(nM) root growth shoot growth
>200 >200
>200 >200
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3. SYNTHETIC STUDIES OF QUASSINOIDS 3.1 Chemical transformations in the quassinoids Quassinoids have also attracted much attention as synthetic target molecules and numerous synthetic approaches have been developed including the total synthetic procedures. Many activities in quassinoid area were due partly to the fact these natural products possess a wide spectrum of biological properties including in vivo antileukemic, antiviral, antimalarial, antifeedant, and amoebicidal activity. Essential requirements for in vivo antileukemic activity are the presence of an epoxymethano bridge between C(8) and C(l 1) or C(8) and C(13), and an a,(3-unsaturated ketol unit in ring A [1]. The efforts at total synthesis quassinoid can be summarized as follows: Problems associated with construction of C(8), C(ll) bridged hemiketal structural array, which is common to numerous naturally occurring quassinoids. To follow is described the synthesis of pentacyclic alcohol (243) which features a protocol for elaboration of ring C functionality found in chaparrinone and related quassinoids. A facile five-step sequence commencing with picrasane derivative (237) has been developed for elaboration of the sensitive ring C hemiketal unit chaparrinone (226) (c.f. pentacyclic alcohol 243) [118].
241
474
The mild and efficient method detailed above for the elaboration of the sensitive C/E ring system of chaparrinone (226) and glaucarubolone should prove exceedingly useful in total synthesis quassinoid [118]. Chaparrin (244), which can be obtained in relatively large amount from Mexican Castela species, lacks these structural features and does not possess antineoplasic activity [119]. Biologically inactive but easily available chaparrin (244) was converted into potent antileukemic C-15 esters of glaucarubolone (245-247) and quassinoid analogs in which C-15 ester side chain has been replaced by an alkyl or alkenyl group. The synthetic methodology developed and described below has been applied to the preparation of C-15 ester derivatives and quassinoid analogs [120]. OH
244
* ykAJs. ,
245 R' = t-BufCH3)2Si(TBDMS) i = TBDMSiCl/DMF/Imidazole (40-45%) or TBDMS-enol ether of 2_ 3_ 2,4-pentanedione/DMF (85-88%) R -R -H ii = TMSTf/Pyridine/CHCl3 (95-98%) 246 R = TBDMS ; R1 = (CH3)3Si(TMS) in = LDA/LHF, -78°C MoOPH and temperature raised to -44°C (40-45%)R3 = H or KHMDS/THF, -78°C, Ph-SOrN-CH-Ph (66-70%) 247 R1 = TBDMS ; R2 = TMS \ / R3 = OH O
Among the antimalarial active quassinoids, bruceolide (248) is one of the representative compounds as a common core skeleton. On the basis of a comparative analysis for stability in mouse serum between 15-O-acetylbruceolide (257) and bruceolide 15-methyl carbonate (258), several 3,15-dialkyl carbonates of bruceolide (251, 252-256) were synthesized and their in vitro antimalarial activity was assessed. Methyl, ethyl, and isopropyl carbonates with pronounced in vitro activity were further evaluated for in vivo antimalarial potency. Both the methyl and ethyl carbonates significantly increased the mice's life span as compared with 3,15-di-O-accetylbruceolide (230) and chloroquine (225) [121].
475
Various other synthetic studies on bioactive quassinoids have been described in the literature [122-124], such as synthesis of a picrasanerelated intermediate from diterpenes constituents from Cupresaceae species, such asJunipems communis [125].
OMe OR i) Ac 2 O, pyr ii) HF-pyr; THF
. CHJCOO'
250R = TES 7 i) TESC1, pyr ii) rc-Bu4NF, CH2C12
O
i) AC2O, pyr ») alkyl chloroformate, pyr
H TESC1, pyr L
-bruceolide ( 248 R = H) *-249R =
230 R = CH 252 R = OCH 3 255 R = OCH(CH 3 ) 2 253 R = OCH 2 CH 3 256 R = O(CH 2 ) 3 CH 3
OH
I O
i) Ac2O, pyr ii) alkyl chtoroforraate, pyr iii)HF-pyr, THF, 0"C
o OMe OOCR
H I H
257 R = CH 3 (bruoeine B) 258 R = OCH 3
3.2 Total synthesis of Quassinoids In recent years, a number of studies on the synthesis of quassinoids have been reported [126]. Among them, total synthesis of quassin (possessing 7 chiral centers) and of castelanolide (possessing 9 chiral centers) both carried out Grieco's group constitute two successful works to synthesize natural quassinoids [1], Below the main total synthesis of some quassinoids are described.
476
3.2.1 (±)-Amarolide Amarolide (267) is one of the representative quassinoid, and was first isolated from Ailanthus glandulosa [127] and A. altissima [128] being its structure established as (267). The total synthesis of (±)-amarolide (267), a quassinoid having 10 chiral centers, was accomplished from the previously reported 12(3hydroxy-pricasan-3-one (259) by 18 step reactions. The synthetic strategy applied by Hirota et al. [129] is outlined above. In order to transform (259) into amarolide (267), there were three points to be considered: /) the transposition of the carbonyl group from C-3 to C-l position (259 -» 264), if) the hydroxylation at C-2 and C-l 1 positions, both with the introduction of an cc-equatorial hydroxyl group (264 -» 265), and Hi) the oxidation of the oxane Dring to give the 6-lactone moiety (265 —» 267). OEE i) PhSeCl/TsOH/AcOEt ii) mCPBH/pyr
o
iii)EtoCH=CH2/PPTS/CH 2 Cl iv) H2O2/NaOH/THF-H2O
i) LDA/LTF; (CH3>3SiCl ii) mC-PBAItm, Ha,THF
lf, = H H * 260
ii)DBU/THF iii) TsOH/AcOEt OH
477
3.2.2 Simalikalactone D Simalikalactone D (282) [130] the major constituent of Quassia africana Baill. have occupied the attention of synthetic chemists for well over years, in part because these cytotoxic substance exhibit potent activity in vivo against the P-388 lymphocytic leukemia in mice (PS). The synthesis of simalikalactone D (282) described below by Grieco et al. [131] commences with the tetracyclic compound (268).
MeO
OMe
478
A cleavage of the fer/-butyldiphenylsilyl ether followed by thermodynamic silyl enol ether formation provided (269), which upon exposure to iV-iodosuccinimide in tetrahydrofuran and subsequent treatment with tetra-w-butylammonium fluoride afforded (270). After reprotection of the lactol, the resulting pentacyclic keto alcohol (271), was transformed into the corresponding tosylhydrazone, which was treated with excess methyllithium to give rise to olefin (272). Acetylation of (272) followed by exposure to osmium tetraoxide gave rise to diol acetate (273). Diol (273) was oxidized, the corresponding diketo acetate was hydrolyzed, and the resulting C(12) hydroxyl was reprotected as methoxymethyl ether, giving rise to (274). Selective formation of the methyl enol ether and reduction of the C(ll) keto function followed by protection of the resultant alcohol as methoxymethyl ether provided (275). Hydroboration of the methyl enol ether in (275) and subsequent oxidation provided crystalline (276). The selective hydrolysis of the protected lactol followed by treatment with methanesulfonyl chloride generated the sensitive dihydropyran (277), which was treated directly with osmium tetraoxide to give (278) and (279). Upon treatment of the mixture of (278) and (279) with excess manganese dioxide in chloroform there was of hydroxy lactone (280), which was treated with excess lithium hexamethyldisilazane and sequential addition of chlorotrimethylsilane and ./V-bromosuccinimide provided ketone (281), and exposure of tetra-butylammonium fluoride provided treated with (Z?)-2-methylbutyric anhydride, DMAP to give to two diastereoisomers, which were deprotected providing two diastereomers (282) (simalikalactone D) and (283) which separated by HPLC. 3.2.3 (±) Chaparrinone
479
Chaparrinone (226) is a major quassinoid found in the fruits of Hannoa klaineana Pierre and Engler [132]. The strategy for the constructing the ABC carbocyclic ring system of chaparrinone (226) was based on a Diels-Alder approach which necessitates, at some point in the synthesis, the inversion of configuration at C(9) (cf. structure 286). OTBEPS
OTBEPS H
I H I
TBDPSCI inridazolcDMF"!
M e O OLMMDS.THF.HMPA, , MejSO, ^ HD,,.. ' ii)B 2 H6,THF
1 * 1
H
i) BjJfc THF.NaOH, H;O2_
i)Bu 2 AIH,THF ii)MeOH,HClTHF
. T , - l OTBDPS i) HC1. Hfi. THF ' ' ' '
o **
& I H I
ii)PCC,NaOAc, C
i)PYHBT;CSA.THF n) LiBr, LizCOj THP, DMF ni)BBr 3 ,CH 2 Cl;
Toward this end, exposure (9 h) of dienophile (284) to 5.0 equiv. of dienoic acid (285) in 3.0 M LiClO-r-diethyl ether gave rise in 72% yield to crystalline keto acid (286), a which was reduction with sodium borohydride in methanol at 0°C by treatment with concentrated hydrochloric acid afforded the crystalline tetracyclic alcohol (287). To set the stage for the inversion of configuration at C(9), tetracyclic alcohol (287) was transformed into tetracyclic enone (291), via tetracyclic ketone (290), which was readily available by a five-step sequence from (287) described above [133].
480
3.2.4 5(R)- and 5(S)-Polyandranes The 5(R)- and 5(,S)-polyandranes (33 and 29), fas-lactones, were isolated from Castela texana and Castela polyandra [17], respectively, and have been shown by single-crystal X-ray analysis to possess the novel carbon skeleton. In view of the structural similarity between the polyandranes and the C20 quassinoids, it has been suggested that (33) and (29) are derived biogenetically from chaparrinone (226) [134]. The synthesis commences with the c/5-fiised dione (302), was prepared in near quantitative yield, and served as the logical starting material for the synthesis of (33) and (29).
iOSIOMCl
i) H;, Pd/C, BOH-lBg IHKr
OMOM H>T8,M«3H.
T l T
0BO*«(^.)
H
aOBtt^c^a,
TH|
>
p-^fHJ a '
""OMe MIR-Hn »«*"»
J3K-Ha UR-H8
481
The quinone Diels-Alder strategy gives rise to the proper stereochemistry at C(8) and C(14) and provides a as-fused BC ring system that ensures selective reduction of the C(7) carbonyl from the convex face of the molecule. Indeed, reduction of (301) with sodium borohydride in ethanol at 15 °C afforded (302). Adoption of the Diels-Alder approach depicted requires that the eventual C(9) configuration be inverted at some point in the synthesis and that the six-member B ring undergo ring contraction. Prior to addressing these two issues, the hydroxyl group in (302) was protected [TBDPSC1, imidazole, DMF, 70%] as its tertbutyldiphenylsilyl ether (303). The remaining of total synthesis of polyandranes, detailed above [135], constitutes the first published account of a total synthesis among this small group of, related Ms-lactones. 3,2.5 (±) Glaucambolone ami (d$ Holacanthone
Since the isolation and characterization of glaucambolone (329) by Polonsky in 1965 [132] and holacanthone (330) by Wall and Wani in
482
1970 [136], only limited of (329) and (330) have been made available from plant species. Attention has been focussed on quassinoids such as glaucarubolone and halacanthone, partly, because they display potent antileukemic activity [137], This is detailed below the first total synthesis of (±) glaucarubolone (329) and (+) holacanthone (330) by Grieco etal [138]. The synthesis of glaucarubolone described above commences with the tetracyclic alcohol (319), which we had prepared in conjunction with a synthesis of chaparrinone (226). The transformation of (319) into glaucarubolone requires: (a) incorporation of a 2-oxo-A3'4 olefin unit into ring A, (b) elaboration of the C(8), C(ll) bridged hemiketal structural array in ring C, and (c) introduction of a C(15) (3-hydroxyl group into the eventual ring D 8lactone, 3,2.6 (±) Shinjulactone C Examination of the bitter principles of Simaroubaceous plants, in particular Ailanthus altissima Swingle led to the discovery in 1983 of an intriguing quassinoid, Shinjulactone C (341), possessing a modified picrasane skeleton [139],
The total synthesis of the quassinoid shinjulactone C (341) is detailed above, which proceeds via pentacyclic lactone [140] and
483
features new protocols for elaboration of the ring A l(3-hydroxy-2oxo-A3'4 olefin unit and the C(8), C(ll) bridged hemiketal array which are present in numerous quassinoids. The synthesis of (341) starts with the know tetracyclic alcohol (331) utilized in synthesis of racemic chaparrinone (226). 3.2.7 (+) Bruceantin OSMDEFT
/
X > y - ^ V ^ OH
iJPCCNuOAcCHjClj.celile ii) T»NHNH;. TiOH, MgS04, THF iii)LDA.Dn>A.1HF ^
OMC
1
358
Bruceantin (234) has been tested in phase II clinical trial as an antitumor agent. However, it has not progressed to drug development yet. Oxidation of the side chain may cause deactivation of (234) and limit its efficacy [141].
484
Since the isolation and characterization of bruceantin (234) by Kupchan and associates over 30 years ago [104], synthetic organic chemists worldwide have been engaged in efforts to prepare (234) and closely related quassinoids via total synthesis. The synthesis of (234) described above for Grieco et al. commences with tricyclic ketone (342), which had been recognized over 20 years ago by number of synthetic groups as logical starting point for a synthesis of bruceantin (234) [142]. 3.2.8 (+)-14P, 15$-Dihydroxyklaineanone Examination of the extracts of Eurycoma longifolia Jack., a slender small tree indigenous to Southeast Asia which has played a major role in folk medicine, led to the isolation and the characterization via spectral methods of a new quassinoid, 14[3,15|3-dihydroxyklaineanone (136) [143],
The total synthesis of (136) described for Grieco et al [144] commences with the tetracyclic lactone (361), which possesses all the carbon atoms of 14p,15(3-dihydroxyklaineanone. Transformation of (361) into (136) requires elaboration of the 1p-hydroxy-2-oxo-A3'4
485
olefin functionality of events leading to the total synthesis of (136) proved to be critical. The total synthesis of the 14(3,15(3dihydroxyklaineanone (136) confirms the structural assignment put forth by Itokawa and coworkers [143]. 4. ABBREVIATIONS DBU DDQ Dess-Martin Oxid. DMAP DME DMF DMSO HMPA KHMDS LDA LiHMDS mCPBA M0MC1 MoOPH NMO PCC PhSO2(NCH)OPh PPTS PPTS Pyr. TBAF TBDM TBDMS TBDMSiCl TBDPSC1 TESC1 THF TMSC1 TMSTf TPAP
= 1, 8-diazabicyclo[5.4.0]undec-7-ene = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone = periodinane = dimethylaminopropylamine = 1,2-dimethoxyethane = dimethylformamide = dimethyl sulfoxide = hexamethylphosphoramide = potassium hexamethyldisilazide = lithium diisopropylamide = lithium 1,1,1,3,3,3-hexamethyldisilazane = Tn-chloroperoxybenzoic acid = methoxymethyl chloride =oxodiperoxymolybdenum-(pyr.)hexamethylphosphoramide = JV-methylmorpholine iV-oxide = pyridinium chlorochromate = 2-phenyl sulfony 1-3 -phenyloxaziridine = pyridinium/?-toluenesulfonate = pyridinium/?-toluenesulfonate = pyridine = tetrabutylammonium fluoride = tar/-butylchlorodiphenyl = /-butylchlorodiphenylsilane = /-butyldimethylsilyl chloride = /-butyldimethylphenylsilyl chloride = triethylsilyl chloride = tetrahydrofuran = trimethylsilyl chloride = trimethylsilyl triflate = tetrapropylammonium perruthenate
486
ACKNOWLEDGEMENTS The authors gratefully acknowledge grants from the Universidade Estadual do Norte Fluminense Darcy Ribeiro; and to CNPq for a research fellowship. 5. REFERENCES [I] [2]
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Moher, E. D,; Collins, J. L,; Grieco, P. A.; Journal of the American Chemical Society, 1992,114, 2764-2765. Polonsky, J.; Bourguignon-Zylber, N,; Bulletin de la Sociiti Chimique de France, 1965,2793. Gross, R. S.; Grieco, P. A.; Collins, J. L.; Journal of the American Chemical Society, 1990,112, 9436-9437. Furuno, T.; Naora, H.; Murae, T.; Hirota, H.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y ; Matsushita, K.; Chemical Letters, 1981,1797. Walker, D. P.; Grieco, P. A.; Journal of the American Chemical Society, 1999,121, 9891-9892. Wall, M. E.; Wani, M. C; Abstracts 7* International Symposium on Chemistry of Natural Products, R i p , USSR, 1970, E138,614. Kupchan, S. M.; Lacadie, J. A.; Howie, G. A.; Sickles, B. R. Journal of Medicinal Chemistry, 1976,19,359. Fleck, T. 1; Grieco, P. A.; Tetrahedron Letters, 1992,33,1813-1816. IsMbashi, M.; Tsuyuki, T.; Hirota, H.; Murae, T.; Takahashi, T.; Itai, A.; Iitaka, Y.; Bulletin of the Chemical Society of Japan., 1983,56,3683, Collins, J. L.; Grieco, P. A.; Gross, R. S.; Journal of Organic Chemistry, 1990,55, 5816-5818. CMen, M. M.; Rosazza, J. P.; Journal of Chemical Society Perkin Trans., 1981, i, 1352. VanderRoest, J. M.; Grieco, P. A.; Journal of the American Chemical Society, 1993,115, 5841-5842. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H.; Tanaka, O.; Chemical Letters, 1990, 749-752. Grieco, P. A.; Cowen ,S. D,; Mohammadi, F.; Tetrahedron Letters, 1996, 57, 2699-2702.
(Ed.) Studies Studies in in Natural Products Chemistry, Chemistry, Vol. Vol. 33 33 Atta-ur-Rahman (Ed.) © 2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©
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THE DITERPENOIDS FROM THE GENUS SIDERITIS FRANCO PIOZZI, MAURIZIO BRUNO, SERGIO ROSSELLI, ANTONELLA MAGGIO Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermo, Italy ABSTRACT: The genus Sideritis consists of 100-150 or more species, growing mainly in the countries around the Mediterranean area. The genus is particularly rich in diterpenoids, occurring in almost all the species, and shows many different carbon skeleta. Several species are still used in traditional medicine. Recent studies indicated the occurrence of interesting biological activities, like anti-inflammatory, antibacterial, antimicrobial, anti-HIV replication, antifeedant, antiulcerogenic, analgesic, and antihypoglycaemic.
INTRODUCTION The name of the genus Sideritis, family Lamiaceae (Labiatae), given by Dioscorides, seems to be derived from the Greek word "sideron" (iron) on account of an alleged efficacy in curing wounds produced with iron swords. The genus comprises at least from 100 to 150 species [1], occurring in temperate and tropical regions of the Northern Hemisphere. The countries around the Mediterranean Sea, and especially Spain, Canary Islands and Middle East, are particularly rich. The taxonomic classification of the genus is rather complex. The chemical investigations on Sideritis were concerned with flavonoids, essential oils, and especially diterpenoids. Diterpenoids occur in almost all the species and show a remarkable variability of carbon skeleta. The chemistry of these diterpenoids was reviewed by Al-Hazimi and Miana [2] in 1987 and by Gonzalez [3] in 1990. We wish to update these former reviews. The description of these natural diterpenoids will follow a chronological order, but their numbering will be done according to the
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different carbon skeleta (kaurane, atisane, beyerane, trachilobane, labdane, pimarane, abietane, rosane), almost all of which having the ent configuration. CHEMISTRY OF THE SECONDARY METABOLITES Chronologically, the first species investigated for diterpenoids was collected on the mountains of Sicily and was indicated as S. sicula Ucria [4,5]; later on, the species was included into S. syriaca L. [6,7] and finally validated as S. italica (Miller) Greuter& Burdet [8]. From its aerial parts, in 1965, two new diterpenes were isolated [9] and their structures elucidated in 1968 as sideridiol (33) and siderol (34) [10]. From the same species, some other new diterpenoids were isolated and characterised as sideroxol (54) [11], sideritriol (40) [12], epoxysiderol (55) [13], sideripol (35) and epoxysideritriol (60) [14], siderone (48) [15]. All these eight products have the en?-kaurane skeleton. This discovery of diterpenoids in this species roused the interest for the genus, especially of researchers in Canary Islands (Tenerife) and Spain (Madrid and Granada): many indigenous species were collected and studied and a considerable number of papers were published. The first following species to be studied was S. candicans var. eriocephala, collected in Canary Islands. Candicandiol (6), epieandicandiol (7) and candidiol (20) were isolated [16-17]; the structures of the first two products were later revised [18]. Some years later, three new diterpenoids were isolated, together with the known epicandicandiol (7), from an unidentified variety of S. candicans: candol A (2), candol B (4) and 7-acetyl-epicandicandiol (8) [19]. These six products also have the enf-kaurane skeleton. Many new diterpenoids, occurring in 5. canariensis, harvested in Canary Islands, have been also isolated. Trachinodiol (152) and trachinol (150) showing the enMrachilobane skeleton [20] were accompanied by epicandicandiol (7) and two known diterpenes: the hydrocarbons entkaur-16-ene (1) and dehydroabietane (161). Moreover enf-trachilobane (149), already known as a synthetic product, was found for the first time in nature. Later, the ketone tiganone (90) with a modified enf-labdane
495
backbone was isolated [21]. Finally, two enf-kauranes were obtained: vierol (62) and powerol (63) [22]. Another paper [23] reported the two new compounds: ribenol (72) (an enf-13-epimanoyloxide) and 7-aceryltrachinodiol (153), and the known enf-13-epimanoyloxide (69). Many years later, another paper [24] reported the occurrence in S. canariensis var. pannosa of eight new products: canadiol (28), acetyl-trachinol (151), formyl ribenol (74), 18-palmitoyl-traehinodiol (154), 7-aeetyl-18-formyltrachinodiol (156), sicanadiol (68), en£-2-oxo-13-epimanoyloxide (71) and en?-3|3-hydroxy-pimara-8(14),15-diene (160). Also sixteen known diterpenoids, that had been isolated in the meantime from other species of Sideritis, were found in this material: episinfernal (45), powerol (63), epicandicandiol (7), 7-acetyl-epicandicandiol (8), 18-palmitoylepicandicandiol (10), candol A (2), candol B (4), trachinol (150), ribenol (72), acetyl-ribenol (73), kauranol (61), trachinodiol (152), 7-acetyltrachinodiol (153), diacetyl-trachinodiol (155), enf-13-epimanoyloxide (69), and enf-3-oxo-13-epimanoyloxide (75). Another species attracting attention was S. leucantha; it grows in South-eastern Spain. A first paper [25] reported the occurrence of six new diterpenoids, all showing the en?-kaurane skeleton: foliol (16), sidol (17), linearol (18), isofoliol (36), isosidol (37) and isolinearol (38): the first three are enf-kaur-16-ene derivatives, the last three en?-kaur-15-ene derivatives. Later on, two other compounds were isolated [26]: leucanthol (24) (an enf-kaur-16-ene) and isoleucanthol (47) (an ent-kauT-15-ene); the complete experimental work was reported in another paper [27]. S. linearifolia, from Spain, yielded [25, 27] foliol (16), sidol (17) and linearol (18), whereas S. angustifolia [28], also from Spain, gave a new product, lagascatriol (158), for which an enf-isopimarane structure was suggested. Two years later, the structure was revised [29] and it was demonstrated that the 10a-Me had migrated on C-9, giving the rare skeleton of enf-rosane. Two more diterpenoids were isolated [30] from S. angustifolia: jativatriol (122) and eonchitriol (121), both having the entbeyer-15-ene skeleton. An en?-atisane, sideritol (143) was also extracted [31]; finally, the enf-atisane minor component isosideritol (146) was isolated and its structure was also elucidated by X-ray analysis [32, 33]. A chemical correlation between sideritol (143) and jativatriol (122) was established [34].
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The investigation of 5. pusilla, again from Spain, gave six ent-beyer15-ene derivatives [35-36]: pusillatriol (134), isopusillatriol (128), pusillatetrol (137), 14-acetyl-isopusillatriol (130), 7-acetyl-pusillatriol (135) and 7-acetyl-pusillatetrol (138). A partial synthesis of pusillatriol (134), starting from sideridiol, was also performed [37]. The structures proposed were confirmed by chemical transformation of 7-acetylpusillatriol into ent-beyerane [38]. Another Spanish species, S. mugronensis, yielded borjatriol (117), a diterpenoid with manoyloxide structure and normal configuration [39]. This was the first case of a normal configuration in the diterpenoids of Sideritis. The structure was amended some years later [40], changing the position of the secondary hydroxy group from 6a to 7(3. From S. grandiflora, also a Spanish species, the known sideridiol (33) and 7-acetyl-pusillatriol (135), and the new tartessol (127) were isolated; the latter product is an enf-beyerene derivative [41]. Eight already known diterpenoids were isolated from S. lagascana (Spain) [42]: foliol (16), isofoliol (36), sidol (17), isosidol (37), linearol (18), isolinearol (38), leucanthol (24), and isoleucanthol (47). Analogously, six already known products were found in 5. valverdei (Spain) [42]: pusillatriol (134), isopusillatriol (128), pusillatetrol (137), 14-acetyl-isopusillatriol (130), 7-acetyl-pusillatriol (135) and 7-acetylpusillatetrol (138). 5. glacialis, collected in Southern Spain, contained [43] the wellknown siderol (34) and epoxy-siderol (55). A subspecies of S. arborescens (Southern Spain) yielded [44] the known enf-kaurane siderol (34) and the new barbatol (89), a derivative of enMnanoyloxide. In a further paper [45], the occurrence of the new entlabdadiene andalusol (104) was reported. Another new enf-labdadiene, 6deoxyandalusol (100) was found [46] later. Other researchers isolated the three known enf-kauranes foliol (16), sidol (17) and linearol (18) [47] from a taxon now indicated as 5. arborescens (arborescens). At last, a new product, enf-llp\18-dihydroxy-kaur-16-ene (29) was extracted from the same taxon [48].
497
From S. serrata (South-eastern Spain) several diterpenoids were obtained [49]: the known lagascatriol (158), jativatriol (122), conchitriol (121), sideritol (143), and the new products serradiol (142) (an entatisane), benuol (119) and tobarrol (120) (both enf-beyeranes), lagascol (157) (an enf-rosane). Some years later, three minor em-beyeranes were isolated from the same source [50]: the known 12-acetyl-jativatriol (124) (see later) and the two new 1,17-diacetyl-jativatriol (126) and 1,12-diacetyl-jativatriol (125). Known compounds were isolated from three other Spanish species [51]: S. chamaedryfolia yielded sideridiol (33), foliol (16), S. hyssopifolia siderol (34), S. luteola foliol (16), sidol (17) and linearol (18), all with the en/-kaurane skeleton. Some years later, S. chamaedryfolia was reinvestigated and the structures of some minor products were elucidated: four known entkauranes: siderol (34), epicandicandiol (7), 7-acetyl-epicandicandiol (8), isofoliol (36); and seven new labdane derivatives: villenol (110), 19acetyl-villenol (111), villenolone (112), 19-acetyl-villenolone (113), villenatriolone (114), villenatriol (115), 19-acetyl-villenatriol (116). It is very interesting that these seven products have the (rare in Sideritis) normal configuration [52]. One year later, a re-examination [53] of this species led to the isolation of a new derivative, ent-11P, 18-dihydroxykaur-15-ene (49). In the eastern region, a Greek species was investigated [54]: S. theezans gave the new epoxy-isolinearol (58) and the known siderol (34), sideridiol (33), isolinearol (38), isosidol (37), sideroxol (54), isofoliol (36). Again from Southern Spain, S. reverchonii contained derivatives of enf-beyerane, enr-atisane, en?-isopimarane [55]: the new 12-acetyljativatriol (124), and the eight already known lagascol (157), lagascatriol (158), tobarrol (120), benuol (119), jativatriol (122), conchitriol (121), serradiol (142), sideritol (143).
498
The species S. gomerae, from Canary Islands, yielded five entlabdanes [56]: the four new eraMnanoyloxides, gomeraldehyde (95), 13epi-gomeraldehyde (87), gomeric acid (96), 13-epi-gomeric acid (88): and the known en?-8a,15-dihydroxy-labd-13(14)-ene (109): this last product had been isolated previously by Jefferies and Payne [57] from an Australian Bey era species. The investigation [58] of 5. paulii from Central Spain led to the isolation of the new epoxy-isofoliol (56) and of the two already known sideridiol (33) and isofoliol (36), all having the e«?-kaurane skeleton. Only the known enf-kaurane foliol (16) was found [59] in S. ochroleuca (Southern Spain). On the contrary, S. biflora (South-eastern Spain) was rich in diterpenoids [60]: the new epoxy-isosidol (57), 3acetyl-leucanthol (25), 18-acetyl-leucanthol (26) were accompanied by ten known products: linearol (18), isolinearol (38), sidol (17), isosidol (37), foliol (16), isofoliol (36), leucanthol (24), isoleucanthol (47), epoxyisolinearol (58), and epoxy-isofoliol (56), all with the en?-kaurane skeleton. Recent extensive HPLC analysis of S. biflora [61] allowed to identify also serradiol (142), conchitriol (121), lagascatriol (158), and andalusol (104). Four Greek species were studied [62]: they contained only known entkauranes. S. euboea gave sideridiol (33), siderol (34), sideroxol (54), epoxy-siderol (55), and isolinearol (38). S. roeseri yielded sideridiol (33), siderol (34), sideroxol (54), epoxy-siderol (55), epoxy-isolinearol (58). S. distans and S. syriaca contained sideridiol (33), siderol (34), sideroxol (54), and epoxy-siderol (55). In a second investigation of S. euboea [63], two new enr-kaurene were isolated, eubol (22) and eubotriol (21). A reinvestigation of 5. distans [64] allowed the isolation of a new kaurane distanol (64). Another paper referred to the reinvestigation of "5. syriaca" collected in Sicily that nowadays is known as S. italica [65]: the new ucriol (53) was isolated. Three other Spanish species were investigated [66]. S. crispata contained the known enf-kauranes linearol (18), isolinearol (38), eubotriol (21), leucanthol (24), 18-acetyl-leucanthol (26), 3-acetylleucanthol (25) and the new compound, 7,18-diacetyl-leucanthol (27). In the extract of 5. ilicifolia, four known diterpenoids were identified:
499
sideridiol (33), sideroxol (54), eubotriol (21), ew?-13-epi-manoyloxid-18oic acid (82). S. tragoriganum yielded five known products, serradiol (142), tobarrol (120), jativatriol (122), conchitriol (121), lagascatriol (158), and two compounds, 1-acetyl-sideritol (144) and 1-acetyljativatriol (123), known as semisynthetic derivatives but never found in nature previously. With the progress of investigations on more other species, it was rather easy to isolate only already known diterpenoids. So a species from North-western Spain (Leon), S. lurida yielded [67] four already known enr-kauranes: siderol (34), sideridiol (33), epicandicandiol (7) and 7-acetyl-epicandicandiol (8). 5. incana subsp. virgata, collected in Andalusia (Granada), contained [68] three known en?-kauranes: sideridiol (33), foliol (16) and isofoliol (36). The species S. scardica, growing in Thrace, gave six enr-kauranes [69]. Five of them were the well-known siderol (34), epoxysiderol (55), isolinearol (38), eubol (22) and sideroxol (54). The sixth product, 18acetyl-leucanthol (26) had been reported [60] as a constituent of 5. biflora, but no data had been given; the structure was confirmed by partial synthesis starting from natural isolinearol. Seven known en?-kauranes occurred [70] in S. funkiana, harvested near Granada: foliol (16), isofoliol (36), sidol (17), isosidol (37), linearol (18), isolinearol (38), and eubol (22). One year later, a re-examination of the same plant yielded [71] two new minor erc^-kaurane derivatives, funkiol (50) and sidofunkiol (51). In the same species, by HPLC analysis [72], the known borjatriol (117) was identified. Also in the case of S. hirsuta, coming from Granada, only ten known enr-kauranes were isolated [73]: siderol (34), sideridiol (33), sidol (17), isosidol (37), linearol (18), isolinearol (38), leucanthol (24), isoleucanthol (47), epicandicandiol (7), and 7-acetyl-epicandicandiol (8). Again from Andalusia came an unidentified subspecies of S. pusilla. Previously, the plant was denoted as S. leucantha var. tragoriganum.
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Moreover, this taxon is very similar to S. tragoriganum, because many products occur in both taxa. In total, seven enf-beyeranes and five entatisanes were identified [74-76]: conchitriol (121), jativatriol (122), 1acetyl-jativatriol (123), 12-acetyl-jativatriol (124), 1,12-diacetyljativatriol (125), 1,17-diacetyl-jativatriol (126), tobarrol (120), serradiol (142), isosideritol (146), 1-acetyl-sideritol (144), 1,17-diacetyl-sideritol (145) (new product), and atisideritol (147) (new product). The investigation of S. foetens, a species growing in some reduced areas of Southeast Spain, led to the isolation [77] of the known andalusol (104) and of the three new 6-acetyl-andalusol (105), 18-acetyl-andalusol (106) and 6-acetyl-isoandalusol (107). Some years later, other researchers studied the micropropagation of 5. foetens and the constituents of its extract: the same four products reported in the previous note [77] were found [78]. However, the reinvestigation of the naturally growing plant also yielded siderol (34) and ribenol (72), whereas 6-acetyl-isoandalusol (107) was missing [78]. Several known enr-kauranes were isolated [79] from 5. flavovirens, a species growing near Murcia (Spain) and botanically close to S. leucantha: foliol (16), isofoliol (36), linearol (18), isolinearol (38), sidol (17), isosidol (37), candol B (4). Indeed, many enr-kauranes were found in S. leucantha var. meridionalis, harvested near Granada [80]: linearol (18), eubotriol (21), leucanthol (2) 3-acetyl-leucanthol (25) and 18-acetyl-leucanthol (26). A group of five Spanish species growing in eastern Andalusia was studied jointly for chemotaxonomic purposes, and gave only known derivatives [81]. S. lacaite contained only siderol (34) and sideridiol (33). S. almeriensis yielded linearol (18), sidol (17) and foliol (16). From S. granatensis subsp. nijarensis, seven products were isolated: linearol (18), sidol (17), foliol (16), leucanthol (24), 3-acetyl-leucanthol (25), 18-acetylleucanthol (26), 7,18-diacetyl-leucanthol (27). 5. zafrae contained sidol (17), linearol (18), candidiol (20), 3-acetyl-leucanthol (25), 18-acetylleucanthol (26) and 7,18-diacetyl-leucanthol (27). Lastly, from S. leucantha var. incana five diterpenes were extracted: linearol (18), sidol (17), foliol (16), leucanthol (24) and 18-acetyl-leucanthol (26). It is
501
notable that all these products have the ent-kaurane skeleton. 5. granatensis, considered as an authentic taxa, contains [82] two entkauranes: 18-acetyl-leucanthol (26) and 7,18-diacetyl-leucanthol (27) and the enf-atisane serradiol (142), but not en?-beyeranes. Another species from Andalusia, 5. hirsuta subsp. nivalis gave [83] many known derivatives: siderol (34), linearol (18), sidol (17), leucanthol (24), 18-acetyl-leucanthol (26), foliol (16), ribenol (72), 6-deoxyandalusol (100), and a new enr-labdane, en?-8a-hydroxylabda-13(16),14diene (98). The same research group in Granada investigated S. varoi, a species rich in diterpenoids [84]. Seven known enf-kaurane derivatives were found: linearol (18), sidol (17), isolinearol (38), isosidol (37), 3-acetylleucanthol (25), 18-acetyl-leucanthol (26), and siderol (34). Entlabdadienes were also present: the known andalusol (102) and 6-deoxyandalusol (100), and the new 6-deoxy-andalusal (101). Six ent-13-epimanoyloxides were found in the plant: the known ribenol (72), and the five new varol (77), varodiol (78), 3-acetyl-varodiol (79), 12-acetylvarodiol (80) and 3,12-diacetyl-varodiol (81). A reinvestigation was performed [85] on the subspecies of S. pusilla previously [74-76] examined by the Granada group and now indicated by them as subsp. flavovirens. There is a serious taxonomic problem concerning four taxa: S. pusilla studied in the years 1973-1975 by the Madrid group [35-38], S. pusilla subsp. flavovirens, S. leucantha var. flavovirens [86] and S. flavovirens [79]. Indeed, 5. pusilla was shown to contain only ent-beyerane derivatives [35-38], whereas S. pusilla (subsp. not identified) gave ent-beyerane and enr-atisane diterpenoids [74-76]. The reinvestigation [85] afforded the isolation of the known ent-beyerane tartesol (127) [41], and of many acetylderivatives of pusillatriol (134)(136), isopusillatriol (129), (131), (132) and pusillatetrol (139)-(141). Moreover, the new enr-beyerane flavovirol (133) and the well-known entkaurane siderol (34) was isolated. A subsequent paper [86] reported a phytochemical comparison of the two species, 5. pusilla subsp. flavovirens and 5. leucantha subsp.
502
flavovirens. The latter contains only three well-known mf-kauranes: linearol (18), sidol (17), foliol (16), but no ent-beyeranes. The former is rich in ent-beyeranes. Therefore, these two taxa are believed to be clearly distinct. In the eastern region, a species collected in Southern Anatolia, S. perfoliata, was investigated. Only one diterpenoid was found [87], the new e«N2cc-hydroxy-13-epi-manoyloxide (70): no ent-kamanes were present. This finding is interesting because all the taxa from Central and Eastern Mediterranean regions examined till that year contained only entkauranes: this was the first exception. S. nutans is a species abundant in the island of Gomera, Canary Islands. In a first communication [88], the occurrence of entnorambreinolide (118) was reported. In a second paper [89], the identification of several diterpenoids was discussed. Some of them are the already known enMnanoyloxides: gomeraldehyde (95), 13-epigomeraldehyde (87), gomeric acid (96), 13-epi-gomeric acid (88) and ent2oc-hydroxy-13-epi-manoyloxide (70); A known enMrachilobane diterpene, 7-acetyl-trachinodiol (153) was also found. Four new entmanoyloxides were isolated: sidnutol (93), gomerol (94), 13-epi-gomerol (86) and 3oc-hydroxy-gomeric acid (97). The taxon S. arborescens subsp. paulii was collected in Andalusia and found to be rich in diterpenoids [90]. Some known eraf-labdanes were found: 6-deoxyandalusol (100), 6-deoxyandalusal (101), 6deoxyandalusoic acid (102), andalusol (104) and ribenol (72); also the ubiquitous ent-k&mane siderol (34) was present. Some new products were identified: the ercf-manoyloxides jabugodiol (92), 13-epi-jabugodiol (84), 13-epi-jabugotriol (85), 18-deoxyandalusol (103) and ent-18-hydroxy15(16)-peroxy-labda-13-ene (108); the latter product could be an artefact. It is difficult to say how much this taxon is similar to S. arborescens [4446] and to S. arborescens (arborescens) [47-48]. Again in Andalusia, the taxon S. varoi subsp. cuatrecasasii was harvested [91]. No m£-kauranes were found, whereas several known entmanoyloxides occurred: ribenol (72), varol (77), varodiol (78), 3-acetylvarodiol (79), 12-acetyl-varodiol (80), 3,12-diacetyl-varodiol (81) and 6deoxyandalusoic acid (102).
503
The extraction of S. varoi subsp. oriensis yielded [92] several known diterpenes: the enf-kaurane sideridiol (33), the en?-labdanes andalusol (104), 6-deoxy-andalusol (100), 6-deoxy-andalusal (101), 18-deoxyandalusol (103), 6-deoxy-andalusoic acid (102) and the 13-epimanoyloxide varodiol (78). Another subspecies, S. varoi subsp. nijarensis contained [93] the known ribenol (72), varodiol (78), 6-deoxyandalusal (101) and the new enf-3|3-hydroxy-manoyloxide (91). S. infernalis, a species from Canary Islands, contained [94] five new enf-kauranes: sinfernal (44), episinfernal (45), sinfernol (43), epoxysinfernol (59), and canditriol (23). Three known enf-kauranes were also present: candidiol (20), candicandiol (6), and candol B (4). Other species from Canary Islands were investigated. S. cystosiphon [95] gave two new ewf-kaurane derivatives, 7,18-diacetyl-epicandicandiol (11) and 18-palmitoyl-epicandicandiol (10). Several known enf-kauranes were also present: candol B (4), 7-acetyl-epicandicandiol (8), epicandicandiol (7), candidiol (20) and episinfernal (45). Some years later, a reinvestigation of S. cystosiphon [96] yielded new esters: 7-acetyl18-palmitoyl-epicandicandiol (12), 18-acetyl-epicandicandiol (9), 7acetyl-sideritriol (41), 7,17-diacetyl-sideritriol (42), and 7-acetylepisinfernal (46). A comparative phytochemical study of some ubiquitous species of Canary Islands reported [97] that S. macrostachya and 5. argosphacelus do not contain diterpenes. S. bolleana contains candicandiol (6), epicandicandiol (7), candidiol (20), 7-acetyl-epicandicandiol (8) and 7acetyl-trachinodiol (153). S. dasygnaphala yielded candol A (2) and epicandicandiol (7). From S. dendrochahorra candicandiol (6), epicandicandiol (7), 7-acetyl-epicandicandiol (8), 7-acetyl-trachinodiol (153) and 13-epimanoyloxide (69) were isolated. From S. candicans seven already known diterpenoids were identified: e«?-kaur-16-ene (1), candol B (4), candicandiol (6), 7-acetyl-epi-candicandiol (8), candidiol (20), 7-acetyl-trachinodiol (153) and dehydroabietane (161). S. candicans var. 3890 instead gave the known compounds candol A (2), candol B (4), epi-candicandiol (7), 7-acetyl-epi-candicandiol (8), candidiol (20), and
504
enM3-epi-manoyloxide (69). A reinvestigation of S. canariensis gave, apart from several already previously reported diterpenoids, the known, but not yet identified in these species, en/-kaur-16-ene (1), candol B (4), enr-8oc,15-dihydroxy-labd-13(14)-ene (109) and dehydroabietane (161). A reinvestigation of S. dendrochahorra [95] yielded another new entkaurane derivative, sidendrodiol (66), with an unusual 11,12 double bond. Two papers were concerned with S. javalambrensis, a species from Central Spain. The occurrence of the new e«f-16-hydroxy-13-epimanoyloxide (83) was reported [98]. Another novel en?-13-epi-12ocacetoxy-manoyloxide (76) was isolated [99]. Again from Canary Islands, 5. sventenii was found; it contained [96] three new enf-kauranes, sventenic acid (30), 7-acetyl-episinfernal (46) and 7-acetyl-sideritriol (41). Also some known enf-kaurane derivatives were found: candol B (4), epicandicandiol (7), 7-acetyl-epicandicandiol (8), episinfernal (45) and sideritriol (40). Another species from Canary Islands, S. ferrensis was very rich in diterpenoids [100]. Five new products were identified: diacetyltrachinodiol (155), enM8-acetoxy-16cc-hydroxy-atisane (148), and the enf-kauranes acetyl-candol A (3), acetyl-candol B (5), and ferrediol (39). Moreover, many known compounds occurred: epicandicandiol (7), 7acetyl-epicandicandiol (8), diacetyl-epicandicandiol (11), 18-palmitoylepicandicandiol (10), 7-acetyl-18-palmitoyl-epicandicandiol (12), candol A (2), candol B (4), 7-acetyl-trachinodiol (153), candidiol (20), candicandiol (6), episinfernal (45), enr-kaur-16-ene (1). A systematic chemotaxonomical study [101] was done on 5. massoniana. This species is rather complex and seems to exist as three different taxa: 5. massoniana forma longifolia in the island of Madeira; 5. massoniana var. crassifolia, again in the island of Madeira; S. massoniana var. pumila in Canary Islands. There is some affinity with S. candicans, specially for the two taxa from Madeira island; sometimes in the past S. massoniana and S. candicans were considered synonyms. All the three taxa contain 7-acetyl-epicandicandiol (8) and 7-acetyltrachinodiol (153); in forma longifolia also epicandicandiol (7), ribenol
505
(72), candol A (2), enf-2oc-hydroxy-13-epi-manoyloxide (70) and the acyclic diterpene trans-phytol (162) occur; var. crassifolia contains also epicandicandiol (7), 18-palmitoyl-epicandicandiol (10), episinfernal (45), dehydroabietane (161), e«?-2oc-hydroxy-13-epi-manoyloxide (70) and the acyclic diterpene 2-hydroxy-isophytol (163); var. pumila yields also candol B (4), 18-acetyl-sidendrodiol (67) and en?-18-acetoxy-16cchydroxy-atisane (148). In the last years, starting from 1996, increasing attention was given to species growing in the Eastern Mediterranean region, mainly by Turkish researchers. A species collected in Anatolia, S. huber-morathii, contained five known eraf-kauranes, siderol (34), sideridiol (33), sidol (17), linearol (18) and candicandiol (6), and a new product, 3,7,18-triacetyl-foliol (19) [102]. Also the 3,18-acetonide of foliol was found, but it could be an extraction artefact. However, a recent paper [103] of other authors pointed out that the product indicated [102] as candicandiol (6) is actually epicandicandiol (7). From 5. caesarea [102], only the known siderol (34) and epoxysiderol (55) were isolated. The species S. athoa, growing in Greece and Turkey, but harvested in Anatolia, gave [104] a new enr-kauranes, athonolone (65) and a product claimed the new en?-3cc,18-dihydroxy-kaur-16-ene (15). Also five known enf-kauranes: linearol (18), foliol (16), sidol (17), epicandicandiol (7), en?-3|3-hydroxy-kaur-16-ene (13), were isolated. Another diterpene, indicated as enf-3|3,7cc-dihydroxy-kaur-16-ene (14), was claimed to be a known product. Recently, these results were questioned [105] by other authors: the "new" enf-3(3a,18-dihydroxy-kaur-16-ene (15) is the known canadiol (28), whereas the "known" ent-3|3,7a-dihydroxy-kaur-16-ene (14) was never reported previously; hence the structure of this product isolated from S. athoa is worthy of further investigations. In a recent paper on the phytochemical analysis of some Turkish Sideritis species, the occurrence of the known beyerene flavovirol (133) in S. athoa was quoted [106]. Five species, collected in Anatolia, were investigated [107]. They contained only known enf-kauranes: 5. akmanii, linearol (18), isolinearol (38), foliol (16), isofoliol (36), sideridiol (33) and sideroxol (54). S. niveotomentosa, linearol (18), foliol (16) and epicandicandiol (7); 5.
506
brevidens, linearol (18), epicandicandiol (7) and sidol (17); S. rubriflora, linearol (18), sideroxol (54), epicandicandiol (7) and sidol (17); 5. gulendamii, linearol (18) and epicandicandiol (7). Another Turkish species, S. argyrea was very rich in known entkauranes [108]: candol B (4), epicandicandiol (7), 7-acetylepicandicandiol (8), foliol (16), linearol (18), sidol (17), 18-acetylepicandicandiol (9), siderol (34) and sideridiol (33). Beside these products, the new enr-6p\8a-dihydroxylabd-13(16),14-diene (99) was isolated; this finding is remarkable, because usually the species originating from the Eastern Mediterranean Basin contain only entkaurane derivatives. Two more Turkish species yielded many diterpenoids [109]. S. sipylea contained the seven known en?-kauranes linearol (18), epicandicandiol (7), sideridiol (33), siderol (34), isolinearol (38), isosidol (37), and epoxyisolinearol (58). S. dichotoma gave six known enr-kauranes: sideridiol (33), siderol (34), sideroxol (54), epoxysiderol (55), eubol (22) and eubotriol (21). The known flavovirol (133) was also present, as another exception to the occurrence of the only en^-kaurane derivative. S. trojana was collected in the Marmara region. It contained [110] six known erar-kauranes: siderol (34), sideridiol (33), epicandicandiol (7), isocandol B (31), acetyl-candol A (3) and en?-7a-acetoxy-kaur-15-ene (32). Moreover, a new en?-7a-acetoxy-15(3,16P-epoxy-kaurane (52) was isolated. Also a new en?-2oc-hydroxy-8(14),15-pimaradiene (159) was found: another exception to the occurrence of only enf-kauranes. In the same paper the authors reported the isolation of flavovirol (133) from 5. argyrea. As for the isolation techniques, the use of HPLC is increasing [61,111] also for qualitative and quantitative determinations. In these papers, several species have been studied or reinvestigated: S. almeriensis [61], S. bourgeana [111], S. cillensis [61], S. incana subsp. incana [111], 5. leucantha subsp. incana [111], S. leucantha subsp. incana var. meridionalis [111], S. leucantha var. serratifolia, S. luteola [61], S. pusilla subsp. almeriensis [111], and S. pusilla subsp. pusilla var. granatensis [111]. In these taxa, several known diterpenoids have been identified and their occurrence is reported in Table 1.
507
As far as we know about synthetic studies, no total synthesis of the natural diterpenoids from Sideritis was reported, whereas many hemisyntheses and correlations are known.
1 2 3 4 S 6 7 8 9 10 11 12
13 14 15 16 17 18 19
R=H R=H R=H R = OH R = OAc R = OH R = OH R = OH R = OAc R = Opalm R = OAc R = Opakn
R=H R=H R = OH R = OH R = OH R = OAc R = OAc
R' = H R1 = p-OH R' = P-OAc R' = H R' = H R' = a-OH R' = P-OH R1 = P-OAc R1 = P-OH R' = P-OH R' = P-OAc R' = P-OAc
R' = H R' = OH R' = H R' = OH R' = OH R' = OH R' = OAc
eni-kaur-16-ene candol A acetyl-candol A candol B acetyl-candol B candicandiol epicandicandiol 7-acetyl-epicandicandiol 18-acetyl-epicandicandiol 18-palmitoyl-epicandieandiol 7,18-diacetyl-epicandicandiol 7-acetyl-18-palmitoyl-epicandicandiol
R" = a-OH R" = a-OH R" = P-OH R" = a-OH R"=a-OAc R" = a-OH R" = a-OAc
en*-3P-hydroxy-kaur-16-ene enf-3P,7a-dihydroxy-kaur-16-ene cn?-3a,18-dihydroxy-kaur-16-ene foliol sidol linearol 3,7,18-triacetyl-folioI
508
OH R'
20 21 22 23 24 25 26 27
R=H R=H R=H R=H R=H R=H R = Ac R = Ac
R' = H R' = P-OH R' = P-OAc R' = oc-OH R' = P-OH R' = P-OH R' = P-OH R' = P-OAc
R"=H R"=H R" = H R"=H R" = OH R" = OAc R" = OH R" = OH
candidiol eubotriol eubol canditriol leucanthol 3-acetyl-leucanthol 18-acetyl-leucanthol 7,18-diacetyl-leucanthol
OH
28 29
R = OH R=H
R' = H R'= OH
canadiol enr-lip,18-dihydroxy-kaur-16-ene
OH
30 sventenic acid
509
R'
31 32 33 34 35 36 37 38
R = OH R=H R = OH R = OH R = OAc R = OH R = OH R = OAc
R' = H R' = OAc R' = OH R' = OAc R' = OH R=OH R' = OH R' = OH
39 40 41 42 43 44 45 46 47
R=H R=H R=H R=H R=H R=H R=H R=H R = OH
R' = H R' = (3-OH R' = P-OAc R' = P-OAc R' = a-OH R' = a-OH R' = P-OH R' = P-OAc R' = P-OH
R" = H R" = H R" = H R" = H R" = H R" = OH R" = OAc R" = OH
isocandol B ent-7a-acetoxy-kaur-15-ene sideridiol siderol sideripol isofoliol isosidol isolinearol
R" = CH2OH R" = CH2OH R" = CH2OH R" = CH2OAc R" = CH2OH R" = CHO R" = CHO R" = CHO R" = CH2OH
ferrediol sideritriol 7-acetyl-sideritriol 7,17-diacetyl-sideritriol sinfernol sinfernal episinfernal 7-acetyl-episinfernal isoleucanthol
510
()H 48 R = O 49 R = H,H
R' = H R' = OH
siderone ent-11P, 18-dihydroxy-kaur-15-ene
OH
50 R = Ac 51
R' = H R' = Ac
funkiol sidofunkiol
R'
52 53 54 55 56 57 58
R=H R = OH R = OH R = OH R = OH R = OH R = OAc
R' = P-OAc R' = cc-OH R' = P-OH R' = P-OAc R' = P-OH R' = P-OH R' = P-OH
R"=H R" = H R" = H R"=H R" = OH R" = OAc R" = OH
enf-7oc-acetoxy-15P,16P-epoxy-kaurane ucriol sideroxol epoxysiderol epoxyisofoliol epoxyisosidol epoxylinearol
511 511
59 60
R = a-OH R = P-OH
R' = CHO R' = CH2OH
epoxysinfemal epoxysideritriol
OH
R'
61 R = H 62 R = OH 63 R = H 64 R = OH
R' = H R' = H R' = OH R' = OH
kauranol vierol powerol distanol
CH2OH
OH OH
65
athonolone
512
66 67 68
69 70 71 72 73 74 75
76 77 78
n 80 81
R = H,H R = H,P-OH R=O R = H,H R = H,H R = H,H R = H,H
R R R R R R
= = = = = =
H H OH OAc OH OAc
R = OH R = OAc R=H
R' = H R' = H R' = OH
R' = H,H R' = H,H R' = H,H R' = H,a-OH R' = H,a-OAc R' = H,a-OCHO R' = O
R1 = R' = R' = R' = R' = R' =
OAc OH OH OH OAc OAc
sidendrodiol 18-acetyl-sidendrodiol sicanadiol
en/-13-epi-manoyloxide enf-2ct-hydroxy-13-epi-manoy loxide ent-2-oxo-13-epi-manoyloxide ribenol acetyl-ribenol farmyl-ribenol enf-3-oxo-13-epi-manoyloxide
enf-12a-acetoxy-13-epi-manoy loxide varol varodiol 3-acetyl-varodiol 12-acetyl-varodiol 3,12-diacetyl-varodiol
513
COOH
82
ent-13-epi-manoyloxide-18-oic acid
CH,OH
83 84 85
R=H R = OH R = OH
86 87 88 89
R' = H R' = H R' = OH
R = CH 2 OH R = CHO R = COOH R = CH 2 OH
e«M6-hydroxy-13-epi-manoyloxide 13-epi-jabugodiol 13-epi-jabugotriol
R' = H R' = H R' = H R' = OH
13-epi-gomerol 13-epi-gomeraldehyde 13-epi-gomeric acid barbatol
514
90
91 92
R=H R = OH
93 94 95 96 97
R R R R R
R' = H R' = OH
=H =H =H =H = OH
R' R' R' R' R'
= = = = =
tiganone
R" = OH R"=H
OH CH 2 OH CHO COOH COOH
ent-3|3 hydroxy-manoyloxide jabugodiol
sidnutol gomerol gomeraldehyde gomeric acid 3a-hydroxy-gomeric acid
515
98 R = CH 3 99 R = CH 3 100 101 102 103 104 105 106 107
R' = H R' = a-OH
R" = H R"=H
e«f-8a-hydroxy-labda-13(16),14-diene enf-6P,8a-hydroxy-labda-13(16),14diene R = CH2OH R' = H R"=H 6-deoxyandalusol R = CHO R"=H R' = H 6-deoxyandalusal R = COOH R " = H 6-deoxyandalusoic acid R' = H R = CH3 R' = P-OH R " = H 18-deoxyandalusol R = CH2OH R = (3-OH R" = H andalusol R = CH2OH R = P-OAc R " = H 6-acetyl-andalusol R = CH2OAc R' = P-OH R" = H 18-acetyl-andalusol R = CH 3 R' = P-OAc R" = OH 6-acetyl-isoandalusol
108
e«?-8a,18-dihydroxy-15,16-peroxy-labda-13-ene
109
e«f-8a,15-dihydroxy-labda-13-ene
516
•CH 2 OH
CH,OR
110 111
villenol 19-acetyl-villenol
R=H R = Ac
CH,OH
CH,OR
112 113 114
R = H R' = H R = Ac R' = H R = H R' = OH
viUenolone 19-acetyl-viUenolone villenatriolone
~CH,OH
OH CH 2 OR
115 116
villenatriol 19-acety 1-villenatriol
R=H R = Ac
H
OH
117
borjatriol
517 517
118
enMior-ambreinolide
CH 2 OH
119 120 121
benuol tobarrol conchitriol
R' = H R' = OH R' - O H
R = OH R =H R = OH
OR1 CH,OR" OR
122 123 124 125 126
R =H R = Ac R =H R = Ac R = Ac
R' = H R' = H R' = Ac R' = Ac R' = H
R"=H R"=H R" = H R"=H R" = Ac
jativatriol 1-acetyl-jativatriol 12-acetyl-jativatriol 1,12-diacetyl-jatrivatriol 1,17-diacetyl-jatrivatriol
518
OR
127 128 129 130 131 132
R =H R =H R =H R =H R=H R = Ac
133 134 135 136
R' = OAc R=OH R' = OH R' = OAc R' = OAc R' = OAc
R" = H R" = OH R" = OAc R" = OH R" = OAc R" = OH
tartessol isopusillatriol 3-acetyl-isopusillatriol 14-acety 1-isopusillatriol 3,14-diacety 1-isopusillatriol 14,18-diacetyl-isopusillatrio
R = OH R = OH R = OAc R = OH
flavovirol pusillatriol 7-acetyl-pusillatriol 14-acetyl-pusillatriol
R"'O
137 138 139 140 141
R =H R =H R =H R = Ac R = Ac
R' = H R' = Ac R' = Ac R' = Ac R=H
R"=H R"=H R"=H R"=H R" = Ac
R'" = H R"1 = H R"1 = Ac R"' = H R1" = H
pusillatetrol 7-acetyl-pusillatetrol 3,7-diacetyl-pusillatetrol 7,18-diacetyl-pusillatetrol 14,18-diacetyl-pusillatetrol
519
"MCH,OR"
142 143 144 145 146
R R R R R
= = = = =
H OH OAc OAc H
R' = R' = R' = R' = R' =
H H H H OH
serradiol R"=H R" = H sideritol R"=H 1-acetyl sideritol R" = Ac 1,17-diacetylsideritol R"=H isosideritol OH
HO/,, Si
147
ICH,OH
atisideritol
R1 OAc
148
ent- 16a-hydroxy-18-acetoxy-atisane
520
R' R
149 150 151 152 153 154 155 156
R R R R R R R R
=H =H =H = OH = OH = Opalm = OAc = OCHO
R=H R' = OH R' = OAc R' = OH R' = OAc R' = OH R' = OAc R' = OAc
enf-trachilobane trachinol acetyl-trachinol trachinodiol 7-acetyl-trachinodiol 18-palmitoyl-trachinodiol diacetyl-trachinodiol 7-acetyl-18-formyl-trachinodiol
OH
157 158
159 160
R = OH R=H
R=H R = OH
R' = H R' = OH
lagascol lagascatriol
em-2a-hydroxy-pimara-8(14),15-diene enf-3P-hydroxy-pimara-8(14),15-diene
521
161
dehydroabietane
OH
162
trans-phytol
OH OH
163
2-hydroxy-isophytol
522 Table 1. Occurrence of diterpenoids in Sideritis taxa 5. almeriensis S. akmanii S. angustifolia S. arborescens S. arborescens subsp. arborescens S. arborescens subsp. pauli S. argyrea S. athoa S. biflora S. bolleana S. bourgeana S. brevidens S. caesarea S. canariensis S. canariensis subsp. pannosa S. candicans S. candicans subsp.?? S. candicans var. 3890 S. candicans var. eriocephala S. chamaedryfolia S. cillensis S. crispata S. cystosiphon S. dasygnaphala S. dendrochahorra S. dichotoma S. distans S. euboea S. ferrensis S. flavovirens S. foetens S. funkiana S. glacialis S. gomerae S. granatensis S. granatensis subsp. nijarensis S. grandiflora S. gulendamii S. hirsuta S. hirsuta subsp. nivalis S. huber-morathii S. hyssopifolia S. ilicifolia S. incana subsp. incana S. incana subsp. virgata S. infernalis
16 [81], 17 [81], 18 [81], 36 [61], 121 [61], 158 [61] 16,18, 33,36,38, 54 [107] 117 [119], 121 [30], 122 [30], 143 [31], 146 [32, 33], 158 [28, 29] 34 [44], 89 [44], 100 [46], 104 [45] 16 [47], 17 [47], 18 [47], 29 [48] 34, 72, 84, 85, 92,100,101,102,103,104,108 [90] 4, 7, 8, 9,16,17,18,33, 34, 99,133 [108] 7 [104], 13 [104], 14 [104], 16 [104], 17 [104], 18 [104], 28 [105], 65 [104], 133 [106] 16 [60,61], 17 [60,61], 18 [60,61], 24 [60], 25 [60], 26 [60], 36 [60,61], 37 [60], 38 [60], 47 [60], 56 [60], 57 [60], 58 [60], 104 [61], 121 [61], 142 [61], 158 [61] 6, 7, 8, 20,153 [97] 16,36,104,142 [111] 7,17,18 [107] 34, 55 [102] 1 [97], 4 [97], 7 [20, 97], 62 [22, 97], 63 [22, 97], 69 [23, 97], 72 [23], 90 [21], 109 [97], 149 [20, 97], 150 [20, 97], 152 [20, 97], 153 [23, 97], 161 [97] 2,4, 7, 8,10, 28, 45, 61, 63, 68, 69, 71, 72, 73, 74, 75,150,151,152,154,155, 156,160 [24] 1, 4, 6,8, 20,153,161 [97] 2, 4, 7,8 [19] 2,4, 7, 8, 20, 69 [97] 6 [16, 17, 18], 7 [17, 18], 20 [17] 7 [52], 8 [52], 16 [51], 33 [51], 34 [52], 36 [52], 49 [53], 110 [52], 111 [52], 112 [52], 113 [52], 114 [52], 115 [52], 116 [52] 16,18, 34,104,142,158 [61] 18, 21, 24, 25, 26, 27, 38 [66] 4 [95], 7 [95], 8 [95], 9 [96], 10 [95], 11 [95], 12 [96], 20 [95], 41 [96], 42 [96], 45 [95], 46 [96] 2, 7 [97] 6 [97], 7 [97], 8 [97], 66 [97], 69 [97], 153 [95] 21, 22, 33, 34, 54, 55,133 [109] 33, 34, 54, 55, 64 [62] 21 [63], 22 [63], 33 [62], 34 [62], 38 [62], 54 [62], 55 [62] 1, 2, 3,4, 5, 6, 7, 8,10,11,12, 20, 39, 45,148,153,155 [100] 4,16,17,18,36, 37, 38 [79] 34 [78], 72 [78], 104 [77, 78], 105 [77, 78], 106 [77, 78], 107 [77, 78] 16 [70], 17 [70], 18 [70], 22 [70], 36 [70], 37 [70], 38 [70], 50 [71], 51 [71], 117 [72] 34, 55 [43] 87, 88, 95, 96,109 [56, 97] 26, 27,142 [82] 16,17,18,24, 25, 26, 27 [81] 33,127,135 [41] 7,18 [107] 7, 8,17,18, 24, 33,34,37, 38, 47 [73] 16,17,18,24, 26, 34, 72,98,100 [83] 7 [102, 103], 17 [102], 18 [102], 19 [102], 33 [102], 34 [102] 34 [51] 16,17,18, 24, 25, 26, 27 [81] 142,158 [111] 16, 33,36 [68] 4, 6, 20, 23, 43,44, 45, 59 [94]
523 S. italica S. javalambrensis S. lacaite S. lagascana S. leucantha S. leucantha subsp. flavovirens S. leucantha subsp. incana S. leucantha subsp. incana var. meridionalis S. leucantha subsp. meridionalis S. leucantha subsp. serratifolia S. linearifolia S. luteola S. lurida S. massonianu subsp. crassifolia S. massonianaf. longifolia S. massoniana subsp. pumila S. mugronensis S. niveotomentosa S. nutans S. ochroleuca S. paulii S. perfoliata S. pusilla S. pusilla subsp. ?? S. pusilla subsp. almeriensis S .pusilla subsp. flavovirens S. pusilla subsp. pusilla var. granatensis S. reverchonii S. roeseri S. rubriflora S. scardica S. serrata S. sipylea S. sventenii S. syriaca S. theezans S. tragoriganum S. trojana S. valverdei S. varoi S. varoi subsp. cuatrecasasii S. varoi subsp. nijarensis S. varoi subsp. oriensis S. zafrae
22 [14], 33 [10], 34 [10], 35 [14], 40 [12], 48 [15], 53 [65], 54 [11], 55 [13], 60 [14] 34 [125], 72 [125], 76 [99, 122], 77 [125], 83 [98, 125], 98 [122] 33, 34 [81] 16,17,18, 24, 36, 37, 38, 47 [42] 16 [25, 27], 17 [25, 27], 18 [25, 27], 24 [26, 27], 34 [25, 27], 36 [25, 27], 38 [25, 27], 47 [26, 27] 16,17,18 [86] 16 [81], 17 [81], 18 [81], 24 [81], 26 [81], 36 [111], 121 [111], 158 [111] 16, 36,104,121 [111] 2,18, 21,25, 26 [80] 17,36,142 [111] 16,17,18 [25, 27] 16 [51], 17 [51], 18 [51], 31 [61], 104 [61], 142 [61], 158 [61] 7, 8,33,34 [67] 7, 8,10,45, 70,153,161,163 [101] 2, 7, 8, 70, 72,153,163 [101] 4, 8, 67,148,153 [101] 117 [39,40, 72] 7,16,18 [107] 70 [89], 86 [89], 87 [89], 88 [89], 93 [89], 94 [89], 95 [89], 96 [89], 97 [89], 118 [88, 89], 153 [89] 16 [59] 33, 36, 56 [58] 70 [87] 120 [111], 128 [35], 130 [36], 134 [35], 135 [36], 137 [35], 138 [36] 120 [76], 121 [75], 122 [75], 123 [76], 124 [76], 125 [76], 126 [76], 142 [76], 144 [76], 145 [76], 146 [76], 147 [74, 75] 16,17,18,34,104,142 [111] 34,127,129,131,132,133,134,135,136,139,140,141 [85] 16,18,121 [111] 119,120,121,122,124,142,143,157,158 [55] 33, 34, 54, 57,58 [62] 7,17,18,54 [107] 22, 26, 34, 38,54, 55 [69] 119 [49], 120 [49], 121 [49], 122 [49], 124 [50], 125 [50], 126 [50], 142 [49], 143 [49], 157 [49], 158 [49] 1,18, 33,34,37, 38, 58 [109] 4,7,8, 30, 40, 41, 45, 46 [96] 33, 34, 54,55 [62] 33, 34, 36,37,38, 54 ,58 [54] 120,121,122,123,142,144,158 [66] 3,7,31,32,33,34,52,159 [110] 128,130,134,135,137,138 [45] 17,18, 25,26,34, 37, 38, 72,77, 78, 79, 80 , 81,100,101,102 [84] 72, 77, 78,79, 80 , 81,102 [91] 72, 78,91,101 [93] 33, 78,100,101,102,103,104 [92] 17,18,20, 25, 26, 27 [81]
524 Table 2. Diterpenoids and their occurrence Kauranes
4 5 6 7
en(-kaur-l 6-ene candolA acetyl-candol A candol B acetyl-candol B candicandiol epicandicandiol
[20],[97],[100], [19],[24],[97],[100],[101] [100],[110] [19],[24],[79],[94],[95],[96],[97],[101],[108] [100] [16],[17],[18],[94],[97],[100],[102] [17],[18],[19],[24],[52],[67],[73],[95],[96],[97],
8 9 10 11 12 13 14 15 16
7-acetyl-epicandicandiol 18-acetyl-epicandicandiol 18-palmitoyl-epicandicandiol 7,18-diacetyl-epicandicandiol 7-acetyl-18-palmitoyl-epicandicandiol enf-3P-hydroxy-kaur-16-ene ent-3 P,7(X-dihydroxy-kaur-16-ene ent-3a, 18-dihydroxy-kaur-16-ene foliol
[19],[52],[67],[97],[108] [96],[108] [24],[95],[100],[101] [95],[100] [96],[100] [104] [104] [104] [25],[27],[42],[47],[51],[59],[60],[61],[68],[70],
17
sidol
[25]i[27]![42]![47]![51],[60],[70],[73],[79],[81],
18
linearol
[25]![27]![42]![47],[51],[6b],[6l'],[70],'[73],[79], [80],[81],[83],[84],[86],[102],[104],[107],[108],
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
3,7,18-triacetyl-foliol candidiol eubotriol eubol canditriol leucanthol 3-acetyl-leucanthol 18-acetyl-leucanthol 7,18-diacetyl-leucanthol canadiol ent-11P, 18-dihydroxy-kaur-16-ene sventenic acid isocandol B £n?-7cc-acetoxy-kaur-15-ene sideridiol
[102]' [17], [81],[94],[95],[97],[100] [63],[66],[80],[109] [14],[63],[69],[70],[109] [94] [26],[27],[42],[60],[66],[80],[81],[83] [60],[66],[80],[81],[84] [60],[66],[69],[80],[81],[82],[83],[84] [66],[81],[82] [24] [48] [96] [110] [110] [10],[41],[51],[54],[58],[62],[66],[67],[68],[73],
34
siderol
[10],[43],[44],[51],[52],[54],[61],[62],[67],[69],
35 36
sideripol isofoliol
[14] [25],[27],[42],[52],[54],[58],[60],[61],[68],[70],
37 38
isosidol isolinearol
39 40 41 42 43
ferrediol sideritriol 7-acetyl-sideritriol 7,17-diacetyl-sideritriol sinfemol
[25],[27],[42],[54],[60],[70],[73],[79],[84],[109] [25],[27],[42],[54],[60],[62],[69],[70],[73],[79], [84],[107],[109] [100]
1 2
3
[96]' [96] [94]
525 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
sinfemal episinfernal 7-acetyl-episinfemal isoleucanthol siderone ent-11 p, 18-dihydroxy-kaur- 15-ene funkiol sidofunkiol ent-7a-acetoxy-15P,16p-epoxy-kaurane ucriol sideroxol epoxysiderol epoxyisofoliol epoxyisosidol epoxyisolinearol epoxysinfemol epoxysideritriol kauranol vierol powerol distanol athonolone sidendrodiol 18-acetyl-sidendrodiol sicanadiol
[94] [24],[94],[95],[96],[100],[101] [96] [26],[27],[42],[60] [15] [53] [71] [71] [110] [65] [ll],[54],[62],[66],[69],[107],[109] [13],[43],[62],[69],[102],[109] [58],[60] [60] [54],[60],[62],[109] [94] [14] [24] [22],[97] [22],[97],[24] [64] [104] [95] [101] [24]
Labdanes 69 70 71 72
ent-13-epi-manoyloxide «n?-2a-hydroxy-13-epi-manoyloxideenf-2-oxo-13-epi-manoyloxide ribenol
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
acetyl-ribenol fommyl-ribenol en/-3-oxo 13-epi-manoyloxide ent-12a-acetoxy- 13-epi-manoyloxide varol varodiol 3-acetyl-varodiol 12-acetyl-varodiol 3,12-diacetyl-varodiol enf-13-epi-manoyloxide-18-oic acid ent-16-hydroxy-13-epi-manoyloxide 13-epi-j abugodiol 13-epi-j abugotriol 13-epi-gomerol 13-epi-gomeraldehyde 13-epi-gomeric acid barbatol tiganone en«-3P-hydroxy-manoyloxide jabugodiol sidnutol gomerol gomeraldehyde gomeric acid
[23],[24],[97] [87],[89],[101] [24] [23],[24],[78],[83],[84],[90],[91],[93],[97],[101], [125] [24] [24] [24] [99],[122] [84],[91],[125] [84],[91],[92],[93] [84],[91] [84],[91] [84],[91] [66] [98],[125] [90] [90] [89] [56],[89],[97] [56],[89],[97] [44] [21] [93] [90] [89] [89] [56],[89],[97] [56],[89],[97]
526 97 98 99 100 101 102 103 104 105 106 107 108
3<x-hydroxy-gomeric acid enr-8a-hydroxy-labda-13(16),14-diene e«f-6P,8a-dihydroxy-labda-13(16),14-diene 6-deoxyandalusol 6-deoxyandalusal 6-deoxyandalusolic acid 18-deoxyandalusol andalusol 6-acetyl-andalusol 18-acetyl-andalusol 6-acetyl-isoandalusol ent-Sa, 18-dihydroxy-15,16-peroxy-labda-13-
[89] [83H122] [108] [46],[83],[84],[90],[92] [84],[90],[92],[93] [91],[90],[92] [90],[92] [45], [61],[77],[78],[84],[90],[92],[lll],[126] [77],[78] [77],[78] [77] [90]
ene
109 110 111 112 113 114 115 116 117 118
enf-8a,15-dihydroxy-labda-13-ene villenol 19-acety-villenol villenolone 19-acety-villenone villenatriolone villenatriol 19-acety-villenatriol borjatriol enf-norambreinolide
[56],[97] [52] [52] [52] [52] [52] [52] [52] [39],[40],[72],[119] [88],[89]
Beyeranes 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141
benuol tobarrol conchitriol jativatriol 1 -acetyl-jativatriol 12-acetyl-jativatriol 1,12-diacetyl-jativatriol 1,17-diacetyl-jativatriol tartesol isopusillatriol 3-acetyl-isopusillatriol 14-acetyl-isopusillatriol 3,14-diacetyl-isopusillatriol 14,18-diacetyl-isopusillatriol flavovirol pusillatriol 7-acetyl-pusillatriol 14-acetyl-pusillatriol pusillatetrol 7-acetyl-pusillatetrol 3,7-diacetyl-pusillatetrol 7,18-diacetyl-pusillatetrol 14,18-diacetyl-pusillatetrol
[49],[55] [49],[61],[55],[66],[76],[111] [30],[49],[55],[61],[66],[75],[lll] [30],[49],[55],[66],[75] [66],[76] [55],[50],[76] [50],[76] [50],[76] [41],[86] [35],[42],[36] [85] [36],[42] [85] [85] [85],[109],[106] [35],[36],[42],[86], [36],[41],[42],[86] [86] [35],[36],[42] [36],[42] [85] [85] [85]
Atisanes 142 143 144 145 146
serradiol sideritol 1-acetyl-sideritol 1,17-diacetyl-sideritol isosideritol
[49],[55],[61],[66],[76],[82],[111] [31],[49] [66],[76] [76] [33],[32],[76]
527 147 148
atisidentol eM-16ct-hydroxy-18-acetoxy-atisane
[74J,[75] [100],[101]
Trachilobanes 149 150 151 152 153 154 155 156
trachilobane trachinol acetyl-trachinol trachinodiol 7-acetyl-trachinodiol 18-palmitoyl-trachinodiol diacetyl-trachinodiol 7-acetyl-18-formyI-trachinodiol
[20],(22],[97] [20],[24],[97] [24] [20],[24],[97] [24]' [24],[100] [24]
Rosanes 157 158
lagascol lagascatriol
[49],[55]
Pimaranes 159 160
e«f-2a-hydroxy-pimara-8( 14), 15-diene ent-3p-hydroxy-pimara-8(14),15-diene
[110] [24]
Abietane 161
dehydroabietane
pmrnciou.
Acyclics 162 163
trans -phytol 2-hydroxy-isophytol
[101] [101]
BIOTRANSFORMATIONS Some microbiological reactions were attempted either on natural kaurane diterpenoids isolated from Sideritis or on their hemisynthetic derivatives, by incubating them with Gibberella fujikuroi. The purpose was the transformation of the products into gibberelline derivatives. However, epicandicandiol (7) yielded no gibbane compounds, only oxidation of the 19-Me occurred, with the formation of the CH2OH and COOH derivatives (A) and (B) [112]. In the analogous way, sideridiol (33), when incubated with G. fujikuroi, showed the oxidation of the 19-Me to the CH2OH group yielding (C) [113].
528
The same treatment on candol B (4) gave two oxidation products: both showed the insertion of a 7(3-OH group; in the first product the 19-Me was transformed into a carboxy group (D), in the second the oxidation of 19-Me was accompanied by the insertion of a 6a-0H with final formation of a 19,6 kaurenolide (E) [113]. On the contrary, candol A (2) was transformed into a mixture of gibberellic acid, fujenal and some gibberellines [113]. In the case of foliol (16) the incubation with G. fujikuroi brought about the hydroxylation of the hexocyclic methylene with the formation of a 16P-Me and 16a-0H system (F) [114]. In these papers, hypotheses are formulated on the effect of the different substituents on the reactivity of the substrates [112-114]. Recently, biotransformations have been performed by means of Mucor plumbeus [103]. The incubation of epicandicandiol (7) yielded foliol (16), sideritriol (40) and the tetrahydroxykaurane (G), while the incubation of candicandiol (6) gave canditriol (23) and the two polyhydroxy derivatives (H) and (I). The difference of activity due to the 7|3-OH or 7a-0H configurations is discussed.
OH
7 epicandicandiol OH
HOOCN
OH
529 529
OH OH
OH
2 candol A
fujenal
530 530 OH
OH OH
16 foliol OH
7
epicandicandiol
40
sideritriol
OH OH
6 candicandiol
23 canditriol
OH
531 531
BIOLOGICAL ACTIVITIES OF SIDERITIS DITERPENOIDS Infusions and decoctions of the aerial parts of some Sideritis species have been used for a long time in traditional medicine in Spain and other countries of the Mediterranean region, for their gastroprotective and antirheumatic properties [115] and also to treat inflammatory conditions [72,116,117]. The extracts of 5. taurica (in petroleum ether, ethanol, dichloromethane, n-butanol) and some of their chromatographic fractions were tested and reported [118] to be relatively non-toxic and to exibit significant anti-inflammatory, antiulcerogenic, analgesic and antihyperglycaemic activities, but not anticonvulsant and antipyretic effects. An interesting anti-inflammatory activity was found in borjatriol (117), a labdane diterpene isolated from S. mugronensis and occurring also in other species [119]. It is active in acute or chronic inflammation models and against oedema [72,120]. The activity was tested using the cotton pellet-granuloma assay: the compound inhibited the granuloma weight and the serum lysozyme activity in a dose-dependent way. £«?-16-hydroxy-13-epi-manoyloxide (83), extracted from S. javalambrensis, also has anti-inflammatory activity against oedema [121]. It was investigated using the carrageenan mouse paw oedema test: the inhibition of oedema was comparable with the effect of phenylbutazone. Anti-inflammatory activity was studied on two labdane derivatives isolated from S. javalambrensis [122-124], that also contains other known diterpenoids [98,99,125]. These two products, en?-13-epi-12a-acetoxymanoyloxide (76) and e«f-8a-hydroxy-labda-13(16),14-diene (98) were evaluated for anti-inflammatory action in vitro in the concentration range 10" M to 10" M and were compared with aspirin, sodium salicylate and indomethacine: neither compound affected superoxide generation or scavenging and nor did inhibit non-enzymatic lipid peroxidation. These two natural products interact with the eicosanoid system, perhaps at the phospholipase level, but do not interfere with the other tested leukocyte functions or with reactive oxygen species, and are essentially non-toxic to
532
leukocytes at sub-maximal doses. It was found that the product 98 inhibits prostaglandin generation in stimulated macrophages. However, it also enhances the release from membrane phospholipids of unsaturated fatty acids such as oleic acid and arachidonic acid. This could be due to inhibition of acyl-CoA-lysolecithin transferase. It was shown that product 98 appears to possess two mutually opposing actions on the eicosanoid system in macrophages: potentiation of delivery of substrate following cell activation, followed by inhibition of conversion of substrate to product. It is clear that this diterpenoid possesses a unique and diverse array of properties that lead to the alteration of proinflammatory enzyme pathways in macrophages. This biochemical profile distinguishes these diterpenoids from the antiinflammatory polyphenolics, such as flavonoids occurring in the genus Sideritis, and suggests that medicinal decoctions of these plants are likely to owe any anti-inflammatory activity to more than one bioactive ingredient. Also andalusol (104), extracted from S. foetens, was able to inhibit acute inflammatory processes induced by carrageenan [126]. It operates also on the induction of nitric oxide synthase NOS in murine cultured macrophages J774 and is able to scavenge NO directly [127-128]. Andalusol also showed remarkable immunomodulating properties [129]. It exhibited a dose-dependent inhibitory effect on the haemolytic activity of the classical complement pathway, and also inhibited lymphocyte proliferation induced by concanavalin at non-cytotoxic concentrations. No effect on the alternative pathway was observed. Therefore, andalusol has immunosuppressive effects in vitro. Several known labdane diterpenoids occurring in S. foetens were isolated also from plants micropropagated in vitro and proved to inhibit basal adenylacetylase activity [78]. The effect of kaurane derivatives like foliol (16) and linearol (18) was analyzed [130] on the activation of NFi 3 in LPS-treated J774 macrophages. Also lagascatriol (158) from 5. angustifolia showed inhibition on COX-2 pathway of PGE2 release in E. coli LPS-stimulated peritoneal macrophages [131].
533
Several beyerane and atisane diterpenoids isolated from S. pusilla and some of their semisynthetic derivatives were found to have an antimicrobial activity towards Gram-positive bacteria (S. aureus, B. subtilis, M. phlei) but were found to be inactive against Gram-negative bacteria (E. coli and P. aeruginosa). The beyerane 1-acetyljativatriol (123) was active against C. albicans. The relationship between the chemical structure of these products and their antimicrobial activity was discussed [132]. The same activity was observed [133] in the kaurane foliol (16) and the beyerane isopusillatriol (128). In a recent paper [106], the antibacterial activity was investigated on 27 known diterpenoids isolated from five species of Sideritis growing in Turkey. A certain activity, expressed as MIC (minimum inhibitory concentration), was observed for epicandicandiol (7) against E. coli and for sideroxol (54) against B. subtilis. The bioactivity on the feeding behaviour of the larvae of some pest insects of seven kaurane diterpenoids isolated from Turkish species of Sideritis was investigated [107]. These seven natural products showed a limited antifeedant activity against Spodoptera frugiperda, the most interesting being sideroxol (54). It is suggested that the presence of the epoxide in 54 might contribute to the activity of this compound. On the contrary, foliol (16) was a potent phagostimulant for Spodoptera littoralis, and none of the other six products showed antifeedant activity on the same insect species. The antifeedant activity was tested on linearol (18) and 20 of its semisynthetic derivatives (obtained by chemical modification of functional groups) against final stadium larvae of Spodopera littoralis. The binary choice test on glass-fibre discs at 100 ppm was used. While linearol did not influence the feeding behaviour of larvae, several of the derivatives showed a good antifeedant activity with a high Feeding Index, and another was a rather strong phagostimulant [134]. Recently, the activity against HIV replication has been tested on the kaurane linearol (18) and 26 of its semisynthetic derivatives [135]. Whereas linearol itself was inactive, five derivatives showed a significant activity against HIV replication in H9 lymphocyte cells with EC50 values
534
in the range 0.1-3.11 (ig/mL. Two of them, with TI values of 163 and 184, are specially promising for further development as potential antiHIV agents. REFERENCES [I] [2] [3]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 33 © 2006 Elsevier B.V. All rights reserved.
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RECENT DEVELOPMENTS IN THE ASYMMETRIC SYNTHESIS OF LIGNANS GIUSEPPE DEL SIGNORE2 AND OTTO MATHIAS BERNERb "Institutfur Organische Chemie, Rheinisch-Westfdlische Technische Hochschule, Professor-Pirlet-Str. 1, 52074 Aachen, Germany b Kemira Fine Chemicals Oy, P.O. BOX330, FIN-00101 Helsinki, Finland ABSTRACT: Lignans constitute a class of natural products with a great diversity in structure. They possess significant pharmacological activities, especially antiviral and antitumor properties, and have thus been the target of extensive synthetic research. This review will mainly focus on the recent advances in the asymmetric synthesis of the most important classes of lignans. Furthermore, biological properties of selected lignans will be presented.
INTRODUCTION Lignans are a class of secondary metabolites widely encountered in the plant kingdom. The term lignan was originally introduced by Harworth [1] to describe a group of plant phenols whose structure was determined by union of two cinnamic acid residues or their biogenetic equivalents, linked B,fi', Fig. (1).
Fig (1). General structure of lignans
542
Several hundred lignans have been discovered in different parts of various plants, including wooden parts, roots, leaves, flowers, fruits and seeds. The range of natural structures encountered is very diverse and can be exemplified with a proposed classification according to their skeleton [2], Fig. (2). Despite of recognizing the distribution of lignans in plants, their biological purpose in nature is still unclear in most cases. It is known, however, that accumulation of lignans in the core of trees is important for the durability and longevity of the species.
dibenzylbutan(diol)es
tetrahydrofurans
furofurans
(OH)
dibenzylbutyrolactones (OR3)
Ar tetralins
naphthalenes
dibenzocyclooctadienes
Fig. (2). Classification of Hgnans
Lignans are also assumed to function as phytoalexins, that is to provide protection for the plants against diseases and pests, hi addition, they may participate in controlling the growth of the plants. From a pharmacological point of view, these compounds display an array of different and interesting biological activities and have a long and fascinating history beginning with their use as folk remedies by many different cultures. No longer a subject for only the botanist, the lignans have thus attracted the interest of various branches of medicine as well as the pharmaceutical industry and have been the target of intensive synthetic studies. Continued research is currently focused on structure optimisation of the active natural lignans to generate derivatives with
543
superior pharmacological profiles as well as broader therapeutic scope. In the last twenty years a plethora of publications have appeared on the subject, including books [3,4] and numerous reviews [5,6,7,8,9]. However, only few reviews are focused on the asymmetric synthesis of lignans [10], the most recent one being that produced by Achiwa and coworkers in 1992 [11]. The aim of this review is therefore to report about the progress and new technologies developed in the asymmetric synthesis of most important lignan classes after 1992. Furthermore, ex-chiral pool syntheses will also be presented. Analytical methods and identification In the last years increasing attention has focused on finding new and appropriate methods for the analysis of lignans from plant sources and in body fluids. Conventional chromatographic methods, including reversedphase HPLC, still remain the most useful and applied techniques. For example, use of HPLC has been reported in the isolation of lignans from the extract of Steganotaenia araliacea [12] as well as from the extract of Agastache rugosa [13]. Three novel C19 homolignans and a new Cig dibenzocyclooctadiene has been isolated from Schizandra arisanensis by successive HPLC purification [14] and a similar technique has been used for the analysis of the extract of Chilean propolis [15]. Reversed-phase HPLC was applied in the isolation of two new diastereomeric, hepatoprotective lignans from Saurus chinensis, sauchinone and episauchinone [16] as well as in the identification of four new metabolities, aglacins A-D, from the methanolic extract oiAglaia cordata [17]. LC/'H NMR has been used to isolate lignans from an antioxidant fraction of Orophea enneandra [18], while GC-MS has been used to analyse trimethylsilyl derivatives of the lignans present in galls and shoots of Picea glauca [19]. Among the novel techniques, supercritical CO2 has been demonstrated to provide an effective method for the extraction of lignans from leaves, seeds and fruits of Schisandra chinensis [20]. Supercritical fluid extraction and supercritical fluid chromatography have also been employed in the isolation and analysis of magnolol and honokiol from Magnoliae Cortex [21]. Radio immuno assay has been developed as a novel method for the analysis of a new lignan-related hypocholestereolic agent in human and urine [22] whereas bioassay has been used in the isolation of lignans from Kadsura matsudai Hayata [23].
544
Finally, micellar electrokinetic chromatography has been recently applied to the separation and determination of podophyllium lignans [24]. Biological and clinical properties A broad range of biological activities have been associated with lignans, including antitumor, antimitotic, antimicrobial and antiviral activities. The aryltetralin lactone, (-)-podophyllotoxin (1), has been under continuos investigation due to its significant pharmacological activity, Fig. (3). Podophyllin (an alcoholic extract from the roots and rhizome of may apple which contains as the main active ingredient podophyllotoxin) was considered such a popular cathartic and choalagogue in America that it was included in the U.S. pharmacopeia. OH
Fig. (3). Structure of (-)-podophyllotoxin
However, in 1942 it was removed from U. S. pharmacopeia because of its severe gastrointestinal toxicity. Currently, it still remains as an effective therapy for treatment of venereal warts [25]. Furthermore, podophyllotoxin and its derivatives have been extensively studied in the last 60 years for their powerful antitumor effects. Podophyllotoxin itself is a powerful microtubule inhibitor. Microtubules are tubular polymers whose protomeric unit (a and (3-tubulin) forms an heterodimer and are the dynamic constituents of the cytoskeleton. The cytoplasm of eukaryotic cells contains a soluble pool of unpolymerised tubulin protomers as well as an organised array of microtubules. Microtubules can be rapidly assembled or disassembled in response to various stimuli with little or no change in the total amount of tubuline. Cytoplasmatic microtubules are
545
implicated in an array of different processes as cellular mobility, intracellular transport, secretion, organization of cytoplasm and proteins, and growth factor signalling. The action of podophyllotoxin basically consists in disrupting the dynamic equilibrium of assembled and disassembled microtubules in vitro and in vivo. The net result is the destruction of the cytoskeletal framework in the cytoplasm and in the spindle fibres causing inhibiton of cell division in the metaphase. This results in the arrest of cell duplication at the miotic stage. However, since podophyllotoxin attacks both normal and cancerous cells, the toxic side effect has limited its application as a drug in cancer therapy. Therefore, analogs of 1 have been prepared in order to reduce its toxic properties and to enhance its pharmacological profile. Particularly successful have been tenoposide (2) and etoposide (3), two semisynthetic derivatives currently used in treatment of small cell lung cancer, testicular carcinoma, lymphoma and Kaposi's sarcoma [26,27]. Another podophyllotoxin derivative, GL-331 (4), was undergoing phase II clinical trials for treatment of various cancers [28], Fig. (4).
H,C^^^-°
Fig. (4). Structures of etoposide, teniposide and GL-331
546
Antitumor activity has been observed in several other classes of lignans, including butyrolactones, for example burseran (5) [29], and dibenzocyelooctadienes such as steganacin (6) or steganone (7) [30], Fig, (5). Other dibenzocyclooctadiene lignans, such as schinzandrin B [31], are responsible for the antihepatotoxic effects of some of Chinese herbal remedies. The tetrahydrofuran lignans magnone A (9) and B (10) [32] have been found active against the bioactive phosfolipid PAF linked to various haematological responses, including aggregation and degranulation of platelets and neutrophils, and is an important mediator of inflammation and asthma.
,0
O
CH 3 O
PCH, OCHq
Fig. (5). Examples of biological active lignans
Finally, some lignans have also showed antimicrobic properties [33] as well as a synergistic effect on a range of insecticides [34]. Biogenesis The biogenesis of lignans is strictly related to the production of the plant polymer lignin. Lignin is a constituent of the plant cell wall and together with hemicellulose cements the cellulose microfibrils thus connecting
547
cells to one another and strengthening them. The chemical structure of lignin challenged scientists for decades. It does not possess an easily hydrolysable linear array of repeating units but rather is a threedimensional polymer incorporating many different stable carbon-carbon bonds and ether linkages between monomeric phenylpropane units. Lignans are presumed to be related to lignin because of the precursors and processes involved in the biogenesis of both compound classes. However, some differences must exist in the two biosynthetic pathways because lignans are almost entirely optically active whereas no optical activity has been found in lignins. Indeed, the first steps in the biosynthesis are common. A sequence of five reactions - deamination, aromatic hydroxylation, O-methylation, CoA-mediated ligation and NADPH mediated reduction - converts phenylalanine to monolignols such as coniferyl alcohol [35]. At this point the two biogentic pathways diverge. Lignins are probably formed by random radical polymerization whereas an enzyme catalysis is proposed to control the dimerization step of monolignols as well as the oxidation step in the biogenesis of lignans. The biosynthesis of lignans in plants has been established at least in the following cases: Forsythia intermedia Podophyllum peltatum, Thuja plicata and Linum Flavum [36,37,38,39,40,41]. The pathway involves a stereoselective coupling of two coniferyl alcohols (10) to afford (+)pinoresinol (11), Scheme (1). The protein responsible for defining the stereoselectivity of this process is not itself capable of bringing about the oxidation and therefore an auxiliary oxidase is required. Sequential stereoselective reductions of (+)-pinoresinol give (+)-lariciresinol (12) and (-)-secoisolariciresinol (13). Two isofunctional forms of reductase which are responsible for this process have been isolated. Both catalyse the sequential NADPH dependent stereospecific reduction of (+)pinoresinol and (+)-lariciresinol and have similar kinetic parameters as well as molecular weights. Stereoselective dehydrogenation of (-)secoisolariciresinol yields (-)-matairesinol (14) which is considered to be the branch point leading to other important classes of lignans such as the podophyllotoxin series.
548
dirigent protein OCH 3
1e~oxidant
OCH3
OCH3 10
12
11
NADPH
NADP*
podophyllotoxin-type lignans
OCH3
pinoresinol/ lariciresinol reductase
H3CO
OCH3
Scheme (1). Biosynthesis of lignans in Forsithya intermedia
Asymmetric synthesis of lignans Lignans have long been recognised as challenging targets for organic synthesis due to their complex and diverse architectures as well as their important pharmacological properties. To date, much effort has concentrated on the asymmetric synthesis of naturally occurring and biologically active compounds in enantioenriched forms as in most cases the biological properties vary between the enantiomers. The majority of approaches used in the asymmetric synthesis of lignans can be divided in four general groups. This review will outline the main features of each approach and furthermore will provide additional recent examples which do not fit into the categorisation.
549
Diastereoselective alkylation of chiral butyrolaetones This is one of the earliest and most utilized approaches which focuses on the use of the chiral monobutyrolactone (15) as a springboard to different classes of lignans, Scheme (2), hi the pioneering work of Koga and coworkers [42,43], the key building block 15 was originally synthesized by a multistep sequence starting from £-glutammic acid in modest to good enantioselectivities. Deprotonation of the monobutyrolactone followed by diastereoselective alkylation or aldol reaction, permitted a straightforward entry into the different skeletons of lignans. During the last ten years significant efforts have been directed towards the development of new and elegant methodologies to produce butyrolactone 15 in higher enantioselectivities and chemical yields.
P
Ar-
L D A
Ar2COCI
r
O' o Ar,
15
(OR3)
Ar
J{
O LDA Ar2CH2Br O
*•
15 dibenzocyclooctadienes
,0
Ar-
A
LDA Ar2CHO *~
Ar.,1
TFA
HO
15 Scheme (2). Transformations of monobufyrolaotone
hi 1992 Yoda et al. reported an ex-chiral pool synthesis of butyrolaetones starting from Z-malic acid (16) [44] and applied their methodology to the
550
synthesis of (-)-enterolactone (21) [45], Scheme (3). Z-malic acid (16) was converted in four steps into the enantiopure lactam 17. Diastereoselective Michael addition of 17 with mmethoxybenzylmagnesium chloride in the presence of Cul and trimethylsilyl chloride proceeded smoothly affording 18 in 41% yield as sole isomer. A three step procedure consisting of desilylation of 18, reduction with NaBH4 and cyclization of the obtained hydroxyamide 19 gave the desired (i?)-y-butyrolactone 20 in 63% chemical yield. Finally, stereo selective alkylation and deprotection of the two methoxy groups concluded the asymmetric synthesis of (-)-enterolactone (21).
OH HO2C
21
(a) m-MeOPhCH2MgCI, Cul, TMSCI, THF, -78°C; (b) Bu4NF, THF; (c) NaBH4, EtOH; (d) TsOH, benzene reflux; (e) LDA, m-MeOPhCH2CI, HMPA, THF, -78° - 25°C; (f) BBr3, CH2CI2, 0°C;
Scheme (3). Asymmetric synthesis of (-)-enterolactone by Yoda et al.
Achiwa et al. [46,47,48,49] further developed the approach to access the monobutyrolactone skeleton by creating a very efficient catalytic asymmetric method. Aryldensuccinic acid mono-methyl esters (23), obtained by Stobbe condensation of dimethyl succinate and the corresponding substituted aldehydes, were enantioselectively hydrogenated using a neutral rhodium (I) complex of (45',55)-MOD-DIOP (22), Scheme (4). The corresponding (i?)-arylmethylsuccinic acid monomethyl esters (24) were obtained in quantitative yields and in
551
excellent optical purity (ee > 93%). Moreover, virtually optical pure products could be obtained by a single recrystallization from isopropylether. Esters 24 were converted in (i?)-arylmethyl-'Ybutyrolactones according to the procedure of Brown. [50]
Ar x HO2C^
Ar.
a
1 CO 2 Me
100%
b,c,d
F
e
G
95-97%
O
93-95% ee
23
24
25
a) H2 (1atm), 0.2% of (4S,5S)-MOD-DIOP(22) + [Rh(COD)CI]2 ,0.3 eq. Et3N, MeOH; b) KOH, MeOH; c) Ca{BHi)2, EtOH; d) dil. HCl Scheme (4). Asymmetric synthesis of monobutyrolactones by Achiwa et al.
The enantiopure butyrolactones were then used as key intermediates in the synthesis of two lignans, Scheme (5). Alkylation of 26 with 2,3,4trimethoxybenzyl bromide and subsequent non-oxidative biaryl coupling afforded (+)-neoisostegane 27 whereas alkylation of 26 with a mixed anhydride followed by dehydrative ring-closure provided (+)-collusine (28). Moreover, the same procedure was applied to butyrolactone 29 to obtain 30 in 56% yield. At this point saponification of the lactone was performed with NaOH in MeOH and the resulting product 31 hydrogenated using 5% palladium on carbon under 20 atm of hydrogen pressure. Final DCC-mediated cyclization afforded (-)deoxypodophyllotoxin (32) and (+)-deoxyisopicropodophyllin (33) in 37% and 25% yields, respectively.
552 OMe MeCL
MeO' O
OMe 26
V-6 28
a) LDA, HMPA, 3,4-CH2(O)2C6H3CO2CO2Et, THF; b) HCI, MeOH; c) 2,3,4-(MeO)3C6H2CH2Br, LDA, HMPA, THF; d) TTFA, BF3Et2O, CF3CO2H, CH2CI2
MeO
a) 3,4,5-(MeO)3C6H2COCI, LDA, HMPA, THF; b) HCI, MeOH; c) NaOH, H2O, MeOH; d) H2, Pd/C, EtOH; e) DCC, CHCI3 Scheme (5). Asymmetric synthesis of lignans by Achiwa et al.
Doyle et al. developed a very interesting catalytic approach [51] in which chiral dirhodium(II) carbaximidate catalysts controlled a highly enantioselective carbene insertion into the unactivated C-H bond of 3aryl-1-propyl diazoacetate (35), Scheme (6). The diazoacetate species were obtained in high chemical yields from the corresponding cinnamic
553
acids (34) by standard methods. Among the different Rh catalysts tested, particularly efficient results were obtained with Rli2(4S-MPPIM) and the corresponding enantiomer Rh2(4R-MPPIM) providing enantiomeric excesses between 91-96%. The chiral butyrolactones (36) were then converted into lignans using standard procedures. [52]
34
a) Rh2(4S-MPPIM) or Rh2(4R-MPPIM), CH2CI2, reflux Scheme (6). Asymmetric synthesis of monobutyrolactones by Doyle et al.
A very different strategy based on enantioselective deprotonation was used by Honda et al. [53] The starting cyclobutanone 38 was prepared in two steps from safrole (37) by adopting a literature procedure, Scheme (7). Enantioselective deprotonation of 38 was carried out by using a chiral base, lithium (S,S')-a,a'-dimethyldibenzylamide, at -100°C and the resulting enolate was trapped with triethylsilyl chloride to provide the silyl enol ether 39 in 77% yield. Compound 39 was further converted into y-butyrolactone 29 by ozonolysis followed by sodium borohydride reduction of the ozonide. The optical purity of 29 was determined as 80%. At this point stereoselective alkylation using Koga's conditions furnished a facile entry into the dibenzobutyrolactones. Moreover, deprotonation of 29 using LDA and subsequent aldol reaction afforded alcohols 40 as a mixture of ihreo and erythro isomers in a 1:1 ratio. The mixture of the alcohols was first treated with methanesulfonyl chloride and then with DBU to give (-)-isohibalactone (41) and (-)-savin (42) in 54 % and 15 % yields, respectively. In addition, (-)-isohibalactone could be isomerised into (-)-savin by treatment with tributyltin hydride in the presence of AEBN and refluxing benzene to give 42 in 82% chemical yield.
554
o 77%
OSiEt3 39 75%
Dibenzobutyrolactones
d
e 7*3-77%
,0
g,h
42
82%
a) CI3CCOCI, POCI3, Zn-Cu, Et2O, r.t; b) Zn, AcOH, reflux; c) (S,S')-a,a'-dimethylbenzylamine, BuLi, THF then Et3SiCI, -100°C; d) i. 0 3 , MeOH -78°C ii. NaBH4 iii. HCI 0.2 M; e) i. LDA, THF, -78°C ii. aryl bromide; f) LDA, piperonal, THF, -78°C; g) MeSO2CI, Et3N, CH2CI2, 0°C; h) DBU, MeCN, rt (54% for 40, 15% for 41); i) Bu3SnH, AIBN, benzene, reflux Scheme (7). Asymmetric synthesis of lignans by Honda and co-workers
Enzymatic desymmetrization of substituted 1,3-propanediols has been used as key step in the synthesis of y-butyrolactones by Itoh and coworkers, Scheme (8) [54].The diols 43 were treated with lipase PS {Pseudomenas sp.) in the presence of vinyl acetate as acyl donor to afford acetates 44 in excellent chemical yields and very high enantiomeric excesses (90-98%). These monoacetates were then converted into hydroxy nitriles 46 using a three step procedure. Tosylation of the hydroxyl group of 44, followed by treatment with potassium cyanide in dimethyl sulfoxide at 80°C gave the corresponding acetates 45. The acetoxy groups of 45 were finally hydrolysed with lithium hydroxide in a
555
solvent system consisting of THF-H2O (3:1) at 0°C to afford 46 in good overall yield. At this point hydrolysis of the hydroxy nitrile 46 furnished the desired y-butyrolactone 47. In a similar way, using Pseudomonas fluorescens and Psedudomonas cepacia lipases for the desymmetrization of 2-(3-methoxybenzyl)-l,3-propanediol, Chenevert et al. accomplished the total synthesis of (-)-enterolactone (21) [55]. AcO
R9 74-90%
ee = 90-98%
100%
93%
47 a) Lipase PS, vinyl acetate, i-Pr:H20 = 1000:1, rt; b)TsCI, pyridine, CH2CI2, rt; c) KCN, DMSO, 60°C;d) LiOH, THF, H2O, rt; e) NaOH, H2O, reflux, 2 h. Scheme (8). Asymmetric synthesis of y-butyrolactone by Itoh
Costa et al. developed an efficient pathway utilizing a chiral auxiliary to enter the monobutyrolactone skeleton, Scheme (9) [56]. The carboxylic acid 48 was transformed into the corresponding acyl chloride and then esterified using a homochiral enantiomeric alcohol 49. The resulting ester 50 was deprotonated with LDA and alkylated with piperonyl iodide providing the alkylated ester 51 in 62% yield and high diastereomeric excess (94% de). Ester 51 was reduced to the homoallylic alcohol 52 and finally transformed into the desired lactone 29 either by ozonolysis of the
556
hemiketal intermediate 53 or by an one-pot oxidation in the presence of sodium periodate and catalytic amount of potassium permanganate. COOR
.0 62%
48
)^P
R = HO-
82%
(Cy) 2 NO 2 S
49 O
0
a) i. oxalyl chloride, benzene, AgCN ii. 49, 80°C, 4h; b) i. LDA, THF ii. piperonyl iodide; c) LiAIH4, THF, 0°C; d) NalO4, KMnO4 cat., t-BuOH, H 2 0, pH 8, 17h; e) 0 3 , CH2CI2, -78°C, Me2S; f) CrO3, pyridine, CH2CI2 Scheme (9). Asymmetric synthesis of (/?)-piperonyl-y-butyrolactone by Costa and co-workers
Charlton [57] demonstrated the use of oxazolidinones as effective chiral auxiliaries: the commercially available (4J?)-benzyl and (45)-isopropyl-2oxazolidinones were 7V-acylated with dihydrocinnamic acid to give Nacyloxazolidinones (54 and 55) in yields greater than 80%, Scheme (10). Diastereoselective alkylation with tert-butylbromoacetate gave in each case principally only one diastereomer (56 and 57, respectively) (de>95%). The oxazolidinone moiety could be removed by utilizing LiOH-H2C>2 without affecting the tert-butyl ester. The crude acid was reduced to the corresponding primary alcohol with BH3THF, then
557
lactonized using TFA to afford the desired benzylbutyrolactones (58 and 29). Furthermore, conversion of 29 into different optically active lignans was also reported: 4'-demethyldeoxyisopodophyllotoxin (60) was synthesized by utilizing a DDQ oxidation of 4'-demethylyatein (59).
AA Ph 58
54
b,c,d 70%
a 80%
P
t-BuO2C — N
29
b
57
55
de > 95% e 65%
H3CO
0CH3
0CH3
a) NaHMDS, tert-butyl bromoacetate; b) LiOH, H2O2, THF, H2O; c) BH 3 THF; d) TFA, CH2CI2, e) DDQ, TFA
Scheme (10). Asymmetric synthesis of lignans by Chariton and co-workers
Sibi et al. [58] employed a similar approach: 4-diphenylmethyl-2oxazolidinone 61 as was chosen as chiral auxiliary in the asymmetric synthesis of both enantiomers of enterolactone (62), Scheme (11).
OMe
steps
61 Scheme (11). Asymmetric synthesis of enterolactone by Sibi and co-workers
62
558
Wakamatsu and co-workers applied the diastereoselective alkylation of butyrolactones to enter the dibenzocyclooctene skeleton. In a spectacular series of publications [59,60,61,62,63,64,65,66,67,68] they demonstrated the extension of this methodology to the asymmetric synthesis of several lignans including schizandrin, kadsurin, gomisin as well as various metabolites. The general outlines of this approach are depicted in Scheme (12). Achiwa's methodology (vide supra) [46-49] was used to generate the occurring homochiral butyrolactone 63, followed by aldol condensation and dehydratation providing the unsaturated dibenzobutyrolactone 65. At this point iron (III) perchlorate mediated oxidative coupling provided the key intermediate 66. With dibenzocyclooctene 66 in hand, various manipulations of the double bond were employed to gain access to different functionalities.
R4O
66
RiO
R2O
R2O
R3O R4O
R4O
Scheme (12). Synthesis of dibenzocyclooctene: Tanaka's and Wakamatsu's approach
R4O
559
For reasons of brevity we report only the principal four syntheses. Dibenzocyclooctene 67 was hydrogenated affording a mixture of two cis lactones (68) and (69) (the second one probably arises due to the isomerization of the double bond), Scheme (13). Reduction of the lactones lead to a single diol 70. Treatment with methanesufonyl chloride in pyridine followed by lithium triethylborohydride reduction completed the synthesis of (-)-wuweisizu C (71) [65].
100%
a) H 2 , Pd/C, AcOEt, rt; b) DIBAL, THF, 0°C; c) MsCI, pyridine, rt; d) LiBHEt3, THF, rt Scheme (13). Asymmetric synthesis of (—)-wuweizisu C by Tanaka and Wakamatsu
The Rh-catalysed isomerization of the double bond in 72 was used as a key step in the synthesis of the metabolite D of gomisin A (75), Scheme (14) [61]. Successive reduction of the obtained butenolide (73) followed by dihydroxylation and MsCl-treatment gave a diastereomeric mixture of 74 in 46% chemical yield. Without separation, the mixture was reduced using LiAlH4 after which the protecting groups were removed via hydrogenation affording the desired product 75 and its isomer 76 in 49% and 18% yields, respectively.
560 560
MeO MeO BnO
MeO
75
49%
MeO 18%
a) Rh(PPh3)CI, Et3SiH, toluene, reflux; b) DIBAL, THF, reflux; c) OsO4 (cat.), NMO; d) MsCI, pyridine; e) LiAIH4, THF, reflux; f) H2, Pd/C, AcOEt, rt. Scheme (14). Asymmetric synthesis of (+)-met D by Tanaka and Wakamatsu
The synthesis of (-)-kadsurin (85) [66] commenced with the preparation of the tetracyclic lactone 77, Scheme (15). Lactone 77 was reduced with diisobutylaluminium hydride to give the allylic alcohol 78 in 97% yield. Stereoselective epoxidation using t-butyl hydroperoxide in the presence of vanadyl acetylacetonate, followed by MsCl-treatment afforded the mesylated oxirane 79. Treatment of 79 with sodium iodide in refluxing methyl isobutyl ketone and subsequent zinc reduction gave the allylic alcohol 82 via a mixture of diiodide 80 and monoiodinated allylic alcohol 81 in 89% yield. PCC oxidation of 82 furnished the enone 83 which was hydrogenated stereoselectively providing ketone 84 in quantitative yield. Finally, sodium borohydride reduction and subsequent acetylation of 84 with acetic anhydride in the presence of/»-toluenesulfonic acid completed the synthesis of (-)-kadsurin (85) accompanied by a small amount of product isomerized at C-5. The synthesis of (+)-schizandrin (88) [64] was accomplished in 4 steps starting from dibenzocyclooctene 86. Manganese (II) acetylacetonate-catalysed hydratation of 86 proceeded with satisfactory stereoselectivity affording the desired hydroxy lactone 87 as the major product in 70% chemical yield together with a minor diastereomer (11%). Successive treatment of 87 with LiAltLt,
561
methanesulfonyl chloride and again LiAltU afforded in straightforward manner (+)-schizandrin (88) in 63% overall. MeO
MeO MeO OH
97%
MeO^
t
MeO' MeO-
MeO
o
MeO--,
b c ' , 77%
MeO
MeO
e 89% from 79
°L.0 MeO
OH 82
f 167%
MeO
MeOMeO' MeO-
V
g
• isomer (1.7%)
100% 0
0
83 67%
a) DIBAL, THF, 0°C; b) t-BuOOH, VO(acac)2, CH 2 CI 2 ; c) MsCI, Et 3 N, CH 2 CI 2 , d) Nal, MIBK, 48 h, reflux; e) Zn, AcOH, MeOH.rt, 86 h; f) PCC, CH2CI2, rt, 4.5 h; g) Pd-C, AcOEt, 4.5 h, rt; h) NaBH 4 , THF, MeOH, i)p-TsOH, Ac 2 O, rt, 3 h .
MeO
(11%)
OH MeO
88
a) O2, Mn(acac)2, PhSiH 3 , /-PrOH, rt; b) LiAIH4, THF, reflux; c) MsCI, pyridine d) LiAIH4, THF, reflux
Scheme (15). Asymmetric synthesis of (-)-kadsurin and (+)-schizandrin by Tanaka and Wakamatsu
562
Recently, Yamauchi and co-workers [69] have extended the ybutyrolactone methodology to the synthesis of a 2,3,4-trisubstituted tetrahydrofuran lignan, Scheme (16). The synthesis started with an aldol condensation of the butenolide 89 with piperonal using potassium bis(trimethylsilyl)amide as base. The erythro aldol product 90 was obtained in 78 % yield as a single isomer. After protection of the hydroxy group with triethylsilyl triflate, the lactone was reduced to diol 91 in quantitative yield using lithium aluminium hydride. A S-^2 cyclization was adopted to produce the tetrahydrofuran ring. Thus, diol 91 was converted into dimesyl derivative 92 by employing methansulfonyl chloride and triethyl amine. Exposing the dimesyl diester 92 to desilylation conditions using tetra-M-butylammonium fluoride, intramolecular S^2 cyclization occurred spontaneously providing 93 in 90% chemical yield. Finally, the monomesyl ester 93 was treated with aq. sodium hydroxide in DMF to give (+)-dihydrosesamin (94) in 68% yield.
MsO 94
93
92
a) piperonal, KHMDS, THF, -75°C, 1 h; b) TESOTf, 2,6-lutidine, CH2CI2, rt, 1 h; c) LiAIH4, THF, 0°C, 1 h; d) MsCI, Et3N, CH2CI2, rt, 1 h; e)n-Bu4NF, THF, 0°C, 1.5 h; f) 1M aq. NaOH, DMF, 120°C, 16 h.
Scheme (16). Asymmetric synthesis of (+)-dihydrosesamin by Yamauchi et al.
563
Diastereoselective conjugate addition to 2(5//)-furanones Koga et al. developed an alternative approach to access lignans based on diastereoselective Michael addition to chiral butenolide 96, Scheme (17) [70,71,72]. Butenolide 96 was easily accessible starting form the chiral butanolide 95 via a two step synthesis. Michael addition of 96 by a sulphur stabilized carbanion 97 followed by alkylation and desulfurization gave the disubstituted hydroxymethylbutanolide 98. Finally, reduction of 98 with L1AIH4, cleveage of the obtained 1,2-diol by NaIC>4 and oxidation with Collins reagent provided dibenzylbutyrolactone 99, a key intermediate in the synthesis of different classes of lignans.
L-glutamic acid
steps '—OH
H3C
J
\
Jj
H3CO
1
,0
O
H3CO (+)-steganacin
~OCH3 OCH3 (+)-burseran
a) i. LDA, HMPA, -78°C ii. PhSeBr at -20°C -> 19h rt; b) NalO4, MeOH/H2O, 30 min.rt; c) i. 97, THF ii. substituted benzyl bromide; d) Ra-Ni, EtOH, reflux; e) THF, LiAIH,,, rt; f) NalO 4 , f-BuOH, rt; g) CrO3, pyridine, CH2CI2, rt.
Scheme (17). Asymmetric synthesis of lignans utilizing a diastereoselective Michael addition to chiral 2(5H)furanones: the Koga approach
Feringa and co-workers [73,74,75] developed an efficient entry into lignans via a diastereoselective 1,4-addition utilizing 5-(menthyloxy)2(5i7)-furanone as chiral Michael acceptor, Scheme (18). The
564
enantiomerically pure 100 and 101 could be prepared in several gram scale from furfural and /- or J-menthol providing access to both enantiomers. The Michael addition of lithiated dithianes 102 with butenolide 100 was followed by quenching of the resulting lactone enolate anions with different benzylbromides to afford in tandem fashion dibenzobutyrolactones 103 in good yields. Moreover, in every case a single diastereomers was observed indicating complete stereocontrol in both steps. An one-pot, highly efficient procedure was devised for desulfuration, removal of chiral auxiliary via acetal hydrolysis, reduction of an aldehyde group and ring closure of the resulting alcohol to give the y-lactones 104. After eventual debenzylation three, different natural dibenzobutyrolactones were obtained in excellent overall yields and optical purity. Moreover, a straightforward reduction of (-)-enterolactone (21) with LiAlH4 afforded (-)-enterodiol (105) in 87% chemical yield.
100
V
\
(5R)-(l)Menthyloxy-2(5H)-furanone
101 (5S)-(d)Menthyloxy-2(5H)-furanone R3
0
/=V H
R1
R3
^cA>menth
50-67% 0
100
0
''"bmenth
103
104
only isomer
PhS SPh
17
R1
a
Li
R2 102
a) i. 102, THF, -80°C ii. TMEDA, substituted benzylbromide at -80°C to -30°C; b) i. NiCI2.6H2O (5 eq.), NaBH4 (20 eq.), THF/MeOH, 0°C ii. 2N KOH (20 eq.) iii. NaBH4 (5 eq.) iv. 2N HCI.
HO
OH
HO—*"
) HO
29 a) LiAIHd, THF, rt.
Scheme (18). Asymmetric synthesis of lignans by Feringa and co-workers
565
This methodology furnished also an efficient access into the furofuran skeleton, Scheme (19). A tandem Michael addition-aldol reaction was used to provide lactone 107 in 62% yield as a mixture of diastereomers in a ratio of 60:40. Dithiane 107 was converted into ketone 108 using HgO in combination with BF3-Et2O. Subsequent reduction using an excess of LiAlELi afforded tetraol 109 in 67% yield. The synthesis of (-)-eudesmin (110) was completed in 16% overall yield by dehydratation of 109 with BF 3 Et 2 0. MeO
OMe
MeO
OMe
OMe —^-— MeO 89%
107 OMe
MeO MeO 44%
V OMe
a) i. 106, THF, -90°C ii. veratryl aldehyde at -80°C to -30°C, 16 h; b) HgO, BF3 Et2O, THF.1 h rt; c) LiAIH4 (4 eq.), THF, reflux, 1 h; d) BF 3 Et 2 0, CH2CI2, 4°C, 16 h.
Scheme (19). Asymmetric synthesis of (—)-eudesmin by Feringa et al.
Recently, Feringa explored also another variant of this methodology by replacing (5i?)-menthyloxyfuran-2(5//)-one with another chiral butenolide, namely (i?)-5-acetoxyfuran-2(57/)-one (113), Scheme (20) [76,77]. The chiral butenolide was obtained in several gram scale by enzymatic esterification of 5-hydroxyfuran-2(5//)-one (111) using lipase R immobilized on hyflo super cell.
O^O
111
'OAc
113 single enantiomer spontaneus racemization
a) Et2O, vinyl acetate, lipase R, rt 10 days.
Scheme (20). Enzymatic synthesis of (S)-5-acetoxyfuran-2(5//)-one by Feringa
112
566
Pelter et al. have successfully used the (5i?)-menthyloxyfuran-2(5//)-one based approach to enter different classes of lignans, Scheme (21) [78,79,80]. Lactone 113 was obtained using the above mentioned tandem Michael addition-aldol reaction procedure as a mixture of diastereomers in a ratio of 1:1. Desulfurization proceeded smoothly by treating 113 with NaBILt and NiCb to give 114 in excellent chemical yield as a sole isomer. Removal of the chiral auxiliary using NaBtU and KOH followed by acidic cyclization afforded the desired aryltetraline lactone 116. In addition, lactone 118, prepared analogous to Scheme (18), was stereoselectively cyclized to tetrahydrobenzocyclooctene 119 using a novel procedure employing DDQ in trifluoroacetic acid. Pn
S
H
pmenth
Pmenth
a)MeOH, NiCI2 (20 eq.), NaBH4 (62 eq.), rt, 1h;b)i. KOH, EtOH, NaBH4 (4 eq.), 30 min. ii. HCIuntilpH3 c)TFA, rt 150 min.
119
a) TFA, DDQ, rt, 2 h Scheme (21). Asymmetric synthesis of lignans by Pelter et al.
567
Bhat and co-workers applied an asymmetric tandem Michel additionaldol reaction to achieve a very short synthesis of (-)-podophyllotoxin (1), Scheme (22) [81]. The S-(-)-piperonyl phenyl sulfoxide (121) was obtained from piperonal (120) in four steps, which included chiral sulfoxidation via the modified Sharpless method reported by Kagan et al. [82]. The tandem conjugate addition-aldol reaction was performed following Pelter-Feringa's procedure providing adduct 122 in 60% yield. Cyclization was accomplished by treating 122 with trifluoroacetic acid and subsequent treatment with HgO and BF3-Et2O gave directly (-)podophyllotoxin (1), albeit in low yield. OH _CHO
120
b,c
121 H3CO
y 0CH3 0CH3
OCH-,
122
a) i. BuLi, THF, -78°C, 1h ii. butenolide,1 h iii. 3,4,5-trimethoxybenzaldehyde, 2 h; b) TFA, 3h, rt; c) HgO, BF 3 Et 2 0, 28 h.
Scheme (22). Asymmetric synthesis of (-)-podophyllotoxin by Bath
Enders and co-workers developed an elegant variation of this methodology using chiral a-amino nitriles as Michael donors, Scheme (23) [83,84]. The asymmetric Strecker reaction using enantiopure secondary amine 123 and different aromatic aldehydes as starting materials furnished the a-amino nitriles 124 in good yields. Michael addition of lithiated 124 to butenolide afforded the 1,4-adducts 125 in good chemical yields and diastereoselectivities. At this point two divergent routes were used: diastereoselective alkylation followed by removal of chiral auxiliary afforded 126 in excellent yields and diastereoselectivities. Alternatively, aldol reaction resulted in a mixture of epimeric alcohols 127.
568 H3C. ,CH3
H3CCH3
oXo
Ar1
123 a
after purification 'Vhen Ar 1 = piperonyl
124
125
o
de = 56 - 80% (94 - 98%)
de = 60 - > 98%, ee > 96%
78-80% e,d
a) Ar1CHO, H2O, HCI, pH = 4-5, KCN, rt; b) i. THF or Et2O, LDA , -78"C, 90 min ii. butenolide, -78°C 3-5 h; c) LDA (2 eq.), THF, -100°C, LiCI (2.2 eq.), Al^CHO; d) AgNO 3 , H2O, THF, 25°C; e) f-BuLi (2 eq), THF, -78°C, Ar^CH2Br, -90 - 0°C.
Scheme (23). Asymmetric synthesis of dibenzylbutyrolactones by Enders et al.
The methodology was particularly successful when piperonal was used as starting material, Scheme (24). Aldol products 128 were obtained in a synlanti ratio of 87:13-93:7 which could be further increased by purification after cleavage of the chiral auxiliary to 96:4-99:1. Alcohols 128 were then converted in furofurans using a four step procedure: reduction of the ketone with L-selectride, reduction of the lactone with LiAlH4, mesylation of the primary alcohols and cyclization. (-)-Methyl piperitol (130), (-)-sesamin (131) and (-)-aschantin (132) were synthesized via this method with perfect stereocontrol and in good overall yields. In addition, ketone 133 was used as a springboard to other classes of lignans. Reduction of 133 with NaBH4 and subsequent catalytic hydrogenolysis gave (+)-yatein (134) in 70% yield. Oxidative coupling using TI2O3 in the presence of BF3-Et2O in neat TFA provided (-)isostegane (135) in very good yield (77%). Finally, reduction of (+)yatein (134) with UAIH4 and subsequent acid-catalysed cyclization of the diol afforded (+)-burseran (136) in 77% yield.
569
OH
a.b 40-51%'
$yn:anti (aldol) > 96:4 - > 99:1 ee > 98%
130 a) L-selectride, THF, -78°C; b) LiAIH,, THF, 6Q"C; c) MsCI, Pyridine 0 •\
a,b t 70%
77%
H3CO 13S
cfe>98%, ee = 97%
CH3O
Y OCH3 OCH3 136
a) NaBH4, MeOH, 2 h, rt; b) H2, Pd/C, cat. HCIO4, EtOH, 4 Atm, rt; c) TI2O3, TFA, BF3-Et20,10 sac, 0°C d) UMH4, THF, rt, 3 h; e) HCI, MeOH, reflux, 48h. Scheme (24). Asymmetric synthesis of Hgnans by Enders et al.
Recently, the methodology has been extended to the synthesis of an aryltetralkie lignan by completing a straightforward synthesis of (+)lintetralin (140), Scheme (25) [85]. Adduct 137 was easily accesible via the Enders methodology in virtually enantiopure form and excellent
570
overall yield. Reduction of 137 afforded a mixture of epimeric triols 138 in a ratio of 2:1. Subsequent Friedel-Crafts-like cyclization afforded diol 139 as sole isomer. Finally, methylation of the hydroxy groups yielded (+)-lintetralin (140) in 92% yield. MeO •
95%
"
\
OH
87%
MeO'
OMe OMe
a) LiAIH4, THF, rt, 5 h; b) BF3-Et20, CH2CI2, 1 h , rt; c) NaH, Mel, THF, 2.5 h Scheme (25). Asymmetric synthesis of (+)-lintetralin by Enders et al.
Use of chiral oxazolidinones As the previous chapters have demonstrated, chiral auxiliaries have found a widespread application in the asymmetric synthesis of lignans. Among them, chiral oxazolidinones have been used extensively due to their ability to produce excellent diastereoselectivities in aldol as well as in numerous other reactions. For example, Kise et al. reported the use of (£)4-isopropyl-3-(phenylacetyl)-2-oxazolidinone (141) in oxidative homocoupling reactions and its application in the asymmetric synthesis of dibenzylbutyrolactones and dibenzylbutandiols, Scheme (26) [86,87]. Treatment of 3-arylpropanoic acid derivative 142 with LDA in the presence of TiCU yielded a mixture of the dimeric compounds 143 in a ratio of 85:15 to 87:13. The major product having (R,R) configuration was converted into dibenzylbutyrolactones 145 in a three step sequence
571
including cleavage of the chiral auxiliary by LiOOH, cyclization of the obtained diacid 144 and subsequent NaBlrLt-reduction to give the desired product. Alternatively, diacid 144 could be reduced to dibenzylbutandiols 146 by LiAlH4 in excellent chemical yield. Moreover, the results were compared with the chiral auxiliary of Helmchen 147 [88], which has been used in an analogous synthesis of (-)-hinokin [89]. Auxiliary 147 gave a better selectivity in the coupling reaction when CuC^/DMPU was used as oxidanting agent (92:8).
43-71%
Ar^> AK^J.,,
X 80-90% A K ^ . O
142
OH 80-90%
'
O
143
Ar = piperonyl, veratryl or 4-metoxyphenyl
Ar
145
de = 85:15-87:13
a) LDA, THF, TiCI4, -78°C-rt, 24h; b) THF, LiOH, H2O2, rt 12-24 h, c) i. AC2O, MeOH -78°C ii. NaBH4, 1 h; d) LiAIH4, THF, 12 h
Ar Ar
45-63% 148
Ar = piperonyl, veratryl or 4-metoxyphenyl
Y
X b Ar X 70-80%
o 149
Y
OH
as above
o 144
de = 88:12-92:8 a) LDA, hexane/THF, CuCI 2 , DMPU, -78°C-rt, 12 h; b) LiOH, THF/H 2 O 2 , reflux 2 4 ^ 8 h.
Scheme (26). Asymmetric synthesis of dibenzylbutyrolactones and dibenzylbutandiols by Kise et al
572
Gordon and co-workers used iV-4-pentenoyloxazolidinone 150 as chiral starting material in the asymmetric synthesis of (-)-sesaminone (159), Scheme (27) [90]. 9. o
0
O
OH
-OH
159 a) Bu2BOTf, CH2CI2, 0°C, Et3N, 0 to -78°C, piperonal; b) LiBH4, H2O, THF, 0°C; c) TBDPSCI, imidazole, THF; d) MOMCI, Et3N, CH2CI2; e) OsO4, NMO, (-BuOH, H2O, THF; f) NalO4, H2O, THF, 0°C; g) 5-lithio-1,3-benzodioxole, THF, -78°C to 0"C; h) MnO2, CH2CI2; i) TBDMSOTf, Et3N, CH2CI2; I) TiCI4, CH2CI2, -78°C; m) 48% HF, C5H5N, MeCN, 0°C.
Scheme (27). Asymmetric synthesis of (-)-sesaminone by Gordon et al.
Asymmetric aldol reaction according to the Evans procedure between 150 and piperonal produced aldol adduct 151 with excellent
573
diastereoselectivity (19:1) which could be further increased via purification to give the product free of isomers in 82% yield. Reduction of the aldol adduct 151 with lithium borohydride in the presence of water afforded diol 152 again in 82% yield. Both alcohols of diol 152 were consecutively protected first with TBDPSC1 and then with M0MC1 to give the diprotected 153. At this point, dihydroxylation of the double bond and subsequent cleveage of the diol 153 provided aldehyde 154. 1,2Addition of 5-lithio-l,3-benzodioxole to 154 proceeded smoothly to give after oxidation of the secondary alcohol the aryl ketone 155 in 76% yield. Aryl ketone 155 was first transformed into a mixture of silyl enol ethers 156 (7:1) and then cyclized to tetrahydrofuran 157 using titanium tetrachloride. Tetrahydrofuran 157 was produced in good yield together with a small amount of a-tetralone 158. Finally, deprotection of 157 with fluoric acid afforded (-)-sesaminone 159 in 84% yield. Yamauchi et al. produced the chiral monoprotected diol 161 analogous to the procedure of Gordon in 4 steps and 56% overall yield, Scheme (28) [91]. Dihydroxylation of the double bond with osmium tetroxide followed by cleavage of the obtained diol and oxidation of the transient lactol with silver carbonate-celite gave lactone 162 in excellent yield. Reaction with methyl chloro formate proceeded stereoselectively to afford 163 as single isomer. Treatment of 163 with lithium aluminium hydride gave the desired triol 165 in 63% yield together with the corresponding hemiacetal 164 (22%). The hemiacetal 164 was converted into 165 via sodium borohydride reduction. The total yield of triol from lactone 163 was 76 %. Cyclization of the triol to the tetrahydrofuran ring-system was achieved by an intramolecular SNI reaction using 10-camphoric acid as catalyst. The desired 166 was obtained as a mixture of diastereoisomers in a ratio of 1:1. Pyridinium chlorochromate oxidation furnished aldehyde 167 which partially epimerized resulting in a 2:3 ratio. Stereoselective ochydroxylation of 167 was performed with triisopropylsilyltrifluoromethane in the presence of DBU and DMAP. The unstable silyl enol ether was oxidated using osmium tetroxide after which the crude product was treated with A^^'-dimethylethylenediamine and finally chromatographed successively on silica gel. The resulting unstable ahydroxyaldehyde 168 was immediately reacted with tetrabutylammonium fluoride to cleave the silyl group affording ( l S ^ S ^ (169) as a single isomer in 78% yield.
574
OH .0
TBDPSO
U ^ 0 > 92%
r
Bn
a.b.c
v
TBDPSO 162
161
160
A ii
47%
HO, HO HO—
OTBDPS
HO—-
X
-OTBDPS
H3CO2C—-
22% 164
63% 165
^—OTBDPS 163
54% 83%
\ HO — ^
/ ^-OTBDMS 166 1:1
CX 0 >
h 72%
0=^N-
i,k 71%
HO 0 = =*
^-OTBDMS sole isomer
OTBDMS
167 2:3
I /
/ 78%
168
^O
) 0
HO")— <""H
169
a) OsO4, NMO, Acetone, (-BuOH, H2O, rt, 24 h; b) NalO4, MeOH, rt, 3h; c) Ag 2 CO 3 , celite, toluene, reflux, 1 h; d) LiHMDS, THF, CICO2CH3, -75°C, 2h; e) LiAIH4, THF, rt, 3 h; f) NaBH4, EtOH, 3 h; g) CSA, CH 2 CI 2 , rt, 24 h; h) PCC, MS 4A, CH 2 CI 2 , rt, 5 h; i) TIPSOTf, DBU, DMAP, CH2CI2, rt, 2 h; k) i. OsO4, NMO, acetone, /-BuOH, H2O, rt, 24 h ii. (CH3NHCH2)2, benzene, reflux, 0.5 h, SiO2; I) n-Bu4NF, THF, rt, 1 h
Scheme (28). Asymmetric synthesis (+)-hydroxysamin by Yamauchi
Sibi and co-workers developed two protocols which utilized 4diphenylmethyl-2-oxazolidinone (170) as chiral auxiliary to enter different classes of lignans. Oxazolidinone 170 was efficiently acylated to
575
cinnamates 171 using n-BuLi and the corresponding acid chlorides, Scheme (29) [92]. Michael addition employing aryl cuprates gave the corresponding adducts 172 in good yields. Treatment of 172 with sodium hexamethyldiazide generated the corresponding anions which could be quenched with tert-butyl iodoacetate introducing the acetic acid chain in respectable yields and excellent diastereoselectivities. Chemoselective reduction of the carbonyl attached to the chiral auxiliary was achieved using lithium aluminium hydride at low temperature. Work-up followed by silica gel column chromatography furnished the butyrolactones 174. However, with the 3,4-methylenedioxyphenyl group the procedure was inefficient, so an alternative sequence was devised. The chiral auxiliary was hydrolyzed using LiOH-EkC^. The resulting acid ester was reduced with borane and subsequently cyclized with /7-toluenesulfonic acid to furnish benzobutyrolactone 174. Finally, alkylation of the butyrolactones 174 using NaHMDS and methyl iodide gave the natural peperomins 175 as a mixtures of diastereoisomers in a ratio of 10:1 - 12:1. 0
X=
0
NH
ypu Ph 170 O 0
If
,CO2(-
0 NH
a
.
^
Ar b 81-89%
O
Ar
171 170
^_c__^Ar\/ \, X X
172
Ar = piperonyl, veratryl, 3,4-methylenedioxy-5-methoxypheny
1
64-74%
I
Jl
Ar O 173 single isomer
80-98% d ore
Ar 175 ds>10:1 a) THF, n-BuLi, CICOAr, -78°C, 3 h; b) Mg, ArBr, CuBr(CH3)2S, THF/(CH3)2S (3:1), -48 to 0°C. 3 h; c) NaHMDS, (-BuO2CCH2l, THF, -78°C to -48°C, 6 h; d) LiAIH4, THF, 20 h; e) i. LiOH/H2O2, THF, 0°C, 5h; ii. BH 3 , THF -10°C, 14 h iii. PTSA, benzene, reflux, 2 h; f) NaHMDS, THF, Mel, -78°C, 3 h.
Scheme (29). Asymmetric synthesis of peperomins by Sibi et al.
576
In a similar way, compound 176 was used as starting material in the synthesis of several lignans, Schemes (30) and (31) [93]. A radical addition was used to introduce the remote aryl group: 176 was treated with 3-methoxybenzyl bromide in presence of an equimolar amount of samarium triflate as lewis acid and BU3S11H and Et3B as radical promoters. Only one diastereomer was detected in the reaction. However, a side product was formed due to the addition of the ethyl group to the double bond which could be removed after purification to give 177 in very good yield. Treatment of 177 with NaHMDS and quenching the resulting anion with 3-methoxybenzyl iodide afforded only the syn product 178. Removal of the chiral auxiliary was then performed utilizing LiOH and H2O2. Reduction of the acid moiety with borane and subsequent cyclization gave the desired dibenzobutyrolactone ring system 180. Finally, treatment with boron tribromide provided (-)-enterolactone 21.
H3CO
COOEt
21
180
a) SmiOTfh, 3-methoxybsnzyl bromide, CHzCI/rHF (4:1), Bu 3 SnH, EtaB/Oj, -78°C; b) NaHMDS, THF, S-MeOCjH^CHal, -78"C to -54"C; 0) LiOH, H2O2; d) BH3/THF, -15"C, 18 h; e) PTSA, benzene, reflux 4 h; f) BBr 3 , CH 2 CI Z , 0'C
Scheme (30). Asymmetric synthesis of (-)-enterolactone by Sibi et al.
577
Using the same approach, the easily accessible lactones 175 and 176 were converted in good chemical yields into (-)-isoaretigenin 177 and (-)arctigenin 178 after removal of protecting groups via hydrogenolysis, Scheme (31). O
0 COOEt
1
V" Ph 176
0
II
0
0 Ph 176
f) H2, Pd/C, EtOAc, AcOH, 1.5 h. Scheme (31). Asymmetric synthesis of (-)-isoarctigenin and (-)-arctigenin by Sibi et al.
Asymmetric cycloaddition reactions Asymmetric Diels-Alder reactions have found widespread use in the total synthesis of complex natural products. However, only few examples have been reported on their application towards the asymmetric synthesis of lignans. Charlton et al. used an asymmetric Diels-Alder reaction as a key step in the total synthesis of (—)-a-dimethylretrodendrin (193), Scheme (32), [94]. The aldehyde 185 was prepared in three steps and in good overall yield starting from 3,4-dimethoxybenzaldehyde. Irradiation of 185 in a benzene solution containing dissolved an excess of SO2 afforded the key intermediate 186. Thermolysis of 186 produced the diene 187 which could be trapped with chiral fumarate 188 yielding a mixture of diastereomers in a ratio of 9:1. After purification, the cycloadduct 189 was isolated in 44% yield. Lactonization of the secondary hydroxy group with y-ester followed by transesteriflcation and opening of the lactone were achieved in an one-pot fashion utilizing sodium methoxide in
578
methanol to give acid ester 191 in excellent yield. Catalytic hydrogenation followed by reduction of the ester group and refluxing the crude product in a benzene/p-toluenesulfonic acid mixture afforded the lactone 192 as sole product in 72% yield. Finally, epimerization of the C2 carbon gave optically pure (-)-a-dimethylretrodendrin (193). CO2R RO ,C
CO2Me "Ph
188
MeO. MeO
OMe OMe 193 a) benzene, pyridine, SO2, hv, 6 h, rt; b) 188, ZnO, toluene, reflux,1 h; c) NaOMe/MeOH, rt, 24 h; d) H2, Pd/C, EtOAc/AcOH, rt, 24 h; e) THF, LiEt3BH, rt 19 h; f) benzene, p-TsOH, reflux, 14 h; g) (-BuONa/f-BuOH, reflux 24 h.
Scheme (32). Asymmetric synthesis of (-)-a-dimethylretrodendrin by Charlton
579
hi addition, a modified version of this methodology was applied some years later to the synthesis of (-)-deoxypodophyllotoxin (202) [95] as well as other aryltetralines [96]. Ketone 194 was synthesized in 9 steps starting from piperonal, Scheme (33).
OCH 3
H3C0 194
197 58%
H3CO"
OCH 3
H3C0
y
0CH3
OCH3
H3CO
OCH3 199
^CH20H '.,., 'CO2H
H3CO
g 30 % overall from 192
198
Q. v. 0
0GH H3CCf ^ f 3 OCH3 202
a) n-BuLi, THF, -78°C; b) toluene, reflux, 196, 44 h; c) BF3Et2O, CH2CI2, -20°C then LiAIH4, -55°C-rt; d) Pd/C, H 2 , MeOH/AcOH, rt, 89 h; e) (CF3CO)2O, reflux, 2h; f) NaBHj, /-PrOH, 15 h, rt; g) benzene, p-TsOH, reflux, 17.5 h.
Scheme (33). Asymmetric synthesis of (-)-deoxypodophyllotoxin by Charlton et at
Treatment of 194 with n-BuLi afforded the benzocyclobutenol 195 in 71% yield. Thermolysis of 195 in refluxing toluene and treatment with methyl (5)-mandelate (196) gave a mixture of cycloadducts via the ahydroxy-a-aryl-o-quinodimethane 197 intermediate. The major
580
cycloadduct 198 was isolated in 58% yield. Reduction of the hydroxy group was acieved using BF3'Et2O followed by LiAlHj treatment. A mixture of three isomers were obtained in a ratio of 15:1:2. Isolation of the major isomer 199 was very difficult due to the similar polarity of the three products. Therefore, the crude was used directly in the next steps without purification. The mandelate group was cleaved by catalytic hydrogenolysis after which refluxing the crude product in trifluoroacetic anhydride gave a product, presumed to be the anhydride 200. Reduction of 200 with NaBH4 resulted in a mixture of y-hydroxy acids in a ratio of 3.3:1 providing the desired y-hydroxy acid 201 as the major product. Finally, lactonization via /?-toluenesulphonic acid concluded the total synthesis of (-)-deoxypodophyllotoxin (202) in 30% overall yield starting from 198. Pelter et al. used (51?)-menmyloxy-2(5/^-furan°ne (100) as chiral dienophile in the asymmetric synthesis of (—)-isopodophyllotoxin (209), Scheme (34) [97]. Treatment of starting material 203 with the chiral dienophile 100 in refluxing toluene gave, via the arylisobenzofuran 204 generated in situ, a complex mixture of 5 products. After purification by silica gel chromatography the major product 205 could be isolated in 37.5% chemical yield. Raney-nickel reduction of the cycloadduct 205 afforded isopicropodophyllotoxin (206), along with a small amount of the C-10 epimer. After cleavage of the menthyloxy group by using a mixture of sodium borohydride and potassium hydroxide in EtOH, products 207 and 208 were obtained in 52% and 44% yields, respectively. Finally, ZnCl2 mediated lactonization of 207 completed the asymmetric synthesis of (-)-isopodophyllotoxin (209),
581
37.5%
MeO
y OMe OMe 205
OMenth
OMe
MeO
y
OMe
OMe 206 + epimer
MeO
y OMe OMe
209 a) toluene, 100, reflux, 9 h; b) Ra-Ni, EtOAc, H2, 60 psi, rt, 15 h; c) NaBH4, EtOH, 20 h; rt; d) THF, ZnCI2, MS 4 A, reflux, 6 h.
Scheme (34). Asymmetric synthesis of (-)-isopodophyllotoxin by Pelter et al.
Jones and co-workers [98] concluded a very efficient synthesis of (-)podophyllotoxin (1) based on an asymmetric Diels-Alder addition to 1aryl-2-benzopyran-3-one [99]. The o-quinonoid pyrone 210 reacted smoothly when (5i?)-menthyloxy-2(5//)-furanone (100) was used as dienophile, Scheme (35). The cycloaddition proceeded with high facial selectivity as well as very high regioselectivity affording 211 as sole isomer in 79% chemical yield. The cycloadduct 211 underwent ring opening with acetic acid to give the acid 212 in 87% yield. Hydrogenation followed by oxidation with lead tetraacetate converted 212 into the
582
acetate 213. Hydrolysis of 213 under carefully controlled acidic conditions gave two epimeric lactols 214 and 215 in a ratio of 1:1 which were separable via chromatography. Brief treatment of the individual lactols with diazomethane followed by facile reduction with LiEtaBH furnished the two methyl podophyllates 216 and 217. The former was readily converted into methyl podophyllate 217 by treatment with hydrochloric acid. Final lactonization using ZnCb completed the total synthesis of (-)-podophyllotoxin (1) in 15% overall starting from 210.
9°2 H p-menth
CH 3 O'
64% a) MeCN, 100, 50°C, 29 b) HOAc, 49°C, 13 h; c) EtOAc, Pd/C, H2, 40 h; d) HOAc, THF, rt, Pb(OAc)4, 3 h; e) dioxane/HCI (3:1). 41°C; f) CH 2 N 2 , Et2O/MeOH (24:1), 0°C; g) THF, LiEt3BH, -78°C, 1h; h)THF/HCI (1:1.5), rt, 3.5 h i) THF, ZnCI2, molecular sieves, reflux, 2.5 h.
Scheme (35). Asymmetric synthesis of (—)-podophyllotoxm by Jones and co-workers
583
Miscellaneous Wirth and co-workers reported on the application of the easily accessible chiral diselenide 218 to the asymmetric synthesis of furofuran lignans, Scheme (36) [100]. Cleaveage of the diselenide group in situ with bromine followed by treatment with silver triflate afforded the electrophilic selenium species 219. Allowing 219 to react with alkene 220 for 15 min followed by addition of 2,3-butadien-l-ol (221) gave 222 in 56% chemical yield and excellent selectivity (16:1). Compound 222 was then subjected to radical cyclization with triphenyltin hydride in the presence of AIBN to give tetrahydrofuran 223 as a mixture of two diastereoisomers in a ratio of 1:1 and 64% yield. Dihydroxylation of the double bond and subsequent oxidation gave the two corresponding aldehydes 224 and 225. Finally, after removal of the protecting group with tetrabutylammonium fluoride, cyclization of the mixture containing both isomers to the hemiacetal occurred spontaneously affording (+)samin (226) in 67% yield.
Et '''OH Se) 2 218
226
a ) i . Br2, Et 2 O,-78°C, 15 min ii. AgOTf iii.-100°C, 220 15 min iv. 221, 3 h; b) Ph 3 SnH, AIBN, toluene, 90°C, 1 h; c) N-morpholine-N-oxide, acetone, t-BuOH, OsO 4 , H 2 O, 4 h; d) NalO 4 , THF, 8 h; e) n-Bu 4 NF, THF, 0°C, 4 h.
Scheme (36). Asymmetric synthesis of (+)-samin by Wirth and co-workers
584
In addition, the strategy was extended to the asymmetric synthesis of diaryl furofurans, Scheme (37) [101]. Compound 227 was synthetized using the same route as above in 23% overall yield. Oxidation of the vinylic double bond with osmium tetroxide and subsequent treatment with periodic acid afforded directly furofuran 228. It is noteworthy that cleavage of the diol to give the aldehyde, removal of the protecting group, isomerization at C-4 and cyclization could be obtained in one pot, albeit in moderate yield. Finally, Grignard addition gave the furofuran lignan (+)-mesembrine (229) in 45% yield. OMe TBDMSO
a
J
-'OH
SeOTf
219
^ 2 3 %
M e O
OMe
227
228
229
a) OsO 4 , N-morpholine-N-Oxide, acetone, f-BuOH, H2O, 10 h; b) H5IO6, THF, H2O, rt, 13 h; c) 4-methoxyphenyl magnesium bromide, THF, 70°C, 3h.
Scheme (37). Asymmetric synthesis of (+)-mesembrine by Wirth
Yamauchi et al. completed an ex-chiral pool synthesis of a samin-type lignan, Scheme (38) [102]. The diprotected tetraol 230 was obtained from Z-glutammic acid by a 15 step procedure in 7-8% overall yield [103]. The diprotected tetraol 230 was treated with boron trifluoride diethyl etherate in dichloromethane to give the tetrahydrofuran 231 in 84%-87% yield. After deprotection with tetrabutylammonium fluoride, the resulting diol was oxidized by dihydridotetrakis(triphenylphospine)ruthenium(II) to provide two lactones 232 and 233 in a ratio of 2:1. Lactone 232 was transformed in samin-type lignan 234 by diisobutylaluminium hydride reduction in 70% yield.
585 MeO.
84-87% H " 4 — H H TBDPSO-"
^"OH
230
TBDPSO"^
231
234
a) BF 3 Et 2 0, CH2CI2, 0°C, 30 min,; b) n-Bu4MF, THF, rt, 1 h; c) RuH2(PPh3}4, acetone, toluene, reflux, 1.5 h; d) DIBAL, toluene, -75°C, 1 h. Scheme (38). Asymmetric synthesis of samin-type Hgnan by Yamauchi
Ohmizu and co-workers explored the use of (S)- and (i?)-3-(2,2-dimethyll,3-dioxolan-4yl)-cw-2 propenoate (235 and 236, respectively) as chiral building blocks in the asymmetric synthesis of lignans, Scheme (39) [104,105]. Michael addition of cyanohydrin 237 to the ester 235 proceeded smoothly in the presence of 2 equivalents of HMPA. The Michael adduct 238 was obtained in 94% chemical yield and very high diastereoselectivity (93%). The 1,4-adduct 238 was then converted into lactone 239 by treatment with sodium metaperiodate in methanol. Aldol reaction of 239 with veratraldehyde was carried out in THF at -78°C using LDA as base. After removal of the silyl group, the ketone 240 was recovered as sole product in 84% yield. Reduction of the ketone 240 with sodium borohydride afforded the diol 241 together with its C-4 stereoisomer in 86% and 8% yields, respectively. Diol 241 was further reduced with LiAlH4 to tetraol 242 and finally cyclized to (+)-fargesin (243). hi addition, synthon 244, accessible by the same procedure in 67% overall yield and 93% de, was used in the asymmetric synthesis of (-)picropodophyllone (247). Reduction of 244 with sodium borohydride in hot THF-methanol followed by selective oxidation of the resulting diol with Fetizon's reagent gave the y-lactone 245 in 67% yield. Aldol reaction of 245 with 3,4,5-trimethoxybenzaldehyde followed by intramolecular Friedel-Crafts like cyclization promoted by trifluoroacetic acid afforded the product 246. Without any purification, 246 was treated
586
with tetrabutylammonium fluoride completing the synthesis of (-)picropodophyllone (247) in 83% overall yield starting from adduct 245.
MeO2C
1 0
MeO2C
Xx
235
T XT 0
236 H
JPMe
243 a) LDA, THF, 2 eq. HMPA, 235, -100°C, 30 min.; b) NalO4, H2SO4, MeOH, 40°C, 24 h; c) LDA, THF, -78°C veratraldehyde, 30 min; d) TBAF, CH2CI2, AcOH, rt, 2 h; e) NaBH4, MeOH, 0°C, 30 min; f) LiAIH4, THF, 60°C, 1 h; g) pyridine, MsCI, 0°C, 6 h. TBDMSO CN
TBDMSO .CN
OMe
TBDMSO CN
67%
>/
A 244
245
\\ O
*OCH3 OCH3 246
<°X)
-A
83% from 237
0
i H 0
H3CO
~OCH 3
OCH3 247
a) THF, NaBH4, reflux, MeOH, 1 h; b) toluene, Ag2CO3, reflux, 14 h; c) LDA, 3,4,5-methoxybenzaldehydeJHF, -78°C, 30 min; d) TFA, CH2CI2, rt, 6 h; e) TBAF, CH2CI2, AoOH, rt, 6 h.
Scheme (39). Asymmetric synthesis of lignans by Ohmizu et al.
Corey et al. completed a very elegant enantioselective synthesis of (-)wodeshiol (255), Scheme (40) [106]. Treatment of vinyl ketone 248 with
587
bromine followed by triethylamine-promoted elimination of hydrobromic acid afforded the a-bromoderivative 249. Enantioselective reduction of 249 proceeded smoothly using catecholborane and (i?)-proline derived Bmethyl CBS catalyst 250 providing allylic bromo alcohol 251 in 84% yield and 88% enantiomeric excess. Treatment of 251 with f-BuLi and subsequent transmetallation in situ with BuaSnCl afforded the organometallic derivative 252 which could be coupled using Pd(PPli3)4 in the presence of copper chloride and copper dichloride to give after purification the desired diene 253 in 82% chemical yield and 99% enantiomeric excess. Bis-epoxidation of the diene 253 using f-BuOOH as oxidant and VO(acac)2 as catalyst furnished 254 as a mixture of two diastereomers in a ratio of 14:1. Finally, pyridinium tosylate-promoted cyclization provided (-)-wodeshiol (255) in 66% yield.
OH Br
SnBu3
84% 251
252
ee = 88% 82% e
HO'-)—("'OH
61%
255
a) Br2, CH2CI2, -78"C, 30 min; b) i. Et3N, Et2O, -78"C, 2 h ii. 0°C, 14 h; c) 20% 250, catecholborane, toluene/CH2CI2 (1:1), -78 - C, 132 h; d) i. f-BuLi, Et2O, -78'C, 2.5 h ii. Bu3SnCI, -40"C, 2.5 h; e) Pd(PPh3)4, CuCI, CuCI2, DMSO, 60'C, 2 h; f) cat. VO(acac)2, f-BuOOH, CH2CI2, 0°C, 14 h; f) PPTS, benzene, 80"C, 22 h.
Scheme (40). Asymmetric synthesis of (—)-wodeshiol by Corey et al.
Sefkow completed the synthesis of a-hydroxylated butyrolactones using commercially available (-)-diisopropylmalate (256) as chiral source, Scheme (41) [107,108]. Alkylation of 256 using LiHMDS as base gave the desired product 257 in high yield and excellent selectivity. Saponification of the ester groups was achieved by treating 257 with 4 equivalents of KOH in ethanol. During the hydrolysis a sluggish
588
epimerization took place and thus two acids 258 and 259 were isolated in a ratio of 11:1. After recrystallization, the desired diacid 259 was obtained in 87% yield as sole isomer. Acid-catalyzed acetalization of diacid 259 with pivalaldehyde proceeded smoothly in benzene using a soxhlet apparatus filled with activated 4 A molecular sieves. Both transdioxolanone and cz's-dioxolanone were obtained in over 80% yield with a ratio of 1:5. The desired cis isomer 260 could be obtained in diastereomerically pure form by recrystallization in 70% yield. For the second alkylation step LiHMDS was selected as base: treatment of 260 with two equivalents of LiHMDS at -72°C in the presence of the appropriate electrophile afforded dioxolanone 261 as single isomer in 71% yield. Reduction of the acid moiety using BH3 followed by treatment of the reaction product with aqueous HC1 provided 262 in 88% yield. Finally, hydrogenolysis of the benzyl ethers gave (-)-wikstromol (263) in quantitative yield. OH ^X—CO2iPr
OH _a_ 80°/i
256 OMe 259 92% 87% after recrystallization
BnO' H3C6 260 70% after recrystallization
a) LiHMDS (2 eq.), THF, 1-(benzyloxy)-4-(bromomethyl)-2-methoxybenzene, -78CC to 8"C, 12h; b) KOH, EtOH, 72 h, rt; c) TsOH, pivaladehyde, benzene, reflux, 8 h, MS 4 A; d) THF, LiHMDS (2 eq.), 1 -(benzyloxy)-4-(bromomethyl)2-methoxybenzene, -78°C, 5 h; e) i. BH3SMe2, Et2O, reflux, 5 h ii. 10*C, aqueous HCI, 3h; f) EtOH, H2, Pd/C, rt, 20 h
Scheme (41). Asymmetric synthesis of (-)-wikstromol by Sefkow
589
Charlton explored a biomimetic, oxidative free-radical coupling in the asymmetric synthesis of (+)-rabdosiin (274), Scheme (42) [109].
= CH 2 CH=CH 2 R, = T B D M S
OR
OH
a) i. AcCI, AICI 3 ii. HCI; b) NaNO 2 , H 2 O; c) H 2 O 2 , NaOH, H 2 O; d) acetone, allyl bromide, K 2 CO 3 , reflux, 22 h; e) CCI 4 , 270, DMAP, Et 3 N, rt 1 h; f) THF, n-Bu 4 NF, rt, 15 min ; g) acetone, FeCI 3 , H 2 O, 23 h in the dark; h) (PPh 3 ) 2 PdCI 2 , AcOH, Bu 3 SnH, 1 h
Scheme (42). Asymmetric synthesis of (+)-rabdosiin by Charlton
(5)-Tyrosine (264) was acylated using a Friedel-Crafts reaction providing acetyl-(5)-tyrosine (265) as hydrochloric salt. The amino acid 265 was then transformed into hydroxy acid 266 with retention of configuration by a two step procedure consisting of diazotization of 265 and subsequent
590
hydrolysis in situ. Baeyer-Villiger oxidation followed by hydrolysis gave directly diphenol 267. At this point, the phenolic and carboxylic groups were protected as allyl ethers by treatment with allyl bromide and potassium carbonate. Esterification of 268 with the acid chloride 270, prepared in 3 steps from caffeic acid 269, occurred in 97% yield. After removal of the silyl groups with fluoride ion, ferric chloride-promoted radical coupling reaction proceeded sluggishly affording the desired aryltetralin 273 together with the isomer 272 in low yields and in a ratio of 1:1.6. Finally, deprotection using tributyltin hydride and bis(triphenylposphine)palladium dichloride under acidic conditions completed the synthesis of (+)-rabdosiin (274). Chenevert synthesized both enantiomers of a dibenzylbutyrolactone lignan using as key step an enzymatic desymmetrization reaction of meso alcohols, Scheme (43) [110]. H ?\
H
^ r
AK.
JL .OMe 98% Ar^ / L .OH 74% Ar^ JL .OAc 48%
(~)MP
a
Ar ^ r
H
Ar
.. 275 Ar = 3,4-dimethoxyphenyl
H 276 i 96% e
OH
H °
b
-
H 277 ee >
'
U
H 278
98%
H
H A
H 279
_ u Ar^S^OAc c,d , r ^ ^ o 80% Ar^/j^OH 48% Ar^^W H Ho 280 281 ee > 98%
a) LiAIH4, CH2CI2:Et20 (3:2), rt; b) CAL, vinyl acetate, benzene, rt, 74%; c) PDC, DMF, rt; d) p-TsOH, HCI 5N, toluene reflux; e) Acetic anhydride, DMAP, pyridine, rt; f) CAL, EtOH, isopropyl etherbenzene (5:2) Scheme (43). Asymmetric synthesis of cw-dibenzylbutyrolactones by Chenevert et ah
Diester 275 was prepared according to the method of Ward [111]. Reduction of the diester with lithium aluminium hydride furnished the desired meso diol 276. Diol 276 was subjected to the enzymatic esterification reaction by treatment with Candida antartica lipase (CAL)
591 591
in benzene using vinyl acetate as acyl donor to give monoester 277 in 74% yield and with an enantiomeric excess greater than 98%. Oxidation of the primary alcohol group and subsequent acidic cyclization concluded the total synthesis of 278. To synthesize the enantiomeric form a modified procedure was used: diol 276 was acylated with acetic anhydride in pyridme in the presence of DMAP to give the meso-diacetate 279. At this point transesterification of 279 by utilizing ethanol and CAL gave 280 as sole isomer in 80% yield. The conversion into the natural product 281 was then achieved as described above. Berkowitz and co-workers disclosed a new synthesis of (-)podophyllotoxin via enzymatic desymmetrization of a meso diacetate, Scheme (44) [112,113]. The bromo acetal 282 was synthesized from piperonal in 80% yield by using a two step procedure. Hydroxymethylation of 282 proceeded smoothly using s-BuLi in the presence of solid paraformaldehyde affording 283 in 82% yield. Treatment of 283 with neat acetylenedicarboxylate under acid catalysis gave the cycloadduct 285 via isobenzofuran 284 in high yield. It is noteworthy that the reaction could be performed in 20 gram scale. Catalytic hydrogenation of 285 occurred exclusively from the less hindered exo-face providing after reduction with LiAlELt and acetylation the meso diacetate 286 in an excellent 87% yield. Desymmetrization of the meso diacetate 286 was conducted under carefully monitored conditions by treating 286 with porcine pancreatic lipase (PPL) in DMSO at pH 8. The reaction could be performed in several gram scale affording monoacetate 287 in 66% chemical yield and 95% enantiomeric excess. The chiral monoacetate 287 was then converted into the silyl-protected aldehyde through a three step procedure commencing with silylation of the primary alcohol followed by deacetylation and finally Swern oxidation to afford the product 288 in 97% yield. Treatment of 288 with base lead to a retro-Michael ring opening giving 289 in 90% yield. The secondary alcohol was protected as 2-(trimethylsilyl)ethoxymethylether and the aldehyde oxidized to acid 290. The acyl oxazolidinone functionality was then introduced by treatment of 290 with carbonyl diimidazole followed by reaction with 2-lithium-oxazolidinone. Compound 291 was recoverd in 60% yield. Michael addition on 291 using ArMgBr and CuCN cleanly produced the 1,4-adduct 292 as sole isomer. Desilylation of 292 with TBAF, followed by spontaneous lactonization provided lactone 293 in 62% yield. Finally, epimerization to
592
the termodinamically less stable trans-lactom under Kende's conditions [114], separation of the two isomers by chromatography and removal of the protecting group completed the total synthesis of (-)-podophyllotoxin
287
288
90% I
OH OTIPS
r
CHO
SEMO
SEMO
OTIPS
OTIPS
m,n 93%
289
290
291 85%
SEMO
H3CO
y
SEMO
OCH3
H3C0
OCH3 293
OTIPS
y 0CH3 0CH3 292
a) n-BuLi, THF, (CH2O)n; b) HOAc, DMAD, 80°C; c) H2, Pd/C; d) LiAIH4, Et2O, reflux; e) Ac2 O, pyr, DMAP, -5°C; f) PPL, 10% DMSO, 50mM KPO4, buffer, pH 8; g) TIPSCI, imidazole, DMF, rt; h) K2CO3, MeOH; i) (COCI)2, DMSO, CH2CI2, Et3N, -78°C; I) NaOMe, MeOH, rt; m) SEMCI, /-Pr2NEt, CH2CI2, rt; n) NaCIO2, NaH2PO4, (-BuOH, 2-methyl-2-butene; o) i. carbonyl diimidazole, THF ii. n-BuLi, oxazolidinone -78°C; p) 3,4,5-trimethoxyphenylmagnesium bromide, CuCN, THF, 10°C, 2.5 h; q) TBAF, THF, 50°C; r) i. LDA, -78°C ii. pyr-HCI; s) MgBr2, EtSH, Et2O-Benzene 4-1, 0°C to rt.
Scheme (44). Asymmetric synthesis of (-)-podophyllotoxin by Berkowitz and co-workers
Uemura et al. investigated the use of (arene)chromium complex in a formal total synthesis of (-)-steganone (7), Scheme (45) [115]. Treatment
593
of 294 with (-)-l,2,4-butanetriol followed by methylation afforded the 1,3-diequatorially substituted dioxane derivative 295 in 73% yield. Chromium complexation proceeded smoothly giving the corresponding arenium complex 296 in 67% yield. A bromine atom was then stereoselectively introduced by lithiation of 296 and subsequent bromination with l,2-dibromo-l,l,2,2-tetrafluoroethane providing 297 in 47% yield and 90% enantiomeric excess. Furthermore, after one fractional crystallization from ether-hexane, the enantioselectivity could be increased to greater than 99%. Acidic hydrolysis of the acetal followed by reduction of the obtained aldehyde furnished the alcohol 298 in 77% overall yield. Suzuki cross-coupling between 298 and 2-formyl-4,5methylenedioxyphenylboronic acid (299) in the presence of Pd(PPli3)4 gave the desired coupled biaryl 300 in 67% yield without formation of the corresponding atropisomer. Protection of the hydroxy group as silyl ether followed by reaction with methyl lithium at -78°C afforded a 5:1 mixture of secondary alcohols 301. Allylation of the secondary alcohol 301 was then performed by reaction with NaH and allyl bromide. After demetallation by air, 302 was recovered in 57% overall yield. Finally, deprotection with tetrabutylammonium fluoride followed by bromination of the primary alcohol and reaction with sodium dimethyl malonate afforded 303 in 45% yield. This concluded the formal total synthesis of (— )-steganone (7) as Meyers et al. had earlier converted 303 into the natural product [116].
594
OMe
H3CO
OCH3 OCH3 *Cr(CO)3 297
ee = 90%
(CO)3Cr
H3CO. H3CO
a) (S)-1,2,4-butanetriol, TsOH, MeOH, reflux, 6 h; b) Mel, NaH, THF, DMF; c) Cr(CO)6, butyl ether, heptane, THF, 130°C, 24 h; d) n-BuLi, toluene, -78°C, 1,2-dibromo-1,1,2,2-tetrafluoroethane; e) 6N HCI, THF, 40°C, 1h; f) MeOH, NaBH4, 5 min, rt; g) 299, Pd(PPh3)4, aq. Na2CO3, MeOH, reflux, 1 h; h) NBuMe2SiCI, imidazole, CH2CI2, i) MeLi, Et2O, -78°C; I) allyl bromide, NaH, THF, DMF; m) hv, O2, Et2O; n) n-Bu4NF, THF; 0) CBr4, PPh3, CH2CI2, 0°C; p) NaCH(CO2Me)2, MeOH.
Scheme (45). Formal total synthesis of (-)-steganone by Uemura
In addition, intermediate 300, produced using Uemura's methodology, was used in another very elegant synthesis of (-)-steganone (7) by Molander and co-workers, Scheme (46) [117]. The primary alcohol of 300 was converted into the corresponding bromide 304 by utilizing
595
methanesulphonic anhydride in the presence of triethylammonium bromide. Stille coupling between 304 and 305 was high yielding using the weakly coordinating AsPli3 as ligand to give the chromium complexed product 306 in almost quantitative yield. At this point, Smk promoted 8endo radical cyclization was achieved using 3.5 equivalents of Smk in fert-butanol in the presence of HMPA. The cyclized product 307 was obtained as a single isomer in 73% yield. PCC-Treatment of 307 buffered with NaOAc in CH2CI2 accomplished the oxidation of the secondary alcohol as well as decomplexation of the chromium complex in one pot. The resulting ketone 308 was obtained in 85% yield as a mixture of two atropisomers in which the absolute stereochemical information of alcohol 307 was retained only at the lactone ring juncture stereocenters. Equilibration of the stereocenters was achieved by treating ketone 308 with DBU in THF at reflux affording (-)-steganone (7) in 82% yield.
r-q 91% "H3CO,
(CO)3Cr 300
OCH3 304
H 3 crj / (CO)3Cr
0 73%
H3CO, H3CQ
OCH3
(CO)3Cr
306
82% H3CO'
a) Ms2O, Et3NHBr, Et 3 N, CH2CI2, 0°C, 1 h; b) 298, Pd2(dba)3, AsPh3, THF, reflux; c) 3.5 eq Sml 2 , 2 eq f-BuOH, THF/HMPA, 0°C; d) PCC, NaOAc, CH2CI2; e) DBU, THF, reflux.
Scheme (46). Asymmetric synthesis of (-)-steganone by Moiander et at
596
Conclusion In the last ten years significant progress has been made in the asymmetric synthesis of lignans demonstrated by the multitude of elegant total syntheses reported in this review. As new lignans with interesting biological activies are isolated at a frequent rate, it is very likely that in the future we will see more exiting total synthesis of this extraordinary class of compounds. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] II1] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
601 601
NATURAL OLIGOSTILBENES MAO LIN, CHUN-SUO YAO Institute ofMateria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China ABSTRACT: More than 200 naturally occurring oligostilbenes isolated from plant kingdom are grouped into I-V structural types. They are a special kind of polyphenolic natural products with multi-faceted bioactivities. This article will review their classification, distribution, spectral characteristics, biological activities (anti-fungal, antibacterial, antioxidant, anti-inflammatory and anticarcinogenic activities, etc.) and mimetic biosynthesis.
INTRODUCTION
The naturally occurring oligostilbenes are a special group of polyphenolic compounds polymerized from resveratrol or other stilbene units (such as isorhapontigenin, oxyresveratrol, etc.). They usually possessed novel and complex structures and are widely distributed in plant families. A number of structures of stilbene oligomers have been elucidated. A conservative estimate suggests that the number of naturally occurring oligostilbene has increased to more than 200 in recent years with the development of modern spectroscopic techniques, especially various kinds of 2D-NMR and mass spectral techniques. Their multi-faceted biological activities including anti-inflammatory, anti-viral, anti-tumor, antibacterial and anti-fungal are attracting the worldwide medicinal chemists. These compounds are considered to play an important role in the protective effects of plants against fungal and bacterial invasion. They also appear to have considerable potential for pharmaceutical uses as chemopreventive and anti-inflammatory agents against neoplastic changes and inflammatory effects in human body. The biomimetic synthesis of oligostilbenes is based on their biogenetic pathway using different stilbene monomers or £-viniferin as precursor in the synthesis of new types of compounds with strong activity for drug development
602
In 1951, the first resveratrol (1) polymer, hopeaphenol (2), was isolated from Hopea odorata and Balanocarpus heimti by King and his co-workers [1]. But its structure was not established as a resveratrol tetramer until 1966, when a single crystal, a dibromide derivative of deca-O-methyl ether of 2 had been prepared and analyzed by X-ray method [2]. Langcake and Pryce reported that 1 existed as a phytoalexin in infected grapevine leaves, since then (+)-s-vinferin (3) and a-vinferin (4) have been isolated from the infected grapevine leaves [3,4]. Thereafter, about 20 oligostilbenes have been isolated from five plant families. In 1993, Sotheeswaran and Pasupathy [5] divided naturally occurring resveratrol
2 hopeaphenol
3 (+)-&-viniferin
4 a-vinferin
oligomers into two major groups: Group A contained at least one oxa-cyclic ring, usually the fra«s-2,3-diaryl-2,3-dihydrobenzofuran moiety, while group B did not contain any oxygen heterocyclic rings. All the oligostilbenes of group A appeared to be formed from resveratrol via the dimer ^-viniferin. However, oligostilbenes of group B were polymerized directly from various kinds of stilbene monomers without s-viniferin being an intermediate. Most naturally occurring oligostilbenes belong to group A, only a few of them belong to group B. With the increase of naturally occurring oligostilbenes, a variety of other oligomers containing different stilbene monomers, especially isorhapontigenin (S), oxyresveratrol (€), as well as their glycosides were obtained from plant resources. Thus, we proposed a new classification method as follows: all naturally occurring oligostilbenes hitherto known could be classified into the following five major groups (I-V) according to the different stilbene monomers present in their structures. Each basic type was subdivided into two groups (A and B) depending upon whether they
603
contained oxygen heterocycles or not. Group I was further divided into group I-A, I-B, I-C and I-D respectively. 1. Classification of Oligostilbene The stilbene monomers that polymerized to oligostilbenes are depicted in the following: Ri
R2
R3
R4
1
resveratrol
H
H
OH
H
5
isorhapontigenin
H
OCH3
OH
H
6
oxyresveratrol
OH
H
OH
H
7
piceatanol
H
OH
OH
H
8
rhapontigenin
H
OH
OCH3
H
9
gnetol
OH
H
H
OH
The numbering of oligostilbenes complies with the following rule: Each
OH
Fig. (1). Numbering of resveratrol (A) and (-)-s-viniferin (B)
stilbene monomer is numbered respectively and the different units are distinguished by a, b, c, etc; as an example, the numbering of 1 and 3 are shown in Fig. (1). I. Oligostilbenes polymerized from compound 1. Most naturally occurring oligostilbenes belong to this type and are further divided into four groups (I-A, I-B, I-C and I-D) according to whether they contain oxa-cyclic rings and the type of polymerization. I-A group: this group contains at least one five-membered, oxa-cyclic ring, usually the 2-aryl-2, 3-dihydrobenzofuran ring. About 60 oligomers
604
10 (-)-s-viniferin
11 ampelopsin A, R=OH 12 ampelopsin B, R=H
14 amuresins C
16 amuresins H
13 miyabenol C
15 amuresins D
17 amuresins"L
18 anigopreissin
belonging to this group have been isolated from nature. (+)-s-Viniferin (3) and (-)-£"-viniferin (10) are the basic units, which form various structures of oligostilbene with a straight chain including dimers, trimers, tetramers and pentamers. For example, 3 and miyabenol C (13) belong to I-A group. But the oligomers containing a moiety of bicyclo [5,3,0] decane or 10-oxabicyclo [6,3,0] undecane ring system, such as ampelopsin A (11) and ampelopsin (12), are not included in this groups. Some oligostilbenes with benzofuran ring also belong to this group. Anigopreissin A (18) was the first oligostilbene containing an unsaturated benzofuran ring [8J. Recently, amuresins C (14), D (15), E, F, H (16) and L (17) have been isolated from Vitis amurermsis [6,7], In the structures of amuresins D, E and F, the locations of 4-hydroxybenzene moiety and 3,5-dihydroxybenzene of those compounds were interchanged compared with those of 14, which are the new type of oligostilbenes.
605
Some oligostilbenes with cis olefinic protons, such as maximol B (19) [9] and gnemonol L (20) [10], have been found in plants. I-B group: Oligostilbenes are polymerized directly from resveratrol monomers but do not contain any oxygen heterocyclic ring in their structures, such as ampelopsis D (21) [11]. Until now, 20 oligomers of this group have been found in plants.
OH OH
20 gnemonol L
19 maximol B
Caraphenols B (22) and C (23) are a pair of isomers with different connectivity recently isolated from Caragana sinica [12].
21 ampelopsin D
22 caraphenol B
23 caraphenol C
I-C group: Oligostilbenes containing at least a bicyclo [5,3,0] decane or bicyclo [6,3,0] undecane ring system in their structures. Compound 11 or 12 is the basic moiety of I-C group. In the past few years, more than 40 oligomers, including trimer (+)-vinferol D (24) [13], tetramer (+)-viniferol A (25) [14], hexamer vaticanols D (26), H, I and heptamer vaticanol J have been successfully isolated from the genera of Vitis and Vateria [15,16]. Recently, the only octamer, vateriaphenol (27), has been isolated from Vateria indica [17].
606
24 (+) viniferol D
25 (+)-viniferol A
27 vateriaphenol A
26 vaticanol D
28 hemsleyanoside F
29 gnemonoside B
30 gnemonoside K
I-D group: this group includes all oligostilbene glycosides. The structures are usually formed by an aglycone of I-A and I-C groups and connected with 1-3 glucoses composing O- glycoside or C-glycoside. Since 2001, about 28 glycosides belonging to this group have been found as natural products. Hemsleyanoside F (28) is an I-C type of stilbene dimer-C-glucoside from Shorea hemsleyama and Gnetum gnemonoides, respectively.
31 gnemonoside H
32 gnemonoside F
607
Two glycosides of resveratrol polymer, gnemonoside B (29) and K (30) have cis olefinic bonds in their aglycones, The aglycone moieties of gnemonoside H (31) and F (32) are dimer and trimer of resveratrol, and each of them is connected with 3 glucoses, respectively [19,20]. Recently, two glycosides of stilbene dimer named compound 1 (33) and compound 2 (34) were obtained from an aqueous extract of the roots of Polygonum cuspidatum. Among them, 34 was a new type of resveratrol dimer possessing a four-membered ring [21].
33 compound 1
34 compound 2
35 shegansu B
II. Oligostilbenes polymerized from compound 5. More than 20 polyphenols of this type have been isolated from plants. II-A group: shegansu B (35) was obtained from Belamcanda chinensis in 1997 [22], which later was also obtained from most of the Gnutum species. Recently, gnetupendin D (36) was isolated as the first isorhapontigenin glycoside from Gnetum species having 35 as aglycone moiety [23]. A pair of isorhapontigenin isomer (gnetuhainin N (37) and O (38)) polymerized from gnetulin (41), and an isorhapontigenin unit (with a dihydrobenzofuran moiety at C-7c,8c,5b and 4b-O positions and different stereochemistry at H-7c and H-8c) were isolated from Gnetum hainanense [24]. II-B group: gnetifolins C (39) and D (40) were obtained by our research group in 1992 [25]. At first, gnetifolin C was assigned a structure with
OCH,
36 gnetupendin D
37 gnetuhainin N
38 gnetuhainin O
608
an eleven-membered ring (39) according to the 2D-NMR spectroscopic methods. For further confirmation of the structure, an oxidative reaction was carried out. Oxidation of acetylated gnetifolin C (39a) with Lemieux-Johnson's method (OsO4/NaIO4 in dioxan-HbO) produced two products, a ketone (a) and an aldehyde (b) Fig (2). The reaction indicated that the structure of gnetifolin C (39) was identical with a known compound,
MeO-
HO
OH
HO
39 gnetufolin C
OCH,
40 gnetufolins D
OH
OCH, •Ms
41 gnetulin
42 gnetuhainin R
gnetulin (41)[27], which has an exo-double bond with a five-membered ring in the molecule. Gnetuhainin R (42), the first isorhapontigenin tetramer isolated from Gnetum hainanense, was attributed to II-B group [28]. Up to now, most of the isorhapontigenin oligomers have been isolated from Gnetum species in China. It may be a characteristic of Gnetum species in China. III. Oligostilbenes polymerized from piceatanol. Only eight piceatanol dimers have been reported up till now.
609 OCH 3
OAc
OCH 3
OAo
OCH
OAc
OCH,
39a Fig. (2) The oxidation reaction of gnetufolin C
III-A group: this group of oligomers is polymerized from two units of 7, with a five-membered oxygen heterocyclic ring or a benzo-dioxane ring. For example, cassigarol D (43) belongs to the former, and cassigarol E (44) belongs to the latter [29a, 29b].
OH
42 gnetuhainin R
43 cassigarol D
OH
44 cassigarol E
III-B group: piceatanol oligostilbenes without any oxygen heterocyclic ring in their molecules. This type of polyphenol was formed directly from two units of 7 through carbon-carbon bond. Cassigarol A (45) is an example for this group. [30]. IV. Oligostilbenes coupling from 1 and 6 units or two units of 6. IV-A group: oligostilbenes containing a benzo-oxygen heterocyclic ring. 17 compounds have been found involving dimers and trimers from plants such as gnetumontanin A (46) [31]. An oxyresveratrol dimmer, gnetuhainin S (47) [28] and parvifol A [32] were isolated from Gnetum hainanesis and Gnetum pravifolium, respectively. Both compounds are identical and have been reported almost simultaneously. Gnemonol A (48) and gnemonol C (49) from Gnetum gnemon are polymerized from 1 and 6, respectively [33].
610
HO
^ ^
OH
45 cassigarol A
46 gnetumontanin A
48 gnemonol A
47 gnetuhainin S
49 gnemonol C
IV-B group: oligostilbenes from 1 and 6 units connected by carbon-carbon bond forming cyclic or chain oligomers. For example, andalasin A (50) coupled with two units of 6 has been isolated from Morus macroua [34]. Gnetuhainin D (51) polymerized from 1 and 6 was obtained from Gnetum hainanense [35].
OH HO
50 andalasin A
51 gnetuhainin D
OH
52 gnetuhainin K
V. Oligostilbenes polymerized by other monostilbene units besides I-IV group. V-A group: gnetuhainin K (52) is a polymer of 5 and 9 isolated from Gnetum hainanense [36]. Recently two oligostilbenes, gneafricanin A (53) and B (54) have been isolated from Gnetum africanum: 53 was polymerized from 5 and 6 units, whereas 54 from 5 and 7 units [37]. V-B group: oligomerstilbenes connected by different stilbenes units with carbon-carbon bonds such as gnetuhainin J (55), which was polymerized from 5 and 6 units [38].
611
53 gneafricanin A
54 gneaf ricanin B
55 gnetuhainin J
2. Distribution of Oligostilbenes Resveratrol oligophenols have been found mainly from five plant families in the past [5]. Owing to the rapid increase in oligostilbenes in the recent years, the distribution in plants have so far been extended to nine plant families, namely Dipterocarpaceae, Vitaceae, Cyperaceae, Leguminosae, Gnetaceae, Iridaceae, Celastraceae, Paeoniaceae and Moraceae. More than 200 naturally occurring oligomerstilbenes have been isolated from these plants. Most compounds are resveratrol dimers, trimers, and tetramers. A few compounds belong to pentamer, hexame,r heptamer and octamer. 3. Biogenesis and Conformations of Oligostilbene Initially it was proposed that all naturally occurring oligostilbenes of I-A with dihydrobenzofuran moieties were formed from 1 via the dimer e-viniferin, such as the proposed biosynthetic route of 13 [38]. This biosynthetic pathway may be the main biogenetic route for oligostilbenes, but other biogenetic precursors seem to exist during biogenesis of oligostilbenes of group A, because many oligostilbenes with various novel skeletons have been separated as in the cases of amurensins B-F and J [39,6,40]. Amurensins B, 14 and J, each with a cis dihydrobenzofuran
56a 7a,8a-cfe-£-viniferin 56b iso-e-viniferin Blg.(3) Plausible intermediate of biogenetic pathways of oligostilbenes
612
12 ampelopsin B 21 (-)-ampelopsis D 57 (+)-ampelopsin F Fig. (4) Plausible biogenetic pathways of 12,21 and 57 from 3 moiety, might be formed from 1 via 7a,8a-cis-ff-viniferin (56a ), while amurensins D-F might be formed via iso-a-viniferin (56b) [6] Fig, (3). Unfortunately, 56a and 56b were not isolated from plants till now. It was concluded that the biogenesis of oligostilbenes is very complex and implies multiplicity.
[A]
613
[AJ + [B]
25
*=a 2 *=0 61 Fig.(5) Plausible biogenetic pathways of stilbenetetramers from 10
In the last few years, Niwa M. et al. reported that oligostilbenes have mainly been isolated from Vitaceae Dipterocarpaceae, Cyperaceae, Leguminosea and Gnetaceae;oligostilbenes isolated from Vitaceaeous plants are chemically different from those from other families as shown in the cases of e-viniferin and hopeaphenol. 3 has only been isolated from Vitaceaeous plants, but 10 has been isolated from plants of other families. Therefore, Vitaceaeous plants have a specific biogenetic pathway distinguished from other plants such as Dipterocarpaceae, Cyperaceae, Leguminosae. 3 seems to be a biogenetically important precursor for many
614
oligostilbenes isolated from Vitaceaeous plants. Niwa M. et al. also designed a transformation of 3 in different acids by the biomimetic pathway leading to obtain a variety of oligostilbenes. Isomerization and/or rearrangement of 3 yielded compounds 12, (+)-ampelopsin F (57) and 21. The difference of products is apparently due to the difference of the position of protonation at the initial stage of the reaction as shown in Fig. (4) [41]. The oxidative coupling along with isomerization may also transform 3 to 2, (+)-vitisin A (58), (-)-vitisin B (59), (+)-vitisin C (60), (-)-isohopeaphenol (61) and 25 as shown in Fig. (5)[42]. All the mentioned transformation and oxidative coupling reactions provide us information of the reactivity based on the biogenetic pathway and further confirmation of absolute structures of the resveratrol oligomers.
4. Spectral Characters of Oligostilbenes The structural elucidation of naturally occurring oligostilbenes depends on a modern spectroscopic evidence, such as MS, NMR, IR and UV. All kinds of 2D-NMR techniques (including COSY, HMQC, HMBC, NOESY) play important roles in the structural elucidation of oligostilbenes. We will mainly summarize their 1 H NMR and B C NMR characteristics in this section.
4-1 tHNMR and13CNMR Characteristics In *H NMR and 13C NMR spectra of oligostilbenes, the signals of various monostilbenes are useful to determine their polymerization degrees. The different monostilbenes could be distinguished by XH NMR signals of ring A Fig. (6). Signals of ring A appear as A2B2 system for resveratrol, ABX system for 5, 6, 7 and 8. There is one methyloxy signal more in 5, 8 than in 6 and 7. Following is an example of the NMR features of resveratrol oligomers. The trans double bonds (H-7, H-8) of resveratrol units appear in three forms after polymerization. 1. Still in trans double bond, the proton signals appear as two doublets at 8 6.4~7.2 with coupling constant of
615
15.0~17.0Hz. The carbon signals appear at 5 128-135 (C-7) and 5 120~125(C-8) respectively. A few of them in cis double bond, the proton signals appear at 8 5.5-6.1(J = 12 Hz). 2. As part of a dihydrobenzofuran moiety Fig. (6), H-7a and H-8a are in trans relationship and C-7a is generally linked to the oxygen atom. The signals of H-7a and C-7a in NMR spectra appear at lower field of 8 5.2-6.0 and 8 85-95, respectively, due to the deshielding effect of the oxygen atom. H-8a and C-8a appear at 8 4.2-4.8 and 8 45-60 respectively. The coupling constant between H-7a and H-8a is within 3.5~8.6 Hz when they are in trans relationship. 3. As members of aliphatic ring, the signals of H-7 and H-8 appear at 8 2.5-4.5 and 8 35-65 respectively.
Fig (6) Polymerization of resveratrol in a dihydrobenzofuran moiety
Most of the 4-hydroxybenzene groups (ring A) in 1 display signals of A2B2 system in aH NMR spectra after polymerization, presenting two coupled doublets ( J = 7.5-9.0 Hz) at 8 7.0-7.5 for H-2 and H-6, and at 8 6.4-6.9 for H-3 and H-5. In 13C NMR, the signals of C-2 and C-6 appear at 8 127-130, and of C-3 and C-5 at 8 114-117. Only a few of them showed signals of ABX system in tetramers and pentamers. The 3,5-dihydroxybenzene group (ring B) in 1 showed signals of AB2 system at 8 5.8-7.0 after polymerization, such as H-lOa, 12a, 14a in Fig. (6). The 1HNMR proton signals of H-12a appear as a triplet with coupling constant between 1.0-2.5Hz and proton signals of H-10a,14a appear as symmetrical doublets with the same chemical shift and coupling constant. In 13C NMR, C-lOa and 14a have the same chemical shift at 8 105-108, and the chemical shift of C-12a appears at 8 100-103. When the 3,5-dihydroxybenzene group was involved in a dihydrobenzofuran moiety, two meta coupled protons such as H-12b and H-14b showed two doublets at 8 6.0-6.6. In 13C NMR, the signals of C-12b appeared at 8 95-100, and of C-14b at 8 105-110.
616
The quaternary carbons of C-l and C-9 (not attached to a double bond) in resveratrol exhibited signals at 8 130~136 and 5 141~149, respectively, after polymerization. The quaternary carbons attached to a hydroxy group in resveratrol oligomers showed signals at 8 155~162. The stereochemistry of H-7a, H-8a and H-7b, H-8b of oligostilbene with bicyclo (5,3,0) or (6,3,0) decan or undecane ring system could be determined by 1 H-NMR spectrum. The coupling constant ( J = 11.0-12.8 Hz) of H-7a and H-8a suggested the stereochemistry between H-7a and H-8a to be trans, such as 11 (J=11.7Hz) and 2 (/=12.5Hz). The chemical
62 (+)-viniferol C.
63 (+) vaticanol B
64 vaticaphenol A
shift value of H-8a at 8 4.25-4.44 indicated the stereochemistry between H-8a and H-7b to be and as shown in the case of 2, 63 and vaticaphenol A (64). This upfield shift is caused by a shielding effect of the aromatic group B at C-7b. The chemical shift value of H-8a at 8 5.0-5.4 indicated the stereochemistry between H-8a and H-7b to be syn as shown in the case of 62. The coupling constant value of H-7b and H-8b (J=11.7 Hz) indicated the stereochemistry between H-8b and H-7b to be trans as shown in the case of 62. A coupling constant of J=3.0-3.9Hz in 63 and 64, which have the same ring system as 62, suggested a cis stereochemistry between H-7b and H-8b. This consideration was further supported by NOE difference experiments and molecular mechanics calculation as well as biogenetical synthesis [14,43]. 4-2. MS Characteristics Since the oligostilbenes have high molecular weights and mutiplet hydroxyl groups, it is generally difficult to obtain their molecular ion peaks in EI-MS except for some dimers or those without a hydroxyl group in the structures. Modern MS techniques, such as FAB-MS, FD-MS, ESI-MS and LSI-MS were used to obtain their molecular ion peaks.
617
4-3. UV Characteristics
The maximum absorption bands of oligostilbenes depend on the conjugation system in their structures. The maximum absorption bands appear at nearly 320 nm for oligostilbenes having trans double bonds, and at about 280 nm for oligostilbenes without trans double bonds in their structures. Oligostilbenes with a benzofuran ring have maximum absorption band at about 340 nm. 4-4. IR Characteristics The IR spectra of oligostilbenes exhibit absorption bands for hydroxyl groups (3200-3500 cm"1), benzene group (1450-1600 cm"1) and double bonds (1610-1670 cm"1). There is a strong absorption band at 965 cm"1 if trans double bonds exist in the structure. An ambiguous absorption band at 730-675 cm"1 will appear in certain cases, if there are cis double bonds in the molecule. 4-5. Absolute Configuration of Oligostilbene In 1990, two chiral carbons of 10 had been established to be 7a/? and 8ai? respectively, by Kurihara et al. [44]. After that, in 1996, 7aS and 8aS had been established for 3. Their CD spectra showed negative and positive Cotton effects at 237nm, respectively. The absolute configuration of a large number of oligostilbenes belonging to I-C group has been established by Ito et al. [46]. The absolute configuration of 11 was
lla
65
66
618
3
V"S
CHO
67
68
established by the following method: methylation by methyl iodide to give a penta-methyl ether (lla), which lead to oxidation giving the corresponding ketone (65). The observation of NOE between H-8a and H-2b (6b) suggested the configuration of the 4-methoxyphenyl group at C-7b to be pseudo-axial against the adjacent carbonyl group in 65. The structures of 65 and 66 show positive and negative Cotton effects respectively. On the basis of octant rule and by comparison of CD spectra, the ketone of 65 showed a positive Cotton effect at 357nm indicating that the absolute configuration of 11 should be represented as in structure 65. The absolute configuration of 12 has been established by comparison of its CD with that of 11. The three positive absorption maxima at 208-211, 233-236 and 287-288nm showed strong resemblance to that of 11. Therefore, the absolute configuration of 12 is identical to 11 as shown in 12. For some tetramer oligostilbenes such as 58, the absolute configuration has been established by the same method. Two parts of degradation products 67 and 68, with aldehyde groups, have been obtained after oxidation. One degradation product, 67 should have the same absolute configuration as 3. The other degradation product 68 was a seven-membered ring aldehyde resembling 65 in wavelengths and the CD spectrum. Thus the entire absolute configuration of 58 could be established by the combination of chemical reactions and NOE as well as C D spectrum [47]. 4-6. X-ray Crystallographic Analysis Most naturally occurring oligostilbenes have been isolated as amorphous powder, therefore it was very difficult to determine their structures by
619
X-ray crystallographic analysis. Hitherto, only the structure of 2 was established by X-ray crystallographic analysis of its bromide. Recently, the structure of 47 isolated from Gnetum hainanense has been verified by X-ray crystallographic analysis [28], 5. Biological Activities Oligostilberies are a large group of phenolic substances isolated from 5 principle plant families. About 70 of more than 200 individual known compounds have been tested for various biological activities. Many studies suggested that this group of constituents exhibited potent biological activities, including antimicrobial, antioxidant, anti-inflammatory, antihepatotoxic, antitumor and other activities. The various in vitro biological activities of oligostilbenes have been tested while their in vivo pharmacological efficacies were less documented in the past studies. 5-1. Antifungal and Antibacterial Activities Since compounds 3 and 4 were first isolated from infected grapevin leaves together with 1, so antifungal activity studies on 1 and its oligomers have been conducted. In the preceding studies, 1 showed moderate antifungicidal and antibacterial activities, while its polymers possessed greater or broader bioactivities [5], Almost 10 compounds have been screened by filter paper disc method (FPDM) in the Mueller Hinton Agar medium using Staphylococcus, Esherichia coli and Staphylococcus aureus etc. in the past years [38, 48, 49, 50, 51, 52], In 2002, Tomoko N. reported the antibacterial activity of the extract prepared from 181 species (75 families) of tropical and subtropical plants, which were screened against various types of pathogenic bacteria. Among the 505 extracts, 53 of them from barks of Shorea hemsleyana and the roots of Cyphostemma bainesii. Some of them showed significant activity in reducing the viable cell number of methicillin-resistant Staphylococcus aureus. The active compounds were all identified as stilbene derivatives. Hemsleyanol D (69), a stilbene tetramer, isolated from S. hemsleyana was the most effective [53].
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5-2. Antioxidant Activity
Various reactive oxygen species (ROS) are generated from enzymatic and nonenzymatic processes during the normal metabolism in cells. If excessive ROS are not eliminated promptly, the accumulated ROS will induce cytotoxicity, which plays important role in pathophysiology of
69 hemsleyanol D
70 gnemonol B
many diseases, such as inflammation, atherosclerosis, reperfusion injury and cancer. Antioxidants can terminate ROS chain reaction by disturbing initiation and propagation steps of the reaction and play important protective role against the damage caused by various diseases. Thus the studies on antioxidant activities have attracted many pharmacologists. Lliya I. et al. [54] found that many stilbenes showed potential antioxidant activities by comparing the antioxidant activities of nine compounds including monomers 2, 5, 6, dimmer 10, trimmers gnemonol B (70), I (71), K (72) and 20 with Vitamin E for lipid peroxide inhibition and super oxide scavenging activity [10]. Oligostilbene dimers 53, 54, gneafricanins D (73), and E (74), isolated from Gnetum africanum, showed inhibition of lipid peroxide and super oxide scavenging activities as shown in Table 1 [37,55].
71 gnemonol I
72 gnemonol K
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73 gneafricanin D
74 gneafricanin E
Ten natural polyphenolic products, i.e. longusone A (75), longusols A (76 ), B ( 77) and C (78), were isolated as the active constituents together with 10 phenolic compounds from the methanolic extract of the whole plant of cyperus longus by a bioassay-guided separation. The antioxidative action for l,l-diphenyl-2- picrythydrazyl (DPPH) radical showed that frans-scirpusin B (79) was the most potent (SCso=2.8|j,M), and the scavenging activity of stilbene dimers (75, 76, 77, 78, 79 and 80) was stronger than those of monomers (1,7) except for pallidol Table 1. [56]. Liu G. T et al. investigated the antioxidation activities of nine compounds including oligostilbenes 16 and 81, monostilbenes and isoflavones by several models in vitro, among them 1, 5, 16 and 81 have potent antioxidation activities in vitro. Furthermore, 16 showed significant protective effects on neurotoxins induced oxidative cell injuries and apoptosis in primary cultured cerebellar granule cells and bovine aorta endothelial cells through inhibiting release of cytochrome c from mitochondria and activation of Caspase-3 [57a, 57b].
OH
75 longusone A
78 longusol C
76 longusol A
79 frans-scirpusin A
77 longusol B
80 rrans-scirpusin B
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Compounds 25, 26 and 27 belonging to I-C group, isolated from Vatica rassak and V. indica, showed antioxidation and SOD like activities, indicating that this group of oligomers also has antioxidation activity and superoxide scavenging activity [58,59, 60]. Two glycosides of stilbene dimer, 33 and 34 were isolated from Polygonum cuspidatum. Pharmacological studies showed that 34 which has a special skeleton showed moderate inhibition (inhibition rate 36%) in the formation of MDAat a concentration of 2 pM [21]. The results of recent research on antioxidation activities have revealed that the radical scavenging activity depends on the structure and multiple phenolic hydroxyls of the oligostilbenes. Table 1 Natural oligostilbenes with antioxidant activity* ^ompoiixiua
Super oxide scavenging activityriCsoTuM)!
lipid peroxide inhibition activity[ICsn(nM)l
16
15
69 59 79 57 20 20 10 33 30 26 29
19 7 50 25 33 34 13 50 32 29 45 81 36
LangusoneA Longusol A Longusol B Longusol C Treres-seirpusin A 7>*aras-scirpusin B Cassigatol E Cassigarol G Pallidol Gnemonol K Gnemonol L Gnemonol B Gnemonol I (-)-*-viniferin Gneafricanin A Gneafricanin C Gneafricanin D Gneafticanin E Gnetin F Bisisorhapontigenin B Compound 1 Compound 2
DPPH radical [SCsoOiM)] 4.6 9.3 4.3 5.0 8.2 2.8 3.2 4.5 29
* DPPH=l,l-diphenyl-2-picrylhydrazyl
Studies on the structure-activity relationship of oligostilbenes drew the following conclusion: (i) the presence of conjugated system with double bonds and para-hydroxy groups, which have better electron-donating properties and are responsible for electron delocalization. This is a radical target; (ii) multiple hydroxy groups on different positions of the molecule, for example, ortte-dihydroxystilbene forming metal chelate complex can
623
increase the radical scavenging capability; (iii) cyclic sizes of oligomer and different attachment of rings have different contributions to radical scavenging capability. Thus, the estimation of oligostilbene as antioxidants has a pharmacological prospect. 5-3. Anti- Inflammation and Anti-Carcinogenic Activities Leukotrienes (LTA4, B4, C4, D4, E4, F4) and prostaglandins (PGS) are a series of important metabolites of arachidonic acid (AA) via 5-lipoxygenase (5-LO) and cyclooxygenase (COX). They were found to play a major role in the pathogenesis, including cell proliferation, inflammation and immune response, platelet aggregation, smooth muscle contraction and maintenance of fluid and electrolyte balance. Therefore, inhibitors of 5-LO and COX are known to have anti-inflammatory and anti-carcinogenic activities. Many oligostilbenes from Gnetum species showed a variety of biological activities on LTs. A stilbene dimer (Gn-3) separated from Gnetum parvifolium exhibited potent inhibitory activities on LTC4 and D4 enzymes and their receptors [61, 62]. Some oligostilbenes polymerized by 1 and 5 obtained from Gnetum or Vitis species were observed to exhibit potent inhibition action on LTB4. Compounds 81,2, 61, 58, (+)-vitisinfuran A (82), amurensin F (83) and 35 showed inhibitory activity on LTB4 at concentrations of 10"5 -10"4 mol'L"1
81 heyneanol A
82vitisinfuranA
with an inhibitory ratio of 76%, 56%, 60%, 63%5 72%, 67% and 98% respectively [22, 40]. 15 and 35 also showed slight activity on biosynthesis
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of LTD4 [6, 22]. In the research for anti-inflammatory and/or cancer chemopreventive activities of natural products, Lee SH et al estimated the inhibitory effects on the COX activities of over 600 species of wild plants in Korea. 4 was isolated as a main active constituent in the dose-dependent inhibition of COX from Carex humilis (originally from Caragana chamlagu), with an IC50 of 7|J.M and 3-4 fold stronger than 1 [64]. Therefore, 4 exhibited anti-inflammatory and cancer chemopreventive activities.
83 amurensin F
84 kobophenol A
A series of oligostilbenes has been tested for various inflammatory models. Among them, compounds 16, 81 and 2 showed strong inhibitory effects both in vitro and in vivo. Their IC50 of inhibition of PMN chemotaxis induced by fMLP were respectively 4.8xlO"13, 4xlO"10 and 1.6xlO"10 mol-L"1 in vitro. They showed significantly the inhibition of inflammatory response with multiple drug administration pathways. In the inflammatory model of croton, oil-induced ear edema and DNFB induced the delayed-type hypersensitivity. The results suggested that 16, 81 and 2 are worthwhile for further studies [65]. 5-3-1. Protein Kinase C (PKC), a family of serine/threonine kinases, is one of the major regulatory enzymes, which involves multiple cellular responses. Since the activation of PKC has been implicated in both inflammatory and proliferative processes, the inhibitors of PKC may be of potential therapeutic value. Compounds 4, 13 and kobophenol A (84) were separated from Caragana sinica by bioassay-guided fractionation, which were evaluated
625
for their PKC activity in vitro. 4 and 13 showed inhibition activity at a concentration of 8-6 l^M range and were more potent than the tetramer, 84. While not showing appreciable isoenzyme selectivity, the three compounds were selective in inhibiting PKC over the cAMP-dependent kinase (PKA). 4 showed modest activity (IC50 47)LIM) in zymosan activated leukocytes in whole blood and also potently inhibited the proliferation of NHEK cells (IC50 0.4 ^M) and MCF-7 mammary tumor cells (IC50 3.6(j.M). The 50-100-fold decrease in activity of 4 after a short-term exposure (8h) in the MTT assay suggested that its anti-proliferative activity is not the result of direct acute cytotoxicity. 4 also showed anti-inflammatory activity in the carrageenin induced mouse hind paw edema model. The research indicated that 4 may be useful in treating hyperproliferative or inflammatory skin diseases [66]. 5-3-2. Tumor Necrosis Factor (TNFa) is one of the pro-inflammatory cytokines, which seems to be involved in the initiation and amplification of the inflammatory process. Thus, searching for an inhibitor or modulator of TNFa is one of the main tasks in the quest for anti-inflammatory leading compounds and drugs. HO
OH
85 gnetupendin C
87gnetumontanin B
86 gnetin D
88 gnetumontanin A
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Two tetramers (59, 60), and gnetupendin C (85), gnetin D (86), gnetumontanin B (87) (dimer) and gnetumontanin A (88) (trimer) were isolated from Vitaceae and Gnetaceae respectively. Pharmacological test revealed that 85, 86 and 87 showed potent inhibitory activity on TNFa with an inhibitory ratio of 59.50, 67.23 and 58.1% respectively at concentration of 10"5 mol-L"1. The IC 50 value of 89 was 1.49xlO"6 mol-L"1, for the inhibition of TNFa production by murine peritoneal macrophages [23, 31].
5-4. Cytotoxic A nti- Tumor A ctivity
Much bioassay-guided research has been conducted to find cytotoxic anti-tumor agents in plants especially those known to be used in folk medicine for this purpose. The choice and number of cell lines used in these bioassays were very variable. OH
HO OH
OH
89 resveratrol trans-dehydrodimer
OH
90 pallidol
Resveratrol frans-dehydrodimer (89) and pallidol (90), constituents of grapes (Vitis spp.), which are formed in response to the microbial attack by the fungal grapevine pathogen Botrytis cinerea, showed modest cytotoxicity against human lymphoblastoid cell (CEM) with IC50 values of 49 |iM, and 32 pM respectively [63]. Malibatol A (91) and B (92) only inhibit cytotoxicity to the host cell (CEMSS), with IC50 values of 13 and 21 i^g-ml"1 respectively [68]. Vatdiospyrodol C (93) from Vatica diospyroides displayed the most potent activity against oral epidermoid carcinoma (KB, EC50 1.0 \ig-m\~1), colon cancer (CoI2, EC50 1.9 M-gml"1), and breast cancer (BC1, EC 50 3.8 \xg-ra\~1) cell lines. This is the first example in which significant cytotoxic
627
91 malibatol A
92 malibatol B
93 vatdiospyrodol C
activity against human cancer cell line has been reported for a tetramer. As the decamethyl derivative of 93 did not show significant activities in the cell lines tested in this study, therefore the hydroxyl groups in 93 play an important role in the cytotoxic activity of 93 [69]. Recently, a patent for cancer remedy and preventive medicines of 93 and 4 has been applied by Linuma, M. et al. [60]. 5-5. Inhibition of Tyrosinase Activity
Melanin biosynthesis inhibitory compounds are useful not only as skin whitening agents but also used in cosmetics and as a remedy for disturbances in pigmentation. Tyrosinase is one of the key enzymes for melanin biosynthesis in plants, microorganisms and mammalian cell [70]. Therefore, many tyrosinase inhibitors have been tested in cosmetics and Pharmaceuticals for preventing overproduction of melanin in epidermal layers. Tyrosinase is also one of the most important key enzymes in the insect molting process. Thus the investigation of inhibitors of this enzyme may provide important clues for developing new insect control agents or whitening agents in cosmetic products and inhibition of the mammalian cancer cell. Artogomezianol (94) and 50 isolated from Artocarpus gomezianus are two oligostilbenes with straight chain. They showed moderate tyrosinase inhibitory activity with IC50 values of 68 and 39 fxM, respectively. The inhibition of 50 is about two times stronger than 94, which possesses only one 4-substituted resorcinol structure (ring C). The relationship of 4-substituted resorcinol skeleton and tyrosinase inhibitory activity has been discussed in reference [71].
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5-6. Antihepatotoxic Activity
There are a variety of crude drugs in China and Japan, which are effective in liver diseases. Ohizumi Y et ah found that some Vvtaceaeous plants such as Ampelopsis brevipedunculata Trautv, A. brevipedunculata Trautv.var. hancei Render and Vitis coignetiae Pulliat et Planch exhibited significant antihepatotoxic activity at a concentration of 1 mg-ml"1 in carbon tetrachloride (CCU), and D-galactosamine-induced cytotoxicity of primary cultured rat hepatocytes [72]. Further studies showed that 3 was a main active principle from the extract of Vitis coignetiae which showed strong hepatoprotective activity against injuries of primary cultured rat hepatocytes induced by CCU and D-galactosamine (D-GalN). 3 also reduced ALT values significantly in CCU-treated mice at a dose of 30 mg-kg"1. Besides 3, 11, ampelopsin C (95), 57, 58 and ds-vitisin A (96) were also present in the extract. Among them, 95 and a mixture of 58/96 increased ALT values approximately 4.1 and 5.7 times respectively, compared to the control value, when given at 30 mg-kg"1 to CCU-treated or non-treated mice, also showing strong hepatotoxins [73]. Surprisingly, the extract of Vitis coignetiae, an Oriental crude drug used in treatment of liver disease, contained two contradictory constituents [72].
94 artogomezianol
95 ampelopsin C
96as-vitisinA
Four oligostilbenes, 21, ampelopsins E (97), H (98) and cis ampelopsin E (99), were obtained from Ampelopsis brevipedunculata var. hancei. Among them, 97 and 99 showed antihepatotoxic activity at a dose of 0.1 mg-ml'1 in the culture medium, reducing the elevation of GPT levels by 64 and 73% respectively [11].
629
97 ampelopsins E
98 ampelopsins H
99 cis ampelopsins E
A stilbene polymer (Gn-3) isolated from Gnetum parvifolium inhibited the development of liver injury in mice caused by CCU, N-acetyl-P-aminophenol (APAP) and Bacillus Calmette-Guerin (BCG) plus bacterial lipopolysaccharide (LPS) at a dose of 50 mg-kg^-d"1 sc administered for 3 d; thus Gn-3 was found to have liver protective effects [74] 5-7. Other Biological Activities
Kim YC et al. found that 4 and 84 have inhibitory activity on acetylcholinesterase (AChE). The cholinergic system is in relation with Alzheimer's disease. A promising therapeutic strategy for re-activating the central cholinergic function has been used for inhibitors of AchE, which is responsible for the metabolic hydrolysis of ACh. 4 and 84 from methanolic fraction of Caragana chamlague inhibited AchE activity in a dose-dependent manner and the IC50 values were 2.0 and 115.8 |uM respectively. However, AChE inhibition of 1 or a glycoside of 8 was not significant. Therefore, 4 might be a valuable AChE inhibitor because it has an appropriate bulky structure that masks AChE and prevents ASCh from binding to AChE in a noncompetitive manner. In contrast, 84, which is a tetramer with a bulky structure, was less active due to the difficulty of accessibility to AChE [67]. Ecdysteroids are the steroid hormones of insects, where they regulate moulding and metamorphosis. As ecdysteroids are essential to the normal development of insects, it was presumed that some plant secondary metabolites might have the ecdysteroid antagonistic activity and be capable of affecting insect development. Such compounds would be useful
630
tools for the elucidation of ecdysteroid gene regulation and as potential lead compounds for the development of new classes of insecticides [75]. Dinan L. reported that three resveratrol trimers, suffruticosols A (100), B (101), C (102), and one monomer cis resveratrol from Paeonia suffruticosa are active as ecdysteroids (antagonists (EDso) 10-50 pM vs. 5xlO"8|iM of 20-hydroxyeedyson), but inactive as agonists in the Drosophila melanogaster BII cell bioassay for ecdysteroids agonists/ antagonists [75]. It was reported that 12 and 4 from the methanolic extract of the seeds of Iris clarkei, antagonized the action of 20-hydroxyecdysone with ED50 of l.OxlO'5 and 3.3xlO"5 M respectively. [76]. The potencies of 12 and 4 are similar to those of 100-102 (ED50,1.4xl0~5-5.3xl0"5M) and eis-resveratrol (EDso at 1.2xlO"5M) [76] as well as three oligostilbenes from the seeds of Carex pendula (EC50 at 31, 19 and 37^M M) [77] indicating that resveratrol oligomers are potentially significant constituents in reducing insect predation on plants.
100 suffrutieosol A
101 suffruticosol B
102 suffruticosol C
6. Biomimetic Pathways The biological effects of some naturally occurring oligostilbenes were usually greater and/or broader than their monostilbenes, but because of the low content, strong polarity as well as similar chromatographic properties, it was quite difficult to isolate the pure compounds for further pharmacological studies. Recently, some biomimetic syntheses have been reported which may be a way to obtain this kind of bioactive oligostilbenes by semi-synthesis and total synthesis. Now, we will submit for discussion in following four sections.
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6-1. Catalysis by Oxidase Langcage P. et al. proposed that the viniferin like compounds could be obtained from resveratrol when resveratrol was treated with horseradish peroxide and H2O2. The major products (103-106) of this reaction were
103
103-106
Rl
R2
R3
R4
R5
H
H
H
H
H
H
OH
OH
H
OH
R6
104
OH
H
105
H
OH
H
H
OH
H
106
H
H
OH
H
H
OH
obtained in 40% yield [78]. Coupling reaction of resveratrol or s-viniferin, with an additional unit of resveratrol could take place by the same process. Similar coupling reactions have been achieved with other 4-hydroxylated stilbenes. Ito and Niwa isolated 3, 57 and 59 successfully from the same source, and synthesized those viniferin oligostilbenes by the same process [79]. The methods were special for the synthesis of oligostilbenes. 6-2. Catalysis by Metal A. Coupling reaction using ferric chloride (FeC^) as oxidant 1, The polymerization from homo-monomer: Compound 16 isolated from Vitis amurensis showed strong biological activity [7, 65] and its biomimetic synthesis was achieved as shown in Fig. (7). Oxidative coupling reaction of 1 with FeCl3 as oxidant produced an intermediate, (±)-£-viniferin (107), by silica gel column chromatography. After acetylation, it was dehydrogenated by treatment with 2,3-dichloro-5,6- dicyano-1, 4benzoquinone (DDQ) to afford an intermediate(108) and the desired compound 16 in 20% yield [7].
107
108 Fig. (7) The mimetic synthesize of 16
16
632
MaO
109 shegansuB
110 111 112 bisisorhapontigeninA bisisorhapontigenin B triisorhapontigeninA
HO
113 bisisorhapontigenin C
^ ^
OH
114 bisisorhapontigenin D
115 tetraisorhapontigenin A
Shegansu B, the isorhapontigenin dimer from Belamcanda chinensis, was synthesized from 5 with FeQa as oxidant [22]. In the reaction procedure, seven compounds (109-115) of dimeric, trimeric and tetrameric polymers were obtained. Their possible formation mechanisms were also deduced respectively [80, 81]. For instance, the mechanism of 115 was described as shown in Fig. (8). 2, The polymerization from hetero-monomer: A group of oligostilbenes polymerized from 1 and 5, such as gnetuhainin J, K, L and Q, isolated from Gnetum hainanesis [36, 35a], represented a type of hetero-monomer stilbenes. In order to study the biogenetic pathway of hetero-monomer oligostilbenes, a probe work was carried out. One molecule of 1 and two molecules of 5 (1:2 mol) were dissolved in acetone and stirred with FeCl3-6H2O (0.12 mol) at room temperature for 24 hrs. The suspension was filtered. The filtrate was evaporated to remove off acetone
633
.OH
-115
Fig. (8) The mechanism of coupling reaction of 115 in vacuo and extracted with EtOAC, which was concentrated to dryness. The residue was chromatographed on silica gel and ODS Rp-18 to obtain nine compounds, which were identified as 107,116,117,118,119,120,
116 Fe-3
117 Fe-4
119 Fe-7
118 Fe-5
120 Fe-8
634
121 Fe-9
122 Fe-10
121, 122 and 114 respectively, by spectra analysis, especially 2D NMR spectra. The desired stilbene dimers, such as gnetuhainin L and Q, were not obtained. But compounds 118-121 were identified as hetero-monomer stilbene oligomers of Compound 119, which has a novel skeleton with one unit of 1 and two units of 5. The skeletons of 120, 121 and 122 were similar to those of the naturally occurring compounds 97 and 15 [11, 6, 821.
Fig. (9) Condensation of 44 and 123 by Ag2O
B. Coupling reaction using silver oxide (Ag2O) as oxidants. Two oligostilbenes of 44 and 123 (III-A group) from Cassia garrettiana were synthesized by Baba K. et al by 7 in acetone with Ag2O as oxidant [29] Fig.(9). C. Coupling reaction using vanadium oxytrichloride (VOCI3) as oxidant
124 miyabenol A
125 miyabenol B
Fig. (10) The oxidation of miyabenol A and B by vanadium oxytrichloride
635
Kawabata J et al. isolated two compounds, miyabenol A (124) and miyabenol B (125), from Carex species. The former belongs to I-A group and the latter to I-C group. In determining the stereochemistries of 124 and 125, oxidative coupling of 124 with VOCI3 as oxidant gave compound 125. Their spectral data were in agreement with those of the naturally occurring compounds as depicted in Fig. (10) [83]. 6-3. Photooxidative Coupling Reaction Using UVas Photooxidant
In the course of polymerization reaction, UV was always used as photooxidant to prepare oligostilbenes. For example, 58 was converted to its cis isomer 96 by photochemical transformation as shown in Fig. (11) [47]. The structure-activity relationship analysis indicated that cis configuration of the stilbene unit is the most important factor in combretastatin group for inhibition of cancer cell growth. In order to obtain oligostilbenes with cis olefinic protons, our research group designed and achieved two oxidative coupling reactions using FeCl; and UV as oxidants respectively. With FeCb as an oxidant, the coupling reaction of 1 at room temperature produced an intermediate (±)-£-viniferin after silica gel chromatography. Irradiation of (±)-e-viniferin in anhydrous
58(+)-vitisinA 96 cis- vitisin A Fig. (11) Photochemical transformation of 58 and 96
alcohol with UV light (254 nm, 200W) as photooxidant for 2.5 hrs afforded two object products in 25% and 20.0% yield after column chromatography, as shown in Fig. (12). Among them, 126 is a
636
czs-£-vinferin with a 51.4% inhibition rate on TNFa at concentration of 10"5 mol-L"1, and 127 is a phenanthrene derivative with a 58.1% inhibition rate on TNFa [84].
OH +
HO
127 resveratrol
7b,8b-cw-£-viniferin
2b,14b-dehydrobisresveratrol
Fig. (12) The synthesis of 126 and 127 by photochemical transformation
6-4. Coupling Reaction with Acid The natural products 2, 12, 21, 57, 58, 59 and 60 have been isolated from Vitaceaeous plants. Niwa M. et al reported the regiospecific and stereospecific transformations of e-viniferin to above oligostilbenes using various acids as polymerizing agents, such as H2SO4, HCl and CF3SO3H, according to the biogenetic pathways of oligostilbenes, see Figs. (3) and (4) [41, 42].
128 y-viniferin
129 y-2- viniferin
Fig. (13) The structure of 128 and 129 verified by HCl
Korhammer S et al. isolated two stilbene tetramers, y-viniferin (128) and y-2- viniferin (129), form Vitis roots. Treatment of 128 with 0.1% HCl in MeOH afforded 129, Fig.(13); their structures were verified as unambiguous [85]. Sotheeswaran S. et al. reported that the formic acid could be used as a cyclization agent [86]. Both resveratrol trimers of stemonoporol (130) and
637
copalliferol A (131) were isolated from Dipterocarpaceae plant. 130 was treated in formic acid to afford 131, which confirmed that the former was a precursor of the latter [Fig.(14)].
HCO2H or toluene-p-sulDhonic
130 stemonoporol 131 copalliferol A Fig. (14) Chemical conversion of 130 and 131 into formic acid
In order to get various cyclo-oligostilbenes, our research group designed a dimerization reaction between natural compounds 1 and 5 using 80% formic acid as catalyst. A series of tetralins, isorhafomicols A, B, C and D (132a-135) and resformicols A, B and C (132b-134b) were obtained this way. All structural assignments were made on the basis of various spectral evidences, including 2-DNMR techniques. To our surprise, during ?L
,OH
OH OH
132aD132bD
133a (133b)
OCH3
134a (134b)
1, R=H 5,R=OCH3
135
Fig. (15) Tetralins of isorhapontigenin and resveratrol by formic acid
638
dimerization both the structures of 134a and 134b were found to lose a substituted phenyl group, i.e, 3-methoxy-p-hydroxy-phenyl and j?-hydroxy- phenyl, respectively. Moreover, 135 successively lost two substituted benzene rings and one methoxy group during the course of trimerization. This type of reaction has not been reported before. The probable mechanisms both cyclodimerization and cyclotrimerization are discussed respectively below [87]. The cases mentioned above indicated that strong, electron-donor substituents lead to the formation of a 6-membered ring. In the investigation of the Diels-Alder reaction from 1 and 5, a side reaction, involving loss of a substituted benzene ring during dimerization or trimerization, driven by ejection of a methoxy group was found. It is a very special reaction we have ever seen. It may be formed through some transition states as shown in Figs. (15) and (16).
134a
CH S O,
639
HO
^"^
OH
135 Fig. (16) The proposed mechanism of 135
CONCLUSION The natural oligostilbene is a type of the important polyphenolic compounds distributed widely in the plant kingdom. A large number of pharmacological tests indicated that these compounds possess strong biological activities, especially for a variety of cytokines. Recently, some cytokine modulators, such as selective blockers of IL-1(3 and TNFot receptors, have been employed clinically. The use of these drugs has some disadvantages regarding their high cost, important side effects and route of administration (subcutaneous injections). The fact that oligostilbenes are widely distributed in plants and plenty of them have important anti-cytokine activities, studies on the development of therapeutic agents would be beneficial. The natural oligostilbenes or its derived agents could be used alone or in association with other available effective drugs, allowing a reduction in costs and /or side effects and possibly leading to an increase in effectiveness. Large-scale studies on biological activity, phytochemistry and biomimetic synthesis reveal that their constituents would have considerable potential for pharmaceutical uses as chemopreventive agents against neoplastic change and inflammation in human body. The review of oligostilbene may assist relevant fields to seek for new, effective and low side effects drugs. ACKNOWLEDGEMENTS The National Natural Science Foundation of China financially supported some studies of relevant oligostilbenes in this review by a grant No. 30,
640
070, 889. The authors also thank Prof. L,N. Li, GT. Liu, G F. Cheng for giving nice suggestions for this review. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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ISOLATION, STRUCTURE ELUCIDATION AND BiOAcnvrnES OF PHENYLETHANOID GLYCOSIDES FROM CISTANCHE, FORSYTHM AND PLANTAGO PLANTS T. DEYAMA,1 H. KOBAYASHICLAIE),1 S. NISHIBE,2 and P. TU3 1 Central Research Laboratories, Yomeishu Seizo Co., Ltd, Mnawa-Machi, Nagano 399-4601, Japan 2 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tohetsu-Cho, IsMkari-Gun, Hokkaido 061-0293, Japan 3 School of Pharmaceutical Sciences, Peking University, Beijing 100083, PR Chirm ABSTRACT: Many Phenylethanoid glycosides were isolated from Cistanche Herb, produced from Cistanche deserticola, C. salsa, and other Cistanche plants, C tubulosa, C sinensis and C. phelypaea (Grobanehaceae). Cistanoside A-I and tubuloside A-E were isolated from C. deserticola and C. tubulosa, respectively, together with known phenylethanoid glycosides; echinacoside, acteoside and isoacteoside. They possessed rhamnosyl (1—»3)glucosyl group. Suspensaside and forsythiaside, possessed rhamnosyl (1—»6)glueosyl group, were isolated as main components from Forsythia suspensa (Oleaceae), not from F. viridissima. On the other hand, acteoside and J3-hydroxyacteoside were isolated from F. viridissima as main components, not from F suspensa. Plantamajoside, isoplantamajoside and hellicoside, possessed glucosyl (1—»3)glucosyl group, were isolated from Plantago asiatica (Plantaginaceae) together with acteoside, P-oxoacteoside, P-hydroxyacteoside, and campenoside. Several phenylethanoid glycosides, forsythiaside, suspensaside, acteoside, |3hydroxyacteoside and plantamajoside, showed an antibiotic activity against Staphylococcus aureus. Forsythiaside, suspensa- side, plantamajoside and isoplantamajoside showed a strong inhibitory activity against cAMP phosphodiesterase, whereas acteoside and P-hydroxyacteoside showed a weak activity. Plantamajo- side, hellicoside exhibited a strong inhibitory activity on arachidonic acid-induced mouse ear edema. Acteoside and isoacteoside showed a weak activity. Suspensaside showed a continuous hypotensive activity on spontaneously hypertensive rat (SHR).
646
INTRODUCTION Cistanche (Orobanchaceae) plants parasite on the root of Chenopodiaceae and Tamaricaceae plants and are distributed in Middle Asia from China to Turkey, Arab and North Africa Cistanchis Herba (Roucongrong in Chinese) has specified as the fleshy stem of Cistanche deserticola Y. C. Ma and used for staminal tonic, treatment of male impotentz, female sterility, and cold sensation in the loins and knees in Chinese Pharmacopeia 2000 [1]. Other Cistanche plants, such as C. salsa (C. A. Mey) G Beck, C. sinensis G Beck and C. tubulosa (Schrenk) Wight have been used for the similar purposes [2-4]. We reported the pharmacognostical studies on Cistanche plants from the morphological, phylogenetic and chemical viewpoints [5-8]. The dried Forsythia fruit has been used since ancient times as an antidote, an anti-inflammatory agent and a diuretic in Japan, Korea and China [9]. In Japan, the dried fruit originating from F suspensa Vahl or F viridissima Lindley (Oleaceae) is listed in the Japanese Pharmacopeia XTV as crude drugs "Forsythiae Fructus". The dried fruit from F. koreana Nakai is also used as crude drugs in Korea On the other hand, the Forsythia leaves are used as health tea for cold in China. The Plantago aerial parts have been used since ancient times as a diuretic, an anti-inflammatory and an anti-asthmatic drug "Plantaginis Herba" in Europe and Asia [10,11]. Plantaginis Herba from Plantago asiatica L. (Plantaginaceae) is listed in the Japanese Pharmacopeia XTV as an important crude drug and an aqueous extract is also used as a medicine. The aerial parts of P. depressa Wild, P. major L. and P. lanceolata L. are also used as herbal medicines in Europe and Asia [10,11]. The seeds of P. asiatica have been used since ancient times as a diuretic, an antitussive, an expectorant and an anti-inflammatory drug in Japan and China [12]. In China, the seeds of P. depressa have been also used for the similar purposes. P. psyllium L. is cultivated in Spain and France for the seeds. The dried ripe seeds from this plant (Psyllium) are used in Europe as domulcents and in the treatment of chronic constipation, while the seeds from P. ovata Forskal (Ispaghula husk) are used for similar purposes in India and Pakistan [13]. We shall describe the isolation and structure elucidation and HPLC analysis of the phenylethanoid glycosides from several Cistanche plants growing in China, Mongolia, Kazakhstan, Pakistan, Turkey, Bahrain and Qatar, and from Forsythia plants and Plantago plants used as important herbal medicines.
647
PHENYLETHANOID GLYCOSIDES FROM CISTANCHE PLANTS Isolation of phenylethanoid glycosides from Cistanche plants Many reports on the phenylethanoid glycosides from Cistanche plants have been published in this twenty years. They are acteoside (verbascoside;l), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A-E (5-9), cistanoside Q H (10,11), salidroside (rhodioloside; 12), decaffeoylacteoside (13), osmanthuside B (14), syringalide A 3'-a-rhamnoside (15), isosyringalide A 3'-a-rhamnoside (16), crenatoside (17), tubuloside A-E (18-22), jionoside D (23) and poliumoside (24), and tentatively named sinenside A (25), sinenside B (26) and are listed in Table 1. 1. Phenylethanoid glycosides from Cistanche salsa (C. A. Mey) Bunge Fourteen phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A-E (5-9), cistanoside G, H (10,11), salidroside (12), decaffeoylacteoside (13) and osmanthuside B (14) were isolated from the whole body of C. salsa in China[14]. Recently, tubuloside B (19) has been isolated from 75% ethanol extract of C. salsa stem[15]. 2. Phenylethanoid glycosides from Cistanche deserticola Y. C. Ma Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), echinacoside (4), cistanoside A-C (5-7) and cistanoside H (11) were isolated from 50% ethanol extract of C. deserticola [16]. 3. Phenylethanoid glycosides from C. tubulosa (Schrenk) G Wight Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), rhodioloside (12), decaffeoylacteoside(13), syringalide A 3'-a-L- rhamnoside (15), and crenatoside (17) were isolated from 95% ethanol extract of C. tubulosa in China [17]. Five new phenylethanoid glycosides, tubuloside A-E (18-22) were isolated and their structures were elucidated, together with six known glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), syringalide A 3'-a-Lrhamn oside (15), isosyringalide A 3'-a-L-rhamnoside (16), from C. tubulosa in Pakistan [18,19]. 4. Phenylethanoid glycosides from Cistanchephelypaea (L.) Cout. Five phenylethanoid glycosides, acteoside (1), 2'-acetyacteoside (2), echinacoside (4), tubuloside A (18) and tubuloside E (22) were isolated from C. phelypaea in Qatar [20]. 5. Phenylethanoid glycosides from Cistanche sinensis G Beck in China Two new phenylethanoid glycosides, tentatively named sinenside A (25) and sinen-
648
OR,
coutn: R=H
HO
caff: R=OH fer: R=OCH3
Table 1. Phenylethanoid Clyeosides from Cistanche plants No.
Name
R3
R4
R5
R«
H
R2 rham(OH)3
caff
H
OH
OH
Ri
1
acteoside (verbascoside)
2
2-acetylacteoside
COCH3
rham(OH)3
caff
H
OH
OH
3
acteoside isomer(isoacteoside)
H
riiam(0H)3
H
caff
OH
4
echinacoside
H
rham(OH)3
caff
5
cistanoside A
H H H H
rham(OH)3
caff
glc glc
OH OH OCH3
OH
rham(OH)3
fa-
glc
OCH3
OH
rham(OH)3
caff
H
OCH3
OH
rham(OH)3
fer
H
OCH3
OH
H
rham(OH)3
H
OCHj
OH
H
H H H
6
cistanoside B
7
cistanoside C
8 cistanoside D 9 cistanoside E 10
cistanoside G
H
rham(OH)3
11
cistanoside H
COCH3
rham(OH)3
12
salidroside(rhodioloside)
H
H
13
decaffeoylacteoside
rham(OH)3
H H H
14
osmaiithuside B
rham(OH)3
OH
H
OH
OH
OH
H
H
OH
H
OH
OH
count
H
H
OH
rham(OH)3
15
syringalide A 3' o-rhamnoside
H H H
caff
H
H
OH
16
isosyringalide A 3' a-rhamnoside
H
rham(OH)3
coum
H
OH
OH
18
tubuloside A
COCH,
rham(OH)3
caff
glc
OH
OH
19
tubuloside B
COCH3
rham(OH)3
H
caff
OH
OH
20
tubuloside C
COCH3
rham(Ac)3
caff
glc
OH
OH
21
tubuloside D
COCH3
rham(Ac)3
coum
glc
OH
OH
22
tubuloside £
COCH3
rham(OH)3
eoum
OH
OH
H H
rham(OH)3
caff
H H
OH
OCHj
rham(OH)3
caff
rham(OH)3
OH
OH
caff
glc glc
H H
OH
caff
23 jionoside D 24
poliumoside
25
sinenside A
H
rham(OH)3
26
sinenside B
COCH3
rham(OH)3
H 2 OH
B HO 17
crenatoside{orobanchoside)
OH
OH
649
side B (26), have been elucidated by LC/MS, as major components together with acteoside (1), 2'-acetylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), eistanoside C (7), tubuloside A (18) and tubuloside B (19), jionoside D (23) and poliumoside (24), from Cistanche sinensis G Beck in China [21,22]. HPLC analysis of phenylethanoid glycosides from Cistanche plants and Cistanchis Herba We reported HPLC analysis of the phenylethanoid glycosides of crude drug Cistanchis Herba, and C. deserticola, C. salsa, C. salsa vat. albiflora, C. tubulosa and C, sinensis in China. They were similar to one another except C. sinensis, which was different from the others; the major phenylethanoid glycosides were sinenside A (25) and sinenside B (26) [8]. We measured the content of acteoside (1), 2'-acetylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), cistanoside C (7) and tubuloside A (18) of C. deserticola in China, C. salsa in China and Turkey, C. tubulosa in China, Pakistan and in Bahrain, and C. phelypaea in Qatar [6]. Further our study on Cistanche plants by HPLC showed that most Cistanche plants have 1-7, osmanthuside B (14) and tubuloside E (22) [23]. HPLC chromatogram of Cistanche plants were shown in Fig. 1. HPLC analysis of phenylethanoid glycosides from callus of Cistanche tubulosa, C deserticola and C. phelypaea Contents of acteoside (1), 2-aceiylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), eistanoside C (7) and tubuloside A (18) in callus and regenerated plants of C tubulosa and C. phelypaea were analysed by HPLC [6\. It is very interesting thai all callus, regenerated plants of C. tubulosa and C phelypaea showed no peak of eistanoside A (5) nor eistanoside C (7), as well as their normal plants. It seemed that 5 and 7 are able to be used as the marker compounds in chemotaxonomy of Cistanche plants. Callus of C desrticola is abundant in echinacoside (4) and poor in eistanoside C (7) and tubuloside A (18). Structure elucidation of phenylethanoid glycosides from Ciatanche plants Structure elucidation of phenylethanoid glycosides were carried out on the bask of spectral analysis and chemical evidences. Assignment of the 13C-NMR chemical shifts of phenylethanoid glycosides were le-examined and shown in Table 2. [24] Two dimensional NMR spectra, such as ^ - ^ ( C O S Y ) , *H-
650 13
C(HMQC and HMBC) directly afforded the importnat structural informatioa Acteoside (verbascoside: 1) was isolated as an amorphous powder. The sonic spray ion mass (SSI-MS) showed the negative ion at m/z 623 (M-H)", 461(M-163)' and 161 in Table 3. By acetylation with acetic anhydride-pyridine, 1 gave the amorphous nonaacetate, suggesting the presence of five alcoholic acetoxyl groups at 8 All O.OIOr
"i"335'W|
la
14 19 18
i 3 ,61
30
40
] I 50
AU 0.100j
TTSl
0.050:
18
J3 10
20
30
2 40
50
UV 335 m
0.100
25
26
0.050
aiC.ckserttoh fiom China b.Csalsa fxm China
19
, 18 10
20
30
LUj 40
50
Fig.1. HPLC Chromatogram of Cistanche plants
c.C.tuhdasa from China ACtubulosa from Pakistan e.Csinensis from China
651 651
1.87,1.94,2.02, 2.08 and 2.10 (3H, each), and four phenolic acetoxyl groups at 5 2.27, 2.28 (3H, each) and 2.30 (6H). By methanolysis[25] with acetyl chloride in methanol, 1 afforded methyl caffeate and 3,4-dihydroxy- phenethyl alcohol, and by acid-hydrolysis with 10% sulfuric acid, 1 gave glucose and rhamnose in a ratio of 1 to 1. The 13C-NMR chemical shifts of 1 were shown in Table 2. Compound 1 was identified as acteoside [26,27] (verbascoside [28,29]) by comparison with authentic sample. 2'-Acetylacteoside (2) was isolated as an amorphous powder. The SSI-MS showed the negative ion at m/z 665 (M-H)", 503 (M-163)", 461 (M-205)" and 161 in Table 3. The spectral data of 2 suggested the structural resemblance to 1 except for the glucose moiety in 13C-NMR spectrum (Table 2) and for the presence of an alcoholic acetoxyl group at 5 1.99 (3H) in ^ - N M R spectrum. Acetylation of 2 gave the octaacetate, which was identified as the nonaacetate of 1. The location of the acetoxyl group in the glucose moiety was determined by its acetylation shift value in the 13C-NMR chemical shifts. The 13C-NMR spectrum of 1 showed the signals at 104.1, 75.9 and 81.6 ppm due to C-l', C-2' and C-3' carbons of glucose moiety, respectively, whereas those of 2 showed the signals of corresponding carbons at 101.6,75.1 and 80.3 ppm, respectively, in Table 2. The differences in the chemical shifts of the corresponding carbon atoms of 2 and 1 were -2.5 (C-l1), -0.8 (C-21) and -1.3 ppm (C-31) in Table 4. The differences showed that an acetoxyl group was linked to the C-2' hydroxyl group of the glucose moiety [30]. Compound 2 was identified as 2'-acetylacteoside [31] by comparison with an authentic sample. Isoacteoside (3) was isolated as an amorphous powder, whose 13C-NMR chemical shifts were shown in Table 2. The SSI-MS showed the negative ions at m/z 623 (M-H)", 461 (M-163)" and 161 in Table 3. Compound 3 was identified as isoacteoside [25] by comparison with authentic sample. Echinacoside (4) was isolated as an amorphous powder. The SSI-MS showed the negative ions at m/z785 (M-H)", 623 (M-163)"and 161 in Table 3. The 13CNMR spectrum of 4 was silimar to that of 1 except for six carbon signals due to a glucose moiety (in Table 2). The respective signals at 74.5 and 69.2 ppm, due to C-5' and C-6' carbons of glucose, showed upfield shift by 1.6 ppm and downfield shift by 6.9 ppm, respectively, indicating that a glucose moiety is linked to C-6' hydroxy group of inner glucose (in Table 5). Compound 4 was identified as echinacoside [32] by comparison with authentic sample. Cistanoside A (5) was isolated as an amorphous powder. The SSI-MS showed negative ions at m/z 799(M-H)", 637(M-163)" and 161 in Table 3 and the field desorption mass (FD-MS) exhibited positive ion at m/z 823 (M+ Na)+. The 'HNMR spectrum of 5 showed the signal at 8 3.84 due to an aromatic methoxyl
652
group. By aeetylation, 5 afforded the undeeaaeetate, whose 'H-ISIMR spectrum exhibited the presence of eight alcoholic acetecy groups at 8 1.87,1.94,2.00,2.10 (3H each), 1.98 and 2.03 (6H each), and three phenolic acetoxyl groups at 5 2.29 Table 2. 13 C-NMR Chemical 1 2 3 Aglycone 1 131.6 131.9 131.4 2 117.2 117.2 117.1 3 146.0 145.9 146.0 4 144.4 144.4 144.6 116.6 116.6 116.3 5 121.4 121.4 121.3 6 72.3 a 72.3 72.5 36.5 36.2 36.6 P Ester 1 127.7 127.7 127.7 2 115.5 115.5 115.1 146.6 146.6 146.7 3 4 149.5 149.6 149.5 116.4 116.4 116.5 5 123.2 123.2 123.1 6 148.0 148.1 147.2 7 114.8 114.7 114.9 8 9 168.3 168.1 169.1 Glucose 104.1 101.6 104.3 75.4 2' 75.1 75.9 3' 80.3 84.0 81.6 70,0 4' 70.3 70.7 75.6 76.1 5' 76.0 6' 62.3 62.2 64.6 Rhamnose V 102.8 103.1 102.7 2" 72.0 72.3 72.1 3' 71.7 72.3 72.1 4' 73.6 74.0 73.8 5' 70.7 70.4 70.7 17.8 6' 18.4 18.4 Glucose
r
1" 2» 3" 4" 5" 6" OCHj CH 3 CO
20.9 171.5
Shifts of Phenylethanoid Glycosides (in Methanol-rfj) 4 7 11 6 8 5 9 10 131.4 131.5 131.6 131.6 131.6 131.6 117.1 113.9 113.9 113.9 114.0 113.9 145.7 148.7 148.7 148.8 148.8 148.8 144.3 145.7 145.9 145.9 145.9 145.9 116.4 116.1 116.2 116.6 116.6 116.2 121.3 122.4 122.4 122.4 122.4 122.4 72.1 72,1 72.2 72.3 72.3 72.3 36.4 36.6 36.6 36.6 36.7 36.7
12
130.7 130.8 116.1 156.7 116.1 130.8 72.3 36.3
131.8 116.2 144,5 145.9 117.1 121,2 72.4 36.3
130.8 130.8 116.2 156.7 116.2 130.8 72.0 36.4
101.8 74.8 83.1 70.3 77.9 62.5
104.4 75.1 78.1 71.8 77.9 62.8
127.5 115.3 146.4 149.5 116.4 123.1 148.1 114.7 168.3
127.6 115.4 146.6 149.5 116.5 123.1 148.1 114.7 168.3
127.6 112.1 150.7 149.3 116.5 124.2 148.0 115.1 168.3
127.7 115.5 146.7 149.6 116.2 123.2 147.9 114.8 168.3
127.2 112.1 150.7 149.3 116.2 124.2 147.8 115.2 168.2
103.9 75.9 81.5 70.2 74.5 69.2
104.1 75.9 81.5 70.3 74.6 69.3
104.1 76.1 81.4 70.3 74.7 69.4
104.1 76.0 81.6 70.3 76.1 62.4
104.2 76.0 81.4 70.3 76.0 62.5
104.2 75.5 84.8 70.1 77.7 62.8
104.1 75.5 84.6 70.1 77.7 62.7
102.7 72.1 72.1 73.7 70.5 18.3
102.8 72.1 72.1 73.7 70.6 18.4
102.8 72.2 72.2 73.7 70.7 18.4
102.8 72.1 72.0 73.8 70.7 18.4
102.8 72.2 72.1 73.8 70.8 18.4
102.7 71.9 72.3 74.0 70.3 17.9
102.7 102.9 72.3 71.6 72.3 72.1 74.0 73.7 70.2 70.3 17.9 17.8
104.4 74.9 77.6 71.3 77.6 62.5
104.5 74.9 77.6 71.4 77.6 62.6 56.6
104.6 75.0 77.7 71.5 77.7 62.6 56.6 56.6
56.6
56.6 56.6
56.6 20.9 171.6
(3H) and 2.31(6H). The 13C-NMR spectrum of 5 showed almost the same chemical shifts as those of 4 except for the signals due to aglycone moiety (in Table
653 653 Table 2. Continued 14 13 Aglycone 1 131.6 130.7 2 116.3 131.2 3 144.6 116.1 4 146.0 156.6 5 117.1 116.1 6 121.3 131.2 72.1 72.3 a 36.2 36.5 P Ester 1 127.1 130.8 2 3 116.8 4 161.1 5 116.8 6 130.8 7 147.5 g 114.8 9 168.2 Glucose 1' 104.1 104.1 2' 75.5 76.0 3' 84.6 81.5 4' 70.2 70.1 5' 77.7 75.9 6' 62.7 62.3 Rhamnose 1' 102.7 102.7 72.2 2' 72.1 3' 72.1 72.3 4' 74.0 73.7 5' 70.2 70.7 6' 18.4 17.9 Glucose 1" 2" 3" 4" 5" 6" Rhamnose 1" 2" 3" 4" 5" 6" OCH3 CH3 CO
15
16
17
18
19
20
21
22
23
24
130.6 130.8 116.1 156.6 116.1 130.8 72.1 36.2
131.4 117.0 146.0 144.5 116.3 121.2 72.2 36.5
129.1 113.8 145.7 145.7 115.5 118.2 77.8 72.3
131.8 117.2 145.9 144.5 116.3 121.4 72.5 36.2
131.7 117.1 145.9 144.4 116.3 121.3 72.4 36.3
131.8 117.1 145.9 144.4 116.3 121.4 71.8 36.2
131.7 117.1 145.9 144.4 116.3 121.3 71.9 36.2
131.7 117.1 145.9 144.4 116.2 121.3 72.5 36.2
132.9 117.1 147.4 144.5 112.9 121.2 72.1 36.5
131.5 117.1 146.1 144.7 116.5 121.3 72.1 36.6
127.6 115.2 146.6 149.6 116.4 123.1 147.0 114.6 168.2
127.1 131.2 116.8 161.2 116.8 131.2 147.5 114.7 168.2
126.9 114.6 146.2 149.2 115.8 122.6 167.3 147.6 113.8
127.6 115.4 146.7 149.8 116.6 123.3 148.3 114.6 168.2
127.7 115.2 146.7 149.5 116.5 123.1 147.2 114.9 169.0
127.4 115.3 146.8 149.9 116.6 123.2 148.4 114.3 168.0
126.8 116.9 131.4 161.5 131.4 116.9 148.0 114.4 168.1
126.9 131.3 116.8 161.3 116.8 131.3 147.7 114.6 168.0
127.7 115.3 146.8 149.8 116.5 123.2 148.0 114.7 168.3
127.7 115.3 146.8 149.8 116.4 123.2 148.0 114.8 168.0
104.1 76.0 81.5 70.3 75.9 62.3
104.1 76.1 81.5 70.2 75.9 62.3
98.4 81.3 76.7 69.7 77.2 61.4
101.6 75.0 80.5 70.7 74.8 69.2
101.8 74.8 82.6 70.3 75.4 64.4
101.6 75.5 80.1 70.5 74.6 69.1
101.6 75.0 80.3 70.7 74.6 69.1
101.6 75.0 80.4 70.7 76.0 62.1
104.2 76.2 81.7 70.4 76.0 62.4
104.4 76.2 81.6 70.5 74.7 67.6
102.8 72.1 72.1 73.7 70.5 18.3
102.8 72.1 72.1 73.7 70.6 18.4
101.5 71.4 71.3 72.9 69.5 17.7
103.9 71.9 71.4 73.6 70.7 18.5
102.8 72.1 71.8 73.7 70.5 17.8
99.5 70.0 71.2 71.4 68.2 18.0
99.6 70.1 71.2 71.4 68.2 18.0
103.1 71.8 71.8 73.5 70.7 18.4
103.0 72.4 72.1 73.8 70.6 18.5
103.0 72.3 72.0 73.8 70.4 18.4
104.6 74.6 77.7 71.8 77.7 62.6
104.6 74.6 77.7 71.9 77.7 62.6
104.6 75.0 77.8 71.9 77.8 62.6
102.3 72.3 72.0 74.0 69.9 18.0 56.5 20.9
20.9
171.5
171.6
20.4 20.9 171.2 171.5
20.4 20.9 171.2 171.5
20.9 171.4
654
2). Compound 5 gave methyl cafifeate and 3-methoxy-4-hydroxyphenethyl alcohol by methanolysis, and afforded glucose and rhamnose in a ratio of 2 to 1, on acid hydrolysis. Consequently, the structure of cistanoside A (5) was determined to be 2-(4-hydroxy-3-methoxyphenyl) ethyl-(>a-L
No. 1 2 3 4 5 6 7 8 9 12 14 15 16 18 19 20 21 22 25 26 27 28
Name acteoside(verbascoside) 2'-aeetyl acteoside acteoside isomer echinacoside cistanoside A cistanoside B cistanoside C cistanoside D cistanoside E salidroside(rhodioloside) osmanthuside B syringalide A 3'-a-L-rhamnopyranoside isosyringalide 3'-a-L-rhamnopyranoside tubuloside A tubuloside B tubuloside C tubuloside D tubuloside E sinenside A sinenside B forsythiaside suspensaside
(M-HT
(M-163T
623 665 623 785 799 813 637 651 475 299 591 607 607 827 665 953 937 649 769 811 623 639
461 503 461 623 637
(M-205)"
461
475
161 161 161 161 161 161 161 161
445 665 503 791
623 461 749
607 649 461
607
161 161 161 161 161 161
161 161 161 161
Cistanoside B (6) was isolated as an amorphous powder. The FD- MS showed ion at m/z 836 (M-1+Na)+. Acid hydrolysis of 6 afforded glucose and rhamnose in a ratio of 2 to 1. Methanolysis of 6 gave methyl feruate and 3-methoxy-4hydroxyphenethyl alcohol. Acetylation of 6 gave a decaacetate as colorless needles, of which ^-NMR spectrum showed eight alcoholic acetoxyl groups at 8 1.86, 1.93,1.98,2.08 (3H each), 1.96 and 2.00 (6H each), and two phenolic acetoxyl groups at 8 2.28 and 2.31(3H each). The 13C-NMR spectrum of 6 showed almost the same chemical shifls as those of 5, except for the signals due to the caffeic acid moiety (in Table 2). The signals at 6 3.75 and 3.78 (3H, each) in the ^-NMR spectrum, and those at 56.6 ppm in the 13C-NMR spectrum showed the presence of two aromatic methoxyl groups. Cistanoside B (6) was determined to be 2-(4hydroxy-3-mfithoxyphenyl) etiiyl-O-a-L-rhamnosyl (l-^3)-O-[p-D-glucopyranosyl (l—*6)] (4-0-feruoyl)-P-D-glucopyranoside.
655
Cistanoside C (7) was isolated as an amorphous powder. The SSI-MS showed negative ions at m/z 637(M-H)" ,475(M-163) and 161, and the FD-MS exhibited ion at m/z 661(M+Na)+. Acid hydrolysis of 7 afforded glucose and rhamnose in a ratio of 1 to 1. Methanolysis of 7 gave methyl caffeate and 3-methoxy-4hydroxyphenethyl alcohol. Acetylation of 7 gave the octa acetate (7a). The 'HNMR spectrum of 7a showed the signals of five alcoholic acetoxyl groups at 8 1.88,1.95,1.98,2.09 and 2.10 (3H each) and three phenolic acetoxyl groups at 8 2.30(3H) and 2.31 (6H). The 13C-NMR spectrum of 7 was very similar to that of 5, except for the lack of six carbon signals due to the glucose moiety (in Table 2). Cistanoside C (7) was determined to be 2-(4-hydroxy-3-methoxyphenyl) ethyl O-a-L-rhamnopyranosyl (1 —^3)-(9-(4-0-caffeoyl)-P-D-glucopyranoside. Cistanoside D (8) was isolated as an amorphous powder. The SSI-MS showed the negative ion at m/z 651(M-H)" in Table 3, and the FD-MS exhibited ion at m/z 675 (M+Na)+. Compound 8 gave the heptaacetate (8a), by acetylation. The 1HNMR spectrum of 8a showed the signals of five alcoholic acetoxyl groups at 8 1.88, 1.96, 1.98 (3H each) and 2.10 (6H), and two phenolic acetoxyl groups at 8 2.31 and 2.33 (3H each). The 13C-NMR spectrum of 8 showed almost the same chemical shifts as those of 7, except for the signals due to the caffeic acid moiety, and also very similar to that of 6, except for the lack of six carbon signals due to one glucose moiety (in Table 2). Cistanoside D (8) was determined to be 2-(4hydroxy-3-methoxyphenyl)ethyl O-a-L-rhamnopyranosyl(l ^•3)-O-(4-O-feruoyl)P-D-glucopyranoside. Cistanoside E (9) was isolated as an amorphous powder. The SSI-MS showed the negative ions at m/z 475 (M-H)" and 161 in Table 3, and the FD-MS exhibited ion peaks at m/z 476 (M") and 499(M+Na)+. The 'H-NMR spectrum of 9 exhibited the signals due to the methyl group of rhamnose at 8 1.25 (3H, d, J=6Oz), benzylic methylene protons at 8 2.84 (2H, t, >7Hz), a methoxyl group at 8 3.82 (3H, s), a glucose anomeric proton at 8 4.50 (1H, d, ^ 8 H z ) , a rhamnose anomeric proton at 8 5.14 (1H, brs) and aromatic protons at 8 6.6-6.9 (3H). Acetylation of 9 gave the heptaacetate (9a), of which the 'H-NMR spectrum exhibited the presence of six alcoholic acetoxyl groups at 8 1.94,1.98,2.01,2.11 (3H each) and 2.07 (6H), and a phenolic acetoxyl group at 8 2.28 (3H). The 13C-NMR spectrum of 9 showed almost the same chemical shifts as those of 7, except for the lack of signals due to the caffeoyl moiety (in Table 2). Cistanoside E (9) was determined to be 2-(4hydroxy-3-methoxyphenyl) ethyl-O-a-L-rhamnopyranosyl (1—»3)-0-p-D-glucopyranoside. Cistanoside G (10) was isolated as an amorphous powder. The 'H-NMR spectrum of 10 showed the signals due to a methyl group of rhamnose at 8 1.26 (3H, d, J=6Hz), benzylic methylene protons at 8 2.84 (2H, t, J=7Hz), a glucose
656
anomeric proton at 8 4.31 (1H, d, ^ 8 H z ) , a rhamnose anomeric proton at 8 5.18 (1H, d, J=lHz) and aromatic protons at 8 6.72 and 7.09 (2H each, d, J=9Hz). Compound 10 gave glucose and rhamnose in a ratio of 1 to 1, on acid hydrolysis. The 3C-NMR spectrum of the aglycone moiety was almost the same as that of 14 [33], and that of sugar moiety of 10 was the same as that of 13 [33] in Table 2. Acetylation of 10 gave the heptaacetate (10a), of which the ! H-NMR spectrum exhibited the presence of six alcoholic acetoxyl groups at 8 1.96, 2.09, 2.11, 2.14 (3H each) and 2.04 (6H), and a phenolic acetoxyl group at 8 2.29 (3H). Cistanoside G (10) was determined to be 2-(4-hydroxyphenyl) ethyl 0-a-L-rhamnopyranosyl (1 —>3)-O-P-D-glucopyranoside. Cistanoside H (11) was isolated as an amorphous powder. The ^ - N M R spectrum showed the signals due to a methyl group of rhamnose at 8 1.22 (3H, d, J=6Hz), methyl protons of acetoxyl group at 8 1.96 (3H,s), benzylic methylene protons at 8 2.66 (2H, t, ^=7Hz), a glucose anomeric proton at 8 4.41 (1H, d, J=8Hz), a rhamnose anomeric proton at 8 5.16 (1H, brs) and aromatic protons at 8 6.4-6.8 (3H). The 13C-NMR spectrum of 11 showed almost the same chemical shifts as those of 13 [33], except for the signals due to the glucose and acetoxyl groups, and exhibited the acetylation shifts due to the linkage of the acetoxyl group to the C-2 hydroxyl group of glucose (in Table 2). Acetylation of 11 gave the heptaacetate (lla), which was identified as known acetate, decaffeoylacteoside octaacetate[34]. Cistanoside H (11) was determined to be 2-(3,4-dihydroxyphenyl) ethyl O-a-L- rhamnopyranosyl (l-+3)-2-0-acetyl-P-D-glucopyranoside. Salidroside (rhodioloside:12) was isolated as prism. The SSI-MS showed negative ion at m/z 299 (M-H)\ The 13C-NMR spectrum was very similar to that of 10, except for the lack of the signals due to the rhamnose moiety (in Table 2). Compound 12 was identified as salidroside [34] by comparison with authentic sample. Decaffeoylacteoside (13) was isolated as an amorphous powder. The 13C-NMR spectrum was almost the same as that of 9, except for the lack of the signals due to the methoxyl group (in Table 2). Partial methylation of 13 with dimethyl sulfate and potassium carbonate gave the dimethyl ether, which was identical with cistanoside E monomethyl ether. Compound 13 was identified as decaffeoylacteoside [34] by comparison with authentic sample. Osmanthuside B (14) was isolated as an amorphous powder. The SSI-MS showed the negative ions at m/z 591 (M-H)~ and 161 in Table 3, and the FD-MS exhibited ion peak at m/z 615 (M+Na)+. Acetylation of 14 afforded the heptaacetate (14a) as colorless needles, mp 135-136°C, of which the 'H-NMR spectrum showed five alcoholic acetoxyl groups at 8 1.87, 1.94, 2.00 (3H each) and 2.09 (6H), and two phenolic acetoxyl groups at 8 2.28 and 2.31 (3H each). Compound
657
14a was identified as osmanlhuside B heptaacetate [33] by comparison with authentic sample. Syringalide A 3'-a-rnamnoside (15) and isosyringalide A 3'-a-rhamnoside (16) were isolated as amorphous powder, and gave the oetaacetates (15a, 16a), respectively, on acetylation. Compounds 15a and 16a were identified as syringalide A 3'-a- rhamnoside octaacetate and isosyringalide A 3'-a-rhamnoside octaacetate [35], respectively, by comparison with the authentic samples. The 1Hand I3C-NMR spectra of 15 and 16 supported those structures (in Table 2). Consequently, compounds 15 and 16 were identified as syringalide A 3'-a- rhamnoside and isosyringaiide A 3'-a-rhamnoside, respectively. Crenatoside (17) was isolated as pale yellow powder, of which FAB-MS showed ions at m/z 663 (M+H)+, 477 (M+H-Rha)+ and 325 (M+H-Rha-Aglyeone)+. The 13C-NMR spectrum was shown in Table 2. Compound 17 was identified as crenatoside [36] by comparison with authentic sample. Tubuloside A (18) was obtained as an amorphous powder. The SSI-MS showed negative ions at m/z 827 (M-H)" ,665 (M-163)', 623 (M-205)' and 161. The 'H-NMR spectrum showed the signals of a methyl group of rhamnose at 6 1.07 (3H, d, ^=6Hz), a methyl signal of an alcoholic acetoxyl group at 8 1.98 (3H, s), benzylic methylene protons at 8 2.70 (2H, t, «£=7Hz), two glucose anomeric protons at S 4.32 and 4.54 (1H each, d, J=8Hz), a rhamnose anomeric proton at 8 5.11 (1H, brs), two trans olefinic protons at 8 6.25 and 7.64 (1H each, *M6Hz) and aromatic protons at 8 6.5-72 (6H). The 13C-NMR spectrum of 18 was almost identical with that of 4, except for the signals due to the glucose linked directly to aglycone and the acetoxyl group (in Table 2). Comparing the chemical shifts of C-l', C-2' and C-3' carbons of glucose moiety of 4 with those of coresponding carbons of 18, glucosylation shifts were observed at C-l', C-21 and C-3' carbons of inner glucose of 18, which suggests that the acetoxyl group is linked to C-2' hydroxyl group of inner glucose moiety (in Table 5). Acetylation of 18 gave the undecaacetate, which was identical with echinacoside dodecaacetate. Tubuloside A (18) was determined to be 2-(3,4-dihydroxyphenyl) eihyl-O-a-L-rhamnopyranosyl (1—»3)-0-|^-D-glucopyranosyl (1—^]-(4-0-caffeoyl)-2-O- acetyl-^-D-glucopyranoside. Tubuloside B (19) was obtained as an amorphous powder. The SSI-MS showed negative ions at m/z 665 (M-H)", 503 (M-163)", 461 (M-205)" and 161. The [ H-NMR spectrum showed the presence of an alcoholic acetoxyl group at 8 1.98 (3H, s). The 13C-NMR spectrum of 19 was similar to that of 3, but differed slightly in the signals due to the glucose moiety and the presence of acetoxyl group at 20.9 ppm (CH3) and 171.6 ppm (C=O) in Table 2. The location of acetoxyl group in the glucose moiety was determined by acylation shifts, in detailed comparison of "C-NMR chemical shifts of 19 with that of 3. The signals at C-l', C-21 and C-3'
658
carbons of inner glucose showed acetylation shifts -2.5 (C-l1), -0.6 (C-21), and -1.4 ppm (C-31), indicating that Ihe acetoxyl group is linked to C-2' hydroxy group of the glucose moiety of 19 (in Table 4). Acetylation of 19 gave the octaacetate, as an amorphous powder, which was identical with nonaacetate of 3 [31]. Tubuloside B (19) was determined to be 2-(3,4-dihydroxyphenyl)ethyl O-a-L- rhamnopyranosyl (1—^3)-(6-O-caffeoyl)-2-O-acetyl-P-D-glucopyranoside. Tubuloside C (20) was obtained as an amorphous powder. The SSI-MS showed negative ions at m/z 953 (M-H)~ ,791 (M-163)", 749 (M-205)" and 161. The ^ - N M R spectrum showed the presence of four alcoholic acetoxyl groups at 81.80, 1.90,1.95 and 2.08 (3H each, s). The 13C-NMR spectrum of 20 was similar to that of 18, except for the Ihe signals due to the rhamnose moiety, suggesting that an acetoxyl group is linked to the C-21 hydroxyl group of glucose moiety. The location of other acetoxyl groups was determined by detailed comparison of the 13C-NMR spectrum of 20 with that of 18. Acetylation of 20 gave the octaacetate, which was identical with undecaacetate of 18. Acetylation shifts were observed in the signals of C-2, C-3 and C-4 carbons of rhamnose moiety in the chemical shifts of 20. Consequently, tubuloside C (20) was determined to be 2-(3,4-dihydroxyphenyl) ethyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl (1—^3)-O-|y9-D-glucopyranosyl (1—> 6)]-(4-O-caffeoyl)-2-O-acetylTff-D-glucopyranoside. Tubuloside D (21) was isolated as an amorphous powder. The SSI-MS showed negative ion at m/z 937 (M-H)~. The 'H-NMR spectrum showed the presence of four alcoholic acetoxyl groups at 81.81, 1.93, 1.96 and 2.09 (3H each, s). Acetylation of 21 gave the heptaacetate, of which ' H - N M R exhibited the prsence of eight alcoholic acetoxyl groups at 8 1.87,1.94,1.96,1.99,2.10 (3H each, s), 2.02 (9H, s) and three phenolic acetoxyl groups at 8 2.27,2.30 and 2.32 (3H each, s). The 13CNMR spectrum of 21 was similar to that of 20, except for the signals due top- coumaric acid moiety in Table 2. Methanolysis of 21 with acetyl chloride in methanol showed the presence of methyl />coumarate and 3,4-dihydroxyphenethyl alcohol. Acid hydrolysis of 21 with sulfuric acid gave glucose and rhamnose. Consequently, tubuloside D (21) was determined to be 2-(3,4-dihydroxyphenyl) ethyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl (1^3)-0-|$-D-glucopyranosyl(l—• 6)]-(4-O-j9-coumaroyl)-2-O-acetyl-y?-D-glucopyranoside. Tubuloside E (22) was isolated as an amorphous powder. The SSI-MS showed negative ion at m/z 649 (M-H)~. The ^ - N M R spectrum of 22 exhibited the signals due to a methyl group of rhamnose at 8 1.07 (3H, d, J=6Hz), an acetoxyl group at 8 1.98 (3H, s), benzylic methylene protons at 8 2.72 (2H, t, J=7Hz), a glucose anomeric proton at 84.54 (1H, d, J=8Hz), a rhamnose anomeric proton at 8 5.00 (1H, brs), two olefinic protons at 8 6.34 and 7.66 (1H each d, J=\6Hz) and aromatic protons at 8 6.50-7.46 (7H). Acetylation of 22 gave the octaacetate, of which the
659
'H-NMR spectrum exhibited the presence of five alcoholic acetoxyl groups at 5 1.87,1.95,2.02 (3H each, s) and 2.10 (6H s), and three phenolic acetoxyl groups at 5 2.27,2.28,2.31 (3H each, s). The 13C-NMR spectrum of 22 was almost identical with that of 2, except for the the signals due to the j>cournaric acid moiety (in Table 2). Tubuloside E (22) was determined to be 2-(3,4-dihydroxyphenyl) ethyl-0-a-Lrhamnopyranosyl( 1 —>3)-2-(9-acetyl-4-O-j!>coumaroyl-P-D-glucopyranoside. Jionoside D (23) was isolated as an amorphous powder and showed negative ion at m/z 637 (M-H)" in FABMS. The 'H-NMR spectrum of 23 exhibited an anomeric proton of glucoe at 8 4.37 (1H, d, ^ 8 . 1 Hz) and that of rhamnose at 8 5.18 (1H, d, J=\2 Hz). The 13C-NMR spectrum was also identical with that of jionoside D [36]. Poliumoside (24) was isolated as an amorphous powder and showed negative ion at m/z 769 (M-H)" in FABMS. The 'H-NMR spectrum of 24 exhibited an anomeric proton of glucoe at 8 4.27 (1H, d, ^ 8 . 0 Hz) and two that of rhamnose at 8 4.52 (1H, d, J=2.Q Hz) and 8 5.08 (1H, d, J=\ .5 Hz) and sujested phenylethanoid glycoside. The 13C-NMR spectrum was also identical with that of poliumoside [37]. Compound 24 was identified as poliumoside [37], by comparison with authentic sample. Sinenside A (25) showed negative ions at m/z 110 (M) ", 769 (M-l)", 607 (M-163)" and 161 in SSI-MS. The UV spectra showed absorption maxima at 233.6 and 329.1 ran. Known phenylethanoid glycosides, compound 1,3-5, and 7 also showed the SSI-MS ions at m/z (M-l)', (M-163)", and 161 (Table 3). Fragment ion at m/z (M-163) " was determined as the cleavage of caffeoyl group (M-C9H7O3)". Sinenside A (25) was tentatively determined to be 2-(4-hydroxyphenyl) ethyl-O-a-L-rhamnopyranosyl(l—»3)-O-|j5-D-glucopyranosyl (1—>6)]4(9-caffeoyl-/?-D-glucopyranoside. Sinenside B (26) showed SSI-MS ions at m/z 811 (M-l)", 649 (M-163)", 607 (M-163-42)" and 161 (Table 3). Fragment ion at m/z 607 was determined as the cleavage of caffeoyl and acetyl groups (M-C9H7O3- COCH2)". Known acetylated phenylethanoid glycosides, 2 and 18-20 also showed the ions at m/z (M-l)", (M-163)", (M-163-42)" and 161(Table 3). Sinenside B (26) was tentatively determined to be 2-(4-hydroxyphenyl) ethyl-O-a-L-rhamnopyranosyl (1—>3)-0-|/?-Dglucopyranosyl (1 —>6)]-(4-O-caffeoyl)-2-O-acetyl-y5-D-glucopyranoside. SPECTROSCOPIC ANALYSIS OF PHENYLETHANOID GLYCOSIDES Phenylethanoid glycosides consist of phenyethyl glucosyl ether, acyl ester (coumaroyl, feruroyl and caffeoyl) and glycosyl groups. The structure of isolated phenylethanoid glycosides was determined or identified by comparison with UV, IR, MS
660
and NMR spectra and chemical evidence. 13 C-NMR chemical shifts of isolated phenylethanoid glycosides were shown in Table 2. At present, two dimensional NMR spectra, such as ^HfCOSY), 1H13 C(HMQC and HMBC) directly afford the importnat structural information. Chemical shifts by methylation, glycosyMon and acylation suggested the information of substituted carbon positions. 13
C-NMR chemical shifts of phenylethanoid gtycosides
Studies on the structure elucidation of phenylethanoid glycosides of 13C-NMR, Hie change of chemical shift value were observed. Tab4e4Acetylat»n Shifts ofCarbon Signals in Methanol-di
104.1
101.6
101.6
-2.5
-Z5
1035
101.6
101.6
8 (184) -23
2
75.9
75.1
75.0
-0.8
-05
755
75.0
75.5
-0.9
-0.4
3'
81.6
803
80.4
-13
-12
81.5
80.5
80.1
-1.0
-1.4
1
2
22
8(2-1)
8(22-1)
4
18
20
8 CSM) -23
TaHe 5. Glycosylatwn Shifts of Carbon Signals inMethanoM,
Glucose
1
4
5
6
8(4-1)
8(5-1)
8(6-1)
2
19
5"
76.1
74.5
74.6
74.7
-1.6
-1.5
-1.4
76.0
74.8
8 (18-2) -12
6"
623
692
693
69.4
65
7.0
7.1
622
692
7.0
Table 6. Methylation Shifts of Carbon Signals in Methanol-rf4
1 Aglycone 2 117.2 4 144.4 5 116.6 6 121.4
7
8
9
113.9 145.9 116.6 122.4
114.0 145.9 116.6 122.4
113.9 145.9 116.2 122.4
5
6
113.9 145.7 116.1 122.4
113.9 145.9 116.2 122.4
8(5-4) -3.2
8(7-1) -3.3
8(8-1) -3.2
8(9-1) -3.3
1.5 0.0 1.0
1.5 0.0 1.0
-0.4
1.5 1.0
Table 6. Continued
4 Aglycone 2 117.1 4 144.3 5 116.4 6 121.3
8(6-4) -3.2
1.4
1.6
-0.3
-0.2
1.1
1.1
8(8-1) -3.4 4.1 1.0
8(8-7) -3.4 4.0 1.0
Table 6. Continued Ester
2 3 6
1
7
g
115.5 146.6 123.2
115.5 146.7 123.2
112.1 150.7 124.2
4 115.3 146.5 123.1
5
6
8(6-4)
8(6-5)
115.4 146.6 123.1
112.1 150.7 124.2
-3.2 4.2 1.1
-3.3 4.1 1.1
661
Acetylation of C-21 position at inner glucose caused the acetyMon shifts of C-l', 2' and 3' carbons of glucose. Their shifts values were about (-2,4) ppm, from (-0.4) to (-0.9) ppm and firni (-1.0) to (-1.4) ppm, respectively (in Table 4). GlycosyMon of C-6' position at glucose caused the glycosylation strife of carbon signals of C-51 and C-6' position, the former changed from (-1.2) to (-1.6) ppm and the latter shifted about (+7.0) ppm, respectively, as shown in Table 5. MethyMon shifts of phenol groups were shown in Table 6. In Table 6, C-2,4, 5 and 6 carbons in aglycone changed in the range from (-2.5) to (-2.7) ppm, about (+4.4) ppm, from (-0.6) to (-1.0) ppm and about (+1.0) ppm, respectively, by methylation of hydroxyl group at C-3 position of 3,4-dihydrojq?phenylethyl group. A similar change of chemical shifts value was observed by methylation of hydroxyl group at C-3 position of eafleoyl group in Table 6. The chemical shifts value of C-2,3 and 6 carbon signals changed about (-3.3), (+4.1) and (+1.0) ppm, respectively.
MS spectra of phenylethanoid glycosides MS spectra of phenylethanoid glycosides from Cistanche plants were measured by the SSI method. Compounds 1, 3-5, 7,14 and 25 showed negative ions at m/z (M-H), (M-163) and 161 in Table 3. Fragment ion at m/z (M-163) was deter mined as the cleavage of caffeoyl group (M-C9H7O3). Compounds 2, 18-20 and 26 showed negative ions at m/z (M-H) ", (M-163)", (M-205) "and 161 in Table 3. Fragment ion at m/z (M-205)" was determined as the cleavage of caffeoyl and acetyl groups (M - C9H7O3 - COCH2).
PHENYLETHANOID GLYCOSIDES FROM FORSYTHM PLANTS Isolation of phenylethanoid glycosides from Forsyihia plants Four phenylethanoid glycosides, forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29), were isolated from the Forsyihia fruit and leaves [39-44]. They are listed in Table 7. 1. Phenylethanoid glycosides from Forsythkt suspensa Forsythiaside (27) and suspensaside (28) were isolated from the methanol extract of fruit [39,40] and forsythiaside (27) from the methanol extract of leaves [43,44], respectively. 2. Phenylethanoid glycosides from Forsythia viridissima Acteoside (1) and P-hydroxyacteoside (29) were isolated from the methanol
662
extract of Suit [41] and aeteoside (1) from the methanol extract of leaves [43,44], respectively. 3. Phenylethanoid glyeosides fiom ForsytMa komana Forsythiaside (27), suspensaside (28), acteoade (1) and pMrydroxyacteoside (29) were isolated from the methanol extract of fruit [42], andfcrsythiaside(27) and aeteoside (1) from the the methanol extract of leaves [43,44], respectively. Structure elucidation of phenylethanoid glyeosides from Forsythia plants The structure elucidations of the novel compounds, fbrsythiaside (27), suspensaside (28) and (3-hydroxyacteoside (29) were carried out on the basis of the analysis of 'H- and I3C-NMR spectra and chemical evidences [39,40]. They are listed in Table 7. Forsythiaside (27) was obtained as pale yellowish powder, C29H36O15, mpl44150 °C, whose molecular weight was confirmed by the observation of m/z 647 (M+Na) +on field desorption mass spectrometry (FD-MS). The spectral data of 27 suggested that 27 bears a marked structural resemblance to aeteoside (1). Alkaline treatment of 27 followed by acid hydrolysis gave caffeie acid, 3,4-dihydroxyphenethyl alcohol, D-glucose and L-rhamnose. The 13C-NMR of 27 indicated the attachment of the cafleate moiety at the C-4' carbon of glucose and of the rhamnose moiety at the C-61 carbon of glucose (Table 8). The chemical shift of the C-a carbon of 27 at 72.2 ppm suggested the linkage of the 3,4-dihydroxyphenethyl moiety to the C-l1 carbon of glucose. Consequently, the structure of fbrsythiaside (27) was established as 3,4Klihydroxyphenethyl-<>«-L-rhamnopyranosyl-(l—*6)4^£>«arreoyl-P-D-glucopyranoside. Suspensaside (28) was obtained as colorless powder, C29H36O16, mpl77-182°C, whose molecular weight was confirmed by the observation of m/z 640 (M4) on FD-MS. The molecular formula of 28 differs in composition by an increment of one unit of oxygen atom relative to that of 27. The lH-NMR spectrum of 28 resembled that of 27 except for the disappearance of the signal (8 2.80,2H, t, J=7 Hz) assigned to two benzyl protons of the phenethyl moiety. The spectral data of 28 suggested that it bears a marked structural resemblance to 27. The chemical reaction and ^C-NMR spectral data suggested that the phenylethanol moiety of 28 consists of |3,3,4-trihydroxyphenethyl alcohol (Table 8). The 13C-NMR of 28 indicated the attachment of the caffeate moiety at the C-41 carbon of glucose and of rhamnose moiety at the C-61 carbon of glucose. The chemical shift of the C-a carbon of 28 at 76.8 ppm suggested the linkage of the P,3,4-trihydroxyphene1hyl moiety to the C-l' carbon of glucose. With regard to the problem of the absolute configuration at the C-P position in 28, the molecular optical rotation differences
663
between 27 and 28 (A[M]D -3.6°) and between deacylforsythiaside dimethyl ether and deacylsuspensaside dimethyl ether ( A | M ] D +3.0°) were compared with the molecular optical rotation of (+)-phenylethane-l,2-diol [(+)-P-hydroxyphenethyl alcohol] (|M|D +83.2°). It was expected that the molecular optical rotation value attributable to the P-hydroxyphenethyl moiety of 28 having the ^-configuration at
caff
No. 1 27 28 29 30 31 32 33 35 36 37 38
Table 7. Phenylethanoid Glycosides from Forsythia and Plantago plants R, R, R2 Name R4 Ri H rham caff H OH acteoside (verbascoside) forsythiaside H rham OH H caff suspensaside H rham H caff OH P-hydroxyacteoside rham H H caff OH plantamajoside glc H H caff OH hellicoside H OH H caff glc glc isoplantamajoside H caff H OH H 3,4-DPCG H caff H OH campenoside rham caff H H OH p-oxoacteoside rham caff H H OH lavandulifolioside rham-arab caff H H OH forsythoside B rham apio caff H OH
Rfi
R?
H H OH OH H OH H H H
H H H H H H H H OCOCH3 =O
H H
H H
CH 2 OR 3
Table 7. continued No. Name 34 plantasioside 17 orobanchoside(crenatoside)
R, H rham
R2 H caff
R3
caff H
C-P position would probably be nearly equal to that of (+)-phenylethane-l,2-diol. The value of the molecular optical rotation differences suggested that P,3,4-trihydroxyphenethyl moiety is a racemate. Consequently, the structure of suspensaside (28) was established as DL-P3,4-trihydroxyphenethyl-(9-a-L-rhamnopyranosyl-(l->6)-4-O-caffeoyl-P-D-glucopyranoside. P-Hydroxyacteoside (29) was obtained as an amorphous powder, C29H36O16, mpl77-183 °C, whose molecular weight was confirmed by the observation of. m/z 663 (M+Na) + on FD-MS. The 'H-NMR spectrum of 29 resembled that of acteoside (1) except for the disappearance of the signal assigned to the two benzyl protons of the phenethyl moiety. The chemical reaction and 13C-NMR spectral data
664
of 29 clearly suggested that 29, like 28, consists of P,3,4-trihydroxy-phenethyl moiety and a rhamno-glucose moiety containing a caffeoyl group (Table 8). The 13 C-NMR spectrum of 29 indicated the attachment of the caffeate moiety at the C-4'carbon of glucose and of the rhamnose moiety at the C-31 carbon of glucose. The chemical shifts of the C-a carbon of 29 at 76.3 ppm and of the C-l' carbon of Table 8. "C-NMR Chemical Shifts of Phenylethanoid Glycosides in (Metlianol-
glucose at 104.0 ppm suggested the linkage of the glucose moiety to the C-a position of p,3,4-trihydroxyphenethyl alcohol. Consequently, the structure of P-
665
hydroxyacteoside(29) was established as P,3,4-trihydroxyphenethyl-O-a-L-rhamnopyranosyl-(l—>3)-4-O-cafFeoyl-P-D-glucopyranoside. With regard to the problem of the absolute configuration at the C-P position of the P-hydroxyphenethyl moiety, the molecular optical rotation difference between deacylacteoside dimethyl ether and deacyl-P-hydroxyacteoside dimethyl ether (A [M]D +14.1°) was compared with that between deacylforsythiaside dimethyl ether and deacylsuspensaside dimethyl ether (A[M]D +3.0°), and a related compound, (+)-phenylethane-l,2 -diol [(+)-P-hydroxyphenethyl alcohol]([M]D +83.2°). The value of the molecular optical rotation difference suggested that the P-hydroxyphenethyl moiety has both S- and i?-configuration in a ratio of approximately 7:5. PHENYLETHANOID GLYCOSEDES FROM PIANTAGO PLANTS Isolation of phenylethanoid glycosides from Plantago plants Thirteen phenylethanoid glycosides, plantamajoside (30), hellicoside (31), acteoside (1), isoplantamjoside (32), 3,4-dihydroxyphenethyl alcohol-6-O-caffeoyl-PD-glucoside (3,4-DPCG) (33), plantasioside (34), P-hydroxyacteoside (29), campenoside (35), P-oxoacteoside (36), orobanchoside (oraposide, crenatoside;17), lavandulifolioside (37) and isoacteoside (3), were isolated from the Plantago herbs [4549]. Two phenylethanoid glycosides, acteoside (1) and forsythoside B (38), were isolated from the Plantago seeds [50,51]. 1. Phenylethanoid glycosides from P. asiatica Plantamajoside (30), hellicoside (31), acteoside (1), isoplantamjoside (32), 3,4dihydroxyphenethyl alcohol-6-O-caffeoyl-P-D-glucoside (3,4-DPCG) (33) and plantasioside (34) were isolated from the methanol extract of herb [45,46] and acteoside (1) from the methanol extract of seed [50], respectively. 2. Phenylethanoid glycosides from P. depressa Acteoside (1), P-hydroxyacteoside (29), campenoside (35), P-oxoacteoside (36) and orobanchoside (17) were isolated from the methanol extract of herb [47] and acteoside (1) from the methanol extract of seed [50], respectively. 3. Phenylethanoid glycosides fromi? major Plantamajoside (30), acteoside (1) and isoplantamajoside (32) were isolated from the methanol extract of herb [48]. 4. Phenylethanoid glycosides from P. lanceolata Acteoside (1), plantamajoside (30), lavandulifolioside (37) and isoacteoside (3) were isolated from the methanol extract of herb [49]. 5. Phenylethanoid glycosides from P. ovata Acteoside (1) and forsythoside B (38) were isolated from the methanol extract
666
of seed [51]. 6. Phenylethanoid glycosides from P. psyllium Acteoside (1) and forsythoside B (38) were isolated from the methanol extract of seed [51]. Structure elucidation of phenylethanoid glycosides from Plantago plants The structure elucidation of the novel compounds, plantamajoside (30), hellicoside (31), plantasioside (34), P-oxoacteoside (36), orobanchoside (17) was carried out on the basis of the analysis of 1 H- and 13C-NMR spectra and chemical evidences [4549]. Another known compounds were identified by comparison with respective authentic samples or comparison of their spectral data with those reported in the literatures. Plantamajoside (30) was obtained as an amorphous powder, whose structure was elucidated as 3,4-dihydroxyphenethyl-O-P-D-glucopyranosyl-(l^'3)-4-Ocaffeoyl-P-D-glucopyranoside on the basis of the analysis of 1H- and 13C-NMR spectra (Table 8) and chemical evidences by Ravn et al [48]. Hellicoside (31) was obtained as an amorphous yellow light powder, whose molecular formula was confirmed by the observation of m/z 679 [M (C29H36O17) + Na] + by positive ion FAB mass spectrometry. The 'H-NMR spectrum resembled that of 30 except for the disappearance of the signal assigned to two benzyl protons of the phenethyl moiety. The 'H-NMR spectrum of acetate of 31 showed the presence of seven alcoholic acetoxy and four phenolic acetoxy groups, and a proton at the benzyl position bearing an acetoxy group. These data suggested that 31 bears a marked structural resemblance in the sugar moiety to 30 and in the phenethyl moiety to 29. Partial hydrolysis of 31 gave desrhamnosyl P-hydroxyacteoside and 4-caffeoylglucose. Total hydrolysis of 31 yielded only glucose. The results clearly indicated that 31 consists of a P,3,4-trihydroxy-phenethyl moiety and a glucose-glucose moiety containing a caffeoyl group. The 13C-NMR of 31 supported the attachment of the caffeate moity at C-4' position of the inner glucose, the glucose moiety at C-3' position of the inner glucose and the linkage of the inner glucose moiety to the C-a position of P,3,4-trihydroxy-phenethyl alcohol (Table 8). Consequently, the structure of hellicoside (31) has been established as P,3,4-trihydroxyphenethyl-O-P-D-glucopyranosyl-( 1 ^3)-4-0-caffeoyl-P-D-glucopyranoside. For the absolute configuration at C-P position of the P-hydroxyphenethyl moiety, the molecular optical rotation difference between31 and 30 was compared with that of (+)-phenylethane-l,2-diol [(+)-P-hydroxyphenethyl alcohol]. The value as expected ( A [ M | D + 9 8 . 1 O ) suggests that the P-hydroxyphenethyl moiety of 31 has the S-configuratioa
667
Plantasioside (34) was obtained as an amorphous powder, whose molecular formula (C23H24O11) was confirmed by the observation of ions at m/z 499 (M + Na) + and m/z 477(M + H) + by positive ion FAB-mass spectrometry. The 'H-NMR spectrum of 34 resembled that of 3,4-dihydroxyphenethyl dcohol-6-O-caffeoylp-D-glucoside (3,4-DPCG) except for displaying signals at 5 4.53 (1H, dd, J= 3, 10 Hz) assignable to a C-p proton and 5 3.93 (1H, dd, J= 3,13 Hz), 5 3.65 (1H, m) assignable to C-<x protons instead of 8 2.77 (2H, t, J= 7 Hz), 5 3.56 (1H, m) and 8 3.73 (1H, m) in the phenethyl moiety. The ^ - N M R spectrum of the acetate showed the presence of two alcoholic acetoxy and four phenolic acetoxy groups but no presence of a proton at the benzyl position bearing an acetoxy group as that of acetate of 29. Hydrolysis of 34 with acid afforded only glucose. These data suggested that 34 bore a marked structural resemblance in the linkage between a glucose and a phenethyl moiety to that of oraposide from Orobanche spp which contains, besides the glycosidic linkage, an ether linkage between a glucose and a phenethyl moiety [36,52]. The 13C-NMR spectrum of 34 was correlated with those of 3,4-DPCG (33) and oraposide (17) (Table 8). The spectrum of 34 supported the attachment of the caffeate moiety at the C-6' position of the glucose moiety and the presence of an ether linkage between a glucose moiety and phenethyl moiety, besides the glucosidic linkage. Consequently, the structure of plantasioside (34) has been established as l',2'-[P (3,4-dihydroxyphenyi)-a, P-dioxoethanol]-6'-O-caffeoyl O-P-D-gJucopyranoside. P-Oxoacteoside (36) was obtained as an amorphous powder, whose molecular formula was confirmed by the observation of m/z 639 \M (C29H34O16) + H] + and m/z 661 [M(C2S)H34Oi6) + Na] + by positive ion FAB mass spectrometry. The ^ - N M R spectrum showed signals due to methyl protons, anomeric protons of a sugar moiety, aromatic protons of a phenethyl moiety bearing a carbonyl group at the P-position and protons of a caffeate moiety. The reaction of 36 in methanol with excess diazomethane gave deacyl-P-oxoacteoside dimethyl ether. Acid hydrolysis of 36 gave P-oxo-P-(3,4-dimethoxyphenyl)-ethanol, caffeic acid , D-glucose and L-rhamnose. P-Oxo-P-(3,4-dimethoxyphenyl)-ethanol was identical with the compound which was synthesized by the reaction of 3,4-dimethoxy-acetophenone with iodobenzene. These results clearly suggested that 36 consists of a P-oxo-P-(3,4dihydroxyphenyi)-ethyl moiety and a rhamno-glucose moiety containing a caffeoyl group. The 13C-NMR spectrum of 36 (Table 8) supported the attachment of the caffeate moiety at C ^ ' position of the inner glucose, the rhamnose moiety at C-3' position of the inner glucose and linkage of the inner glucose moiety to the exposition of P-oxo-P-(3,4-dihydroxyphenyl)-ethanol. Tthe structure of P-oxo-acteoside (36) has been established as P-oxo-P-(3,4-dihydroxyphenyl)-ethyl-O-a-Lrhamnopyranosyl-(l^'3)-P-D-(4-O-caffeoyl)-glucopyranoside.
668
Orobanchoside (oraposide, crenatoside:17) One of phenylethanoid glycosides from P. depressa was identical with orobanchoside from Orobanche rapumgenistae, whose structure was elucidated as P,3,4-trihydroxyphenethyl-O-a-L-rhamnopyranosyl-(1^2)4-0-caffeoyl-P-D-glucopyranoside by C. Andary et al [28]. In the process of structural elucidation of 30, it was found that the chemical shifts at the C-a and P positions of the phenethyl moity in the 13C-NMR spectrum of orobanchoside isolated from Plantago were consistent with those of 34 and oraposide [52], but 2different from that of 29. In addition , the positive ion FAB-mass spectrum of orobanchoside gave only the ion at m/z 645 as (M + Na)+, which was consistent with that of oraposide (molecular formula C29H32O14) [52]. Thus, the structure of orobanchoside should be replaced by that of oraposide. Furthermore, the 13C-NMR spectra of oraposide, and crenatoside (17) which was isolated from Orobanche crenata by Afifi et al. [36], were compared. As a result, the 13C-NMR spectra of orobanchoside, oraposide and crenatoside (17) in methanol-^ were completely superimposed (Table 8). The spectral data of orobanchoside were also in agreement with those of oraposide and crenatoside (17), and the X-ray analysis of orobancho- side supported these spectral data [52]. It was concluded that orobanchoside, orapo- side and crenatoside (17) are the same compound, that is, l',2'-[p(3,4-dihydroxyphenyl)-a,P-dioxoethanol]-4'-0-caffeoyl-0-a-L-rhamnopyranosyl-(1^3)-(9-P-Dglucopyranoside. BIOLOGICAL ACTIVITIES Antibacterial activity It has been known for many years that the Forsythia fruit is effective in the treatment of skin diseases. The antibacterial activities of the phenyletanoid glycosides from Forsythia fruit were examined [40,42]. A higher antibacterial activity against Staphylococcus aureus was observed in phenylethanoid glycosides. The minimum inhibitory concentration (MIC) of phenylethanoid glycosides against Staphylococcus aureus Terashima was determined by a broth dilution method. Forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29) exhibited MIC activities of 3.2 mM (2.0 mg/ml), 4.1 mM (2.6 mg/ml), 3.2 mM (2.0 mg/ml) and 2.0 mM (1.3 mg/ml), respectively. This suggests that the antibacterial activity of Forsythia fruit is attributable to the phenylethanoid glycosides present in them. Plantamajoside (30) from Plantago herb also exhibited MIC activity of 4.1 mM (2.7 mg/ml) [45].
669
Inhibitory effect on cyclic AMP phosphodiesterase It is known that cyclic AMP inhibits the release of a chemical mediator fiom the mast cell. So when cyclic AMP phosphodiesterase is inhibited by some inhibitor, the concentration of cyclic AMP is increased to inhibit the release of the chemical mediator from the mast celL Inhibitors against cyclic AMP phosphodiesterase may be useful in the therapy for allergic diseases. In addition, by the presence of 5-lipoxygenase, free arachidonic acid is converted to leukotrienes, which is one of the chemical mediators known as a slow reacting substance of anaphylaxis. Therefore, specific inhibitors of 5-lipoxygenase may be useful in the therapy for allergic diseases. Phenylethanoid glycosides were assayed for their inhibitory effect on beef heart cyclic AMP phosphodiesterase [53]. Forsythiaside (27) and suspensaside (28) from F sumensa showed a high inhibitory effect with IC50 of 11.0 x 10'5 mol/1 and 18.3 x 10 mol/1, respectively. On the other hand, the IC50 of both acteoside (1) and fJhydroxyacteoside (29) from F. viridissima was over 50 x 10"5 mol/1. Plantamajoside (30) and hellicoside (31) from P. asiatica showed a high inhibitory effect with IC50 of 16.0 x 10"5 moM and 16.9 x 10"5 mol/1, respectively [45]. Inhibitory effect on 5-lipoxygenase Phenylethanoid glycosides fiom ForsytMa fiuits were assayed for their inhibitory effect on 5-lipoxygenase from rat peritoneal cells [54]. Forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29) showed a high inhibitory effect with IC50 of 2.50 x 10"* M, 7.97 x 10 4 M, 5.27 x 10* M and 19.3 x 10"6 M, respectively. Phenylethanoid glycosides from Pktntago herbs were assayed for their inhibitory effect on 5-lipoxygenase from RBL-1 cells [45] Plantamajoside (30), isoplantamajoside (32), hellicoside (31), acteoside (1) and P-hydroxyacteoside (29) showed a high inhibitory effect with ICJO of 3.73 x 10"7M, 0.42 x 10"7 M, 3.16 x 10'7 M, 13.6 x 10"7 M and 49.8 x 10"7 M, respectively. The anti-inflammetory and anti-asthmatic action of Forsythia fruits and Plantago herbs may be ascribed to the inhibition of cyclic AMP phosphodiesterase and 5-lipoxygenase by the phenylethanoid glycosides contained in these herbal medicines. Antihypertensive activity The antihypertensive activity in the aqueous extracts of F smpensa has been
670
clinically reported [9]. The blood pressure in anesthetized spontaneously hypertensive rats (SHR) was therefore directly measured by carotid cannuration [54]. In fact, the aqueous extracts of F. suspensa showed antihypertensive activity (35 mm Hg, 10 mg/kg, i. v.). The most potent activity occurred in suspensaside (28), which showed high inhibitory activity on cyclic AMP phosphodiesterase. These facts suggested the possibility that there is some correlation between the therapeutic effect of herbal medicines and biological activities of phenylethanoid glycosides.
Antiallergic activity Plantamajoside (30) from P. asiatica and acteoside from P. lanceolata were tested for inhibitory effect on arachidonic acid-induced mouse ear edema [49]. Plantamajoside (30) showed a high inhibitory effect with inhibitions of 12 % at 1 mg / ear and of 25 % at 3 mg / ear. On the other hand, acteoside (1) showed a weak inhibitory effect with inhibitions of 6 % at 1 mg / ear and of 14 % at 3 mg / ear. This result showed good correlation with the inhibition of cyclic AMP phosphodiesterase and 5-lipoxygenase. Analgesic activity Acteoside (1) was isolated as an analgesic principle by activity-guided separation, from the leaves and stems of Lippia triphylla (L'Her) O. Kuntze (Verbenaceae), called Cedron and has been used as a calmative and carminative for stomachache in Peru. Acteoside (1) showed analgesia on acetic acid-induced writhing mice by 300mg/kg, p.o. and 2mg/kg,z.v and exhibited a weak sedative activity on the prolongation of pentbarbital-induced anesthesia and on the depression of locomotion enhanced by methanphetamine [55]. Antistress effect Cistanchis Herba has been used for staminal tonic and treatment of male impotentz [1]. The effect on sex (licking, mounting and intromission) and learning behaviours were studied in the chronic hanging stressed-adult male mice. The phenylethanoid glycosides fraction of Cistanchis Herba (20mg/kg, p.o.) showed the marked protective effect against decrease of sex and learning behaviours. Acteoside (1), cistanoside A (5) and cistanoside B (6) also exhibited the same effect by the administration of 2Qmg/kg,/>.0. Echinacoside (4,10mg/kg,/>.o.) showed the effect against decrease of sex behaviour, but little effect on learning behaviour [56].
671 671
Inhibitoiy effect on lipid peroxidation in rat liver microsomes Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A (5), cistanoside C (7) and tubuloside A (18) showed a strong inhibitorey effect on lipid peroxidation in phospolipid-ascorbic acid system. Their IC50 values were 1.82,7.10,1.73,4.63,1.92,4.50 and 2.20 / M , respectively [57]. Kadota et al reported that acteoside (1), 2-acetylacteoside (2), isoacteoside (3) and tubuloside B (18) significantly suppressed NADPH/CCU-induced lipid peroxidation in rat liver microsomes and prevented cell damage induced by exposure to CCU or D-galactosamine [58]. Neuroprotective effect Tubuloside B (19) significantly attenuated MPP+-induced cytotoxicity, DNA fragmentation and intracellular accumulation of reactive oxygen species (ROS). These results indicated that tubuloside B (19) prevent MPP+-induced apoptosis and oxidative stress and may be applied as an anti-Parkinsonian agent [59].
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H.; Tu, P.; Chem. Pharm. Bull., 2002,25,218-222. Tu, P.; Wang, B.; Deyama, X; Zhang, Z.; Lou, Z.; Acta Pharmaeutica Smica, 1997,32,294-300. Jiangsu New Medical College. Chinese Materia Medica Dictionaiy. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 111-1113. Hansel, R.; Keller, K.; Rimpler, H.; Schneider, G Hagers Handbuch der Pharmazeutischen Praxis 5. Heidelberg, New York; Springer Verlag; 1994, pp. 221-239. Jiangsu New Medical College. Chinese Materia Medica Dictionary. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 401-403. Jiangsu New Medical College. Chinese Materia Medica Dictionaiy. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 403-405. Stuart, M. The Encyclopedia of Herbs and Herbalism. London: Qrbis Publishing; 1979, p. 242. Following our pharmacognostical studies on Cistanchis Herba and research for Citanche plants in China, comercial Cistanchis Herba is mainly produced from C. deserticola, and so Kobayashi's work of Cistanchis in our laboratory should be done witht C. deserticola: a) Kobayashi, H.; Karasawa, H.; Miyase, T; Fukushima, S.; Chem. Pharm. Bull, 1984,32, 3009-3014; b) Idem ibid., 1984, 32, 3880-3885.; c) Idem ibid., 1985, 33, 1452-1457.; d) Karasawa, H.; Kobayashi, H; Takizawa, N ; Miyase, T.; Fukushima, S.; Yakugaku Zasshi, 1986,106,562-566.; e) Idem ibid., 1986, 106,721-724. Lei, L.; Tu, P.; Wu, L.; Proceeding of The Second Symposium on Herba Cistanchis and Desert Medicinal Plants, Xinjiang Hetian, China, May, 2002,p76-78. Xu, W.; Qui, S.; Zhao, J; Xu,Y; Wan, K.; Yan, Q.; et d; Zkmgcaqyao (Chinese Thaditional and Herbal Drugs), 1994,25,509-513. Song, Z.; Tu, P.; Zhao, Y; Zheng, J.; Zhongpaoyao ((Chinese Traditional and Herbal Drugs), 2000,31,808-810. Yoshizawa, F.; Deyama, T; Takizawa, N ; Usn^nghani, K; Ahmad, M.; Chem. Pharm. Bull., 1990,38,1927-1930. Kobayashi, H; Oguchi, H.; Takizawa, N.; Miyase, T; Ueno, A.; Usmanghani, K.; Ahme4 M.; Chem Pharm Bull, 1987,35,3309-3314. Deyama, T; Yahikoaawa, K.; Al-Easa, H.; Rizk, A.; Qatar Univ. Sci J. 1995,15,51-55.
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Kitagawa, S.; Tsukamoto, H.; Hisada, S.; Nishibe, S.; Chem. Pharm. Bull, 1984,32,1209-1213. Kitagawa, S.; Nishibe, S.; Baba, H.; Yakugaku Zasshi, 1987,107,274-278. Kitagawa, S.; Hisada, S.; Nishibe, S.; Phytochemistry, 1984, 23, 1635-1636. Kitagawa, S.; Nishibe, S.; Benecke, R.; Thieme, H.; Chem. Pharm. Bull, 1988,36,3667-3670. Ravn,H.; Nishibe, S.; Sasahara, M.; Li, X.; Phytochemistry, 1990, 29, 3627-3631. Nishibe, S.; Tamayama, Y; Sasahara, M.; Andary, C ; Phytochemistry, 1995,38,741-743. Nishibe, S.; Sasahara, M.; Jiao, Y; Yuan, C ; Tanaka, T.; Phytochemistry, 1993,32,975-977. Ravn, H.; Brimer, L.; Phytochemistry, 1988,27,3433-3437. Murai, M.; Tamayama, Y; Nishibe, S.; Planta Med., 1995,61,479-480. Kawamura, T.; Hisata, Y; Okuda, K.; Hoshino, S.; Noro, Y; Tanaka, T; Kodama, A.; Nishibe, S.; Natural Medicines, 1998,52,5-9. Nishibe, S.; Kodama, A.; Noguchi, Y; Han, Y; Natural Medicines, 2001, 55,258-261. Andary, G; Wylde, R.; Maury, L.; Heitz, A.; Dubourg, A.; Nishibe, S.; Phytochemistry, 1994,37,855-857. Nishibe, S.; Kitagawa, S.; Hisada, S.; Baba, H.; Yasui, S.; Narita, T; Yoshioka, K.; J. Pharmacobio-Dyn., 1987,10, s48. Kimura, Y; Okuda, H.; Nishibe, S.; Arichi, S.; Planta Med., 1987, 2, 148-153. Nakamura, X; Okuyama, E.; Tsukada, A.; Yamazaki, M.; Satake, M.; Nishibe, S.; Deyama,T; Moriya, A.; Maruno, M.; Nishimura, H.; Chem. Pharm. Bull, 1997,45,499-504. Sato, X; Kojima, S.; Kobayashi, K.; Kobayashi, H.; Yakugaku Zasshi, 1985,105,1131-1144. Moriya, A.; Theses of Graduate School of Agricuture, Gifu University, Gifu, Japan, 1995. Xiong, Q.; Hase, K.; Tezuka, Y; Tani, T; Namba, X; Kadota, S.; Planta Med, 1998,64,120-125. Sheng, G; Pu, X.; Lei, L.; Tu, P.; Li, C ; Planta Med, 2002,68,966-970.
(Ed.) Studies Studies in in Natural Products Chemistry, Chemistry, Vol. Vol. 33 33 Atta-ur-Rahman (Ed.) © 2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©
675 675
PHARMACOLOGICAL ACTIVITIES OF PHENYLPROPANOIDS GLYCOSIDES MARINA GALVEZ, CARMEN MARTIN-CORDERO, MARIA JESUS AYUSO. Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain ABSTRACT: The pharmacological assays and activities of natural phenylpropanoid glycosides, extracted from a variety of plants are summarized in this review, such as antioxidant, anti-inflammatory, healing, antimicrobial and antitumoral-chemopreventive. Structureactivity relationships are also discussed.
INTRODUCTION The phenylpropanoid glycosides (PPGs) are a group of derivatives of phenylpropane, distributed on Gamopetalas (Lamiales, Oleales, Asterales...) and located in most of the vegetable tissues and on the pollen, [1-4]. This group is characterised by having a caffeoyl and hydroxyphenylethyl moieties, both of which are linked to (3-glucose by ester (C-4) and glucosidic (C-l) linkages. Other sugars such as rhamnose, xylose or arabinose may be attached to C-3, C-4 or C-6 of the glucosyl residue [2]. This group of compounds has also been defined as phenethyl glycosides (PhGs), phenyletanoid glycosides or caffeoyl phenylethanoid glycosides (CPGs). We are going to use the term phenylpropanoid glycosides (PPGs) Fig- (1). Various plants used in traditional medicine contain significant amounts of PPG: For example, different species of Scrophularia genus, have been traditionally used for several skin inflammatory ailments. The leaves of species of Buddlej'a have been applied topically as a poultice or lotion for the healing of wounds and ulcers. Ballota nigra is commonly used for their neurosedative activity and Pedicularis sp. are a Chinese folk medicinal h erb, found e specially i n T ibet, u sed to t reat m alignant s ores, collapse, exhaustion, and relieves uneasiness of body and mind.
676 Fig. (1). Chemical structures of PPG (phenylpropanoids glycosides) mentioned. .OR,
R1 H P-D-api ara ara ara ara P-D-xyl P-D-api a-L-rha H caf H H H P-D-glu a-L-rha p-D-api fer caf H fer Ac caf fer H H a-L-rha P-D-api P-D-api H H H a-L-rha H H H H p-D-xyl P-D-glu caf H
Name 2' -Acetilverb ascos ide Alyssonoside Angoroside A Angoroside B Angoroside C Angoroside D Arenarioside Ballotetroside Brandioside Calceolarioside A Calceolarioside B Cistanoside C Cistanoside D Cistanoside F Echinacoside Forsythiaside Forsythoside B Isomartynoside Isoverbascoside Lavandulifolioside Leucosceptoside A Luteoside A Luteoside B Luteoside C Martynoside Myricoside Pedicularioside A Pedicularioside A Pedicularioside M Persicoside Phlinoside A Phlinoside C Poliumoside Rossicaside A Samioside Teucrioside Trichosanthoside A Trichosanthoside B Tubuloside A Tubuloside B Verbascoside
R2 caf fer caf fer fer fer caf caf caf caf H caf fer fer caf caf caf H H caf H caf H H fer caf caf fer fer caf caf caf caf caf caf caf caf caf H H caf
0 H
Caffeoyl
3OO^Y Feruloyl
R3 a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-arab (1-2) a-L-rha a-L-rha H H a-L-rha a-L-rha a-L-rha a-L-rha H a-L-rha a-L-rha a-L-rha a-L-ara (1-2) a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha (1-3) P-D-api P-D-api a-L-rha a-L-rha P-D-glu P-D-glu (1-2) a-L.rha a-L-rha (1-2) a-L.rha a-L-rha P-D-glu (l-4)a-L-rha P-D-api (1-4) a-L-rha a-L-lyx (1-2) a-L-rha P-D-xyl (l-4)a-L-rha P-D-xyl (l-4)a-L-rha a-L-rha a-L-rha a-L-rha 0 ||
OH
Ho'SZ^Sj ^ O H Rhamnose
R4 H H H H H H H H H H H H H H H H H H H H H H H H CH 5 H H CH , H H H H H H H H H H H H H
R5 H H H H CH, H H H H H H CH, CH, CH, H H H H H H H H H H H
H H H
H H H H H H H H H H H H H
OH H0
R6 Ac H H H H H H H H H H H H H H H H H H H H P-D-api P-D-api p-D-api H H H H H P-D-glu H H H H H H H H Ac Ac H OH
k5~° A V^H -0
iOH HC .HO Arab mose
H
Apiose
677
Roles in Plant Physiology It has been detected that the plants increase the phenylpropanoids synthesis in different situations, like, defense against herbivores; protection of microorganism attack or invasion by other species. Besides, some external factors that can increase the free radicals levels, such as stress, high light (more UV incidence), low temperatures, pathogen infections, nutrient deficiency [5], drought or ozone exposition [6], can induce a higher production of PPG by the plants. On the other hand, the PPG are precursors for other complex molecules, also useful to the plants [7, 8]. From these phenylpropanoids, other phenols have been synthesized such as flavonoids, coumarins, lignans, stilbenes, psoralenes, pterocarpanes, tannins, which also act against the oxidative stress [9]. All these functions that have the phenylpropanoids, have conduced to the genetic engineer to modify the biosynthetic pathways, hi that way, it has induced illness resistance, or foraged plants digestibility increase [10].
METHODOLOGY The data has been obtained after a revision of the literature available by different data basis, such as MedLine or Chemical Abstracts, and by the references in the literature. The order of the tables is based on the alphabetical order of the family, following "The International Plant Name Index", by The Royal Botanic Gardens, Kew, The Harvard University Herbaria and Australian National Herbarium (www.ipni.org/index.html), for the nomenclature. RESULTS The data is presented in table 1 divided in five columns that include the family of the plant specie where the phenylpropanoid glycoside (PPG) has been isolated, the specie, some of the popular uses of the plant, the trivial name of the PPG, the assayed activity with the result, also including the negative results, and the reference.
Globulariaceae
Globularia trichosantha
Mussatia sp.
Diuretic, laxative, carminative, tonic.
Euphoric effect
Table 1. Pharmacological activities of PPG. Species Popular use Family Acanthaceae Asthma, Barleria prioniits Fever, Cold Bignoniaceae Markhamia lutea Diarrhea, Asthenia, Infections
Crenatoside (=oraposide) Rossicaside A Trichosanthoside A Trichosanthoside B Verbascoside
Mixture of 4cinnamoylmussatioside + 4dimethylcaffeoylmussatioside + 4-pmethoxycinnamoylmussatioside
Luteoside C Verbascoside
Luteoside A Luteoside B
Isoverbascoside
Verbascoside
PPG
• • • •
Active Active Active Active
Antioxidant: Scavenging activity (DPPH) (TLC) • Active
• Inactive as inhibitor of prostaglandin biosynthesis. Antimicrobial: • No effect in the range 0,05-5 mg/strip. (microorganisms not shown on the paper) Cardiovascular effects 1 No effect on blood pressure and heart rate. Platelet agregation • Inhibition of rat ADP-induced platelet aggregation. It might be through cAMP-phosphodiesterase inhibition.
Antiviral: • Active against RSV. • Inactive against VZV, mCMV, HSV 1 and 2. • Active against RSV. • Active against RSV. • Inactive against HSV-1 and 2. • Active against RSV. • Active against RSV. • Inactive against HSV-2 and CMV. Anti-inflammatory:
Antiviral: • Active against RSV.
Assayed activity
[14]
[13]
[13]
[13]
[13]
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References
00
OS
678
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Lamiaceae Ballota nigra Flu, cold, nervousness.
(+)-E-caffeoyl-L-malic acid Arenarioside Ballotetroside ForsythosideB Verbascoside
(+)-E-caffeoyl-L-malic acid Arenarioside Ballotetroside ForsythosideB Verbascoside
(+)-(E)-caffeoyl-Lmalic acid Alyssonoside Angoroside A
(+)-(E)-caffeoyl-Lmalic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside Verbascoside
PPG
• • • •
Inactive Inactive Inactive Inactive
• Active • Active • Active • Active -Methal chelation (Cu 2+) • Inactive
• Inactive • Inactive Antioxidant: -Inhibition of in vitro Cu (2+) - induced LDL peroxidation • Active
Inactive Inactive Active Inactive Active Inactive Active E. faecalis; P aeruginosa; E. coli; E. aeroenes; K. pneumoniae; K. oxytoca • Inactive
• • • • " • • •
Antibacterial: • S. aureus; S. aureus (methicillin- resistant); P. miral • Inactive
Assayed activity
[16]
[16]
[15]
[15]
References
679
Leonurus cardica
Leonotis nepetaefolia
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Lamiaceae (cont.) B. nigra (cont.)
Lavandulifolioside
Lavandulifolioside
Lavandulifolioside Martynoside Verbascoside
(+)-E-caffeoyl-Lmalic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside Verbascoside
(+)-E-caffeoyl-L-nialic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside
PPG
• Active • Active • Active • Active • Active • Active • Active Antioxidant: Scavenging activity {DPPH) • Active • Active " Active Chronotropic • Negative Hypotensive; • Active
• Active • Active • Active • Active • Active • Active Sedative: BZD; D;n receptor agonists, • Active
Antioxidant (cont.): -Scavenging activity in vitro and in vivo against Oa"" OH • Active
Assayed activity
, 0HC1,
[19]
[19]
[18]
[17]
[17]
References
680
Phlomis armeniaca
Nepeta uaraintca
Table 1. Pharmacological activities of PPG, (cont.) Popular use Family Species Lamiaceae (cont.) Marruhium vulgare
Verbascoside
Verbascoside
Leucosceptoside A Martynoside Phlinoside B
Forsythoside B
Verbascoside
(+)-E-eaffeoyl-Linalic acid Arenarioside Ballotetroside Forsythoside B Verbascoside
(+)-E-caffeoyl-Lmalic acid Arenarioside Ballotetroside Forsythoside B Verbascoside
PPG
• COX-2 inhibitor • COX-1 inhibitor • COX-2 inhibitor • Selective COX-2 inhibitor Immunomodulatory: • Increases chemotactic activity. • Positive effect on respiratory burst of neutrophils. Antitu moral: - Cytotoxic activity against RLh-84; S-I80; P-388; HeLa and hepatocytes • Cytotoxic on dRLh-84, S-180 and P-388/dl. • No cytotoxic on hepatocyte • No cytotoxic • No cytotoxic • Cytotoxic on dRLh-84, S-180. • No cytotoxic on hepatocyte • Cytotoxic on dRLh-84, S-180 and P-388/dl. • No cytotoxic on hepatocyte - Apoptosis induction • Active on HL-60
• Active • Active • Active • Active Enzymatic inhibition (anti-inflammatory): • Inactive.
Antioxidant: Inhibition of Cu (2+)- and AAPH induced LDL oxidation • Active
Assayed activity
[24]
[23]
[22]
[21]
[20]
References
681
Sideritis licia
P. samia
P. pungens
P. physicalyx
Table 1. Pharmacological activitieg of PPG, (cont.) Family Species Popular use Lamiaceae (cont.) P. monocephala
Lavandulifolioside Leucosceptoside Martynoside Verbascoside
Samioside Verbascoside
Samioside
Samioside Verbascoside
Forsythoside B Alyssonoside
Forsythoside B Leucosceptoside A Martynoside Physocalycoside Verbascoside Wiedemannioside C
Alyssonoside Forsythoside B Verbascoside
PPG [25]
Antioxidant: Scavenging activity (DPPH) • Active • Active • Active Antioxidant: Scavenging activity (DPPH) • Active • Active • Active • Active • Active • Active Vasocontracting: against free radical-induced impairment of endothelium-dependent relaxation. • Active • Active Antibacterial: S. aureus; S. epidermidis; E. cloacae; E. coli; K. pneumonia: P. aeruginosa. • Active • Active Antifungal-.Candida albicans, C. glabrata, C, tropicalis • Active Antioxidant: Scavenging activity (DPPH) " Active • Active Anti-inflammatory: against carrageenan-induced paw edema • Active • Active • Active • Active
[29]
[28]
[28]
[28]
[27]
[26]
References
Assayed activity
682
Loganiaceae
Inflammations, wounds and ulcers
Conjunctive congestion, clustered nebulae.
B. globose
B. officinalis
Buddleja cordata
Teucrium polium
Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Lamiaceae (cont.) Stachys sieboldii
AngorosideA Calceolarioside Campneoside Echinacoside Forsythoside B Verbascoside
Verbascoside
Verbascoside
Echinacoside Verbascoside
Verbascoside
Verbascoside
Poliumoside
Verbascoside
Stachysoside C Verbascoside
PPG
Active
Healing: Fibroblast-protector against ROS. • Active • Active Antibacterial • Active Antitu moral •Active Enzymatic inhibition: In vivo COX and 5-LOX inhibition. -Inactive "Inactive "Inactive "Inactive -Inactive -Inactive
•
Antibacterial: • Active against S aureus. • Mechanism: Affecting protein synthesis Antibacterial:
Anti-anoxia: Inhibition of the KCN-induced anoxia in mice • Active • Active Anti-nephritic: • Preventive of nephritis and glomerulonephritis An titu moral: Cytotoxic activity against dRLh-84, S-180, p-388/Dl, HeLa • Cytotoxic on dRLh-84, S-180, p-388/Dl.
Assayed activity
[36]
[37]
[37]
[35,36] [35]
[34]
[33]
[32]
[31]
[30]
References
00
683
Oleaceae
Myoporaceae
Forsylhia sp.
Fraxinus sieboldiana
Allergic and inflammatory diseases
Poliumoside Verbascoside
Cold, headache, sores chest pain
E. gilesii
Verbascoside
p-hydroxyverbascoside Suspensaaside
Forsythiaside
Calceolarioside B Calceolarioside A Verbascoside
Verbascoside
Verbascoside
AntivirahHIV • Active • Inactive • Inactive Antiinflamatory: Kffect on arachidonic acid metabolism •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L "Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L
• Active Cardiotonic • Chronotropic positive • Inotropic positive. • Increase of coronary perfusion rate by increase of c AMP Hypo-Hypertension: •No effect on arterial pressure Inhibition of platelet aggregation: • Active • Active Inhibition of serotonin release: • Active
[44]
[43]
[42]
[39-41]
[39-41]
Verbascoside Antimicrobial
[38]
Neuroprotective: Against MMP-induced apoptosis and oxidative stress in PC12 neuronal cells. •Active. Parkinson prevention.
Fever, headache, pain, conjuntivitis, inflammation.
References
Assayed activity
PPG
Eremohyla ahernifolia
Table 1. Pharmacological activities of PPG, (cont.) Popular use Family Species Loganiaceae (cont.) B. officinalis (cont.)
2
684
Oleaeeae (cont.)
Sores, headache, pain, hypertension
Inflammations
Ligustrum robustum
L. vulgare
Forsythia sp. (cont.)
Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Antioxidant: DPPH scavenging activity •Active •Active
Assayed activity
Verbascoside
Osmanthuside B Verbascoside
Q O
• Inactive • Active Vasocontracting • Active (Inhibition of NO production)
•Active •Active Antioxidant: Protection against hemolysis of red blood cells induced by AAPH Ligupurpuroside A • Active Ligupurpuroside B • Active LigurobustosideM • Active LigurobustosideN • Active LigurobustosideO • Active Osmanthuside B • Active Osmanthuside B6 • Active Verbascoside • Active Antioxidant: - Inhibition of in vitro Cu (2+)- induced LDL peroxidation Isoverbascoside • Active Ligupurpuroside A • Active Verbascoside • Active - Inhibition of in vitro peroxyl radical-induced LDL peroxidation Isoverbascoside • Active Ligupurpuroside A • Active a's-Ligupurpuroside B • Inactive ftww-Ligupurpuroside • Inactive
Forsythiaside P-hydroxyverbascoside Suspensaaside Verbascoside
PPG
[48]
[47]
[46]
[45]
[44]
References
00
en
685
Orobanchaceae
Calceolaria kypericina Cistanche deserticala
Syringa vulgaris
Malaria
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Oleaceae (cont.J_ Olea europaea
2*-acetyl-verbaseoside Cistanoside A Cistanoside F Echinacoside Isoverbascoside Syringalide A 3*-arhamnopiranoside Tubuloside A Tubuloside B Verbascoside
Verbascoside
2'-acetyl-verbascoside Isoverbascoside Tubuloside B Verbascoside
Calceolarioside A
Verbascoside
Verbascoside
PPG
• Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger.
Platelet aggregation: • Active Anti-hepatotoxic: In vitro protection against CCU and D-GalN • Active • Active • Active • Active Inhibition of LPS and D-GalN-induced apoptosis on hepatocytes. • Active Antioxidant: -DPPH and superoxide anion scavenging activity • Active as DPPH,s uperoxide anion and NO scavenger. " Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH and superoxide anion scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH and superoxide anion scavenger.
Antioxidant :Scavenging activity (ATBS) • Active Antiviral: * Inactive against-HI¥ Heart rate effect: • Decrease of heart rate. Antihypertensive effect: • Active
Assayed activity
[55,56]
[54]
[53]
[52]
[51]
[51]
[50]
[49]
References
686
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Orobanchaceae(cont.) C. deserticola (cont.) 153]
Antioxidant: -Inhibition of NADPH-CCU -induced lipid peroxidation • Active • Active • Active • Active -Inhibition of ascorbic acid and Fe(2+)-induced lipid peroxidation • Active " Active • Active • Active • Active • Active
[55]
156]
[55]
References
Assayed activity
• Active " Active • Active Enzymatic inhibition: inducible Nitric Oxide synthase (iNOS) 2 '-acetyl-verbascoside • Inactive Cistanoside A • Inactive Echinacoside • Inactive Isoverbascoside • Inactive Tubuloside A • Inactive Tubuloside B • Inactive Verbascoside • Inactive. Xanthine oxidase (XOD) 2'-acetyl-verbascoside • Inactive Cistanoside A • Inactive Cistanoside F • Inactive Echinacoside • Inactive Isoverbascoside • Active
2 '-acetyl-verbascoside Cistanoside A Cistanoside F Echinacoside Isoverbascoside Syringalide A 3'-ccrhamnopiranoside Tubuloside Tubuloside A Verbascoside
2'-acetyl-verbascoside Isoverbascoside Tubuloside B Verbascoside
PPG
687
Pedaliaceae Harpagophyium procumbens
O. hypericina
O. hederae
Orobanche caerulescens
C. salsa
Inflammatory degenerative diseases,skin lesions, fever, tonic.
Kidney deficiency and neurasthenia Tonic for impotence, inflammation, cystitis, feces softener
Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Orobanchaceae (cont.) C. deserticolci (cont.)
6'-0-acetylVerbascoside Isoverbascoside Verbascoside
Calceolarioside A Calceolarioside B Calceolarioside C
Verbascoside Orobanchoside
Caerulescenoside 3'-methyl crenatoside Isoverbascoside Verbascoside Campneoside II Crenatoside Derhamnosyl verbascoside
Verbascoside
Syringalide A 3'-arhamnopiranoside Tubuloside A Verbascoside Tubuloside B
PPG
• Active • Inactive
Enzymatic inhibition: Elastase • Strong activity
Platelet aggregation inhibition: • Active • Active Platelet aggregation inhibition: • Active • No effect • No effect
Antioxidant: Inhibition of LDL oxidation • Active • Active " Active • Active • Active • Active • Active
• Inactive • Inactive • Active. Neuro-protecting effect against MMP • Active by inhibition of caspases activation
Enzymatic inhibition: Xanthine oxidase (XOD) (cont.) • Inactive
Assayed activity
[59]
[58]
[58]
[58]
[57]
[55]
References
688
Schrophulariaceae
Polygonaceae
Brandisia hancei
Polygonum lapatifotium
Necrotic osteitis, rheumatoid arthritis, hepatitis, hyperlipemias
Dysentery, articular pain, inflammations
Same as P. lanceolata
P. major P. media
Wound healing, respiratory problems, cancer
P. lanceolata
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Plantaginaceae Plantago cynops
2' -acetyl verbascoside Brandioside Poliumoside Verbascoside
Vanicoside B
Lapathoside A Vanicoside B
Lapathoside A Vanicoside B
Homoplantaginine Verbascoside
Plantamajoside
Cistanoside F Isoverbascoside Lavandulifolioside Plantamajoside Verbascoside
Verbascoside
PPG
two-stage
two-stage
skin
skin
Antioxidant:Inhibition of free radical-induced hemolysis of red blood cells and superoxide radical generation. • Active • Active • Active • Active
Antitumoral-chemopreventive Inhibition of EBV-EA induction by TPA " Active • Active Anti-tumor-promoting effects on mouse carcinogenesis induced by DMBA and TPA • Active • Active Anti-tumor-promoting effects on mouse carcinogenesis induced by NO donor • Active
[60]
Antibacterial • Active Anti-inflammatory:-Inhibition of arachidonic acid-induced edema • Inactive " Inactive • Inactive • Active " Active Antibacterial: against S. aureus; E. coli • Active Antitumoral-chemopreventive: EGFR, TK and tumor growth inhibition • Active • Active
[65]
[64]
[64]
[64]
[63]
[62]
[61]
References
Assayed activity
689
MonochAsthma savatierii
Digitalis purpurea
Castilleja linariaefolia
Table 1 • Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) B. hancei (cont.)
Dehydroverbascoside Verbascoside
Calceolarioside A Calceolarioside B Forsythoside Plantainoside
Isoverbascoside Verbascoside
2'-acetylverbascoside Brandioside Poliumoside Verbascoside
2'-acetylverbascoside Arenarioside Brandioside Isoverbascoside Verbascoside
2'-acetylverbascoside Brandioside Poliumoside Verbascoside
PPG Antitumoral: Antiproliferation on A7r5 cells • Active • Active • Active • Active Enzymatic inhibition: Xanthine oxidase • Inactive • Inactive " Inactive • Competititve inhibitor • Inactive Prevention against arteriosclerosis • Active " Active • Active • Active Antitumoral: Cytotoxic activity against P-388 • Active • Active Enzymatic inhibition:PKCa • Active • Active • Active • Active Enzymatic inhibition: Aldose reductase in vivo. • Inactive • Strong activity
Assayed activity
[70]
[69]
[68]
[66]
[67]
[66]
References
690
P. plicata
P. lasiophyris
P. alashanica
Schrophulariaceae (cont,) Pedicularis sp. Tonic for treatment of debility, collaptse, exhaustion, swating, seminal emission, senility
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular
Verbascoside Martynoside
Martynoside
Cistanoside D
Leucosceptoside A Martynoside
Cistanoside C Pedicularioside A Verbascoside
Cistanoside D Echinacoside Isoverbascoside Pedicularioside A Verbascoside
Cistanoside Echinacoside Isoverbascoside Pedicularioside A Verbascoside
PPG
Antioxidant: -Inhibition of autoxidation of Hnoleic acid in CTAB • Active • Active • Active • Active • Active -Protection against oxidative hemolysis • Active • Active • Active • Active • Active Antitumor-chemopreventive: DNA adducts-reparation: dAMP+; dAMP-OH; dAMP(NH) dGMP+; dGMP-OH; PoliG-OH; TMP -Active • Active • Active Antioxidant: Scavenging activity against Ch~ and OH • Active • Active Antioxidant: Protection against oxidative hemolysis • Active Chemopreventive: DNA adducts-reparation: T Active Motor level: Retardation of skeletal muscle fatigue. • Active • Active
Assayed activity
[77, 78]
[76]
[72]
[75]
[73,74]
[72]
[71]
References
691
P. striata
Asthenia
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) P. spicata
Pedicularioside A Verbascoside
Verbascoside
Isoverbascoside
Isoverbascoside Verbascoside
Pedicularioside A Pedicularioside M Pedicularioside N Verbascoside
Echinacoside Pedicularioside A Verbascoside
Isoverbascoside PermethylVerbascoside Verbascoside
Cistanoside C
PPG
• Active Protection against oxidative hemolysis • Active • Active • Active Scavenging activity (OH" and O2~) • Active • Active • Active • Active Antitumoral-chemopreventive: Induction of cellular differentiation on: • SMMC-7721 and MGc803 . MKN45 and MGc803 Antiproliferation on MGc803 cell line • Active Telomerase inhibition on MKN45 cell line • Active DNA adducts-reparation: T-OH • Active • Active
Chemopreventive: DNA adducts-reparation: T' • Active Antioxidant: Inhibition of FeSO,t-induced lipid peroxidation • Active • Inactive
Assayed activity
[87]
[85]
[83,84] [85,86] [84]
[75, 82]
[81]
[72]
[80]
[79]
References
692
Scrophularia albida
Rehmannnia glutinosa
Penslemon linarioides
Tonic, antianemic, antipyretic
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) P. striata (cont.) Antitumoral-chemopreventive (cont.): DNA adducts-reparation: T" • Active • Active • Active • Active DNA adducts-reparation: dGMP-OH • Active Repair effect on DNA damage Enzymatic inhibition: "Inactive as xanthine oxidase inhibitor. "Inactive as xanthine oxidase inhibitor. Metal chelating effect: (ferric ion) • Active • Inactive
Assayed activity
• Active Enzymatic inhibition: PKCa Leucosceptoside A • Active Poliumoside " Active Verbascoside • Active Immunosuppressive: Study of hemolytic plaque forming cells in mice. Cistanoside F •Active Isoverbascoside •Active Jionoside A-l •Active Jionoside B-l •Active Purpureaside C •Ative Verbascoside •Active Antitu moral: Cytotoxic activity against dRLh-84; S-180; P-388/D1; HeLa (+) syringaresinol-o-p• Active D-glucopyranoside
Isoverbascoside Permethyl Verbascoside Verbascoside
Pedicularioside A Verbascoside
Verbascoside Verbascoside
Leucosceptoside A Pedicularioside M Pedicularioside N Verbascoside
PPG
[32]
[90]
[69]
[80]
[72,75]
[88]
[76]
References
693
S. ningpoensis
S. scorodonia Skin inflammatory ailments
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) S. scopolii
Angoroside C Verbascoside
Verbascoside
Verbascoside
Isoverbascoside
Angoroside D
Angoroside C
Angoroside A
Verbascoside
Angaroside A Angaroside B Angaroside C
PPG [32]
Antitumoral: Cytotoxic activity against dRLh-84; S-180; P-388/D1; HeLa •Active •Active "Active Antiviral: against HSV and VSV "Inactive Anti-inflammatory: Inhibition of PGE2, LTC4 TXB2 and NO release in calcium ionophore-stimulated mouse peritoneal macrophages and human platelets. •Active as inhibitor of PGE2,TXB2, LPS-induced TNF-a and LPS-induced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. •Active as inhibitor of PGE2,TXB2 and LPS-induced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. "Active as inhibitor of PGE2, LPS-induced TNF-a and LPSinduced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. "Active as inhibitor of PGE2, LPS-induced TNF-a and LPSinduced NO release •Active as inhibitor of PGE2 (COX-2) accumulation. •Active as inhibitor of TXB2, LPS-induced TNF-a and LPSinduced NO release •Active as inhibitor of PGE2 (COX-2) accumulation. Antitumoral: Cytotoxic activity against: HeLa, Vero and BHK-21 •Inactive Antitumoral-chemoproventive DNA adducts-reparation: d-AMP-OH and d-GMP •Active •Active
[93]
[91]
[92]
[91]
References
Assayed activity
694
Verbenaceae
Clerodendron bungei and C. tnchotomum
Caryopteris incana
Veronica persica
Verbascum macrurum
Malignan lung cancer, rheumatism, rheumatic articular pain
Table 1. Pharmacological activities of PPG, (cont) Family Species Popular use Schrophulariaceae (cont,) Scutellaria armeniaca Assayed activity
Deshamnosylisoverbascoside Deshamnosylverbascoside Isoverbascoside Verbascoside
Incanoside Isoverbascoside Phlinoside A Verbascoside
• Cytotoxic on B16F10 • Cytotoxic on B16F10
• Cytotoxic on B16F10
Antioxidant: Scavenging activity against DPPH, OH' and O2". • Active • Active • Active • Active Antitumoral: Cytotoxic activity againstB16F10, MK.-1 and HeLa • Cytotoxic on B16F10
Antitumoral: Cytotoxic activity against dRLh-84, S-180, P-388/dl HeLa and hepatocytes. Teucrioside • Cytotoxic on all cell lines.. Verbascoside • Cytotoxic on dRLh-84; S-180 and P-388, Leucosceptoside A • No cytotoxic. Martynoside • No cytotoxic. Antioxidant: DPPH scavenging activity and preserving activity 6'-O-a-Lagainst sunflower oil oxidative rancidity. arabinopyranasyl•Active martynoside + 6'-O-PD-xylopyranosylmartynoside Martynoside •Active Verbascoside •Active Antioxidant: -Scavenging activity against DPPH Persicoside • Active Verbascoside • Active
PPG
[97]
[96]
[95]
[94]
[23]
References
695
Premna subscandens
L. multiflom
Lippia dulcis L. canescens
Lantana camara
Lantana camara
Malaria
Cough and bronchitis
Table 1. Pharmacological activities of PPG, (rout.) Popular use Family Species Verbenaceae (cont.) C. trichatamum Hypertension
Verbascoside
Verbascoside
Arenarioside Diacetyl- martynoside Isoverbascoside Leucosceptoside A Martynoside Verbascoside
Verbascoside
Verbascoside
Isomartynoside Isoverbascoside Leucosceptoside Martynoside Verbascoside
Isomartynoside Isoverbascoside Jionoside D Leucosceptoside A Martynoside Plantainoside C Verbaseoside
PPG Antiviral: Inhibition of HTV-1 integrase • Inactive • Active • Inactive • Inactive • Inactive • Inactive • Active Enzymatic inhibition: ACE • Active • Active • Active • Active • Active Antitumoral: Cytototic activity on L-1210 • Active Enzymatic inhibition: PK.C • Active, by catalytic domain Antitu moral: Antiproliferative effect against B16F10, MK-1 and HeLa • Active on B16F10. • Inactive • Active on B16F10 • Inactive • Inactive • Active on B16F10 Anti-inflammatory: TXA3 synthesis inhibition • Inactive Healing: Promotion of collagen network formation in vitro • Active
Assayed activity
[104]
[103]
[101,102]
[100]
[100]
[99]
[98]
References
696
Not given on the paper
Verbena oficinalis
Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Verbenaceae (cont.) Stachytarpheta Liver cayennensis diseases, flues, cough, arthritis Verbena litoralis
Verbascoside
Purpureaside C
Purpureaside B
Purpureaside A
Isoverbascoside
2' -acetyl verbascoside Desrhamnosil Verbascoside
Isoverbascoside Jionoside Verbascoside
Verbascoside
PPG
Antiviral against virus Aujeszky Antitumoral-chemopreventive: •Induction of HL-60 differentiation Antimicrobial against a E. coli Antiviral against virus Aujeszky Antimicrobial against a E. coli Not antiviral against virus Aujeszky Antimicrobial against a E. coli Not antiviral against virus Aujeszky Chemopreventive: "Scavenging activity against O2" and lipid peroxidation •Antimetastatic effect on lung metastasis with B16 melanoma. •Inhibition of tumor-induced angiogenesis through downregulation of MMP expression in vitro. "Active against nephritis, through the inhibition of cellular proliferation.
Chemopreventive No increase of NGF • Active • Active • Active Anti-inflammatory • Active against carrageenan-induced mouse paw edema Antimicrobial against a E. coli
Anti-inflammatory: • Active against carrageenan-induced mouse paw edema • Inhibition of bradykinin and histamine effects.
Assayed activity
D'3]
[111] [112]
[HO]
[108]
[108] [109]
[107] [108]
[106]
[105]
References
697
698
DISCUSSION Before starting to discuss the pharmacological activities shown by PPGs, it is interesting to take into account that it is difficult to compare all the results due to the different experimental models used. Moreover, in each assay, different kinds of PPGs with more than one structural difference have been compared, therefore being hard to understand, which moieties or structure modifications could contribute to each activity. Nonetheless, it has been found that some structure-activity relationships, which are discussed below, and based on this, some suggestions for further analysis of PPGs are given. Antioxidant activity An antioxidant can exert its activity through different mechanisms of action such as: Radical scavenging or free radicals (ROS), metal chelating, inhibition of lipid peroxidation or inhibition of endogenous enzymes that generate ROS [72]. PPGs can act as antioxidants through different mechanisms. They possess in vitro scavenging activity against superoxide, hydroxyl, hydrogen peroxide hippochlorite and nitric oxide radicals, tested out of and within cell systems tests [17, 55, 56, 65, 75, 82, 96, 110]. Curiously, some of them (pedicularioside M, N and martynoside) have shown an antioxidantprooxidant dual effect, being hydroxyl scavengers at high concentrations but becoming radical generators at low doses. This dual effect could be due, in part, to the capacity of binding to iron III, reducing it to iron II. However, others P PGs s uch as v erbascoside, h ave o nly s hown s cavenging p roperties [75]. Even so, it has been concluded that there is a structure-activity relationship between the different PPGs. There is a direct relationship between the antioxidant activity and the number of hydroxyl groups, and an inverse relationship with the increasing of higher monosaccharides moieties. Detailing what was mentioned above, in both, enzymatic and cellular models, it has been proven that verbascoside is the compound with the highest scavenging power. It has published different potency orders, such as:
699
Verbascoside > Forsythoside > Arenarioside > Ballolletroside or, Verbascoside > Pedicularioside A > Pedicularioside M > Leucosceptoside A > Martynoside N > permethylverbascoside [75]. There was only one assay where two PPGs, brandioside and poliumoside, were better superoxide scavengers than verbascoside. This fact could be due to the rhamnosyl moieties, that might be more potent against superoxide radical [65]. All these scavenging capacities are based on one fact: The polyphenol compounds are attacked by superoxide or hydroxyl radicals predominantly at the o-dihydroxy site. The semiquinone radicals formed from the reaction of the o-dihydroxy structures with the radicals are quite stable, probably because of the presence of hydrogen bonding. However, if the PPG has a methoxy radical at the ortho position, the aroxyl radical derived from its reaction with superoxide or hydroxyl radical is less stable owing to the absence of intramolecular hydrogen bonding [114]. Despite this being known, it might be interesting to try to develop some of the assays described in bibliography, under the same conditions, with all the possible variations, and including isoverbascoside and derivatives. There is some controversy in the bibliography about the chelating properties of the PPGs. Some authors found that some are iron chelants [80], whereas others have proven the opposite for ferric [75] and cupric [16] irons. Another antioxidant mechanism of action is the inhibition of the lipid peroxidation. It can be detected by different methods, but, in most of them, the peroxidation is induced by F enton reaction, in which hydroxyl radicals are generated. Therefore, it would be logical to think that those PPGs that have shown hydroxyl radical scavenging, will be active as lipid peroxidation inhibitors. But it is necessary to develop these kind of assays, because, the absorption properties of these compounds into cells or the ways in which they may offer protection to the exterior of the cell wall, still remain unknown [35]. All the PPGs tested showed activity [53, 55, 80], being isoverbascoside, an isomer of verbascoside, a slightly higher than verbacoside. On the other hand, it has been studied that some PPGs affects the expression or activation of endogen enzymes that produce free radicals, like xanthine oxidase [72, 82] or inducible nitric oxide synthase [56]. But, the PPGs assayed (2-aeetylverbascoside, cistanoside, echinacoside, forsythoside
700
isoverbasocoside, pedicularioside, tubuloside, and verbascoside) do not affect those enzymes, except isoverbascoside, which is a competitive xanthine oxidase inhibitor [55, 67]. Therefore, most of the PPGs only reduce the ROS levels acting as radical scavengers. This antioxidant activity, and to be more exact, the scavenging activity, is one of the responsible m echanisms of other pharmacological activities that have been described for PPGs, as anti-inflammatory, healing and antitumoral-chemopreventive activities. Anti-inflammatory activity The inflammatory response involves many types of tissues and cells. These cells produce some common modulators like: eicosanoids, cytokines, ROS, and nitrogen intermediates. The eicosanoids are classified into three big groups, prostaglandins and prostacyclins, leukotrienes, and thromboxanes. The prostaglandins are lipid mediators implicated, not only in inflammation, but also, in other pathological processes, such as edema, fever, hyperalgia, cancer or Alzheimer's disease. The cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of PGE2, TXB2 and prostacyclines from arachidonic acid. Leukotrienes are involved in immune-regulation, asthma, inflammation, and various allergic conditions. In the presence of 5-lipooxygenase (5-LOX), free arachidonic acid is converted to 5-HPTE, which is then reduced to 5HETE or dehydrated to a non-stable intermediate LTA4 [115]. LTA4 is further converted enzymatically to leukotrienes, LTB4 and LTC4 [116, 117]. The LTC4 is a leukocyte chemotaxic that participates in cell adhesion, superoxide production, calcium translocation and the release of different enzymes. Therefore, there are different targets to attack the inflammatory process. Due to this, the study of the anti-inflammatory activity by the PPGs has been studied following different experimental models, trying to justify their mechanism of action as anti-inflammatory agents. Verbascoside has shown carrageenan-induced rat paw edema inhibition [105].
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Liao and cols.,[36] published that different PPGs, as, angoroside A, calceolarioside, capnoepside, echinaeoside, forsythoside, and verbascoside, do not inhibit to COX or 5-LOX on rat peritoneal leukocytes at 50 [ig/mL. However, has recently been studied the effect of PPG on COX enzymes in two different publications. On one hand, Sahpaz and cols. [21] found that arenarioside, forsythoside, and verbascoside, were the strongest COX-2inhibitors at 100 uM. Moreover, these compounds did not exhibit any significant inhibition on COX-1 at the same concentration. The authors defended the existence of a structure-activity relationship: the possession of two o r t hree s ugar u nits i n t heir s tructure c ould c ontribute t o t he se lective inhibition on COX-2. On the other hand, ballotetroside, with four sugar units, exhibited a weaker activity, and, interestingly is more active over COX-1 than COX-2. They think that the addition of a sugar unit (in this case arabinose on position C-2 of rhamnose) increases the steric hindrance, which prevents the molecule easily getting to the active site of the enzyme. The study of forsythoside and arenarioside is important because they have the same PPG structure, with the only difference in the kind of third sugar moiety joined to C-6 of glucose. Comparing the activity showed by each active compound, we also would hypothesize even more that apiose moiety joined to C-6 of glucose seems to contribute to COX-2 inhibition more than xylose moiety. Nevertheless, the presence of xylose on this position exerts the same effect on COX-2 inhibition than verbascoside, a diglycoside PPG. Diaz and cols. [92] studied more PPGs (angoroside A, C and D, isoverbascoside and verbascoside), as anti-inflammatory compounds, and their effect not only over COX-1 and 2, but also over TX-synthase, and NO generation. Angoroside A, C and D and isoverbascoside exhibited inhibitory activity on COX-1 in A23187-stimulated macrophages. The inhibition was more evident with angoroside C and A. Of all tested compounds, only angoroside A, C and verbascoside showed a significant effect on TXB2release. All compounds strongly inhibited LPS-induced NO production, being more active angoroside A, D and isoverbascoside. All compounds except angoroside C also inhibited the accumulation of PGE2 that means that they are active inhibiting COX-2. All compounds, except angoroside C,
702
strongly inhibited LPS-induced TNF-a production, being more potent in the case of angoroside D and isoverbascoside. These authors concluded that verbascoside is the most active compound on TX-synthase inhibition; therefore, caffeoyl moiety is an important function for this activity. The replacement by a feruloyl radical, leads to a complete loss of this activity. The attachment of a caffeate moiety at C-6 of glucose (case of isoverbascoside) appears to be favorable for COX-1 activity and TNF-a release inhibition, although it is detrimental for TX-synthase inhibition activity. On the other hand, the attachment of arabinose is favorable for NO activity and detrimental for TNF-a activity, but, if in this case, the caffeoyl is replaced by a feruloyl moiety, it is favorable for NO and TNF-a activity. Comparing these results with those showed by Sahpaz and cols. [21], we also can conclude that, it should be interesting to develop the assays of Diaz and cols [92], on forsythoside and arenarioside as well, and study the effect of the methoxylation (feruloyl radicals) on diglycosides like leucosceptoside and martynoside, not only on triglyeosides as angoroside. Also it might compare the activity of forsythoside with poliumoside and angoroside A, all of them with three sugars moieties, but, differing on the last one, apiose, rhamnose and arabinose, respectively, joined at the same position of the second sugar moiety, rhamnose. Other authors have been studying other aspects of the inflammatory process. Kimura and cols. [44] assayed different PPGs as forsythiaside, suspensaside, verbascoside and P-hydroxyverbascoside, on the 5-HETE and LTB4 inhibition. They conclude that two adjacent phenolic hydroxyl groups (caffeoyl radicals) are essential for potent inhibition of the formation of 5HETE and LTB4. The inhibition of 5-LOX enzyme is reversible and noncompetitive. With these results, we also suggest that a hydroxylation in position (3 could decrease this activity. Besides, it seems that there is no influence of the position of the second sugar moiety (rhamnose on position C-3 or C-6 of the glucose) or the metoxilation of the hydroxyl groups. But, surprisingly, despite the compounds inhibited the formation of 5-HETE at concentrations 10" -10"3 M, at concentrations between lO^-lO"4 M they stimulated the formations of TXB2 and 6-keto-PGFa.
703
Xiong and cols.,[56] also studied the influence on the inhibition of nitric oxide (NO) by PPGs in activated macrophages, because during inflammatory reactions NO is also produced by iNOS in different cells, such as macrophages, hepatocytes and renal cells. NO acts as a defense and regulatory m olecule with h omeostatic a ctivities [118]. H owever, i t i s a lso pathogenic when it is excessively produced. NO, per se, is a reactive radical, damaging directly to functional normal tissue [119-123]. The PPGs assayed 2'-O-acetylverbascoside, cistanoside A, echinacoside, isoverbascoside, and tubuloside A and B, and verbascoside, could specifically scavenge NO radical at high concentration (200 JJM) without attenuation of iNOS RNA-expression or iNOS protein levels or iNOS activity. The compounds that had a disaccharide moiety (2'-0acetylverbascoside, isoverbascoside, tubuloside B, verbascoside) showed a better inhibitory potency than those with a trisaccharide (cistanoside A, echinacoside, and tubuloside A). This result suggests that an increase in the number of monosaccharide units in glycosylated sugar attenuates the scavenging activity of phenylethanoids for maerophage-generated NO radical [56]. Healing activity Another interesting activity to take into account is the healing properties exerted by some PPGs. The process of wound healing involves a variety of processes such as inflammation, cell proliferation and contraction of the collagen lattice formed [124]. After wounding, several types of cells are recruited into the site of the injury to carry out the processes of repair. Following the neutrophils and monocytes, fibroblasts are attracted into the site to initiate the repair proliferating phase. They are the cells that secrete the collagen fibers and the glycosaminoglycans of the new granulation tissue, and subsequently effect the remodeling of the granulation tissue into mature dermis. They also secrete different growth factors that stimulate proliferation, differentiation and migration of other cells involved in the wound healing [125]. Mensah and cols.[35] showed the effect of two PPGs, echinacoside and verbascoside, both differentiated on the number of sugar moieties, as
704
protectors on human dermal fibroblasts against hydrogen p eroxide-induced oxidant injury. This activity could be attributed to the antioxidant property that has this kind of compounds. Therefore, this antioxidant effect may be one of the mechanisms that facilitate the wound healing. Furthermore, Sudo and cols. [104] studied the effect on collagen by different PPGs. The collagen protein plays diverse important roles, forming connective tissues, basement membranes and core proteins for bone formation. However, the production of an excess amount of collagen in the liver and lung causes flbrosis and eventually, liver cirrhosis. For wounding, the rapid formation of collagen is required in the early phase and effective resorption of excess collagen in the later phase. In each case, a proper turnover of the collagen protein is necessary for the maintenance of homeostasis. They found that verbascoside, at a concentration of 20 |ig/mL formed a more complex network of collagen fibers. These authors compared the activity shown by verbascoside with martynoside, which loose the collagen fibers formation, concluding that the caffeoyl moieties must be required for the expression of the biological activity. Moreover, verbascoside made thinner and densely distributed collagen fibers. This activity could be another mechanism of action in the wound healing activity, since production of minute networks with more slender collagen fibers is favorable for rapid granulations and for avoidance of formation of ugly scars which result from thick collagen fibers. Antimicrobial activity Some PPGs have been tested against Gram-positive and Gram-negative bacteria. Didry and cols. [15] performed the assay over five strains Staphylococcus aureus including one methicillin-resistant strain, five strains of Enterococus faecalis, three strains of Pseudomonas aeruginosa, five strains of Escherichia coli, three strains of Proteus mirabilis and one of each strain of Enterohacter aerogenes, Klebsiella pneumoniae, K. oxytoca with, , arenarioside, ballotetroside, forsythoside and verbascoside. These compounds do not possess antimicrobial activity up to 128 |a,g/mL. Verbascoside, forsythoside, arenarioside possessed a moderate inhibitory
705
effect against S. aureus and P. mirabilis. Arenarioside showed the most significant results from all of them. But, in this publication, the authors did not show the effect that the tested PPGs have on the rest of the bacteria that we have mentioned above. Kyriakpoulou and cols,[28] discovered that samioside, is more active than verbascoside against S. aureus, S. epidermidis, Enterobacter cloaceae, E. coli, K. pneumoniae and Pseudomonas aeruginosa. This result could make to think that an additional sugar moiety, in this case, apiose, at C-4 of rhamnosa could contribute to the antibacterial activity. To confirm this, it would be interesting to study some other PPG that have modifications on the sugar moieties, or feruloyl radicals in stead of caffeoyl ones. Despite the PPG showing a weak activity, it has developed further antimicrobial assays. Pardo and cols, reported [34] the MIO= 400 p.g/mL and MBC= 800 jxg/mL of verbascoside. Moreover, Avila and cols. [33] studied its mode of action in vitro. There is no evidence of inclusion of [3H]-leucine into the cell, whereas [3H]-thymidine and [3H]-uridine were not observed. That resulted in the conclusion that the mode of action of verbascoside is through the inhibition of protein production, since leucine is an important metabolite in protein synthesis. Antitumoral-chemopreventive activities The PPGs exert a cancer chemopreventive activity through different mechanisms: Repair ofDNA Adducts:
DNA damage can be caused by the environmental agents, such as ionizing radiation, UV light, and a variety of chemicals as well as normal metabolism in which reactive oxygen species are formed as side-products. With regard to ionizing radiation, there are direct and indirect ways to damage DNA. With direct effect, ionization occurs within the DNA itself and generates base radical cations and base radical anions. Indirect damage is caused by
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hydrated electrons and hydroxyl radicals produced by ionization taken place in close vicinity to DNA reacting with DNA. DNA gives up base electron adducts and base radical anions [126-128]. Considering the scale of electron affinity of the different nucleosides, pyrimidine is found to be a much better electron acceptor than purine. On other hand, the purine bases, with the electron-rich imidazole, react with hydroxyl radical (OH*) faster than the electron-deficient pyrimidine [127]. This fact reflected the electrophilic nature of the hydroxyl radical. Concerning cellular DNA, a two-component hypothesis has been developed. According to this hypothesis, the e lectron loss c enters (radical cations) end up with the purines, particularly with the guanine moiety, whereas the final site of deposition of the ejected electron is with the pyrimidines, particularly with thymine [127]. The two-components hypothesis implies that in DNA there are mechanism of electron and positive hole transfer by which the initially generated and randomly distributed electron gain and loss centers are tunneled into the T and G "traps" respectively. The PPGs tested have been active both in purine and pyrimidine adducts, reducting or oxidizing radicals [73,76, 87, 88, 93,126, 129]. In table (2) it is summarized the different PPG that have been tested against some DNA base adducts, explaining the mechanism of action.
707 Table (2): PPG repairing DNA adducts activity and mechanism of action. Base adduct Mechanism Repair potency Ref. dAMP is rapidly protonated to Cis> Ver > Ped for [74] NH2 NH2 NH 2 give N-protonated radicals, dAMP*" [73] strong reductants. PPGs can Verb > Ped> Cis react with hydrated electron for dAMP(-H)' due to the electrophilic phenyl-substituted unsaturated dAMP- and dAMP(-H)' carboxylic group It may undergo a dehydration Cis > Ped [129] reaction by which a very weak oxidizing radical is converted into a strong oxidizing radical, which leads to a neutral Ncentered radical, that is dAMP-4-OH oxidizing. This can be repair by PPGs NH2 There is a ring-opening Cis > Ped [129] reaction, to give a strong ,OH reducing radical, which is converted into FAP. Can be repaired if it goes dAMP-g-OH under dehydration. This adduct mainly exists in Cis > Ped [129] the oxidizing stats and can be reverted to dGMP or hydrated 'dGMPbyPPGs.
-u:->
i,JL>
dGMP-4-OH ^H 1
1
\
^
HN
%
>
HjiN
N
•
N H
R
N *
N
R
dGMP-5-OH
i
K,
I
R
y-R
OH
It is a reducing radical because there is a little unpaired spin density on the nitrogen. But, if it goes under dehydration, becomes an oxidizing radical. PPGs can repair it by donating electron. Predominantly it is a reducing radical. The repaired proportion by PPG is very little.
Cis > Ped
[129]
Cis > Ped
[129]
R
dGMP-8-OH
oi
IT R
TMP radical anion
PPGs react through the electrophilic phenyl substituted unsaturated carboxylic group.
Cis > Ped > Verb [126]
708
The PPGs are mainly nucleophilic, (electron donors) that react easily with the oxidizing adducts (electrophilics), and thanks to the non-saturated carbonyl- phenyl moiety, which is electrophilic, they can act as electron acceptors too. In a wide sense, we can conclude that the PPGs are able to repair the DNA due to their phenol groups and to the electron transfer process by the formation of a complex in minor groove of the double helix [89]. Due to the two-components hypothesis mentioned above, it is reasonable to say that by repair TMP radical anion, PPGs can repair indirectly other base radical anions produced in cellular DNA by radiation. In case of dAMP and dGMP-8 OH, there is redox ambivalence. This is a general property of radicals since they are in-between two stable oxidation states; Therefore, they can be oxidized or reduced depending on their reaction partner. The proportion repaired, depends not only on the reducing activities of PPGs but also on their concentration in the repaired system. Finally, there has been found a structure-activity relationship, the repair activities of PPGs toward oxidizing hydroxyl adducts of dGMP and dAMP are also positively related to the number of phenolic hydroxyl groups.[129]. Prevention against tumor formation induced by carcinogens
The PPGs were able to inhibit the tumor formation induced by different carcinogenic agents, such as galactosamine, [63, 64] lipopolysaccharide [54] due in part to the increase of their elimination. Induction of cell differentiation:
Verbascoside and isoverbascoside are able to induce differentiation on different cell lines. In case of verbascoside, on MKN45 [85] and human gastric adenocarcinoma (MGc803) [86] cell lines and for isoverbascoside, human hepatocellular carcinoma (SMMC-7721) [83], MGc803 [84], and human promyelocytic leukemia (HL-60) [109]. Despite the mechanism by which these two compounds induce differentiation in these cell lines remains to be investigated, some of the observed effects are the accumulation of cells
709
in GQ/GI phase, Mid the decrease in S phase. This arrest is a common phenomenon in the cells undergoing induction of differentiation [130]. Besides, isoverbacoside induces the upregulation of protein expression of p53, p21/WAF, and pl6/INK4, as well as the suppression of C-myc expression [84]. The cyclin-dependent kinase inhibitors such as p21 and pi6 proteins play central roles in this process. These proteins exert their functions by combining with cyclins/CDKs complexes, conducting finally to the blockage of the cell cycle at Gl-S checkpoint [131,132]. p53 is a tumor suppression gene, that can exert its cell cycle arresting function by up-regulating the expression of p21/WAFl protein [131, 132]. On the other hand c-myc is an oncoprotein, that acts as a nuclear transcription factor, and regulates the cell cycle by promoting the transcription of DNA synthesis related genes and therefore, impelling cell cycle into S phase [131,132]. Another important fact related to the induction of the cell differentiation is the antioxidant activity that these compounds have. The tumoral cells have lower antioxidant levels; therefore, ROS scavengers might induce this differentiation. Another mechanism closely connected to the antitumoral activity is the inhibition of oncologic signals and enzymes implicated on proliferation, differentiation and transformation processes. Verbascoside and homoplantaginin inhibit EGFR [63] and tirosin-kinase (TK). Calceolarioside A and B, forsythoside, leucosceptoside A, plantainoside, poliumoside, and verbascoside inhibit a-isoform of protein-kinase C (PKC) [69, 100]. The potency of this activity might be related to the number of sugar moieties in an inverse way, but it required more assays with other PPG to find the relationship. It has been found that verbascoside interacts with the catalytic domain of this enzyme, but a structure-activity relationship was not discussed. Apoptosis activation:
Verbascoside is also able to induce apoptosis [24]. The apoptosis is the programmed cell death functioning to conserve tissue homeostasis. It is
710
induced by the activation of cysteine proteases (caspases), ceramide formation by sphingomyelinase, activation of MAPK cascade and generation ofROS. Their mechanism of action still remains unknown, but it has been observed that those PPGs that also have pro-oxidant activity, can exert apoptosis activation as well, probably due to a generation of DNA damage through the hydrogen peroxide generation. This DNA damage might lead to the apoptosis process because of the accumulation of DNA fragments. Surprisingly, better results have been obtained with in vivo than with in vitro assays. Telomerase inhibition:
Verbascoside is able to inhibit another enzyme related to tumor cells, the telomerase [85]. The tumoral cells express this enzyme that elongates the 3' ends of telomere [133]. Telomere shortening and telomerase activity have been detected in almost all human tumors but not in normal somatic tissues [133, 134]. Zhang and cols, [85] showed that the telomerase inhibition by verbascoside may involve telomere-lenght regulation. Although verbascoside can arrest tumor cell growth, repair DNA oxidative damage and induce cell apoptosis and differentiation, whether telomerase inhibition by verbascoside may lead to tumor cell apoptosis, and whether all the cell cycle changes exist as a common mechanism, is not yet known. Anti-angiogenic and anti-metastasis
Finally, the effect of verbascoside on others carcinogenic steps, angiogenesis and metastasis, have been studied [111, 112], where ROS may also be important. This activity has been tested in vivo. Cytotoxicity:
Surprisingly, of all the cell lines studied, verbascoside only showed a cytotoxic effect on the cells with a murine origin [23, 32, 91]. Other PPGs
711
have been studied and it seems to be a structure-activity relationship, where the possession of feruloyl radicals, decreases the activity. We can conclude suggesting more deep assays that contribute to the knowledge of the wide antitumoral activity that verbascoside and isoverbascoside have, because they seem to be promising antitumoralchemopreventive agents. Moreover, it is thought that more than one mechanism of action is implicated. Besides, it should be interesting to develop all these assays with other PPGs, to compare their activity and be able to find more exact structure-activity relationship. ABBREVIATIONS A7r5 AAPH B16F10 BZD cAMP
CCUCOX CTAB dAMP dAMP(NH) dAMP+ dAMP-OH D-Gal dGMP dGMP+ dGMP-OH DMBA DPPH dRLh-84 EBV-EA EGFR H2O2 HeLa
=
= =
=
Rat aortic smooth muscle cells. 2,2'-azo-bis(2-amidinopropane)dihydrochloride Murine melanoma. Benzodiazepine cyclic 3',5'-adenosine monophosphate Carbon tetrachloride Cyclooxygenase Cetyl trimethylarnmonium bromide 2' -deoxyadenosine 5' -monophosphate dAMP N-protonated radical adduct dAMP radical cation dAMP hydroxyl radical adduct D-Galactosamine 2'-deoxyguanosine 5'-monophosphate dGMP radical cation dGMP hydroxyl radical adduct 7,12-dimethylbenz(a)anthracene 1,1 ,-diphenyl-2-picrylhydrazyl radical Rat hepatoma Epstein-Barr virus early antigen Epithelial growth factor receptor Hydrogen peroxide Human epithelial carcinoma
712
HeLa HIV HL-60 HSV-1 HSV-2 L1210 5-LOX LPS mCMV MDA MGc803 MK-1 MMP MPP NO Of OH P-388 P-388-D1 RSV S-180 SMMC-7721 T" TK TLC TMP TNF-a T-OH TPA
Human uterus carcinoma. Human immunodeficiency virus Human promyelocytic leukemia Herpes simplex vims type 1 Herpes simplex virus type 2 Lymphocytic mouse leukemia 5-lipooxygenase Lipopolysaceharide Murine Cytomegalovirus Malondialdehyde Human gastric adenocarcinoma Human gastric adenocarcinoma Metaloproteinases l-methyl-4-phnylpyridinium ion Nitric oxide. Superoxide Hydroxyl radical Murine lymphocytic leukemia. Mouse lymphoid neoplasma Respiratory syncytial virus Sarcoma Human hepatocellular carcinoma TMP radical anion Tirosin kinase Thin layer chromatography. Thymidine-5' -phosphate Tumor necrosis factor Thymine-hydroxyl radical adduct 12-0-tetradeeanoylphorbol-13-acetate
713
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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DEVELOPMENT OF TUBULIN INHIBITORS AS ANTIMITOTIC AGENTS FOR CANCER THERAPY S. MAHBOOBI1, A. SELLMER1 and T. BECKERS2 Department of Pharmaceutical Chemistry I, University ofRegensburg, D-93040 Regensburg, Germany, Germany; phone: (+49) 941-9434824, Fax: (+49) 941-9431737, E-mail:
[email protected] 2
Therapeutic Area Oncology, ALTANA Pharma AG, D-78467 Konstanz, Germany, Phone:(+49) 7531 842974; Fax:(+49) 7531 8492974; E-mail: Thomas. Beckers@altanapharma. com ABSTRACT: Cancer represents one of the most threatening diseases of mankind. Within the last decade, our understanding of malignant cell growth and regulation of the cell cycle machinery has offered several new molecular targets with the promise of higher selectivity in human cancer therapy. Within mitosis, aP-tubulin heterodimers, building up the mitotic spindle, are still an attractive target in the development of anticancer drugs. In the following article, we review the recent advances in the development of tubulin interfering agents. These agents are divided according to their mode of action into colchicine site binder, vinca-alkaloid related drugs and those interacting with the Taxol binding site and functioning as stabilising agents. Since clinically used compounds such as Paclitaxel or Vincristine are facing severe disadvantages, namely a small therapeutic window, restrictions in bioavailability and solubility, a complex synthesis and most importantly development of drug resistance in patients, special emphasis is laid on the development of synthetic small molecule tubulin inhibitors (SMTIs). These new agents offer promise for the rational design of new chemotherapeutic drugs by their simple structures and potential broad applicability in 2nd and 3 rd line standard chemotherapy regimens towards resistant tumours. In this regard, most SMTIs are not P-glycoprotein substrates. Most tumours can only grow beyond a critical size by inducing the formation of new blood vessels, a process called neovascularisation. SMTIs as well as natural tubulin inhibitors have been described to interfere with this angiogenic process and some, like combretastatin A4 phosphate, are even described as selectively damaging tumour vasculatures. Finally, the present review emphasises the preclinical and clinical status of tubulin inhibitors in cancer therapy.
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INTRODUCTION Cancer represents one of the most threatening diseases of mankind. In spite of great efforts in basic research, cancer is still the second leading cause of death in industrialised nations and this picture will get even worse in near future. Within the last decade a better understanding of the cell cycle machinery offered several new targets with the promise of higher selectivity in cancer therapy. Uncontrolled proliferation is a hallmark of cancer cells, thus selective interference with the M-phase of the cell division cycle is most important. Within mitosis, after condensation of nuclear chromatin and disruption of the nuclear envelope, the mitotic spindle is formed which segregates the daughter chromatids. Cyclindependent protein kinases (CDKs) and their inhibitors (CKIs) [1] are key regulators of the cell cycle machinery and microtubules [2] and microtubule associated proteins (MAPs), building up the mitotic spindle, are of high interest in anticancer drug development. In the present review, we report about recent development of compounds that interfere with the dynamics of tubulin polymerisation and depolymerisation by direct binding to tubulin (for earlier review see [3,4, 5,6,7]. Tubulin, a heterodimer of closely related and tightly linked globular a- and p-tubulin proteins, is the essential structural element of the mitotic spindle, a- and P-tubulin heterodimers are polymerised parallel to cylindrical axis-building, helical, hollow tubes called microtubules, forming the mitotic spindle. The mitotic spindle is a bipolar, self-organising machine that gathers energy from nucleotide hydrolysis to segregate sister chromatids accurately into daughter cells [8,9]. The rapid switch between growing and shortening states of microtubules in the process of dynamic instability and driven by P-tubulin dependent GTP hydrolysis is essential for the movement of chromosomes [10]. A modification of microtubule properties is induced by binding of microtubule associated proteins (MAPs) [11,12]. Besides their function in mitosis, microtubules are also associated with important cellular processes, namely axonal transport and cell movement [2]. Tubulin binding agents interfere with the dynamic instability of microtubules and thereby arrest mitotic cells in the M-phase of the cell division cycle, finally leading to induction of apoptosis. Most, if not all, tubulin inhibitors bind within B-tubulin to distinct epitopes. They are
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categorised according to their tubulin binding sites or to the first compound described (e.g. colchicines or vinca alkaloid like tubulin binding agents). Colchicine itself and colchicine analogues predominantly bind to a high affinity site, the so-called colchicine binding site located at the intradimer interface between a - and p-tubulin, facing the lumen of the microtubule. Colchicine (1) Fig. (1) inhibits microtubule formation and disrupts microtubules as a tubulin destabilising agent. HjCO.
/^O
H 3 CO'
0
OCH3 1 Colchicine
3
Taxol INN Paditaxe!
Fig. 1: Chemical structures of Colchicine (1), Vinblastine (2), Vincristine (2a) and Taxol (3).
Vinblastine (2) and several vinca-related drugs bind to a different site, as do a number of other drugs that bind competitively with each other, but do not compete with the colchicinoids. Like the colchinoids, vinca alkaloids destabilise microtubules. The Taxol (3) binding site is located in a pocket that is lined by several hydrophobic residues and is well defined from crystal structures of ap*-tubulin. It represents the putative binding site for other microtubule stabilising drugs like epothilones [2,11]. Other binding sites have been postulated according to tubulin interfering agents with a binding behaviour distinct to that of taxanes, vinca alkaloids and colchinoids.
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From a therapeutic point of view, compounds of low molecular weight with oral bioavailability and high therapeutic index are very attractive as lead structures. By inhibition of tumour cell proliferation and neovascularisation, tubulin inhibitors are potent anticancer drugs. Nevertheless, clinically available compounds such as Paclitaxel or Vinchristine are facing severe disadvantages. [13]. hi the following we therefore focus on different structures with low molecular weight, socalled "small molecules tubulin inhibitors" or SMTIs. Most of these SMTIs can be placed in the category of colchicine-site binders. THE FAMILY OF COLCHICINE-SITE BINDING TUBULIN INHIBITORS The family of colchicines-site binders includes compounds of diverse structure unified by interference with the colchicine binding epitope. The respective compounds can be clustered according to their chemical structure mainly into I) colchicines and compounds with colchicine like substructures, II) combretastatins and phenstatins, III) compounds having an indole core structure, IV) quinolones, V) sulphonamides and VI) naturally occurring as well as synthetic compounds. Colchicinoides Colchicine (1), the first known tubulin binding agent isolated from the plant colchicum autumnale, is too toxic to be used as an anticancer agent. Nevertheless it has been used in the treatment of gout and other inflammatory diseases since the 6th century a.c. [14]. Binding of colchicine induces an alteration in tubulin dimer structure and hinders micro tubule assembly. The drug (1) additionally can bind to tubulin at a second, lower affinity site in a reversible manner [15]. Another highly active and naturally occurring colchicine analogue is cornigerine (la) Fig. (2), derived from colchicum cornigerum [16]. In order to reduce the toxicity, a lot of efforts have been made to provide new analogue as potential anticancer drugs. Previous experimental observations as well as structure evaluation programmes (SAR) suggested that the colchicine binding site of B-tubulin has very stringent structural requirements. The three methoxy groups in the A-ring
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seem to be essential for high binding affinity. The seven-membered ring and the C (7) side chain do not seem to be essential for binding to tubulin. Furthermore, the seven-membered C-ring may also be exchanged by a benzene ring, as shown by the high biological activity of allocolchicine (lb) (IC50 = 1 . 4 |jM in inhibition of tubulin polymerisation) and (lc), which was shown to inhibit the tubulin polymerisation reversibly and much more potently than colchicine itself without enhanced toxicity (for review see [17,18] ). Further allocolchicinoids, exhibiting high tubulin binding affinity and potent inhibitory activities against solid human tumour cell lines, have been reported [19]. ZD 6126 Ongoing research has resulted in ZD 6126 (Id) [20], the water-soluble phosphate prodrug of N-acetylcolchinol, a novel tubulin binding agent directly targeting tumour vasculature. It is currently developed by AstraZeneca in phase II clinical trials in solid tumour patients and was inlicensed from Angiogene Pharmaceuticals Inc. in 1999. In preclinical studies, single doses of ZD 6126 (200mg/kg i.p.) induced haemorrhage and necrosis in the PC14PE6 NSCLC nude mice metastasis tumour model with some selectivity towards tumour endothelial cells [20]. In a 2nd preclinical study, ZD6126 showed significant effect on tumour vasculature in different nude mice xenograft models at well-tolerated doses up to 16fold below the MTD of « 400mg/kg (i.p. or i.v.). In the Calu-6 NSCLC model, lOOmg/kg ZD 6126 (i.p. for 5d) in combination with 4mg/kg Cisplatin had more than additive effects on tumour growth delay [21]. Phase I clinical trials for definition of a MTD, DLT and circulating endothelial cells (CECs) as a surrogate marker has been finished recently, showing reasonable tolerability, a rapid clearance of Nacetylcolchinol with t\a = 2-3h and a 2 fold increase in CECs 4-6h after ZD6126 infusion [22,23].
724
H3C OPO 3 H 2 H3C 1a
Cornigerine
1b R = CO 2 CH 3 Allocolchidne 1c R = COCH3
1e ZD6126
EtO. H3C
4 2-Methoxyestradiol (NSC-659853)
Ring expanded homologes of 4 exhibit structural similarity to colchicine
Fig. 2: Colchicinoide derivatives and synthetic steroids exhibiting antiproliferative activities
2-Methoxyestradiol / NSC-659853 2-Methoxyestradiol / NSC-659853 (4), a cytotoxic human metabolite, binds to the colchicine site of tubulin with an affinity of IC50 = 4.7 \iM. On the basis of a hypothetical relationship to the colchicine structure, a series of B-ring expanded 2-ethoxyestradiol analogues were synthesised, in which the B-ring of the steroid was replaced by the B-ring of colchicines. While the resulting analogues showed significant affinity to the colchicine binding site consistent with the proposed structural resemblance, derivatives having a ketone at C-6 surprisingly behaved like Paclitaxel [24]. Further investigations on 2-methoxyestradiol have been performed and shown anti-proliferative effects, both on hormone dependent and independent breast cancer cells [25] as well as on antiangiogenetic activity [26]. The pharmacological profile has furthermore been shown to be especially dependent on steric and electronic influences of the substituent in position 2 [27]. Phase II trials with solid tumours, sponsored by EntreMed, are ongoing (National Cancer Institute, Maryland, USA, web site:
[email protected]).
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Combretastatins, Heterocombretastatins and Phenstatins Combretastatin A-4 A second group of tubulin inhibitors is represented by compounds exhibiting the combretastatin structure. Combretastatin (5) itself, as well as combretastatins A-l (5a), A-4 (5b) and A-2 (5d), have first been extracted from a South African tree Combretum caffrum by Pettit et al., depicting affinity to the colchicine binding site [28]. Syntheses of various analogues of this simple molecule, thereafter, have been reported [16,29, 30] (see also Scheme 1).
CBr4, PPh3
Bu3SnH Pd(PPh3)4 H3C
H3C
B(OH)2 Pd(PPh3)4 Na 2 CO 3
H 3 C . '' ° CH 3 H3C
(Suzuki Cross-Coupling) Combretastatin -A4
Scheme 1: Synthesis of Combretastatin-A4 according to Gaukroger [36]
Combretastatin-A4 (5b) is one of the most potent antimitotic agents [31] also active towards multi-drug-resistant (MDR) cell lines [18]. Research on combretastatin A-4 (5b), which has so far reached phase II in clinical development, has focussed on the structure dependency of antimitotic combretastatin agents [32], improvement of water solubility and in vivo activity [16]. One of the most important findings has been the introduction of an amino group instead of the phenolic hydroxy group as shown by the amino stilbene AC-7728 (5e), increasing water solubility and efficacy [31]. Based on the structural features of (5e), different heterocombretastatins (e.g. 5f-5h) have been prepared retaining IC50 values for inhibition of tubulin polymerisation in the low nanomolar range [33].
726
The water-soluble prodrug of combretastatin A-4, Combretastatin-A43-O-phosphate (CA4P) (5c) was licensed by Oxigen Inc. from Arizona State University in 1997. Like ZD6126 (le), CA4P acts as a tumour vascular targeting agent leading to tumour necrosis by shut-down of blood flow [34] (for review see [35]). CA4P was investigated in three phase I clinical trials and the results from the Ireland Cancer Centre Trial have been published recently [36]. CA4P currently is evaluated in phase II clinical trials [37], monitoring tumour blood flow as the critical parameter for optimal dosing of tumour vasculature targeting drugs. A-289099 and A-l05972 The recently developed oxadiazoline A-105972 (5i) displayed reasonable cytotoxic activity towards a panel of human cancer cell lines in vitro [38], but its utility in vivo was limited by a short half-life. Further efforts led to the identification of the indolyloxozoline A-259745 (5k) [38] that demonstrated a better pharmacokinetic profile and three times increased survival of tumour bearing nude mice upon oral dosing. The mechanism of tubulin interference and the enantio selective synthesis of A-289099 (5k, S-enantiomer) have been investigated, confirming the competition with colchicine [39]. After the preclinical development no further investigation has been reported. Another group of closely related compounds is the phenstatins. Phenstatin (6a) and hydroxyphenstatin (6b) resulted from a SAR-study on combretastatin A-4 and exhibited potent inhibition of cancer cell growth [29]. Further modifications have been performed, resulting in highly potent agents, namely 243869 (6c) with in vitro toxicity of IC50 = 0.58 nM towards HeLa cervical carcinoma cells [40,41]. SAR information about a series of 3-aminobenzophenone compounds (e.g. 6d and 6e), based on the mimic of the aminocombretastatin molecular skeleton, revealed that by the introduction of an amino group instead of a hydroxy group inhibition of tubulin polymerisation through binding to the colchicine binding site could fully be preserved or even improved [42].
727 727 H3C O H3C
NH2
H3C
H3C H3C
5a Ri = R2 = OH, R3 = R4 = CH 3 Combretastatin A-1 5b R-i = H, R2 = OH, R3 = R 4 = C H 3 Combretastatin A-4 5c R, = H, R2 = OP(O)(ONa)2, R3 = R4 = CH 3 Combretastatin A-4-3-O-phospate 5d R-) =H, R2 = OH, R 3 and R4 briged byCH 2 Combretastatin A-2 5e Ri = H, R2 = NH 2 , R3 = R4 = CH 3 AC-7739/AVE-8063
5f
'
x
5g
"
x
_
N=N
NH 2
5i A-105972
-CH 3
5k S-enantiome: A-289099 Racemate: A-259745
51 Heterocombrestatines e.g. 2-benzoylindoles
6 6a 6c 6d 6e
R-i = H , R2 = OH, R 3 = O C H 3 Phenstatin R, = R2 = OH, R3 = OCH3 H ydroxyphenstati n R1 = H; R2 and R3 = methylendioxy R1 = H, R2 = NH 2 , R3 = OCH 3 R1 = H, R2 = NH 2 , R3 = OC 2 H 5
Fig. 3: Chemical structures of different combretastatins and phenstatins
Incloles Several compounds possessing an indole moiety as a core structure have been developed during recent years Fig. (4).
728 728
8 7
Dlarylindoles
6-Methoxy-2-(4-methaxyphenyl)3-(3,4,5-trimetoxybenzoyl)benzo[b]thiophene
9
X = S, Benzthiaphene-derivatives X = NH, R = H, OCH 3 , F e.g. 6-Methaxy-2-(4-methoxyphenyl) indole
C4H9 10
12-Formyl-5,6-dihydro-indolones
N-Pyndin-4-yIH1-(t-chIorbenzyl)lndol-3-yl]glyoxyl-am(d (D-24851)
12 R = H, D-84131 12a R = OCH 3 , D-64144 12b R = F, D-8118?
13 Nocodazole
Fig. 4: Tubulin polymerisation inhibitors possessing an indole moiety as a core structure
Diarylindoles, e.g. (7), classified as heterocombretastatins with respect to the ethylene bridged diaryl structure and the 3,4,5-trimethoxy substitution pattern, displayed cytotoxicity especially towards leukaemia, non-small cell lung and CNS cancers [33, 43]. A modification of the tubulin inhibitor (8) [44] led to the thiophene and indole analogue (9) with remaining high cytotoxicity (IC50-values in the range of 10 to 100 nM) [45]. The structure of (9) also represents a fragment of the tetracyclic tubulin inhibitor (10) [46] that showed similar activity.
729 729
D-24851 (INN: Indibulin) A structurally related member of this group is presented by N-(pyridin4yl)-[l-(4chlorbenzyl)-indol-3yl]glyoxylamide / D-24851 or Indibulin (11), a compound currently in phase I clinical development by Baxter Oncology. D-24851 destabilises microtubules and blocks cell cycle transition specifically at the G2-M phase by binding to B-tubulin, competing with colchicine for binding [47]. D-24851 is highly cytotoxic in vitro towards a panel of established human tumour cell lines including SKOV3 ovarian cancer, U87 glioblastoma, ASPC-1 pancreatic cancer cells as well as towards tumour cell lines with various resistance phenotypes. hi vivo, a complete tumour regression in rats bearing Yoshida AH13 sarcomas was observed. D-24851 displayed no neurotoxic effects at efficacious doses in rats as studied by a deficit in motor function and reduction of nerve conductance velocity. No data on D-24851 analogue, developed by Zentaris GmbH, have been published so far. 2-Aroylindoles Another recently published class of potent antimitotic agents is 2-aroylindoles (12-12b), exhibiting ICso-values in a low nanomolar range (IC50 = 20 to 75 nM) towards various carcinoma cell lines. 2-Aroylindoles bind to the colchicine site of (3-tubulin, however, and in contrast to colchicine (1), vincristine (2a), nocodazole (13) or taxol (3), they showed no significant influence on the GTPase activity up to 10 fiM [48,49]. In addition, angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay was inhibited by D-64131 and analogue. Since 2-aroylindoles are easy to synthesise (Scheme 2), the role of the indole substitution pattern was studied in more detail. hi terms of SAR information, substituting the indole structure (azaindoles; X or Y = N) yielded inactive or compounds of low cytotoxicity. The same holds true for N-alkylation or substitution of the methoxy-group in the indole core structure of (12a) vs. a methyl group. In summary, the results from the cellular cytotoxicity screening suggested the benefit of the 5-methoxy-indolyl-group for potent antitumour activity. The 3-methoxyphenyl- (12a) (IC50 < 3.2 pM) and the 3-fluorophenyl(12b) derivatives showed even higher cytotoxic activity than the unsubstituted compound (12). An insignificant electronic effect of the substituents in the aromatic ring is observed. The respective aroylindoles
730
have furthermore been shown to be no MDR/MRP substrates with no cross resistance with various resistance phenotypes [50]. In xenograft studies, no signs of systemic toxicity were observed after p.o. dosages of up to 400 mg/kg of D-64131 [13].
X and Y = C or N
Scheme 2: Synthesis of 2-aroylindole derivatives and the respective precursors. Conditions: i: THF, - 78 °C; ii: Pyridiniumchlorocromate (PDC), CH2C12, 20 °C; iii: THF, -78 °C; iv: NaOH, EtOH, A; v: Tetrabutylammoniumfluoride (TBAF), THF, A.
Quinolones Quinolones and related structures represent another class of compounds affecting the colchicine-binding site of tubulin. The synthetic 2-phenyl-4-quinolones (14) Fig. (5) structurally related to naturally occurring anti-mitotic flavonoids (15) [17] displayed promising activity and impressive differential cytotoxicity towards human tumour cell lines with IC50 values in the low micromolar to nanomolar range, comparable to that of colchicine. The most active compounds from this series were obtained by introducing of functional groups with non-bonding electrons e.g. -NRR', -OCH3, Cl or F at the 6'position of the A-ring and the 3'position of the C-ring. The structurally related 2-phenyl-l,8naphthylpyridin-4-ones (16), containing an additional nitrogen position 8 of the aromatic system, also exhibited potent cytotoxicity in particular towards the ovarian cancer 1A9 and the P-gp-expressing, vincristine resistant HeLa/KB-VIN cell lines.
731
14
2-Phenylquinolones e.g.R = N-pyrrolidine
14a
Fluorinated 2-Phenylquino!ones e.g. NSC 656158
15
Flavones
16
related quinazolones
14 b
Fig. 5: Chemical structure of quinolones, flavones and quinazolones
Further investigations based on SAR-studies resulted in fluorinated analogue (e.g. 14a and 14b), exhibiting high cytotoxicity against renal and melanoma tumour cell lines with ICso-values in the in vitro tubulin polymerisation assay of 0.46 |j,M (14b). The biaryl system, composed of rings A and C, being probably analogous to the biaryl-system occurring in many antimitotic agents, as well as the ketone functional moiety have been shown to be essential for a strong interaction with tubulin [51,52,53, 54]. Sulphonamides E7010 / ABT-751 Sulphonamides as E 7010 (17) inhibit tubulin polymerisation by binding to the colchicine site, exhibiting potent anti-proliferative activity in vitro (IC50 = 0,2 - 40ng/ml), currently investigated in clinical phase II studies [55,56] by Eisai/Abbott in patients with solid tumours. The pharmacophore structure responsible for tubulin binding seems to be the 3-pyridinyl-4-methoxybenzenesulfonamide substructure [57] Fig. (6). New analogue have been published recently by Abbott having a higher cytotoxicity but are disadvantageous because of short half-lives of t V2 < lh [58]. Closely related structures being active against multidrugresistant-cancer cells are an illustrative example of the different modes of
732 732
anticancer-drug-action that may result from small pharmacophore modifications. T138067 (Batabulin) Tl38067 (18), currently in phase III clinical trials in therapy of liver cancer, inhibits tubulin polymerisation by irreversible binding to cysteine239 on (3-tubulin isoforms 1, 2, and 4. The covalent modification of Ptubulin inhibits the polymerisation of the a(3-tubulin heterodimer into microtubules, leading to cell arrest followed by apoptosis induction [57,59]. A recently described analogue of T138067, exhibiting an amide structure instead of fluorine substituent in the B-ring and less lipophilicity, retained the same mechanism of action and an increase in potency towards HeLa and MDR expressing and non-expressing human mammary carcinoma cells [60]. E7070 (INN: Indisulam) hi contrast, the antimitotic compound E7070 (19), synthesised by Eisai Co. Ltd (Tsukuba Research Laboratories, Ibaraki, Japan) disturbs P388 murine leukaemia cells by disrupting the cell cycle progression and not by inhibition of tubulin polymerisation. This compound, structurally closely related to E7010 and Tl 38067, caused accumulation of cells in the Gl but not in the M-phase, as typically observed for tubulin inhibitors and can therefore be classified as cell cycle inhibitor. Human tumour xenograft models demonstrated that E7070 could cause a tumour regression in three of five colorectal and two out of two lung cancer models [57,61]. Phase II clinical trials are ongoing.
H2NSO:
17
E7010
18 T138067
Fig. 6: Structures of antimitotic sulphonamides. Dependent on the pharmacophore core structure the respective compounds can be classified as reversible (17) or irreversible (18) tubulin inhibitors respectively as cell cycle inhibitor (19).
733 733
Miscellaneous Compounds Affecting the Cokhicine Binding Site: Indanones, RPR112378 andRPR115781 (E)-2-Benzylidene-l-indanones (20) e.g. indanocine/NSC698666 [62] and the water-soluble prodrug SDX103 are in preclinical development by Salmedix. Indanocine (20) is a synthetic indanone that has been identified by the NCI Developmental Therapeutics Programme. Indanocine interacts with the colchicine binding-site of B-tubulin, potently inhibiting tubulin polymerisation in vitro and inducing apoptosis in cancer cells at concentrations that do not impair the viability of normal non-proliferating cells [63]. RPR112378 (21) and RPR115781 (21a) extracted from the Indian plant Ottelia alismoides were identified in a screening programme for new anti-mitotic drugs by a research group at Rhone-Poulenc Rorer / Aventis Pharma. The more potent destabilising tubulin inhibitor (21) (IC50 =1.2 \iM, tubulin polymerisation in vitro) competes with Colchicine for binding and reacts with the sulfhydryl groups of tubulin. RPR-112378 furthermore was shown to be cytotoxic with IC50 = 20nM towards the HeLa / KB human cervical carcinoma cell line [64]. No further preclinical data, or development, have been published on this new class of compounds until now. OH HO' CH3
20 Indanones, e.g.
21
RPR112378
21a
RPR115781
Indanocine NSC 698668
Fig. 7: Chemical structures of indanocine, RPR112378 and RPR 115782, compounds affecting the colchicine binding site
SYNTHETIC SMALL MOLECULES OF DIFFERENT STRUCTURE TARGETING BINDING SITES DISTINCT FROM THAT OF COLCHICINOIDS COBRA-0 and COBRA-1 Based on the structure of ap-tubulin dimer, resolved by electron crystallography of zinc-induced tubulin sheets, a previously unrecognised
734 734
hydrophobic binding pocket on the surface of a-tubulin was identified. A novel synthetic drug targeting this unique binding cavity in a-tubulin was synthesised, designated Cobra-0 because of its mono-THF head portion attached to a long Cn-chain, resembling the shape of a cobra. COBRA-0 (22) and the analogue COBRA-1 (22a), the first enantiomerically pure prototype compounds targeting this cavity, bind to a-tubulin and exhibit weak cytotoxicity in concentrations > 100 fiM. Treatment of human breast cancer and glioblastoma cells with COBRA-1 caused destruction of microtubule organisation and induction of apoptosis [65]. SPIKET-Compounds SPIKET-P (23), a novel synthetic spiroketal Pyran [66], represents another pharmacophore identified by docking simulations [67] with the marine product Spongistatin 1 (24), a macrolide polyether [68]. SPIKET compounds target the spongistatin binding site of [3-tubulin and exhibit potent cytotoxicity with IC50 values against the NCI panel of human cancer cell lines in the sub-nanomolar range (for review see [5]). The preclinical and clinical status of the compounds as mentioned in the sections before is summarized in table 1. H25C1:
OH
22
WHI-261 /Cobra 0
22a
OH
Cobra 1 OH
13 H
23 SPIKET-P1 AcO'
CH, H3C
OAc
OH
24 Spongistatin 1
Fig. 8: Synthetic Small Molecules of Different Structures Targeting Various Binding Sites
735
Table 1: SMTIs currently investigjated in preclinical / clinical studies Compound Name (Nr.) ZD-6126 N-Acetylcolchinolphosphate (le) 2-Methoxyestradiol NSC-659853 (4) CA4P (Prodrug) (5c)
Status in Company ic 50 development values in vitro Phase II n.p. Astra Zeneca ongoing
Phase II ongoing Phase II
A-289099 (5k)
Ceased
D-24851 (11)
Phase I
D-64131 (12) E-7010/ ABT-751 (17) T-13 8067/ Batabulin(18)
4.7 uM
0.9-3 nM Oxigene INC (drug) 7nM
36-285 nM Preclinical 24 - 144 nM Phase II 0.2-40 ng/ml Phase III 11-165 (liver cancer) nM
Indanocine NSC-698666 (20)
Preclinical
EntreMed
< 20 nM
Abbott Laboratories Baxter Oncology Baxter Oncology Abbott Laboratories Tularik Inc.
Salmedix
Remarks
Ref.
vascular targeting agent
[22,23]
angiogenesis inhibitor vascular targeting agent
[24,26, NCI website] [30,34,36 37] [39] [47]
angiogenesis inhibitor
[50] [56]
Irreversible P-tubulin binder
[59]
[63]
VINCA SITE BINDING AGENTS Vinca Alkaloids Vinblastine (2) and Vincristine (2a) are the leading compounds of the widely recognised antimitotic Vinca alkaloids, which represent a chemical class of major interest in cancer chemotherapy. The natural compounds are only present in minute quantities in the leaves of the Madagascan periwinkle, Catharanthus roseus. Several hundred analogues have been synthesised and evaluated for their pharmacological profile. The recent success of vinorelbine in human chemotherapy has encouraged the search for analogues with a new activity and tolerability profile. The clinical trial on Vinflunine has been reported recently (25). Vinflunine is the bifluorinated analogue of Vinorelbine (25a), which has used in the treatment of NSCLC and advanced breast cancer since 1992 [69].
736
Vinflunine was also successfully investigated in a phase-I trial by Pierre Fabre for use in the therapy of metastatic breast cancer in combination with Doxetaxel [70]. In comparison with Vinorelbine (25a), by the use of Vinflunine (25), MDR related drug resistance is developed less readily and the overall response rate in vivo with 64% is superior [71, 72, 73]. Phase III studies in NSCLC compared to standard 2nd line therapy with Doxetaxel and treatment to resistant bladder cancer are ongoing. Cryptophydns The Cryptophycins are a unique family of 16-membered macrolide antimitotic agents originally identified in blue-green algae (cyanobacteria) belonging to Nostocaceae (for review see [74]). The parent compounds of the series, Cryptophycin-1 or Cryptophyem-A (26) were found to block cells at mitosis in the low picomolar concentration range [75]. CH3
H3C
.. . CH 3
COOCH3 25
Vinflunine
25a
H
OCOCH 3 COOCH3
Vinorelbrine
26
R ~H Cryptophycin-1 (Cryptophycln-A) 2Sa R = CH 3 Cryptophyein 52 (LY 355703)
Fig. 9; Chemical structures of different Vinca site binding agents
Cryptophycin-52 (26a), a member of the cryptophyein family developed by Eli Lilly and produced by total chemical synthesis [76], was selected from diverse synthetic analogues displaying superior potency, stability and amenability of clinical formulation [77,76]. Cryptophycin-52 in vitro binds non-covalently to tubulin at a single high affinity site, which presumably overlaps with the Vinblastine binding site [75]. Proliferation of diverse tumour cell lines was inhibited with IC50 values of 13 pM to 232 pM, minimally affected by P-gp or MRP overexpression [78]. Data from phase I and II trials showed severe toxicities as long
737
lasting neuroconstipation and neurosensory toxicity without objective tumour responses leading to trial suspension [79,80]. Dolastatms The Dolastatins are cytotoxic cyclic pentapeptides isolated from the marine shell-less mollusk Dolabella auricularia [81].
27
27a
27b
Dolastatin 10 (NSC-376128)
Dolastatin 15
Cematodin (LU-103793)
27c
Synthadotin (LU-223651 / ILX -651 / LU223851)
Fig. 10: Dolastatins as potent inhibitors of tubulin polymerisation.
They were first described in 1990 as inhibitors of tubulin polymerisation (IC50 = 1,2 joM for Dolastatin 10) mediated through high affinity binding to the vinca binding site [82]. The Dolastatins include Dolastatin-10 / NSC-376128 (27), the analogue Auristatin / TZT-1027, as well as Dolastatin-15 (27a) and the analogues Cematodin / LU-103793 (27b) and ILX-651/ LU-223651 or synthadotin (27c). In a phase I study on Dolastatin 10, patients with advanced solid tumours developed peripheral sensory neuropathy, which was not dose limiting [83]. hi phase II studies with patients having metastatic prostate and colorectal carcinoma, Dolastatin 10 was very well tolerated but lacked significant clinical efficacy [84, 85]. Cematodin, the synthetic and water soluble
738 738
analogue of dolastatin 15 does not inhibit the binding of vinblastine to tubulin and is taken into account in this context due to similarity in structure. Scatchard analysis of Cematodin binding to tubulin indicates that there are two affinity classes and spindle dynamics are effected through a distinct molecular mechanism by binding to this novel site in tubulin [86,87]. Table 2: Preclinical / clinical status of Vinca site binding agents Compound Name (Nr.) Vinflunine (25)
Cryptophycin (LY-355703) (26a) Dolastatin 10 NSC-376128 (27) Cematodin LU103793 NSC D-669356 (27b) Synthadotin ILX-651 LU223651 (27c)
Status in development Phase III (NSCLC, bladder) Phase II suspended
Ref.
ICso-values in vitro 18nM
Company Pierre Fabre
[73]
13-232pM
Eli Lilly
[79,80]
Phase II ongoing
0.5 nM (L1210 cells)
NCI
[83]
Phase II finished, further investigation recommended Phase II
0.1 nM
Abbott
Abbott Ilex Oncology
Remarks
No vinca-site binding
[87,90]
[88,91, Company Info]
Investigations in a phase I trial determined the MTD with 2.5 mg/m and neutropenia, peripheral oedema as well as liver function test abnormalities as dose-limiting toxicities [89]. Phase II trails in patients with malignant melanoma noted only a small percentage of responders, but significant duration of response in patients with liver metastases [90]. Only a few data are published on ILX-651 developed by Ilex Oncology. Data from a phase I study have been reported showing reasonable tolerability but short plasma half-life [91]. ILX 651 is currently in multicentric phase II studies in advanced melanoma or NSCLC patients with additional trials scheduled.
739
TAXOL, SEMISYNTHETIC ANALOGUES AND TAXOL RESEMBLING COMPOUNDS Paclitaxel (Taxol) (3) is one of the most broadly used as anti-cancer agents with therapeutic value in the treatment of breast, ovarian carcinoma and NSCLC. However, despite high initial response rates to Paclitaxel, many patients relapse and develop drug resistance. Another limitation of Paclitaxel in the clinical use is the poor solubility and the toxicity exerted by its vehicle, i.e. Cremopher EL containing polyoxygenated castor oil [92]. Doxetaxel (Taxotere) (28), a semisynthetic analogue displays considerable activity against several types of solid tumours including those of the breast, lung, head and neck as well as ovary. Compared to Paclitaxel, Doxetaxel has broader activity [93,94]. Having impact on the clinical setting, analogues with a broader efficacy, higher tolerability and no cross-resistance are urgently needed [95]. BMS-184476 and BMS-188797 Data on two of these new taxanes, namely BMS-184476 (29) and BMS188797 (29a), were published by Bristol-Myers-Squibb (BMS), which also developed Paclitaxel [96]. In a comparative preclinical study, both analogues were found to have cytotoxic potency similar to Paclitaxel but overcome two different forms of Paclitaxel resistance. BMS-184476 was found to be clearly superior to Paclitaxel especially on A2780 ovarian carcinoma, HCT/pk, a moderately Paclitaxel-resistant colon carcinoma and L2987 lung carcinoma. BMS 184476 is currently in phase II clinical trials in breast, NSCLC, oesophageal and gastrointestinal cancers [97].
740
X2H2O
OH O
OCOCH
30a RPR 109881A 29 R = CH2SCH3, BMS-184476 29a R = H.BMS-188797
O
31
O
QH
IDN 5109
Fig. 11: Semisynthetic Taxol analogues
TXD-258 and RPR-109881A Two taxane analogues being in clinical development by Aventis Pharma are TXD-258 (30) (phase II) and RPR-109881A (30a) in phase III. RPR109881 was prepared as a single diastereomer by partial synthesis from 10-deacetylbaccatin III, the major natural taxoid extracted from the needles of taxus species. It has been shown to be active against tumours sensitive to Doxetaxel. In tumour models being poorly sensitive to Doxetaxel, the activity of RPR-109881 was similar. Nevertheless, RPR109881 had substantially lower affinity as Doxetaxel for P-gp in highly resistant cell lines and was found to be active in several cell lines moderately resistant to taxoids and Vincristine. In a phase I dose finding study, the recommended dose of RPR-109881A given as a one hour infusion on days 1 and 8 of a 21-day cycle [98] was determined to be 45mg/m2. Currently, phase III studies in metastatic breast Cancer are ongoing (Company info 2004). TXD-258, after i.v. and p.o. administration, has been reported to be effective towards various human tumour xenografts including MDR
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positive and taxane resistant models, and to cross the blood-brain-barrier [99]. TXD-258 is currently in clinical phase II trials, but so far no data have been published. IDN-5109 Substitutions in the 14fl-hydroxy-10-deacetylbaccatin III (14-OHDAB) synthon, a diterpene present in the needles of Taxus wallichiana, led to a new class of taxanes from which IDN-5109 [13-(N-boc-Bisobutylisoserinyl)-14hydroxybaccatin-l,14-carbonate] (31) was selected because of its enhanced antiproliferative activity and lack of cross resistance in tumour cell lines expressing the MDR phenotype [92]. IDN5109 is a poor substrate of P-gp, hence is highly active towards MDR positive cancer cell lines and has oral bioavailability of about 50% [100,101]. IDN-5109 given p.o. was highly active towards the human ovarian carcinoma xenograft 1A9 and HOC 18 in vivo (90-100 % tumour regressions) and showed activity towards the Paclitaxel-resistant MNBPTX1 xenograft (10 % tumour regression) [92]. The oral efficacy of this taxane, likely related to the inability to be a substrate of the P-gp, allowed an adequate intestinal absorption and is a unique feature among the taxanes and presumably a benefit for clinical use [101]. IDN-5109 was first described by Indena, licensed to Bayer AG in March 2000 and is currently in phase II clinical trials in patients with aggressive refractory non-Hodgkin's lymphoma. Recently clinical data on the toxicity and efficacy profile have been published [102] (for review see also [103]). Paclitaxel Prodrugs Further efforts in reducing the dose-limiting side effects of Paclitaxel are reflected by the synthesis of Paclitaxel prodrugs, e.g. (32), designed for bioreductive activation. Blocking the C2'-OH group, which is important for activity, has recently been employed to generate several Paclitaxel prodrugs exhibiting diminished cytotoxicity [104].
NATURAL COMPOUNDS OF DIVERSE STRUCTURE WITH TAXANE-LIKE Several natural compounds of diverse structure, shown in Fig. (12), affecting the taxane-binding site on tubulin are currently developed in clinical trials, namely (+)-Discodermolide, Epothilone B, BMS-247550 (Azaepothilone B, NSC-710428) and Eleutherobin.
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(+)-Discodermolide (XAA-296) The lactone-bearing polyhydroxylated alkatetraene (+)Discodermolide (33), isolated from the sponge Discodermia dissoluta, was described as a stabilising tubulin inhibitor with up to 100 fold higher activity as Taxol [105]. (+)-Discodermolide competitively inhibits the binding of [3H] Paclitaxel (3) to tubulin, is a poor P-gp substrate and effective towards Paclitaxel resistant ovarian carcinoma cells with mutated B-tubulin isotypes [106,107]. In a study using the taxane-resistant NSCLC cell line A549-T12, requiring taxol for normal cell division, (+)Discodermolide could not be substituted for taxol [108]. Further studies showed a strong synergism between Taxol and (+)-Discodermolide, which was a surprise and could not be observed with Epothilones or Eleutherobin. Thus, new combination regimens with (+)-Discodermolide might be feasible for clinical use [106]. (+)-Discodermolide is currently developed in a phase II clinical trial in solid tumour patients by Novartis. CH 3
CH 3
5H 3
CH 3
^
C H z
R^
HO,,,
'''CH 3 33
OH
(+)-Discodermolide
OH
= H,X = O Epothllone A 34a R = CH 3 , X = O Epothilone B 34b R = CH 3 , X = NH BMS-247550
34
H3C
O-Ri H3«
CH
3
"2
O _"A
R, - CH 3 , R2 = o K3 - un Eleutherobin
s ^%
H
° V " TJ^CH
35 (yH 2
,0
36a R-, = H, R2 = COOCH3, R3 = H Sarcodictyin A 16b R-I = H, R2 = COOCH2CH3, R3 = H Sarcodictyin B 36c R1 = H, R2 = COOCH3, R3 = OH Sarcodtatyin A 36d R, = H, Rj = COOCH3, R3 = H Sarcodictyin D
n
• Jrocanoyl 3
!
E E E E Z
Fig. 12: Natural compounds of diverse structure affecting the taxoid site.
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Epothilone B (Patupilone, EpoB) Epothilones A (34) and B (34a) (CGP-47906, EpoB, EPO-906), developed by Novartis, are 16-membered macrolides isolated from the mycobacterium Sorangium cellulosum. Epothilones were originally isolated and structurally resolved by G. Hoefle and colleagues in 1996 [109]. Epothilones A and B act by stabilising microtubules, competing with Taxol for binding to B-tubulin. These agents showed slightly higher in vitro cytotoxicity towards taxane resistant tumour cell lines [107,110]. In contrast to Taxol, Epothilones A and B have no endotoxin like properties [111]. Initial data on a phase I and II studies with EpoB have been published showing alopecia, neuropathy, nausea and diarrhoea as most prominent toxicities [112,113]. In preliminary results from a phase II study, EpoB was well tolerated at 2.5 mg/m2 i.v. [114], improving diarrhoea control which was observed being the DLT [113]. Currently Phase II clinical trials in patients with breast, colorectal, ovarian, NSCLC, renal and ovarian cancer are on-going. BMS- 247550 (Azaepothilone B, NSC-710428, INN: Ixabepilone) BMS-247550 (INN: Ixabepilone) (34c) is a semisynthetic analogue of Epothilone B developed by Bristol-Myers Squibb currently in phase II/III clinical trials. Like Epothilone B, BMS-247550 is a stabilising tubulin antagonist with broad anti-tumour activity [115].The drug is active towards Taxol sensitive and insensitive cell lines, including A2780Tax ovarian carcinoma cells with defined B-tubulin mutations (F270 -» V or A364 -> T; [116]. BMS-247550 has a potent tubulin polymerisation capacity (2,5 fold more potent as Taxol), a broad cytotoxicity (mean IC50 of 3.9 nM) and is efficacious in vivo after i.v. and p.o application [115]. A MTD with neutropenia as DLT (without G-CSF co-treatment) was determined and objective responses in breast and cervical cancer patients refractory to prior taxane therapy were observed [117]. In a clinical trial, the formation of microtubule bundles in peripheral blood mononuclear cells (PBMCs) was monitored and cell death occurred after peak microtubule bundle formation [118]. Eleutherobin The anti-mitotic diterpene Eleutherobin (35) and Sarcodictyins A - D (36a-d) are structurally related, natural compounds isolated from the marine soft coral Eleutherohia spec, and Sarcodictyon roseum,
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respectively [119], whose chemical synthesis has been published [120]. Eleutherobin is a competitive inhibitor of Taxol binding to tubulin, thereby enhancing tubulin assembly and stability. The in vitro cytotoxicity towards human tumour cell lines (IC50 10 nM - 60 nM) is comparable to that of Epothilone A with Sarcodictyins A and B being at least 10 times less potent [119]. Eleutherobin is a substrate of P-gp, whereas there are conflicting results concerning the cross resistance towards Taxol resistant ovarian carcinoma cell lines with the mutated B tubulin isotype M40 [116,119,121]. Eleutherobin, identified by researchers at the Scripps Institution of Oceanography in La JoUa, was licensed to Bristol-Myers Squibb and entered clinical trials but clinical development was not performed. Table 3: Status of several natural / semisynthetic compounds of diverse structure, affecting the taxoid site currently in clinical development Compound Name (Nr.) BMS-188797 (29a) TXD-258 (30) RPR-109881A (30a) IDN-5109
Status in development
IC50-values in vitro
Company
Phase II
Bristol-Myers partial Taxol Squibb resistance Aventis Pharma
Phase III
Aventis Pharma
Phase II
Bayer / Indena
Phase II
(31)
(+) Discodermolide (33) Epothilone B CGP-47906 EpoB / EPO 906 (34a) BMS-247550 Azaepothilone B NSC-710428 (34c) Eleutherobin (35)
Remarks
Phase II
8 - 36 nM
Novartis
Phase II ongoing
0.2 - 0.8 uM
Novartis
Phase II/III
Ceased
Bristol-Myers Squibb
[97] [99] [98, Company info 2004] [92,102, 103]
No P-gp substrate, no Taxol resistance [106,108, Synergism with Taxol, no Taxol Company web page] cross resistance No Taxol cross [107,110-114] resistance
3.9 uM (mean) Bristol-Myers No Taxol cross Squibb resistance
10-60nM
References
No P-gp substrate
[115,118]
[119]
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The abbreviations used are: cdk, cyclin dependent kinase; cki - cdk inhibitor; CEC, circulating endothelial cells; DLT, dose limiting toxicity; GTP, guanosinetrisphosphate; MAP - microtubule associated protein; MDR, multidrug resistance; MRP, multidrug resistance protein; MTD - maximal tolerated dose; NSCLC - non-small cell lung cancer; PBMCs, peripheral blood mononuclear cells; P-gp - P-glycoprotein; SAR, structure-activityrelation; SCLC, small cell lung cancer; SMTI, small molecule tubulin inhibitor; REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
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Atta-ur-Rahman (Ed.) (Ed.) Studies Studies in in Natural Products Products Chemistry, Chemistry, Vol. Vol. 33 33 Atta-ur-Rahman ©2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©
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CHOLESTEROL BIOSYNTHESIS INHIBITORS OF MICROBIAL ORIGIN HYUN JUNG KIM1, IK-SOO LEE1* and SAM SIK KANG2* 'College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea 2 College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul 110-460, South Korea ABSTRACT: Coronary heart disease is the leading cause of morbidity and mortality in the developed countries, which is due to abnormal deposition of lipids in the inner walls of coronary arteries. Higher levels of low-density lipoprotein (LDL) cholesterol are believed to be a major risk factor of coronary heart disease. Thus, inhibition of de novo cholesterol biosynthesis has been known to be one of the most efficient approaches in the regulation of LDL cholesterol levels. Many attempts have been made to find cholesterol biosynthesis inhibitors for development as hypocholesterolemic agents. Microbial secondary metabolites have been used as valuable natural sources in the development of novel cholesterol biosynthesis inhibitors. Mevastatin and lovastatin were isolated from the fungi, Penicillium citrinum and Aspergillus terreus, respectively, as potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which is involved in the rate-limiting step of cholesterol synthesis in mammals. These findings have led to the development of 'statins', which are drugs of choice in the treatment of hypercholesterolemia. HMG-CoA reductase inhibitors have also been shown to decrease the synthesis of other biologically important isoprenoid compounds derived from mevalonate, including ubiquinone and dolichol. And there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Cultured animal cells and intact animals have generally been used for the screening of cholesterol biosynthesis inhibitors. Coverage of the review includes chemical and biological aspects of the cholesterol biosynthesis inhibitors originated from microorganisms and their semisynthetic and biotransformed analogues reported to the present.
INTRODUCTION Cholesterol (C27H46O) (1) is the most widely occurring sterol in all the animal tissues as a constituent of animal membranes. Since it was first isolated from human gallstone, this compound was named "cholesterol" from the Greek word for "bile solids". Cholesterol is synthesized from
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five-carbon isoprene units in the liver [1,2]. The structure of cholesterol is shown in Fig. (1). It constitutes a major part of the membranes of the central and peripheral nervous systems. In addition, it is an important precursor of specific biological products including bile acids, steroid hormones and vitamin D [1,2].
Fig. (1). Structure of cholesterol
The lipids including cholesterol and triglycerides are transported in the blood as part of large molecules called lipoproteins. There are four major families of blood (plasma) lipoproteins. Chylomicrons transport exogenous triglycerides and cholesterol from the gastrointestinal tract to the tissues. They are degraded by lipoprotein lipase and free fatty acids are absorbed in the peripheral tissues. The remnants of chylomicrons are taken up in the liver cells, where cholesterol is stored, or metabolized to the bile acids, or else released into very low-density lipoproteins (VLDLs). VLDLs transport cholesterol and de novo synthesized triglycerides from the liver to the peripheral tissues. VLDL remnants, namely, low-density lipoproteins (LDLs) still contain much cholesterol, some of which are absorbed into the peripheral tissues or taken up in the liver on endocytosis via LDL receptors. High-density lipoproteins (HDLs) absorb cholesterol derived from the peripheral tissues including arteries, and then, transfer it to VLDLs and LDLs. HDLs also are transported to hepatocytes via HDL receptor, SR-B1 [2,3]. Although cholesterol is one of the most important physiological constituents in mammals, higher total cholesterol levels, more specifically, increased low-density lipoprotein (LDL) cholesterol levels are known to be a major risk factor of coronary heart disease [4-7]. Elevated levels of circulating cholesterol, specifically LDL cholesterol, result in the migration and penetration of LDL into the arterial walls, and lead to lipid
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accumulation in smooth muscles and in macrophages (forming foam cells). Accumulated LDLs are oxidized, which also increases proliferation of smooth muscles and deposition of connective tissue components in response to growth factors and cytokines. These deposits result in a disease process called atherosclerosis, which can cause blood clots to form that will ultimately totally stop blood flow. If this happens in the coronary arteries, a heart attack will occur. Coronary heart disease has been the leading cause of death in the developed countries for the past half century [3,8]. The clinical studies have indicated that the lowering of total and LDL cholesterol levels reduces the risk factor of coronary heart disease [4-7]. Endogenous and exogenous pathways determine plasma levels of cholesterol and lipoproteins. Plasma cholesterol levels and coronary heart disease risk can be reduced by decreasing cholesterol biosynthesis in hepatocytes, increasing its catabolism to bile acids, increasing excretion with bile acids, and by reducing its absorption from the intestine [3,6,7,9,10]. The more profound knowledge about cholesterol absorption, biosynthesis and metabolism has allowed the development of several cholesterol-lowering drugs with different mechanisms of action, with the purpose of reducing both morbidity and mortality associated with coronary heart disease [3]. CHOLESTEROL BIOSYNTHETIC PATHWAY Cholesterol is formed biosynthetically from isopentenyl pyrophosphate (active isoprene). The majority of cholesterol in the body derives from de novo biosynthesis in the liver [1,2]. Cholesterol synthetic pathway has been assumed to occur primarily in the cytoplasm and endoplasmic reticulum (ER). However, more recent evidences have suggested that the enzymes, except squalene synthase, squalene epoxidase and oxidosqualene cyclase, are partly localized in the peroxisomes, which are essential for normal cholesterol synthesis [11]. Acetoacetyl-CoA thiolase catalyzes the initial step in cholesterol biosynthesis, the condensation of two molecules of acetyl-CoA into forming acetoacetyl-CoA, which is then followed b y its condensatio n with a third molecule of acetyl-CoA to produce the six-carbon compound HMG-CoA via HMG-CoA synthase [12-15]. HMG-CoA is converted to mevalonate by HMG-CoA reductase. HMG-CoA reductase requires two
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molecules of NADPH as a donor of two electrons during the conversion of HMG-CoA to mevalonate. This reaction catalyzed by HMG-CoA reductase is the rate-limiting step, which is the major point of regulation on the cholesterol biosynthetic pathway [1,2]. In the next step, C-3 and C5 hydroxyl groups of mevalonate are reactivated by successive phosphorylations with three ATP molecules, to yield 3-phospho-5pyrophosphomevalonate. Then, the phosphate group at C-3 and the carboxyl group at C-l are removed, yielding isopentenyl pyrophosphate (IPP), a five-carbon activated isoprene molecule. By isomerization, isopentenyl pyrophosphate is converted into dimethylallyl pyrophosphate (DAPP), another activated isoprene unit. A head-to-tail condensation of IPP and DAPP generates the ten-carbon isoprene, geranyl pyrophosphate (GPP). GPP further undergoes another head-to-tail condensation with IPP, yielding farnesyl pyrophosphate (FPP), the fifteen-carbon isoprenoid. The NADPH-requiring enzyme, squalene synthase, catalyzes the tail-to-tail condensation of two molecules of FPP, forming 30-carbon isoprenoid, squalene. Squalene undergoes a two-step reaction to yield lanosterol. The first step is catalyzed by squalene monooxygenase, which requires NADPH as a cofactor to add one oxygen atom at the end (between C-2 and C-3 positions) of squalene to yield an epoxide. In the next step, squalene-2,3-oxide is cyclized by squalene-2,3-oxide cyclase to lanosterol [1,2]. Lanosterol is converted into cholesterol in a series of nineteen enzyme reactions [18]. The production of cholesterol from lanosterol involves the reduction of the double bond at C-24, demethylations of ge/w-dimethyl at C-4 and a tertiary methyl at C-l4, and isomerization of the double bond from C-8 to C-l. Two major pathways involving the same enzymes have been proposed [16-18]. Cholesterol biosynthetic pathway is outlined in Fig. (2).
755
acetyl CoA
acetaacetylCoA thiolase
acetoacetyl CoA HMG-CoA synthase
3-hydroxy3-methylglutaryI CoA (HMG-CoA)
mevalonate
HMG-CoA reductase
phosphorylation decarboxylatlon
isopentenyl pyrophosphate
isopentenyl adenosine
isomerase dimethylallyl ' pyrophosphate (DAPP)
geranyl pyrophosphate (GPP)
famesyl pyrophosphate (FPP) X2 dolichol, ubiquinone
squalene squalene monooxygenase
squalene-2,3-oxide squalene-2,3-oxide cyclase
lanosterol
756
lanosterol
A24-sterol reductase^
lanosterol 14ademethylase
4,4,14-trimethylcholest8-en-3B-ol lanosterol 14ademethylase
4,4-dimethylcholesta8,14-dien-3p-ol
4,4-dimethylcholesta8,14,24-trien-3p-ol
A14-sterol reductase
4,4-dimethylcholest8-en-3B-o! I t
4,4-dimethylcholesta8,24-dien-3B-ol
C4-methyl sterol oxidase decarboxylase 3-ketosterol reductase
I » I
cholesta-8,24-dien-3p-ol (zymosterol)
cholest-8-en-3B-ol
sterol A8-A7 isomerase
cholest-7-en-3p-ol (lathosterol)
cholesta-7,24-dien-3p-ol A5-desaturase
7-dehydrocholesterol
7-dehydrodesmosterol
A -sterol reductase
desmosterol A24-sterol reductase
cholesterol
Fig. (2). Cholesterol biosynthetic pathway [1,2,16,17]
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INHIBITORS OF CHOLESTEROL BIOSYNTHESIS Inhibition of endogenous cholesterol biosynthesis has been known to be one of the most efficient approaches in the regulation of LDL cholesterol levels. Based on the several screening assay systems on enzymes, cell lines and laboratory animals, a number of cholesterol biosynthesis inhibitors have been isolated and identified from microorganisms as the lead compounds, some of which have been developed as therapeutic agents to regulate cholesterol metabolism. Hydroxymethylglutaryl-CoA (HMG-CoA) Reductase Inhibitors An important class of active agents that potently inhibit HMG-CoA reductase has evolved from extensive studies for microbial secondary metabolites. Since Brown and Goldstein have reported that the rate of cholesterol biosynthesis is determined by the activity of HMG-CoA reductase [19,20,21], this enzyme has been known to be a prime target for discovery of novel therapeutics against hypercholesterolemia. Several fungal secondary metabolites were isolated as useful inhibitors of endogenous cholesterol biosynthesis and developed as commercially available hypolipidemics. Endo and Hasumi have extensively reviewed natural, semisynthetic and synthetic HMG-CoA reductase inhibitors in 1993 [22].
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In 1976, mevastatin (ML-236B, 6-demethylmevinolin) (2) was first reported as a potent competitive inhibitor of HMG-CoA reductase from the culture of Penicillium citrinum [23,24], which is identical with compactin, an antifungal compound isolated from P. brevicompactum [25,26]. Lovastatin (mevinolin, monacolin K) (3) has been isolated from the cultures of Aspergillus terreus [27] and Monascus ruber [28,29], separately.
R= H mevastatin (2) R= CH 3 lovastatin (3)
Fig. (3). Structures of mevastatin and lovastatin
In addition to mevastatin and lovastatin, several other related derivatives showing inhibitory activities against HMG-CoA reductase were isolated from cultures of the fungi mentioned above. ML-236A (4), ML-236C (5) and dihydrocompactin (6) have been isolated from P. citrinum [23,30]. o0H
ML-236A (4)
ML-236C (5)
dihydrocompactin (6)
Fig. (4). ML-236A, ML-236C and dihydrocompactin isolated from Penicillium citrinum
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Monacolins J (7), L (8), M (9) and X (10), dihydromonacolin L (11) and 3a-hydroxy-3,5-dihydromonacolin L acid (12) were isolated from cultures of M. ruber [31-34]. Mevinolinic acid (13) and dihydromevinolin (14) were produced by A. terreus [27,35]. Of them, HMG-CoA reductase inhibitory activities of dihydrocompactin and dihydromevinolin were comparable to those of mevastatin and lovastatin [36].
monacolin L (8)
monacolin J (7)
monacolin M (9)
,,OH
'"OH monacolin X (10)
dihydromonacolin L (11)
3a-hydroxy-3,5-dihydromonacolin L acid (12)
Fig. (5). Monacolins J, L, M, X, dihydromonacolin and 3oc-hydroxy-3,5-dihydromonacolin L acid from Monascus ruber
760
mevinolinic acid (13)
dihydromevinolin (14)
Fig. (6). Mevinolinic acid and dihydromevinolin isolated from Aspergillus terreus
The structural feature of these compounds is the hexahydronaphthalene ring system which is functionalized with a-methylbutyric acid ester and a P-hydroxy-8-lactone linked by an ethylene bridge. The p-hydroxy-8lactone portion of these compounds can be easily opened, and it is converted to the 3',5'-dihydroxyheptanoic acid [22]. This hydroxy acid portion of their structures, which resembles the HMG portion of the HMG-CoA, is responsible for the activity, and it is known to interact competitively with the HMG binding domain of the enzyme active site [37]. A systematic investigation of structure-activity relationship (SAR) has shown that the introduction of an additional aliphatic group on the carbon a to the carbonyl group in a-methylbutyric acid ester linkage of lovastatin increases its potency. This result has led to the synthesis of simvastatin (15) and this modification increased the intrinsic inhibitory activity of lovastatin by 2.5 times [38]. o 0H
simvastatin (15) Fig. (7). Structure of simvastatin, a semisynthetic analogue of lovastatin
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Microbial transformation method along with SAR studies has provided some novel metabolites that inhibit cholesterol biosynthesis. The phosphorylated derivatives (16 and 17) were produced by the conversion using several fungi [39]. Also, L-669262 (18) has been produced by microbial transformation of simvastatin, which is more active than simvastatin, its parent compound [40].
R= H (16) R=CH3(17)
Fig. (8). Microbial transformation products of statins
Pravastatin (CS-514) (19) has been produced by microbial transformation of mevastatin (compactin, ML-236B) [41,42]. Cytochrome P-450sca from Streptomyces carbophilus was described in detail as being responsible for this conversion [43]. It differs from lovastatin in that it contains a P-hydroxyl group at C-6 position of the hexahydronaphthalene ring. This modification makes it more hydrophilic than lovastatin. In
762 762
addition, the p-hydroxy-8-lactone ring has been opened to afford 3',5'dihydroheptanoic acid, the active form. Lovastatin, simvastatin and pravastatin are all widely used in the treatment of hypercholesterolemia and are known to be the first generation statins.
pravastatin (19)
Fig. (9). Structure of pravastatin
Lovastatin (Mevacor™) (3), simvastatin (Zocor™) (15), pravastatin (Pravachol™) (19), atorvastatin (Lipitor™) (20), cerivastatin (Baycol™, withdrawn on August 1, 2003) (21), and fluvastatin (Lescol™) (22) were introduced to lower total cholesterol levels, and especially LDLcholesterol levels to prevent coronary heart disease. These HMG-CoA inhibitors inhibit de novo synthesis of cholesterol in the liver. The ratelimiting enzyme in cholesterol synthesis is HMG-CoA reductase, which catalyzes the conversion of HMG-CoA to mevalonate. The resulting decrease in hepatic cholesterol synthesis leads to increased synthesis of LDL receptors and thus increased clearance of LDL-cholesterol in plasma [44,45]. Recently, two new and more potent statins (also called "superstates") have been studied for their LDL cholesterol-lowering ability, toxicity and drug-drug interaction [46,47]. Rosuvastatin (23) is a highly potent, selective and relatively hydrophilic statin with a low propensity for muscle toxicity and drug interaction. Pitavastatin (24) is known to be another statin with a high oral bioavailability. Since its catabolism is not mediated by cytochrome-P450, it reduces the potential of drug-drug interaction [46,47]. Rosuvastatin was approved for marketing in the U.S.A. in August, 2003 [48].
763 HOOC
,OCH3
COOH OH
atorvastaiin (20)
OH
cerivastatin (21)
OH
OH
COOH
fluvastatin (22)
COOH
COOH
msuvastatin (23)
pitavastatin (24)
Fig. (10). Structures of the synthetic statins
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Squalene synthase inhibitors The findings of HMG-CoA reductase inhibitors resulted in the development of clinically effective hypocholesterolemics. However, HMG-CoA reductase inhibitors have also been shown to decrease the synthesis of other biologically important isoprenoid compounds derived from mevalonate including, ubiquinone, dolichol and isopentenyl f-RNA. Thus, there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Squalene synthase is one of those enzymes involved in the later stages of cholesterol biosynthesis, which has been targeted recently for the development of new therapeutic agents [49]. Screening of a number of fermentation cultures of microorgamsms afforded potent inhibitors of squalene synthase. Squalestatins 1 (25), 2 (26) and 3 (27) were isolated from cultures of Phoma sp. C2932 [50-52]. All of these three compounds possess the hydrophilic core unit, [15(la,3a,4p,5a,6a,7P)]-4,6,7-trihydroxy-2,8-dioxabicyclo-[3.2.1]octane3,4,5-tricarboxylic acid. These compounds showed potent inhibitory activities against rat liver squalene synthase (IC50 = 15.2,15.1 and 5.9 nM, respectively) [52].
OAc
4
X
COOH
OH squalestatin 1 (zaragozic acid A) (25)
OH
OH squalestatin 2 (26)
765 OAc
HOOC HOOC OH squalestatin 3 (27)
Fig. (11). Structures of squalestatins 1-3
Zaragozic acids A (25), B (28) and C (29) were also isolated from the cultures of MF5453 (ATCC 20986), Sporormilla intermedia and Leptodontium elatius, respectively [53-56], Among them, absolute stereostructure of zaragozic acid A is identical to that of squalestatin 1 [53]. These compounds exhibited dose-dependent inhibition of cholesterol biosynthesis as potent competitive squalene synthase inhibitors. Moreover, zaragozic acid A (squalestatin 1) showed inhibitory activity against cholesterol biosynthesis in freshly isolated rat hepatocytes (IC50 = 39 nM), and reduced the serum cholesterol level in mammals including marmosets, mice and rats in vivo [56,57]. It was postulated that zaragozic acids mimic effectively the intermediate presqualene diphosphate in the enzyme reaction catalyzed by squalene synthase [56].
zaragozic acid B (28)
766
OAc
zaragozic acid C (29)
Fig. (12). Sturctures of zaragozic acids B and C
Extensive studies on the structure-activity relationships (SAR) in a series of compounds derived from squalestatins 1 (zaragozic acid A) (25) and 3 (27) have been performed, focusing on the role of the carboxylic acids at C-3, C-4 and C-5 positions of [lS-(la,3a,4p,5a,6oc,7p)]-4,6,7trihydroxy-2,8-dioxabicyclo-[3.2.1]-octane-3,4,5-tricarboxylic acid [5861]. Consequently, the modification of the carboxylic acid at C-5 in squalestatin 1 analogues was not tolerated, but the carboxylic acids at C-3 and C-4 were not absolutely required for the retention of squalene synthase inhibitory activity [58]. In further SAR study for C-3 heterocycle-substituted derivatives of squalestatins 1 and 3 [61], the inhibitory activities of squalestatin 3 analogs showed a greater dependence on the nature of the C-3 substituent, which is different from those of squalestatin 1 analogues. Potent squalene synthase inhibitory activities equivalent to those of squalestatins 1 and 3 were retained in C-3 analogues substituted with a tetrazol-5-yl functionality which closely mimics the parent carboxylic acid (see Table 1). Also, electrostatic potential maps studies showed that squalene synthase inhibitory activity similar to that of the methyl ester (IC50 = 220 nM) was retained only in those C-3 heterocycle-substituted squalestatin 3 analogues for which electrostatic potential maps of the C-3 substituent were closely similar to that of a methyl ester [61]. Squalene synthase inhibitory activities of several analogues substituted with a heterocyclic moiety at C-3 are shown in Table 1.
767 767 Table 1. In vitro inhibitory activities of the synthetic analogues of squalestatin 1 (R! = COOH, R 2 = 4,6dimethyl-2-octenoyl) and squalestatin 3 (Ri = COOH, R 2 = H), substituted with a heterocyclic moiety at C-3, against rat squalene synthase |61]
R2O,
OAc
HOOC HOOC synthetic analogues of squalestatins 1 and 3 ICso (nM)
R2 Ri
R2
4,6-dimethyl2-octenoyl
H
COOH
12
26
COOMe
7
220
R.
A
4,6-dimethyl2-octenoyl
H
92
505
63
-
SyN
Am ri NyN
4
25
mA
-
785
72
310
SyN
146
1181
357
2875
172
1940
57
442
NH 2
Me
A NyO
NHMe
10
147
NHMe
Me 43
156
Me
H N N
N'NH Me
d Aa SH
-
312
768
7"-Hydroxylated analogue (30) of zaragozic acid A was produced from zaragozic acid A by microbial transformation using Streptomyces cyanus ATCC 55214, which inhibited the human squalene synthase in a dosedependent manner with an IC50 of 0.091 nM in HepG2 cells [62].
OAc
Fig. (13). Microbial transformation product of zaragozic acid A
Zaragozic acids D (31) and D2 (32) were further isolated from cultures of the keratinophilic fungus Amauroascus niger as potent inhibitors of rat liver squalene synthase with IC50 values of 6 and 2 nM, respectively. Structures of zaragozic acids D and D2 are related to those of the previously described zaragozic acids A, B and C, which contain 4,6,7trihydroxy-2,8-dioxabicyclo~[3.2. l]octane-3,4,5-tricarboxylic acid core unit and hydrophobic alkyl and acyl side chains [63].
OH zaragozic acid D (31)
769
OH zaragozic acid D 2 (32)
Fig. (14). Zaragozic acids D and D2 isolated from Amauroascus niger
Four zaragozic acid derivatives, F-10863 A (33), B (34), C (35) and D (36), were recently isolated from culture broths of the fungus Mollisia sp. SANK 10294. These compounds also contained a 4,6,7-trihydroxy-2,8dioxabicyclo-[3.2.1]octane-3,4,5-tricarboxylic acid core like previously reported zaragozic acid compounds. However, they contained two hydrophobic acyl side chains. It was found that F-10863 A is identical to zaragozic acid D3 (33) which was produced by Libertella sp. [54]. All the four compounds exhibited potent inhibitory activities against rat liver microsomal squalene synthase (IC50 values of 0.7, 1.3, 1.6, 2.0 nM, respectively) in a dose-dependent mode. Also, the compounds F-10863A and B showed inhibitory activities against sterol biosynthesis in freshly isolated rat hepatocytes (IC5o= 6.7 and 11 nM). Furthermore, F-10863A exhibited in vivo serum cholesterol-lowering activity in marmosets and hamsters [64].
F-10863A(33) (zaragozic acid D3)
770
F-10863B(34)
F-10863C(35)
Fig. (15). Structures of zaragozic acids F-10863A through D isolated from Mollisia sp.
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Isolation and structure elucidation of a novel family of cholesterollowering constituents, viridiofungins A (37), B (38) and C (39), from Trichoderma viride, were reported and they were potent in vitro inhibitors of squalene synthase (IC50 values = 41.6, 19.3 and 0.29 |a.g/mL) in HepG2 cells. These compounds also showed broad-spectrum and potent antifungal activities [65,66].
HO
viridiofungin A (37)
H3CO
O H3CO
viridiofungin B (38)
772
viridiofungin C (39) Fig. (16). Structures of viridiofungins A through C from Trichoderma viride
Bisabosqual A (40) was isolated from the culture broth of Stachybotrys sp. RF-7260. Its structurally related compounds, bisabosquals B (41), C (42) and D (43) were also isolated from Stachybotrys ruwenzoriensis RF6853. All the bisabosqual compounds inhibited squalene synthases of microbial and mammalian origins. Bisabosqual A showed significant inhibitory activity against the microsomal squalene synthases from HepG2 cells and rat liver. Its IC50 values were 0.95 and 2.5 JJ. g/mL, respectively. Bisabosqual A also showed broad-spectrum antifungal activity in vitro. Bisabosqual C exhibited inhibitory activities similar to those of bisabosqual A [67,68].
....OH O
OHC CHO bisabosquat A (40)
CHO bisabosqual B (41)
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H
t •
H1
OHI 0
OHC
OHC
CHO
CHO
Disabosqual C(42)
bisabosqual D (43)
Fig. (17). Structures of bisabosquals A-D from Stachybotrys sp.
CJ-15,183 (44) has been isolated from the fermentation culture of the fungus, Aspergillns aculeatus CL38916 as a squalene synthase inhibitor. The compound potently inhibited rat liver and human squalene synthases. In addition, it showed antifungal activities against filamentous fungi and yeast. The structure was elucidated to be an aliphatic tetracarboxylic acid compound consisting of an alkyl y-lactone, malic acid and isocitric acid moieties by spectroscopic analyses [69].
o COOH HOOC'
Y
O
COOH
CJ-15,183 (44)
Fig. (18). Structure of CJ-15,183 from Aspergillus aculeatus
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CJ-13,981 (45) and CJ-13,982 (46) were isolated from the fermentation broth of CL15036, an unidentified fungus, as new squalene synthase inhibitors. They inhibited human liver microsomal squalene synthase with IC50 values of 2.8 and 1.1 uM, respectively. Based on the spectroscopic analyses, the structures of CJ-13,981 and CJ-13,982 were elucidated to be highly carboxylated fatty acids, 3-hydroxy-3,4dicarboxy-15-hexadecenoic acid (45) and 3-hydroxy-3,4-dicarboxyhexadecanoic acid (46), respectively [70],
COOH
CJ-13,981 (45)
COOH
CJ-13,982 (46)
Fig. (19). Structures of CJ-13,981 and CJ-13,982 isolated from CL15036
Recently, a Streptomyces species microorganism that produces squalene synthase inhibitors was screened from soils, and two active inhibitors were isolated from its culture broth. Structures of these inhibitors were identified as macrolactins A (47) and F (48) on the basis of spectral analyses. The IC50 values for macrolactin A were 0.11, 1.66, and 1.08 uM for the squalene synthases of pig liver, rat liver and Hep G2 cells, respectively. Macrolactin F also showed significant inhibitory activities with IC50 values of 0.14, 1.53, and 0.99 uM, respectively. Kinetic results for macrolactins A and F showed that they appear to be
775
non-competitive inhibitors of rat liver squalene synthase. However, both macrolactins A and F exhibited no inhibitory activity against the squalene synthase of the fugus Candida albicans [71],
macrolactin A (47)
macrolactin F (48)
Fig. (20). Structures of macrolactins A and F
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Cholesterol Biosynthesis Inhibitors Screened by Using Cell Lines A family of 10-membered lactones was detected by chemical screening for inhibition of cholesterol biosynthesis. Taxonomic studies and fermentation conditions of the screened microorganisms have demonstrated that they belong to the species Penicillium simplicissimum and Penicillium corylophilum [73]. Decarestrictines A (49), B (50), C (51) and D (52) were isolated as active constituents. In vitro tests using the HepG2 cell assay showed the decarestrictines to be inhibitors of cholesterol biosynthesis [72,73], which exhibited inhibition effects of about 40%, 20%, 30% and 50% in the concentration of 100 nM, respectively. Decarestrictines were highly selective in that they exhibit no significant antibacterial, antifungal, antiprotozoal and antiviral activities [74]. Hypolipidemic activity of decarestrictine D was also confirmed in rats in vivo [72,73]. Of them, decarestrictine D (tuckolide) was selected for further study. Toxicity studies revealed that decarestrictine D displays good tolerability, showing a lack of change in a standard set of defined safety parameters [75]. Synthetic studies of this compound have been performed to provide a clue for the design of new cholesterol biosynthesis inhibitors [73-75].
OH decarestrictine A (49)
OH
o
decarestrictine B (50)
HO1" OH decarestrictine C (51)
OH decarestrictine D (52) (tuckolide)
Fig. (21). Decarestrictines A-D
777 777
The purification, structure elucidation, and antihyperlipidemic activities of nine compounds of the decarestrictine family, descarestrictines E to M (53-61), of P. simplicissimum were also reported. All of the compounds exhibited inhibitory effects on cholesterol biosynthesis in HepG2 liver cells in vitro [76].
O
H 3 CO
0
decarestrictine E (53)
OH decarestrictine H (56)
OH decarestrictine K (59)
0
HO decarestrictine F (54)
OH decarestrictine I (57)
decarestrictine G (55)
OH decarestrictine J (58)
OH decarestrictine L (60)
Fig. (22). Decarestrictines E-M
decarestrictine M (61)
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Enzyme inhibition assays in vitro led to the isolation and synthesis of a variety of therapeutic agents and biologically active constituents as potent inhibitors of cholesterol biosynthesis [22,49]. Also, the cultured human liver cell lines including HepG2 and Chang liver cells have been traditionally used as an effective model for screening of cholesterol biosynthesis inhibitors from natural sources including plant materials, foods and microorganisms [77-81]. The methods using human liver cells have been shown recently to provide valuable profiles for the evaluation of inhibition mode against cholesterol biosynthesis based on the analysis of incorporation and distribution of radio-labeled precursor into nonsaponifiable lipids [80,81]. In a more recent search for new cholesterol biosynthesis inhibitors from microorganisms, a modified assay system was established in vitro to screen cholesterol biosynthesis inhibitors using Chang liver cell line [82,83]. Of a total of 83 microbial EtOAc extracts screened, the EtOAc extract of the fungus Hormoconis resinae showed significant inhibitory activity in the in vitro assay system. Bioactivity-guided fractionation of the extract led to the isolation of a cholesterol biosynthesis inhibitor, and the structure of this compound was elucidated as a new ergostane-type steroidal analogue, 3 P-hydroxy-1,11 -dioxo-ergosta-8,24(28)-diene-4aearboxylic acid (62) [84].
27
(62)
Fig. (23), Structure of 3p-hydroxy-l,l l-dioxo-ergosta-8,24(28)-diene-4a-carboxylic acid isolated from Hormoconis resinae
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Details of its structure elucidation and biological activity are presented herein. The [M+H]+ peak at mlz 471,3118 (calcd. mlz 471.3110) shown in HR FABMS of 62 was assigned the molecular formula. The TH and 13C NMR spectra showed an exomethylene signal [8H 4.67 (brs), 4.73 (d, J=1.0 Hz); 8 C 107.0,157.1], two tertiary methyl signals [8H 0.71, 1.51; 8C 12.3, 19.8], a secondary methyl [S H 0.96 (d, J=6.5 Hz); 8C 18.9], two isopropyl methyl signals [8H 1.02 (d, J=7.0 Hz), 1.03 (d, J=7.0 Hz); 8C 22.3, 22.4], a hydroxymethme [SH 3.95 (ddd, J=6.3, 10.3, 11.0 Hz); 8C 75.3] and a tetrasubstituted double bond [8C 136.1,160.7,201.0], a ketone [Sc 211.8], and a carboxyl group [8c 180.8]. These findings suggested that this compound is an oxygenated tetracyclic steroid analogue. The structure of 62 was established by analysis of HMBC and " H - ' H COSY spectral data. The proton signals of isopropyl group at H-25, H-26 and H-27, and the methylene protons at H-23 showed correlations with the exomethylene signals at C-24 and C-28. The methylene protons at H-2 were correlated to the carbons at C-l, C-3 and C-10. The methine protons at H-3 and H-4 showed long-range couplings with the carboxyl group at C-29. The proton signals at H-7p\ H-14 and H-12 were respectively correlated to the carbons at C-8 and C-9 and C-ll in a conjugated enone. Also, H-12, H-14 and H-17 protons showed correlations with C-18. Another tertiary methyl, H-19 was correlated to the C-l, C-5, C-9 and C10 carbons. The secondary methyl protons at H-21 also showed longrange couplings with C-l7, C-20 and C-22 carbons. The connectivities of C-14, C-15, C-16 and C-17 were clarified by the 'H-'H COSY correlations. The stereochemistry of 62 was determined by NOESY and selective NOE correlations, and unambiguous structure of this compound was assigned to be 3p-hydroxy-l,ll-dioxo-ergosta-8,24(28)-diene-4acarboxylic acid as shown in Fig (23). The compound exhibited moderate to significant cholesterol biosynthesis inhibitory activity with an IC50 value of 8 ng/mL when compared with that of mevastatin (8 ug/mL), a commercially available HMG-CoA reductase inhibitor. However, this compound showed an inhibitory profile different from that of mevastatin. Analysis of the extracts of cells treated with 62, revealed the presence of accumulated lanosterol in the sterol fractions as evidenced by TLC, whereas no labeled sterol intermediate was detected in case of mevastatin under the same condition.
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CONCLUSIONS Inhibition of cholesterol biosynthesis constitutes an important strategy to the lowering of higher blood total and LDL cholesterol levels. Several in vitro assay systems have been used as screening methods for developing novel leads for cholesterol biosynthesis inhibitors. Two major enzyme inhibition assays, targeting for HMG-CoA reductase and squalene synthase, respectively, have led to the isolation and synthesis of a variety of therapeutic agents and biologically active constituents as potent inhibitors of cholesterol biosynthesis. The findings of HMG-CoA reductase inhibitors resulted in the development of anti-hypercholesterolemic agents that could effectively lower the serum LDL cholesterol level. Microorganisms have been used as valuable sources for developing therapeutic agents to regulate the cholesterol metabolism. An important class of active agents that potently inhibit HMG-CoA reductase has evolved from extensive studies for microbial secondary metabolites. Lovastatin, simvastatin and pravastatin are all widely used in the treatment of hypercholesterolemia and are known to be the first generation statins. On the other hand, HMG-CoA reductase inhibitors have also been shown to decrease the biosynthesis of other biologically important isoprenoid compounds derived from mevalonate. Thus, there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Zaragozic acid derivatives were isolated from several microbial species and they exhibited dose-dependent inhibition of cholesterol biosynthesis as potent squalene synthase inhibitors. Viridiofungins, bisabosquals, macrolactins, CJ-15,183, CJ-13,981 and CJ-13,982 were also isolated as mammalian squalene synthase inhibitors of microbial origin. In addition, 10-membered lactone compounds, namely, decarestrictines, were identified by chemical screening for inhibition of cholesterol biosynthesis using in vitro and in vivo assay systems. 3(3-Hydroxy-l,lldioxo-ergosta-8,24(28)-diene-4a-carboxylic acid was isolated as an inhibitor of cholesterol biosynthesis using human Chang liver cell system.
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Atta-ur-Rahman (Ed.) (Ed.) Studies Studies in Natural Products Products Chemistry, Chemistry, Vol. 33 33 Atta-ur-Rahman © 2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©
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STRUCTURE-ACTIVITY RELATIONSHIPS OF CURCUMIN AND ITS ANALOGS WITH DIFFERENT BIOLOGICAL ACTIVITIES1 LI LIN and KUO-HSIUNG LEE* Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill Chapel Hill, NC27599-7360, U. S. A. ABSTRACT: Curcumin is a constituent of the yellow pigments isolated from Curcuma longa, which is commonly named turmeric and is the major ingredient of the spice curry. Curcumin possesses many bioactivities, such as anti-oxidant, anti-inflammatory, antiviral, antifungal, cancer chemopreventive, and cancer chemotherapeutic properties. This review paper is prefaced by a general introduction to the origin and bioactivity of curcumin, followed by general synthetic routes to curcumin and its analogs. The major part of this paper examines the current structure-activity relationship studies of curcumin and various curcumin analogs relating to different bioactivities and distinct biological targets of interest to scientists worldwide. Finally, the advantages of curcumin and related compounds, which have multiple biological targets in cancer treatment, are discussed.
INTRODUCTION Plants of the Zingiberaceae family have been used as spices and indigenous medicine in Asia for thousands of years. The rhizome of Curcuma longa (Zingiberaceae), which is commonly named turmeric, is used as a spice (e.g., curry), flavoring agent, food preservative, coloring agent, and medicine for treatment of inflammation and sprain in India, China, and other Asian countries. Curcumin (1) [diferuloyl methane; 1,7bis-(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione] is the major constituent of the yellow pigments isolated from Curcuma longa (Zingiberaceae) and other Curcuma species. The main components of turmeric include curcumin, demethoxycurcumin (2), and bisdemethoxycurcumin (3), together referred to as curcuminoids, Fig. (1). Curcumin was first isolated in 1870. Its chemical structure was determined • To whom correspondence should be addressed. Tel: (919) 962-0066. E-mail:
[email protected] f Antitumor Agents 241.
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in 1910 [1] and subsequently confirmed by synthesis. Curcumin has a unique conjugated structure including two methoxylated phenols and the enol form of a heptadiene-3, 5-diketone linking the two phenols, giving a bright yellow color. OH
o OCH,
2 Demethoxycurcumin OH
OH
O
3 Bis-demethoxycurcumin
Fig. (1). Structures of curcuminoids in Curcuma longa
Over the last few decades, curcumin has received increasing interest from researchers worldwide. In addition to its well-known antiinflammatory application, curcumin has been found to possess multiple therapeutic effects, such as reducing blood cholesterol, preventing LDA oxidation, inhibiting platelet aggregation, suppressing thrombosis and myocardial infarction, suppressing symptoms associated with type II diabetes, rheumatoid arthritis, multiple sclerosis and Alzheimer's disease, inhibiting HIV replication, enhancing wound healing, increasing bile secretion, protecting from liver injury, cataract formation, and pulmonary toxicity and fibrosis, showing antileishmaniasis and anti-atherosclerotic properties, as well as preventing and treating cancer [2]. Besides its numerous reported therapeutic effects, curcumin is non-toxic even at high dosages. It has been classified as "generally recognized as safe" (GRAS) by the National Cancer Institute [3]. Many curcumin analogs have been developed from the lead compound curcumin based on structure-activity relationship (SAR) studies and optimization of compounds as drug candidates.
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Curcumin has been reviewed from the aspects of its biology and mechanisms of action [2, 4]. In this paper, a comprehensive review will be presented with respect to SAR of curcumin and its analogs regarding different bioactivities.
GENERAL SYNTHETIC ROUTES TO CURCUMIN AND ITS ANALOGS Certain curcumin analogs differ structurally from curcumin only in their phenyl ring substitutions. The general synthetic strategy for this analog series is the condensation of substituted benzaldehydes with 2,4pentanedione [5], Fig. (2). Boron oxide is used in this condensation reaction to form a boron complex with 2,4-pentanedione and protect C-3 from Knoevenagel condensation. Other analogs are l,5-diphenyl-l,4diene-3-ones, which are synthesized by coupling aromatic aldehydes with acetone or cyclic ketones under acidic conditions [6].
o
o
0,7 eq, B 2 O 3
sV 0
Vp
o
o
OH
1.2eq. ArCHO, 2 eq. 2- i. 5 eq.BuNH 2 3 . HC1
O
R'
0 5 eq
HC1
R
(n= 0,1,2)
O
R' ^
HC1
R' ^
v
R
Fig. (2). General synthetic routes to curcumin and selected analogs [5]
STRUCTURE-ACTIVITY RELATIONSHIPS DIFFERENT BIOLOGICAL ACTIVITIES
(SARs)
FOR
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Curcumin exhibits diverse biological activities and has numerous biological targets [2]. Various research groups have studied the SAR of curcumin and its analogs with respect to different activities, hi this part of the review, the relationships between the structures of curcumin and its analogs and their targeted activities will be discussed and summarized. Anti-inflammatory activity Turmeric has been used for the treatment of inflammation for thousands of years in Asian countries. The active constituents were found to be curcuminoids, including curcumin as well as its two natural analogs, demethoxycurcumin and bisdemethoxycurcumin [7]. Demethoxycurcumin showed the most potent anti-inflammatory activity among these three natural curcuminoids [8]. According to present studies, curcumin acts by diverse anti-inflammatory mechanisms and at several sites along the inflammation pathway, which are summarized in Fig. (3) [9]. Stimulus
J J-
Cell membrane disruption Phospholipase )
COX II | —
|—
NO | —
Lipo-oxygenasi — | LOXI
Leukotnene
T"^
Prostaglandins
T
| Thromboxane
T
Prostacylin
T
Collagenase, elastase, hyaluronidas MCP1, Interferon-inducible protein, TNF, IL-12 I—
: Indicates inhibition by/ (curcumin
Fig. (3). Sites of action of curcumin along the inflammation pathway [9] MCP-1, monocyte chemoattractant protein-1; COX-II, cyclo-oxygenase-II; TNF, tumor necrosis factor; IL-12, interleukin-12.
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Sodium curcuminate, tetrahydrocurcumin, and curcumin were reported to have greater anti-inflammatory activity than phenylbutazone at low doses using carrageenin-induced rat paw edema and cotton pellet granuloma assays [10]. Various semi-synthetic analogs of curcumin, Fig. (4), were screened for anti-inflammatory activity using the same animal assay [11, 12]. Diacetylcurcumin (5), diacetoxycurcumin (10), and tetrabromocurcumin (4) were the most potent analogs among those studied. The study concluded that the presence of the j3-diketone moiety is important for the anti-inflammatory activity and addition of alkyl groups at ortho positions of the phenyl rings decreased potency. OH
O
Br
O
O
Br
H,C
H,CO AcO
HO
Fig. (4). Curcumin and semi-synthetic curcumin analogs tested for anti-inflammatory activity [11, 12]
Nurfina et al. designed and synthesized symmetrical curcumin analogs [13]. Anti-inflammatory activity was evaluated by inhibition of the carrageenin-induced swelling of rat paw. The structures of these synthetic analogs and their inhibitory potencies are shown in Table 1. The rank order of their potencies was 14 > 15 > 24 > 1 (curcumin) > 21 > 16 > 3 > 20 > 19. Other analogs showed no anti-inflammatory activity in this assay. Based on comparison of the structures and potencies, the following SAR conclusions were made: 1) The unsubstituted compound 11 showed little inhibitory activity. Therefore, appropriate substitutions on the phenyl rings are necessary for the anti-inflammatory activity. 2) Proper substitution at para positions of the phenyl rings is also crucial. Analogs without para substitution (11, 12,and 17) did not show anti-inflammatory activity. A para phenolic hydroxy group is essential for potent anti-inflammatory
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activity, as analogs without the para phenolic hydroxy group exhibited weak or no anti-inflammatory activity. 3) Likewise, substitution at positions ortho to the phenolic hydroxy group is important for the activity. Di-methyl substitution (14) enhanced the activity most, followed by diethyl (15), di-methoxy (24), and then di-isopropyl (16). However, ditetrabutyl substitution (22) diminished the anti-inflammatory activity, probably due to its bulkiness. Table 1. Curcumin derivatives tested in an anti-inflammatory bioassay [13] R2
_Comgmind___ 1 (curcumin) 3 11 12 13 14 15 16 17 IS 19 20 21 22 23 24
R2 H H H H H
H H H OCH3 H H H H H H
H
R3 OCH3 H H OCH, OCH3 CH3 CH 2 CH 3 i-C 3 H 7 H H
H H OCH3 t-C 4 H 9 E OCH3
OH
O
R4 OH OH H H OCH3 OH OH OH H Cl OCH3 CH3 OCH3 OH E OH
R2
RS
H H H H OCH3 CH3 CH2CH3 i-C3 H7 H H H H H t-G,H 9 H OCH3
__EDsoJimg/^}
38±4 73 ±5 — — --
13+2 22 ±6 58 ±21 — — 82 ±7 80 ±18 50 ±22 —
28±5
Cyclovalone (25), which differs from curcumin in the linker between the two phenyl rings, and three analogs showed anti-inflammatory activity based on inhibition of the enzyme cyclooxygenase [14], Fig. (5). Curcumin was used as a reference, and compounds 26, 27, and 28 were more potent than curcumin. Compounds 26 and 28 showed greater inhibition than compounds 25 and 27, respectively. Therefore, in this series, di-methyl substitution enhanced the ability to inhibit cyclooxygenase more than mono-methoxy substitution. Compound 25 showed greater inhibitory activity than compound 27; however compounds 26 and 28 had similar activity. Hence, the authors concluded that modification of the cyclic ketone from cyclohexanone to
791
cyclopentanone increased the inhibitory activity if the phenyl substitution was methoxy, but had no significant effect on analogs with di-methyl substitution.
H,CO OH
HO CH,
Fig. (5). Cyclovalone and its three analogs [14]
hi addition to curcumin, many other constituents in the Zingiberaceae family possess anti-inflammatory activity [15]. Some of these constituents, such as yakuchinones A and B isolated from Alpinia oxyphylla Miquel [16], Fig. (6), are diarylheptanoids and structurally similar to curcumin. H,CO
29 Yakuchinone A
30 Yakuchinone B
Fig. (6). Structures of yakuchinoine A and yakuchinone B [16]
Anti-oxidant activity Many of the therapeutic effects of curcumin are attributed to its strong antioxidant property. Most natural antioxidative compounds can be classified into two types: phenolic compounds and /S-diketone compounds [17]. Sesaminol from sesame belongs to the former type, and ntritriacontan-16,18-dione from the leaf wax of Eucalyptus belongs to the latter type. However, few antioxidative substances possess both phenolic hydroxy and /3-diketone groups in one molecule, and curcumin has both features.
792
Curcumin has both antioxidant and pro-oxidant effects in oxygen radical reactions, acting as a scavenger of hydroxy radicals or a catalyst in the formation of hydroxy radicals, depending on the experimental conditions [18-20]. The antioxidant effect of curcumin presumably arises from scavenging of biological free radicals. The antioxidative activity of the three natural curcuminoids and their hydrogenated analogs, Fig. (7), were examined in three antioxidative bioassay models, the linoleic acid auto-oxidation model, rabbit erythrocyte membrane ghost system, and rat liver microsome system. The results obtained from the three models were consistent, and revealed that curcumin was the strongest antioxidant among the natural curcuminoids and tetrahydrocurcumin had the strongest antioxidative activity among the hydrogenated curcuminoids [17]. Among all six compounds, tetrahydrocurcumin showed the highest potency, implying that hydrogenation of curcumm and demethoxycurcumin increased their antioxidative ability. Absence of one or both methoxy groups resulted in decreased antioxidant activity in both natural curcuminoids and tetrahydrocurcummoids. A second paper also reported that the presence of methoxy groups in the phenyl rings of curcumm enhanced the antioxidant activity [21]. OH
O OCH3 HjCO
HjCO.
HO"
1 Curcumm OH
OH
OCH,
HO
OH
31 Tetrahydroureumin O
O
O
H 3 CO.
HO'
2 Demethoxycurcumin OH
HO
32 Tetrahydro-demethoxycurcumin O
O
3 Bis-demethoxycurcumin
OH
HO
v
O
v
"OH
33 Tetrahydro-bis-demethaxyeurcumin
Fig. (7). Curcuminoids and tetrahydrocurcuminoids
Venkantesan et al. [22] used three models to investigate the importance of the phenolic hydroxy groups, as well as other substituents in the phenyl rings of curcumin and curcumin analogs, to antioxidant
793
activity. The curcumin analogs used in the bioassays are shown in Table 2. The three antioxidant bioassay models were inhibition of lipid peroxidation, free radical scavenging activity by the DPPH method, and free radical scavenging activity by the ABTS+* method. Table 2. Chemical structures of curcumin analogs [22] OH
O
QO OCH,
31 Tetrahydrocurcumin
Cmpd
Ri
R2
R3
Lipid peroxidation inhibition IC50 (jiM)
DPPH scavenging IC50 0«M)
ABTS*'scavenging TEAC 3 min
1 3 5 11 13 14 19 20 21 22 31 34
IS min
20.02 1.30 3.37 2.61 3.09 32.08 3.04 2.19 4.96 4.31 1.33 2.01 NA 1.85 2.33 >250 NA 3.36 1.57 2.78 NA 1.90 2.98 15.32 3.43 0.89 21.75 1.28 1.13 0.63 2.14 2.04 NA >250 2.05 0.67 1.52 NA >250 1.95 >250 2.67 1.86 2.49 NA 23.72 t-C 4 H 9 1.07 0.81 0.98 6.48 t-C 4 H 9 18.22 2.52 2.08 2.37 1.83 3.32 2.36 3.07 30.32 OH OEt H 1.11 NA till 90 35 H 1.09 NA SCH 3 H --IC50 is the concentration required for 50% inhibition of lipid peroxidation or scavenging of DPPH radical. TEAC is the trolox equivalent antioxidant capacity, which is defined as the mM concentration of a trolox solution having the antioxidant capacity equivalent to a 1.0 mM solution of the substance under investigation. OCH3 H OCH3 H OCH3 CH3 H H OCH3
OH OH OCOCH3 H OCH3 OH OCH3 CH3 OCH3 OH
9 min
H H H H OCH3 CH3 H H H
In all the three models, the phenolic curcumin analogs were more potent than the non-phenolic analogs. This result indicates that the phenolic groups are important to the antioxidant activity. The presence of methoxy groups in curcumin increased antioxidant activity in all the three models, which was in agreement with previous reports. Ethoxy substitution also enhanced the activity to some extent. Compound 14, which has two methyl groups at ortho positions relative to the phenolic hydroxy groups, showed the highest inhibitory activity in the lipid peroxidation model. However, compound 22, with bulkier ^-butyl groups flanking the phenolic group, was ten-fold less active than compound 14.
794
The decreased potency of 14 probably resulted from the offsetting influences of the electron donating and steric effects. In the three models, tetrahydrocurcumin showed comparable activity with curcumin, which implies that the enhanced electron delocalization of the double bonds may not be essential in terms of curcumin's antioxidant activity in these three bioassay models. l,5-Diphenyl-l,4-pentadiene-3-ones and cyclic analogs were synthesized by Sardjiman et al. [23]. The structures and antioxidant activity (inhibition of lipid peroxidation) of these analogs are shown in Table 3. Table 3. 1, 5-Diphenyl-l,4-pentadiene-3-ones and cyclic analogs and antioxidant activities [23]
Compound 25 26 36 37 38 39 40 41
OCH3 CH3 H
R2 H CH3 H
C2H5 i-C 3 H 7 t-C 4 H 9 OCH3
C2H5 i-C 3 H 7 t-C 4 H 9 OCH3
Cl
Cl
Ri
Lipidjjcroxidation inhibition ICso^yM)^ >4 2.8 ±0.0 » 4
2.0 ±0.3 4.4 ± 1.1 » 4
1.6 ±0.4 Inactive 11.0 ± 1.3
Curcumin
Compound 27 28 42 43 44 45 46 47
R, OCHj CH3 H
R2 H CH3 H
C2H5 i-C 3 H 7 t-C 4 H 9 OCH3
C2H5 i-C 3 H 7 t-C 4 H, OCH3
2.2 ±0.2
Cl
Cl
» 4
Anti-oxidant activity IC 5 0 (|JM)
>4 2.5 ±0.2 » 4 >4
» 4 0.9 ±0.2
795
Compound 48 49 50 51 52
•
Ri
Rj
H OCH3 CH3 OCH3 Cl
H H CH3 OCH3 Cl
Anti-oxidant activity IC50((J.M) »4 >4 •
1.3 ±0.4 2.9 ±0.4 Inactive
Compared with the structure of curcumin, these synthesized analogs retain para hydroxy groups in the phenyl rings but lack one carbonyl group and methylene group in the linker between the phenyl rings. The bioassay results revealed the following SAR information. 1) Electron withdrawing substitutions in the phenyl rings (41, 47, and 52) resulted in loss of antioxidative activity. 2) Bulky alkyl substituents (e.g. isopropyl, fe/t-butyl) at ortho positions relative to the phenol groups (38, 39, 44, and 45) retarded the antioxidative activity. 3) Small alkyl groups (e.g. methyl, ethyl) and electron donating groups (e.g. methoxy) at ortho positions (26, 37, 40, 28, 43, 46, 50, and 51) enhanced the activity. Dimethoxy substitution potentiated the antioxidative activity more than monomethoxy substitution. Several analogs were more potent than curcumin. Therefore, the truncation of the linker did not result in decreased antioxidative activity, but rather, enhanced the activity. Recently, Youssef et al. [24] reported the synthesis of curcumin analogs as potential antioxidant and cancer chemopreventive agents. The general structures of the synthesized analogs are shown in Fig. (8). These compounds were tested for scavenging ability of DPPH free radicals and in an ATP chemiluminescence assay. The SAR conclusions from the results were mainly consistent with the prior conclusions drawn by other researchers. In addition, they found that di-substitution of the central methylene group resulted in decreased antioxidative activity.
796
R'
X R R' R"
XO R"
= = = =
H, CH 3 CH 3 , C 2 H 5 , C3H7 H, CH 3 , OCH3 H, CH 3 , OCH 3 , OC 2 H 5
R"
Fig. (8). General structures of curcumin analogs synthesized by Youssef et al. [24]
The antioxidant mechanisms of curcumin have been under extensive investigation. The predominant conclusion is that curcumin is a classical phenolic chain-breaking antioxidant, which donates H atoms from the phenolic groups [25-30]. However, some conflicting results suggest that H atom donation takes place at the active methylene group in the diketone moiety [31, 32]. Ligeret et al. published the latest report [33]. They evaluated the effects of curcumin derivatives (Table 4) as well as curcumin on the mitochondrial permeability transition pore (PTP), which can release apoptogenic factors from mitochondria to induce apoptosis. The authors postulated that PTP opening is closely related to the antioxidant property of curcumin. From the data on mitochondria swelling, O2* and HO* production, thiol oxidation, and DPPH* reduction, the authors concluded that the phenolic hydroxy groups are essential for activity and are more effective at the para position than at the ortho position, hi addition, an electron-donating group at the ortho position relative to the phenolic group is also required for activity, but bulky substituents, such as f-butyl, are not favorable. In contrast, electron-withdrawing substitution, such as NO2, reduced activity. Based on the observations that antioxidant activity was attenuated when the P-diketone moiety was replaced by a cyclohexanone ring, but was not found with ferulic acid, they concluded that the p-diketone could not induce, but did contribute to the activity of curcumin derivatives. Their conclusions agree with the prevailing SAR for antioxidant activity.
797
Table 4. Structures of curcumin derivatives [33]
o
Compound 1 (Curcumin) 3 11 13 22 24 53 54 55 56 57
o
o
R2 OH OH
Rl OCH3 H H OCH3 t-C4H, OCH, OH
H
OCH3 OH OH OCH3 OCH2O OC4H, OH
OCH3 NO2
R3 H H H OCH3 t-C 4 H 9
OCH3 H H H H
NH
HN
Compound 25 36 39 40 58 59 60 61 62 63 64
o
R2 OH OH OH OH H OCH3
R3 H H t-C4H, OCH3 H
OCH3
OCH3
OCH3
OCH3 NO2
OC4H, OH
H H
Rl OCH3 H t-C4H, OCH3 H OH OCH2O
H H
However, a curcumin analog without phenolic and methoxy groups was reported to be as active as curcumin in terms of scavenging hydroxy radicals and other redox properties [34]. Considering the variety of test free radicals, solvents, and pH ranges used in the literature, Wright employed theoretical chemistry to interpret the controversy [35]. First, he explored the stable conformer of curcumin,
798
pointing out that the enol form is the most stable, followed by the transdiketo form, and then the cw-diketo form, Fig. (9). Calculations showed that the phenolic O-H is the weakest bond in the curcuminoids. This theoretical result favors the necessity of a phenolic OH group for the antioxidant activity of curcumin and its analogs. However, the C-H bond of the methylene group becomes active when radicals with very high bond dissociation enthalpy, such as methyl and ?-butoxyl radicals, are used. Thus, the variety of the attacking radicals in the bioassay system is likely responsible for differing experimental results. OH
o
0CH3 OH cu-diketone form
trans-diketone form
Fig. (9). Three forms of curcumin
Anti-HIV activity In addition to reverse transcriptase and protease, HIV-1 integrase has become a new target being explored in order to find more effective treatments for AIDS. HIV-1 integrase is the enzyme catalyzing integration of the double-stranded DNA of HIV into the host chromosome. Curcumin was reported to have inhibitory activity against HIV-1 integrase [36]. Other classes of compounds have also shown inhibited HIV-1 integrase in enzyme assays, but few have shown specificity against HIV-1 integrase, and even fewer were active in cell-based assays [37]. However, curcumin was reported to have moderate activity in cell-based assays, in addition to its activity in enzyme assays [38]. Therefore, modified curcumin analogs were developed for anti-HIV potency evaluation, as well as mechanism of action study [37, 39]. Mazumder et al. [39] synthesized curcumin analogs (Table 5) as probes for an anti-HIV-1 integrase mechanism study. Evidence suggested that curcumin does not bind to the DNA-binding domain of HIV-1 integrase [40] or the same binding site of another HIV-1 integrase
799
inhibitor, NSC 158393 [41]. Compounds 11 and 19, which have no hydroxy group in the phenyl ring, did not inhibit HIV-1 integrase. Therefore, hydroxy groups in the phenyl rings are apparently essential for the inhibitory activity. Compounds 65 and 66 exhibited greater activity than compound 1 (curcumin). Hence, replacing one or two methoxy groups of curcumin with hydroxy groups increased the activity. Compound 31 did not show inhibitory activity in this bioassay, suggesting that the unsaturated linker also contributes to the inhibitory activity. Three compounds (65, 66, and 67) showed very high potency. They have at least one catechol (3,4-dihydroxybenzyl) substructure as a common feature. More conclusions about this SAR study were obtained from further investigation of curcumin analogs as inhibitors of HIV-1 integrase. In addition to the catechol unit and an unsaturated linker, the syn disposition of the C=C-C=O moiety in the linker and the coplanarity of the structures are important to the integrase inhibitory activity of curcumin analogs [37]. The SAR conclusions drawn from experimental data are consistent with QSAR studies performed with MOE and Cerius2 programs [42]. Fig. (10) summarizes the anti-HIV-1 integrase SAR of curcumin analogs. However, no therapeutic index of these tested compounds was reported. Table 5. Structures of curcumin analogs and anti-HIV-1 integrase ability [39] OH
o
H3CO
Compound
Rl
R2
R3
R4
1 2 3 11 19 31 65 66 67
OCH3 H H H H
OH OH OH H OCH3
OCH3 OCH3 H H H
OH OH OH H OCH3
OH OCH3
OH OH
OH OH
OH OH
3-Processing
Strand transfer
IC 50 0*M) 150 140 120
IC 50 (jiM) 140 120
>300 >300 >300 6.0± 1.5 18.0+9.0 9±7
80±20 >300 >300 >300 3.1±0.1 9.0+ 3.0 4.0± 1.5
800 Coplanarity
syn disposition of enone
,,----.N
unsaturated linked 1
catechol unit
J
>
catechol unit
Fig. (10). Schematic picture of structural features favoring anti-HIV-1 integrase activity
Chemopreventive activity Chemoprevention is a relatively new concept. It attempts to use natural and synthetic compounds to intervene at early stages of cancer before invasive disease begins [43]. Nontoxic agents are administered to otherwise healthy individuals who may be at increased risk for cancer. Some potential diet-derived chemopreventive agents include epigallocatechin gallate in green tea, curcumin in curry, and genistein in soya. Curcumin has demonstrated wide-ranging chemopreventive activity in preclinical carcinogenic models of colon, duodenum, forestomach, mammary, oral cavity, and sebaceous gland and skin cancers. It is under Phase I clinical trial as a chemopreventive agent of colon cancer by the National Cancer Institute. The mechanisms of the chemoprevention induced by curcumin are pleiotropic. It enhances the activities of Phase 2 detoxification enzymes of xenobotic metabolism, such as glutathione transferases [44] and NAD(P)H: quinone reductase [45]. It also inhibits procarcinogen activating Phase 1 enzymes, such as cytochrome P450 1A1 [46]. With regard to the mode of chemopreventive action in colon cancer, curcumin exhibits a diverse array of metabolic, cellular, and molecular activities, including inhibition of arachidonic acid formation and its further metabolism to eicosanoids [47]. In order to search for new chemopreventive enzyme inducers and to elucidate the structural features responsible for the ability of curcumin to induce Phase 2 detoxification enzymes, Dinkova-Kostova et al.[48, 49] examined some natural as well as synthetic curcumin analogs for ability to induce NAD(P)H: quinone reductase (a prototype for phase 2 detoxification enzymes) in murine hepatoma cells. Compounds
801 801
containing a Michael reaction acceptor, such as phenyl-C=C-C=O, were expected to have phase 2 enzyme inducer potency [49]. Fig. (11) shows the structures of the curcumin analogs and their potencies in the QR assay in murine hepatoma cells. OH
o
H3CO
o
OH
OCH,
OCH3
HO
1 Curcumin CD = 7.3
OH HO
2 Demethoxyeurcumin CD = 9.5 OH
OH
O
3 Bisdemethoxycureumin CD =11.5 OH
OH
O OCH, OH
31 Tetrahydrocurcumin CD = 35.7
72 2,4-pentadione Inactive
75 Inactive
Fig. (11). Structures of curcumin analogs and their inducer potencies (CD//iM) in the QR assay in Hepa lc lc.7 murine hepatoma cells [48] (CD value is the concentration required to double the quinone reductase specific activity.)
Dibenzoylpropane (71) was inactive as an inducer probably due to its lack of an enone moiety. Dibenzoylmethane (69) was quite active with a CD value of 0.8 fiM; the keto-enol tautomerization of the /3-diketone can provide the Michael reaction acceptor feature. The three natural curcuminoids showed similar CD values; therefore, the methoxy group in the phenyl ring does not greatly affect the potency. However, the CD
802
values of compounds 11 and 68 indicated that the introduction of orthohydroxy groups on the phenyl ring dramatically enhanced the chemopreventive potency. Tetrahydrocurcumin (31), which as previously described is a potent anti-oxidant, possessed moderate potency (CD=35.7 juM) in the QR assay. Its potency may be due to the contribution of the keto-enol tautomerization of the /3-diketone moiety. 2,4-Pentadione (72) and l,l,l,5,5,5-hexafluoro-2,4-butanedione (73), which do not possess a phenyl ring, were inactive. Therefore, although the presence of a j8diketone moiety in the structure could enhance the activity, it is not sufficient to induce activity. Anti-prostate cancer Prostate cancer is the most common cancer in males living in developed countries and the second leading cause of cancer death. Curcumin has been reported to have anti-prostate cancer activity in vitro and in vivo [5052]. Although mechanisms of action have been postulated [53], they are not yet fully understood. The androgen receptor (AR) plays an important role in prostate carcinogenesis; therefore, anti-AR reagents such as hydroxyflutamide are widely used for the treatment of prostate cancer. Sixty-one curcumin analogs were synthesized or isolated from natural sources in our laboratory (Table 6) [54-58] and evaluated for AR inhibitory activity in prostate cancer cell lines. Among these analogs, compounds 2 and 9 exhibited the greatest inhibitory activity against the transcription of AR in three prostate cancer cell lines LNCaP, PC-3, and DU-145. Other analogs exhibited lower activity, and the rest showed no inhibition [55, 56]. Based on the bioassay results, the present SAR of curcumin analogs as anti-AR reagents is as follows. 1) A conjugated (3diketone moiety is crucial for the activity. Converting the /3-diketone moiety to pyrazole (105-108) resulted in decreased activity, and saturating (31, 109-115) or eliminating (116-121) the C=C bonds resulted in decrease or loss of activity. 2) When the methylene group in the linker was not substituted, replacing the phenolic hydroxy groups with methoxy (2) or methoxycarbonylmethoxy (4) groups resulted in significant increase in the inhibitory activity. 3) When the phenyl ring substitution of curcumin was retained, adding an ethoxycarbonylethyl group to the central methylene group (9) greatly increased the anti-AR activity. 4) All electron-withdrawing substitutions in the phenyl rings resulted in the loss of anti-AR activity. 5) The cyclic diarylheptanoids did not show
803 803
significant anti-AR potency; thus, the acyclic structure may be required for this activity. It is noteworthy that curcumin (1), which was reported to have anti-prostate cancer activity, was found to be inactive in the anti-AR bioassay. Therefore, curcumin does not inhibit the growth of prostate cancer cell through an anti-AR mechanism. The mechanism for how the synthetic curcumin analogs inhibit the transcription of AR remains unknown. Further modifications are ongoing in our laboratory to expand the SAR and optimize anti-AR activity. Table 6. Curcumin analogs synthesized for anti-AR assay [55, 56] A) Symmetric aryl rings with unsaturated diketone linker OH
O OCH, N
OCHj
H 3 C(H 2 C) 2 O
Compound 1 (Curcumin) 3 13 21 76 77 78 79 80 81 82 83 84 83 86 87 88 89 90 91 92 93 94 95 96 97
Rl H H H H H CH3 (CH2 ) 2 COOH (CH 2 ) 2 COOH (CH2 ) 2 COOH (CH2 ) 2 COOEt (CH 2 ) 2 COOEt (CH 2 ) 2 COOEt (CH2 ) 2 COOEt (CH2 ) 2 COOEt (CH 2 ) 2 CH3 H H H H H H H H H H H
R2 H H H H H H H H H H H H H H H H H F H CF3 H F F F NO2 H
R3 OCH3 H OCH3 OCH3 OCH3 OCH3 H OCH3 OCHj OCH2 OCH3 OCH3 OCH3 N(CH3)2 OCH3 H F H F F H H H H H NO2
R4 OH OH OCH3 OCH3 OCH2 COOCH3 OCH3 H OH H OH H OCH3 OCH3 OCH3 O(CH2 ) 2 CH3 F H H OCH3 H OCF3 F OCH3 H OCH3 OH
R5 H H OCH3 H H H H H H H H H OCH3 H H H H H H H H H H H OCH3 OCH3
MS H H H H H H H H H H H H H H H H H H H H H H H OCH3 H H
804 98 99 100 101 102
H H H
H H H
NO2
N(CH3)2
OCHj OCH3
OEt
OEt
OCH3
H H
B) Asymmetric aryl rings with unsaturated diketone linker OH
O
R,,
Compound 2 103 104
Rl H OCHj OCH3
Rl'
R2
R2'
OCH3
OH OCH3 OH
OH OH OCH2COOCH3
OCH3
C) Symmetric/asymmetric aryl rings with unsaturated pyrazole linker HN
_Comgound______ 105 106 107 10g
Rl OCH3 OCH3
N
Rl' OCHj
H
H H
OCH3
OCHj
R2' OH OH OH OCH3
R2 OH OH OH OCH3
D) Symmetric aryl rings with saturated linker
H3CO
Compound 31 109 110 111 112 113 114 115
OCH,
Rl OH OH OH OCHj OCHj OCHj OCH3 OCHj
R2
R3
R4
=0 =0 -OH =0 =O -OH =0 -OH
=0 -OH -OH =O -OH -OH -OH -OH
H H H H H H CH3 CH3
H H H
805 805 D) Symmetric phenyl rings with diketone linker R,
0
O
R2
Compound
R2 OCH3 H H H H H
Rl
H OCH3 H H
116 117
118 119 120 121
H
H
R3 OCH3 OCH3 OCH3 NO2 NO2 H
R3' OCH3 OCH3 NO2 NO2 NO2
H
H
CH2 COPh
E) Cyclic diarylheptanoids R,0
Compound 122 123 124
Rl
R2 H H OH
H H CH3
JR3_ C=O C=N-OH C=O
H3CO
_jCojnpjound_
Rl H CH 3 CH3 Ac
R2
R3
125 126 127 128
H H CH3 Ac
c=o c=o c=o
129
H
H
130
C=O
0
o
R4
H H Br Br
806
Anti-angiogenesis In order to maintain growth, a tumor must develop new blood vessels to obtain a constant supply of oxygen and nutrients and remove waste. Angiogenesis is the growth of new blood vessels from preexisting vasculature and is stimulated by biochemical signals. Inhibiting angiogenesis is a therapy to starve the cancer cells, Curcumin has demonstrated anti-angiogenic activity in vitro and in vivo [45, 59, 60]. The molecular mechanisms of anti-angiogenesis of curcumin were postulated to be the down-regulation of the expression of proangiogenic genes [61] and the down-regulation of matrix metalloproteinase [62]. The design of curcumin analogs as potential anti-angiogenic agents has been explored [63-66]. Hydrazinocurcuminoids modified from eurcurninoids by Shim et al. [64] showed greater anti-proliferative potency than curcuminoids in bovine aortic endothelial cells (BAECs) (Table 7). In contrast to curcumin, which inhibited the proliferation of several epithelial and fibroblast cells non-selectively, hydrazinocurcuminoids showed specificity against endothelial cells. Thus, proper modification on the linker between the two phenyl rings cannot only enhance the anti-angiogenic activity but also the specificity. The rank order of the potencies of the curcuminoids was 1 > 2 > 3. The hydrazinocurcuminoids showed the same trend. Therefore, the elimination of methoxy groups from the phenyl rings resulted in decreased anti-angiogenic potency. Robinson et al. [66] designed and synthesized curcumin analogs as anti-angiogenic agents. They postulated that the two aromatic rings of curcumin might be crucial for its binding to the potential receptor; therefore, they retained these rings and replaced the unsaturated j3diketone linker of curcumin with an enone or dienone linker. The impact of substitutions in the aromatic rings was also explored by preparation of chlorinated analogs. The structures of the analogs and their percent inhibition of endothelial cell proliferation are shown in Table 8. (Because curcumin was not tested in parallel, no conclusion could be drawn regarding to the relationship between structure and activity of curcumin.) The introduction of /?ara-methoxy groups on the phenyl rings increased the anti-angiogenic potency (cf. compounds 134 and 135). The introduction of two ortho-chloto groups on one phenyl ring also enhanced the inhibitory activity against the proliferation of endothelial cells (cf. compounds 135 and 136). Replacing one phenyl ring with a naphthyl ring
807
had little effect. In the dienone series, the most potent analog was compound 48, which has no substitution in the phenyl rings. Replacing the dienone moiety with a diene-cyclohexanone moiety, replacing the phenyl rings with pyridyl rings, or substituting the phenyl rings with 4-hydroxy-3methoxy moieties or two ortho-chlorines, all resulted in a decrease in the anti-angiogenic activity. Table 7. Structures of curcuminoids and hydrazinocurcuminoids and their inhibitory potencies against BAECs [64] Compound
Rl
R2
Inhibition of BAECs ICSOQtM)
1 2 3
OCH3 OCH3
OCH3
15±3 22 ±5 53 ±6
105 106 107
OCH3 OCH3
131
OCH3 OCH3
132
H
H
133
JU
H H OCH3
H H OCH3
H
0.52 ±0.04 1.8 ±0.3 5.8 ±0.2 0.93 ±0.04 2.4 ±0.1 8.7 ±0.2
lAnH
Table 8. Percent inhibition of in vitro endothelial cell proliferation by curcumin analogs [66] Structure
3 fig/ml
25 H 3 CO.
.OCH,
90.1
96.0
96.6
97.7
94.4
97.7
98.2
98.1
48
134
808 808 135
92.9
97.5
92.8
94.4
92.2
94.7
89.1
96.9
92.9
96.7
87.1
90.4
136
137
138
139
140
a
cr
CONCLUSION Curcumin is a natural product that possesses multiple biological activities, has various biological targets, and modulates numerous signal transduction pathways. Most cancer biologists suggest that, because tumor cells always have multiple pathways to escape the host defense mechanisms, a drug that is specific for modulation of one signal transduction pathway in the tumor cells may not be adequate [2]. Curcumin is an ideal anti-cancer drug candidate that affects multiple pathways and yet is still pharmacologically safe. However, due to its natural occurrence and long history of dietary use, curcumin cannot be patented. Hence, curcumin is a good lead compound for the development of better analogs that are patentable and more potent in the targeted activities [58, 67-70]. ACKNOWLEDGEMENTS This work was supported by National Cancer Institute Grant CA-17625 awarded to K. H. Lee.
809
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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THE VINCA ALKALOIDS: FROM BIOSYNTHESIS AND ACCUMULATION IN PLANT CELLS, TO UPTAKE, ACTIVITY AND METABOLISM IN ANIMAL CELLS MARIANA SOTTOMAYOR1 AND ALFONSO ROS BARCELO2 Department of Botany of Faculty of Sciences and Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre, 823, 4150180 Porto, Portugal, 2 Department of Plant Biology (Plant Physiology), University ofMurcia, E-30100 Murcia, Spain. ABSTRACT: The leaves of Catharanthw roseus (L.) G. Don (formerly Vinca rosea L.) were used in traditional medicine as an oral hypoglycemic agent and investigation of this activity ultimately led to the serendipitous discovery of the cytostatic terpenoid indole alkaloids vinblastine and vincristine. These compounds were the first natural anticancer agents to be clinically used and, together with a number of semisynthetic derivatives, are universally known as the Vinca alkaloids. Due to its important pharmaceutical alkaloids, C. roseus has now become one of the most extensively studied medicinal plants and much has been discovered about the biosynthetic pathway of terpenoid indole alkaloids, the regulation and compartmentation of the pathway, and the mechanisms of accumulation of those compounds inside the plant cell. The biosynthesis of vinblastine involves more than twenty enzymatic steps, nine of which are now well characterized at the enzyme and gene level and, recently, regulatory genes of the initial part of the pathway (ORCAs) have been cloned, in what consists a highly promising strategy for the manipulation of the pathway. On the other hand, the activity of vinblastine and vincristine in human cells has been thoroughly studied. The cytostatic activity has been shown to result from interference with tubulin, but the precise mechanism of action is still not perfectly understood. Uptake and extrusion in human cells has been characterized, specially the extrusion mechanism responsible for resistance to the drugs, and their metabolism in the human body has also been studied. Together, the above mentioned studies enable to establish some interesting evolutionary links between the enzymes involved in plant biosynthesis of the anticancer alkaloids and the enzymes involved in animal metabolism of the drugs, and also, possibly, between their vacuolar transport in plant cells and multidrug resistance in human cancer cells.
INTRODUCTION The so-called Vinca alkaloids are dimeric terpenoid indole alkaloids well known by their antimitotic activity, which has made them extremely
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useful drags in cancer therapy for more than thirty years. This designation includes the natural products vinblastine and vincristine, Fig. (1), extracted from the leaves of the plant Catharanthus roseus (L.) G. Don (previously Vinca rosea L.)» and a number of semi-synthetic derivatives like vindesine, vinorelbine and the recently developed vinflunine, Fig. (1). OH
CH31
Vinblastiue
, ,,, OOOCH3 I H J 'COOOJ3 0H CII3
CHO
*
ocoai3 'OOOCH3 "
Fig. (1). Structure of natural and semi synthetic Vfnca alkaloids. Shaded areas indicate the structural differences from vinblastine.
Catharanthus roseus, known as the Madagascar periwinkle, was used in traditional medicine as an oral hypoglycemic agent in the treatment of diabetes mellirus, and investigation on this activity ultimately led to the discovery of the anticancer alkaloids, almost simultaneously, by two totally independent groups: the group of Noble and collaborators in Canada [1,2] and the group of Svoboda and collaborators from the EM
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Lilly Company, Indianapolis, in the United States [3]. The chain of events leading to the discovery of vinblastine by the group of Noble is particularly interesting and represents a paradigmatic example of the role serendipity often plays in scientific discoveries. A full description of the discovery, including the casual details that started the whole process, was published by Robert Noble in 1990 [4]. After their discovery, the Vinca alkaloids became the first natural anticancer agents to be clinically used, and they are still an indispensable part of most curative regimens used in cancer chemotherapy nowadays. On the other hand, the plant producing these alkaloids, C. roseus, has become one of the most extensively studied medicinal plants. The levels of vincristine and vinblastine in the plant revealed to be extremely low and, for pharmaceutical production, approximately hah0 a ton of dry leaves is needed to obtain 1 g of vinblastine [4]. This fact stimulated intense investigation in alternative methods for the production of vinblastine and vincristine, namely chemical synthesis and plant cell cultures. However, chemical synthesis showed not to be viable due to the high number of transformations involved, and the anticancer alkaloids were never detected in cell cultures, which express alkaloid metabolism very poorly [5, 6]. The biosynthetic pathway of terpenoid indole alkaloids in C. roseus has also been intensively studied with the objective of developing a manipulation strategy to improve the levels of the anticancer alkaloids in the leaves of the plant [5, 7-10]. This review intends to put together what is known about the biosynthesis and accumulation of Vinca alkaloids in the plant Catharanthus roseus, with what is known about their uptake, mechanism of action and metabolism in animal cells. This will enable to highlight the curious matching of some of the enzymes involved in alkaloid biosynthesis in plant cells with some of the enzymes involved in alkaloid metabolism in animal cells, as well as to highlight the likely relation between the putative alkaloid accumulation mechanism in the vacuole of plant cells, and the transport mechanism responsible by multidrug resistance in animal cells. These similarities suggest that, during plant/herbivore co-evolution, plants have developed toxic chemical defenses against herbivores, like the Vinca alkaloids in C. roseus, by recruiting the same type of enzymes and transport mechanism that animal herbivores have, on their side, recruited for the defense against the very same compounds.
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THE VINCA ALKALOIDS AND THEIR CLINICAL USES The first Vinca alkaloid to be discovered was isolated in 1957 by Noble and collaborators from the Western University of Ontario, London, Canada, who named the alkaloid vincaleukoblastine, in view of its origin and its effect on immature white cells - leukoblasts [1, 2]. Later, the name was shortened to the less cumbersome vinblastine. Almost at the same time, the group of Svoboda and collaborators, at the Eli Lilly Research Division in the United States, detected two compounds with antitumour activity in C. roseus [3]. One of them was the already identified vinblastine, the other one was named leurosine. The two groups came in touch in a conference held by the New York Academy of Sciences in 1958, and worked in close collaboration thereafter. Clinical trials confirmed the usefulness of vinblastine in the treatment of Hodgkin's disease, lymphoma and other cancers, and the drug was introduced in the clinic shortly after. Leurosine was proved to be unsuitable for cancer therapy due to its toxicity, but Svoboda later isolated another alkaloid, which was also cytostatic and suitable for therapy [11]. This compound was first named leurocristine, then vincaleukocristine and finally vincristine. Vinblastine and vincristine have now earned a place among the most valuable agents used in cancer chemotherapy. They are dimeric terpenoid indole alkaloids differing only in that vincristine has a formyl group at a position where vinblastine has a methyl group, Fig. (1), but, although their chemical structure is very similar, they differ markedly in the type of tumors they affect and in their toxicity. The basic structure of terpenoid indole alkaloids includes an indole nucleus derived from tryptophan, via tryptamine, and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (see biosynthesis below). The anticancer alkaloids are built from two different terpenoid indole units derived from the precursors vindoline and catharanthine, this later suffering a rearrangement during the dimerization reaction to give rise to the so called velbenamine or cleavamine part of the dimeric molecule, Fig. (2). The direct product of the dimerization reaction is the dimer oc-3',4'anhydrovinblastine whose potential for cancer therapy is also being investigated [12]. However, apart from the reference cited, no report about the anticancer action of anhydrovinblastine was found in indexed
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scientific publications.
CH3 CH 3
Vindolme
Vinblastine Fig. (2). Biosynthesis of vinblastine from the monomeric precursors catharanthine and vindoline. Anhydrovinblastine is the direct product of the dimerization reaction and the precursor of the anticancer drugs. Shaded areas indicate the structural differences between the precursor catharanthine and the deavamine part of anhydrovinblastine.
Vinblastine, with the commercial names Velbe®, Velban® and Vinblastine®, is used alone and as a component of combined regimens with other anticancer drugs in the treatment of Hodgkin's disease and other lymphomas, in advanced carcinoma of the testis, in Kaposi's sarcoma and histiocytosis X. It can also be used in the treatment of breast carcinoma and choriocarcinoma. The use of vinblastine is mainly limited by its hematological toxicity due to destruction of the bone marrow [1315]. Vincristine, with the commercial names Oncovin , Vincasar , Vincrisul®, Pericristine®, and Kyocristine®, is used as a component of combination therapy in the treatment of Hodgkin's disease and lymphomas, and also in acute leukemias, sarcomas and carcinomas. Because of its relative lack of hematologic toxicity it is widely used as a component of many chemotherapeutic regimens. Combined with prednisone it produces complete remission in up to 90% of children with
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acute lymphocytic leukemia. The major and dose-limiting adverse effect of vincristine is neurotoxicity, specially to the peripheral nervous system [13-15]. Soon after the introduction of vinblastine and vincristine in clinical usage, during the 1970's, intensive chemical research was undertaken in order to try to obtain semi-synthetic derivatives of Vinca alkaloids showing higher activity, lower toxicity, and a wider spectrum of anticancer efficacy (for reviews see [16, 17]). The Eli Lilly company developed several series of derivatives with modifications in the vindoline part of the dimeric structure, culminating with the approval of vindesine, Fig. (1), for clinical treatments. Vindesine, with the commercial name Eldisine® and Enisone®, has a vincristine-like spectrum of activity, and is used mainly in the treatment of melanoma, acute lymphoblastic leukaemia and advanced non-small cell lung cancer [13, 14, 16]. Vindesine is approved in Europe and other areas but, in the United States, vindesine is approved only for investigational use [15]. In 1975, Potier and collaborators proposed that, inplanta, the dimeric vinblastine type alkaloids resulted from the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19]. This reaction resulted in the formation of an iminium dimer which, after reduction with NaBKj, yielded a-3',4'-anhydrovinblastine, Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine, Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21]. Vinorelbine demonstrated important antitumour properties associated with reduced toxic side effects and its application was developed during the 1980's by the French pharmaceutical company Pierre Fabre Medicaments, under the commercial name Navelbine . Vinorelbine is now widely used in the treatment of non-small cell lung cancer and breast cancer, and several other potential indications are under clinical investigation, like lymphoma, esophageal cancer and prostatic carcinoma [16, 22, 23]. Furthermore, it has been proved that vinorelbine is well absorbed orally with no unpredictable toxic effects and an oral formulation of the drug was registered in France in 2001 [13, 16]. The main side effect of vinorelbine is hematological toxicity.
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Pursuing the effort to obtain new useful Vinca alkaloids, the research divisions of Pierre Fabre produced a new family of derivatives using superacidic chemistry, from which vinflunine, a difluorinated derivative of vinorelbine, was selected for detailed preclinical investigations. Results showed that vinflunine is more active than vinorelbine, vinblastine or vincristine against a number of murine tumours and human tumour xenografts, and it entered phase I clinical trials in 1998, phase II in 2000, and is entering phase III in 2003 [16, 23, 24] . For a review on preclinical anticancer properties of vinflunine see [25]. The vinflunine case demonstrated that the Vinca alkaloids remain a drug family where it is still possible to identify new members with unprecedented and promising pharmacological properties. When the ongoing research on the mechanisms of action of Vinca alkaloids unravels the precise relation structure/function of the dimeric molecules, it should be possible to rationally design a new generation of Vinca alkaloids with new therapeutic properties. BIOSYNTHESIS OF VINCA ALKALOIDS IN CATHARANTHUS ROSEUS After the discovery of the anticancer properties of vinblastine and vincristine, the elucidation of their structure, Fig. (1), was a natural step achieved in the early 60s [26, 27], and it was shown that they were dimeric terpenoid indole alkaloids - as already stated above. Simultaneously, further studies of the plant C. roseus revealed that this plant is an amazing chemical factory, producing more than 100 different terpenoid indole alkaloids, including two other with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative [6, 28]. Terpenoid indole alkaloids (TIAs) comprise a large family of secondary metabolites, with around 3000 members identified, including several with important biological activity, like the Vinca alkaloids, the rat poison strychnine, and the antimalarial drug quinine [7], [29, 30]. They are almost restricted to four plant families of dicotyledones: Apocynaceae, Loganiaceae, Rubiaceae and Nyssaceae [9, 31]. In the plant, alkaloids are thought to play a defense role, mainly as deterrent factors against herbivorous pests, and some have been shown to be toxic against certain fungi and bacteria [32, 33]. A number of reports about the
820
antibiotic or antifeedant activity of the TIAs present in C. roseus has been published [34-40]. (7YTOPI.ASM
.tryplamine
— strictosidinc RER
16-hydroxv. ' ™ tabereomne
... strict™dmc aglvcome Vincristine
tabersonine "
cat haranthine
Anhydrovinblastinc deacety] vindol i ne
vindoline
Fig. (3). Compartmentalization of the biosynthetic pathway of terpenoid indole alkaloids in plant cells. G10H: geraniol 16-hydroxylase; SLS: secologanin synthase; TDC: tryptophan decarboxylase; STR: strictosidine synthase; SGD: strictosidine (3-D-glucosidade; T16H: tabersonine 16-hydroxylase; OMT: S-adenosyl - Lmethionine : 16-hydroxytabersonine - 16-O-methyltransferase; NMT: 5-adenosyl - L-methionine : 16-methoxy - 2,3-dihydro-3-hydroxytabersonine - A'-methyltransferase; D4H: desacetoxy vindoline 4-hydroxylase; DAT: acetylcoenzyme A : 4-O-deacelylvindoline 4-O-acetyltransfcrase; PRX: peroxidase.
The great pharmacological importance of the dimeric alkaloids, allied with its low availability, stimulated intense research in the biosynthesis of
821 821
TIAs in C. roseus and in the regulation of the pathway, with the aim of eventually manipulating plant metabolism in order to obtain higher levels of the anticancer alkaloids. The biosynthesis of vinblastine has shown to be highly complex, involving more than twenty enzymatic steps, and a great deal is already known about the pathway, the enzymes and genes involved, and about their regulation. However, considerable parts of the pathway remain relatively hypothetical, and enzymatic characterization is still lacking for many steps. The biosynthetic pathway of vinblastine revealed to be highly compartmentalized inside the cell, since a number of enzymes were shown to be localized in different cellular compartments, either experimentally, or by inference from the presence of targeting signal peptides in their aminoacid sequences, Fig. (3) [8, 41]. Enzymes and genes involved in branching points of the early stages of biosynthesis, like geraniol hydroxylase, tryptophan decarboxylase and strictosidine synthase, Fig. (4), have been thoroughly characterized, and the last 6 steps in the path leading to vindoline, Fig. (6), one of the monomeric precursors of vinblastine, Fig. (2), have received much attention as well, with several enzymes/genes of these late steps being characterized. This part of the pathway is not expressed in cell suspension cultures, what possibly accounts for the absence of dimerics in this system. The dimerization step itself has received considerable attention and is thought to be mediated by a class III plant peroxidase [42-44]. In spite of all this, the pathway from primary metabolism to vinblastine and vincristine still includes many transformations that remain to be characterized, even at the level of the biosynthetic intermediates. Previously, the biosynthesis of TIAs has been reviewed in [7, 8, 10, 45]. Biosynthetic pathways What is known about the biogenetic routes leading to the biosynthesis of the dimeric akaloids vinblastine and vincristine in C. roseus is represented in Fig. (4) to (6). Enzymes and genes that have been characterized are indicated, and the subcellular compartmentalization of the pathway is presented in Fig. (3). The basic structure of TIAs includes an indole nucleus derived from tryptamine, the decarboxylation product of the aminoacid tryptophan, and a versatile C9 or CIO terpenoid unit arising from the iridoid glucoside secologanin, Fig. (4).
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Tryptophan is a product of the shikimate pathway and is converted into tryptamine by tryptophan decarboxylase (TDC), Fig. (4). TDC is a cytosolic soluble enzyme that occurs as a dimeric protein, and it was shown to exhibit a high substrate specificity and to be under posttranslational control [46-52]. A cDNA clone encoding TDC was isolated by DeLuca et al. [53] and the foil gene was characterized by Gooddijn et al. [54, 55] who found that TDC is encoded by a single copy gene without introns. The Tdc promoter has also been cloned and its regulation characterized [56, 57]. Glyceraldehyde / pyruvate pathway Geraniol
OH
Shikimate pathway
I
COOH
10-Hydroxygeraniol OH
L-Tryptophan
lOGlu NH
Tryptamine
Secologanin
Fig. (4). Early steps of the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus. Triple arrowheads indicate multiple steps. G10H: geraniol 16-hydroxylase; TDC: tryptophan decarboxylase; STR: strictosidine synthase.
The terpenoid portion of TIAs is derived from secologanin, whose monoterpene precursor geraniol, is produced by the recently discovered Rommer or triose phosphate / pyruvate pathway, responsible for the synthesis of isoprenes like geraniol in the plastids [58-60].
823
The first committed step in the biosynthesis of secologanin is the hydroxylation of the C-10 position of geraniol by geraniol 10-hydroxylase (G10H), Fig. (4), which was one of the first cytochrome P-450 monooxygenases to be characterized in plants [61-63]. The enzyme and the associated NADPHxytochrome P-450 reductase were purified to homogeneity from cell suspension cultures of C. roseus and characterized [64, 65]. G10H was shown to be able to hydroxylate both geraniol and its cis isomer nerol. The end product alkaloid catharanthine was proved to be a reversible, linear, noncompetitive inhibitor of G10H, while vindoline and vinblastine were less inhibitory but still interfered with activity [66]. G10H was found to be localized in provacuolar membranes [67], although the same authors state later that what they had characterized as vacuolar membranes could in fact represent a differentiated form of endoplasmic reticulum [61]. In spite of this ambiguity, G10H is considered by most reviewers to be localized in the vacuolar membrane [5, 8, 41]. The NADPHxytochrome P-450 reductase (CPR) was found to be similar to the mammalian enzyme and both G10H and CPR were cloned and the genes characterized [68-70]. The pathway leading from 10hydroxygeraniol to secologanin has been relatively well characterized [8, 10, 29, 71] and the enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, secologanin synthase (SLS), Fig. (4), was also shown to be a cytochrome P450 [72, 73]. The stereospecific condensation of tryptamine and secologanin under the action of strictosidine synthase (STR), Fig. (4), is the first committed step in TIAs biosynthesis, and it yields the glucoalkaloid 3-a(S)strictosidine, which is the central biogenetic precursor of all TIAs [7477]. STR was first purified by Treimer and Zenk [78, 79] and Mizukami et al. [80] from cell cultures of C. roseus. The enzyme was found to have a high substrate specificity, and to suffer no inhibition by end-product alkaloids, such as vindoline and catharanthine [78-80]. It was observed that STR occurred as different isoenzymes [81, 82], and the subcellular localization was determined to be the vacuole [83]. The complete mRNA sequence of Str was determined by Pasquali et al. [84], who also showed that STR is encoded by a single-copy gene, indicating that the above mentioned isoenzymes are formed post-translationally from a single precursor. Comparison of the primary structure of the STR protein with the amino acid sequence deduced from the Str mRNA showed the
824
presence of a signal peptide of 31 amino acids in the amino-terminal sequence. This signal peptide appears to be essential for vacuolar targeting of STR, according to results obtained with transgenic N. tabacum [83]. The promotor of Str has been studied in great detail and has enabled the identification of transcriptional factors involved in the regulation of TIAs biosynthesis [9, 85, 86] (see section "Regulation..." below). Strictosidine is the general precursor of several divergent pathways leading to the multitude of TIAs accumulated by C. roseus. Somewhere, downstream of strictosidine formation, the pathway of TIAs suffers several ramifications mostly uncharacterized. The less ill characterized branches are the ones leading to catharanthine and vindoline, the monomeric precursors of the Vinca alkaloids, Fig. (2). Those branches will be the ones discussed here.
21
" lH ,,oau
OH
4,21-Dehydrogeissoschizine
Strictosidine aglycone
CH3OOC
CH 2 OH
Stemmadenine
CH 3
COOCH3
Tabersonine Fig. (5). Biosynthesis of catharanthine and tabersonine from strictosidine, the central precursor of all terpenoid indole alkaloids. SGD: strictosidine p-D-glucosidade.
825 825
The first step following strictosidine synthesis is the removal of its glucose moiety by strictosidine p-D-glucosidade (SGD) with formation of an unstable aglycone, Fig. (5) [87]. SGD is encoded by a single copy gene in C. roseus and is most likely associated with the ER, as suggested by in vivo staining and by the presence of a putative ER signal sequence in the protein [88]. Deglucosylated strictosidine is converted via several unstable intermediates into 4,21-dehydrogeissoschizine from which catharanthine and vindoline are believed to derive, Fig. (5). This part of the pathway has been scarcely characterized - it includes an undetermined number of steps, seems to involve the intermediate stemmadenine, and the branching point for the 2 paths giving rise to catharanthine and vindoline has been proposed to be dehydrosecodine by Blasko and Cordell [71], and to be stemmadenine by Verpoorte et al. [89]. The 6 last biosynthetic steps leading to the production of vindoline from the intermediate tabersonine have been thoroughly characterized and are represented in Fig. (6) [45, 90]. The first step in the conversion of tabersonine to vindoline is hydroxylation of the C-16, which is catalyzed by the enzyme tabersonine 16-hydroxylase (T16H), Fig. (6). Characterization of T16H indicated the enzyme is a cytochrome P-450 monooxygenase [91], what was confirmed by the molecular analysis of the isolated cDNA [92]. Southern analysis suggests the presence of at least two T16H genes in C. roseus. The following step in the biosynthesis of vindoline is the Omethylation of 16-hydroxytabersonine to yield 16-methoxytabersonine by the enzyme S-adenosyl-L-methionine: 16-hydroxytabersonine-16-0methyltransferase (OMT), Fig. (6) [45, 93]. Only a preliminary identification of OMT has been carried out in crude desalted extracts from C. roseus leaves [91, 94]. O-methylation of 16-hydroxytabersonine is followed by an uncharacterized hydration step, and then by iV-methylation of the Nindole by the enzyme S-adenosyl - L-methionine : 16-methoxy - 2,3dihydro - 3-hydroxytabersonine - iV-methyltransferase (NMT), originating desacetoxyvindoline, Fig. (6). NMT has been roughly characterized by DeLuca et al. [95], and partially purified by Dethier and DeLuca [96] from young leaves of 6 months old C. roseus plants. Subcellular localization studies indicated that NMT is specifically associated with the membranes of thylakoids [46].
826
COOCH3
Tabersonine
16-Hydroxytabersonine
NMT €H3
„
H
COOCH3
16-Methoxvtabersonine
D4H
<^'., OH COOCH3
16-Methoxy-2,3-dihydro-3 -hydroxy tabersonine
OCH3
I
CH3
CH3
Desacetoxy v indo line
OH boOCH3
Deacety tv indoline If DAT
Vindoline «
OCOCH3 'COOCH3
CH3
Fig. (6). Biosynthesis of vindoline from tabersonine. T16H: tabersonine 16-hydroxylase; OMT: S-adenosyl i-methionine : 16-hydroxytabersonine - 16-O-methyltransferase; NMT: S-adenosyl - I-methionine : 16methoxy - 2,3-dihydro-3-hydroxytabersonine - iV-methyltransferase; D4H: desacetoxy vindoline 4hydroxylase; DAT: acetylcoenzyme A : 4-O-deacetylvindoline 4-O-acetyltransferase.
The second to last step in vindoline biosynthesis is hydroxylation of the C4 of desacetoxyvindoline by a 2-oxoglutarate-dependent dioxygenase, desacetoxyvindoline 4-hydroxylase (D4H, EC 1.14.11.11), Fig. (6). D4H has an absolute requirement of 2-oxoglutarate and molecular oxygen, and its activity is enhanced by ascorbate [97]. Twodimension electrophoresis resolved the purified D4H into three isoforms, all showing a high affinity for desacetoxyvindoline. The authors suggested that this may partially explain the low concentration of desacetoxyvindoline found inside the cell. Furthermore, D4H did not show inhibition even by high concentrations of the hydroxylation product deacetylvindoline [98, 99]. D4H seems to be localized in the cytosol [97]. Molecular characterization of cDNA and genomic clones of D4H showed the presence of a single-copy gene in C. roseus and that D4H belongs to a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin [100].
827
The last step in vindoline biosynthesis is catalyzed by acetylcoenzyme A : 4-0-deacetylvindoline 4-0-acetyltransferase (DAT, EC 2.3.1), a reversible 0-acetyltransferase that transfers acetate from acetylcoenzyme A to deacetylvindoline. Fig. (6) [101, 102]. DAT was purified and characterized and was first thought to consist of two subunits with molecular weights between 20 and 30 kDa [101, 103, 104]. However, more recently, work developed while cloning DAT cDNA proved that the cellular form of the enzyme is actually a single polypeptide of 50 kDa, indicating that the protein had been cleaved during purification [105]. The purified enzyme is strongly inhibited by tabersonine and the product coenzyme A, but not by up to 2 mM vindoline. This means that the rate of this reaction may be regulated by the level of free coenzyme A in the cell, while remaining unaffected by vindoline accumulation. This could again explain why also deacetylvindoline does not accumulate in C. roseus leaves [103, 104]. Subcellular localization studies indicated that DAT is a cytosolic enzyme [46]. Vindoline and catharanthine are the last monomeric precursors of the dimeric anticancer alkaloids of C. roseus, and they are also the two major alkaloids accumulated in the leaves of the plant [106, 107]. The study of the dimerization biosynthetic step stems in early work on the chemical synthesis of the dimeric alkaloids and it has involved much discussion. Moreover, the chemical dimerization reaction has industrial application in the synthesis of vinorelbine and vinflunine. Due to its potential regulatory importance for the production of the dimeric Vinca alkaloids in the plant, and to the much that is known about the chemical and biosynthetic reactions, the dimerization step will be presented in particular detail here. The dimerization step In face of the structural similarities unraveled during the 1960s of vindoline and catharanthine with the dimeric alkaloids, and due to their great abundance in the plant, these two compounds were immediately considered the most likely monomeric precursors of the Vinca alkaloids, although the cleavamine moiety of vinblastine presented some differences from catharanthine, namely a fragmentation of the C5-C18 bond, Fig. (2). The natural big abundance of vindoline and catharanthine made also a semisynthetic process for the synthesis of the dimerics very attractive and, after several failed attemps by other groups, Potier et aL in 1975 [19] reported for the first time the synthesis of a dimer with the natural
828
configuration through a modified Polonovski reaction. In this reaction, catharanthine N-oxide was treated with trifluoracetic anhydride in the presence of vindoline, leading to C5-C18 skeletal fragmentation of catharanthine, which was followed by nucleophilic attack of the C18 position by vindoline, and formation of the coupling bond. The simultaneous presence of vindoline was essential to obtain the natural configuration, and the authors proposed that the reaction leading to the natural epimer proceeded through a concerted process in which vindoline was involved in displacing the C5-C18 bond, originating the natural stereochemistry. The formation of the unnatural epimer, in rates dependent on experimental conditions, was explained as resulting from a stepwise reaction [18, 19, 108, 109]. The modified Polonovski reaction first used in [19] and later called the Potier-Polonovski reaction, resulted in the formation of an iminium dimer which, after reduction with NaBHU, yielded a-3',4'-anhydrovinblastine, Fig. (2). Thus, this was the first dimeric Vinca alkaloid with the natural configuration to be synthesized. This method allowed, subsequently, the development of approaches to the synthesis of other natural dimerics like vinblastine, vincristine, leurosidine and leurosine [110-114], and more recently, to the semisynthetic vinorelbine and vinflunine [16, 22]. The chemical coupling of catharanthine and vindoline to yield anhydrovinblastine led to the obvious hypothesis that this compound might also be the first product of dimerization in the plant, and the dimeric precursor of vinblastine and vincristine. For three years it was not possible to find anhydrovinblastine in the plant, until Scott et al. in 1978 [115], by modifying the established methods for extraction and purification of alkaloids, isolated anhydrovinblastine from C. roseus plants, with incorporation of radiolabelled catharanthine and vindoline, thus proving that anhydrovinblastine was actually a natural product. In 1979, Langlois and Potier [116] proposed that anhydrovinblastine could be the precursor of most, if not all, dimeric alkaloids of C. roseus, and feeding studies indicated the enzymatic incorporation of anhydrovinblastine into vinblastine and other dimeric alkaloids [117-120]. Incorporation studies were fiirther confirmed by experiments with cell free homogenates of C. roseus cell suspension cultures [121, 122]. Several biosynthetic routes were proposed in which either anhydrovinblastine or its iminium were the pivotal intermediates of all dimeric alkaloids [114,
829
117, 118, 123, 124] but the reactions that really occur in the plant and the respective enzymes have not been characterized. The search of the enzyme responsible for the dimerization reaction, i.e. for the biosynthesis of anhydrovinblastine, resulted in the finding that peroxidase-like activities extracted from cell suspension cultures were capable of performing the coupling of catharanthine and vindoline into anhydrovinblastine [125-127]. Horseradish peroxidase, a commercial plant peroxidase, was also capable of performing the coupling reaction [128]. At this point, anhydrovinblastine had been proved to actually be a major alkaloid present in C. roseus leaves [106, 107] representing together with catharanthine and vindoline the three major alkaloids of the plant. This indicated the presence of high in vivo anhydrovinblastine synthase activity in leaves and that this was the appropriate biological material to search for the enzyme. Work with leaves started at the laboratory of Prof. Frank DiCosmo from the University of Toronto, Canada, and has mostly been developed in our labs, at the University of Murcia and the University ofPorto. We have characterized and purified a basic peroxidase from C. roseus leaves with a-3',4'-anhydrovinblastine synthase activity. This enzyme was, on the one hand, the single peroxidase isoenzyme detected in C. roseus leaves and, on the other hand, the single anhydrovinblastine synthase activity detected in C. roseus extracts, and was thus considered to be the most likely in vivo responsible for the synthesis of anhydrovinblastine [42, 43]. Moreover, vacuole isolation and peroxidase cytochemical detection showed that the enzyme was localized in the vacuole, the same subcellular compartment where both substrates and product of the dimerization reaction are accumulated [42, 44]. The mechanism of the peroxidase mediated dimerization reaction was investigated and it was shown that both vindoline and catharanthine are suitable electron donors for the oxidizing intermediates of the basic peroxidase, compound I and compound II, and it was proposed that the coupling reaction proceeds by a radical propagated mechanism [43, 44, 129]. Recently, we have discussed the assignment of a specific ftmction, such as the synthesis of anhydrovinblastine, to a multiftmctional enzyme, such as the basic peroxidase of C. roseus leaves, and we have proposed a channeling mechanism for the peroxidase-mediated-vacuolar synthesis of anhydrovinblastine [44]. We have now characterized the cDNA and
830
genomic sequence of this anhydrovinblastine synthase-peroxidase in collaboration with Mark Leech from the John Innes Centre, UK [130]. Regulation of the biosynthesis biosynthesis in the plant
of terpenoid
indole
alkaloid
The TIAs pathway in C. roseus has been shown to be under developmental regulation, showing also cell-, tissue-, and organ-specific expression [90, 131]. Particularly interesting is the differential localization of early and late stages of vindoline biosynthesis in particular cells of leaves shown by in situ RNA hybridization and immunocytochemistry studies. Those experiments suggest the involvement of at least two cell types in the biosynthesis of vindoline and the existence of intercellular translocation of a pathway intermediate [131]. The pathway from tabersonine to vindoline, specifically, has been shown to be under developmental and light regulation, a fact that was pointed as connected with the absence of vindoline in cell suspension cultures [90, 93, 132]. On the other hand, and since alkaloids constitute a defense against certain environmental stresses, it is not surprising that the TIA pathway shows induction by several biotic and abiotic stress factors [6, 133-137]. Among those, the induction by fungal elicitors has been particularly well characterized, together with the regulation by the plant stress hormone methyljasmonate [5, 9]. Regulation studies of the promotor of Str, Fig (4), enabled the identification of an autonomous jasmonic acid-responsive sequence JERE (jasmonate- and elicitor-responsive element) present in the promotor [138]. The JERE was shown to interact with three jasmonic acid-responsive transcription factors - the ORCAs (octadecanoid responsive Catharanthus AP2-domain proteins) [85, 138]. ORCAs belong to the AP2/ERF family of transcription factors, which are unique to plants. ORCA3 was shown to positively regulate the TIAs biosynthesis genes Tdc, Str, Cpr and D4h, belonging to both early and late steps of the pathway [139]. Moreover, ORCA3 also regulates genes from the primary metabolism pathways involved in the biosynthesis of tryptophan and geraniol, and was thus considered a master regulator of metabolism [9, 139]. However, several genes in the TIA and secologanin pathways are not regulated by ORCA3, meaning that overexpression of this transcription regulator alone is not sufficient to enhance the levels of
831 831
TIAs in C roseus cell suspension cultures or plants. Nevertheless, the transcriptional factor approach bears a great potential for manipulation of the TIA pathway, and discovery of a few more regulators like 0RCA3 may, in the future, enable to induce/increase the biosynthetic pathway of vinblastine in cell cultures and/or plants. ACCUMULATION OF THE VINCA ALKALOIDS IN THE PLANT VACUOLE Vinca alkaloids and other TIAs produced by C. rosetts are toxic, not only to animal cells, but also to plant cells, and even to the plant cells that produce them [140-142]. This is actually the common situation with many secondary metabolites produced by plants. Plant cells are able to accumulate high levels of those compounds because they are removed from the cytosol and sequestered inside the vacuole. The vacuole of plant cells may occupy up to 90% of the cell volume and performs a number of important functions like regulation of turgor pressure with a role in cell growth, detoxification of xenobiotics, storage of many useful compounds, ion homeostasis, hydrolysis of various molecules and macromolecules, and accumulation of secondary metabolites that may act in defense against herbivores, pathogens, UV light, etc. [143,144]. In C. roseus, Vinca alkaloids and other TIAs are thus transported across the vacuohr membrane, the tonoplast, and accumulated inside the vacuole, as has been shown in a significant number of reports. Deus Neumann and Zenk [145] showed that serpentine is exclusively stored within the vacuoles of C. roseus cells, and that C. roseus vacuoles can uptake and accumulate ajmalicine, catharanthine and vindoline. Ajmaficine and serpentine accumulation inside the vacuoles of suspension cells was confirmed by [146], and immunocytochernical localization of vindoline indicated its major presence in the central vacuole and in small vesicles of mesophyl cells [147]. If TIAs are stored inside the vacuole, they must cross the tonoplast and concentrate inside the organelle. The mechanisms for transport of TIAs across the tonoplast and of their accumulation inside the vacuole have been the subject of much controversy during the end of the 1980s. For critical reviews see [143, 148]. Since that time, hardly any report on TIAs transport across the tonoplast has been published, meaning that this is still an unsolved problem.
832
Here, we will review the controversial work done about TIAs tonoplast transport and discuss possible directions for future work. Two types of mechanisms have been proposed for TIA transport across the tonoplast: i) a highly specific carrier mediated mechanism and ii) a variety of more or less unspecific "trapping" mechanisms which assume that transport through tonoplast occurs by passive diffusion and that inside the vacuole alkaloids suffer some transformation that lowers their activity or permeability and thus favors their accumulation. It has been further proposed that membrane vesicles may be involved in the biosynthesis and/or packaging of the alkaloids and their transport to the central vacuole [147, 148]. Among trapping mechanisms, the most discussed has been the "iontrap" model [146, 148-153]. This model is based on the assumption that, being low molecular weight amines, the neutral form of the alkaloids is lipophilic and will thus freely diffuse across the lipidic phase of biologic membranes, while the protonated alkaloid formed in acidic pH will have a much lower permeability coefficient. Under these conditions, the ion trap model implies that when several compartments exist, the neutral base will diffuse across membranes and will be present at the same concentration in each compartment, while the cation will remain trapped and will thus accumulate in the more acidic compartment. Accumulation will depend on the pH gradient across the compartment membrane and on the dissociation constant (pKa) of the alkaloid - the higher the ApH and the pKa are, the higher the accumulation will be. This model is supported by a number of experiments performed mainly with ajmalicine and with some alkaloids not present in C. roseus, which confirmed the postulated low specificity of the ion trap model [146, 149, 151-153]. Nicotine, an alkaloid which is not produced by C. roseus, accumulated in C. roseus vacuoles to concentrations 12 times higher than ajmalicine, in agreement with its higher pKa of 8.0 in comparison with 6.3 for ajmalicine [153]. The low specificity observed, the insensivity of accumulation to ATP, the absence of saturation in short-term assays, the identical pattern of influx and efflux curves, and above all, the linear dependence of accumulation on the external pH, were interpreted as strong evidence supporting the ion trap model [149,151-153]. Other trapping mechanisms, which have been suggested for indole alkaloids and could work in complementation with the ion trap mechanism, are binding to other vacuole components like phenolics, or to
833
the inner side of the tonoplast, which seems to be the case of serpentine [148]. A very efficient trapping was observed by Hauser and Wink [154] in vacuoles of Chelidonium majus containing high concentrations of ehelidonic acid, which was shown to readily complex alkaloids, including vinblastine. The authors showed that vinbkstine could easily cross the tonoplast by diflusion and accumulate against a concentration gradient, apparently due to complexation to cheKdonic acid. A similar complexation mechanism with meconic acid has been proposed for accumulation of morphine in Papaver latex vacuoles. Further metabolization of alkaloids inside the vacuole is another possibility of trapping. Blom et aL [146] showed that ajmalicine is oxidized by peroxidase into the charged serpentine inside the vacuole, creating a trap that retains alkaloids more efficiently within the organdie. The group of Renaudin [152, 153] observed that two pools of ajmalicine were present in C roseus cells: one pool which could rapidly move to and from the vacuole, corresponding to molecules accumulated by ion trapping (quickly exchangeable pool) and a second pool also exchangeable in both directions but within a much longer time scale (slowly exchangeable pool). The authors hypothesized that this could either be due to the presence of two populations of vacuoles with different transport characteristics, or to binding of ajmalicine to other vacuole components like those referred above, meaning the existence of two different trapping mechanisms. Possibly related with these results, McCaskill et aL [149] observed that accumulation of vindoline, ajmalicine, tabersonine and vinblastine by C. roseus protoplasts was biphasie, with an initial burst of uptake followed by a slow, prolonged phase of accumulation, while accumulation of nicotine was monophasic. The data presented suggested that the initial burst for vindoline and ajmalicine and the accumulation of nicotine were driven by the pH gradient between the vacuole and the external medium, through an ion trap. For ajmalicine, the second phase of uptake was not inhibited by azide and the authors suggested it could be due to complexation with organic counterions or phenolics inside the vacuole. In the case of vindoline, it was observed that azide inhibited the second phase of accumulation and the authors concluded that transport of vindoline across the tonoplast should also involve a specific energyrequiring uptake. This idea that indole alkaloids transport across tonoplast couM be mediated by a highly specific energy dependent carrier was proposed and
834
defended by Deus-Neumann and Zenk [145, 155]. In their experiments, Deus-Neumann and Zenk observed vacuolar uptake to be very specific for the endogenous alkaloids [145]. Vindoline, catharanthine and ajmalicine were taken up only by vacuoles of C. roseus and not by vacuoles of other alkaloid accumulating plants, like Nicotiana tabacum and Papaver somniferum. Inversely, C. roseus vacuoles did not accumulate nicotine or morphine, in contrast to what has been observed by Renaudin and McCaskill et al. [149, 153]. Moreover, transport was saturable and exhibited dependence on pH with an optimum at pH 6.5; transport was sensitive to temperature and was inhibited by the ATPase inhibitor DCCD (iV^-decyclohexylcarbodiimide). Transport was not, however, stimulated by ATP. KM values of 1.5 uM for vindoline, 2.5 uM for catharanthine and 1.67 uM for ajmalicine were determined. In further experiments with Fumaria capreolata and the isoquinoline alkaloids reticuline and scoulerine, Deus-Neumann and Zenk observed again an absolute specificity for the alkaloids indigenous to the plant, and even for the natural («S)-enantiomeric form of the alkaloids[155]. Stimulation by ATP and inhibition by a protonophore was observed, and alkaloid efflux was shown to have the same characteristics as uptake. The authors proposed that alkaloid uptake and release through the tonoplast is a highly specific carrier-mediated and energy-dependent proton antiport system, and they postulated that "for every alkaloid group, maybe even for every single alkaloid molecular species, there is a highly specific alkaloid carrier, or specific binding sites of a general carrier, present in the tonoplast membrane". Mende and Wink obtained results similar to DeusNeumann and Zenk for the uptake of the quinolizidine alkaloid lupanine by Lupinus polyphyllus vacuoles [156]. In view of the contradictory results and proposals concerning alkaloid transport, Guern et al. [148] tried to reconcile the two opposing concepts by proposing that the ion-trap and carrier models are not necessarily exclusive, but most likely coexist. The relative importance of the two systems would depend on the physieoehemical properties of the alkaloid molecule, i.e., its lipophilicity and basicity, and on the physiological conditions concerning the driving forces for vacuolar accumulation, i.e., the trans-tonoplast pH gradient. Under this light, Guern et al. [148] reinterpret some of the opposite results obtained by defenders of the two models. According to these authors, and based on measurement of the vacuolar pH using the 31P-NMR technique, vacuoles isolated using NaCl
835
as an osmoticum, as done by Deus Neumann and Zenk [145], present a higher pH (6.35) compared to vacuoles isolated in sorbitol (pH 5.33), the technique used by the group of Renaudin. Thus, in the vacuoles isolated by Deus Neumann and Zenk [145], which had presumably lost their acidity, uptake of alkaloids should proceed mainly through the highly specific binding component of transport, while the ion-trap component could not exert its action. Also Wink [143], in his outstanding review about the plant vacuole, stresses that the two mechanisms are compatible. Nevertheless, the discrepancy of results obtained is still puzzling and the transport of indole alkaloids across the tonoplast and their accumulation in the vacuole is still an unsolved problem. Experiments should be designed in a way that enables the distinction between transmembrane transport and possible subsequent intravacuolar events, like complexation or metabolism, and the pH gradient across the tonoplast should be monitored all through the experiments. Membrane permeability to the neutral and ionized forms of alkaloids should also be precisely determined, since this is a fundamental question in the problem, and data available is controversial. If membranes are significantly more permeable to the neutral form of the indole alkaloids than to the protonated form, it is reasonable to think that at least in cells with a vacuolar pH as acid as 3 [150] even alkaloids with a low pKa as vindoline (5.5) will accumulate by ion-trapping. Interestingly, Yoder and Mahlberg [157] observed that alkaloids accumulated in specific cells which showed a lower vacuolar pH than other mesophyll cells, as determined by neutral red staining. It is also interesting to notice that carrier mediated transport has been characterized by Deus Neumann and Zenk [145] mostly for vindoline (pKa=5.5), which has a pKa significantly lower than ajmalicine (6.3) and catharanthine (6.8), and thus would always be trapped less efficiently by the pH gradient alone. It was also for vindoline that McCaskill et al. [149] detected, apart from an ion-trapping, a specific energy-requiring uptake component. But the possible involvement of specific carriers, at least for vindoline transport, lacks confirmation, namely the final proof which is the purification of the carrier or carriers. Although no fiirther work has been published on transport of indole alkaloids across the tonoplast, much work has since been published about tonoplast transporters, which may add some relevant clues. It was shown that in maize, the last step in anthocyanin biosynthesis involves its conjugation to glutathione and this conjugate is then recognized for transport into vacuoles by the glutathione pump [158]. This pump is
836
responsible for the vacuolar compartmentalization of glutathione conjugates of xenobiotics, thus enabling its detoxification. In view of their results, the authors further suggested that many of the naturally synthesized plant secondary metabolites could also be recognized and transported by the glutathione pump. Hortensteiner et al. [159] identified in vacuoles of Hordeum vulgare a transport system capable of actively transport the bile salt taurocholate across the tonoplast. The pysiological substrate(s) for this transporter of plants have not been identified, but vinblastine inhibited the taurocholate transport, and kinetic analysis of the inhibition revealed that vinblastine could be a substrate for the transporter [160]. Glutathione pumps and the bile salt transporter of Hordeum vulgare belong both to the ATP-binding-cassette (ABC) transporter superfamily that also includes the multidrug resistance protein, P-glycoprotein, responsible by resistance of cancer cells to drugs such as the Vinca alkaloids (see section "Uptake..." below). Knowledge about the function of ABC transporters in plants is still scarce but the vacuolar uptake of a number of compounds has been associated to these transporters, such as: glutathione conjugates of both exogenous (xenobiotic) and endogenous (secondary metabolites) compounds; clorophyll catabolites; glucuronides and glucosylated herbicides [161]. The Arabidopsis ABC transporter gene family has been shown to contain 131 members, exceeding the 48 reported for Homo sapiens [162], and Jasinski et al. [162] correlate the diversity and evolution of plant ABC transporters with the diversity of plant secondary metabolism, pointing out the requirement of adapted transporters to transfer those metabolites into the vacuole or the external medium. In view of all this, we believe that TIA transport across the tonoplast may indeed be mediated by one of the several types of ABC transporters, and that this hypothesis should be further investigated. Supporting this idea, an ABC protein has been shown to be responsible for the transport of the benzylisoquinoline alkaloid berberine across the plasma membrane of Coptis japonica [163]. Moreover, a gene encoding an ABC transporter has been cloned in C. roseus, whose expression in cell cultures is enhanced by the addition of citokinins, methyl jasmonate and auxin suppression, all conditions that increase the production of terpenoid indole alkaloids by the same cell suspensions [164].
837
MECHANISM ALKALOIDS
OF
ACTION
OF
THE
CATHARANTHUS
Vinca alkaloids are cytotoxic to most cells (including plant cells) with a stronger effect in actively dividing cells like cancer cells. They have been shown to interfere with several distinct cellular processes like protein synthesis and degradation, lipid metabolism and calcium movements [13, 165, 166], but, until now, their main target in what concerns their cytotoxicity to cancer cells is still considered to be the microtubules, with consequent effects in mitosis. After the discovery and clinical application of vinblastine and vincristine as anti cancer drugs, it was soon pointed out that the anticancer action of these compounds seemed to be due to microtubule depolymerization and mitosis arrest as a result of the absence of mitotic spindle [167-169]. The Vinca alkaloids were thus classified as mitotic blockers with their primary site of action being M phase of the cell cycle. However, more recently, it has been shown that although Vinca alkaloids depolymerize microtubules at ^imolar concentrations, they actually have a more subtle effect at low concentrations (nanomolar range), stabilizing microtubules due to inhibition of normal microtubule dynamics [170, 171]. This effect, in actively dividing cells, seems to be able to induce a program of cell death [172-174]. The antineoplastic activity of Vinca alkaloids in the clinical treatment of cancer may thus arise from perturbation of a variety of microtubule-dependent processes, including the cell cycle, ultimately inducing programmed cell death. The complexity of microtubule behavior and the difficulty in determining exactly how occurs the interference of Vinca alkaloids with that complexity has made it very difficult to characterize the precise anticancer mechanism of action of these drugs, which is still not fully understood. A thorough and clear review on what is known about microtubule behavior and the way Vinca alkaloids interact with microtubules and the cell cycle has been published very recently by Mary Ann Jordan [172]. Here, a more general overview will be presented, trying to highlight adequately the main points of this difficult subject.
838
Interaction of Vinca alkaloids with tubulin and microtubules Microtubules are highly dynamic protein polymers present in all eucaryotic cells, with important roles in the determination of cell shape, in cell signaling, in cellular and intracellular movements, and in cell division. Microtubules are built from dimeric subunits composed of two similar globular proteins, a- and P-tubulin, that are stacked in 13 linear chains arranged in parallel to form hollow tubes with 25 nm in diameter and a variable length that may reach many u.m. Cells contain a mixture of free tubulin dimers and microtubules that undergo continual remodeling by constant assembly and disassembly of tubulin subunits at the microtubule ends. During mitosis, the microtubules become organized in two arrays that form the mitotic spindle, responsible for the precise distribution of chromosomes between the two daughter cells - during this process the microtubules suffer constant and dramatic reshaping [175]. Vinblastine can bind with high affinity to the dimeric tubulin subunits and the microtubule ends (Kd =1-2 uM), and with lower affinity (Kd = 0.25-0.3 mM) to tubulin sites located along the sides of the microtubule cylinder [172, 176]. This binding of the Vinca alkaloids along the length of the microtubule must be the responsible for their ability to depolymerise microtubules at high concentrations (> 1 uM). Among vinblastine, vincristine and vmorelbine, vincristine demonstrates the highest overall affinity for tubulin and vinorelbine the lowest [177]. Vinflunine has been shown to bind to tubulin with even lower affinity than vinorelbine [178]. Affinity is thus ranked vincristine > vinblastine > vinorelbine > vinflunine, parallel to drug toxicity, but not to therapeutic potential. These values correlate inversely with drug doses used in clinical treatments, since vincristine is used at the lowest dose and vinorelbine at the highest. Binding of Vinca alkaloids to tubulin is reversible, and the precise location and number of binding sites in tubulin dimers and microtubule ends is not clear. Rai and Wolff [176], using a fluorescent vinblastine derivative, detected and characterized a single high affinity binding site in P-tubulin and also detected the presence of several low affinity sites that were not possible to characterize. More recently, nuclear magnetic resonance analyses revealed the presence of three binding sites in the oc/ptubulin dimer for vinorelbine and vinflunine [179]. At 30° C, binding of
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vinflunine to tubulin was hardly detected, in agreement with the significant lower affinity of this drag to tubulin. In reconstituted microtubule systems and in cells, the interaction of Vinca alkaloids with microtubules results in different effects depending on drug concentration: i) at low concentrations (< 1 uM = nanomolar range), the drugs diminish microtubule dynamics increasing the time spent in the "resting state"; ii) at intermediate concentrations (1-2 uM), they depolymerize microtubules and inihibit assembly; and Hi) at high concentrations (> 10 uM), they induce self-aggregation of tubulin with formation of large paracrystals and other aggregates [172, 176]. In all cases, normal microtubule function is compromised. In spite of all that is known about the interaction between Vinca alkaloids and tubulin, the precise molecular location and chemical bonds established during that interaction are not known what prevents a rationale design of new Vinca alkaloids. The different therapeutic profiles and toxicities of the Vinca alkaloids are thought to be partially due to the different affinities they show towards tubulin [172, 177], namely towards different tubulin isotypes that may have tissue specific expression [180]. It is interesting to remark that, although vinorelbine and vinflunine show significant lower affinity to tubulin than vincristine and vinblastine, a fact correlated with their lower toxicity, they actually present a higher anticancer therapeutic action, a feet correlated with their higher intracellular accumulation (see section "Uptake..." below) [23, 25, 181]. This means that vinorelbine and vinflunine present a differential effect between normal cells, where lower affinity to tubulin prevents toxic effects in spite of Mgh intracellular concentrations, and cancer cells, where the weak interaction of the high concentrated drugs is sufficient to induce a strong effect. There is no explanation for this feet but Ngan et al. [181] consider that nontumor cells, with normal checkpoint proteins, may tolerate better the relatively less powerful inhibitory effects of vinflunine and vinorelbine on microtubule dynamics, than cancer cells, with abnormal cell cycle regulation. Mechanism of inhibition of cell proliferation The mechanism of action of the Vinca alkaloids was initially thought to involve the depolymerization of spindle microtubules and induction of
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paracrystalline tubulin-F/«ca alkaloid aggregates, the effects observed at intermediate and high concentrations of the drugs [167-169]. However, the more recent observation that, at low concentrations, the Vinca alkaloids inhibit the microtubule dynamic behavior raised two questions: i) whether this effect could also block mitosis and ii) which of the two situations is actually involved in anticancer therapy with this group of drugs. Investigating this question, Jordan and collaborators [182] observed that, at the lowest effective concentrations of five Vinca alkaloids, inhibition of cell proliferation and blockage of HeLa cells at metaphase occurred with little or no microtubule depolymerization and no spindle disorganization. With increasing drug concentrations, the authors observed that the organization of microtubules and chromosomes started to deteriorate. Dhamodharan et al. [183] also observed that low vinblastine concentrations (nM levels) block mitosis in BS-C-1 cells, in association with suppression of microtubule dynamics but in the absence of appreciable changes in microtubule mass. Recently, it has been shown that the precise parameters of microtubule dynamics that are inhibited at low concentrations by vinorelbine and vinflunine differ significantly from vinblastine [181]. In spite of those differences, further investigations showed that all the three drugs produced remarkably similar effects on spindle organization. In all cases, proliferation inhibition seemed to be induced my mitotic block at the metaphase/anaphase transition with formation of aberrant spindles, consistent with induction of block by suppression of microtubule dynamics [23]. As a whole, the results presented above indicate that low concentrations of Vinca alkaloids, probably similar to therapeutic concentrations, have an antiproliferative activity that is due to inhibition of mitotic spindle function by changing the dynamics of microtubules rather than by depolymerizing them. A growing body of evidence seems to indicate that, specially in cancer cells, where mitosis regulation is already disrupted, the suppression of microtubule dynamics with mitosis arrest induces a signaling cascade leading to cell death by apoptosis (a type of programmed cell death) [172-174]. Other cellular targets of Vinca alkaloids Vinca alkaloids are toxic molecules that easily cross cellular membranes due to their lipophilicity and interfere with a multitude of cell targets.
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Although tubulin and microtubules are undoubtedly the main target responsible by their antieancer action, other targets may also contribute to this action, while some others may be associated with their toxic side effects. Recently, it has been shown that proteasomes, the proteolytic machinery of the ubkjuitin/ATP-dependent proteolysis pathway, can also be considered a target of vinblastine. Proteasomes have a crucial role in the regulation of the cell cycle, and proteasome inhibitors can block cell cycle progression and induce apoptosis in certain cell lines. Vinblastine seems to have an inhibitory effect on proteasomes and could thus interfere with mitosis also through this path [165]. Another target of Vinca alkaloids seems to be DNA. Tiburi et al. [184] showed that vinblastine, vincristine and vinorelbine all had significant genotoxicity, as assayed by the wing Somatic Mutation and Recombination Test (SMART) of Drosophila. All the three drugs caused increments in the incidence of mutational events and somatic recombination. An effect of Vinca alkaloids that may also be important in their anti tumour activity is their antivascular action. It has been shown that vincristine and vindesine are able to reduce the capillary network formation by HUVEC cells cultured on Matrigel at non-cytotoxic concentrations, while vinblastine and vinorelbine produce anti-angiogenic effects by direct cytotoxicity [185]. Vinflunine seems to have an antivascular activity consistently superior to that of vinorelbine [25]. UPTAKE AND EXTRUSION ANIMAL CELLS
OF
VINCA
ALKALOIDS
IN
Vinca alkaloids are lipophilic molecules that can readily cross membranes by simple diffusion [186]. Experiments performed with several human cancer and tissue cell lines have shown in all cases rapid uptake of every one of Vinca alkaloids [23, 187-189]. Uptake is thought to occur by diffusion although energy dependence or independence of the uptake is seldom mentioned in reports. However, for instance in cultured human promyelocytic leukemia HL-60/C1 cells it has been shown that rates of uptake of vinblastine were unaffected by depletion of cellular adenosine triphosphate, reinforcing that uptake is not mediated by an energydependent system [189].
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When incubated with human hepatocytes, vinorelbine was the most rapidly and intensely accumulated Vinca alkaloid followed by vinblastine, vindesine and vincristine, as would be suggested by the lipophilicities of the molecules [187]. Vinflunine is even more ipophUic than vinorelbine and accumulates fester inside cells [23]. Although uptake is considered to occur by diflusion, the Vinca alkaloids accumulate inside animal cells to concentrations many times higher than extracellular concentrations. Vinblastine and vincristine have been shown to accumulate more than 100 fold in cultured human promyelocytic leukemia HL-60/C1 cells [189]. Addition of 10 nM vinblastine to the culture medium of HeLa cells resulted in a 40 fold accumulation, while addition of 100 nM vinblastine resulted in a 31 fold accumulation [182]. In BS-C-1 cells, 32 nM vinblastine accumulated 284 fold [183]. This intracellular accumulation is thought to result, at least in part, from the binding of the drugs to tubulin and microtubules [172], For example, the maximum vinblastine intracellular levels observed in HeLa cells are similar to the intracellular levels of tubulin [182]. However, the existence of other intracellular reservoirs for drug accumulation is not discarded and, recently, some evidence has been obtained that suggest that vinorelbine and vinflunine may be sequestered inside the ceE by other mechanism than binding to tubulin [23]. The authors characterized uptake of 1 nM vinblastine, 3 nM vinorelbine and 30 nM vinflunine by HeLa cells. The concentrations used for each drug were the ones inducing the same effect in cells, i.e., a 50% inhibition of mitosis. As predicted from their lipophilicity, uptake rate was much higher for vinflunine, followed by vinorelbine, and the peak concentration for the three drugs was 4.2 pM for vinflunine (140 fold), 1.3 uM for vinorelbine (430 fold) and 130 nM for vmblastine (130 fold). In these conditions, mitosis was blocked but microtubules were not disassembled. Since micromolar concentrations of vinorelbine and vinflunine significantly reduce microtubute polymerization in vitro [181], the authors suggest that not all intracellular vinflunine and vinorelbine is available to bind to tubulin and must be sequestered in other cellular reservoirs such as membrane compartments [23]. In animal models, concentrations in tissues can also exceed those in plasma, and it has been observed that, in certain tissues, the drugs are retained for prolonged periods, sometimes related to the specific therapeutic indications of each drug [190]. In mice, vinblastine is
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selectively retained in genetic tract and lymphatic tissues, a fact that may be the basis of the activity of this alkaloid against malignant transformations with an origin in these tissues [191]. Likewise, in rats, the lungs were among the organs with higher accumulation of vinorelbine, which is used in the treatment of small cell lung cancer [190]. This differential retention may result in some cases from different relaxation times of the binding of the drug to different tissue isotypes of tubulin, but, in other cases, it seems that the tissues not retaining the alkaloids possess effective means of extruding the drug when plasma levels decrease [191]. Meaning that extrusion of the Vinca alkaloids from animal cells may occur not only by diffusion but also as a result of a more efficient transport mechanism. In many cases, the Vinca alkaloids are just released by diffusion after exposure to the drug ends [189], with a rate dependent on the strength of their binding to tubulin and/or on the release rate from other intracellular sequestration mechanisms. However, as stated above, some tissues possess a more effective way of extruding the drugs, namely cancer cells that have become resistant to chemotherapy. In fact, the best characterized mechanism of resistance of cancer cells to chemotherapy drugs, like the Vinca alkaloids, is the phenomenom known as multidrug resistance (MDR), which is due to the overexpression of the mdrl gene coding for the membrane localized P-glycoprotein, that actively pumps the drugs out of the cell [15, 192]. P-glycoprotein is constitutively overexpressed in various normal tissues including the renal tubular epithelium, the adrenal medulla, the liver, and the blood brain barrier, where it is thought to protect cells from toxic agents/xenobitotics [193, 194]. P-glycoprotein belongs to the family of ABC transporters (see section "Accumulation ..." above) and it is localized in the plasma membrane, being able to extrude from the cell a variety of structurally diverse drugs, drug conjugates and metabolites. Extrusion of these compounds by P-glycoprotein is ATP-dependent and can take place against considerable concentration gradients [192]. P-glycoprotein expression may occur in tumour types derived from tissues that normally express the protein, like renal cell cancer, but its overexpression may also be induced by the treatment with anticancer drugs. All Vinca alkaloids used in cancer therapy can induce the expression of P-glycoprotein and the associated multidrug resistance phenotype, due to the capacity of P-glycoprotein to pump out of the cell a
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number of unrelated anticancer drugs, preventing therapeutic intracellular concentrations to be achieved [15]. METABOLISM OF VINCA ALKALOIDS IN ANIMAL CELLS Vinca alkaloids are metabolized primarily by the liver, and metabolites are eliminated by biliary excretion [15]. When incubated with human hepatocytes in suspension, vinblastine, vincristine, vindesine and vinorelbine are rapidly taken up and intensely metabolized by the cells in a number of unidentified products [187]. On the other hand, the capacity of cancer cells to metabolize these drugs is usually limited. For example, HPLC analysis of extracts of human promyelocytic leukaemia HL-60/CI cells incubated with growth-inhibitory concentrations of labelled vinblastine and vincristine indicated little or no metabolism of either drug by cells or culture fluids [189]. In the liver, the only and possibly main enzymatic system that has been shown to be involved in metabolism of Vinca alkaloids is the cytochrome P450 monooxygenase CYP3A4 [195-197]. The large interpatient variability in the sensitivity to the Vinca alkaloids has been frequently associated to individual disparities in the levels of CYP3A4. Recently, it has been shown that tumour CYP3A4 may also contribute to the development of drug resistance during chemotherapy [196]. Another enzyme that has been implicated in the metabolism of vincristine in acute myeloblastic leukaemia (AML) cells is myeloperoxidase. The fact that AML is resistant to vincristine has been related to the presence of mieloperoxidase, which is able to catalize the vincristine's oxidative breakdown [198, 199]. The compounds resulting from metabolism of the Vinca alkaloids are little characterized. The main hepatic metabolite of vinblastine, vincristine, vinorelbine and vinflunine seems to be, for each of the compounds, the respective 4-O-deacetyl alkaloid. Other metabolites have also been detected but only very seldom were structurally characterized [15, 191, 200-203].
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THE VINCA ALKALOIDS FROM PLANTS TO ANIMALS - THE EVOLUTIONARY LINK The Vinca alkaloids metabolism and transport in the producing plant cells and in the treated animal cells illustrate some interesting aspects of how evolution can be winding and parsimonious in the solutions it creates. Plants possess an incredibly diverse biosynthetic capacity leading to the production of a myriad of compounds that, although not having an apparent function for fundamental life processes (growth, development and reproduction), seem to have vital roles as mediators of ecological interactions, being very important for the survival of plants. This chemical wealth is the basis of the use of plants in medicine, and is still largely unexplored. One example of application of the so called plant secondary metabolites are the terpenoid indole alkaloids of Catharanthm roseus, used in cancer therapy, and known as the Vinca alkaloids. In the plant, the biosynthesis of the Vinca alkaloids involves more than 20 enzymatic steps including several cytochrome P450 monooxygenases and one class III plant peroxidase. Removal of the toxic alkaloids from the cytoplasm to the vacuole of plant cells is made by an uncharacterised transport mechanism that we suggest may be an ABC transporter, as already shown for several other plant secondary metabolites. In human and model animal cells, metabolism of the exogenously applied Vinca alkaloids is carried out by a cytochrome P450 monooxygenase and in some cases by myeloperoxidase, and removal of the toxic alkaloids from the cytoplasm to the extracellular compartment is made by an ABC transporter. The mechanisms involved in metabolism and transport of the Vinca alkaloids in animals are thought to have developed, at least in part, as a defence against the toxic compounds present in the plants that animals eat [204]. The similarities between mechanisms involved in production and accumulation of toxic defence compounds in plants, and metabolism and transport of the same compounds as xenobiotics in animals, mean that essential building blocks of such complex and divergent organisms as plants and animals have a very ancient common origin, and that, through evolution, they were sometHnes recruited to functions that oppose to each other in nature. The P450 superfemiry, for example, is found in all groups of organisms, including Archae, and is believed to have originated in an
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ancestral gene that existed over 3 billion years ago [205]. The ABC transporter superfamily is also referred as one of the biggest multigene families and it has been shown to exist in bacteria, fungi, plants and animals [161, 194, 206]. Single ancestor genes have thus suffered major duplication events and evolved to result in a panoply of functions. To construct the amazing diversity of life, evolution has sometimes played with a small number of pieces to construct the biochemical survival strategies inherent to that diversity. ABBREVIATIONS D4H DAT PRX G10H NMT OMT ORCA SGD SLS STR T16H TDC TIA
desacetoxy vindoline 4-hydroxylase acetylcoenzyme A : 4-O-deacetylvindoline 4-0acetyltransferase peroxidase geraniol 16-hydroxylase S-adenosyl - I-methionine : 16-methoxy - 2,3-dihydro-3hydroxytabersonine - iV-methyltransferase S-adenosyl - Z-methionine : 16-hydroxytabersonine - 16O-methyltransferase octadecanoid responsive Catharanthus AP2-domain protein strictosidine p-D-glucosidade secologanin synthase strictosidine synthase tabersonine 16-hydroxylase tryptophan decarboxylase terpenoid indoe alkaloid
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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THE CHEMISTRY OF OLEA EUROPAEA Armandodoriano Bianco, Alessia Ramunno Scuola di Specializzazione in Chimica e Tecnologia delle Sostanze Organiche Natvrali - Universita di Roma "La Sapienza ". Piazzale Aldo Moro 5, Roma, Italy. ABSTRACT: This review summarises the chemistry of Olea europaea, in particular of hydrophilic components, which are seen as the key compounds responsible for several properties, identified as the organoleptic characteristics of foods derived from O. europaea and as the biological properties attributed to this plant, like ipotensive, anti-oxidising, etc. In fact, two groups of polar compounds can be identified in O. europaea: terpenenoids, as oleuropein; phenols, as hydroxy-phenyl ethanol derivatives; and compounds related to these two categories. Oleuropein and its metabolites are terpenoids present in all parts of plant and represent the chemotaxonomic markers of O. europaea. Tyrosol, hydroxytyrosol and related compounds represent the main characteristic phenolic fraction of O. europaea and are recognised as very powerful anti-oxidising agents. The chemistry of oleuropein and of other phenolic components of O. europaea is described, in relation also with the biological activity of these compounds. O. europaea represents, in fact, a key plant in the economy of Mediterranean region, and olives are the key component of Mediterranean diet. Olives, olive oil in particular, represent a unique food in the scenario of Mediterranean region and they certainly have contributed to the evolution of civilisation of this part of the world. 1. Introduction 2. Chemistry of Olea europaea 2.1. Molecular composition 2.2. Molecular modifications 3. Biological activity and Pharmacology of Olea europaea 3.1. Antioxidative and radical scavenging effects of olive phenols 3.2. Effects of oleuropein on gastric mucosa 3.3. Effects of olive components on glycaemic and blood pressure controls. 3.4. Antimicrobial activity 4. Bibliography
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1. INTRODUCTION Olea europaea is a typical plant of Mediterranean region created, according to the classical tradition, by Athena, the Greek goddess of Knowledge. The history of olive-tree is a fascinating story. We could think that an olive branch announced to Noe that the Deluge was finished (Genesis 8, 10-12). An olive-wreath had been reward for the winner in Olympia's games in Greece and was present in lustral ceremonies in Rome. Olive-tree was not only the symbol of Justice (Psalms 54,10) and Knowledge (Siracide 24, 14) for Jews, but was also the image of prosperity and beauty (Geremia 11,16). Honour is given to men and gods by the olive oil (Giudici 9, 9). We can find olive-tree and olive oil in every important moment of Jesus Christ's history. Jesus prayed in the Garden of Olives, Maddalena (Luca 7, 36-56) as Maria of Betania (Giovanni 12,1-3) covered Jesus's feet with olive oil. Islam also considers olive-tree a sacred plant (Holy Koran, sura XCV, 1; sura LXXX, 29; sura XVI, 11). The olive has followed the evolution of the civilisation, starting from the Middle East to the Mediterranean region until the New World. Olive still symbolises the Peace. Literature too considers it. Dante in the Divina Commedia talks about tiie olive for three times: twice in the Purgatory and once in the Paradise but never in the Hell. So this plant appears to be at the borderline between research and history. Why? In our opinion, it is because olive tree is a living organism, having a rather deep significance. Following these considerations, it is easy to understand that studying O. europaea is not a simple molecular recognition but rather it needs a widespread research. 2. CHEMISTRY OF OLEA EUROPAEA In the O. europaea, we may contemporaneously observe two aspects of natural products chemistry: the study of molecular structure (the compounds present in the olive), and the study of the chemical reactivity (as we will describe later, these same compounds are more or less modified during the technological process of olive oil production).
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In the olive, two main groups of compounds are present: fats, as glycerides and lipids, and phenols and related substances. This article discusses about phenols and their related polar compounds that are typical of this plant and that have been until now slighted respect to those constituting the main part of the olive, such as glycerides and fatty acids. Before 1960, one of main components of olive, the oleuropein, was studied in Roma. Panizzi's research group established the structure of this monoterpene glucoside, the leader of the group, that only several years later was identified as secoiridoids. Panizzi's group has isolated two other compounds: the oleuropeic acid that is a monocyclic monoterpene, and its saccharose derivative. That was the beginning of the scientific discovery of the olive tree. In fact, in the pioneering researches of Panizzi, the polar and phenolic fraction of olive was forgotten. In 1993, we began to face up the isolation of olive glycosidic fraction from a systematic point of view, using a specific protocol for polar compounds, and succeeded in the isolation of many glycosidic components present in olive. In the olive, there are two main compounds, besides that reported in the literature. The first one is oleuropein, the first secoiridoid isolated in 1960 by Panizzi in Roma, as mentioned above. The second one is the cornoside that is a hemiquinone glucoside, structurally and biogenetically related to hydroxy-tyrosol that is the principal free phenol in the olive. It should be remembered that, depending on the olive cultivar, the oleuropein can be prevalent against the cornoside, or it could be the contrary, until reaching the equality. With the same protocol, many minor components were also isolated. Some of them are new compounds, while other known. Without dealing here with the problems of isolation and purification, we will list their molecular structures that generally were identified using all possibilities offered by NMR, coupled with chemical manipulation. Firstly, we report on the three hydroxy-tyrosol glucosides that are present in the plant. Other minor glycosidic components are glucosides of tyrosol. In addition, we isolated the verbascoside, a phenyl-propanoid glycoside that is characterised by the presence of hydroxy-tyrosol moiety. Different analogues of oleuropein, as the isomer of oleuropein at the double bond in 8-9 position, were isolated. We have found also polar, but non-glycosidic compounds, i.e. free tyrosol and hydroxy-tyrosol and the aglycone of cornoside, halleridone. Finally, one more thing to mention is that phenols, in the olive, are not only linked to a polar substrate, but to non-polar ones
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as well. Recently, we have isolated an oleil derivative of tyrosol that is present in significant amounts in the olive. 2.1. Molecular Composition The molecular composition of O. europaea, with respect to the phenolic and polar fractions, is quite characteristic, being based on the presence of a phenolic moiety that can be inserted in a terpenoid skeleton, as in oleuropein and related secoiridoids, or may be alone with the presence or absence of a sugar unit. 2.1.1. Oleuropein. Oleuropein 1 is the first secoiridoid whose structure was recognised in 1958-65[l-3], but only several years later it was classified as secoiridoid, when this class of monoterpenoids was constituted.
Figure 1. Structure of oleuropein 1
Oleuropein structure was determined by Panizzi et al.[2] on the basis of the results obtained from an accurate degradation of this compound. Only the absolute configurations of C-l and C-5 remained undetermined, as the cis/trans configuration of C-8/C-9 double bond. Hinouye determined some years later [4], the absolute configuration of chiral centres of the secoiridoid oleuropein 1, relating 1 to the iridoid asperuloside 2. Bisdeoxy-acetyl asperuloside 3, prepared by hydrogenolysis from asperuloside 2, was opened by a series of oxidative steps to the two
863
secoiridoid dimethyl esters 4a and 4b» having racemie C-8 centre with hydroxyl in both R (4a) and S (4b) configurations. Dehydration of these two secondary alcohols afforded two possible olefins: 5-ateohol 4b afforded the lf-olefin 5b, while Jl alcohol 4a gave the Z-olefin 5a.
Figure 2. Absolute configuration of Oleuropein 1
A simple work up on oleuropein 1, transesterification with methanol of ester function at C-7 afforded the J£-olefin 5b, so demonstrating the absolute configuration of the C-8/C-9 double bond, as well as all chiral centres of oleuropein 1. A similar approach was used for the partial synthesis of oleuropein 1 [5] that was depicted in Figure 3, The chiral starting product was a
864
glucosidic iridoid, the bisdeoxy-asperuloside 6. The partial synthesis starts with the protection of compound 6 with benzyl groups that can easily be eliminated at the end of the synthesis in mild hydrogenolytic conditions that do not interfere with ester functions of oleuropein. After protection, compound 6 is osmilated to diol 7. COOCHj
COOCH, 3 !
COOCH,
RO
HO OH
Figure 3. Partial synthesis of oleuropein 1.
This last compound was selectively oxidised at the vicinal diol function with sodium periodate and successively with Jones's reagent, affording the desired acid 8. Direct esterification of compound 8 with dioxy-phenyl-ethanol 14, having phenolic functions protected with benzyl moieties, gave the key intermediate secoiridoid 9. The last step of the synthesis is the stereoselective reduction of 9 that afforded the alcohol 10
865
as a single product, with the desired (S) absolute configuration at C-8 centre. The compound 10 was then dehydrated, giving the C-8/C-9 double bond with the correct configuration. The final removing of the protecting benzyl groups by hydrogenolysis afforded the oleuropein 1. Oleuropein 1 has been a recognised chemo-taxonomic marker of O. europaea; however its presence in olives is attributed to the ripening stage of fruits. Our recent experimental data were achieved for some Spanish, Portuguese and Italian cultivars, which were examined in different stages of ripening (green, cherry and black) [6,7]. These data confirmed the previously obtained results [8,9], with a decrease of oleuropein 1 during olive maturation and a contemporary increase of oleuropein derivatives. 2.1.2. Cornoside. Cornoside 11 is a glucoside that could be biogenetically related to hydroxy-tyrosol, the main phenolic component of O. europaea.
Figure 4. Cornoside 6
Its presence in olive was reported for the first time in 1993 by Scarpati et al. [10], who isolated 11 from leccino cultivar. Successively Bianco et al. demonstrated that cornoside 11 (Fig.4) constitutes, together with oleuropein 1, one of the main glucosidic phenolic components present in O. europaea [6,7]. Depending on the olive cultivar, the oleuropein 1 can be prevalent against the cornoside 11, or it could be the contrary, until reaching the equality [6,7]. Oleuropein 1 appears to be present in larger quantities in olives original of Italy and Greek (27-28%) compared to the quantities (18.521%) detected in olives of Spain and Portugal [7]. In addition, 1 appears to be the main glucosidic component of olives original of Italy ("Taggiasca" "Carolea" and "Cassanese" cultivars) and Greece ("Thasos" and "Conservolia" cultivars). The main glucosidic component of olives, originated from Spain and Portugal, appears to be, on the
866
contrary, cornoside 6 (24.5% in "Hojiblanca" and 23.5% in "Douro" cultivars) [7]. The determination of the profile of the phenolic fraction was performed for the first time by Bianco et al. [7] by nuclear magnetic resonance of proton, on different olive cultivar samples, appropriately selected. Obviously, the proposed NMR determination of the phenolic profile is not an alternative to HPLC procedures, but constitutes a rapid, alternative methodology to examine the phenolic contents in relation to the main components. In fact, the sensibility of NMR technique does not evidence components that are present in quantities less than 5% of the total. Comoside 11 was first isolated from Forsythia genus of Oleaceae by Jensen et al. [11]. Its presence in O. europaea and its structure were demonstrated by spectroscopical methods, examining, in addition to NMR date of 11, date, of the compounds mixture also obtained by enzymatic hydrolysis of 11 (Fig. 5) [10].
Figure 5 Enzymatic hydrolysis of cornoside 11
The aglycon 12 of cornoside 11 is in fact in equilibrium with an addition product 13 that is known as rengyolone [12] or halleridone [13], and it seems to be a natural compound and not an extraction artefact. We consider comoside 11 to represent, together with oleuropein 1, the chemotaxonomic marker of oleaceae, being present in several species and always constituting the main glucosidic component [7], 2.1.3. Tyrosol and Hydroxy-tyrosol Glucosides. Tyrosol and hydroxy-tyrosol glucosides (Fig.6) constitute the principal part of minor phenolic components of olive. In fact, hydroxy-tyrosol 14 and tyrosol 18 are present in all parts of O. europaea. It seems that glucose links ail hydroxyls of phenol moiety without an evident
867
preference. This can be related to the necessity of allowing a high hydro solubility to this phenolic fraction of olive, as in the general concept of glycosylation in natural products. Hydroxy-tyrosol glucosides were isolated from different olive cultivars, and their relative quantities seem to be in relation with the olive organ considered [14]. 1 OH
14 OH HO
OH
OH
Figure 6. Hydroxy-tyrosol glucosides
Compound 15 was first identified in Osmanthus asiaticus, a plant of Oleaceae family [15]. Compound 16 was isolated in Primus grayana, a plant of rosaceae family [16] and in Ricciocarpus natans, Ricciaceae [17]. Compound 17 was assumed to be present in O. europaea [18,19], on the basis of chromatographic considerations and was identified [14], together with 15 and 16[14], in carolea cultivar. Structures of compounds 15-17 were determined by spectroscopical method, essentially 'H- and 13C-NMR spectroscopy. Comparison between 13 C-NMR data of glucosides 15-17 and hydroxy-tyrosol allowed to determine the site of glucosylation and therefore the structure of these compounds. Tirosol glucosides (Fig. 7) are also present as minor components in olive, but their relative quantities do not seem related to the olive organ considered [20].
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Compound 19 is also known as salidroside. The isolation of salidroside from O. europaea had been already described [21-22] but needed an unambiguous identification.
Figure!. Tyrosolglucosides
The structure of 19 was determined by proton and carbon magnetic resonance experiments. 'H-NMR and I3C-NMR spectra unambiguously determined the nature of the aglyconic and the glucosidic parts of the molecule and the relative positions of glucose moiety. In particular, the 'H-NMR spectrum revealed, besides the aromatic protons resonances in the range 6.69-7.15 ppm, the -CH2-CH2- methylene part as triplets centred at 2.80 and 3.75 ppm. Moreover, the spectrum showed a doublet at 4.38 ppm due to the anomeric proton of glucose moiety that produces resonance signal in the range 3.10-3.70 ppm. The site of glycosylation was determined, comparing the 13C-NMR spectrum of compound 19 to the tyrosol one. In the 13C-NMR spectrum of 19, the deshielding of the primary alcoholic function (about 10 ppm) and the shielding of the C-lp (about 1 ppm) typical of the glycosidation effect were noticed. Compound 20 [23] was also detected in comparable quantities with respect to 19, and its structure was determined, as did for 19, by spectroscopical methods. 2.1.4. Oleuropein Related Compounds. Several minor oleuropein related compounds were isolated in olive, reported in Figure 8. Compound 21, demethyl-oleuropein, is a hydrolysis product of conjugated methyl ester present in oleuropein. The presence of this compound increases during the olive ripening [6-9]. Compound 22, ligstroside, is very similar to oleuropein from which it differs for the presence of a tyrosol unit instead of hydroxy-tyrosol [24].
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Compound 23, oleoside (see Figure 9), is the dimethyl ester, corresponding to oleuropein, where methyl alcohol esterifies the carboxyl group at C-7 also [25]. COOMe HO
XJ
COOH
l
4
21
OH
Figure 8. Oleuropein related compounds
In compound 24, oleuroside, a modification at the level of monoterpenoid unit is present. In fact, the C-8/C-9 double bond of oleuropein is shifted to C-8/C-10 position, as in the secologanin. The occurrence of 24 in O. europaea seems to be in contrast with the biogenetic trend of this plant that appears to be devoted to the a molecular structure that was classified as oleuropein-type [26,27]. MeO
COOMe
HO
0
Y ° COOMe
HO
23
24
Figure 9. Oleuropein related compounds (continue)
There are also some non-glucosidic iridoids related to oleuropein and have been isolated from O. europaea. Two of them are aldehydes 25 and 26 (see Figure 10) that probably arise from the hydrolysis of the
870
glucosidic moiety of oleuropein with a subsequent rearrangement of the obtained aglycone [28]. OOCH3 25 R=H R'=CH3 26 R=CH3 R'=H
Figure 10. Oleuropein related compounds (continue)
Two other non-glycosidic secoiridoids (see Figure 11) have been isolated from O. europaea. Compound 27 is the elenolic acid, described by Panizzi et al. [2], whereas compound 28 is its methyl ester whose total synthesis was accomplished by MacKellar et al [29], so demonstrating the absolute configuration of these two compounds. OOCH3 27 R=OH
OHC'X 6
28R=OCH3
H3O 'H Figure 11. Oleuropein related compounds (continue)
The o-diphenolic compound 29 (Figure 12) was obtained from ripe black olives by Scarpati et al. [30] and successively reisolated by PaivaMartinsetal. [31].
29 Figure 11 Oleuropein related compounds (continue)
These authors demonstrated that in unripe green olives, oleuropein 1 is present as the major o-diphenolic compound, while in ripe olives, demethyloleuropein 21 predominates. Both these glucosides disappear from olive juice, as they are hydrolysed by native P-glucosidases.
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Demethyloleuropein aglycon rapidly eliminated the carboxyl group, giving 29. 2.1.5. Tyrosol Related Compounds and Other Phenolics. Recently, the major C6-C2 phenolic compounds found in the vegetation water of fruits of O. europaea, Dritta and Cipressino cultivars, were 4-hydroxyphenylethanol 18 and 3,4-dihydroxy-phenylethanol 14, A further hydroxylated compound, the 3,4-dihydroxyphenylglyeol 30 appears to be the 2-hydroxy-derivative of 14. This latter compound (see Figure 13), the formal metabolite of norepinephrine, is first reported as a major component of the olive phenolic fraction by Bianchi et al [32].
30
Figure 13. 2-hydroxy-tyrosol
Phenolic fraction of O. europaea is also constituted by lignans and related compounds. Recently, two new lignan glucosides, (+)-l-acetoxypinoresinol 4"-Me ether 4'-(3-D-glucoside and (+)-l-hydro3y-pinoresinol 4'-P-D-glucoside, together with 2 known glucosides, (+)-acetoxypinoresinol 4-P-D-glucoside and esculin, have been isolated from the bark of O. europaea africana [33]. Flavonoids are also present. De Laurentis et al. [34] isolated and identified a series of flavonoid compounds from the dried leaves of blooming cultivars of O. europaea: hesperidin, rutin, luteolin-7-Oglucoside, apigenin, apigenin-7-O-glucoside, quercetin, kaempferol 2.1.6. Oleuropeic Acid and Oleuropeil Saccharose. In the early researches on O .europaea, [35], Panizzi demonstrated that oleuropeic acid 31, the 4-(l.hydroxyisopropyl)-l-cycloexene-l-carboxylic acid, occurs in the root bark of 0. europaea, mainly as a sucrose ester, i.e. the 6-O-oleuropeil saccharose 32 (see Figure 14). The structure of 31 was determined by comparison with an authentic sample [36-38]. The demonstration of the position of ester linkage in oleuropeil saccharose 32 was achieved with a combination of enzymatic and chemical reactions [39].
872
K>i°
HO
32
31
HO
OH
Figure 14 Oleuropeic acid and oleuropeil saccharose
Alkaline hydrolysis of 32 afforded saccharose and oteuropeic acid, indicating the nature of the glycosidic moiety of 32. Selective hydrolysis of interglycosidic linkage of saccharose with invertase allowed to isolate free fructose together with glucose esterified with oleuropeic acid The esterification site was revealed through periodate oxidation and confirmed by NMR data that showed the classical esterification downfield shin for the protons geminal to the primary alcoholic function of glucose. 2,1.7. Verhascoside. Verbascoside 33, whose structure is reported in Figure 15, is present in O. europaea, as in other oleaceae, demonstrating the ubiquous occurrence of this compound in nature [40]. J3H
33 HO Figure 15. Verbascoside
Verbascoside revealed also a significant antioxidant activity in vitro, indicating that this compound boosts the antioxidant activity of O. ewopaea derivatives.
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2.1.8. Phenolic Esters, The less polar fraction of O .europaea appears to be constituted by phenols esterified by a fat acid moiety, Esterified phenols are not completely soluble in water medium where they give rise to a micellar phase. The main and first component isolated from this fraction resulted to be the oleil ester of tyrosol 34, shown in Figure 16.
Figure 16.1-oleoyl-tyrosol
The structure of 1-oleyltyrosol [41] was determined by simple chemical and spectroscopic methods. The nature of the fatty acid moiety was deduced, besides spectroscopic considerations, by alkaline hydrolysis that allowed to isolate oleic acid. Alkaline hydrolysis allowed to isolate also tyrosol thereby determining the phenolic component The site of esterification was determined by analysing of the 'H-NMR spectrum of this phenolic ester, in which the usual deshielding of about 1 ppm of primary alcoholic protons appeared to give a triplet at 6 4.60; the 13 C-NMR spectrum also shows a deshielding of a carbon of the primary alcoholic function of tyrosol of about 2 ppm typical of esterification
effect 2.2. Molecular Modification Give the molecular composition of the olive's polar fraction, there began an investigation of the molecular modifications of these compounds. Also because this chemical problem is strictly linked to that of the production of the oil and of other foods derived from olive. We in fact know mat the olive is the key-plant in the agrifood system of Mediterranean region countries. Now-a-days the olive oil is a functional food, because it contains not only the fat material, but also high quality compounds, such as phenols. And these compounds come out
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from a complex sequence of chemical reactions that we began to analyse step by step. The particular aspect of the olive oil is the extraction procedure from fruits of the olive. Even if this procedure has undergone several technological innovations, its ground scheme is always the same. We can distinguish three different phases of this extraction: the first one consists in the pounding of the olive; the scutching follows, it consists in making a mix of the olive's paste with water for a variable time (generally for half an hour) at 30-40 degrees. The last step is the separation, with different methods, generally by pressure, of liquid phase from solid residue, and successively of the oil phase from the water phase. This allows to realise a series of several enzymatic reactions. In the first fragmentation's phase of the olives, all the substances present in the fruit are together with the available enzymes. Efficient hydrolases exist in the olive that cleave both glycosidic and ester bonds, letting reactive structures to be free, as the 1,5-dialdehydic functions, inside the oleuropein structure. We observed quickly the hydrolysis of oleuropein, making an NMR study, consisted in putting some drops of the liquid obtained by cutting a ripe olive. On the contrary, in the second phase, the scutching, non-enzymatically catalysed reactions are prevalent, essentially acid catalyses, because in the pounding step, hydrolysis of ester functions produced free carboxylic groups. Last step of olive oil preparation is similar to the process of phase separation from the solid and liquid parts in the first section, and successively from the fat and aqueous phases. These separations are not very accurate and we obtain a product, the olive oil, saving little quantities of water that transfer the phenolic fraction from the olive to the oil. The study of these reactions allowed to acquire a series of data on the chemical modification of oleuropein that is particularly rich in functional groups, and of phenol present in O. europaea. 2.2.1. Chemical Modification of Oleuropein. Enzymatic hydrolysis of glucosidic function allows to free aglycon that exists in different forms, which we revealed by following the hydrolysis of oleuropein 1 in NMR tube [42-43]. The hemiacetalic 35a structure (see Figure 17) is formed as a consequence of the hydrolysis of glucose that appeared to be in equilibrium with the dihaldeidic structure 35b. In the
875
same hydrolytic medium, the hydration of the C-3 formyl occurred giving rise to a compound 35c.
~V^
CHO 35b
OH
CHO 35c
OH
25/26
Figure 17. Oleuropein aglycon rearrangements
Aglycon 35 gives rise also to rearrangement products; the more important ones are 25-26 that are derived by the addition of the enolic form of aglycon to the exocyclic double bond. Gariboldi et al. [28], however, seem to demonstrate in our paper that these compounds are natural ones, deriving from a biogenetic pathway as that followed by Bianco et al [42-43] by enzymatic hydrolysis in NMR tube. After the hydrolysis of esters functions, the hydrolysed aglycons 36a and 37a are, as above, in equilibrium with the dihaldeidic forms, 36b and 37b. A series of non-enzymatic reactions may be observed, as the decarboxylation of the beta-carbonyl acid, with the formation of the decarboxylated aglycon 38a in equilibrium with 38b, (see Figure 18).
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COOH
COOH ^"CHO
^_
CHO
CHO 38a
OH
38b
^CHO
37b
Figure 18. Oleuropein aglycon rearrangements (continue)
Figure 18 shows compounds that are transferred in the oil. Some people insists on searching oleuropein in the oil, cutting of compounds that are more abundant, thinking that oil preparation should be a simple transfer of compounds from olive to oil, instead of a complex series of enzymatic and non-enzymatic reactions. The presence of the various forms of oleuropein aglycon was demonstrated by their isolation from olive oil [42- 43,23]. 2 2 . 2 Chemical Modification ofCornoside. A simple transformation undergoes the cornoside 11 (see Fig 19) that consists in the formation of aglycone 12, followed by two different rearrangements [10,23]. HO /
\ HO
Figure 19. Cornoside rearrangements
This usually happens in mild acid conditions. These two rearrangements are: the first one is hydroxy tyrosol 14, that arises from a
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transposition of tertiary hydroxyl and the second one is halleridone 13, that derives from the addition of a primary alcoholic function to the unsaturated carbonyl function. These reactions were observed in NMR tube. 3. BIOLOGICAL ACTIVITY AND PHARMACOLOGY OF OLEA
EUROPAEA Epidemiological studies in the Mediterranean region evidenced that a diet rich in grain, legumes, fruits and vegetables, wine and olive oil has beneficial effects on human health. In fact, these foods are rich in antioxidant vitamins, flavonoids and polyphenols that play an important role in prevention against cancer and coronary heart disease. It has been now recognised that the phenolic profile of the foods, along with high intakes of the monounsaturated fatty acids, as oleic acid mainly, confers its health-promoting properties to the Mediterranean diet. In fact, olive oil is the main source of unsaturated acids and polyphenols that constitute a complex mixture in olive fruits and in its derived products. In addition, in O. europaea fruits, phenolic profile and content are important factors to consider in order to evaluate virgin olive oil quality. To remain that they are also partly responsible for autoxidation stability and organoleptic characteristics of olive oil. Elevated use of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants, as well as squalene, oleic and other unsaturated acids, should afford significant protection against cancer (colon, skin, breast), coronary heart disease and ageing by inhibiting oxidative stress [44]. In Europe, epidemiological data demonstrated that mortality from breast and colorectal cancer is considerable by lower in countries where olive oil consumption is high (such as Greece, Italy and Spain) than in those where the consumption is low (such as Scotland, England and Denmark) [45]. Pharmacological studies were mainly focussed on oleuropein that represents the key phenolic compound in O. europaea, and on related phenolic compounds such as tyrosol and hydroxy-tyrosol. Therefore, the following discussion will be focussed on reported biological activity of these compounds.
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Oleuropein and hydroxy-tyrosol, derived from oleuropein hydrolysis [46], possess several biological properties certainly mediated by their antioxidant and tree radical-scavenging ability (antimicrobial, hypotensive, vasodilatator and hypoglycaemic activities) [47]. 3.1 Antioxidative and Radical Scavenging Effects of Olive Biphenols In the last years, natural products have been used as antioxidative, melanogenesis inhibitors and sunscreen [48]. Lipid peroxidation is related to aging, membrane damage, heart disease, stroke and cancer in living organism. This oxidative mechanism could be stopped by the addition of synthetic anti-oxidants, but now it has been recognised that natural antioxidants are safe compared to the synthetic compounds [49]. Well-known natural anti-oxidants are represented by ubiquinones, tocopherols and related compounds, flavonoids, cinnamic acid derivatives, Hcopene and related tetraterpenoids, and also by phenolic compounds [50]. The antioxidative effect of oleuropein and hydroxy-tyrosol (see Fig 20) was investigated by Saija et al. [51], in a model system consisting of dipalmitoyl-phosphatidyl-choline/linoleic acid unilamellar vesicles and a water-soluble azo-compound as a free radical generator.
OLEUROPEIN
^ , ^7\L0H
HYDROXYTYROSOL
Figure 20. Oleuropein and hydroxy-tyrosol
This model system studies the antioxidant potential against the attack of oxygen radicals on biomembranes from aqueous phase [52]. Antioxidant effects of olive phenols depend on their interaction with model membranes [53], e.g., oleuropein interacts with DMPC (dimyristoyl-phosphatidyl-choline) membranes. Oleuropein contains a sugar moiety needed to prevent drug access to lipid membranes [54]. In
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fact, lipophilicity of drugs is evidently related to their incorporation with lipids in the model membranes. The interaction observed between oleuropein and DMPC liposomes may be due to the introduction of lipophilic molecules into the ordered structure of the lipid bilayer [55].
Figure 21. lipid bilayer
Drug molecules act as a spacer in such a structure causing a destabilisation of the lipid mosaic. The modification of the fluidity of the model membrane is an important factor for cell membranes functions (Figure 21). Oleuropein appears to interfere with some biological processes such as lipoprotein oxidation, platelet aggregation, platelet and leukocyte eicosanoid production and cardiovascular control too. As previously described, oleuropein and hydroxy-tyrosol are characterised by a catechol moiety that appears to be needed for their scavenger and antioxidant activities. In fact, it was demonstrated that these compounds prevent thermally initiated autoxidation of methyl linoleate in homogenous solutions [56], protect LDL from oxidation [57] and inhibit production of
880
isoprostanes and other markers of lipid peroxidation, occurring during LDL oxidation [58]. The free radical-scavenging capacity of oleuropein and hydroxy-tyrosol was tested, using some radical generators. As a radical initiator, AAPH (2,2'-azobis (2-amidinopropane)-hydrochloride) was used for peroxyl radicals generation, because it is a common free radical found in the body [59] and has often been used in several antioxidant activity assays [60]. It appears to be slightly less reactive than OH-radical [61]. R-+O 2
•
ROO • +LH
• ROOH + L •
L +O2
-LOO-
LOO-+LH
• LOOH + L •
2 LOOLOO-+InH LOO- +In R-N=N -R
—
•
ROO •
N o radical products
• LOOH + In _^ No radical products •
? p -
Table 1. R-N=N-R radical initiator, LH linoleic acid, L linoleic radical, LOO linoleic peroxy radical, InH inhibitor.
Other experimental studies carried out with a different radical, DPPH {l,l-diphenyl-2-picrylhydrazyl radical), revealed that oleuropein and hydroxy-tyrosol elicit a good, concentration-dependent, scavenging effect. Incubation of DPPC/LA {dipalmitoylphosphatidylcholine/linoleic acid) LUV with AAPH increased the accumulation of LOOH (linoleic peroxy acid) formed from LA peroxidation. When the tested biphenols were added, a reduction in the amount of LOOH formed was observed and thus oleuropein proved more effective than hydroxy-tyrosol [62]. The cited works demonstrated that oleuropein and hydroxy-tyrosol are potent antioxidants against lipid peroxidation in phospholipid bilayers, induced by aqueous oxygen radicals. These results may be very interesting, because biphenols could have important applications in human diseases caused by free radical damage. Polyphenols are clearly present in the technological products of olive, such as olive oil. The phenolic contents are related to the peculiar procedure of oil production. In fact, this manufacturing process consists in
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traditional steps that began with washing the olives to remove dirt and other rubbish adhered to the fruit. Then, olives are crushed and the juice is homogenised before pressing. Olive oil is produced under pressures and successive filtration to separate oil from water. After centrifugation, extravirgin olive oil (VOQ) is obtained. High quality oils are bottled directly, but low quality oils (high acidity) are processed once more to obtain refined virgin oil (RVO). Finally, the oil extracted from the residual juice or husk with organic solvents, such as hexane, yields a low-quality refined husk oil (RHO). Several experimental data supported that VOQ contains a higher concentration of three phenolic antioxidants classes, simple phenols, secoiridoids and lignans, and squalene than RVO and seed oils. For these reasons, in the Mediterranean region, where olive oil is an essential constituent of the diet, there is a lower incidence of cancer and heart disease [63]. Probably olive oil components may reduce oxidative stress via inhibition of lipid peroxidation, an interesting mechanism responsible for diseases such as cancer, heart ailment and ageing. The soluble fat should have chemopreventive effects against breast cancer and other diseases. Recent epidemiological studies demonstrated that the components of dietary olive oil might have an important role in the disease prevention, over all against breast and colorectal cancer development [64]. Therefore, it is necessary to establish the olive oil components that are responsible for their protective effects. Once again, there is a significant difference in the concentration of phenolic compounds between VOQ and RVO. Secoiridoids in olive oils is higher in VOQ than in RVO and the concentration of lignans is higher in VOQ than in RVO. The antioxidant effects of natural phenols in olive oils was studied by Papadopoulos [46], adding extracts of a VOQ, having polyphenols, to a sophisticated bleaching oil, washed-out of phenolic antioxidants and evidenced a significant inhibition of auto-oxidation over time in comparison with samples without such an addition. On the other hand, it has revealed that this effect is more pronounced in hydroxy-tyrosol than caffeic acid and protocatechuic acid Reactive oxygen species are associated to xanthme/ipoxanthine system. In fact, the oxidative stress caused by hydrogen peroxide and xanthine oxidase is repressed in the presence of hydroxy-tyrosol, though, unexpectedly, it was inactive in concentrations under 500 umol/L. The
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antioxidant effect of hydroxy-tyrosol is probably due to a dihydroxy substituted phenol ring [65]. Xanthine is one of our organism amino acids and may be catabolised by the xanthine oxidase. This enzyme can attack the amino acid in artery walls so that cholesterol could deposit to heal the lesions. Using the hypoxanthine/xanthine oxidase model to generate reactive oxygen species and comparing VOQ and RVO oils with seed oils, it has been evidenced that scavenging of the hydroxyl radical was higher in olive oil extracts than those of seed oils. In addition, the samples of studied olive oils are also found to be potent inhibitors of xanthine oxidase activity. It is important to underline that phenolic compounds purified from olive oil have a chief antioxidant power than vitamin E and dimethylsulphoxide, standard free radical scavengers both in vitro and in vivo. CO OH .OH
m' 2,5-DHBA
URIC ACID Figure 22. Salicylic acid and its hydroxylated xanthine/hypoxanthine oxidase system.
metabolites produced
by
The studies carried out by Owen [65] revealed not only the difference between the oils in the phenolic fraction composition, but also the degree of diphenol (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid)
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produced by hydroxyl radical attack on salicylic acid. This experiment was executed monitoring the concentration of each phenolic component, the hydroxylation of hypoxanthine and at last the hydroxylation of salicylic acid. Thus, the olive oil samples analysed were dissolved in a phosphate buffer and the end products of the enzyme or free radical reactions were quantitated (see Figure 22). In the olive oil extract, especially in VOQ, lignans were also identified. Experiments carried out on animals have been shown to inhibit cell growth in cancer of skin, colon, and breast. Probably, the lignans anticancer activity is due to the structural similarities with oestradiol and the synthetic antioestrogen tamoxifen; as a result, the lignans have been evidenced to inhibit MCF-7 human breast carcinoma cells increase, induced by oestradiol. Other studies have revealed the squalene existence in the olive oils. The activity of this compound is resulted one of the principal protective agents against skin cancer, probably due to the scavenging singlet oxygen generated by ultraviolet light [66]. These results are confirmed by studies that show a lower incidence of this neoplasm in the Mediterranean population. Furthermore, olive cake, the material left after compression of the fruits, contains triglycerides, oleanane derivatives and some phenolic compounds. Using BHT (butylated hydroxytoluene), ascorbic acid and a-tocopherol as standards for comparison, Amro et al [67] carried out some experimental tests to identify the olive compounds responsible for antioxidant and radical scavenging activities. After a testing period, fraction containing oleuropein showed a significant antioxidant activity, even if the auto-oxidation of the representative fraction of material cake of tested olive was less than those treated with BHT [67]. Moreover, reducing power of olive compounds contained in the examined fractions was greater compared to ascorbic acid, which is known as a good reducing agent. In this case, only fractions containing ferulic acid, cinnamic acid and caffeic acid showed a reducing power higher than the ascorbic acid, because these compounds may act as electron donors and react with free radicals to convert them into more stable products. Table 2 contains the IC5o values of the compounds isolated from olive cake butanol extract and their scavenging effects. The IC» values of BHT, ascorbic acid and a-tocopherol are 37,0 ug/ml, 28,4 ug/ml and 34,2 ug/ml, respectively.
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Protocatechuic acid
ICso 27ug/ml
Coumaric acid Ferulic acid Cinnamic acid Caffeic acid
IC50 135ug/ml IC50 4,5ug/ml IC50 7 ug/ml ICs, 4,5 ug/ml
Oleuropein
IC50 25 ug/ml
Table 2 ICm of phenolic component ofO. Europaea
These results showed that oleuropein has a good antioxidative activity. The beneficial health effects of Mediterranean diet are probably related to non-nutrient components present in foods, such as olive oil, as well as wine and others. In fact, oleuropein and hydroxy-tyrosol, both green and black tea components and gallic acid inhibit androstenedione 6-phydroxylase activity. This enzyme is a CYP3A marker of human liver microsomes and has an important role in the metabolism of xenobiotic substrates [68]. Metabolic reactions occur in the hepatic microsomial cytochromes P450 (CYP) that represent an important source of oxygen radicals, such as superoxide anions, O2", and hydrogen peroxide, H2Q2. In feet, more enzymes needed for chemical compounds metabolism are in the lipophilic membrane of the hepatic reticuloendoplasmatical system. Microsomial oxidation reactions of chemical and natural products occur in an interesting enzymatic system that includes cytochrome P450, cytochrome P450 reductase, NADPH and O2. Cytochrome P450 oxidation cycle has been described in the following Figure 23. Enzymes of cytochrome P450 are haem-proteins related to NADPHcytochrome P450 reductase. All these biological factors are needed for the oxidative reactions. CYP1, CYP2, and CYP3 constitute the P450 enzyme family. Assays carried out in human and in rat liver microsomes by some olive oil phenols demonstrated inhibition of cytochrome P450 activity, specifically of CYP3A and CYP2C11 markers and of reactive species generation.
885 885
Figure 23. Oxidative reactions of cytochrome P4S0
In human liver microsomes, it was evidenced that inhibitors of reactive oxygen species generation are, sequentially, gallic acid, hydroxy-tyrosol and 3,4-dihydroxyphenylacetic acid; while, in rat liver microsomes, the most potent inhibitors were, in the growing order, gallic acid, caffeic acid, pyrogallol, oleuropein, 3,4-dihydroxyphenylacetic acid and hydroxytyrosol. The presence of phenolic hydroxyl groups, for the scavenging radical activity, seems very important, which 3,4-dimethoxyphenethyl alcohol does not for DPPH scavenging activity. For these reasons, a classification of antioxidants has been done: those that inhibit the generation of reactive oxygen species, and those that scavenge the reactive oxygen species generated. Some results evidenced that human CYP3A have a very good capacity to generate reactive oxygen species; therefore, there were interesting results in examining the effects of dietary phenols, such as oleuropein and hydroxy-tyrosol, known CYP3A inhibitors [69].
886
However, it is chiefly underlined that olive oil phenols, such as hydroxy-tyrosol, and others food-derived compounds, such as gallic acid, work as free radical scavengers. Recently, free radicals have been correlated to several diseases for human health. Hydroxy-tyrosol (DPE: 3,4-dihydroxyphenylethanol) is a liposoluble and hydrosoluble compound present in high concentration in extra virgin olive oil, in free or esterified form, such as oleuropein aglycone [42-43]. Several chemical and epidemiological studies confirmed that olive oil's beneficial effects are related to high concentration of oleic acid and the presence of vitamin and non-vitamin antioxidants, such as DPE. This compound shows different biological actions; one of them is the inhibitory effect on peroxynitrite dependent DNA base modification and tyrosine nitration. Furthermore, DPE counteracts cytotoxicity, caused by reactive oxygen species, ROS, in Caco-2 cells and in erythrocytes [70]. In particular, the Caco-2 cells imitate, in vitro, the food-intestinal tract interaction. In fact, these cells have often been used in laboratories to study the molecular mechanism of DPE intestinal transport. To evaluate DPE transepithelial transport cultured Caco-2 cells were used. In this system, cells grow and differentiate on a membrane of polycarbonate, in which the luminal part of the polarised epithelium is oriented. During the experiment, adding [14C] DPE to the compartment, a transepithelial flux of DPE was observed. Moreover, other data suggest that DPE is quantitatively absorbed following its oral administration. The only characterised DPE metabolite was the HMPE (3-hydroxy-4-methoxyphenylethanol), a methylated product of intestinal COMT (catechol-O-methyltransferase) activity. In vitro, DPE, in the presence of a methyl donor S-adenosylmethionine and purified COMT from porcine liver, has a Km lower than that of endogenous substrate, such as dopamine. For these reasons, DPE may be a favoured substrate for COMT in vivo. The obtained data revealed, first of all, the high ease of use of DPE and the relationship between nutritional positive effects of olive oil and the high content of DPE and its precursor, oleuropein aglycone [71].
887
3.2 Effects of Oleuropein on Gastric Mucosae Oleuropein, the major active substance contained in O. europaea, was shown to inhibit prostaglandins biosynthesis and release (see Figure 24). On the other hand, the drug possesses anti-inflammatory action and very interesting effects on gastric mucosae [72]. Prostaglandins (PG) belong to eicosanoid family of poly-unsaturated fatty acids oxygenated products. These compounds show great biological activity; in fact, prostaglandins are chemical mediators that are released during allergic and inflammatory processes. PG, in particular PGE, inhibits the gastric acid secretion. In fact, PGE and its analogues reflect a pronounced protection against steroids and NSAIDs-induced (non-steroidal anti-inflammatory drugs) gastric ulcers. Acetylsalicylic acid (ASA), the pioneer of NSAIDs group, is distinguished from others, as it blocks COX (cyclooxygenase) with irreversible acethylation reaction (Figure 24). Acetate
ASA
^JX (attive)
Acethytated COX (inattive)
Figure 24 Cox's acetylation by ASA
PGE2 and PGF2a stimulate synthesis of protective mucus in stomach and in small intestine. When ASA is administrated, prostaglandins and prostacyclins synthesis is blocked and acid secretion is increased, reducing mucosae protection. COX has two different isoforms: COX-1, expressed in most tissues and implicated in the regulation of normal homeostatic functions such as gastric acid secretion; and COX-2, induced often by inflammatory processes. As a result, COX-2 is implicated in the production of pro-inflammatory eicosanoids (see Figure 25). In fact, one of the therapeutic actions of ASA et similia NSAIDs has been the inhibition of COX activity to reduce pro-inflammatory eicosanoids [73].
888
Arachidonic acid
OOH PGG,
* PGEsmthase
PGE, , PGE S-dietoredutase
COOH
Figure 25. Prostaglandins and tromboxans biosynthesis
In one of the test animals groups, ASA, in 1% carboxymethylcellulose (CMC) suspension, was administrated orally at 150 mg/Kg/24h. In three other test groups, O. europaea extract was given respectively at 25, 50 and 100 mg/Kg/24h one hour before ASA ingestion. The treatment lasted for three days (see Table 3). During this period, they were only allowed to take water. Twenty four hours after the last treatment, all animals were sacrificed and their stomachs removed to evaluate the ulcers developed. Histologic examination has shown that most of the recorded lesions were antral gastritis, hypertrophic gastritis and fundus cystic dilatation. In all these cases, inflammatory signs have been noted, but pre-treatment with O. europaea extract resulted in preventing the lesions induced by ASA.
889
Treatment
Number rats
of Mean scores
% of rats
Ulcer index
presenting ulcers
ASA 150 mg/Kg/24h. for 3
10
1,3
70
91
10
0,8
60
48
10
0,6
60
36
10
0,6
50
30
days ASA 150 mg/Kg/24h. for 3 days + Olea eur. 25 mg/Kg/24h 50 mg/Kg/24h 100 mg/Kg/24h
Table 3. Results of digestive tolerance study
This effect doesn't seem to be dose dependent, especially in animal groups treated by 25 and 50 mg/Kg/24h of the extract, where histologic examination respectively showed 5 and 6 regeneration signs out of 10 surface of epithelium. Probably, this fact corresponds to the activation of epithelium cells multiplication. In group treated with higher doses (100mg/Kg/24h), histologic study indicated a normal mucosae. Since oleuropein was demonstrated to antagonise acetylcholine and E2 prostaglandin's actions on smooth muscle, the anti-inflammatory activity of O. europaea extract was investigated. Moreover, as compounds with oleanic structure, contained in O. europaea leaf, exert anti-ulcer activities, so they were studied for their effect on the rat gastric mucosae. 3.3 Effects of Olive Components on Glycaemic and Blood Pressure Controls The effects of oleuropein on NO release in cell culture and its activity on nitric oxide synthase (iNOS) were also studied. NO (nitric oxide) is a reactive free radical, characterised by numerous important biological functions. Nitric oxide is also an important chemical mediator for vertebrates [74].
890 NH,
NHOH NADPH *O,
NH3 ARGINYL
,
NADP+
XJ.—„ NOS A
L-NMMA
NH 3 N-0-HYDRO XYARGINYL
NOS
NO
CITRULLINE
NITRIC OXIDE
Vasodllatation Immunity regolation Inhibition of platelet aggregation
Figure 26. Nitric oxide activity
NO is both cytostatic and cytotoxic for some pathogens, such as Plasmodium falciparum, Schistosoma mansoni, Leishmania major and Toxoplasma gondii [75]. It is generated by nitric oxide synthase that, in murine macrophages, is induced by LPS (lipopolysaccharide). During incubation of murine macrophagic cells line with LPS and oleuropein, there was a marked production of nitrites. In fact, some reported data show that oleuropein increases the response of macrophages to bacterial lipopolysaccharides, probably due to an increase in iNOS activity. Oleuropein activity on NO production is very interesting and may resolve some pathological problems such as platelet aggregation, thrombosis, vasorelaxation, etc [76]. In fact, NO exerts several effects on cardiovascular system, exhibits vasorelaxant activities and reacts with superoxide to form peroxynitrite (see Figure 26). Flavonoids, phenolic compounds occurring ubiquitously in vascular plants, show a broad spectrum of pharmacological effects. There are a
891
number of medicinal plants used for their anti-inflammatory, spasmolytic, anti-carcinogenic, vessel stabilising or diuretic actions. It is well known that flavonoids interfere with several enzymes, such as adenosine deaminase, cAMP phosphodiesterase, trypsin or cytosolic amino peptidase. There was carried out a screening of flavonoids, phenylacrylic acids and various hydroxylated phenyl acetic acids, urinary metabolites of flavonoids, against three metallopeptidases, containing Zn as cofactor, to study their mechanism of activity in vitro [77]. These ectoenzymes, neutral endopeptidase (NEP), angiotensinconverting enzyme (ACE) and amino peptidase N (APN) are located at the outer membrane of different cells, especially in mammal atrium, are endowed with natriuretic and diuretic properties, and are capable to release the vassal musculature. APN is constituted by 151 amino acids and is synthesized in the cardiac atrial cells; the atrial straining seems to be the most important factor entangled in ANP liberation. This peptide increases in some pathological conditions, such as congested heart failure, renal failure and insufficient ADH secretion. GIU386
Glu-352
Hta-3«5
J
^
Tyr-471
Figure 27. ANP (amino peptidase N).
In fact, ANP increases elimination of Na and urinary flux, glomerular filtering, thus inhibits the secretion of renin, vasopressin and aldosteron. These actions cause an arterial pressure reduction (see Figure 27).
892
NEP inactivates a series of renal and CNS-active peptides, such as substance P, bradykinin, enkephalins and atrial natriuretic factor. It has been well underlined that inhibitors of NEP should be useful in the treatment of pain because of having a large spectrum of activities similar to that of opioid analgesics. In fact, inhibitors of NEP protect the endogenous atrial natriuretic factor degradation and enhance the typical renal effects of ANF on diuresis and natriuretic response (see Figure 28). Converting enzyme is a dipeptidyl-carboxypeptidase that catalyses dipeptide separation from carboxylic terminal of a series of peptides. Its important substrates are angiotensin I that is transformed in angiotensin II, and bradykinin that is inactive (see Figure 28). Angiotensinogen
Kininogen jRcnin
t-PA
/
Kallikrcin/
Angiotensin I *
\ CAGE
* Bradykinin
I
~
Angiotensin II
AT,
receptor
•Vamconttrictioa •EmtoUwlin production
—
[ Degradation products
A T 2 receptor
Bj receptor
B 2 receptor
•V»««
•Vasoeoimnction
•Vuod.maion «NO produclion •PrMt*gl«M)in production
•Collular growth f
Figure 28. ACE (angiotensin-converting enzyme).
Flavonoids have a different inhibitory potency on NEP that depends on chemical characters. In fact, neighbouring hydroxyl groups in 4', 5' instead of 3', 4' reduce inhibitory activity. NEP is inhibited by a free OHgroup at 7 position, because 7-O-glycosides did not show any influence on NEP activity. A reduction of atherosclerotic complications is the major objective of long-term therapy with antihypertensive agents [78]. Several studies were carried out on the effects of oleic acid and its congeners, such as elaidic and stearic acids, and on the structural properties of membranes.
893
Fatty acids and derivatives are abundant in biological membranes as components of phospholipids and cholesterol esters. In fact, their presence, in free or bound form, modulates the lipid membrane behaviour. It was demonstrated, with X-ray diffraction, that oleic acid produced important concentration-dependent alterations of the lipid membrane structure. Oleic acid is capable of altering markedly the phospholipid mesomorphism [79]. Increased visceral fat accumulation is a strong predictor of arterial hypertension. Moreover, the hypothesis that an increase of blood pressure increases also the hepatic portal venous free fatty acid release was explored, which may explain the relation between hypertension and obesity [80]. A clinical assay of O. europaea aqueous extract was carried on two groups of patients suffering with essential hypertension. After three months, a significant decrease of blood pressure and a little decrease of glycaemia and calcaemia was observed for both groups [81]. Obviously the interference of antihypertensive agents with cellular lipid metabolism may modify the atherosclerotic risk of individuals. Therefore, the effects of the Ca2+ antagonists, such as verapamil, diltiazem, m'fedipine, and P-blockers, such as propanolol and metapralol on low density lipoprotein (LDL) receptor activity, cholesterol esterification time, oleate incorporation in triglycerides and sterol synthesis were studied in freshly isolated human leukocytes and in HEP 62 cells (Hepatoma cells). The monounsaturated fatty acids (MUFA) have beneficial effects on glycaemic tolerance in vivo due to increased glucagons-like peptide-1 (GLP-1) release. Lean Zucker rats were subjected to a synthetic diet, containing 5% fat derived either from olive oil (74% MUFA) or coconut oil (87% saturated fatty acids ; SFA) for 2 weeks. This test revealed that the olive oil group had better glycaemic tolerance than the coconut oil in both oral and duodenal glucose tolerance tests. It was also demonstrated that an increased secretion of gut glucagons-like immunoreactivity (gGLI) was needed to value the GLP-1 levels, in the olive oil rats compared to coconut oil rats. Nevertheless, issue levels of GLP-1, plasma insulin and glucagon were the same between two rats treated groups. To demonstrate the mechanism by which the olive oil diet enhanced GLP-1 secretion, oleic acid (MUFA) and palmitic acid (SFA) were added
894
in GLP-1 secreting L line culture for incubation for 24 h. Later, this cells line was challenged with GEP that is known as stimulator of the L-cell. From this test, it was established that oleic acid but not palmitic acid appreciably increased GIP-induced GLP-1 secretion. Hence, diet therapy with MUFAs may be useful for the treatment of patients with impaired glucose tolerance and type 2 diabetes during increased GLP-1 secretion [82]. Q-3 fatty acids lower blood pressure, improve lipids and reduce other cardiovascular disease risk factors [83]. Polyunsaturated fatty acids (PUFA) of the (0-3 or ©-6 series have the potential to regulate serum triglycerides and cholesterol levels that are considered important risk factors in cardiovascular pathologies. These fatty acids play an important role also in the amelioration of autoimmune diseases, such as arthritis, and in the inhibition of the rapid proliferation of cancer cells. However, PUFA in membrane lipids are vulnerable to free radical-initiated oxidation, generated by xenobiotics or normal aerobic cellular metabolism that results in the formation of lipid peroxides [84]. During a study, there has been evaluated the effect of supplementation with a low dose of ra-3, obtained by olive oil, on the oxidative modification of low density lipoprotein (LDL) in a group of healthy volunteers, for 16 weeks. Oxidative modification of LDL was assessed measuring the concentrations of free cholesterol, cholesteryl esters and cholesteryl linoleate hydroperoxide in LDL, following copper-induced lipid peroxidation for 0, 2, 3 and 4 h. LDL eicosapentaenoic acid and docosahexaenoic acid compositions were significantly lower in the group treated with a»-3 olive oil than the group treated with eo-3 fish oil. The levels of palmitic acid, palmitoleic acid, stearic acid and oleic acid increased in both groups, after 4 h of copper-oxidation. While concentrations of cholesteryl oleate, cholesteryl linoleate, cholesteryl arachidonate and cholesteryl docosahexanoate were reduced, following copper stimulated oxidation, in both groups [85]. In conclusion, a regular dietary intake of (a-3 PUFA didn't have any influence on the susceptibility of LDL to copper-induced oxidation, including foods rich in ©-3 PUFA reduced in vivo lipid peroxidation and hence urinary F2 isoprostane excretion [86].
895 (Wiphs-ol TtawT>
LIVER
Figure 29. Triglyceride (TG) absorption transport and metabolism.
As reported in the Figure 29, fat is absorbed and packaged as chylomicrons (CM), and lipoprotein lipase (LPL) releases TG to yield the CM remnants. Very low density lipoproteins (VLDL) synthesised by the liver are broken down by LPL to yield intermediate density lipoprotein (DDL) and ultimately low-density lipoprotein (LDL). High-density lipoprotein clears cholesterol from the cell through lecithin-cholesterol acyltransferase (LCAT). 3.4 Antimicrobial Activity Oleuropein, the secoiridoid responsible for the bitter taste of olives, was studied in vitro for its antimicrobial activity. Recently, it was investigated against Mycoplasma hominis, Mycoplasma fermentas, Mycoplasma pneumoniae, Mycoplasma pirum [87]. O. europaea is a plant resistant to microbe and insectan attack. In particular, oleuropein has been shown to inhibit or stop the growth time of a serial of bacteria and microfungi [88]. In general, this compound is used as a food additive.
896
Oleuropein and its aglycone inhibit Lactobacillus plantarum, Pseudomonas fragi, Staphylococcus carnosus, Enterococcus faecalis, Bacillus cereus, Salmonella enteritidis and, moreover, a serial of fungi [89]. The phenolic compounds act on the exoprotein secretion of the Staphylococcus aureus too [90]. In vitro, there has been observed the possible antimicrobial activity of oleuropein against human pathogenic bacteria. Moreover, the phenolic compound also confirmed its action on gram-positive and gram-negative bacterial strains such as Salmonella spp., Vibrio spp. and Staphylococcus aureus [91]. Experimental data indicated that M. pneumoniae, M hominis, Mfermentans and M. pirum are vulnerable to oleuropein [92]. Phenolic compounds, indicated as surface-active agents, are able to denature microbial proteins and are generally harmful [93]. These moieties damage the cell membranes or the cell peptidoglycan, causing escape of cytoplasmic constituents such as proteins, glutamate or potassium and phosphate ions [94]. In fact, bacterial cell is characterised by three important structures: cellular wall, cytoplasmic membrane and cytoplasm (Figure 30).
Figure 30. Bacterial membrane.
The phenolic compounds actions are concentration dependent and at sublethal concentrations enhance the leakage of cell constituents [95]. In particular, oleuropein causes a significant outflow of glutamate, potassium and inorganic phosphate ion form, e.g., E. coli; the same
897
compound has no influence on the velocity of glyeolysis but decreases the ATP contents of the cells. Gleuropein is more toxic for gram-positive than for gram-negative, because the glycosidic group impedes the drug penetration on the outer membrane or the contact on the target site [96]. The o-diphenol system is certainly responsible for antimycoplasmal activity of oleuropein. It is difficult to control such mycoplasma! infections with suitable broadspectrum antibiotics. Moreover, tetracycline and erythromycin, two important chemotherapeutic agents, as well as quinolones, are needed for the efficacy of an integral host immune system to eliminate the mycoplasmas. Therefore, the recent studies on activity carried on oleuropein indicate that this phenolic compound might be considered as an interesting and potential new natural drug for the treatment of mycoplasmal infections. In fact, oleuropein showed also activity against M. fermentans and M hominis strains, naturally resistant to erythromycin and tetracyclines [97]. Moreover, several studies carried out on olive fermentation showed that naturally occurring antimicrobial substances are needed to be removed in order to initiate fermentation [98]. In particular, at effective concentrations of oleuropein, the supplements affected both spore germination and subsequent increase. The inhibition appears during the transformation of the phase bright spore to a phase dark form. It was demonstrated that the addiction of oleuropein at various stages during the germination caused inhibition of the outgrowth of the germinated spores. During the development, important changes occur in the initiation of RNA, protein and membrane syntheses [99]. In fact, adding phenols to bacteria, sporulated or not, also affected membrane synthesis or inactivated cellular enzymes [100]. As reported before, oleuropein attacks the cytoplasmic membrane, destroying its permeability and causing outflow of intracelMar constituents such as glutamate, potassium and phosphorus. This action is crucial for me spores while the effectiveness of germination inhibitors may depend upon their capability to permeate the spore coat and may obstruct germination-promoting sites [101]. Furthermore, an interesting bactericidal activity of oleuropein against nine strains of Lactobacillus plantarum isolated from green olive fermentation brines was observed [102]. Lactobacillus plantarum [103] is a facultative heterofermentative, asporogenous, gram-positive rod and is responsible for Spanish-style
898
green olive fermentation. During ibis process, by opportune acidity, Lactobacittus plantarum metabolises sugars eluted from olives in brines. Moreover, phenolic compounds are responsible for the stoppage or the variability in lactic acid fermentation, and some phenolics related to oleuropein cause delay or inhibition in the growth of various bacteria, including Lactohacillus plantarum. Several experimental studies have demonstrated mat the morphological characteristics of these bacteria change after heat-treated or untreated oleuropein incubation, such as cells become longer in size and wider. These changes are observed after 30-60 minutes of incubation in both oleuropein solutions. Probably, oleuropein promotes disruption of the lactobacillus peptidoglycan. Acknowledgements We thank to all members of my team: researchers, PhDs, technicians and students. Financial support of MIUR. and CNR is acknowledged. 4. BIBLIOGRAPHY [1] LPanizzi, MX.Scarpati, G.Oriente, Ricerca Sci, 28,994 (1958). [2] LPanizzi, M.L.Scarpati, G.Oriente, Gazz.Chim.Ital, 90,1449 (1960). [3] LPanizzi, M.L.Searpatis C.Trogolo, Gazz.Chim.ltal, 95,1279 (1965). [4] Rlnjouye, T.Yoshida,S.Tobita, K.Tanaka, T.Nishioka, Tetrahedron Letters, 2459 (1970). [5] A.Bianco, G.Naccarato, P.Passacantilli, G.Righi, M.L.Scarpati, J.NatProd, 55,760 (1992). [6] A.Bianco, N.Uccella, Food Research International, 33,475 (2000). [7] LBastoni, A.Bianco, F.Piccioni, N.Uccella, Food Chemistry 73,145151 (2001). [8] J.P Donaire., A.J Sanchez., J.Lopez-Gorge, L.Recalde, Phytochemistry, 14,1167 (1975). [9] MJ.Amiot, A,Fleuriet, J.J.Macheix. Phytochemistry, 28,67 (1989). [10]A.Bianco, R. Lo Scalzo, MLScarpati, Phytochemistry, 32, 455 (1993).
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[51] A. Saija, D. Trombetta, A. Tomaino, R. Lo Cascio, P. Princi, N. Uccella, F. Bonina, F. Castelli, International Journal of Pharmaceutics, 166,123-133 (1998). [52] D.Fiorentini, M.Cipollone, M.C.Galli, A.Pugnaloni, G.Biagini, L.Landi, Free Rod. Res., 21,329-339 (1994). [53] A. Saija, F.Bonina, D.Trombetta, A. Tomaino, L.Montenegro, P.Smeriglio, F.Castelli, Int. J. Pharm., 124,1-8 (1995). [54] A.K.Ratty, N.P.Das, Biochem. Med Metab. BioL, 39,69-79 (1998). [55] MKJain, Order and dynamics in bilayers. Solute in Bilayers, in: Jain M.K. (Ed.), Introduction to Biological Membranes, John Wiley and Sons, (1988), New York, 122-165. [56] B. Le Tutor, D.Guedon, Phytochemistry, 31,1173-1178 (1991). [57] F.Visioli, G.Bellomo, G.F.Montedoro, C.Galli, Atherosclerosis, 117, 25-32 (1995)) [58] M.Salami, C.Galli, L.De Angelis, F.Visioli, Pharmacol Res., 31, 275-279 (1995). [59] B.Halliwell, G.M.C.Gutteridge, Arch. Biochem. Biophys., 280,1-8 (1990). [60] H.Wang, G.Cao, R.L.Prior, /. Agric. Food Chem., 45,304-309 (1997). [61] KX.Horan, B.S.Lutzke, A.R.Casers, J.M.McCall, D.E.Epps, Free Rad Bio. Med, 17,587-596 (1994). [62] A. Saija, D. Trombetta, A. Tomaino, R. Lo Cascio, P. Princi, N. Uccella, F. Bonina, F. Castelli, International Journal of Pharmaceutics, 166,123-133 (1998). [63] M.Gerber, Olive oil and cancer, in: Hill M.J., A.Giacosa, C.P.G.Caygill,, Eds. Epidemiology of diet and cancer, Chichester: Ellis Horwood, (1994), 263-275. [64] W.ROwen, A.Giacosa, W.E.Hull, KHaubner, G.Wurtele, B.Spiegelhalder, HBartsch, The Lancet Oncology, 1,107-112 (2000). [65] W.ROwen, AGiacosa, W.E.Hull, RHaubner, G.Wurtele, B.Spiegelhalder, H.Bartsch, European journal of cancer, 36,1235-1247 (2000). [66] M.Serraino, L.U.Thompson, Nutr. Cancer, 17,153-159 (1992). [67] B.Amro, T.Aburjai, S.Al-Khalil, Fitoterapia, 73,456-461 (2002).
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[68] LStupans, G. Stretch, P.Hayball, Journal of Nutrition, 130,23672370 (2000). [69] LStupans, M.Murray, A.Kirlich, K.L.Tuck, P.J.Hayball, Food and chemical toxicology, 39,1119-1124 (2001). [70] C.Manna, P.Galletti, V.Cucciolla, O.Moltedo, A.Leone, V.Zappia,/. Nutr., 127,286-292 (1997). [71] C.Manna, P.Galletti, G.Maisto, V.Cucciolla, S.D'Angelo, V.Zappia, FEBS Letters, 470,341-344 (2000). [72] B.Fehri, J.M.Aiache, S.Mrad, S.Korbi, J.L.Lamaison, Bollettino Chimico Farmaceutico, 135,42-49 (1996). [73] M. Demasi, G.E. Caughey, M.J. James and L.G. Cleland, Inflammation Research, 49, 737-743 (2000). [74] F. Visioli, S. Bellosta, C. Galli, Life sciences 62, 6, 541-546, (1998). [75] I. Vouldoukis, V. Riveros-Moreno, B. Dugas, F. Ouaaz, P. Becherel, P. debre, S. Moncada, M.D. Mossalayi, Proc NatlAcadSci USA, 92 7804-7808 (1995). [76] J.M. Wong, T.R. Billiar, Regulation and function ofinducible nitric oxide synthase during sepsis and acute inflammation, L. Ignarro and F. Murad Eds, 155-170, Academic press, San Diego 1995. [77] RBormann, M.F.Melzig, Pharmazie, 55,129-132 (2000). [78] H.Naegele, B.Behnke, A.Gebhardt, M.Strohbeck, Clinical Biochemistry, 31(1), 37-45 (1998). [79] S.S.Funari, F.Barcelo, P.V.Escriba, J. LipidResearch, 44(3), 567575 (2003). [80] RJ.Grekin, AP.Vollmer, R.S.Sider, Hypertension, 26(1), 193-198 (1995). [81] S.Cherif, N.Rahal, M.Haouala, B.Hizaoui, F.Dargouth, M.Gueddiche, Z.Kallel, G.Balansard, K.Boukef, J. De Pharmacie De Belgique, 51(2), 69-71 (1996). [82] AS.Rocca, J.LaGreca, J.Kalitsky, P.L.Brubaker, Endocrinology, 142 (3), 1148-1155(2001). [83] RJ.Woodman, T.AMori, V.Burke, I.B.Puddey, G.F.Watts, LJ.Bellin, American Journal ofClinical Nutrition, 76(5), 1007-1015 (2002). [84] T.F.Slater, Biochem. J, 111, 1-15 (1984).
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[85] S.Higgins, S.McCarty, B.M.Comdan, HMRoehe, J.M.W.Wallace, N.M.O*Brien, P.A.Morrisey, Nutrition Research, 20(8), 1091-1102 (2000). [86] T.A.Mori, B.Q.Bao, V.Burke, LB.Puddey, L.J.Beilin, Hypertension, 34,253-260(1999). [87] P,M. Fumeri, A. Marino, A. Saija, N. Uccella, G, Bisignano, International Journal of antimicrobial agents, 20,293-296 (2002). [88] N.Paster, B.IJuven, RHarshermesh, J. Appl BacteriolM, 293-297 (1988). [89] RCapasso, A.Evidente, L.Schivo, G.Orru, M.A.Marcialis, G.Cristinzio, J. Appl. Bacteriol, 79,393-398 (1995). [90] H.S.Tranter, C.C.Tassou, G. J,E.Nychas, J. Appl. Bacterial., 74,253259 (1993). [91] G.Bisignano, A.Tomamo, R.Lo Cascio, G.Crisafi, N.Uccella, A.Saija, J. Pharm.Pharmacol, Sl 5 971-974 (1999). [92] P.M. Fumeri, A. Marino, A. Saija, N. Uccella, G. Bisignano, IntemationalJournal of antimicrobial agents, 20,293-296 (2002). [93] S.P.Denyer, G.S.A.B.Stewart, Int. Biodet. Biodeg., 41,261-268 (1998). [94] BJuven, Y.Henis, BJacoby, J. Appl Bad, 35, 559-567 (1972). [95] H.P.Fleming, W.Walter, J.L.Etchells, Appl. Microbiol, 18, 856-860 (1969). [96] G.Bisignano, A.Tomaino, R.Lo Cascio, G.Crisafi, N.Uccella, A.Saija, J. PharntPharmacol, 51,971-974 (1999). [97] CBebear, MDupon, RRenaudin, B.de Barbeyrac, Clin. Infect Dis, 17 Suppl 1,202-207 (1993). [98] HRFleming, J.L.Etchells, Appl. Microbiol., 15,1178-1184 (1967). [99] V.Vinter, J. Appl. Bacteriol., 30,50-59 (1970). [100] RMLDavidson, A.L.Branen, J. FoodScL, 45,1607-1613 (1980). [101] F.K.Cook, M.D.Pierson, Food TecknoL, 37,115-116 (1983). [102] J.L. Ruiz-Barba, A. Garrido-Fernandez, R. Jimenez-Diaz, Letters in Applied Microbiology, 12,65-68 (1991). [103] R.H.Vaughn, H.C.Douglas, J.RGilliland, Cat. Agricultural and Experimental Station Bulletin 678 (1943).
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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THE CHEMISTRY OF THE GENUS CICER L. PHILIP C. STEVENSON1*2 & SHAZIA N. ASLAM1 x
Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, UK. 2 Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey, TW9 SAB, UK. ABSTRACT: The genus Cicer L. consists of 43 species of annual and perennial herbs in the monogeneric tribe Cicereae (Leguminosae). The genus affords considerable interest because C. ariettnum, the most well studied and most vulnerable species to disease and pests, is the chickpea, an important crop and food for resource-poor farmers, especially in Asia. More than 200 natural products have been identified from the genus. This chapter describes the chemistry of Cicer and the function of those compounds with known biological activities especially in the context of micro-organisms and insects that are natural pests and diseases of the cultivated crop. Some compounds occur constitutively in healthy chickpea plants that present inbuilt defence to infection by a range of pathogens and insects. For example, although the pterocarpans, maackiain and medicarprn occur constitutively they also increase in concentration when the plant is attacked by a fungus, nematode or bacterium. These induced compounds (phytoalexins) are either biosynthesised de novo or as recent work suggests are accessible from glycosides stored in vacuoles. While these phytoalexins occur in all species of Cicer a few produce a wide array of isoflavonoids including recently discovered compounds from the rare isoflav-3-ene and arylbenzofuran classes all of which show varying degrees of biological activity against pathogens of chickpea and all are inducible. The chemical ecology of the plant especially with relevance to agricultural applications is discussed. We also describe their distinct occurrence among Cicer species and indicate how knowledge about their biosynthesis can inform the taxonomy of the genus. The recent synthesis of the arylbenzofuran cicerfuran from novel stilbenes precursors has been achieved along with several analogues. Their importance beyond the defensive value to the plant has become apparent through biological activity testing against different micro-organisms and shows that these compounds may provide valuable novel activity against bacteria, fungi and protozoans. The chapter will also discuss some aspects of chemical synthesis of flavonoids of structural relevance to Cicer L.
1-INTRODUCTION The genus Cicer L. occurs in the monogeneric tribe Cicereae in the sub-family Papilionoideae of the Leguminosae. It contains 43 species of annual and perennial herbs [1] with a geographic distribution from the Himalayas to the Ethiopian Highlands, and has centres of diversity across central Asia, An isolated species, C. canariense occurs on the Canary Islands in the Atlantic Ocean [2]. The genus affords considerable research interest since one species, C. arietinum L., the cultivated species
906
known variously as chickpea, garbanzo and channa, is an important food crop and so has been well studied, although the chemistry of 22 other species in the genus has also been investigated [ 3 - 6 ] . A summary of the plant chemistry of Cicer was published in 2001 [7]. Interestingly, C. arietinum is not known in the wild [8] but is a major source of human and domestic animal food, particularly in the semi-arid tropics where its production is concentrated [9] and it provides a considerable portion of the daily diet of some of the world's poorest people. It is especially important in South Asia, where it comprises a large part of a primarily vegetarian diet. It is also a highly versatile crop providing gram flour, the principle ingredient for many biscuits, breads, sauces and sweets as well as its more familiar role in vegetable dishes. Because it is used in such a wide variety of geographical locations it is considered the world's third most important pulse crop after dry beans (Phaseolus vulgaris L.) and dry peas (Pisum sativum L.) [10]. Its value as a crop has focused interest on the genus and much of the work on related species has been conducted in the hope that phenotypic characters can be found that, if transferable to the cultivated species, might enhance productivity. Perhaps the most important of these characters is natural resistance to agricultural pests, especially fungal diseases. More than 50 species of fungus have been reported to occur on chickpea [11], although only a few of these have the potential to cause serious crop damage and thus pose a threat to production, notably Aschochyta blight (Aschochyta rabiei), Botrytis Grey Mould (Botrytis cinerea) and Fusarium wilt (Fusarium oxyspomm f. sp. ciceri). Chickpea is also attacked by various insect pests most notably the pod borer Helicoverpa armigera (Lepidoptera : Noctuidae) and some work has focused on identifying chemical components in Cicer that influence food choice of this insect as well as the determination of components that affect the behaviour of ovipositing adults [12,13]. Some of these studies have also led to the identification of chemotaxonomic markers that support recently proposed sub-generic systematic restructuring and groupings within the genus [6]. Over 200 compounds have been identified so far in the genus although approximately 120 of these are simple hydrocarbons and relatively ubiquitous in the plant kingdom. Nonetheless they may be important as possible recognition chemicals and mediators for oviposition by insect pests [14]. This chapter lists all the known compounds that have been isolated from the genus Cicer under 5 sections in the text. It also ascribes a biological activity to the compounds where applicable to the ecology of Cicer. Where possible, semi-systematic names have been
907
used along with the trivial names since IUPAC approved names for many compounds are too cumbersome and are rarely used in the literature. Section 6 describes the biosynthesis of the flavonoids, the main group of natural products in Cicer, and section 7 highlights some areas of synthesis relevant to structures in Cicer, notably those describing the synthesis of the aryl benzofurans. 2- ALIPHATIC ACIDS. One of the most characteristic physical attributes of Cicer is the profuse exudation from leaf hairs of some species especially C. arietinum. The composition of this exudate is highly acidic (pH <2) and has been considered an important component in the resistance of the plant to insect pests [13]. A comprehensive analysis of the exudate revealed the presence of 7 organic acids [15]. The exudate was adsorbed from leaf surfaces by wiping them with cotton wool plugs from which it was transferred to vials and analysed directly by HPLC and HPTLC. The principal component of the exudate was identified as malic acid (1) although oxalic acid (2) was also abundant with malonic acid (3), citric acid (4), succinic acid (5), oxaloacetic acid (6) and fumaric acid (7) also identified [15]. The authors also report the presence of a high concentration of glucose-6-phosphate (8) in the exudate and surprisingly suggest that 8 was responsible for the low pH even though it would seem more likely to be associated with the presence of the organic acids. The roots are also known to release an exudate which on analysis revealed the presence of the same organic acids with the exception of 2, 3 and 6 which thus appear to be restricted to the leaf exudate [16]. Gluconic acid (9) was also detected in the same root exudates and stimulates a positive chemotaxis (movement by a cell or organism in reaction to a chemical stimulus) toward Rhizobium. This suggests an ecological role for 9 in the nitrogen fixation potential of chickpeas. The authors proposed that chemotaxis was a major factor in host-symbiont specificity and motility guided by chemotaxis facilitates infection. The ability of chickpea to fix nitrogen is a highly valuable bonus to resource poor farmers by reducing fertilizer inputs for subsequent crops - especially rice. Thus, potential avenues for optimising nitrogen fixation are being sought. Malonic acid (3) is particularly abundant in the Leguminosae although its function is still not entirely clear. Li and Copeland [17] described a study of the temporal and spatial distribution of organic acids in chickpea plants and investigated the different kinds of abiotic stress on organic acid
908
content. They determined that the most abundant acid in the roots and nodules of chickpea was 3 where, they argued, its role lies in defence, although they demonstrated that it did not accumulate as a primary response to stress. They also detected and quantified levels of 1, 5 and 7 and determined that 1 was the most abundant organic acid in the leaves [17]. O
HO'
X
r Y° OH
HO ~ \
o
3
0 -OH
Y 0
^
O
HO
11
II
HO'
HC
2
s.
o
O
0
1
X
o
V-OH
H
Y~V °
°
0H
OH
HO b HO
"OH
The role of these organic acids as disruptors in the feeding behaviour of pest insects has often been presumed but this has only been investigated fairly recently. In a biological assay, recording the feeding behaviour of the pod borer (H. armigera) on chickpeas, washed leaves were tested against unwashed leaves [18]. Insects eating the unwashed leaves developed more slowly than those feeding on washed leaves indicating that the inhibition was caused by a leaf surface component. Further analysis identified 1 and 2 as the major components of the water soluble leaf surface extract as suggested previously [15] but only 1 inhibited insect development when tested in isolation in bioassays. More recently effects on the oviposition by H. armigera females have been studied [19]. Compounds 1 and 2 were presented to ovipositing female H. armigera on nappy liners, a favoured ovipositing material in culture. Compound 1 stimulated oviposition at a concentration of 0.6 /mnol cm"2 but inhibited it at 3.4 /xmol cm"2. Compound 2 showed neither stimulation nor inhibition of oviposition at 0.25-1.7 /xmol cm"2. Interestingly, during the podding stage, when the greatest number of eggs is laid, there was no significant correlation between either egg density or pod damage and levels of 1 suggesting they have little impact on the insect predation. However, a significant negative correlation between pod damage and
909
levels of 2 was reported and it was concluded that 2 has an important role in resistance to this pest in chickpea and is a potential target compound in the development of insect resistant chickpeas [19].
HO.
OH
L
i—N
X
O OH
OH
10
Pangamic acid (6-0-(dimethylaminoacetyl)-D-gluconic acid) (10) has also been isolated from C. arietinum and is reported to have anti-stress and anti-hyperlipidemic activity [20].
3 - AROMATIC ACIDS: Phenylpropanoids. Phenylpropanoids (also known as cinnamic acids) are relatively simple secondary metabolites that are derived from the shikimic acid pathway via phenylalanine and tyrosine in some plants [21]. For example, the elimination of ammonia from phenylalanine by phenylalanine ammonia lyase (PAL) gives cinnamic acids. This group of compounds occurs very widely in the plant kingdom and not surprisingly several have been identified in Cicer. Two mono-hydroxy derivatives of cinnamic acid, 3-hydroxy and 4-hydroxycinnamic acid (11 and 12) have been found together with caffeic acid (3,4 dihydroxycinnamic acid) (13), ferulic acid (14), 5-0-caffeoylquininc acid (chlorogenic acid) (15) and 3-(4hydroxyphenyl)propanoic acid (phloretic acid) (16) [22], although 4hydroxycoumaric acid was first identified in Cicer by Wong [23]. Singh et al., [24] identified the simplest phenylpropanoid cinnamic acid (17) together with 14 and 15 in leaves treated with Sclerotium rolfsii, a soil borne disease that causes collar rot in many crop plant species including C. arietinum. The production of these compounds by the plant was induced when the plant was treated with the culture filtrate of the fungal culture. Although the specific ecological role that these compounds play in Cicer is not fully understood it has been suggested that 14 may prevent
910
infection by S. rolfsii. In vitro bioassays showed that 14 inhibited mycelial growth at concentrations as low as 250ug ml"1 and caused complete inhibition at concentrations as high as lOOOug ml"1. Furthermore, higher amounts of 14 in the stems and leaves in infected plants suggested an induced response to the infection [25].
12
O
HO,
MeO
OH
Phenylpropanoids have been associated with crop resistance to H. armigera together with flavonoids discussed below and further work may elucidate whether they have a role in the development of resistance in deer since similar compounds have been shown to be responsible for resistance to folivorous insects on other legume crops including groundnuts [26]. Compound 15 isolated from Arachis paraguariensis retarded development of H. armigera in artificial diets as well as increasing the number of days larvae took to pupate, a desirable character in integrated pest management since this increases the time that larvae are exposed to predation and parasitism by beneficial insects and other biocontrol agents. Interestingly, the development of insects was also retarded by caffeic acid the hydrolysed phenolic moiety of 15 suggesting that the activity was associated with the phenylpropanoid part of the molecule [26]. There is considerable evidence that the dihydroxy group of both caffeic acid and chlorogenic acid is responsible for its activity since similar activity is also reported against larvae of a related noctuid
911
Spodoptera litura for 15, quercetin (3,5,7,3',4'-pentahydroxyflavonol) and rutin, its 3-0-rhamnoglucoside, both of which have 3',4'-dihydroxy substitution on the B ring [27].
Benzoic acids. From the cinnamic acids or phenyl propanoids described above, /3oxidation and truncation of side chains yields a variety of benzoic or simple phenolic acids [28]. Rao et ah, [22] identified gallic acid (18), gentisic acid (19), protocatechuic acid (20), /»-hydroxybenzoic acid (21), oc-resorcyclic acid (22), vanillic acid (23) and salicylic acid (24) in C. arietinum and showed that overall, leaf content of all phenolic compounds was much greater than in roots and stem. They postulated that the production of these compounds may enhance the activity of indole acetic acid oxidase or may express antimicrobial properties when leached into the soil. However, Singh et ah [24] showed that the production of both 18 and 24 by C. arietinum was induced when treated by the culture filtrate of Sclerotium rolfsii along with the phenyl propanoids 14, 15 and 17 mentioned above.
HO-
OH HO 20
,0 HO OH
21
MeO
23
912
Interestingly, oxalic acid (2) detected in the exudate of Sclerotium fungal culture also induced production of 18, and as described above, 2 is one of the principal constituents of the leaf hair exudate if C. arietinum. Further work has found that two plant growth promoting rhizobacteria, Pseudomonas fluorescens and P. aeruginosa protected chickpea seedlings from infection by S. rolfsii owing, according to the authors, to induced systemic resistance resulting from the induction of 24 and associated salicylic acid dependent pathways [29]. Thus there may be a complex process of signalling associated with defence. Unfortunately these authors did not look further at other compounds such as the pterocarpans known to be produced by Cicer species [6] and discussed in detail below.
4 - FLAVONOIDS. Flavonoids constitute one of the largest groups of naturally occurring phenols and are virtually ubiquitous in plants. It is not surprising therefore that several have been identified from the genus Cicer. All contain fifteen carbon atoms in their basic skeleton and these are arranged in a C6-C3-C6 assembly with two aromatic rings linked by a three carbon atom unit which may (flavonoid) or may not (chalcone) form a third ring. These rings are labelled A, B and C as indicated in Fig. (1), but the modified numbering systems for the different groups of flavonoids are shown for the first representative structure of each listed in the text. The flavonoids are all linked by common biosynthetic pathways that incorporate the shikimic acid pathway and the acetate malonate pathway and this will be discussed in section 6.
Fig. (1). Basic structure of flavonoids with numbering.
913
Isoflavones & isoflavanones. The isoflavonoids are a large subclass of the flavonoids and constitute the greatest area of phytochemical interest in the genus Cicer. They have a skeleton that is biogenetically derived by rearrangement of the 2-phenyl of the flavonoids to a 3-phenyl substitution of the C ring via a 1,2-aryl migration. Their biosynthesis will be described in more detail in section 6, but for now it suffices to say that structural variation encountered in isoflavonoids is considerable and several of the subgroups of isoflavonoids are represented in Cicer. They generally occur more frequently as aglycones than as glycosides and are substituted most frequently by glucose and never more than diglycosylation. This contrasts sharply with the frequency of glycosylation in the ubiquitous flavonoids in which sugar substitution is much more common. These variations arise not merely from the number and complexity of substituents on the basic isoflavonoid skeleton but also from different oxidation levels of the two aromatic rings and subsequent additional cyclisation (5 and 6 membered) arising usually from the dehydration of adjacent hydroxyl groups. The numbering systems follow those of the flavonoids above with the exception of pterocarpans, coumestanes and arylbenzofurans which are discussed after the present section. The first phytochemical study of Cicer was published in 1945 and reported the occurrence of three crystalline compounds, biochanin A (25), biochanin B (26) and biochanin C (27) from the sprouted germs of Cicer arietinum [30]. These trivial names were derived from the Hindi/Urdu word for chickpea - chana, and the fact that these compounds were biochemicals; hence biochanin. The structure of 25 (5,7-dihydroxy-4'methoxyisoflavone) was later clarified by Bose & Siddiqui [31], who went on to determine the structure of Biochanin B as formononetin (7hydroxy-4'-methoxyisoflavone) (26) [31-33]. There appears to have been no further work on the component originally described as Biochanin C with the exception of that of Warsi & Kamal [34] who looked again at the original work of Siddiqui and identified three products, biochanin A and C being identical to those described by Siddiqui. Biochanin C was found to have the molecular formula C6Hi3 N3O4) but biochanin B (C15H12O4) could not be found. Instead they identified another compound (C16H12O4) which they called neochanin and described as a monohydroxy monomethoxy flavone. It appears from subsequent literature in which the biosynthesis of these isoflavonoids was studied [35] that the consensus
914
from early literature is that formononetin, biochanin B and neochanin are in fact synonyms and are referred to as 26 in this text [36].
OH
o
OMe
OMe
25R = H 30 R = Glc 36 R = Malonylglc
26 34 35 37 38
R = R, = R2 = H R = Glc, Rf = R2 = H R = Malonylglc, R^ = R2 = H R = R2 = H, R, = OH R = Ri = H, R2 = OH
O OMe 28R = H 31 R = Glc
OH
O
32R = H 33 R = Glc
OH 40
Daidzein (7,4'-dihydroxyisofiavone) (28) and pratensein (5,7,3'trihydroxy-4'-methoxyisoflavone) (29) were identified in seedlings of C. arietinum along with biochanin A 7-0-glucoside (sissotrin) (30); the first report of an isoflavonoid glycosides in deer [23]. Subsequently
915
daidzein-7-O-glucoside (31) was also reported to occur in C. flexuosum and C. baldshuanicum [4]. Many isoflavonoids and flavonoids in Cicer occur as glycosides for what appears to be storage purposes, especially in the roots and often in larger quantities than the aglycones. For example, genistein (5,7,4'trihydroxyisoflavone) (32) has also been found in C. arietinum together with genistein-7-O-glucoside (33) and the 7-0-glucoside of formononetin (ononin) (34) and biochanin A (sissotrin) (30) and the glucose was reported to be esterified by malonic acid to give formononetin 1-0glucoside-6"-0-malonate (35) and biochanin A 7-0-glucoside-6"-(9malonate (36) which are considered to be the most abundant compounds in Cicer [37]. The occurrence of these isoflavonoids as glycosides is probably for storage since they appear to occur most abundantly in the roots and stems in this form [37] but only the aglycone occurs elsewhere in the plant [6]. The hydroxlated derivatives of 26, namely 2'-hydroxyformononetin (37) and 3'-hydroxyformononetin (calycosin) (38) have been identified in germinated cotyledons of C. arientinum that were treated with an Aschochyta rabiei elicitor [38], indicating that they are produced as phytoalexins. The same study identified the only other isoflavone from C. arietinum, pseudobaptigenin (7-hydroxy-3',4'-methylenedioxyisoflavone) (39). Compound 39 is particularly noteworthy being one of only two isoflavones to be substituted with a methylenedioxy bridge - a feature that is of importance in other isoflavonoid groups represented in the genus and discussed in section 6. Compounds 37-39 are also especially important as intermediates in the biosynthesis of pterocarpans. Another isoflavone with a methylenedioxy bridge has been identified in C. cuneatum as cuneatin (7-hydroxy-2'-methoxy-4',5'methylenedioxyisoflavone) (40) [3]. This compound is also notable in that it indicates the presence of a 2'-O-methyltransferase which in other genera e.g., Ateleia may account for the absence of pterocarpans [39]. Once the 2-0-position has been methylated it can no longer be converted to the five membered ring characteristic of pterocarpans. C. cuneatum then is then capable of both transformations at the 2' position on ring B. Compounds 25 and 26 have also been detected in many other species of Cicer, notably C. anatolicum, C. bijugum, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, C. macranthum, C. montbretii, C. pinnatifidum, C. pungens, C. rechingeri, C. reticulatum, C. songaricum and C. yamashitae [3] and later in C. canariensis, C.
916
microphylum, C. nuristanicum and C. oxyodon [6]. Compounds 28, 29, 30 and 34 have also been found in C. songoricum [5] and 29, 30 and 34 found in C. anatolicum, C. bijugum, C. canariensis, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, C. microphylum, C. macranthum, C. nuristanicum, C. oxyodon, C. pinnatifidum, C. pungens, C. reticulatum and C. yamashitae [6], 31 in C. macranthum [4] and 30 in C. mogoltavicum [40]. It is noteworthy that none of the studies on species other than the cultivated C. arietinum contained isoflavone-glucoside malonates. The ecological function of these isoflavones in chickpea plants is not clear although 25 and 26, previously identified from clover roots {Trifolium repens), stimulated colonization by vesicular-arbuscular mycorrhizal (VAM) fungi and subsequently growth of clover [41]. Compound 26 increased the number of VAM vesicles in roots of asparagus and reduced the percent root lesions caused by Fusarium spp. when compared with the non-treated controls. Compound 26 improved growth and reduced disease of Asparagus in replanted Asparagus and is considered useful in reestablishing Asparagus in abandoned Asparagus fields [42]. Since chickpea is also attacked by a species of Fusarium, 26 may play a role in fungal defense in Cicer spp. In clover, 26 accumulates in nodule primordia and accelerates the breakdown by peroxidase of auxin, suggesting that local changes in flavonoid accumulation could regulate local auxin levels during nodule organogenesis and mediate this important process [43]. Cook et al. [44] also recorded the production of 26 in clover roots and showed that its production was stimulated by infection from the root nematode Ditylechnus dipsaci and that the accumulation of 26 was associated with the race specific resistance. Unfortunately they did not actually show that 26 was active against D. dipsaci in bioassays so were unable to add substantive weight to their argument. Wang et ah, [45] provide a convincing argument that 26 along with 25 and 32, their corresponding 7O-glucosides, biochanin A 7-0-glucoside-6"-0-malonate (36) and genistein 7-O-glucoside-6"-O-malonate from Trifolium subterraneum were deterrent to the red-legged earth mite {Halotydeus destructor) and may well provide a natural defense mechanism to this pest [45]. Isoflavanones differ from isoflavones in that the C ring is fully saturated. Only two of these compounds, homoferreirin (5,7-dihydroxy2',4'-dimethoxyisoflavanone) (41) and cicerin (5,7-dihydroxy-2'methoxy-4',5'-methylenedioxyisoflavanone) (42) have been positively identified in Cicer, and were shown to be induced products in seedlings of
917
C. arietinum treated with the reduced form of glutathione reductase (GSH). They were found together with their esters homoferreirin-7-0glucoside-6"-0-malonate (43) and cicerin 7-0-glucoside-6"-0-malonate (44) [46]. Although this was the first identification of the compounds in the whole plant, 42 was earlier proposed to be inducible in cell suspension cultures of C. arietinum [47]. The induced products identified by Armero et al. [46] were shown to be released into the surrounding media and not into plant root tissue and then only by 4 day old seedlings. They may have a specific ecological role that is currently not known. The authors also reported that the constitutive compounds 25 and 26 were also induced by treatment with GSH. It is worth noting that two peptides have recently been isolated from C. arietinum, one of which was given the trivial name cicerin and this may cause some confusion when searching the literature [48]. In fact cicerin was first used to describe an induced antifungal product from C. arietinum by Kunzru and Sinha [49] and although the product was never characterised there is some thought that they were describing a pterocarpan [50].
OH 41 R = H 43 R = Malonylglc
OMe
OH 42 R=H 44 R = Malonylglc
Pterocarpans Arguably the most important compounds to be identified in the genus deer are the two pterocarpans medicarpin (3-hydroxy-9methoxypterocarpan) (45) and maackiain (3-hydroxy-8,9methylenedioxypterocarpan) (46), owing to their reported role in fungal defence against Fusarium wilt, [51] (and references therein), Botrytis Grey Mould (BGM) [52] and Achochyta rabiei the pathogen causing Aschochyta blight [53 & 38]. This attribute has prompted a considerable amount of research, most notably under W. Barz and co-workers on the biosynthesis of these and related precursors (see [54]). Note that the numbering system for pterocarpans differs from isoflavones.
918
45 R = H 47 R = Glc 49 R = Malonylglc
46 R = H 48 R =
Q
C
50 R = Malonylglc
Pterocarpans are formed from the ring closure of a 2'hydroxyisoflavone [38] although this will be discussed in more detail in section 6. The most notable attribute is their role as a phytoalexin which requires de novo synthesis in response to a stress, usually infection with a pathogen, and toxic activity towards the organism which induces the response. This phenomenon was first shown when cut stems of C. arietinum were treated with spores of the fungus Helminthosporium carbonum and with the exception of the work of Kunzru et al. [49] is the first report of medicarpin and maackiain in Cicer [50]. The two pterocarpans were later also shown to occur as medicarpin 3-O-glucoside (47), maackiain 3-O-glucoside (trifolirhizin) (48), medicarpin 3-0glucoside-6"-O-malonate (49) and maackiain 3-Oglucoside-6"-0malonate (50) [55]. Compounds 45 and 46 also occur in many other species of Cicer notably C. anatolicum, C. bijugum, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, C. macranthum, C. montbretii, C. pinnatifidum,, C. pungens, C. rechingeri, C. reticulatum, C. songaricum and C. yamashitae [3] and were later also found in C. canariensis, C. microphylum, C. nuristanicum and C. oxyodon [6] and 46 was also found in C. mogolativicum in which it is described as inermin and found to occur as the 3-O-glucoside [56]. The 3-C>-glucoside-6"-Omalonate of 46 has also been identified in the roots of C. anatolicum, C. bijugum, C. canariensis, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, C. microphylum, C. macranthum, C. nuristanicum, C. oxyodon C. pinnatifidum, , C. pungens, C. reticulatum, and C. yamashitae albeit in trace amounts [6], and similarly the 3-0glucoside (48) in C. anatolicum, C. bijugum, C. canariensis, C. chorassanicum, C. cuneatum, C. judaicum, C. macranthum and C. pinnatifidum [6]. The apparent absence of the 3-O-glucoside of 46 (47) is unlikely to be of taxonomic or other significance since 50 the malonated glucoside occurs in all the species listed above only in trace amounts so the absence of the 3-O-glucoside may be attributable to its presence at such low amounts as to be undetectable [6].
919
The role of pterocarpans in deer was first suggested by Keen [57] based on work on germinating seedlings but later Ingham [50] treated the cut stems of C. arietinum with Helminthosporium carbonum spores and was able to show that this process of infection directly caused the production of 45 and 46 and that they were anti-fungal to the organism. It should be noted that this fungus is not a natural or field pathogen of Cicer. It was not until Weigand et ah, [53] showed that these compounds were produced in response to treatment with the spores of Aschochyta rabiei, the fungus that causes Aschochyta blight in field crops of chickpea, that any conclusions about the actual ecological role for these compounds could be reached. Later, after showing that the root exudates of C. arietinum were inhibitory to the fungal spores of Fusarium oxysporum f. sp. ciceri [58], Stevenson et ah, [51] demonstrated that wilt resistance in resistant varieties was conferred by the induced production of 45 and 46. Histological studies showed that the root xylem of plants showing wilt were heavily occluded by hyphae as far as the fifth internode whereas the roots of resistant varieties had been penetrated by hyphae but the hyphal growth was very slow and there was no evidence of hyphae in the xylem. Furthermore, there was no localised cell death (hypersensitive response) or gross structural changes (lignification) in the vicinity of those slow growing hyphae, suggesting that the inhibition of growth was dependent upon a chemical mechanism [51]. Furthermore, the production of medicarpin and maackiain was induced in plants growing in soil inoculated with the spores of the fungus, and both inhibited the germination of spores and hyphal growth of F, oxysporum at concentrations at which they occurred in the plant [51]. It is worth noting that pathogens are able to combat these pterocarpan defences. Several races of the Aschochyta blight pathogen were shown to catabolise and so inactivate 46 to a 2'-hydroxyisoflavan and by further oxidation to a lahydroxy-pterocarp-l,4-diene-3-one [59]. The occurrence of 46 as its 3-O-glucoside and 6"-0-malonyl-3-(9glucoside in much greater concentrations than the aglycone in the roots in C. bijugum, C. judaicum and C. pinnatifidum was suggested to be a clue to the role of the glycoside as a rapidly accessible storage form of the fungi toxic defence compound [60 & 61]. Isoflavonoid malonylgluco sides have the potential for rapid turnover [38] so simple cleavage of the glucose could release 46 although this was still not solid evidence for this ecological role. However, a substantial increase in the concentration of 25 and a decrease in the concentration of the malonylgluco side (36) was reported which suggested that the production
920
of 25 may come directly from the cleavage of the glycosyl moiety from the isoflavonoid ester [38]. More recently, Soriano-Richards et al., [62] ran time-course experiments in which the amounts of phytoalexin in hypocotyls of the resistant bean cultivar Flor de Mayo (Phaseolus vulgaris L.) were determined after infection with Colletotrichum lindemuthianum and phaseollin and phaseollidin were shown to accumulate as a defence response. They noted that phaseollidin accumulated earlier than phaseollin and at greater concentration. Like 46 in Cicer, phaseollidin was found conjugated as a glucoside, specifically in vacuoles of Phaseolus vulgaris, and fungal treatment caused a decline of approximately 50% in phaseollidin conjugate only 7 h after infection. Treatment of the vacuoles or isolated protoplasts (where phaseollidin glucoside occurred) with a fungal elicitor also produced a decrease, by half, of the glucoside suggesting a significant contribution of the preexisting pools of phaseollidin glycoside to the accumulation of the aglycone phytoalexins. Thus the glycoside and malonylglucoside of 46 could be constitutive stores of the phytoalexin maackiain in Cicer, in a similar manner to phaseollidin in P. vulgaris.
R = H Phaseollidin R = Glc Phaseollidinglucoside
Resistance in C. bijugum to Botrytis grey mould (BGM) caused by Botrytis cincerea was also shown to be associated with high concentrations of 46 when compared to three susceptible species. The two BGM resistant accessions of C. bijugum contained 46 at between 200 300 mg g"1 of foliage whereas the BGM susceptible species C. arietinum, C. echinospermum and C. reticulatum contained less than 70 mg g"1. Furthermore, the concentration of 46 increased to more than 400 mg g"1 in the resistant wild species after being inoculated with spores of the BGM pathogen whereas no significant increase was recorded in the susceptible
921 921
species. The germination of spores of Botrytis cinerea, treated with 46, was inhibited in a dose dependent manner; less than 10% of spores germinated when treated with 500 mg g"1 suggesting that 46 may also be an important component in BGM resistance and that this is enhanced in the presence of the pathogen [52]. Unfortunately, foliar mechanisms of resistance to the disease are only marginally important in protecting the damage the disease can cause to a crop. It is infection on flowers and the subsequent flower drop caused by BGM preventing sufficient pod formation that really needs to be tackled, and this is still only possible with fungicides since 45 & 46 do not occur in this plant tissue [6]. Isoflav-3-enes, coumestanes & arylbenzofurans. It has already been noted that there is a considerable structural variation among isoflavonoids and at least three other structural types are represented in the genus deer. Note that the numbering systems for these variants differ from each other. Isoflav-3-enes conform to the usual flavonoids numbering system but coumestanes and arylbenzofurans are different.
MeO 51 R = H
54 R = H
52 R = Glc
55 R = Glc
53 R = MalonylGIc
Isoflav-3-enes
Isoflav-3-enes are a rare sub-group of isoflavonoids. To date there are only 21 examples known of which five are restricted to three species in the genus Cicer; C. bijugum, C. judaicum and C. pinnatifidum [6]. As such they have interesting taxonomic implications that will be discussed under the section on biosynthesis. Judaicin (7-hydroxy-2'-methoxy-4',5'methylenedioxyisofiav-3-ene) (51) was the first of these compounds to be identified and was isolated from C. judaicum [60], it should not be confused with a diterpene occasionally referred to as judaicin from Artemisia vulgaris (mugwort), Artemisia judaica, Artemisia taurica and Achillea pratensis which is also known as vulgarin, barrelin, tauremisin
922
A and tauresmin [63]. Compound 51 was also found to occur as the glycoside judaicin-7-O-glucoside (52) and the ester judaicin-7-O-(6"-Omalonylglucoside) (53) in C. judaicum, the first and still the only report of glycosylation in this rare group of isoflavonoids [60] and 51-53 were also found later in C. pinnatifidum and C. bijugum [6]. They were also reported to occur with 48 and 50, and along with the glycosides of the isoflavenes constituted the major flavonoid components of the roots of these three species [60]. A further and very rare substitution for isoflavonoids and the only occurrence in the isoflavenes is 2-O-substitution which was found in 2methyoxyjudaicin (54) the fourth isoflavene to be reported from the genus and which was first found in C. bijugum [64]. It was also later found to occur in C. judaicum and C. pinnatifidum [6] although not completely characterised owing to limited material it was found to co-occur with 2methoxyjudaicin 7-0-glucoside (55) [64]. Since the identification of these compounds only a further 6 isoflav-3-enes have been reported, bringing the total including the 6 known from Cicer to 21 6 & 65-69]. Their ecological role will be discussed below with those attributed to arylbenzofurans. Coumestanes
Two coumestanes, 9-O-methylcoumestrol (7-hydroxy-9methoxycoumestan) (56) and medicagol (7-hydroxy-11,12methylenedioxycoumestan) (57) have been identified in several species of Cicer but were first identified in C. arietinum [70] although 56 was originally described as 12-0-methylcoumestrol but the numbering of coumestanes has since changed. In a survey of isoflavonoids in the genus the distribution of the two coumestanes was distinct. Compound 56 was found in all the species studied namely C. anatolicum, C. canariensis, C. chorassanicum, C. cuneatum, C. echinospermum, C. microphylum, C. macranthum, C. nuristanicum, C. oxyodon, C. pungens, C. reticulatum, and C. yamashitae with the exception of C. bijugum, C. pinnatifidum and C. judaicum, the three species which produce the isoflav-3-enes 51 and 54. The biosynthetic and taxonomic link between the species is discussed in more detail in section 6. Compound 57 was found in C. anatolicum, C. bijugum, C. canariensis, C. chorassanicum, C. cuneatum, C. judaicum, C. nuristanicum, C. oxyodon C. pinnatifidum, C. pungens, C. reticulatum, and C. yamashitae but not in C. echinospermum, C. macranthum, C.
923 923
microphylum, and C. reticulatum [6], No biological function or activity has been attributed to these coumestanes.
9
OMe
57
56
Arylbenzojurans
The arylbenzofurans are marginally more common than isoflav-3-enes but they still constitute an unusual and infrequently occurring group of isoflavonoids. Cicerfuran (2-(2'-niethylenedioxyphenyl)-6hydroxybenzofuran) (58) is the only example that has been found to occur in the genus Cicer and was isolated first from C. bijugurn [61] although like the isoflav-3-enes was later identified in C. judaicum and C. pinnatifidum [6]. MeO
This co-occurrence has implications for the biosynthesis of the compounds and subsequently the taxonomy of the genus [71]. Although it occurred at very low concentrations constitutively its production was induced in the presence of soil borne spores of F. oxysporum f.sp. ciceri the fungus causing wilt disease. Subsequent bioassays showed that 51 and 58 were potently antifungal towards F, oxysporum f. sp. ciceri and more so than 46 suggesting that these compounds play a significant role in the natural resistance of the three species to soil born fungi. None of the glycosides 48, 50, 52, 53 or the aglycone 54 were active against the fungus. This may also indicate that the glycosides of 46 and 51 are in fact
924
storage forms of the defence compounds that are thus easily accessible. Clearly, this rare 2-0- substitution affects the activity buts no other ecological role of 54 is known. Maackiain (46) and judaicin (51) were also shown to deter larval feeding by Helicoverpa armigera at 100 ppm and retained their antifeedant activity at 50 ppm and 10 ppm, respectively [72]. These concentrations are low for plant compounds and indicate potent activity and potential for investigating the development of natural resistance in chickpea to the pod borer based on these compounds. The isoflavonoids were tested in combinations, and with 15, which enhanced the activity of the isoflavonoids tested. As observed with the fungi, both the glucoside of 51 and the 2-methoxy derivative 54 showed much lower activity towards the insects than 46 and 51. Interestingly, H. armigera was the only one of four noctuids tested to be deterred by all four isoflavonoids. Spodoptera littoralis was deterred by 51 alone and S. frugiperda by 46 alone. Heliothis virescens and S. exigua were not deterred from feeding by any of the isoflavonoids. Furthermore, when incorporated into artificial diet, the isoflavonoids decreased the weight gain of early stadium larvae of//, armigera more than they did later stadia, and 46 and 51 were again the most potent compounds. Thus, 46 and 51 could play a role in decreasing the susceptibility of Cicer to attack by H. armigera [72]. Compound 58 has recently been synthesised [73-75], and the product was tested on a range of organisms including bacteria, fungi and protozoans. Several analogues that were synthesised with 58 together with some of the stilbenes that were intermediaries in their synthesis were also tested. Compound 58 was shown to be active ^against plant (Botrytis cinerea) and mammalian pathogens (Aspergillus niger) [76] and later the effect of 58 on the growth of Leishmania parasites was evaluated [77]. Promastigotes from cultures of L. aethiopica, L. major and L. tropica were tested in the exponential phase of growth. All compounds were active at a concentration of 100 /xg ml"1 within 6 hours. However, three of the stilbene intermediates from the synthesis of 58 as well as 58 itself were the most potent. One of the compounds, 2-hydroxystilbene, showed activity at a concentration of <1 jxg ml"1, with an IC50 of 3-5 /ig ml"1 after 48 hours of incubation. Based on the variation in structures among all the compounds tested including several methylated analogues it was postulated that the potency of anti-leishmanial activity was related to the presence of hydroxyl groups [77].
925
Flavonols , flavanones and chalcones. Flavonols The flavonols are the largest subclass of the flavonoids yet constitute a relatively small amount of natural products literature for Cicer. Nonetheless, several are known to occur. They have the typical 2phenylchroman skeleton and occur with considerable variety of structures owing to the variety of glycosides. The first compounds from this class identified in Cicer was garbanzol (3,7,4'-trihydroxyflavanol) (59) which was found along with 3,7,4'trihydroxyflavone (5-deoxykaempferol) (60), 7,4'-dihydroxyflavanone (liquiritigenin) (61) and the chalcones, isoliquiritigenin (4,2',4'trihydroxychalcone) (62) and isoliquiritigenin-4'-glucoside (63) [23]. No ecological function has yet been attributed to these compounds yet. In the same study it was shown that radiolabelled garbanzol converted to garbanzol-7-O-glucoside (64) and 60 by chickpea seedlings [23] indicating that 59 plays a role as both an intermediate in flavonoids biosynthesis and as an end product. Jaques et al., [38] found naringenin (5,7,4'-trihydroxyflavanone) (65) and its 7-0-glucoside naringin (66) which differs from 61 only in the 5-0 substitution. This is important in indicating the source of more advanced isoflavonoids.
OH
59R = H 64 R = Glc
O 65R = H 66 R = Glc
926
62R = H 63 R = Glc
The flavonols kaempferol (3,5,7,4'-tetrahydroxyflavone) (67), quercetin (3,5,7,3',4'-pentahydroxyflavone) (68) and isorhamnetin (3,5,7,4'-tetrahydroxy-3'-methoxyflavone) (69) were isolated and identified from the aerial parts of C. arietinum together with kaempferol 3-0-glucoside (astragalin) (70) and kaempferol 3-0-glucoapioside (71) [78]. It is now known that these flavonols also occur in Cicer as malonated conjugates as demonstrated by the identification of kaempferol 3-O-malonylglucoside (72) and kaempferol-3-O-apiosylmalonylglucoside) (73) [79]. A thorough study of several species of Cicer from Central Asia led to the identification of several new flavonoids for the genus including kaempferol 7-O-glucoside (74), and isorhamnetin-3-Oglucoside (75) in C. arietinum and also in C. pungens and C. flexuosum [4]. In the same study, C. macranthum, was shown to contain 67, 68 & 70 and 2 new compounds for Cicer, quercetin 3-0-glucoside (76) and quercetin 3-O-galactoside (77). C. pungens contained 68, 69 and their respective 3-O-glucosides, C. flexuosum contained kaempferol and kaempferol 3-glucoside (75 & 76), C. baldshuanicum and C. kopetdaghense contained 68 and 76 and C. songaricum contained 67 and 69 along with their 3-O-glucosides [4 & 5] and 75 in C. arietinum. A more recent study identified two new flavonoids in the genus Cicer in the epigeal parts of C. mogoltavicum; luteolin (5,7,3',4'-tetrahydroxyflavone) (78) and kaempferol 3-O-glucuronide (79) along with 67 [40], and later still identified 68, 69 and 78 in C. flexuosum [80] . A comprehensive study of the flavonoids in species of Lathyrus attempted to assign chemical characters to related genera in the two tribes Cicereae and Viceae [81]. Cicer was formerly classified in Viceae before being assigned to Cicereae [82]. Analysis of three species, C. arietinum, C. glaucum and C. montbretii revealed the presence of four flavonoids, 60, 67, 68 and 5-deoxyquercetin (fisetin) (80) in all three species [81]. Interestingly the authors claim that this agrees with the earlier work of
927
Wong et al. [23] but careful reading indicates that although Wong et al. did identify 67 this later study was the first report of 80 in Cicer [81]. They also reported the occurrence of 3-O-glucosides of these flavonoids as a general observation so from their work it is not possible to determine whether this would have included fisetin-3-O-glucoside [81].
OMe
OH O 69R = H 75 R = Glc
OH 1
II
R-i
O
67 R = R2 = H, R-, = OH 70R = H, R2 = Glc, R^ = OH 71 R = H,R 2 = Glc, R ! = O H 72 R = H, R2 = Malonylglc, R^ = OH 73 R = H, R2 = Apiosylmalonylglc, R-| = OH 74R = Glc, R2 = H , R 1 = O H 79 R = H, R2 = GIcA, Ri = OH
OH
II
OH
O 68 R = H 76 R = Glc 77 R = Glc
Flavonoids are known to have a wide variety of biological activities particularly against insects and these have been reviewed recently [83].
928
This work is important in indicating potential compounds that may be useful in the development of natural resistance to key insect pests of chickpea. The potential for these compounds to be manipulated has also been investigated. For example, Butron et al. [84] identified molecular markers for the production of maysin, a flavonoid in Zea mays. Maysin is responsible for the anti-insect activity of the some varieties of maize against Heliothis zea, a species of insect closely related to H. armigera. Thus the breeding of varieties using these selection markers can lead to the development of resistant varieties. The flavonoid, 68 and its glycosides that occur in C. arietinum as described above also occur in wild species of groundnuts {Arachis hypogea) including Arachis paraguariensis [85] and A. chacoensis [86] and are potent developmental inhibitors of both Spodoptera litura [85] and the podborer H. armigera [26]. Crosses of A. hypogea (cultivated susceptible species) and A. chacoensis (resistant wild species) also contained high levels of the entotoxic flavonoids suggesting a transfer of resistance mechanism. But this potential value in the development of insect resistant varieties of groundnut {A. hypogea) has now been realised in the recent production of wild and cultivated species crosses from A. kaempf-mercadoi X A. hypogea in which the Fl offspring not only contains high levels of the flavonoids but also expresses resistance to the insect [87]. Thus, studies on the resistance mechanisms of wild relatives of crops has real and valuable potential to be manipulated for the production of insect resistant germplasm.
4-VOLATILE COMPOUNDS A study to determine the absolute content of the volatile component of C. arietinum was conducted after the pod borer (H. armigera) was shown to be attracted to chickpea seed volatiles [14, 88]. GC-MS analysis of headspace material was collected from ground C. arietinum and identified 132 compounds from mass spectrum analysis retention times or Kovats indices (Table 1) along with a further 22 compounds which are not included here because their identities could not be confirmed [89]. The most abundant components were aliphatic hydrocarbons but the next most abundant class was the terpenoids constituting approximately 35% of the volatile fractions of the floured seed. The most abundant compound of all was a-pinene which comprised 12.6% of the total mass of volatile compound. The source of the many aliphatic hydrocarbons in this
929
analysis was unclear although it was argued that they could not be artefacts because they were absent from the control samples [89].
122
121
120
125
129
123
126
130
124
128
131
Sixteen of the most prominent compounds from this mixture of volatile components were tested for activity against the first instars of Helicoverpa pod borer of which 84, 121, 124 & 126 were attractive and thus may play a role in mediating the behaviour of this pest. The synthetic chickpea kairomone (compound produced by one organism that benefits the behaviour of another species) composed of 84, 121, 124 & 126 was attractive to egg laying H. armigera moths in a wind tunnel and this effect was more pronounced with a mixture containing the terpenes and 84 than lures containing the individual compounds [90]. Conversely, neither unmated females nor males responded to the kairomone but field experiments where traps were set up with a kairomone lure containing the 84, 121, 124 & 126 mixture caught almost exclusively, mated females. Thus, selection of varieties that are low in these compounds could provide a route to novel pest management techniques in which oviposition is avoided by reducing attraction to the plant. The carbonyl profile of freshly ground C. arietinum grain was studied later and 18 different compounds were identified [91] of which ten were new to those identified in the study by Rembold [89]. They confirmed the presence of 98, 99, 100, 105, 107, 108, 109, 110 but also identified formaldehyde (213), acetaldehyde (214), butanal (215), pentanal (216), octanal (217), octanone (218), octenal (219), nonenal (220), decenal (221) and decadienal (222) [91]. They noted that the concentration of total
930 Table 1. Volatile compounds from ground C. arietinum seed identified by GC-MS. (from Rembold et al, [89]) No.
Compound
No.
Compound
No.
Compound
Aliphatic alcohols 81
Ethanol
87
Propan-2-ol
93
2-Methylpropan-1 -ol
82
Propan-1-ol
88
Butan-2-ol
94
2-Methylbutan-l-ol
83
Butan-1-ol
89
Pentan-2-ol
95
3-Methylbutan-l-ol
84
Pentan-1-ol
90
Hexan-2-ol
96
l-Penten-3-oi
85
Hexan-1-ol
91
Heptan-2-ol
97
l-Octen-3-ol
86
Heptan-1-ol
92
2-Methy1propan-2-ol
Aliphatic aldehydes 98
Hexanal
102
2-Methyibutanal
105
(E)-2-Pentanal
99
Nonanal
103
3-Methyibutanal
106
(E)-2-Hexanal
100
Decanal
104
(E)-2-Butenal
107
(E)-2-Heptanal
101
2-Methylpropanai
108
Acetone
111
Hexan-2-one
114
l-Octen-3-one
109
Butan-2-one
112
Heptan-2-one
115
3-Octen-2-one
110
Pentan-2-one
113
2-Methylpentan-2-one
116
Methylacetate
118
119
y-Butyroiactone
117
Ethylacetate
129
P-Phellandrene
Aliphatic ketones
Aliphatic esters Butylacetate
terpenoids 120
a-Thujene
125
a-Phellandrene 3
121
a-Pinene
126
A -Carene
130
Limonene
122
Camphene
127
ct-Terpinene
131
y-Terpinene
128
p-Cymene
132
Terpinolene
123
P-Pinene
124
Myrcene
133
n-Pentane
137
n-Nonane
141
«-Tridecane
134
H-Hexane
138
w-Decane
142
n-Tetradecane
135
n-Heptane
139
n-Undecane
143
«-Pentadecane
136
H-Octane
140
«-Dodecane
144
n-Hexadecane
n-alkanes
Branched Alkanes 145
2-Methylhexane
158
3-Methyldecane
166
5-Methyldecane
146
2-Methylheptane
157
3 -Methylundecane
167
5-Methylundecane
931 Table 1 Contd. 147
2-Methyloctane
158
3-Methyldodecane
168
5-Methyldodecane
148
2-Methylnonane
159
4-Methylheptane
169
6-Methylundecane
149
2-Methyldecane
160
4-Methyloctane
170
6-Methyldodecane
ISO
2-Methylundecane
161
4-Methylnonane
171
2,2,4-Trimethylpentane
151
2-Methyldodecane
162
4-Methyldecane
172
2,3 -Dimethylhexane
152
3-Methylhexane
163
4-Methylundecane
173
2,4-Dimethylhexane
153
3 -Methylheptane
164
4-Methyldodecane
174
2,5 -Dimethylhexane
154
3-Methyloctane
165
5 -Methylnonane
175
2,4-Dimethylheptane
155
3 -Methylnonane
176
Ethylcyclopentane
179
Propylcyclohexane
181
Pentylcyclohexane
177
Methylcyclochexane
180
Butylcyclohexane
182
Decahydronaphthalene
178
Ethylcyciohexane
183
1-Hexene
184
185
Benzene
192
Isopropylbenzene
198
anethol
186
Toluene
193
Propylbenzene
199
1 -Methyl-3 -ethylbenzene
187
Ethylbenzene
194
Methylethylbenzene
200
1 -Methyl-2-ethylbenzene
188
p-Xylene
195
Benzaldehyde
201
1,3,5-Trimethylbenzene
189
m-Xylene
196
Benzonitrile
202
1,2,4-Trimethylbenzene
190
Styrene
197
Estragole
203
1,2,3-Trimethylbenzene
191
o-Xylene
204
2-Methylfuran
207
2-Propylfuran
210
2-Pentylfuran
205
2-Ethylfuran
208
2-Butylfuran
211
2-Hexylfuran
206
Dimethyldisulphide
209
Tetrahydrofuran
212
Trichloromethane
Cycloalkanes
Alkenes 1 -Octene Aromatic compounds
Furans and others
carbonyls increased in storage with most of the additional aldehydes formed from the degradation of linoleic and linolenic acids which decreased in storage [91].
932
5- MISCELLANEOUS COMPOUNDS Polyamines. Gallardo et al, [92] investigated alterations in the amine content of seeds of C. arietinum during the onset of germination and identified putrescine (223), spermidine (224), spermine (225) and cadaverine (226). They showed that the embryonic axis contained more of these compounds than the cotyledons themselves and larger amounts of 223, 224 and 225 than embryonic axes excised from the whole seeds before germination. The embryotomised cotyledons showed far higher levels of the amines indicating that there is at least a partial source of polyamines in the cotyledons for the embryonic axis.
NH, 225
226
Saccharides The study of the saccharide content of grain legume seeds is an important food quality determining factor. For example, the presence of raffinose saccharides in grain legumes has been associated with flatulence and increasing galactosyl cyclitol levels and decreasing raffinose saccharide levels in maturing soybean seeds have been proposed as a way of developing desiccation-tolerant soybean cultivars, without the flatulence associated with high levels of raffinose saccharides [93]. In a study of several grain legumes for saccharide content and overall quality a new trisaccharide, (9-a-D-galactopyranosyl-(l-» 6)-O-a;-galactopyranosyl(l-»2)-l-D-4-0-methyl-c/zz>o-inositol (227) was isolated from the grain of C. arietinum [94] and was given the trivial name ciceritol. The compound was also found in the grain of lentil {Lens esculenta) and lupin {Lupinus albus) but its effect on quality was not determined.
933
.OH
227
Coumarins In a study of C. arietinum to identify flavonoids the ground up aerial parts were extracted and analysed. In addition to 14 and 69 already previously identified in the species the authors made the first record of coumarins in the genus, specifically scopoletin (7-hydroxy-6methoxycoumarin) (228) and umbelliferone (7-hydroxycoumarin) (229) [95].
MeO1 228
No ecological or other functional role was suggested for coumarins although elsewhere in plant chemical studies they have a variety of proposed activities. Mandavia et al. [96] have suggested that 229 plays a part in the defence of cumin against Fusarium wilt and thus it may contribute a similar role for chickpea if it were shown to occur in the roots. Furthermore, 228 was tested in vitro for its effect on the germination of proso-millet (Panicum milliaceum L.) seed; mycelial growth of the sweetpotato fungal pathogens Fusarium oxysporum f. sp. batata, F. solani, Lasiodiplodia theobromae, and Rhizopus stolonifer and
934
growth and mortality of diamondback moth [Plutella xylostella] larvae on artificial diet. Compound 228 inhibited seed germination and larval growth but at much higher concentrations than were measured in the tissues. Mycelial growth of the four pathogenic fungi was inhibited at concentrations occuring in some sweetpotato clones indicating a role in plant defence against Fusarium wilt [97]. Compound 228 has also been shown in cell tissue cultures to be induced by F. solani elicitors suggesting again that they can be produced in response to fungal attack [98]. Lactones. The only lactone described so far from Cicer is 2-methyl-2,3,4trihydroxybutanoic acid-l,4-lactone (230) which was identified in the leaves of C. arietinum that had been deprived of water but could not be detected in well watered plants [99]. The author supposes that the compound may therefore be part of a plant growth regulatory mechanism involving feedback inhibition of the biosynthesis of valine which comes into operation during water stress situations.
230
6- BIOSYNTHESIS OF CICER FLAVONOIDS Flavonoids are classified according to their biosynthetic origin with some compounds being both intermediates as well as end products. These include chalcones which are the first C15 structures to be synthesised from 3 molecules of malonyl coenzyme A molecules and pcoumaroyl coenzyme A [100]. Chalcones represent the convergence of two biosynthetic pathways - the acetate providing the A ring and the shikimate leading to the B ring and are therefore the foundation of flavonoid biosynthesis [Fig. (2)]. The only chalcone found so far in deer is isoliquiritigenin (62) which occurs with its glycoside (63). Furthermore, the occurrence of garbanzol (59) & 5-deoxykaempferol (60) alongside 62 with their related substitution patterns suggests that they are
935
intermediates of the flavonoid biosynthesis of the more advanced flavonoids and isoflavonoids. Compound 62 is commonly found throughout the Leguminosae and while it has become well known for its anti-cancer properties [101, 102] its function in Cicer is not known. Its occurrence as a glycoside, however, as with 59, suggests a function more significant than simply as an intermediate in the biosynthesis of other flavonoids since this suggests it is stored in cell vacuoles. The greatest interest in biosynthesis in the genus Cicer has come from work on the production of the isoflavonoids biochanin A and formononetin (25 & 26) and from the pterocarpans medicarpin and maackiain (45 & 46) owing respectively to their occurrence as malonylated glucosides and their role in the defence of the plant against fungi. The earlier contributions to this comprehensive study have been reviewed [103]. Compounds 25 and 26 represent the foundations of the diversity of isoflavonoids in Cicer and are the products of the enzyme catalysed 1,2-aryl migration of the B ring from the 2 to the 3 position of ring C of the flavanone [Fig. (2)]. Compound 26 derives directly from 62 [Fig. (2)] and the presence of the hydroxylated derivatives of 26 (37 & 38) which were identified in germinated cotyledons of C. arientinum that were treated with an Aschochyta rabiei elicitor [38] suggest that these compounds were identified as intermediates in the biosynthesis of defence compounds, e.g., 45 & 46. They are not reported to occur in other plant parts [6]. The presence of 62 even as a stored glycoside may simply be to provide the precursor for rapid isofiavonoid biosynthesis via 61 to 28 and following 4'-0-methoxylation to 26, itself stored in greatest quantities as a glycoside and malonated glycoside as described above. Interestingly, there is no record of chalconaringenin (2',4',6',4-tetrahydroxychalcone) despite the occurrence of 25, 29 and 32 with the 5-OH group that would indicate a chalconaringenin precursor and 65 the flavanone identified in Cicer by Jaques et al., [38]. This may simply be an oversight by phytochemical studies of Cicer but if it does occur it would confirm the presence of two independent but parallel biosynthetic pathways from the chalcones [Fig (2.)] to the various isofiavonoid end products. In the first, 62 undergoing ring closure to produce 61 and subsequent 1,2-aryl migration to give 28 and following 4'-methylation to 26 [Fig (2)]. In the second, chalconaringenin undergoes similar ring closure and rearrangement to produce 32 via 65 and following similar 4'-methylation to 25.
936
3 x Malonyl-CoA
HO.
OH
'OH OH 'OMe
O 66
Fig. (2). Biosynthetic scheme for flavonoids and isoflavonoids in Cicer L. CHS, chalcone synthase; CHI, chalcone isomerase; IFS+IFD, 2-hydroxyisofiavanone synthase + dehydratase; IOMT, Isoflavone 4'-O-methyI fransferase; FLH, flavone 3-hydroxylaae; FLS, flavonol synthase
937
HO
OMe
OMe
OMe
OMe
HO.
-o Fig. (3). Proposed biosynthesis of pterocarpans in Cicer L. IFH, isoflavone 2'-hydroxylase; IFR, isoflavone reductase; PTS, pterocarpan synthase
938
However, it is the subsequent position of phenyl ring substitutions that indicates a divergence for these two biosynthetic pathways. The occurrence of 2'-hydroxyformononetin but no similar 2'-O- substitution in the 5-0 substituted isoflavonoids (25, 29 and 32) suggests that the ring closure from the 2'-hydroxy to form pterocarpans (45 & 46) and coumestanes (56 & 57) only occurs via formononetin as illustrated for the pterocarpans in Fig. (3). Similarly, cytochrome p-450 mediated 3'hydroxylation of 26 e.g., 38 leads to the formation of the methylenedioxy moiety found in 39, 40, 46, 51, 54, 57 & 58. Thus, most of the isoflavonoid structural diversity e.g., the coumestanes, pterocarpans, benzofuran, isoflavenes etc., that provide the genus with its characteristic isoflavonoid profile, appears to derive from 62 via 28 and 26 rather than from chalconaringenin via 32 & 25. The only 5-0-substituted isoflavonoids found are the three isoflavones 25, 29 and 32. Thus high levels of formononetin glycosides may well be a storage precursor for the formononetin derived defence compounds such as pterocarpans and arylbenzofurans in Cicer whereas biochanin A glycosides must have a different role. Significant differences have been shown in the isoflavonoid profiles of 15 species of Cicer [6] that have significant taxonomic implications. The four isoflavones 25, 26, 30 and 34 and three pterocarpans 45, 46 & 50 were found in the roots of 15 species from 6 different sections within 3 subgenera of Cicer. In fact, 25 & 26 and their respective glycosides are the most abundant isoflavonoids in the roots of all species with the exception of the three annuals C. bijugum, C. judaicum and C. pinnatifidum in which 46, 48, 49, 51, 52, 53 and 54 dominate as described previously for C. judaicum [62]. The high concentration of the maackiain glycosides in these three species may, as described above, be indicative of their potential value as sources of improved resistance to root diseases [61]. But their restricted distribution appears to highlight striking chemosystematic differences among the annual species of subgenus Cicer. The 5 isoflav-3-enes and the 2-arylbenzofuran 58 were only found in C. bijugum, C. judaicum and C. pinnatifidum and with 46 and its glycosides constitute the major components of their phenolic profiles. Furthermore, 56 was common to all species except C. bijugum, C. judaicum and C. pinnatifidum. These qualitative and quantitative observations afford a significant distinction between these species and all others in this sub-genus. Moreover, it suggests that C. bijugum, C. judaicum and C. pinnatifidum, are more distantly related to the other species in series Cicer (C. arietinum, C. echinospermum and C.
939
reticulatum) than published taxonomy suggests [8]. This distinction has been reported previously according to other genotypic and phenotypic characters. For example a unique postzygotic reproductive barrier mechanism exists between members of the so-called Group II members (C bijugum, C. judaicum and C. pinnatifidum) and those in Group I (C. arietinum, C. echinospermum and C. reticulatum) [104, 105]. Group II species are further distinguished according to seed storage protein profiles [106], characteristics of trypsin and chymotrypsin inhibitors [107] and isozyme data using Nei's genetic distance analysis [108] a study further supported by earlier isozyme analyses [105, 109]. In addition, chromosome banding pattern analysis supports this distinction, in part, because C. judaicum and C. pinnatifidum have small chromosomes and similar patterns [110]. However, the same study grouped C. bijugum and C. cuneatum together according to their relatively high heterochromatin content. C. cuneatum, however, has a very distinct isozyme profile [111] and morphology and is also the only species known to produce the phytoalexin cuneatin [3]. There appears to be no other evidence however to support a closer genetic relationship between C. bijugum and C. cuneatum than between C. cuneatum and any other deer species. It is worth mentioning that the leaves, seeds and stems of C. bijugum, C. judaicum and C. pinnatifidum did not contain 29, 48, 50, 51, 52, 53, 54, 56, 57 and 58 and excluding the roots only the leaves contained 45 and 46 constitutively. Thus the unique occurrence in C. bijugum, C. judaicum and C. pinnatifidum of isoflav-3-enes, their glycosylated forms and the 2arylbenzofuran 58 indicates that these species are more closely related to each other than to C. arietinum, C. echinospermum and C. reticulatum. hi a revision of the genus a new series Pinnatifida was proposed within Section Cicer containing C. judaicum and C. pinnatifidum [111]. Stevenson and Veitch [6] proposed that based on isoflavonoid profiles that C. bijugum should also be included in this series. Bearing these taxonomic conclusions in mind, an overview of likely biosynthetic relationships between the isoflavonoids discussed in this chapter was proposed [6] and is represented in Fig. (4). This emphasises those compounds whose occurrence is restricted to the 3 species in the proposed series Pinnatifida (C. bijugum, C. judaicum and C. pinnatifidum) and attempted to address the biosynthesis of these compounds, most notably arybenzofurans which are still missing from the latest versions of the isoflavonoid biosynthesis schemes because of the lack of persuasive biogenetic evidence. A 2'-hydroxyisoflavanol, the immediate precursor of isoflav-3-enes and pterocarpans, is shown as the first intermediate. Its
940
biosynthesis has been discussed in a review [112]. Compounds 46, 51, 54, 57 and 58 all possess equivalent A-ring hydroxy and methylenedioxy groups and these were included on all structures in the scheme Fig. (4) as the precise moment of their addition does not affect the proposed pathways. As discussed above C. bijugum, C. judaicum and C. pinnatifidum are the only species to contain isoflav-3-enes and the 2arylbenzofuran. The authors argued that this could be a direct consequence of C-2' methyl transferase activity, the regulation of which may be important in allowing compounds of these classes to accumulate [6]. Two routes have been suggested for the biosynthesis of 2arylbenzofurans, either loss of C-6 from a coumestan such as 57 in Cicer, or loss of one carbon from the phenylalanine-derived C-ring [C-2 of the immediate precursor of 58 in Fig. (4)] [113]. Investigation of the biosynthesis of the phytoalexin arylbenzofuran vignafuran supported the latter route in the cowpea Vigna unguiculata through labelling studies [111]. Consideration of the isoflavonoids present in C. bijugum, C. judaicum and C. pinnatifidum, also supports this biosynthetic route for the formation of 58, as loss of C-6 from the coumestan 57 would give 58. In addition, treatment of 2-hydroxy-isoflav-3-ene with acid also affords a 2arylbenzofuran and a mechanism for this chemical conversion involving loss of one carbon has also been described [114] also suggesting also more direct route from the isoflav-3-ene 51 to 58. This reaction scheme was suggested as a plausible chemical analogy for the corresponding biosynthesis of 2-arylbenzofuran, and a new biosynthetic scheme depicting 2-hydroxy-isoflav-3-ene as the possible common intermediate for both 2-arylbenzofuran and 5-arylcoumarin was proposed [114].
941 HO.
Fig. (4). Summary of proposed biosynthetic relationships among isoflavonoids found in roots of Cicer L. commencing with a 2' hydroxyisoflavanol the precursor of isoflavans, isoflavones and pterocarpans [112].
942
7- SYNTHESIS OF FLAVONOIDS OF STRUCTURAL RELEVANCE TO CICER L. Flavonoids Banerji & Goomer [116] introduced a general method for the synthesis of flavones in which the key step of the synthesis was the aroylation of O-hydroxy-acetophenones (236) to /?-ketones (237) using very mild reaction conditions [Fig. (5)]. Subsequent cyclization of 237 yielded flavones (238) and the authors succeeded in preparing a series of 7 flavones in yields of between 84-91 %. More recently Bois et al., [117] using a similar approach but with improved reaction conditions synthesised 5-hydroxyflavones in a single step reaction from 2,6dihydroxyacetophenone by treating with aroyl chloride in the presence of excess base.
238
x2
6
X1, X2 = H, OMe, X4, X5, X6 = H, OMe, OCH2O
Fig. (5). Synthesis of flavone by Banerji and Goomer [116]; (i) (a) diisopropylamide, THF, -28°C; (b) benzoyl chloride, -78°C; (c) HC1; (ii) AcOH/H2SO4
A very quick and efficient synthesis of flavonols was reported where flavonols were synthesised in 70-90% yields in less then 10 minutes by treating corresponding 2-aryl-3-nitrochromene with base as shown in [Fig (6)] [118]. The formation of flavonols was suggested to occur via the base
943
catalyzed rearrangement of the intermediate epoxide (not shown in Fig. (6).).
(i)
OH
240
Ri, R2, R3 = H, OMe
Fig. (6). Synthesis of flavonols after Deshpande et al., [118]; Reagents and conditions: 15% H2O2, NaOH, CH3OH
In an attempt to probe the biological mechanism of action of natural flavonols and to identify the relevant receptors for flavonols two analogues of kaempferol were synthesised by Tanaka et al, [119]. The target flavonols (242) were prepared by intramolecular dehydrative condensation of 241 with potassium carbonate in pyridine as shown in Fig. (7). o
.A.
R=
Fig. (7). Synthesis of flavonols by Tanaka et al. [119] (TBDPS = tert-butyldiphenylsilyl) (i) (a) K2CO3, pyridine; (b) TBAF, THF; (ii) H2, Pd/C
944
Isoflavonoids Coumestan and Coumestrol
Laschober and Kappe [120] reported the synthesis of coumestan (245) and coumestrol (246) the demethoxy derivative of 9-O-methylcoumestrol (56) reported in Cicer, using an intramolecular Heck reaction as the key reaction step. Thermal rearrangement of the iodonium ylides (243) gave the starting material (244) for the Heck reaction as shown in Fig. (8). Palladium-catalyzed cyclization of (244) gave the required coumestan 245 and the coumestrol 246 was obtained by acid treatment of 245. MeO
O
MeO
O
Fig. (8). Synthesis of coumestan and coumestrol [120] (i) DMF, reflux (ii) PdCl2, NEt3, reflux (iii) HBr/AcOH, reflux.
Kamara et ah, [121] reported the conversion of chalcones (247) to the coumestans (249) for the first time and the coumestan medicagol (57), found in Cicer was synthesised by this approach along with two other analogues. Conversion of 2,2'-dihydroxychalcone (247) to the corresponding coumestan (249) was achieved with thallium(III) nitrate in methanol and subsequent acid treatment as shown in Fig. (9).
945
(i)
MOMO = H, OMe, R2 = H, Me, Ac
(ii)
Fig. (9). Scheme 5; Synthesis of coumestanby Kamara etal, [121]. (i) TI(NO3)3, MeOH; (ii) MeOH, 10% HC1, reflux.
Recently Al-Maharik and Botting [122] reported a very simple and two step synthesis of coumestrol (253). The first step involved condensation of phenyl acetate (250) with benzoyl chloride (251) and the second step required demethylation of 252 and resulted in simultaneous intramolecular cyclization to give the desired product (253) as shown in Fig (10). MeO,
OMe
MeO2C
OMe CO2Me
MeO.
MeO'
250
252
(ii)
253
\__J
Fig. [10]. Synthesis of coumestrol by Maharik & Bottin [122]. (i) n-BuLi, i-Pr2NH, THF (ii) BBr3) CH2C12
OMe
946 Isoflavones
Farkas et al., [123] synthesised different natural isoflavanones (255) and isoflavans (256) by oxidative rearrangement of 2-hydroxy chalcones (254) with thallium nitrate in methanol followed by acid-catalyzed cyclization as shown in Fig. (11).
256
Fig. (11). Synthesis of isoflavonoids by Farkas et al. [123] (i) (a) T1(NO3)3, MeOH; (b) H+ (ii) H2
A simple one-step methodology was reported for the synthesis of isoflavones (259) by Pelter & Foot [124]. The isoflavones were synthesised by reacting deoxybenzoin (257) with dimethoxymethylaminomethane (258) as shown in Fig. (124). H3C
OMe
M
O
257
H3C
OMe
258
259 O
R, R' = OH, OMe
Fig. (12). Synthesis of Isoflavones by Pelter & Foor [124] (i) C6H6, reflux
A single step synthesis of isoflavanones was reported by Gandhidasan et ah, [125] where the required isoflavones (261) was obtained by treating
947
benzyl-2-hydroxyphenyl ketone (260) with p-formaldehyde in the presence of secondary amines in ethanol as shown in Fig. (13).
R2
O
260
261
Rh R 2 ~ H, OMe, R3, R4 = H, OCH 2 O
Fig (13).Scheme 9 Synthesis of isoflavanones by Gandhidasan et a/.,[125] (i) CH2O, NH(CH3)2, MeOH Pterocarpans.
van Aardt et al. [126] reported the synthesis of pterocarpans (267a, 267b) using an isoflav-3-ene (264) as the key structure. The isoflav-3-ene (264) was synthesised via a 4-step reaction scheme involving reduction, cyclization, oxidation and thermal elimination from propanoates (263). The resulting isoflav-3-ene (264) was then treated with OsO4, and subsequent removal of tertabutyldimethylsilyl group followed by selective mesylation gave (6a, 1 la)-cw-6a-hydroxypterocarpan as shown in Fig. (14) in good yields. Thus, provided appropriate substitution patterns could be assured this scheme could be used to produce 45 or 46. MeO,
MOMO
OMe
OMe
Fig. (14). Synthesis of pterocarpans by van Aardt et a/.,[126] (i) LiAlB, (ii) TPP-DEAD (iii) NaIO4 (iv) 60°C (v) OsO4 (vi) TBAF (vii) Ms2O, pyr.
948 A tylbenzofuran
Nova'k et al, [75] synthesised 58 by palladium-catalyzed coupling of halogenated phenols (269) and acetylene (268). The resulting diaryl acetylene (270) underwent simultaneous cyclization upon removal of protecting groups as shown in Fig. (15).
RO
OMe 268
269
^^
OR
270
R = Ac, H
OMe
MeO
(II)
58
O"
Fig. (15). Synthesis of 58 by Nova'k et al. [75] (i) Pd(OAc)2, P;Bu3, Oil, DIPA, 50°C (ii) KOH,aqMeOH. RO.
OMe
OMe
58 OMe
273
Fig. (16). Synthesis of 58 by Astern et a/.,[74,75] (i) MCPBA, CH2C12 (ii) CuCl 2 2H 2 O,(CH 3 ) 2 O/H 2 O, reflux; (iii) p-toluenebezoic acid, CH3C1
More recently 58 was synthesised in our laboratory via a three step reaction scheme by the epoxidation of stilbenes (271), followed by deprotection (272) and acid catalyzed cyclization (273) as shown in Fig.
949
(16). During the study 5 other novel arylbenzofuran analogues were also synthesised along with the key stilbenes which were synthesised by Wittig and Heck approaches [73,74]. 8- NATURAL PRODUCT LIST FOR CICER L. (excludes those in Table 1) (1) malic acid (2) oxalic acid (3) malonic acid (4) citric acid (5) succinic acid (6) oxaloacetic acid (7) fumaric acid (8) glucose-6-phosphate (9) gluconic acid (10) pangamic acid (6-0-(dimethylaminoacetyl)-D-gluconic acid) (11) 3-hydroxycinnamic acid (12) 4-hydroxycinnamic acid (13) caffeic acid (3,4-dihydroxycinnamic acid) (14) ferulic acid (15) 5-O-caffeoylquininc acid (chlorogenic acid) (16) 3-(4-hydroxyphenyl)propanoic acid (phloretic acid) (17) cinnamic acid (18) gallic acid (19) gentisic acid (20) protocatechuic acid (21)/?-hydroxybenzoic acid (22) oc-resorcyclic acid (23) vanillic acid (24) salicylic acid (25) biochanin A (5,7-dihydroxy-4'-methoxyisoflavone) (26) formononetin (7-hydroxy-4'-methoxyisoflavone) (27) biochanin C (28) daidzein (7,4'-dihydroxyisoflavone) (29) pratensein (5,7,3'-trihydroxy-4'-methoxyisoflavone) (30) biochanin A-7-O-glucoside (sissotrin) (31) daidzein-7-O-glucoside (daidzin) (32) genistein (5,7,4'-trihydroxyisoflavone) (33) genistein-7-O-glucoside (genistin)
950
(34) formononetin 7-O-glucoside (ononin) (35) formononetin 7-0-glucoside-6"-0-malonate (36) biochanin A 7-0-glucoside-6"-0-malonate (37) 2'-hydroxyformononetin (38) 3'-hydroxyformononetin (calycosin) (39) pseudobaptigenin (7-hydroxy-3',4'-methylenedioxyisoflavone) (40)cuneatin(7-hydroxy-2'-methoxy-4',5'-methylenedioxyisofiavone) (41) homoferreirin (5,7-Dihydroxy-2',4'-dimethoxyisoflavanone) (42) cicerin(5,7-dihydroxy-2'-methoxy-4',5'-methylenedioxyisoflavanone) (43) homoferreirin 7-0-glucoside-6"-0-malonate (44) cicerin 7-0-glucoside-6"-O-malonate (45) medicarpin (3-hydroxy-9-methoxypterocarpan) (46) maackiain (3-hydroxy-8,9-methylenedioxypterocarpan) (47) medicarpin 3-0-glucoside (48) maackiain 3-0-glucoside (trifolirhizin) (49) medicarpin 3-O-glucoside-6"-O-malonate (50) maackiain 3-0-glucoside-6"-0-malonate (51)judaicin(7-hydroxy-2'-methoxy-4',5'-methylenedioxyisoflav-3-ene) (52) Judaicin 7-O-glucoside (53) Judaicin 7-0-(6"-0-malonylglucoside) (54) 2-methyoxyjudaicin (55) 2-methoxyjudaicin-7-0-glucoside (56) 9-0-methylcoumestrol (7-hydroxy-9-methoxycoumestan) (57) medicagol (7-Hydroxy-11,12-methylenedioxycoumestan) (58) cicerfuran (2-(2'-methylenedioxyphenyl)-6-hydroxybenzofuran) (59) garbanzol (3,7,4'-trihydroxyflavanonol) (60) 3,7,4'-trihydroxyflavonol (5-deoxykaempferol) (61) 7,4'-dihydroxyflavanone (liquiritigenin) (62) isoliquiritigenin (4,2',4'-trihydroxychalcone) (63) isoliquiritigenin 4'-O-glucoside (64) garbanzol 7-O-glucoside (65) naringenin (5,7,4'-trihydroxyflavanone) (66) naringenin 7-Oglucoside (naringin) (67) kaempferol (3,5,7,4'-tetrahydroxyflavone) (68) quercetin (3, 5,7,3',4'-pentahydroxyflavone) (69) isorhamnetin (3,5,7,4'-tetrahydroxy-3'-methoxyflavone) (70) kaempferol 3-O-glucoside (astragalin) (71) kaempferol 3-O-glucoapioside (72) kaempferol 3-O-malonylglucoside
951
(73) kaempferol 3-0-apiosylmalonylglucoside (74) kaempferol 7-0-glucoside (75) isorhamnetin 3-0-glucoside (76) quercetin 3-0-glucoside (77) quercetin 3-0-galactoside (78) luteolin (5,7,3',4'-tetrahydroxyflavone) (79) kaempferol 3-0-glucuronide (80) 5-deoxyquercetin (fisetin) (213) formaldehyde (214) acetaldehyde (215) butanal (216) pentanal (217) octanal (218) octanone (219) octenal (220) nonenal (221) decenal (222) decadienal (223) putrescine (224) spermidine (225) spermine (226) cadaverine (227) 0-O!-D-galactopyranosyl-(l^> 6)-O- a-galactopyransoyl-(l-* 2)-1-D4-0-methyl-c/jzVo-inositol (228) scopoletin (7-hydroxy-6-methoxycoumarin) (229) umbelliferone (7-hydroxycoumarin) (230) 2-methyl-2,3,4-trihydroxybutanoic acid-l,4-lactone ACKNOWLEDGEMENTS. The authors acknowledge grants from the UK Higher Education Funding Council for England. Also, we are indebted to Dr. Nigel Veitch and Professor David Hall for advice in the preparation of this manuscript.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
957
NEW RESEARCH AND DEVELOPMENT ON THE FORMOSAN ANNONACEOUS PLANTS YANG-CHANG WU Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan To honor one of the pioneers of Taiwanese natural product chemistry-late Professor Sheng Teh Lu
INTRODUCTION The Annonaceae, a pantropic family, is well developed in the tropics and the subtropics of both new and old world. Only a few species are distributed in warm temperature (Asiminid) and the islands of the Pacifica. There are ca 130 genera and over 2200 species in the world, and the greatest concentration of genera and species is in Indo-Malaysian area of Asia [1]. Economically this family is an important source of edible fruits, edible oils, raw material for perfumery, and folk medicine for various purposes [2]. In Taiwan, there are 21 species (8 genera) of Annonaceous plants, where three of them are native: Fissistigama glaucescens (Hance) Merr.; F. oldhami (Hemsl.) Merr.; and Goniothalamus amuyon (Blanco) Merr. [3]. Phytochemistry research on Annonaceae in Taiwan began in 1934. An alkaloid, annomontine, was isolated from Annona montana by Nozoe [4]. There were some alkaloids reports in 1970s by T. S. Yang et al. [5] which then continued by our group in 1980s. Most of the early studies focused on alkaloids. Afterwards, many non-alkaloids, Annonaceous acetogenins, diterpenoids, styrylpyrones and so on also were isolated from various species. Some of them showed interesting biological activities. This review discusses recent investigations on the chemistry and pharmacological activities of four major groups: alkaloids, annonaceous acetogenin, diterpenoids, and styrypyrones.
958 Table. 1. The Investigated Condition of Formosan Annonaceous Plants
unripe fruit
fru ts
'
'eaves stem
bark
root
Annona O
A. cherimola Mill. 0
A. glabra L.
O O
A. artemoya (A. cherimola X A. squamosa)
0
A. montana Macf.
O
O
A. muricata L.
0
0
A. reticulata L.
O
0
A. squamosa L.
O
O
O
0
o
0
o
A. purpurea L.
0
0
Artabotrys O
A. uncinatus (Lam.) Merr.
o
Cananga Cananga odorata (Lam.) Hook. F. & Thomas
O
0
Fissistigma F. glaucescens (Hance) Merr. F. oldhami (Hemsl.) Merr
O
0
0
0
0
0
0
O
o
O
0
Goniothalamus G. amuyon (Blanco) Merr. Polyalthia P. longifolia Benth. et Hook. F. P. longifolia Benth. et Hook. F. 'pendula' P. suberosa Hook. f. P. liukiuensis Hatusima Rollinia R. mucosa Bail
0
Uvaria U. rufa Bl
O means the phytochemical research was done or ongoing by our lab.
0
0
959
NITROGEN-CONTAINING CONSTITUENTS OF FORMOSAN ANNONACEOUS PLANTS Annonaceous plants in Taiwan contained a large amount of alkaloids. Among the alkaloids, mostly are benzylisoquinoline-type ones. General benzylisoquinoline alkaloids are a large group of natural products consisting of more than thousands defined structures and also showed diverse biological functions including the analgesics [6], muscle relaxant [7], anti-tumor [8], and antibiotic activity [9]. The biosynthetic of general benzylisoquinolines begins with decarboxylation, or^o-hydroxylation, and deamination of L-tyrosine into both dopamine and 4-hydroxyphenylacetaldehyde (4-HPPA). Norcoclaurine synthase catalyzes the condensation of dopamine and 4-HPAA to form (5)-norcoelaurine, which represents the first committed pathway intermediate with a benzylisoquinoline nucleus [10,11].
deamination |
COOH
Tyrosine
Dopamine
t hydroxylation|
decarboxylation | HO
H2N COOH
-*.
Hi
1 DOPA
* Norcoclaurine synthase
Benzylisoquinoline plays an important role as a biosynthesis centre for Annonaceous alkaloids such as isoquinolones, protoberberines,
960
proaporphines, aporphines, phenanthrene, aristolactam and litebamine alkaloids.
isoquinolone
morphinandienone
tetrahydroprotoberbenne
tetrahydrobenzylisoquinolme
dioxoxaporphine
litebamine
aristolactam
Six benzylisoquinolines were isolated from Formosan Annonaceous plants. Reticuline (A-l), the most frequently occurring
961
benzyltetrahydroisoquinoline, along with coclaurine (A-2) and iV-methylcoclaurine (A-3) were isolated from A. squamosa [12]. (+)-Orientaline (A-4), together with two new compounds, annocherine A (A-5) and annocherine B (A-6), were isolated from the stems of A. cherimola [13,14],
H A-l, Rj= CH 3 , R2=OH, R3=OCH3
A-S, R=CH3 A-6, R=H
A-2, R t = H, R2= H, R3= OH A-3, R,= CH3, R2= H, R3= OH A-4, R,= CH3, R2=OCH3, R3=OH
Simple Isoquinolones No biogenetic details of simple isoquinolone were elucidated, but it is likely to yield the isoquinolones from the oxidation of benzylisoquinolmes. There are a few isoquinolones in the Annonaceae. Only three simple isoquinolones, doryphornine (A-7), cherianoine (A-8) and thalifoline (A-9), were isolated from the stems of A. cherimola [14,15]. CH 3
H R A-7
A-8, R=H A-9, R=OCH 3
Protoberberines and Tetrahydroprotoberberine Protoberherines: The protoberberine alkaloids are the most widely distributed benzylisoquinoline alkaloids. Hundreds of alkaloids of this series are discovered that they were yielded from the tetrahydroisoquinoline catalyzed by BBE [16], the berberine bridge
962
enzyme. Berberine has general antimicrobial properties [16]. It inhibits reverse transeriptase [17], intercalates with DNA [18], and inhibits aldose reductase [19,20]. Five protoberberines were presented in Formosan Annonaceous plants. Among them, palmatine (A-10) was isolated from G. amuyon [21]. Fissisaine (A-11), eolumbamine (A-12), and dehydrodescretamme (A-13) were isolated from F. balansae (materials collected in Mainland China) [22]. (-)-8-Oxopolyalthiaine (A-14) was derived from P. longifolia [23].
A-10 A-11 A-12 A-13
Ri OCH3 OCH 3 OH OCH 3
R2 OCH3 OCH3 OCH 3 OH
Rj H OH H H
R4 OCH3 OH OCH3 OH
A-14
Tetrahydroprotoberberines (4H-protoberberines): There are four 4H-protoberberines described among Annonaceous plants in Taiwan: Discretamine (A-15) was obtained from F. glauscense [21]; Tetrahydropahnatine (A-16), from G. amuyon [A16], thaipetaline (A-17), from F. balance [22]; and (-)-Kikenamine (A-18), from the stems of A. cherimola and F. balance [13,22]. Most of these 4H-protoberberines are 2,3,9,10-oxygenated derivatives and only A-17 was 2,3,4,9,10oxygenated derivative.
A-15 A-16 A-17 A-18
Ri R2 OH H OCHj H OH OH OCHj H
R3 OH OCH, OH OH
963 963
Proaporphines Proaporphines is a small group of compounds among the Formosan Annonaceous plants; it was regarded as the direct precursor of aporphines [16]. In previous studies, proaporphines demonstrated antivirus [24], anesthetic and weak anticancer activities [16]. Three proaporphines, including stepharine (A-19), glaziovine (A-20) and promucosine (A-21), were extracted from the leaves of A. purpurea [25,26]. Among them, A-21 was the first naturally occurring TV-esterficated proaporphine.
R,
R2
A-19 OCH3
CH3
A-20 OH
CH3
A-21 OCH3 COOCH3
Aporphinoids Aporphines: Aporphines, the simplest derivatives of the tetrahydrohydrobenzylisoquinoline, are the most abundant alkaloids in Formosan Annonaceous plants. There are several different biogenetic pathways of aporphines [16]. In view of the related derivatives, the biogenetic origin of aporphine alkaloids in Annonaceae could be regarded as benzylisoquinolines or proaporphines, which result in the different substitution of aporphines. The 1,2,9,10- and 1,2,10,11- substituted aporphines were synthesized via the ortho-para or ortho-ortho phenolic coupling, respectively. The 1,2- or 1,2,10- substituted ones were formed directly from proaporphines [16]. Most aporphine alkaloids are toxic. They exhibit antagonistic effects to dopamine. Many of them have anticonvulsant activity or induced convulsions in animals [27,28] and cytotoxic activity [29]. They are distributed in all the known Formosan Annonaceous species. Thirty-three aporphines (A-22-A-54) were isolated and eleven of them were identified as new compounds. Among these aporphines, iV-methoxycarbonyl aporphines (A46-A-54), isolated as novel //-substituted aporphines, were firstly described by our lab. Their structures and the occurrences are listed in the following table.
964
Table, 2. The Structures of Aporphines, (* New compound)
Compounds A-22 annonaine
A-23 anolobinc
A-24 artabonatine* A-25 asimilobine
-OCH2O-
-OCH2O-
-OCH2OOH
OCH3
R,
R,
R,
R»
H
H
H
H
H
H
H
OCHj
H
H
R>
Occurrence
Ref.
H
A. cherimala
13
A, glabm
30
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
A-26 calycine
-OCH2O-
H
H
H
OCHj
H
OH
A-27 crebanine
-OCHjO-
H
H
OC
OGH3
H
H
G. amuyon
2i
P. longifolia
23
A. cherimala
13
F. glauscetice
32
F. oldhamii
21
A. uncinatus
33
F. oldhamii
21
A, uncinatus
33
F. oldhamii
34
F. glaucescens 21
H, A-28 glaucine
OCH3
OCHj
H
H
H
OCH3
OCH3
H
CH3
A. purpurea
A-29 isocorydine
OCH3
OCH3
H
H
H
H
OCH,
OH
CH3
A. eherimofa
13
A. purpurea
26
A-30
laurotetanine
A-31 Hrinidine A-32
michelahine
A-33 iV-ibnnylanonaine* A-34 iV-fonnylpurpureine*
25
OCHj
OCH3
H
H
H
OH
OCH3
H
H
A, cherimala
13
OCH3
OH
H
H
H
H
H
H
CHj
A. purpurea
26
H
OH
H
H
H
H
H
A. cherimola
13
C. adorata
35
-OCHjO-
-OCH2OOCH3
OCHj
H
H
H
H
H
H
CHO
A. gtabra
30
OCH3
H
H
OCH3
OCHS
H
CHO
A. purpurea
25
965 A-35 A'-mediyllaurotetanine
H
H
H
OH
OCHj
H
CH,
A. cherimafa
13
H
H
H
H
H
H
CHj
A. cherimah
13
H
H
H
H
H
F. otdhamii
21
OCH3
H
H
H
-OCHjO-
H
H
A, gtabra
30
A-39 norisocorydine
OCH, OCH,
H
H
H
H
OCH3
OH
H
A. cherimala
15
A-40
nornuciferine
OCH3
OCH3
H
H
H
H
H
H
H
A. glabra
31
A-41 norpurpureine
OCH3
OCH3
OCH, H
H
OCHj OCH,
H
H
A. purpurea
26
OH
OCH3
H
H
H
OCHj OCH,
H
CH,
P. longifatia
23
A-43 thabaicalidine
OCH3
OCH3
OH
H
H
OCH3
OCH,
H
CH,
A. purpurea
25
A-44 thalicimidine
OCH3
OCH,
OCHj
H
H
OCHj
OCH3
H
CH3
A, purpurea
26,36
H
OH
H
H
H
H
CH,
A, cherimola
13
C, odorata
35
A-36 /V-methylasimilobine A-37
norannuradhapurine
A-38 nordometicme
A-42
predecentrinc
A-45 ushisunine
A-46 romucosine*
OCH, OCH3 OH
OCHj
-OCH2OOH
-OCH2O-
-OCH2O-
OH OCHj
F. giuucescvm 21
H
H
H
H
H
H
COOCH, R. mucma
37
A-47 romucasine A*
OH
OCH3
H
H
H
H
H
H
COOCH3 R. mucma
38
romucosine B*
OH
OCH3
Cl
H
H
H
H
H
COOCH3 & mucasa
38
A-49 romucosine C*
OCH3
OCH3
H
H
H
H
OH
H
COOCH3 /?, mucma
38
A-50 romucosine D*
OCH3
OCHj
H
H
H
H
OCH3
H
COOCH, R. mucosa
38
romucosine E*
OCH3
OCH3
H
H
H
H
H
H
COOCH, R. mucosa
38
A-52 romucosine F*
OCH,
OCH3
Cl
H
H
OCHj
OCH3
H
COOCH, R, mucosa
26
A-53 romucosine G*
OCHj
OCH,
OCH3
H
H
OCH3
OCH,
H
COOCH3 A. cherimala
14
romucosine H*
OCHj
OCH,
H
H
H
H
OCH3
OH
COOCH, R. mucasa
14
A-48
A-51
A-S4
Aporphine-N-oxides: Aporphine-iV-oxides, the iV-oxidation products of aporphines, were also isolated. Four Aporphine-iV-oxides were obtained from P. longifolia, including (+)-O-methylbulbocapnine-a -iV-oxide (A-S5), (+)-O-methylbulbocapnine- (3-iV-oxide (A-56), (+)-JV-methyl nandigerine-p-iV-oxide (A-57), (+)-oliveroline-P-iV-oxide (A-58). A-55-A-57 were isolated as new compounds [39].
966
A-55 A-56 A-57 A-58
R, R2 H OCH3 H OCH3 H OCR, OH H
R.i
OCH3 OCH3 OH H
orientation ofiV-oxide a
P P P
orientation of 6a-H a a a
P
Oxoaporphines: Oxoaporphines, the other oxidation products of aporphines, were also widely distributed among Annonaceous plants. Fourteen oxoaporphines were isolated and three of them were new compounds. Among them, liriodenine (A-59) is the most ubiquitous and isolated from several species of Formosan Annonaceous plants: A. cherimola [13], A. montana [40], A. reticulate [41], A. squamosa [12], R. mucosa [37], A. uncinatus [42], F. glauscense and G. amuyon [21]. Annolatine (A-60) was isolated from A. montana [40]. Artherospermidine (A-61) was isolated from A. cherimola [13] and A. uncinatus [42]. Atherospermidine A (A-62) was isolated from A. uncinatus [42]. Fissiceine (A-63) was yielded from F. glaucesens [32], kuafumine (A-64) whereas oxocrebanine (A-65) was isolated from F. glauscense [21,32,43]. Oxoanolobine (A-66) and oxoasimilobine (A-67) were isolated from A. cherimola [13,15]. Oxonantenine (A-68) was derived from A. reticulate [12]. Oxoglaucine (A-69) was isolated from A. cherimola [13] and A. purpurea [25,26]. Oxonuciferine (lysicamine, A-70) was isolated from A. cherimola [13], A. glabra [30], A. purpurea [25,26], and C. odorata [35,44]. Oxopurpureine (A-71) yielded from in A. purpurea [25,26], and oxoxylopine (A-72) from A cherimola [13] and F. glaucescens [32].
967
Table. 3. The Structures of Oxoaporphincs. (* New compound)
Ri A-59 A-60
R2
-OCH 2 OOCH 3
OH
R3
R4
R5
R*
H
H
H
H
H
OCH,
OCH3
H
A-61
-OCH 2 O-
OCH3
H
H
H
A-62
-OCH2O-
CH3
H
H
H
A-63
-OCH 2 O-
H
OH
OCH3
H
A-64
-OCH2O-
OCH,
OCH,
OCH,
H
A-65
-OCH2O-
H
OCH3
OCH3
H
A-66
-OCH 2 O-
H
H
OH
H
A-67
OH
OCH3
H
H
H
H
A-68
OCH3
OCH3
H
H
OCH,
OCH3
H
H
A-69
-OCHjO-
-OCH2O-
A-70
OCH3
OCH,
H
H
H
H
A-71
OCH,
OCH3
OCH3
H
OCH,
OCH3
H
H
OCH3
H
A-72
-OCH2O-
6a,7-Dehydroaporphines: 6a,7-Dehydroaporphines is a small group of aporhinoids with only four compounds were isolated from A. purpurea, including 7-formyl-dehydrothalicsmidine (A-73), 7-hydroxydehydrothalicsmidine (A-74), 7-hydroxy-dehydroglaucine (A-75) and dehydrolirinidine (A-76) [25,36].
968
A-73 A-74 A-75 A-76
R, OCH3 OCH3 OCH3 OCH3
R2 OCH3 OCH3 OCH3 OH
R3 OCH3 OCH3 H H
R4 OH CHO OH H
R5 R6 O C H , OCH3 OCH3 OCH3 OCH3 OCH3 H H OCH3
CH3O.
CH,
CH 3
HO'' A-79
A-80, R=OCH 3 A-81,R=OH
OCH,
A-83
A-82
CH3O
A-85, R O H A-86, R O C H 3
A-84
969
Besides the typical aporphinoids, several aporphinoids with interesting structures were also isolated. Two /?-quinone-aporphines, (-)-fissilandione (A-77) and (-)-norfissilandione (A-78), were only isolated from F. balansae [22]. Artacinatine (A-79) and artabonatine C-F (A-80-A-83), were isolated from A. uncinatus [42,45]. Annobraine (A-84) was isolated from A. glabra [30]. Noraristolodione (A-85) and norcepharadione B (A-86) were isolated from F. balansae [22]. Phenanthrenes Most phenantherenes here are aminoethylphenantherene derivatives (open aporphines). There are eight phenantherenes distributed in two genera of Formosan Annonaceous, Annona and Fissistigma. Fissicesine (A-87), fissicesine-A^-oxide (A-88), atherosperminine (A-89), iV-noratherosperminine (A-90), and 7V-methylatherosperminium (A-91) were isolated from F. glauscense [21,46]. Argentinine (A-92) and annoretine (A-93) were isolated from A. montana [40]. Thalicpureine (A-94) was yielded from the leaves of A. purpurea [25]. Among these isolates, A-87~A-88 and A-91~A-93, were isolated as new compounds and A-93 as a novel litebamine-type skeleton which was firstly described by our lab [47,48].
A-87 , R=N(CH3) A-88 , R=N+(CH3)2O"
A-91
A-89, R=CH3 A-90, R=H
OCH
CH3I
A-92
A-93
A-94
970 970
Aristolactams Aristolactams were regarded as intermediates in the biosynthetic pathway of aristolochic acid, which was produced by aporphines via oxidation process. Eleven aristolactams were isolated from Fissistigma. Piperolactam A (A-95), piperolactam C (A-96), aristolactam Allla (A-97), aristolactam BII (A-98), and agoniothalactam (A-99) were isolated from F. balansae [49]. Aristolactam FII (A-100), stigmalactam (A-101), aristolactam All (A-102), enterocarpam I (A-103), and velutinam (A-104) were isolated from F. oldhamii [49]. Aristolactam Bill (A-105) was isolated from both the species [49].
Table. 4. Structures of Aristolactams in Annonaceae
R.
R:
R3
R4
R5
R«
A-95
H
OCH3
OH
H
H
H
A-96
OCH3
OCH3
OCH3
H
H
H
A-97
H
OH
OCH,
OH
H
H
A-98
H
OCH3
OCH3
H
H
H
A-99
H
OCH3
OCH3
OH
H
H
A-100
OCH,
OH
OCH3
H
H
H
A-T01
OCH3
OCH3
OCH3
OH
H
H
A-102
H
OH
OCH,
H
H
H
A-103
H
OH
OCH3
H
OCH3
OCH,
A-104
OCH,
OCH3
H
H
OH
H
A-105
H
OCH,
OCH3
OCH3
H
H
971
Amides Eight acyl-amides, cherinonaine (A-106), a novel dimeric acyl-amide, along with dihydro-feruloytyramine (A-107), Af-m-caffeoyltyramine (A-108), A^nms-feruloymethoxytyramine (A-109), N-cisferuloymethoxytyramine (A-110), jV-fraHs-feruloytyramine (A-lll), iV-frvms-caffeoyltyramine (A-112), and iV-p-coumaroyltyramine (A-113) were isolated from ,4. cherimola [50,51]. A - l l l was also isolated from C. odorata.
CH 3 O.
CH 3 O'
A-107
H
,-OMe "OH
HO
HO
OMe
A-108
A-109, *= trans A-110, *~cis
OH
A-lll, R=OCH3 A-112, R=OH
A-113
Azafluorenes As an observation of our previous studies, azafluorenes were only distriuted in Polyalthia. The biogenetic origin of azafluorenes was proposed: it arises from the degradation of oxoaporphines into 1-azaanthraquinolone and then the loss of CO group converts the 1-azaanthraquinolone into azafluorenes [52]. There are four azafluorenes-
972
polylongine (A-114), darienine (A-115), polyfothine (A-116), and isooncine (A-117)- isolated from P. longifolia [39,53].
Ref. 47
R, A-114
R2
R3
A-115 OCH3 OCH3
OH
A-116
H
OCH3
OCH3
A-117
H
OCH3
OH
Other Alkaloids An indole alkaloid, cheritamine (A-118), and perlolidine (A-119), which possessed a novel skeleton, were isolated from A. cherimola [50] and A. squamosa [54], respectively. Rollipyrrole (A-120), a propentdyopent derivative, was isolated from R. mucosa [55]. Glaucenamide (A-121), a terpene alkaloid, was isolated from F. glaucescens [32]. Cananodine (A-122), a guaipyridine sesquiterpene alkaloid, was isolated from C. odorata [44]. Uncinine (A-123), a (3-utenolide alkaloid, was isolated from A. uncinatus [45]. A-118-A123 were isolated as new compounds. One morphinandienone, O-methylflavinantine (A-124), was isolated from F. oldhamii [21]. Adenosine (A-125) and uridine (A-126) were isolated from A. cherimola [13,15]. Cleistopholine (A-127) was isolated from C. odorata [35]. Squamolone (A-128) was isolated from A cherimola [13] and A purpurea [26].
973 H (CH 2 ) 2 2 -
2
O
A-118
O.
NH2 A-121
A-119
O
O
CH, H3O
A-122
NH2
A-123
OH
O
o
Ho'
A-125
A-126
A-128
Bioactivities of Annonaceous Alkaloids Cytotoxicity: In our published studies, several alkaloids were screened including benzyl isoquinolines, protoberberines, aporphines, and amides. Palmatine [56], norannuradhapurine (A-37) [56], kuafumine (A-64) [43], liriodenine (A-59) [56], atherosperminine (A-89) [56], argentinine (A-92) [40], annoretine (A-93) [40], A^rans-femloytyramine (A-111) [44,57], A^-^ran^-caffeoyltyramine (A-112) [57], A^-p-coumaroyltyramine (A-113)
974
[57], cananodine (A-122) [44] and cleistopholine (A-127) [44] were cytotoxic. Among them, liriodenine was the most potent alkaloid against P-388 (murine lymphocytie leukaemia), KB (human nasopharyngeal carcinoma), and HT-29 (human colon carcinoma) cells [56]. Antiplatelet Aggregation Activity: Alkaloids from Annonaceous plants were reported to inhibit platelet aggregation. According to our previous studies, more than twenty compounds were examined and most of the alkaloids were found inhibiting arachidonic acid (A.A) induced platelet aggregation [41]. Phenanthrene-alkaloids inhibited platelet aggregation initiated by four inducers (arachidonic acid, PAF, collagen, and thrombine) [58] whereas iV-oxide derivatives of aporphines showed slightly reduced efficacy as compared to the parent aporphines [59]. 6a,7-Dehydroaporphines showed better performance than the aporphines and three of them specifically inhibited platelet aggregation induced by PAF [25,36]. Recent studies of iV-methoxycarbonyl aporphines also revealed significant activities [38]. Cardiovascular Activity: Vasorelaxation can be induced by liriodenine (A-59) [60,61], isocorydine (A-29) [41], coclaurin (A-2) [58], iV-methyleoclaurine (A-3) [58], atherosperminine (A-89) [58] and iV-methylatherosperminium (A-91) [58]. Liriodenine was also found to have many bioactivities including antiarrythmic efficacy [62] and antimuscamic activity [60,61]. Antimicrobial activity was displayed by phenanthrenes, benzylisoquinolines and aporphines [63]. (-)-Discretamine (A-15) was examined as an antagonist of a-adrenoceptor, (XiD-adrenoceptor and 5-HT receptors [64,65]. Trachealis relaxation was induced by atherospermidine (A-61) [65].
ANNONACEOUS ACETOGENINS Annonaceous acetogenins are a bio-potent class of natural compounds isolated from Annonaceae plants. Since the first bioactive Annonaceous acetogenin, uvaricin, was found from the roots of Uvaria accuminata (Annonaceae) by Mad et al. in 1982 (Fig. 1) [66], more than 350 Annonaceous acetogenins have been isolated in the last two decades. Generally, it has been reported that Annonaceous acetogenins possess a broad spectrum of bioactivities, such as anticancer, antiparasitic, insecticide, and immunosuppressive effects.
975 OAc
erythro
trans | trans threo
O
Fig. (1). Uvaricin
The common structural features of Annonaceous acetogenins are a terminal y-lactone ring (Fig. 2) and a terminal aliphatic side chain connected with some oxygen-bearing moieties, such as one to three tetrahydrofuran rings, several hydroxyls, acetoxyls and/or ketones (Fig. 3). Depending on the substitutes in the long aliphatic chain, Annonaceous acetogenins were classified into several subtypes, including 1) acetogenins with mono-tetrahydrofuran (THF) ring, 2) ones with adjacent bis-THF rings, 3) ones with non-adjacent bis-THF rings, 4) ones with non-adjacent THF rings or tetrahydropyran (THP) rings, 5) ones with adjacent tris-THF rings, and 6) miscellaneous, which includes those substituting for epoxide or/and double-bond [67].
Fig. (2). Types of the terminal y-lactone rings systems in Annonaceous acetogenins.
976 976 OH
Mono-THF
OH
OH
O
ring Bl
B2
B3
B4
Bis-adjacent THF ring
OH
Non-adjacent
,
THF ring B5 Tri-adjacent
OH
O.
.0.
THF ring
OH
HO.
OH
THP rings
B7
B8 O
Epoxide B9
Bll
BIO
977 Linear (double
OH bond
or diol)
Fig. (3). Types of the oxygen-bearing moiety systems in the aliphatic side chain of Annonaceous acetogenins
All Annonaceous acetogenins contain multiple stereocenters, the elucidation of which often presents stereochemical problems. Because of their waxy nature, Annonaceous acetogenins do not form crystals suitable for X-ray crystallographic analysis. Relative stereochemistries of ring junctions have typically been determined by comparison of natural compounds with synthetic model compounds, and such methods are proven invaluable to the study of acetogenins. Recently, the absolute stereochemistries of the carbinol centers of acetogenins were determined with the help of synthetic model compounds and high field nuclear magnetic resonance (NMR) analysis of their methoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters) [68]. In the past two decades, our laboratory has focused on the investigation of the bioactive constituents in the family plants in Taiwan. A series of acetogenins (AA01-AA75) were isolated from two genera of Formosan Annonaceous plants, Annona and Rollinia. Among them, twenty-seven acetogenins were published as new compounds [69]. Most of them include one or two tetrahydrofuran ring(s), an a»(3-unsaturated-y-lactone as the main structure with functional groups such as -OH, =0, C=C, and adjacent diols on the long chain. According to the structural features, Formosan Annonaceous acetogenins are classified as 1) acetogenins with mono-tetrahydrofuran (THF) ring, including six subtypes (Al-Bl, A1-B2, A2-B1, A2-B2, A3-B1, and A7-B1), 2) ones with adjacent bis-THF rings, including four subtypes (A1-B3, A2-B3, A2-B4, and A3-B1), 3) ones with non-adjacent bis-THF rings, including three subtypes (A1-B5, A2-B5, and A3-B5), and 4) miscellaneous, including those substituted with epoxide and/or double-bond (A1-B9/B11, A2-B9, and Al-Bl 2). The bioactivities of these compounds have been studied and will be discussed later. The following sections will introduce the Annonaceous acetogenins from Formosan Annonaceous plants according to their structural classification.
978
Annonaceous Acetogenins with Mono-Tetrahydrofuran (THF) Ring Mono-THF compounds are the largest group of Annonaceous acetogenins. This class of acetogenins possesses a THF ring with one or two flanking hydroxyls and various terminal lactone rings. To date, thirty-eight mono-THF acetogenins were isolated from Formosan Annonaceous plants. They were classified into six subtypes according to the terminal lactone rings and the number of the flanking hydroxyls, including 1) Al-Bl, six acetogenins with a terminal oc,p-unsaturated y-lactone ring and two hydroxyls flanking with the THF ring (AAOl-07), 2) A1-B2, two acetogenins with a terminal a,|3-unsaturated y-lactone ring and one hydroxyl flanking with the THF ring (AA08, 09), 3) A2-B1, fifteen acetogenins with a terminal a,P-unsaturated y-lactone ring with a hydroxyl at C-4 and two hydroxyls flanking with the THF ring (AA10-24), 4) A2-B2, nine acetogenins with a terminal a,P-unsaturated y-lactone ring with a hydroxyl at C-4 and one hydroxyl flanking with the THF ring (AA25-33), 5) A3-B1, five acetogenins with a ketolactone terminal and two hydroxyls flanking with the THF ring (AA34-39), and 6) A7-B1, one acetogenin with a terminal oc,P-unsaturated y-lactone ring flanking with a THF ring and two hydroxyls flanking with the THF ring (AA40). These compounds are listed as follows (see Table 1). Among these compounds, muricins A and B were isolated and elucidated as stereoisomers. Their absolute configurations were determined through analyses of their Mosher ester derivatives. Muricin B (AA30) is the first example of Annonaceous acetogenin that possesses a hydroxyl group of the S-configuration substituted at C-4. Muricin C is the first example of an Annonaceous acetogenin in which the THF ring begins with another odd position at C-l 7. Muricins D and E are the first Annonaceous acetogenins that possess a C33 skeleton. Table S. Mono-THF Acetogenins Isolated From Formosan Annonaceous Plants
No
Compound Name
THF and Hydroxyl Positions
THF Relat. Config.
Molecular Formula
Molecular Weight
thltlth
CSSHMO,
564
thltlth
C37HftsOs
592
thltlth
CJJHMO,
580
la Al-Bl without free OH group AA01
Solamin
AA02
Reticulatacin
lb Al-Bl with free OH group AA03 Longifolicin
mono-THF 15-20 mono-THF 17-22
mono-THF
979
AA04
Corossolin
AA05
ei'.v-Corossolin
AA06
Corossolone
AA07
c/'.s-Corossolone
10, 13-18 mono-THF 10,15-20 mono-THF 10, 15-20 mono-THF 10=O, 15-20 mono-THF 10=O, 15-20
thltlth
CJSHMOS
580
th/c/th
C35H64O6
580
thltlth
C3.sH62Ofi
578
th/c/th
C35H62O6
578
t/th-th
CJSHMCX
580
t/th-th
C35H64O6
580
thltlth
C35H64O8
612
thltlth
C35HMO7
596
thltlth
C35HMO7
596
thltlth
C 35 H 62 O 7
594
thltlth
C.WHMO7
596
thltlth
C35HMO7
596
thlclth
C35H64O7
596
thltlth
C 3 7Hfis07
624
thltlth
CH^O,
624
thlclth
C57H68O7
624
thltlth
C35H62O7
594
thltlth
C.,5HMO7
596
thltlth
C35H64O6
580
thltlth
CJSHMO?
594
thltlth
C35HHO8
612
t/er-th
1 CJSHMO?
596
2 A1-B2 with free OH group AA08
Vluricin H
AA09
Muricin I
mono-THF -19,24,25 mono-THF -19,24,25,28=29
3 A2-B1 with free OH groups AA10
Annomonicin
AA11
Xylopianin
AA12
Annoreticuin*
AA13
Annoreticuin-9-one*
AA14
Gonithalamicin
AA15
Annonacin
AA16
Cis-annonacin
AA17
Xylomaticin
AA18
Annomontacin
AA19 AA20
Cis-annomontacin* Annonacinone =Annonacin-10-one
AA21
Rolliacocin *
AA22
Murisolin
AA23
Muricin G*
AA24
Annomurilin*
mono-THF 4,8, 13, 15-20 mono-THF 4,8,15-20 mono-THF 4,9, 15-20 mono-THF 4,9=0,15-20 mono-THF 4, 10, 13-18 mono-THF 4, 10, 15-20 mono-THF 4, 10, 15-20 mono-THF 4, 10, 15-20 mono-THF 4, 10, 17-22 mono-THF 4, 10, 17-22 mono-THF 4, 10=O, 15-20 mono-THF 4, 11, 15-20 mono-THF 4, 15-20 mono-THF 4, 10, 15-20,23=24 mono-THF 4, 16-21,28,29
4 A2-B2 with free OH groups AA25 iMuricatetrocin A
mono-THF
I
980
AA26
Muricatetrocin B
AA27
Muricin E*
AA28
Muricin D*
AA29
Muricin A*
AA30
Muricin B*
AA31
Annocatalin*
AA32
Muricin C*
A A3 3
Muricin F*
4,-16, 19,20 mono-THF 4,-16,19,20 mono-THF 4,-16,22,23 mono-THF 4,-19,22,23 mono-THF 4,-19,26,27 mono-THF 4,-19,26,27 mono-THF 4,-19,28,29 mono-THF 4,-21,24,25 mono-THF 4,-21,24,25,28=29
tler-th
C35H64O7
596
tlth-th
C35HMO7
596
tlth-th
C35H64O7
596
tlth-th
CJSHMO,
596
tlth-th
C3 5 H M O 7
596
tlth-th
C35H&1O7
596
tlth-th
C35H64O7
596
tlth-th
C35H62O7
594
thltlth
C35H64O6
580
thltlth
C35H64O7
596
thltlth
C35HMO7
596
thltlth
C35H62O7
594
thltlth
C3 5 H 62 O 7
594
thltlth
C35HMO<,
578
t-thltler
C35H60O7
592
5 A3-B1 AA34
Murisolinone*
AA35
Isoannonacin
AA36
Isoannoreticuin
A A3 7 Isoannonacin- 10-one A Squamone A A3 8 (isoannonacin-9-one) AA39
Isomurisolenin
the ketolactone terminal and mono-THF 15-20 mono-THF 10, 15-20 mono-THF 9, 15-20 the ketolactone terminal and mono-THF 10=O, 15-20 the ketolactone terminal and mono-THF 9=0, 15-20 the ketolactone terminal and mono-THF Cl 1=C12, 15-20
6 A7-B1 AA40
Aromin
mono-THF 9=0,15-20
Annonaceous Acetogenins with Adjacent Bis-THF Rings This class of acetogenins possesses adjacent bis-THF rings with one or two flanking hydroxyls and various terminal lactone rings. To date, fifteen adjacent bis-THF acetogenins were isolated from Formosan Annonaceous plants. They were classified into four subtypes according to the terminal lactone rings and the number of the flanking hydroxyls, including 1) A1-B3, five acetogenins with a terminal a,(3-unsaturated y-lactone ring and two hydroxyls flanking with the THF rings (AA41-45), 2) A2-B3, seven acetogenins with a terminal a,(3-unsaturated y-lactone ring with a hydroxyl at C-4 and one hydroxyl flanking with the THF rings (AA46-52),
981
3) A2-B4, two acetogenins with a terminal a,(3-unsaturated y-lactone ring with a hydroxyl at C-4 and two hydroxyls flanking with the THF rings (AA53,54), and 4) A3-B1, one acetogenin with a ketolactone terminal and one hydroxyl flanking with the THF rings (AA55). These compounds are listed as follows (see Table 2). Table 6. Adjacent Bis-THF Acetogenins Isolated From Formosan Annonaceous Plants
No
Compound Name
THF and Hydroxyl Positions
THF Relat. Config.
Molecular
Molecular
Formula
Weight
la A1-B3 without Hydroxyl groups AA41
Neoannonin
adjacent bis-THF 13-22
thltlthltler
CSSHKC
578
AA42
Desacetylyvaricin
adjacent bis-THF 15-24
thltlthltler
C37H66O6
606
AA43
Isodesacetylyvarcin
adjacent bis-THF 15-24
thltlthltlth
C37H6GO6
606
adjacent bis-THF 15-24,28
thltlthltler
C37H66O7
622
thltlthltler
C37H66O8
638
thltlthltler
CJSH^O,
594
thltlthltlth
C 3 5 H62O 7
594
thltlthltler
C 37 H«,O 7
622
thltlthltler
C37H«*O7
622
thltlthltlth
C 3 7 H66O 7
622
thltlthltler
C37H«,O7
622
lb A1-B3 with Hydroxyl groups AA44
Squamocin
AA45
Rollimusin *
adjacent bis-THF 10, 15-24,28
2a A2-B3 without free hydroxyl groups AA46
Molvizarin
AA47
Parvi florin
AA48
4-hydroxy-25hydroxyneorollinicin (Rolliniastatin 1)
AA49
Annoglaucin
AA50
Asimicin
AA51
Bullatacin
adjacent bis-THF 4, 13-22 adjacent bis-THF 4, 13-22 adjacent bis-THF 4,15-24 adjacent bis-THF 4, 15-24 adjacent bis-THF 4, 15-24 adjacent bis-THF 4, 15-24
982
2b A2-B3 with free hydroxyl group AA52 3
thltlthltlth
CJTHKAS
638
tlthltlth
CJSHSZOB
578
cIMclth
C3sH«aO«
578
thltlthitler
C 3 ? H«O 7
622
A2-B4
AA53
Annoeatacin A*
AA54
Annocatacin B*
4
adjacent bis-THF 4,10,15-24
1O-hydroxyasimicin
adjacent bis-THF 4, - 2 3 adjacent bis-THF 4,-23
A3-B1 the ketolactone terminal
AA55
Bullatacinone and adjacent bis-THF 15-24
Annonaceous Acetogenins with Non-Adjacent Bis-THF Rings This class of acetogenins possesses non-adjacent bis-THF rings and various terminal lactone rings. To date, eleven non-adjacent bis-THF acetogenins were isolated from Formosan Annonaceous plants. They were classified into three subtypes according to the terminal lactone rings, including 1) A1-B5, four acetogenins with a terminal a,p-unsaturated y-lactone ring (AA56-60), 2) A2-B5, five acetogenins with a terminal a,p-unsaturated y-lactone ring with a hydroxyl at C-4 (AA62-64), 3) A3-B5, two acetogenin with a ketolactone terminal (AA65, 66). These compounds are listed as follows (see Table 3). Table 7. Non-adjacent Bis-THF Acetogenins Isolated From Formosan Annonaccous Plants
THF Relat
Molecular
Molecular
Conflg.
Formula
Weight
non-adjacent bis-THF -16,19-24
tith-tUtler
C3 7 H«O 7
622
C-12,15-ew-Squamostatin-D*
non-adjacent bis-THF -16,19-24
clth-thltler
C37H*OT
622
AA58
Squamostatin-A
non-adjacent bis-THF -16,19-24,28
tlth-thltler
C37Hfi6Os
638
AA59
C-12,15-cM-Squamostatin-A*
non-adjacent bis-THF -16,19-24,28
clth-thltler
C37H66O8
638
Compound Name
THF and Hydroxyl Positions
AA56
Squamostatin-D
AA57
No A1-B5
983
A2-B5 AA60
Sylvaticin
AA61
Bullatalicin
AA62
C-12,15-cfe-bullatalicin
AA63
Bullatanocin
AA64
C-12,1 S-ris-builatanocin
non-adjacent bis-THF 4,-16,19-24 non-adjacent bis-THF 4,-16,19-24 non-adjacent bis-THF -16,19-24 non-adjacent bis-THF 4, -16,19-24 non-adjacent bis-THF -16,19-24
tlth-thtder
C37H66Og
638
tlth-thltler
C37H«Os
638
clth-thltler
COHMOS
638
tlth-thltlth
C 3 7H«O g
638
clth-thltlth
CSTHSGOS
638
A3-B5 AA65
Bullatalicinone
the ketolactone terminal and non-adjacent bis-THF-16,19-24
th-thltler
CJJHKA,
638
AA66
C-20,23-cis-bullatalicinone*
the ketolactone terminal and non-adjacent bis-THF -16,29-24
th-thlcler
CJTHMOS
638
Miscellaneous, Including Those Substituting with Epoxide or/and Double Bond This class of acetogenins is named as linear acetogenins because it is composed of a terminal a,p-unsaturated y-lactone ring and a terminal aliphatic side chain with the substituents of the oxygen-bearing moieties, such as epoxide, and hydroxyls with and/or without double bonds. To date, eight linear acetogenins were isolated from Formosan Annonaceous plants. They were classified into two subtypes according to the oxygen-bearing moieties, including 1) A1-B9/B11, three linear acetogenins with epoxide moieties (AA67-69), 2) A2-B5, five acetogenins with hydroxyls at the terminal aliphatic side chain (AA70-74), and 3) miscellaneous, one acetogenins with terminal lactone rings at both ends (AA75). These compounds are listed as follows (see Table 4). Table 8. Miscellaneous Acetogenins Isolated From Formosan Annonaceous Plants
No
Compound Name
THF and Hydroxyl Positions Diol Relat. Config.
Molecular Molecular Formula Weight
A1-B9/B11 AA67
Diepoxymontin*
AA68
Epomusenin B*
AA69
Epomusenin A*
epoxy 11-12 (eposide), 13-14 feposide) epoxy 15-16 (ej>oside2j_19=20 epoxy 17-18 {eposide), 21=22
-
C35H62O4
546
c
CMH^OJ
558
c
C37HMO3
558
984
A1-B12 AA70
Cohibin A
AA71
Artemoin-D*
AA72
Artemoin-C*
AA73
Artemoin-B*
AA74
Artemoin-A*
linear 15, 16, 19=20 linear 15,16 linear 17, 18 linear 19,20 linear 21,22
th-c
C«H64O 4
648
C35HG6O4
550
C 3 5 H«O 4
550
C35H66O4
550
C 3 5 H«O 4
550
C22H36O6
396
—
-
Miscellaneous AA75
Rollicosin*
terminal lactone 15,16-19(19=0)
th
Chemotaxonomy In our investigation of the cytotoxic constituents of Formosan Annonaceous plants, the Annonaceous acetogenins were isolated from five species of this family, Annona muricata, A. reticulata, A. atemoya, A. montana, and Rollinia mucosa. Most of the mono-THF acetogenins were isolated from A. montana and A. muricata, while most of bis-THF acetogenins were isolated from A. reticulata, A. atemoya and R. mucosa (see Table 5). A. atemoya is a hybrid species of A. squamosa and A. cherimola. Compared with the reviews by McLaughlin et al. and Cave et al. [67], in which there was no report on the isolation of bis-THF acetogenins from A. muricata or A. montana, it clues that the genus Annona could be separated into two subgenus by the evidence of the chemotaxonomical researches; one of which takes a biogenetic pathway to generate mono-THF acetogenins and the other takes a resembling but different pathway to generate bis-THF acetogenins. Table 9. Annonaceous Acetogenins Isolated from Formosan Annonaceous Plants
No. AA53 AA54 AA31
Compound Annona muricata A reticulata name Annocatacin A* leaf Annocatacin B* seed Annocatalin* leaf
AA49
Annoglaucin
AA18 AA10
Annomontacin Annomonicin r/.?-Annomontac in* Annomurilin*
AA19 AA24
A. atemoya
Rollinia mucosa
A. montana
unripe fruit seed
seed leaf seed leaf
seed
A. cherimola
985 AA15
Annonacin cir-Annonacin Annonacinone AA20 =Annonacin-10one AA12~~~1 Annoreticuin* Annoreticuin-9AA13 one* AA40 Aromin AA71 Artemoin-D* AA72 Artemoin-C* AA73 __] Artemoin-B* AA74 Artemoin-A* Asinricin AA50 AA51
Bullatacin
AA61
Bullatalicin
<\A62 AA63 AA64
seed, leaf
unripe fruit seed
seed, leaf
unripe fruit seed, leaf seed,leaf seed seed seed seed seed seed
seed
C-12,15-CM-BU1
Bullatacinone
4A6S
Bullatalicinone
C-20a3-c«-bulI atalicinone* Cohibin A AA70 AA04 Corossolin AAOS " 1 ew-Corossolin AA06 Corossolone A A 0 7 _ _ cis-Corossolone Desacetylyvaric AA42 in Diepoxymontin AA67 #
seed seed unripe fruit unripe fruit unripe fruit
seed
AA66
\A69
Epomusenin A*
\A68
Epomusenin B*
4A14
Gonithalamicin 4-hydroxy-25hydroxyneorolli nicin (Rolliniastatin
AA48
seed seed leaf seed, leaf leaf seed
AA3S
10-hydroxyasim icin Isoannoancin |lsoannonacin-10 _J_ -one A
unripe fruit unripe fruit unripe fruit unripe fruit seed unripe fruit
J)
AA52
unripe fruit unripe .fruit
seed
latalicin Bullatanocin C-12,15-c«-BuI latanocin
AA55
seed
unripe fruit leaf unripe fruit
986 AA36 ,
AA29 AA30 AA32 AA28 AA27 AA33 AA23 AA08 AA09 AA22 AA34 AA41 AA47 AA02
I Isoannoreticuin ! lsodesacetylyva 1 rein 1 Isomurisolenin ! Longifolicin i Molvizarin Muricatetrocin A Muricatetrocin B Muricin A* 1 Muricin B* Muricin C* Muricin D* Muricin E* Muricin F* Muricin G* Muricin H* Muricin I* Murisolin Murisolinone* ! Neoannonin Parvi florin Reticulatacin
AA21
Rolliacocin *
AA75
Rollicosin*
AA45
Rollimusin *
AA39 AA03 AA46 AA25 AA26
AA01
Solamin
AA44
Squamocin
AA38 AA58 AA59 AA56 AA57
leaf seed seed seed seed seed seed seed seed seed seed seed seed seed seed seed seed,leaf seed seed seed leaf unripe fruit unripe fruit unripe fruit leaf
seed
Squamone (isoannonacin-9 -one) Squamostatin-A C12,15-CM-Squa mostatin-A* Squamostatin-C C12,15-c«-Squa mostatin-D*
AA60
Sylvaticin
AA17 AA11
Xylomaticin Xylopianin
leaf seed
unripe fruit
leaf seed seed seed seed unripe fruit seed seed
*: new compounds
Bioactivity Many Annonaceous plants are employed in folk medicine. Since Jolad al. isolated the first Annonaceous acetogenins, uvaricin, which et
987
possessed a potent activity of 157% test/control (T/C) at 1.4 mg/kg in the PS test system in 1982 [67], a series of Annonaceous acetogenins have been isolated. Annonaceous acetogenins were proven to possess a broad spectrum of bioactivities, such as anticancer, antiparasitic, insecticide, and immunosuppressive effects. Meanwhile, Annonaceous acetogenins generally exhibited more potent cytotoxic activity than other constituents from Annonaceous plants against tumor cancer [69-78]. They are even considered to be the major active principle of the Annonaceous constituents. However, although most Annonaceous acetogenins have significant bioactivities, their mechanism of action is still unclear. Many hypotheses have been offered, including the inhibitor of mitochondrial NADH-ubiquinone reductase [79], the inducers of programmed cell death [78], and the ionophore-like stimulator on Ca2+-Activated K+ Current in Cultured Smooth Muscle Cell [80], etc. Cytotoxicity and Structure-Activity Relationships Generally, Annonaceous acetogenins isolated from Formosan Annonaceous plants possess significant cytotoxicity against cancer cell lines. In the preliminary bioassay, ten kinds of cancer cell lines, including Hep G2 (human hepatoma cell), Hep 2,2,15 (human hepatoma cell transfected HBV), C6 (Rat glioma cells), KB (human nasopharyngeal carcinoma), CCM2 (human colon tumor cell), CEM (human T-lymphoblastoid cell), P388 (mouse lymphocytic leukemia), A-549 (human lung adenocarcinoma), HT-29 (human colon adenocarcinoma), and MCF-7 (human breast tumor cells), were chosen for screening the bioactivities of Annonaceous acetogenins. The bioactivities of fifty-eight acetogenins are listed in Tables 6, 7, and 8. Based on the results, Annonaceous acetogenins exhibited significant potent bioactivities against hepatoma cell lines, and showed certain bioactivities against seven kinds of cancer cell lines, C6, KB, P-388, A-549, and HT-29; however, these types of natural products did not show the activity against CEM cancer cell line. The structure-activity relationships (SAR) of these tested compounds are discussed below by consideration of four chemical portions: the THF ring moieties, the ^lactone ring moieties, the spacer moiety linking the two rings, and the terminal alkyl side chain attached to THF rings, which often has a diol group and ends with the terminal methyl, and the free hydroxyl groups.
988 Table 10. Cytotoxicity IC50 Values of Annonaceous Acetogenins Isolated from Formosa Plants
Cell Lines Compounds Longifolicin (AA03) Corrosolin (AA04) Corrosolone (AA06) cis-Corossolone (AA07) Muricin H (AA08) Muricin I (AA09) Xylopianin (AA11) Annoreticuin (AA12) Annoreticuin-9-one (AA13) Annonacin (AA14) Xylomaticin (AA17) Annomontacin (AA18) cis-Annomontacin (AA19) Annonacinone (AA20) Rolliacocin(AA21) Murisolin (AA22) Muricin G (AA23) Annomurilin (AA24) Muricatetrocin Ai IB (AA25+26) Muricin E (AA27) Muricin D (AA28) Muricin A (AA29) Muricin B (AA30) Annocatalin(AA31) Muricin C (AA32) Muricin F (AA33) Murisolinone (AA34) Isomurisoline (AA39) Neoannonin (AA41) Desacetyluvaricin (AA42) Isodesacetyluvancin (AA43) Squamocin (AA44) Rollinusin (AA45) Molvizarin (AA46) Annoglaucin (AA49) Asimicin (AA50) Bullatacin(AA51) 10-OH asimicin (AA52) Annocatacin A (AA53) Annocatacin B (AA54)
Hep 2.2.15.
HepG2 4
4.04xl0" 3.53x10' 4.80x10' 1.65x10' 9.51 xlO 2 2.98x10"' 5.49x10"3 6.35 xlO"5
3
C6 D2xl0"2 7.21 21.20 9.7 9.20 36.50 7.71
8.40x10"4 3.43xl0"3 3.49x10"' 2.98x10"' 8.73x10' 6.44x10"' 3.32x10"'
4.90x10" 2.34x10"' 2.84x10"' 4.76x10"2 1.18xl0"2 2.22x10' 6.73 xlO"2 7.25x10"5 5.40x10"3 6.68x10"3 4.20x10"2 8.00x10"3 1.62xlO"2 4.65 xlO"3 1.75xlO"2 9.07x10"2
1.27
8.17x10"'
23.47 2.20x10"' 18.97
4.95 xlO"2
4.83*10°
2.00*10"3
6.60x10"3 5.04 1.78
4.80x10"2 5.13xlO"3 4.29 xlO"3 3.48x10'3 3.87*10"3
9.10X10"4
5.7 4.99x10"' 4.28x10"2 5.73x10"' 2.70x10"2 6.40x10"5 6.20x10"5 6.52x10"5 5.47x10"4 2.15xlO"2 6.62xlO"5 8.88x10"' 6.28x10"5 6.30x10"5 12.11 3.35xlO"2
3.86X10"3 6.58x10"2
2.20x10"3 7.30x10"5 7.10xl0"5 6.94x10"5 9.23 xlO"4 1.45xlO"3 7.80x10"5 1.73 xlO"2 6.60x10"5 6.90x10"5 6.70x10"3 8.17x10"' 2.22x10"'
8.26x10"' 6.79 10.20 27.60
6.70 1.56x10"' 32.60 5.20x10"2
•2 4.65 2.00x10"2
3.47x10"'
1.90xl0"2 8.72 29.3
989 Bullatacinone (AA55) Squamostatin D (AA56) 12,15-cis-squamostatin D (AA57) Squamostatin A (AA58) Bulatalicin (AA61) Bullatalicinone (AA65) cis-20,23-cis-bullatalininone (AA66) Cohibin A (AA70) Rollicosin (AA75)
7.90x10"2 1.50xl0"4 2.20x10"4 7.40x10"5 4.66x10 2 1.79xl0"2
1.38x10"' 1.5OxlO"5 3.10xl0"3 7.88 xlO"5 6.25 xlO"2 3.83xlO"3
5.3OxlO3
5.74x10"2
8.23 1.00x10"'
1.17x10"' 2.06x10"2
2.36
Table 11. Cytotoxicity IC5o Values of Annonaceous Acetogenins Isolated from Formosa Plants Cell Lines Compounds
KB
CCM2
P388
CEM
Annoreticuin-9-one (AA13) Annonacin (AA15) Isomurisoline (AA39) Neoannonin (AA41) Desacetyluvaricin (AA42) Isodesacetyluvaricin (AA43) Squamocin (AA44) Molvizarin (AA46) Asimicin (AA50) Bullatacin(AA51) Bullatacinone (AA55) Squamostatin D (AA56) 12,15-cis-squamostatin D (AA57) Squamostatin A (AA58) Bulatalicin (AA61) Bullatalicinone (AA65) cis-20,23-cis-bullatalininone
3.10 7.96x10"3 5.80x10"' 1.46 xlO"4 1.35xlO"4 7.52xlO"5 2.70x10"' 9.00x10"5 8.16xlO"5 1.17xlO"4 4.80x10"' 3.90x10"5 4.05 xlO"4 9.00x10"5 8.93 6.38
l.OOxlO"2 5.50 4.90x10"2 9.34 23.5 5.72 1.60xl0"2 5.46 13.77 1.41x10"' 8.00x10 3 20.00
2.0x10"' 1.40xlO"5
136.7
15.84 5.90x10"2 1.02 xlO"2
4.15
5.93 xlO"3
(AA66)
6.40x10"2 7.76x10"2
1.72xlO"2
300.61 100 73.56 93 96.59 101.43 104.91
6.11xlO" 2
147.44
2.40x10"4
185.09
9.80x10"3 2.53xl0"2
Table 12. Cytotoxicity ICM Values of Annonaceous Acetogenins Isolated from Formosa Plants
Compounds
Cell Lines P-388 2
A-549
KB
HT-29
Solamin (AA01)
4xlO"
-
3x10"'
-
Reticulatacin (AA02)
-
3.49
-
4.66
Annomonicin (AA10)
2.4x10"'
-
1.73
Annoreticuin (AA12)
1.0
3.37
2.28
Annoreticuin-9-one
2x10"'
4x10"' 10,-2
4.66
1.32
MCF-7 2.91
990 (AA13) io- 4 io- 2
3
-
Annonacin-10-one (AA19) 10"
io-3 io-1
1
-
Isoannonacin (AA35)
3
2xlO" 2
-
2xlO"3
-
Isoannoretocuin (AA36)
3.6
4x10''
6.96
3.06
-
5x10"'
7xl0" 2
-
9x10°
-
5.6
1.34
-
1.50
2.14
2.29
6.4x10"'
-
-
<10"3
<10"5
Annonacin (AA15)
io- 5 6
Isoannonacin-10-one (AA37) Squamone (AA38) Rolliniastatin 2 (AA48)
<10"
Bullatacin (AA51)
-
5
<10"
3
<10"5
Hep G2 : Human hepatoblastoma Hep 2.2.15 : Clone cells derived from Hep G2 cells that were transfected with a plasmid containing HBV DNA C6
: Rat glioma cells
KB : Human nasopharyngeal carcinoma CCM2 : Human colon carinoma P388 : Rat leukaemia cell line CEM : Human leukaemia cell line
The THF Ring Moiety: The bis-THF rings moieties enhance the bioactivity of Annonaceous acetogenins more than the mono-THF ring moiety. Moreover, the adjacent bis-THF rings moiety is slightly better than the non-adjacent bis-THF ring moiety in raising the cytotoxicity of Annonaceous acetogenins. For example, the compound with the terminal a,|3 unsaturated y-lactone ring, molvizarin (AA46), is more active than bullatalicin (AA61) and murisolin (AA22), and the compounds with the terminal ketolactone ring, bullatacinone (AA55), and bullatalicinone (AA65), are more active than murisolinone (AA34). And the Annonaceous acetogenins without THF ring did not show any activity against cancer cell lines. The structure-activity relationship (S AR) of Annonaceous acetogenins with bis-THF rings were analyzed in two ways: 1) the role of hydroxyls flanking with bis-adjacent THF rings, 2) the role of conformation of the THF rings. First, the hydroxyls flanking with two sides of bis-adjacent THF rings would enhance the cytotoxicity of the group of Annonaceous acetogenins because asimicin (AA50) was more active than annocatacin A (AA53) in both cancer cell lines. Moreover, Annonaceous acetogenins with the cis-THF rings was more selective cytotoxicity than ones with the trans-THF rings, especially, toward Hep G2 cell line because annocatacin
991 991
B (AA54) becomes 360 times more selectively active against Hep G2 than annoeatacin A (AA53). Our observations appear to be consistent with Landolt's findings, in which they indicated that acetogenins with the bis-adjacent THF ring or the bis-nonadjacent THF ring are about ten times more active than those with the mono-THF ring after a more complete research in screening 20 acetogenins by mitochondrial inhibition assay in 1995 [81]. The 'y-lactone Ring Moiety: There are two major types of the terminal lactone moieties of Formosan Annonaceous acetogenins, the a,(3 unsaturated y-laetone ring moiety and the ketolactone ring moiety. Totally, there are only ten Annonaceous acetogenins with the terminal ketolactone ring. In the mono-THF acetogenin system, the compounds with the ketolactone ring show the same activity as or even better activity than the ones with the a,|3 unsaturated y-lactone ring, for example, murisolin (AA22) and murisolinone (AA34) have the similar activity against Hepatoma cells; in the bis-THF acetogenin system, the compounds with the a,f$ unsaturated y-lactone ring are more active than the ones with the ketolactone ring, for example desaeetyluvaricin (AA42) and squamostatin A (AA56) are more active against Hepatoma cells than bullatacinone (AASS) and bullatalicinone (AA65). In the a,p unsaturated y-lactone ring acetogenin system, the presence of the hydroxyl group at C-4 seems to play an important role in the cytotoxicity of these types of compounds. The hydroxyl group at C-4 enhances the bioactivity of the mono-THF Annonaceous acetogenins. For example, annonacin (AA15) and annonacinone (AA20) are more active than corrosolin (AA04) and corrosolone (AA06), respectively, against hepatoma cell lines. The stereochemistry of the hydroxyl group at C-4 also affects their bioactivity against cancer cell lines. For example, muricin A (AA29) and muricin B (AA30) are the epimers to each other, in which the only difference is the orientation of the hydroxyl group at C-4. The comparison of their bioactivity indicates that the compound with the (J?)-hydroxyl group at C-4 is slightly more potent than one with the (5)-form. The Role of the Spacer Moiety Linking the Two Rings: In comparing the cytotoxic values (IC50) of muricin C (AA32) and muricin D (AA28) against the Hep 2,2,15 cell line, it appeared that the shorter length of the spacer moiety was associated with weaker potency, however, that was not
992
the case with Hep G2. Comparison with muricatetrocin A (AA25) and muricatetrocin B (AA26) suggested that the appropriate length, approximately 12 carbons (from C-3 to C-14), should be more potent than Hep G2. The Terminal Alkyl Side Chain and the Free Hydroxyl Group: The comparison of the cytotoxic values (IC50) of xylopianin (AA11), annoreticuin (AA12), annonacin (AA15), rolliacocin (AA21) and murisolin (AA22), indicated that the hydroxyl group at C-8, C-9, or C-10 seems to enhance the bioactivity against Hepatoma cells. It could be relative with the improvement of the polarity and solubility of Annonaceous acetogenins with more hydroxyl groups. In the comparison of the cytotoxic values (IC50) of annomurilin (AA24), muricin A (AA29), muricin B (AA30), annocatalin (AA31), the series of compounds found to have a diol in the terminal aliphatic chain and also showed a specific selective activity against Hep 2,2,15 cell line. Against Hep G2, comparisons with muricins A (AA29), B (AA30), and D (AA28) indicated that increasing the length between the THF ring and the diol group leads to weaker potency. Of the annonaceous acetogenins containing a mono-THF ring with one flanking hydroxyl group, muricin F (AA33) was the most cytotoxic against both Hep G2 or 2,2,15, suggesting that the presence of the double bond may enhance the bioactivity. The Mechanism of Action In 1991, Londershausen et al. investigated their mechanism of action [79]. They found that squamocin was a remarkable inhibitor of mitochondrial NADH-ubiquinone reductase, namely Complex I. Those assembled experimental results were confirmed by the work of Lewis el al. in American and Degli et al. in France [82,83]. In 1994, Friedrich et al. found that the inhibition of Complex I by Annonaceous acetogenins was not purely competitive [84]. They thought that Annonaceous acetogenins affect the electron-transfer step from the high-potential iron-sulphur cluster to ubiquinone by directly acting at the ubiquinone-catalytic site in Complex I. In 1998, McLaughlin et al. proposed a model for the mechanism of action of Annonaceous acetogenins [85]. In their model, the lactone ring alone interacts directly with the binding in Complex I, and the THF rings with flanking OH groups functions as a hydrophilic anchor at the membrane surface to allow lateral diffusion (or random distribution) of the lactone ring in the membrane interior. To verify the model, Kuwabara
993
et al. synthesized a series of analogues with two terminal y-lactone rings [86]; however, the bioassay result did not show that these analogues work twice as effectively as Annonaceous acetogenins. In our preliminary study on the mechanism of action of Annonaceous acetogenins, some evidences indicated that Annonaceous acetogenins seem to inhibit cancer cells by apoptosis induction [79]. For example, bullatacin (AA51) induced cytotoxicity of Hep 2.2.15 cells in a time- and dose-dependent manner. ED50 on day 1 of exposure to bullatacin was 7.8± 2.5 nM for 2.2.15 cells. In addition, most of the bullatacin-induced cell death was found to be due to apoptosis, as determined by double staining with fluorescein-isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI). Moreover, in the Ca2+-activated K+ current in cultured smooth muscle cells of human coronary artery, squamocin (AA44) showed 2+ significantly stimulatory effect, which the Ca' cations were taken up largely [80]. Fig. (4). Structures of Annonaceous acetogenins isolated b y our group
Solamin (AA01)
Reticulatacin (AA02)
..Me
longifolicin (AA03)
Corossolin (AA04)
cis-Corossolone (AA05)
994
Corossolone (AA06)
?H
c/s-Corossolone
(AA07)
Muricin H OH
OH
(AA08)
Muricin 1 (AA09)
(AA11)
Annoreticuin (AA12)
(AA13)
IH
OH
Gonithalamicin (AA14)
Annonacin (AA15)
995 <j>H
OH
c/s-Annonacin (AA16)
Xylomaticin
Annomontacin (AA18)
cis-Annomontacin (AA19)
OH
OH
Annonacinone =Annonacin-l 0-one (AA20)
Rolliacocin (AA21)
Murisolin
Me
MuricinG (AA23)
Annomurilin
Muricatetrocin A
996 OH
OH
'
Me
Muricatetrocin B
Muricin E
OH
31 OH
33
Muricin D (AA28)
OH
Muricin A (AA29)
Muricin B (AA30)
Annocatalin 6H
(AA31)
,Me OH
Muricin C
OH
Muricin F (AA33)
- A6
Murisolinone (AA34)
Isoannonacin (AA35)
997
33 « 35
o
Isoannoreticuin (AA36)
lsoannonacin-10-one A (AA37) Squamone (isoannonacin-9-one) (AA38) Isomurisolenin (AA39) Aromin (AA40) Neoannonin (AA41) Desacetylyvaricin (AA42) Isodesacetylyvarcin (AA43) Squamocin (AA44) Rollimusin (AA45)
Molvizarin (AA46)
Parviflorin (AA47)
998 4-hydroxy-25hydroxyneorollinicin (RolUniastatin 1) (AA48) Annoglaucin OH
0
—0
(AA49)
37
v^ OH 0
/ —0
Asimicin (AA50)
Bullatacin
OH
10-hydroxyasimicin
Annocatacin A (AA53)
Annocatacin B (AA54)
Bullatacinone (AA55)
\
Squamostatin-D
0
(AA56)
999 C12-15-dsSquamostatin-D (AA57)
.--' OH
OH
OH
0
Squamostatin-A (AA58)
C12-15-cisOH
OH
OH
Squamostatin-A (AA59)
Sylvaticin OH
OH
OH
OH
(AA60)
Bullatalicin (AA61)
C-12,lS-cisOH
OH
Bullatalicin (AA62)
Bullatanocin OH
OH
(AA63)
C-12,15-cisBullatanocin (AA64)
1000
Bullatalicinone
C-20,23-cisOH
Bullatalicinone
OH
(AA66)
Diepoxymontin (AA67)
Epomusenin B (AA68)
Epomusenin A (AA69)
" 0
Cohibin A (AA70)
35
.
Artemoin-D (AA71)
Artemoin-C (AA72)
35
0
Artemoin-B (AA73)
1001
Artcmoin-A O
22
2t
DITERPENOID
(AA74)
Rollicosin
,••*
Ei¥r-KAURANE
/ —0
(AA75)
- NMR
SPECTRUM
AND
BIOLOGICAL ACTIVITIES era*-Kaurene is a precursor, in turn, of many other structural types of diterpenes. These include enmeins from Lamiaceae (Labiatae), gibberellic acids (found in all plants and in some fungi), and grayanotoxins, extremely poisonous compounds from the family Ericaceae. This precursor is also a source of stevioside, a sweetener from the asteraceous plant Stevia rebaudiana. The biological activity of medicinal plants, such as exhibiting antimicrobial, anti-inflammatory, cardiovascular, diuretic, anti-HIV, and cytotoxic effects, can sometimes be attributed in part to the presence of e«l-kaurane diterpenoid constitutes [86]. There were ca. 60 e«*-kauranes isolated from the Annonaceous plants [13,87-109]. Most of them were isolated from Annona and Xylopia species. T l was the first ewf-kaurane isolated from Formosan Annonaceous plant {A. squamosa and A. glahra) [93,100]. Today, we have isolated 43 enf-kauranes (including 15 new compounds) from Annona and Rollinia specie (Table 13). The NMR spectrum data and biological activities will be discussed herein. NMR en^-Kaurane diterpenoids from Formosan Annonaceous plant all belong to low oxygenated enf-kauranes. Most substitutes were attached on C-4, C-16, and C-17. There are several substitutes on the C-4, such as methyl, carboxylic acid, aldehyde, oxymethylene, methyleneacetate, carboxylic ester, hydroxyl, formic ester, and hydroperoxide group. The NMR chemical shifts of the positions around C-4 are useful tool to assign what kind of substitute attach on C-4 and its stereochemistry (Table 14). When the substitute is on position 19 (axial-a orientation), the NMR chemical shifts of 18-methyl group (equatorial-^ orientation) are more downfleld than 20-methyl group (axial-a orientation), because of the anisotropic effect of the enf-kaurane A-ring. On the other hand, the
1002
chemical shifts of both axial-a methyl groups (in position 19 and 20) are very close and there is a NOE correlation between these two methyl groups. 13C NMR signal of C-5 is another important feature to propose that -OOH or -COOH should be in position 18 or 19. The y-gauche effect of the 18-OOH or -COOH results in a shielding effect on the C-5 (from ca. 555 to ca. 550) signal [102,112]. There are two rare substitutes attached on C-4 of e«/-kauranes: formic ester (T40), and hydroperoxide group (T41 and T42). 13C NMR chemical shifts of C-4 of T40-T42 are more downfield than C-4 of 4-hydroxy nor-en/-kauranes (about 10 ppm) [102].
CH, CH,
T2 16ff-hydro-e^-kauran-1 ?, 19-dioic acid
T3 16a-hydro-eH/-kauran-17,19-dioic acid
•t, CH,
NAME
T1 effif-kaut*' 16-en-19-oic acid
n
COOH
COOH
COOH
K.
COOH
H
AI6, 17
R.,
H
COOH
R,
PARTI
A. %labr&
A, sttuamom
STEM
STEM
A. stflmmasa STEM, FRUIT
SP1.
Table 13. ent-Kaumne Diterpenoids from Formosan Annonaceous Plants
T1-T42
-
A. glahra
sn.
SP$.
FRUIT
-
-
-
STEM, FRUIT A. ckerimala
FART2
T43
iZ-o
-
STEM
PART3
s
o
1003
STEM STEM STEM STEM
A. glabra A. glubra A. glabra A. squamosa
CH2OAc OAc CH,OH H
A15, 16 COOH
CH,
T16 16a-hydro-19-al-enr-kauran-17-oic acid CHO
COOH
COOCH,
CH,
T13 16(5,17-diacetoxy-£7W-kauran-19-oic acid
STEM
A. glabra
OH
T15 17-hydroxy-f m-kaur-15en-19-oic acid
CH,
T12 16a-hydroxy-e«/-kauran-l 9-oic acid
STEM
A. glabra
OCH,
CH,
COOH
COOH
CH,
T11 16a-methoxy-en/-kauran-19-oic acid
STEM
A. squamosa
CH2OAc
OH
CH,
COOH
CH,
T10 160-hydroxy-17-acetoxy-em-kauran-19-oic acid
STEM, FRUIT
A. squamosa
OH
CH,OH
T14 methyl-16a-acetoxy-17-oate-en(-kauran-19-oic acid
COOH
CH,
16a,17-dihydroxy-en/-kauran-19-oic acid
T9
-
-
STEM, FRUIT
-
-
A. glahra
FRUIT
FRUIT
STEM
STEM
STEM
STEM
STEM
A. f^labra
A. glabra
A. glahra
STEM, FRUIT
A. squamosa
CH,OH
OH
OAc
COOH
CH,
163,17-dihydroxy-enMcauran-19-oic acid
T8
A. cherimola
LEAVE
A. reliculata
H
CH,OAc
COOH
COOH
CH,
16a-hydro-17-acetoxy-ew-kauran-19-oic acid
T7
A. cherimola
FRUIT
A. squamosa
CH,OAc
H
CH,
COOH
CH,
16(3-hydro-17-acetoxy-e«/-kauran-19-oic acid
T6
A. cherimola
FRUIT
A. squamosa
H
CH,OH
CH,
COOH
CH,
16a-hydro-17-hydroxy-en/-kauran-19-oic acid
T5
STEM, FRUIT A. cherimola
A. squamosa
CH,OH
H
COOH
COOH
CH,
16f}-hydro-17-hydroxy-ew-kauran-19-oic acid
T4
-
-
-
_
_
STEM
LEAVE
-
-
-
-
A. cherimola
A. reliculata
-
-
LEAVE
LEAVE
A. reliculala
A. reticuhita
1004
CHj CH, CH, CH, CH, CH, CH, CH, CH,
T18 16a-hydro-17-hydroxy-e/M-kauran-19-a!
T19 nethyl-16a-hydro-19-al-en/-kauran-17-oate
T20 163,3 7-dihydroxy-e«/-kauran-19-al
T21 16a, 17-dihydroxy-e«/-kauran-19-al
T22 163-hydroxy-17-acetoxy-en/-kauran-19-al
T23 nethyl-163-acetoxy-19-al-en/-kauran-17-oate
T24 nethyl-16a-acetoxy-19-al-efW-kauran-17-oate
T25 enMcaur-16-en-19-ol
T26
CH, CH,
T28 eM-kaur-15-en-17,19-diol
T29
6a-hydro-19-acetoxy-enf-kauran-17-oic acid
CH,
T27 enf-kauran-16,17,19-triol
6cc-hydro-19-ol-en/-kauran-17-oic acid
CH,
T17 163-hydro-17-hydroxy-
CH2OAc
CH2OH
CH2OH
CH;OH
CH2OH
CHO
CHO
CHO
CHO
CHO
CHO
CHO
CHO
COOH
AIS, 16
OH
COOH
A16, 17
COOCH,
OAc
OH
CH2OH
OH
COOCH,
CH2OH
H
H
CH,OH
CH2OH
H
OAc
COOCH,
CH2OAc
OH
CH2OH
H
H
CH,OH
A. glabra
A. glabra
A. squamosa
A. glabra
A. squamosa
A. glabra
A. glabra
A. squamosa
A. squamosa
A. squamosa
A. glabra
A. cherimola
A. squomosa
FRUIT
STEM, FRUIT
FRUIT
STEM
FRUIT
STEM
FRUIT
FRUIT
STEM
STEM, FRUIT
STEM, FRUIT
STEM
A. cherimola
FRUIT
-
R. mucosa
LEAVE
-
A. reticulata
FRUIT
-
-
-
-
-
-
A. glabra
-
-
STEM -
-
-
-
FRUIT
-
-
A. glabra
-
-
-
-
-
_
-
STEM
-
STEM, FRUIT A. cherimola
1005
OOH
CH,
T39 16(i-acetoxy-17-hydroxy-19-nor-
6(J-hydroxy-17-acetoxy-18-nor-
CHj
T38 16a-hydro-17-hydroxy-19-nor- ert/-kauran-4a-ol
T42
CH,
T37 methyl-16a-acetoxy-19-nor-e/i/-kauran-4oc-ol-17-oate
OOH
CH,
T36 19-nor-CTiMcauran-4a, 163,17-triol
6|3,17-dihydroxy-18-nor-en/-kauran-4P-hydroperoxide
OH
CH,
T35 19-nor-enMcauran-4a-ol-16a-hydro-17-oic acid
T41
OH
CH,
T34 16oc-hydro-ert/-kauran-l7-oic acid
CH,
CH,
CH,
T33 16[l-hydro-en/-kauran-]7-oic acid
6|3-hydroxy-17-acetoxy-19-nor-£VT/-kauranj4a-formate
COOCHj
CH,
T32 nethyl-16(J, 17-dihydroxy-enMcauran-19-oate
T40
COOCHj
CH,
T31 dimethyl- 16a-hrdry-tvi/-kauran-17.19-dioate
CH,
CH,
OCHO
OH
OH
OH
CH,
CH,OAc
CH,
T30 16|}-hydroxy-17,19-diacetoxy-enf-kaurane
A. glabra A. zlahra A. squamosa A. squamosa A. filubra A. squamosa
COOH H H CH2OH OAc H
COOCH, CH,OH
OH
OH
OH
OAc
OH
COOH
COOH
H
STEM
A. squamosa A. squamosa
CHjOH CH2OAc
STEM
STEM
STEM
STEM
STEM
STEM, FRUIT
STEM
STEM, FRUIT
FRUIT
LEAVE
STEM
STEM
A. squamosa
CH,OAc
A. squamosa
A. reticulata
CH2OH
OH
CH2OH
A. f>labru
H
COOCH,
A. cherimola
CH,OAc
OH
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
STEM, FRUIT
-
-
-
-
A. glabra
-
-
-
-
_
-
-
1006
1007
Some pairs of diastereomeric en^-kaurane diterpenes were identified such as T2-T3, T4-T5, T8-T9, and T20-T21 [102]. Crucial NMR shift values are listed in Tables 15 to 6 to help distinguish these diastereomers. In Table 4, H-16 *H NMR resonance signals of T2 and T3 are significantly different, because the anisotropic effect of the ercf-kaurane C-ring made the H-16oc shift downfield. It also caused the 13C NMR chemical shift of the 17-carboxylic acid to the upfield. On the other hand, the anisotropic effect of the carboxylic acid affected the resonance of C-15 (by about 5 ppm). The difference offers important evidence to distinguish this type of diastereomer. In the NOESY spectrum of T3, correlations between H-16a and H-13 or H-16a and H-14 proved its stereochemistry. The H-16(3 stereochemistry of T2 was reconfirmed by the NOE correlation between H-16pandH-ll. Table 14. NMR Data of enf-Kaurane Diterpenoids with Different Substitutes in C-19.
3
Rl
13
C
4 "C
19
18 'H
CDCI3 37-39 42-45 1.20-1.24
13
C
'H
28-29
20 "C
'H
"C
183-186
0.93-0.99 14-16
COOH
CHO
C5D5N 38-40 42-45 1.34-1.37 29±0.5
-
180-182
1.18-1.24 15±0.5
CDCI3 34-36 47-49 0.98-0.99 24±0.5
9.7
204-206
0.83-0.88 16±0.5
CDC13 35-36 39-40 1.00-1.01 27±0.5
3.65,4.03
64-66
0.95-0.96 18±0.5
36-37 39-^0 1.20-1.22 28±0.5
3.66,4.01
64-66
0.99-1.04 18±0.5
CHjOH C5D5N
CH 2 OAc CDCI3 36-37 39±0.5 0.95-0.96 27±0.5 COOCH3 CDClj 37-39 4 3 ^ 5 1.11-1.12 27±0.5 CH3
CDCI3 42±0.5 33±0.5 0.99-1.00 33±0.5
2.10,3.87,4.21 21, 170-172 1.00-1.01 18±0.5 3.6 0.79-O.80
51, 174-176 0.75-0.77 15±0.5 21 ±0.5
0.79-0.80 17±0.5 0.95-0.97 16±0.5
CDCI3 37-38 72±0.5 1.00-1.10 22±0.5 OH C5D5N
38-43 69-72 1.29-1.30 28±0.5
-
-
0.97-1.00 17±0.5
The key NOE correlation between H-17 and H-ll suggested the stereochemistry of 17-hydroxy-16a-e«/-kauran-19-oic acid (T5). The anisotropic effect of the ent-kaurane C-ring shielded the H-17 signal of T5 and deshielded the H-17 signal of T4 (see Table 16).
1008
Resembling methods used to elucidate the NOE correlation between H-17 and H-11 or H-17 and H-13 helped establish the stereochemistries of the 16,17-dihydroxy-e«?-kaurane diterpenes. Relative to the 17-hydroxy-16a-e«£- kaurane diterpenes, a 16a-hydroxy group results in the *H NMR chemical shift of the 17-oxymethene more deshielded, but a 16|3-hydroxy group makes the 13C NMR shift of C-17 more deshielded. Diagnostic carbon signals of C-13 and C-16, especially C-13, are also key points to identify this type of diastereomer (see Table 17). Similar results (Table 18) were observed from the NMR data measured in different solvents. Table IS. 'H (400 MHz) and I3C (100 MHz) NMR Data of Compounds T2 and T3"
position 13 15 16 17
T3 8 H (JinHz) 2.74, brt (4.8) 3.16, dt (12.0, 6.0)
8c 39.5 40.8 45.3 176.9
T2 5H(JinHz) 2.79, brd (3.6) 2.93, d (5.6)
8C 41.6 45.2 46.2 179.7
° Spectra recorded in pyridine-rfs. Table 16. 'H (400 MHz) and 13C (100 MHz) NMR Data of Compounds T4 and T5"
T5 position 17
MJinHz) 3.40, d (7.0)
5c 67.4
T4 8 H (J in Hz) 3.72, d (7.0)
8c 64.2
" Spectra recorded in CDC13. Table 17. *H (400 MHz) and "C (100 MHz) NMR Data of Compounds T8 and T9 *
T9 position 13 16 17
I5H(- n]Hz) 4 .07, d (1 1.2) 4 .17, d (1 1.2)
' Spectra recorded in pyridine-cfc.
8c 45.9 81.7 66 9
T8 nHz) 3•78, d (H.2) 3.86, d (11.2) i5H(-
Sc 41.7 78.8 70 4
1009 Table 18. 'H (400 MHz) and "C (100 MHz) NMR Data of Compounds T20 and T21'
position 13 16 17
T21 8 H (/inHz)
5c 45.6 81.7
3.63, d (11.2) 3.75, d (11.2)
66.3
T20 8 H (/inHz)
3.39, d (11.2) 3.49, d (11.2)
5c 40.6 79.8 69.7
" Spectra recorded in CDClj.
Annomosin A (T43) is the first dimeric of the two e«f-kaurane monomeric units isolated from the Annonaceae. In previous reports on Annonaceae plants, dimeric ent-kaurane diterpenoids have been reported composed of one enf-kaurane monomer and one labdane monomer, and were isolated only from Xylopia species [109-111]. T43 consists of 19-al-e«/-kauran-17-oic acid and 16,17-dihydroxy-ewf- kauran-19-al. In the NOESY spectrum of T43, a correlation between H-12 and H-16 confirmed that the stereochemistry of H-16 was (3. The stereochemistry of OH-16'P was established by the NOE correlations between H-177H-13' and H-17VH-14'. On the other hand, the two aldehyde groups in C-19 and C-19' were confirmed unambiguously to be in an axial orientation from the NOEs between Me-18/H-3(3 and Me-187H-3'p, respectively. Thus, the structure of T43 was determined to be 16(3-hydroxy-19-al-e«^-kauran -17-yl 16(3-hydro-19-al-e^-kauran-17-oate. This type of dimer e«/-kauranes have ever been isolated from the traditional Chinese medicine "Bei-Mu", which were prepared from the bulbs of Fritillaria thunbergii (Liliaceae) and relative species [113-114]. Recently, there isolated dimer e«?-kaurane, annonebinide A, from the stems of Annona glabra [115].
OH
annonebinide A
1010 1010
Biological Activities Although there were some ent-kaumnes isolated from Annona species with significant cytotoxicity [95], we never found any cytotoxic en^-kauranes from Formosan Annonaceous plant. In anti-HIV screen of enf-kauranes isolated by our lab., T4 and T19 exhibited mild activity against HIV replication in H9 lymphocyte cells (therapeutic index > 5, = 4, respectively) [90,99], T5 showed mild inhibition of HIV-reverse transcriptase (46% inhibitory effect at 33 jig/mL) [90]. In anti-platelet aggregation assay, T16 showed complete inhibition of platelet aggregation induced by AA and collagen at 200 \sM, and T l showed complete inhibition of platelet aggregation induced by collagen at the same dose. Also, at 200 \)M, T2 showed moderate inhibition of platelet aggregation induced by collagen [100]. CLERODANE AND £iVT-HALIMANE DITERPENOID In Annonaceae, most clerodane and e«?-halimane were isolated from Polyalthia species. We majored on two of four Polyalthia species, in Taiwan: P. longifolia [117] and its variation, P. longifolia var. pendulla [118]. In the former plant, we only studied the alkaloid layer of MeOH extract. Seven diterpenes (T44, T47, T49, TS8, T59, T66, T67) were isolated from P. longifolia var. pendulla, including two new compounds (T49 and T66). The chemical constituents of P. longifolia and P. longifolia var. pendulla are significantly different, especially in enr-halimane type (Table 19). We also isolated a diterpene T67, which is the third labdane diterpene from Polyalthia species. Table 19. Ditcrpenoids from P. longifolia and P. longifolia var. penduln. Diterpenoids
P. longifolia
n Clerodane diterpenoids F44 16-hydroxycleroda-3,13-dien-15,16-olide
V
T45 16a-hydroxycleroda-3,13Z-dien-15,16-olide
V
TM 16p*-hydrt>xycleroda-3,13Z-dien-15,16-olide T47 clerda-3,13£-dien-15-oic acid (kolavenic acid)
V
T48 16a-methoxycleroda-3,13Z-dien-15,16-olide
V
P. longifolia var. pendula V
Ref.
117,118 118,120
V
123,124
V
118,119, 121,122 123
1011
T49 6-hydroxycleroda-3,13-dien-15-oic acid
V
117 118,119,
6-oxocleroda-3,13£-dien-l5-oic acid
V
6-oxocleroda-3,13Z-dien-l 5-oic acid
V
120
T52 cleroda-3,13-dien-15,16-olide
V
118
T53 6-hydroxycleroda-4( 18), 13-dien-15,16-olide
V
118
T54 clerda-4( 18), 13£-dien-l 5-oic acid
V
118
T55 16-oxocleroda-4( 18), 13Z-dien-l5-oic acid
V
118
T56 cleroda-4( 18), 13 -dien-15,16-olide
V
118
rsi
V
121,122
T57 2-oxokolavenic acid
V
124
T58 3,12£-kolavadien-15-oic acid-16-al
V
117
T59 (4-»2)-abeo-(« and S)16-2,13Z-kolavadien-15,16-olide-3-al
V
117
enf-Halimane diterpenoids T60 16-hydroxy-en/-halima-5( 10), 13-dien-15,16-olide
V
118
T61 e«;-halima-5( 10), 13£-dien-15-oic acid
V
118
T62 16-oxo-en/-halima-5(10),13£-dien-l5-oic acid
V
118
T63 en?-halima-5(l 0), 13£-dien-l 5,16-olide
V
118
T64 en?-halima-l(10),13£-dien-l5-oic acid
V
118
T65 en/-halima-1 (10), 13£-dien-15,16-olide
V
118
F66 3P,5P, 16-trihydroxy-en?-halima-l 3(14)-en-15,16-olide
V
117
V
117
Labdane diterpenoid T67 labd-13£-en-8-ol-l 5-oic acid
Two major secondary metabolisms of P. longifolia var. pendula, T44 and T59, are clerodane hydroxybutenolides. The NMR data indicated that both are 1:1 epimeric mixtures at C-16. It is interesting to note that the 165 configuration was assigned to T45 from the leave and stem bark of P. longifolia [119,120], whereas the \6R configuration was assigned to T46 isolated from P. longifolia var. pendula and P. viridis [121,122,125]. The difference in 13C NMR data for epimeric mixtures, T44 and T59, is not significant (0.1-0.3 ppm). The optical rotation was not suitable to propose the configuration of C-16 because within an enantiomeric clerodane series the optical rotation signs depend on the substituents [118]. The new
1012 1012
e«?-halimane T66 also has hydroxybutenolides and there is only one configuration in C-16. To solve the final stereochemistry, the CD spectrum of T66 was measured [117]. Upon comparison to the Cotton effect data of known butenolides, the absolute configuration at C-16 was found correlated to the sign of the Cotton effects of the n-;r* (235-250 nm) and 7i-7£* (200-220 nm) transitions. The positive n-;z* transitions at 246 nm and negative TI-T? transitions near 200 nm suggested that C-16 has the S configuration, and thus the a-orientation of the hydroxyl group at C-16 could be confirmed. COOH
COOH
o.
CHO
HOOC
O.
O
T59
T58
T49
T47
T44
..OH
COOH
T67
Most halimane diterpenes are halima-l(10),13-diene, halima -5(10),13-diene, halima-l(10),14-diene, and halima-5(10),14-diene type. There is no double bond in A1'10 or A5'10 of T66. The A/B c/s1 junction was assigned by NOE correlation unambiguously. In biogenesis, T66 should be original from halima-5(10),13-diene type.
1013
NOESY correlations of T66
In the previous study, some clerodane hydroxybutenolides showed significant biological activities as antifeedants, antimicrobials, and cytotoxicity to tumor cell cultures. Two clerodane hydroxybutenolides and e«£-halimane hydroxybutenolides were evaluated against a panel of human cancer cell lines [117]. Only T44 displayed moderate cytotoxicity against AGS and HA59T cell lines (IC50= 26.9, 23.6 (iM, respectively). STYRYLPYRONES - CD SPECTRUM AND CYTOTOXICITY The styrylpyrone skeleton is the most important in a number of primitive Angiosperm families, such as the Lauraceae, Piperaceae, Ranunculaceae, Zingiberaceae and Equisetaceae. According to previous studies, styrylpyrones have significant cytotoxic activity against human tumors [126]. The first styrylpyrone goniothalamin, found within the family Annonaceae, was isolated from several species of Goniothalamus [126]. Today, there are more than 40 styrylpyrons that have been isolated from Goniothalamus. Goniothalamus consists of 115 species, distributed throughout the tropics and the subtropics [128], and some of them are widely used as traditional medicines [129]. Goniothalamus amuyon (Blanco) Merr., indigenous to southern Taiwan near the coastal region, is the only specie in Taiwan. The seeds of G. amuyon were reported to be useful for the treatment of edema and rheumatism [130]. In bioactivity-directed studies of the leaves and stems, nine cytotoxic styrylpyrones- goniothalamin (SI), goniothalamin epoxide (S2), (6#,7i?,8#)-goniodiol-7-monoacetate (S3), (6/?,7/?,8i?)-goniodiol-8monoacetate (S4), (6i?,7fl,8/?)-8-methoxy goniodiol (S5), (6i?,7i?,8/?)-8chlorogoniodiol (S6), (55',6/?,7/?,8/?)-goniotriol (S7), (5S,6RJS,8S)goniotriol (S8), (+)-9-deoxygoniopypyrone (S9)- were isolated [131-133].
1014
Fig. (5). ORTEP plots of (6«,7V?,8fl)-8-methoxygoniodiol (S5) and (6«,7/?,8/J)-8- chlorogoniodiol (S6)
.R2
R,
o
R3
R,
R4
R5
51
H
A7,8
52
H
7,8-epoxide
53
H
OAc
H
OH
H
54
H
OH
H
OAc
H
55
H
OH
H
OCH3
H
56
H
OH
H
Cl
H
57
OH
OH
H
OH
H
58
OH
H
OH
H
OH
The relative configuration of styrylpyrones was confirmed by optical rotation values, and X-ray crystallographic data. To check the absolute configuration, several diol- and triol- series styrylpyrones, S1-S9, were subjected to the circular dichroism measurement. The CD value of goniothalamin showed positive Cotton effect at 251 nm, due to n-rc* transition. In CD spectra, all diol-series styrylpyrones appeared as positive absorptions at 251, 262, and 272 nm. It indicated that only minor changes of CD spectra might be effected by the other chromophore, the benzene moiety. In triol system, the data (positive, 250-270 nm) were compared
1015
with the literature data of (+)-osmundalactone, which has the same conjugated system of lactone moiety and relative configuration [134]. It suggested that the stereochemistries of both triol-styrylpyrones are 55 and 6R. Because the relative stereochemistry can be determined by NMR, we assigned the absolute configuration of two known triol-styrylpyrones to be 5S,6R,7R,8R, and 5S,6R,7S,SS, respectively. The benzene moiety also has no great effect in the CD spectra of triol-styrylpyrones.
Fig. (6). The CD spectra of diol- (right) and triol- (left) styrypyrones
S3 demonstrated potent cytotoxicity against KB (human epidermoid carcinoma of the nasopharynx), P-388 (murine lymphocytic leukemia), RPMI (human melanoma), and TE671 (CNS carcinoma) with EDsoDO.l (ig/mL [131]. S4 showed cytoxicity against KB, P-388, A549 (Human lung cancer), HT-29 (Human Colon Cancer), and HL-60 (Human leukemia cancer) with ED50 values of 4.85, 1.68, 4.79, 3.99, 1.85 |ig/mL, respectively [132]. Compounds S2-S6 were also subjected to evaluation for cytotoxicity against HONE-1 (human nasopharyngeal carcinoma) and NUGC (human gastric cancer) (In Table 20). S5 with an 8-methoxyl substitution showed a reduced activity relative to the other substance's atom. S6 is the first styrylpyrone possessing a chlorine atom and the cytotoxicity diverted and demonstrated only to HONE-1. On the other hand, S3 and S4 also showed significant cytotoxicity against the NUGC and HONE-lcell lines [133].
1016 Table 20. Cytotoxic Activity of Selected Styrylpyrones against MICC and HONE-1 Cell Lines
IC 50 (ug/mL) compound c
NUGC
HONE-l ft
SI
4.12 ±0.71
5.69 ± 0.67
S2
5.02 ± 0 . 4 3
6.09 ± 1 . 5 8
S3
167.63 ±12.50
239.73 ±72.15
S4
31.00 ± 9 . 7 0
4.87 ± 0.80
S5 32.05 ±4.91 36.27 ± 6.62 " NUGC: Human gastric cancer. h HONE-1: Human nasopharyngeal carcinoma. c Positive control: Actinomycin CONCLUSION AND FUTURE The above survey summarizes major studies on the chemical constituents from Formosan Annonaceous plants and their pharmacological activities published in the last two decades from our lab. Including these four types of compounds described above, we have found ca 300 compounds from Formosan Annonaceous plants and some of them are potentially useful bioactive compounds for the further development of new drugs [5]. In alkaloids, although this field is investigated to almost exhaustion, still there are new substitutes and novel skeletons we found. The substitution-diversity of Annonaceous alkaloids showed bioactivity -diversity and so inspires us to study the medicinal chemistry. We have preliminary results on total synthesis of some bioactive compounds and derivatives preparation for S.A.R.[135]. The cytotoxicity of Annonaceous acetogenin and styrypyrones are the hot topics in the phytochemical researches of Annonaceae. The stereochemistries of these two skeletons attracted attention of organic chemists. In our lab, some interesting compounds are under preparation for pharmacological mechanism studies. The selective cytotoxicity of acetogenins toward specific cancer cells was detected recently. We hope the further investigations of these two fields would help developing of cancer therapies. e«/-Kauranes were produced from several materials and studied for a long time. In previous literatures, most active ewf-kauranes belong to high oxygen substituted ewf-kauranes which were isolated from Isodon genera. There is little pharmacological results have been reported on the other e«/-kauranes. Lately, some
1017
bioactivities of e«?-kauranes were reported especially the antiimflammatory activity and anti-HIV. The excellent anti-inflammatory results of e«f-kauranes detected by us encouraged us to study deeply. On the other hand, in the cooperation works with National Health Research Institute of Taiwan, we found some fractions of Polyalthia longifolia possess strong cytotoxicity on two unusual cancer cell lines. In the present study of P. longifolia, we got abundant quantity of several pure components. We hope these compounds are the source of the selective cytotoxicity. The chemotaxonomy of Formosan Aoonoaceous plants is also an interesting subject. As shown in Table 21, some types of natural products are important markers to distinguish various genera, such as e«/-kauranes, styrypyones, and clerodanes. There are still many topics of Formosan Annonaceae uninvestigated. We will continue this research to make the studies complete and maximum the utility of these treasure plants.
Annona
a.
e '£ a.
i
A. cherimola
V
V
V
V
A. glabra
V
V
V
V
A. cherimola x A. squamosa
V
A. montana
V
A. muricata
V
A. reticulata
V
V
A. squamosa
V
V
4. purpurea
V
V V
V
V
V
V
V
V
V
Ariabotyrs A. uncinatus Cananga C. odorata
| Azafluorene
s <
i
Aristolactam |
1 s
Oxoaporphine |
e Kaurane
iS^eletons Species ^\^^
Clerodane
|
Table. 21 Chemotaxonomy in Formosan Annonaceous plant
1018
Fissistigma F. glaucescens
V
F. oldhami
V
V
V V
Goniothalamus G. amuyon
V
V
V
V
V
V
V
V
V
V
V
V
Polyalthia P. longifolia P. longifolia varpendula
V
Rollinia R. mucosa
V
V
ACKNOWLEDGMENTS The investigations were supported by grants from the National Science Council and the National Health Research Institutes of the Republic of China. REFERENCES [I] [2]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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STRUCTURAL AND FUNCTIONAL ASPECTS OF FUNGAL GLYCOSPHINGOLIPIDS ELIANA BARRETO-BERGTER*, MARCIA R. PINTO, MARCIO L. RODRTGUES Institute de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral. Universidade Federal do Rio de Janeiro, Cidade Universitdria, CCS, bloco I - Ilha do FundSo. Rio de Janeiro, RJ- Brazil CEP: 21941-590. * Author for correspondence. Email:
[email protected] ABSTRACT: During the last decade, significant progress has been made on the field of biosynthesis and structural elucidation of fungal glycosphingolipids (GSLs), molecules composed of a hydrophobic ceramide moiety linked to one or more sugars. Ceramide monohexosides (CMHs) from several fungal species were characterized in detail, all of them presenting a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoetadecanoic or 2-hydroxyhexadecanoic acids, and a carbohydrate portion consisting of one residue of glucose or galactose. CMHs seem to be associated with growth or differentiation of fungal species such as Pseudallescheria boydii, Candida albicans, Cryptococcus neqformans, Aspergillus nidulans, A. fiimigatus, and Schizophyllum commune. A ceramide dihexoside (CDH) was recently described in Magnaporthe grisea. This glycolipid presented phytosphingosine as the long chain base, and this observation revealed the existence of alternative pathways of ceramide glycosylation in fungal cells. Glycoinositolphosphoryl ceramides (GIPCs), a class of acidic glycosphingolipids expressed by plants and certain parasitic organisms, but not in higher animals, were also identified in fungi, representing potential targets for the development of antifungal agents. The structural characterization of GIPCs, CMHs and, more recently, CDHs from fungal cells allows therefore the design of experimental models for studies involving biosynthesis and function of these molecules.
INTRODUCTION Glycosphingolipids (GSLs) are amphipathic molecules consisting of a ceramide (JV-acylsphingosine) lipid moiety linked to a glycan chain of variable length and structure. These molecules have been implicated in many fundamental cellular processes including growth, differentiation, and morphogenesis. GSLs may also modulate cell signaling by controlling the assembly and specific activities of plasma membrane proteins [1,2] GSLs are present in fungi of the most primitive class of Phycomycetes [3] as well as in the most complex Basidiomycetes [4].
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Neutral and acidic GSLs have been characterized from fungal cells and, currently, GSLs are considered as potential targets for the development of antifungal drugs, since many of them differ in structure and function from those observed in human or animal cells. Currently available antimicrobial drugs for serious fungal infections have essentially two molecular targets, 14 a demethylase (azoles) and ergosterol (polyenes) [5]. The former is a fungistatic target, vulnerable to resistance development; the latter, while a fungicidal target, is not sufficiently different from the host to ensure high selectivity. The search for additional efficient targets for the action of new antinfugal dmgs is clearly necessary. In this context, several natural product inhibitors of sphingolipid biosynthesis have been discovered in recent years [5], some of which act at a step unique to fungi and have potent and selective antifungal activity. In addition, the functional elucidation of fungal monohexosylceramides in the last three years indicates that new alternatives for the antifungal therapy are emerging [6-8]. This chapter will focuses on the current knowledge of GSLs in fungal cells, discussing their structural and functional attributes. Ceramide monohexosides (cerebrosides) Cerebrosides are neutral glycosphingolipids that contain a monosaccharide, normally glucose or galactose, in 1-ortho-beta-glycosidic linkage with the primary alcohol of an iV-acyl sphingoid (ceramide). In plants the monosaccharide is normally glucose and the sphingoid usually phytosphingosine. In animals, the monosaccharide is usually galactose, though this may vary with the tissue and the sphingoid is usually sphingosine or dihydrosphingosine. Since cerebrosides contain one sugar unit, they are also called ceramide monohexosides (CMHs), differing from gangliosides in that the latter contain at least one sialic acid residue. CMHs also differ from globosides in that these glycolipids contain multiple sugar moieties, whereas cerebrosides only contain one. CMHs have been widely detected in fungal cells. The current literature indicates that cerebrosides seem to be present in almost all the fungal species studied so far, with Saccharomyces cerevisiae representing a well-known exception. Fungal cerebrosides contain very conserved structures, in which modifications include different sites of unsaturation as well as the varying length of fatty acid residues in the ceramide moiety and occurrence of glucose or galactose as the sugar unit (Table 1). In general, therefore, fungal CMHs contain a ceramide moiety containing 9-methyl4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2hydroxyhexadecanoic acids, and a carbohydrate portion consisting of one
1027
residue of glucose or galactose. The long chain base 9-methyl-4,8sphingadiene was first described in monohexosylceramides from A. oryzae [9] and subsequently isolated from Schizophyllum commune [10], the plant pathogen Fusicoccum amygdali [11], and the edible fungi Clitocybe geotrope and C. nebular is [12]. CMHs were further characterized in lipid extracts from the fungal species Magnaporthe grisea [13, 14], Cryptococcus neoformans [6], Pseudallesheria boydii [7], Histoplasma capsulation [15], Paracoccidioides brasiliensis [16], Aspergillus fumigatus [17, 18], A. versicolor [18], A. niger [19], Fusarium sp [20], Sporothrix schenckii [21], Fonsecaea pedrosoi [22], Colletotrichum gloeosporioides [23], Candida albicans [24], Pichiapastoris [25], Rhynchosporium secalis [25], Saccharomyces klyuyverri [26], Zygosaccharomyces cidri [26], Z fermentati [26], Kluyveromyces lactis [26], /f. thermotolerans [26], and AT wa/ri/ [26], all of them presenting a ceramide moiety containing 9-methylsphingobase in amidic linkage to Ci6- or Cig- a-hydroxy fatty acids, and a carbohydrate moiety consisting of one residue of glucose or galactose. In addition, cerebrosides from S. kluyveri present an extremely rare tryhydroxy sphingoid base as a unique characteristic [26]. Table 1. Fungal CMHs: ceramide and carbohydrate moieties. Fungal specie
A. fumigatus A. versicolor
Long chain base
Major fatty acid
Sugar
Reference
9-methyM,8-sphingadienine
C 18:1(OH)
Glucose / Galactose
17,18
9-methyl-4,8-sphingadienine
C 16:0(OH)
Glucose
7
Glucose
20
Glucose
16
Glucose
15
A.ftavus P. boydii
C 1&0(OH)
Fusarium sp F. oxysporum F. solani
9-methyl-4,8-sphingadienine
P. brasiliensis
9-methyl-4,8-sphingadienine
C 16:0(OH) C 18:0(OH) C 18:1(OH)
C 18:0(OH) C 18:1(OH)
H. capsulatum
9-methyl-4,8-sphingadienine
C 18:0(OH) C 18:1(OH)
C. neoformans
9-methyl-4,8-sphingadienine
C 18:0(OH)
Glucose
6
C. albicans
9-methyl-4,8-sphingadienine
C 16.0(OH)
Glucose
24
M. grisea
9-methyl-4,8-sphingadienine
C 18:1(011)
Glucose
13,14, 87
S. schenckii
9-methyl-4,8-sphingadienine
t ' 18:0(OH)
Glucose / Galactose
21
C 18:1(OH)
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Analysis ofCMHs Here we will describe the methodology routinely used in our laboratory for CMH extraction and purification in the last decade, but it must be clear that different methods are available in the current literature for isolation and purification of CMHs [6-26]. For preparation of CMHs, a lipid extract from fungal cells is obtained by successive extractions with chloroform/methanol (2:1 and 1:2 v/v). These extracts are usually combined and dried, yielding a crude lipid mixture. The crude extract is subsequently partitioned according to Folch et al. [27], in which the lower phase containing neutral glycosphingolipids is taken for further analysis. CMHs, present at the Folch's lower layer, are purified by chromatographic methods, initially on silica columns. Glycolipids are recovered by elution with chloroform, acetone and methanol. The acetone and methanol fractions, containing the glycosphingolipids, are further purified on another silica gel column, which is sequentially eluted with chloroform / methanol with increasing concentrations of methanol (95:5, 9:1, 8:2 and 1:1 vol/vol) and finally with 100% methanol. The presence of CMHs is monitored by high performance thin-layer chromatography (HPTLC), on silica plates developed with chlorofonn/methanol/ water (65:25:4 vol/vol). The separated glycolipids are visualized with iodine vapor and by spraying with orcinol/ sulfuric acid. Fractions containing CMHs, usually those eluted with chloroform/methanol (9:1 and 8:2 vol/vol), can be further purified by chromatography on Iatrobeads RS 2060, using the same elution system, normally yielding a purified glycosphingolipid fraction. A typical example of CMH purification is given in Fig. (1).
1029 Fungal cells 4 Chlorofbrm/methanol (2:1 and 1:2 vA/)
•CMH
Crude lipid mixture 4 Foleh's partition
Upper layer
+ Lower layer
4 Silica gel column chromatography
4
u
S
Partially purified glycolipids (exemplified in A)
4 latrobeads column chromatography HPTLC GC-MS Purified glycolipid(exemplified in B) FAB-MS Acetylation =* FAB-MS NMR Figure 1. Schematic presentation of the usual steps of purification of fungal CMHs, modified from references 6 and 7.
Purified CMH fractions can at that step be submitted for structural determinations. The sugar composition is achieved by hydrolysis of glycosphingolipids with 3M trifluoroacetic acid at 100°C for 3 h, with preliminary analysis of the resulting monosaccharides by thin layer chromatography. Sugar quantification is determined by gas chromatography (GC), after chemical conversion to the form of alditolacetate derivatives [28]. Fatty acid components are prepared as their methyl ester derivatives, by acid methanolysis using 1 mL of toluene:methanol (1:1 vol/vol) containing 2.5% concentrated sulfuric acid (overnight at 70°C). Samples are diluted in deionized water and extracted twice with hexane: chloroform (4:1 vol/vol), followed by combination of extracts and trimethylsilylation by treatment with 100 |uL of bis(trimethylsilyl)trifluoracetamide / pyridine. Samples are then analyzed by the combination of gas chromatography with mass-spectrometry (GC-MS). The particular use of mass spectrometry techniques was of fundamental relevance in the structural determinations of CMHs from different species, including analytical variations as fast atom bombardment mass spectrometry (FAB/MS), electronspray ionization (ESI-MS) and low energy collision-induced dissociation tandem mass spectrometry (ESI-
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MS/CED-MS). Nuclear magnetic resonance (*H and 13C) has been also successfully used in CMH structural analyses. The combination of these techniques is usually satisfactory for a complete structural elucidation of CMHs, and a vast and detailed literature is available in mis regard [6-26]. CMHs as bioactive components from fungal cells. Although largely distributed in fungi and, in addition, presenting highly conserved structures, the understanding of the functions of CMHs in fungal cells is only beginning to emerge. The old concept that cerebrosides and other glycosphingolipids are membrane structural components with exclusive filling gap roles [29] is obviously simplistic, since it is now clear that such molecules are involved in cell growth, differentiation and signaling [30]. In fungal cells, CMHs have been characterized as bioactive molecules with several distinct roles. For instance, the phytopathogen M grisea produces active elicitors of the hypersensitive response in rice [13, 14] that were identified as monohexosylceramides. Treatment of rice leaves with M. grisea CMHs induced the accumulation of antimicrobial compounds, cell death, expression of pathogenesis-related proteins in rice leaves, and effectively protected rice plants against fungal infection. Fungal cerebrosides were also characterized as antigenic molecules directly or indirectly involved in cell growth or differentiation in 5. commune [10], C. neoformans [6], P. boydii [7], C. albicans [7], A, nidulans [8] and A. fumigatus [8]. Most of these reports, which are discussed below, are very recent in the current literature and represent an open and new field on the biology of fungal glycosphingolipids. We will summarize these studies, mainly focused on the cellular distribution of fungal CMHs and their association with growth or differentiation. CMHs: antigenic structures possibly involved with fungal growth GSLs were shown to be antigenic in different infectious agents. For instance, GSLs from Trypanosoma cruzi epimastigotes react with sera from patients with Chagas' disease and this reactivity is modulated by the ceramide structure [31]. ScMstosome glycolipids are recognized by IgE, which may have a role in immunity against Schistosoma mansoni [32]. In P, brasiliemis, a gakctofuranose-eontaining GSL is reactive with antibodies from patients with paracoccidioidomycosis [33]. Such reactivity was attributed to the nonreducing galactofuranosyl residue in the carbohydrate chain.
1031 1031
As extensively described before, fungal cerebrosides are very similar in that they all contain a 9-methyl-4,8-sphingadienine in combination with N-2'-hydroxy fatty acids that are saturated or unsaturated. Hydroxylation at position 2 of the fatty acid is apparently important for antigenicity of the CMH, and possible epitopes involve both glucose and the hydroxylated fatty acid, with modulation by the sphingosine-derived base. Conformer 4 of glucosylceramide as studied by Nyholm & Pascher [34, 35], which is allowed in a membrane layer, and further stabilized by a hydrogen bond between the 2-OH group on the fatty acid and the 6-OH group on the glucose residue, in addition to the hydrogen bond between glucose 05 and the amide hydrogen, is a candidate for carrying epitopes reactive with antibodies to CMH. In the human pathogen C. neoformans, a major CMH was characterized by our group as a P-glucosylceramide, containing the conserved base 9-methyl-4,8-sphingadienine in amidic linkage to 2hydroxyoctadecanoic acid [6]. This molecule was recognized by sera from patients with cryptococcosis and a few other mycoses, indicating that CMHs are immunogenic glycolipids that induce the production of human antibodies during fungal infections. Aiming the determination of the cellular distribution of CMHs in C. neoformans, we purified the specific antibodies from patients' sera, by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography, to be used in immunofluorescence experiments. Interestingly, antibodies to CMH reacted with the cryptococcal surface mostly at the sites of cell division. Confocal microscopy confirmed that the cryptococcal glucosylceramide in fact accumulates mostly at the budding sites of dividing cells [6] with a more disperse distribution at the cell surface of nondividing cells. In these experiments, the increased density of sphingolipid molecules seemed to correlate with thickening of the cell wall, hence with its biosynthesis. These results raised the possibility that fungal CMHs were involved in fungal growth, which was supported by further experiments using human antibodies to glucosylceramide. The addition of these antibodies to the culture medium of C. neoformans yeasts generated an extensive inhibition of fungal budding and, consequently, growth [6]. An association between the expression of CMHs in fungi and growth or differentiation is supported by other reports. For instance, Kawai and Ikeda [10] showed that fungal glucocerebrosides had fruiting-inducing activity in bioassays with S. commune. The intact 9-methyl-4,8sphingadienine but not the P-glucopyranosyl residue was essential for this activity. Accordingly, following observations indicated that a monoclonal anti-glucosylceramide antibody reacted preferably with the conidiophore of
1032
A. fumigatus [36]. In this context, we investigated whether CMHs and related antibodies interfered with cell growth or differentiation of additional fungal species. As mentioned above, a serological reactivity between cryptococcal CMHs and sera from patients with cryptococcosis, histoplasmosis, aspergillosis and paracoccidioidomycosis was observed [6]. The recognition of a glucosylceramide from C. neoformans by sera from individuals with different mycosis was suggestive that, during fungal infections, human antibodies are produced against similar antigens from distinct species. In this context, antibodies to CMH could interfere with cell division processes in different CMH-containing fungal cells. Conserved CMHs from P. boydii are antigens recognized by antibodies from a rabbit infected with this fungus [7]. These antibodies were purified as described before and used in immunofluorescence analysis. Interestingly, reactions of these antibodies and P. boydii conidial forms were absent or very weak, while mycelia and pseudo-hyphae were strongly reactive [7]. These results suggest that CMHs are differentially expressed in P. boydii according with morphological phase. Biosynthesis, expression or chemical structures of CMHs seems to be modified during the conidium —» mycelium transition, which suggests a role for CMHs in fungal differentiation. In accordance with this is the observation that antibodies to CMH were able to inhibit the formation of germ tube-like structures in P. boydii, although they did not influence mycelial growth [7]. We have shown (unpublished data) that germ tubes are induced after the contact of P. boydii conidia with animal cells, a step preceding efficient fungal invasion. Germ tube formation is also recognized as a crucial event in tissue invasion by C. albicans [37], a fungus that synthesizes CMHs [24] structurally similar to those previously described in other fungi [6-26] and to the characterized molecule from P. boydii. In this context, the influence of antibodies to CMH on C. albicans differentiation was also evaluated. As with P. boydii, anti-CMH antibodies inhibited germ tube formation in C. albicans [7]. The involvement of CMHs in fungal development was further confirmed by experiments using a family of compounds known to inhibit glucosylceramide synthase in mammals. Two analogs, D-threo-l-phenyl-2palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3P,4P-ethylenedioxy-P4, strongly inhibited germination and hyphal growth of A. nidulans and A. fumigatus [8]. However, the mechanisms by which fungal CMHs act on cell growth or differentiation of fungi are not known, and there is controversial evidence in this field of research. For instance, P. pastoris glucosylceramide synthase null mutants are viable and grow like their
1033
parental cells in vitro [38]. In addition, C. albicans null mutants were able to grow in both yeast and filamentous forms, indicating that CMHs do not play essential roles during growth and differentiation of these organisms [38]. These observations could be initially explained by the occurrence of species-specific functions of CMHs and related enzymes in fungal cells. However, the cellular distribution of CMHs in fungi suggests the participation of complementary surface structures possibly involved in the antifungal mechanisms generated after blocking CMHs with antibodies, as discussed later. The mechanisms by which anti-CMH antibodies inhibit fungal growth and/or differentiation remain to be established, but there is a possibility that CMHs are associated with enzymes involved in the hydrolysis and synthesis of the cell wall and / or with GPI-anchored precursors during cell differentiation and division. In this context, binding of antibodies to CMHs could impair the action of CMH-associated functional proteins inhibiting cell wall synthesis. Fungal CMHs: are they exclusive membrane components? In many cell types, cerebrosides were thought to be exclusively membrane components, due to their hydrophobic properties. However, the presence of CMHs as structural components of the cell wall of C. neoformans was clearly demonstrated by electron microscopy of yeast cells labeled with immunogold-antibodies [6]. An abundant deposition of gold particles was observed on the cryptococcal wall rather than on the plasma membrane, Fig. (2), indicating mat the antibody-reactive epitopes of CMH may be sterically accessible only after transfer of the glycosphingolipids to the cell wall. Points of transport of the presumed CMH-containing vesicles from the plasma membrane to the cell wall, were also suggested [6], Fig. (3). The association of CMHs with the cryptococcal cell wall was confirmed by immunochemical analysis, which showed that, by thin layer chromatography, orcinol-reactive bands with RF similar to that of purified CMHs were detected in extracts from cell wall preparations [6]. These bands were recognized by antibodies to CMH, suggesting that cerebrosides actually make part of the fungal cell wall components. What would be the explanation for the presence of CMHs at the fungal wall? Glycosphingolipids form, with sterols and GPI-anchored proteins, detergent-insoluble lipid rafts on the plasma membrane [39-41]. They are required for the processing of GPI-anchored proteins in yeasts, making part of vesicles that link the RES to Golgi to the plasma membrane [42-44]. For the synthesis of the cell wall structural network it has been
1034
proposed mat GPI-anchors have a pivotal constitutive role [45], A truncated GPI anchor which no longer contains inositol and glucosamine is the substrate for a phosphate-linked P-l,6-glucan extension [46, 47]. GPIanchors can be liberated in the periplasmic space by the action of phospholipase C (PI-PLC) as present in & cerevisiae [48] and abundantly expressed in P. brasiliensis [49], or could be transported to the cell-wall in vesicles. This may happen due to the inability of GPI-anchor cleavage by PI-PLC, a property of inositol-acylated molecules found in C. neoformans [50], or to a more generalized process in which precursor molecules and enzymes are transferred to the cell wall in vesicles originating from the plasma membrane. Assuming then that glycosphingolipids closely associated with GPI precursors as in lipid rafts and presumably also biosynthetic enzymes are transported to the cell wall in vesicles, CMHs could accumulate on the fungal cell wall, Fig. (3).
-
mtr
Figure 2. Transmission electron miwoscopy showing an extensive binding of antibodies to CMH to me cell wall of C. neoformans. Bar represents 0.5 nin.
This hypothesis could provide an explanation for the antifungal action of antibodies to CMH. Binding of antibodies to cell wall components could interfere with the biosynthesis and organization of the cell wall polymers. In Fusarium sp [51], treatment with wheat germ agglutinin (WGA), which has a known affinity for cMtin, resulted in alterations in
1035
germ tube formation and caused cell lysis. As a consequence, fungal infection did not spread with lectin-treated Fusarium. The inhibitory activity of antibodies to CMH may involve a different mechanism, since they could impair the utilization and reactivity of the carried components. Antibody inhibition of budding can also be correlated with the increased secretion of enzyme-containing vesicles during bud formation [52].
A
r •V
Figure 3. Possible CMH-containing vesicles are seen (arrow) in C. neoformans cells. These vesicles, which are recognized by antibodies to CMH, can move across the periplasmic space and deposit cell membrane constituents on the cell wall. Bar represents 0.1 urn.
Glycoinositolphosphoryl ceramides Glycoinositolphosphoryl ceramides (GIPCs) belong to a class of glycosphingolipids that are distinguished from the classical GSL already described in that they contain their sugar portion linked to ceramide via an inositol phosphate bridge. Phosphorylinositol-containing sphingolipids, which are absent in animals, have been reported in many plants, fungi, and protozoan [53], as well as in the parasite nematode Ascaris suum [54]. In plants, the complexicity of the inosiltolphosphorylceramides (InsPCers) is comparable to that of the GSLs in animals. The pioneering studies on these molecules were carried out by Carter and co-workers [55], who described the
1036
occurrence of "phytoglycoMpids" in seeds of corn and soybean. In fungal cells, the first report of an InsPCer was that of Wagner and Zofcsik [56], who characterized a so-called "mycoglycolipid" in S. cerevisiae and C. utilis, as ceramide-P-inositol- mannose. Subsequent work with S. cerevisiae [57] indicated that the mycoglycolipid above described resulted from strong alkaline hydrolysis of a major sphingolipid, mannose(inositol-P)2 ceramide, M(IP)2C. Further studies [reviewed in reference 53] allowed the conclusive identification of three classes of GIPCs in S. cerevisiae. These classes consist of inositolphosphoceramides (IPCs), mannosylinositol phosphoceramides (MIPCs) and the major sphingolipid, M(IP)2C, which contains two inositolphosphates with a mannose unit attached to one of the inositols. Based on NMR analysis, M(IP)2C has been shown to have a structure consisting of Ins-l-P-6-Man/? al-»2Ins-l-P-l-Cer. Diversity in the long-chain base, degree of hydroxylation and chain length of the fatty acids from the ceramide portion, give rise to several members of these classes [58]. In addition, non-glycosylated IPCs have been isolated from the phytopathogenic fungus Phytophthora spp [59] and Neurospora crassa [60]. GIPCs from pathogenic fungi • Histoplasma capsulatum Five inositolphosphosphingohpids (compounds II, III, V, VI and VIII) have been isolated, purified and analyzed from the yeast phase of//, capsulatum [61], a dimorphic fungus that causes histoplasmosis. Cells uniformly labeled with [3H] inositol and [32P] were treated with 5% TCA at 0°C and extracted according to Hanson and Lester's method [62]. Isolated lipids were purified by preparative Hquid chromatography (HPLC) on silica gel columns and utilized for structural analysis.
1037 Table 2: Structural diversity of GIPCs from pathogenic fungi1 Fungal pathogen
Compound
GIPC Structure IWCyl
77
i2
Manal -»3Manal-»4Gal pl-»6Manal ->2-Ins -P-Cer p-Xyl 1
C. neoformans
Ref
Manal-»6Manal-»3Manal-»4Galpl-»6Manal-»2-:ihs-P-Cer*
GIPC A
G1PCB
0-Xyl 1
i
Manal -*6Manctl-»6Manal-»3Manal-»4Galpl--»6Manal->2-]hs-P-Cer*
GIPCC
P-Xyl
i
Manal -»2Manal->6Manal->6Manal-»3Mmal->4Galpl-*6Manal-*2-Ins-P-Cer''
H. capsulatum
Manal-»3Manal-»2 or l-»6Ins-P-Cer
V
Manal->3. ";Manal->2 or l->6Ins-P-Cer Gal/al-^6
VI
Manal->3 X Manal->2 or l-»6Ins-P-Cer Galpl^4
C. albicans
GIPCD
(Manp 1 -^2)»Manal-»P-O^fiManal -»2Ins-P-Cer
vm PLM
Manal-*6Bis-P-Ctr
69
74, 76
Manal ->3Mioaal —»-6Ins -P-Cer
S. schenckii
61
Maiial ->6Manal -»3Manal ->3Manal -*6Ins -P-Ce r Manal ^2Manal->6Manal->3Manal->3Manal->6Ins-P-Cer Manal->3Manal->6G1 cNHj al->2Ins-P-Cer
Manal -»3Manal ->2Ins -P-Cer P. brasiliensis
ManalH>3 x J, Manal ^2Ins-P-Cer GalyPl->6
Pb-2
70
Pb-1
'All the sugar units are represented in their pyranose form, except for the cases ofH. capsulatum and P. brasiliensis GIPCs. These species present galactofiiranose-containing GIPCs, which are highlighted with the symbol/ *Molecules characterized in an acapsular mutant (Cap67) of C. neoformans.
Two of these compounds, II and III, were inositolphosphorylceramides similar to those previously isolated from S.
1038
cerevisiae [63], while compounds V, VI and VIII were novel structures presenting an identical kositolphosphoceramide core but differing by the glycosyl substitution on the polar head groups (Table 2). Compound V seems to represent a biosynthetic precursor to compounds VI and VIII since both contain the same trisaccharide core as V, but with the addition of a galactofuranose residue at the 6-position of mannose (compound VI) and a galactofuranose at the 4-position of mannose (compound VIII). The description of compound VI represented the first report on the occurrence of galactofuranose in glycosphingolipids. Compound VHI was the major InsPCer in both yeast and mycehal phase of H. capsulatum, whereas compounds V and VI were unique to the yeast phase. The ceramide portions were composed mainly of C18 phytosphingosine and C24 hydroxy fatty acids. Compounds V, VI, and VIII were recognized by antibodies from sera of histoplasmosis patients, indicating mat, as described for CMHs, GEPCs are antigenic molecules against which antibodies are produced during fungal infections in the human host. • Candida albicans C. albicans is the prominent opportunistic fungal pathogen causing frequent and severe disseminated infections in humans [64]. With the exception of a glucosylceranride previously described [24] almost nothing is known about its sphingolipids. Inositolphosphoryl ceramide-type sphingolipids were isolated from hyphal forms of C. albicans and three classes of molecules were fractionated by preparative HPLC on silica gel [65]. Molecular species in each class differ in the composition of long chain bases and fatty acids; the most abundant long chain bases were C18 and C20 phytosphingosines, while hydroxy and non-hydroxy C24 , C25 and C26 were the most abundant fatly acids. The proposed structures, Ins-P-Cer, Man-Ins- P-Cer and Ins-P-Man-Ins-P-Cer, closely resemble the sphingolipids identified in S. cerevisiae [53]. A phospholipomannan (PLM) antigen was recently isolated from C. albicans [66], Fig. (4). PLM seems to have a role in pathogenesis, since it is shed by C. albicans in contact with macrophages [67] and displays potent activity on the innate immune response [68]. The complete structure of PLM was elucidated by methanolysis, gas chromatography, mass spectrometry and nuclear magnetic resonance [69]. Mass spectrometry together with analysis of the methanolysis products demonstrated the heterogeneity of the ceramide moiety. C18/20 phytosphingosine and C25, 26 or maMy C24 hydroxy fatty acids were found in all PLM molecules, irrespective to the degree of polymerization of their glucan moiety. NMR
1039
analyses (COSY, !H-13C and *H-31P HMQC correlaction and ROESY experiments) have confirmed the P-anomery of the hnkages of the sugar units, their sequence and their linkage with inositol and phytosphingosine. The spacer Unking the (3-1,2-Man polymer to the phytosphingosine of the ceramide moiety was identified as a unique structure, -Man-P-Man-Ins-P(Table 2). In contrast to the major class of membrane glycosphingolipids represented by M(IP)2C derived from MIPC by addition of inositol phosphate [65], PLM seem to be derived from MIPC by the addition of mannose phosphate. PLM appears to be a novel mannose inositol phosphorylceramide which is extensively glycosylated through a unique spacer. The hydrophilic properties conferred by an unsual linear mannan polymer of p-l,2-linked mannose (up to 19 mannosyl residues in length) allow PLM to diffuse into the cell wall and together with mannan present these P-l ,2-oligomannosides to host cells.
C. albicans CHCI 3 /CH 3 OH(21 vtv) CHCI3 / CH 3 OH/water (10:10:1
pellet
lipid extract butanol/water
I butanol phase
I water phase I phenyl-sepharose chromatography
purified PLM
i TLC
I western blot analysis
f Methanolysis
I GC-MS
Figure 4. Steps of fractionation and analysis of PLM.
I ESI-MS
NMR (1Dand2D)
1040
• Paracoccidioid.es brasiliensis Two major GIPCs (Pb-1 and Pb-2) have been extracted from P. brasiliensis [70], a dimorphic fungus endemic to rural areas of South and Central America [71]. Their complete structures were elucidated by XH and P nuclear magnetic resonance spectroscopy (NMR), ESI-MS and MS/CDD mass spectrometry, chemical and enzymatic degradation. These analyses revealed that Pb-1 consists of mannose and galactose in a ratio of 2:1, myoinositol, phosphate and a ceramide consisting of phytosphingosine (tl8:0) and C24:0 hydroxy fatty acid. Glycolipid Pb-2 appeared to be similar, except that it lacked the galactose residue. The linkage structure of the terminal trisaccharide of Pb-1 was confirmed by detection of derivatives 2,3,5,6-tetra-OMe-Gal and 2,3,4,6tetra-O-Me-Man, corresponding to non-reducing end units of galactofuranose and mannopyranose along with 3- and 6-0-substituted mannopyranosyl units. In Pb-2, the absence of derivatives 2,3,5,6-tetra-OMe-Gal and 2,4-di-O-Me-Man and the appearance of 2,4,6-tri-O-Me-Man, supported the proposed Pb-2 structure. The anomeric linkages of the Man/? residues were obtained by a-mannosidase treatment of the free glycoinositols obtained after GIPCs ammonolysis. All these data support the structures for Pb-1 and Pb-2 shown in Table 2. These structures are analogous to those of two GIPCs isolated from H. capsulatum [61]. These observations are in agreement with the fact that, as described for GIPCs from Histoplasma, Pb-1 is antigenic and reactive with sera from patients with paracoccidioidomycosis. It seems likely that the internal linkage in the H. capsulatum antigens is Man/? al->2Ins and that Pb-2 is identical to structure V [61], while Pb-1 differs from Structure VI [61] only in the anomeric configuration of the terminal galactofuranose residue. • Sporothrix schenckii S. schenckii is a dimorphic fungus that causes sporothricosis, a subcutaneous disease that can evolve to a disseminated mycosis in immunosuppressed patients [72]. Novel structures of mannosylinositolphosphoryl ceramides from yeast and mycelial forms of S. schenckii were described by Toledo and co-workers [73] and Loureiro-yPenhaetal. [74]. Two different procedures were used for the isolation and purification of these molecules. Loureiro-y-Penha and co-workers [74] used a 45%(v/v) hot aqueous phenol extraction followed by Bio-Gel P-60 column chromatography, while Toledo et al. [73] extracted GIPCs using
1041
isopropanoVhexane/water (55:20:25 v/v) at room temperature followed by DEAE-Sephadex A-25 column chromatography and preparative HPLC. The structures of purified GIPCs from yeast or mycelial forms of 5. schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. Five triplets of deprotonated molecules at m/z ranging from 908,6 to 1750.9, separately by a multiple of 162 was detected in the negative ion MALDI-TOF mass spectrum, suggested the presence of glycan chains containing 0, 1, 2, 4 or 5 hexose residues [74], The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine Nacylated by hydroxy C24 fatty acid (80%), C24 (15%) or 2,3-hydroxy lignoceric acids (5%). GDPC-derived oligosaccharides from S. schenckii were liberated by ammonolysis and analyzed by methylation analysis and NMR. The position on the inositol to which the phosphorylceramide group is attached was deduced by a combination of methylation and periodate oxidation studies. The ceramide moiety was linked to position 1 via a phosphodiester bridge. The oligosaccharide structures are shown in Table 2. All are unbranched mannooligosaccharides containing a core sequence Manp od-»6Ins, a feature that has not been reported in the glycolipids of eukaryotic cells. The Man/xxl-»2-Ins motif has been identified as a common core structure of all GIPCs expressed by S. cerevisiae [53], H. capsulatum [61], P. brasiliensis [70] and C. neoformam [75]. Further analyses of S. schenckii GBPCs, revealed that an additional motif containing the linkage GlcNHa od-*2Insl-P, which has not been previously observed in any glycolipid, is also present [76]. • Cryptococcus neoformans Ceramide-(phosphoryhnositol)2-mannose, ceramide-phosphoryl inositolmannose and eeramide-phosphorylinositol have been identified in C. neoformans [75], an encapsulated fungus mat causes cryptococcosis and cryptococcal meningitis. Similar compounds have also been identified in yeasts and pathogenic fungi already cited in mis review. More complex GIPC structures were found only in pathogenic fungi as P. brasiliensis [70] and S. schenckii [73,74], Complex GIPCs have been purified from encapsulated (WT) and acapsukr (Cap67) cells of C. neoformans [77]. Their structures were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance specfroscopy, methylation analysis, GC-MS, and chemical degradation. Chemical analysis of the crude GIPCs isolated from
1042
wild type cells and the Cap67 mutant of C. neoformans showed the existence of significant differences between these molecules. A higher molar ratio of Man was found in the mutant compared with WT. GC and GC-Ms analysis of the ceramide portion of cryptococcal GIPCs showed the presence of C18 phytosphingosine and 2-hydroxy C24, C25 and C26 fatty acids for WT-GIPC, and 2-hydroxy C24, and 2,3-dihydroxy C24, C25 and C26 for the GIPC from the acapsular mutant. The chemical composition was in agreement with the mass spectrometric analysis. The negative-ion spectrum of WT contained a major signal [M-H] at m/z 1705.3 consistent with one pentose and four hexose residues linked to 2-hydroxy tetracosanoyl-C18:0 phytosphingosine-phosphoryl inositol. However, the MALDI-TOF mass spectrum of crude Cap67-GIPCs was more complex, with 5 triplet groups of deprotonated molecules. Each triplet group, separately by 162 mass units is consistent with the presence of 3 molecular species of InsPCers, differing in their ceramide moieties and containing 1 pentose and 4,5,6,7 or 8 hexose residues. The difference of 16 units between the two most abundant 12C peaks within each triplet cluster is consistent with the replacement of the monohydroxy by the dihydroxy fatty acids, detected by GC-MS analysis. The structural diversity in the ceramide portions from GIPCs from WT and mutant cells was confirmed by the fact that, in common with the majority of fungi studied so far, WT cells syntliesize GIPCs based on C18 phytosphingosine N-acylated with hydioxy C24 fatty acid, whereas the Cap67 mutant synthesizes GIPCs which are Nacylated with 2,3-dihydroxy C24 or 2-hydroxy C24 fatty acids. The structural diversity between GIPCs from WT and acapsular cells is also present in their glycan moieties. NMR spectroscopy of the Insoligosaccharides released from WT-and Cap67-GIPCs by ammonolysis showed the presence of additional a-Man/> residues to Ins-pentasaccharides of WT-GIPC that correspond to GIPC-B, GIPC-C and GIPC-D, respectively (Table 2). One possible explanation for the additional Man units in Cap67-GIPCs is that the putative al—»6 mannosyl transferase, which adds the first Man residue, may be inactive in the WT strain. GIPCs from higher fungi An early report by Byrne and Brennan [78] described the presence of glycolipids in Agaricus bisporus, although these molecules were not structurally characterized. More recently, novel glycoinositolphosphosphingolipids, designated basidiolipids, were extracted from A. bisporus and A. campestris using aqueous organic solvent mixtures [79]. A homologous series of four glycolipids, designated Bl-1,
1043 1043
Bl-2, Bl-3 and Bl-4 was purified by anion-exchanged chromatography and their structures have been determined by MALDI-TOF-MS and NMR spectroscopy analyses of the whole molecules, combined with methylation analysis, periodate oxidation, acid and enzymatic hydrolysis. From these data, four structures were proposed (Table 3). A detailed characterization of GIPCs present in six basiodimycetes, Amanita virosa (engl.death cup), Cantharellus cibarius (chanterelle), Lentinns edodes (shiitake), Plenrotus ostreatus (oyster-mushroom), Calvatia exipuliformis (engl, puffball), and Leccinum scabrum (engl. red birch boletus) were further carried out [80]. All the basidiolipids analyzed presented the same LCB, phytosphingosine (tl8:0). The majority of the fatty acids were hydroxy C22, C24 and C24:l. hi addition the ceramide of C. cibarius contains a di-hydroxy C24 fatty acid. The predominance of hydroxylated fatty acids may reflect a requirement for basiodiolipidcontaining membrane stabilization under physico-chemical stress conditions. In contrast with the ceramide portion, the oligosaccharide structures varied according to the basiodiomycete specie. Using NMR spectroscopy, methylation analysis, and exoglycosidase cleavage, the oligosaccharide structures obtained after ammonolysis of GIPC were elucidated. Table 3 shows the structures of GIPCs from different mushrooms. Differently from previous studies from Jennermann et al. [79], the a- anomeric configuration for the Man residue was confirmed. The core structure of the basiodiomycetes, Manpa (l-»2)Ins-P-Cer, is common to several fungi studied as 5. cerevisiae [53], C. albicans [65], and C. neoformans [75]. Further modifications of this core structure by addition of a -Man/?, ct-Glcp, a/p- Gal/? , giving rise to different GIPCs, were found in several mushroom species (Table 3) and have also been reported in H. capsulatum, P. brasiliensis and in a novel family of GIPCs from C. neoformans. Apart from these already mentioned GIPCs, other similar molecules containing structurally novel oligosaccharides are present in certain basidiomycetes (see Table 3).
1044 Table 3: Structural diversity of GIPCs from higher fungi1
Mushroom
GIPC Structure
Compound
Ref
Manpl-»2-Ins-P-Cer
BL-1
79
2 Galal-»6 Gal pi-*6Maa|31 ->2-Ins-P-Cer
BL-2
A. bisporus a-Fuc
I
A. campestris
a-Fuc 1
I
2 Galal-*6Galal-»6Gal|31->6Man|31->2-Ins-P-Cer
BL-3
a-Fuc 1
i
2 Galal-^6Galal-»6Galal^6Galpl-^6Mm|31-»2-Ins-P-Cer
BL-4
Ot-Fuc 1
80
I
A. virosa
2 Galotl-»3Gal|31->6Manal-»2/4-]hs-P-Oer a-Fuc 1
I
2 Galal-»3 Gala 1-^2 Gala 1-^3 Gal (51->6Manal-^2/4-Ihs-P-Cer 6
t
a-Fuc Manal-»2/4-Ins-P-Cer C. exipuliformis
80
Galpl-»6Manal->2/4-Ins-P-Cer Galal^6Gal pl-»6Mancri->2/4-Ins-P-Cer a-Fuc 1
i
2 Galal->3Galal-»6Gal|M->6Manal-»2/4-:iiis-P-Cer
Manal-» 3/6 Manal->2M-Ins-P-Cer C. dharius
Manal-»2/4-Ins-P-Cer L. edodes
-
80
Manal -» 3Mstnal -» 3/6 Manal -*2/4-Ins -P-Cer
a-Fuc 1
I
2 Manal ->6 Galal^3 Gal pl->6Manal -^2/A-lns -P-Cer a-Fuc 1
J-
2 Manal->2Manal->6Galal-^3Galpl->6Manal->2/4-In!-P-Cer
80
1045
80
I L. scahrum
4 6 MmctJ-*3Fueal-»2 Gal p l -
_
80
1
P. ostreatus Gaial-J-3GalaI-»
2
'All the sugar units are represented in then pyranose fonn.
Biosynthesis of glycosphingolipids in fungal cells As already described in this chapter, sphingoHpids represent a structurally and functionally diverse class of natural products found in many ceEular types. They are essential components of the cytoplasmic membrane in both mammalian and fungal cells but differ in their structure and biosynthesis. In feet, several inhibitors of fungal sphingolipid synthesis, all natural products and most of them non-toxic to mammalian cells, have been reported in the last decade. The understanding of GSL biosynthesis is therefore fundamental for the development of antifungal drugs and complete knowledge of lipid function in fungal cells. Sphingolipids are derivative of the sphingoid bases (long-chain bases, LCBs), which can be considered as the defining structural unite of sphingoHpids. They are long-chain aliphatic amines, containing two or three hydroxyl groups, therefore consisting of 2-amino-l,3-dihydroxy linear alkanes. LCBs are used in the synthesis of ceramides, the building blocks of sphingolipids. Ceramides consist of a LCB linked to a fatty acid via an amide bond. The formation of ceramides is a key step in the biosynthesis of all the complex sphingolipids, in which the terminal primary hydroxyl group is, for instance, linked to carbohydrate or phosphate units. The comparison of sphingolipid biosynthesis in diverse biological systems revealed the existence of common and distinct steps [81, 82]. In general, synthesis and expression of sphingolipids seems to be essential for normal processes in microbial and animal cells and, in the specific case of fungi, their synthesis is emerging as an attractive target for the action of antifungal drags [5]. Fungal cells possess some exclusive pathways for sphingolipid biosynthesis, some of which are crucial for cell viability. Tn addition, GSLs are antigenic [6, 7, 70] or involved with pathogenesis in
1046
different fungal models [6-8], which stimulate studies on their functions and biosynthesis. Most of the knowledge on sphingolipid biosynthesis comes from studies using the model yeast S. cerevisiae. Several genes involved with file metabolism of spbingolipids were identified in this organism [reviewed in references 81 and 82] and, in this context, S, cerevisiae represents an excellent model for studies on biosynthesis and expression of fungal GSLs. However, there are clear differences between the expression of glycosphingolipids in S. cerevisiae and other fungal species; for instance, monohexosylceramides are commonly detected in pathogenic and nonpathogenic fungi, but not in 51 cerevisiae. For that reason, sphingolipid functions and metabolism in S. cerevisiae, which are the subject of several previous reviews, will be used here as the archetype for the biosynthesis of GSLs in fungal pathogens, always taking into consideration the known structural diversity of GSLs in these cited models. The process resulting in the synthesis of ceramide begins with the condensation of palmitoyl-CoA and serine in the endoplasmic reticulum. This reaction, which occurs in both animal and fungal cells, is catalyzed by serine palmitoyltransferase (SPT), resulting in the generation of the intermediary compound 3-ketodmydrosphingosine (3-ketosphinganine). In S. cerevisiae, three genes are required for optimal SPT activity: the homologue genes LCB1 and LCB2, which are involved in the yeast response for heat stress, and TSC3, a member of the family of temperaturesensitive suppressors of calcium sensitivity (TSC) [81, 82]. The condensation of serine and pahnitoyl-CoA is followed by the reduction of 3-ketosphinganine to the LCB dihydrosphingosine (DHS, sphinganine). This step also occurs in the endoplasmic reticulum and involves the action of 3-ketosphinganine reductase, whose deletion renders S. cerevisiae cells unable to grow in the absence of exogenous LCBs. 3-ketosphinganine reductase, coded by the TSC10 gene, also belongs to the TSC family [81, 82]. The generation of sphinganine gives rise to the first branching point in fungal sphingolipid synthesis. This LCB is hydroxylated, to generate phytosphingosine and afterward inositolphosphorylceramide, or directed for the synthesis of monohexosylceramides. These distinct pathways of the sphingolipid metabolism will be discussed below in more details and are summarized in Fig, (5).
1047
Sphingolipids with phytosphingosine-containing ceramides In mammalian cells, sphinganine is acylated to generate dihydroceramide. The latter is then reduced, resulting in the synthesis of ceramide. This observation diverges from the corresponding pathways observed in yeast cells, in which sphinganine can be hydroxylated to form phytosphingosine, that is then converted to phytoceramide by transfer of acyl groups. Alternatively, sphinganine can be first acylated, generating dihydroceramide, and then hydroxylated, finally forming phytoceramide. In S. cerevisiae, the enzyme encoded by the gene SUR2/SYR2 catalyzes the hydroxylation of either dihydrosphingosine or dihydroceramide [81, 82]. Acylation of LCB and consequent synthesis of ceramide also represents a difference between mammalian and fungal cells, since the latter appear to exclusively transfer a-hydroxylated very long-chain fatty acids (VLCFAs) to phytosphingosine. VLCFAs are formed through the action of the enzymes encoded by ELO2 and ELO3, responsible for the sequential elongation of smaller fatty acids to 24 carbons (Elo2p) and conversion of 24C to 26C fatty acids (Elo3p) [81, 82]. The enzyme responsible for transferring these fatty acids to LCB is called ceramide synthase, encoded by LAG I and its homologue LAC I, and its action is inhibited by the fungal toxin fumonisin [81, 82]. Steps subsequent to phytoceramide formation are unique to fungi and involve the sequential addition of phosphorylated inositol to form inositolphosphorylceramide (IPC), mannose-IPC (MDPC) and, specially in S. cerevisiae, mannose-inositolphosphoryl-IPC (M(IP)2C). Such compounds are frequently glycosylated to produce most complex glycosphingolipids, generating the fungal glycoinositol phosphorylceramides (GIPCs). To form IPC, the Cl-hydroxyl of phytoceramide is linked to phosphoinositol by a phosphodiester bond. This reaction is catalyzed by IPC synthase, (Ipcl), encoded by the AUR1 gene [83]. Because Ipcl activity is both vital and unique in fungi, it has emerged as an attractive target for antifungal drugs [5]. The antifungal peptide aureobasidin A (AbA), produced by Aureobasidium pullulans, presents a strong activity against many pathogens and its molecular target was identified in S. cerevisiae as the essential gene AUR1. This gene was found to be required for the expression of Ipcl and formation IPC in yeast. Therefore the AUR1 gene is also called IPCL Currently, two additional antifungal agents (khafrefungin and rustmicin) targeting Ipcl are known [81, 82]. IPC I was the first gene of the sphingolipid pathway with a role in fungal pathogenesis. IPCI modulated virulence traits of C. neoformans,
1048
such as melanin pigmentation, since its overexpression induced more melanin production, whereas down-regulation decreases melanin pigmentation [84]. One major factor for C. neoformans to produce infection is its ability to grow inside macrophages and, therefore, in acidic conditions, which are to those in the phagolysosome. Down-regulation of IPC1 generated a strain no longer pathogenic in a rabbit model of cryptococcal meningitis. In addition, a decreased Ipcl level impaired the C. neoformans growth into a macrophage-like cell line and in an acidic environment. These results therefore clearly indicate an important role for Ipcl in pathogenesis by modulating different virulence traits of C. neoformans. Concomitant to IPC formation, Ipcl also produces diacylglycerol (DAG) and consumes phytoceramide. The importance of Ipcl therefore may be due not only to the formation of IPC itself, one of the more abundant sphingolipids in the membranes, but also to the regulation of phytoceramide, implicated in growth arrest and yeast stress responses [85, 86], and DAG, a well-established mitogen and activator of protein kinase C (PKC). In S. cerevisiae, IPC is mannosylated to yield mannose-inositolphosphoceramide (MIPC), a reaction that requires the SUR1 and CSG2 genes [81]. Similar reactions should occur in several other fungal species, which appear to use MIPC as the precursor for more complex GSLs. The human pathogen Sporothrix schenckii seems to represent an exception, since a novel GSL containing a glucosamine-inositol-phosphoceramide motif has been described, in addition to GSLs containing the conventional MIPC domain [81]. In S. cerevisiae, the terminal step in sphingolipid synthesis consists of the addition of inositol phosphate to MIPC. This reaction, which requires the product of the IPT1 gene, results in the formation of M(IP)2C [81,82]. Several fungal species proceed with sphingolipid biosynthesis by adding several sugar residues to IPC (as in the case of S. schenckii) or MIPC (as in the case of the pathogens C. albicans, C. neoformans, S. schenckii, H. capsulatum, P. brasiliensis, A. fumigatus, and the higher mushrooms A. virosa, C. exipuliformis, C. cibarius, L. scabrum, L. edodes, and P. ostreatus). The resulting structures are the acidic GSLs glycosylinositol phosphorylceramides, which represent a major class of fungal lipids characterized by the presence of a myo-inositol-l -phosphate spacer between glycan and ceramide. As already mentioned, this class of molecules is synthesized by fungi, plants, and certain parasitic organisms, but not by mammal cells or tissues. The detailed structural characterization of GIPCs from different fungal species revealed a relatively high diversity
1049 1049
(Tables 2 and 3), which requires the use of several still uncharacterized glycosyltransferases. Sphingolipids with 4,8-diene-9-methyl-sphingobase-containing ceramides All sphingolipids in S. cerevisiae are classified as IPCs [81]. Several other fungal species, however, add one or more sugar residues to the C-l of ceramide to form a second class of sphingolipids referred to as glycosylceramides. CMHs, which are the most common examples of such neutral GSLs, were characterized in detailed in several fungal species (Table 1), all of them presenting a ceramide moiety containing 9-methyl4,8-sphingadienine in amidic linkage to C18 or C16 a-hydroxy fatty acids and a carbohydrate unit. These molecules are formed through the action of UDP-glycosyl ceramide glycosyltransferases (glycosylceramide synthases, GCS), which may also act in the synthesis of ceramide dihexosides (CDHs) [87]. Molecular studies using GCS from different organisms [26, 38] provided new insights into the biosynthesis of sphingolipids, as described below. Sarini • paMtoyl CoA Ccmdensaton-SPT I Lcei.LCai,rera 3-krto dlhydruphlngoilnt Redudion-3-ketoredudase I r e c »
Cor^.acyMnn
""""'"""•
rscw
.^^^
HytaylataJsum/SW!
C9.me»iylalta|
VLCF* • Phyto.phln9o.ln. C,,orC,,aaialon-ceratndesynftase l u s t u o
SHnttliyUjHphfni.dl.niMcontilnlngcanmldi
Phyfocinmid* IPCsynthase I • rO-MWIM^, .1msl"°'GWH
flysi
__ "'""' "
^.narrsKraseffj ^.__i.C°"
ta*C.r(PO
9-melhyt-4,8-sph>igadienine
,,.,„.,
2-OHC,,o,C,,l.tlyacicl
l*iivlni-P-Cir(«PC)
•
•
*
ln^P-M.tvlnrf-Cer
Man4MHaivlli>#-Cir
SaMhn-lnt-P-Ctr
(S. ce^ewswe)
(c. alblcans)
(C. neofomxtns, Agerlcus sf^i)
I Variable steps of glyrajlalion
I
(S. diencktl H. capsulatum, P. brasillensis, A fumigitti)$ ,
I
I
CompkiSPCt
Figure 5. Biosynthesis of fungal glycosphingolipids. For details of each biosynthetic step, see "Biosynthesis of glycosphingolipids infungal cells".
Ceramide backbones with C16 or C18 fatty acids linked to the 4,8diene-9-methyl-sphingobase are exclusively precursors for CMH synthesis, whereas ceramide backbones containing VLCFAs and phytosphingosine
1050
are preferentially used as substrates for the synthesis of inositol-containing sphingolipids. However, through a molecular systematic analysis of a glycosyltransferase gene family with members of animal plant, fungal, and bacterial origin, Leipelt and co-workers [38] suggested the occurrence of previously unknown steps of ceramide synthesis and glycosylation, which was concluded from the occurrence of some unexpected sphingolipids produced by S. cerevisiae and P. pas tor is transformed with GCS from different sources. In this study, GCS null mutants of P. pastoris and C. albicans were generated. Both mutants were still viable and grew like the parental strains on different culture media. GCSs from Homo sapiens, Gossypium arboreum,P. pastoris, C. albicans, and M. grisea were then expressed in the P. pastoris GCS null mutant strain, which resulted in the formation of structurally diverse GlcCer molecules. Yeast cells expressing the human GCS, for instance, produced five different GlcCer molecular species, with ceramide backbones corresponding to 18:0-18:0, 18:0(2-OH)18:0, 18:0-18:lM, 18:0-18:2A4'8, 18:0(2-OH)- 18:1 M , and 18:0(2-OH)18:2A4'8, which may all be regarded as biosynthetic precursors of 18:0(2OH)-18:2A4>89m, which is the major ceramide moiety in CMHs from many fungal species. If this hypothesis is correct, it is possible to suggest a sequential modification of the sphingoid base starting with the introduction of the A4-double bond followed by the A8-unsaturation and a final methylation at C9. However, it is not possible to conclude whether these modifications occur at the free sphingobase, in its acylated form, or even after glycosylation of the ceramide. Structural analysis revealed that, in the transformed cells described above, ceramide backbones containing phytosphingosine and a VLCFA molecule were also detected. This was a very significant finding, since such fungal ceramides were thought to be exclusively used for the synthesis of inositol-containing sphingolipids. This observation confirmed a sole previous report, which was further supported by our group, as described below. Glycosylceramides presenting phytosphingosine as the long chain base Recent studies from our group demonstrate that phytoceramide can be alternatively glycosylated to finally form ceramide dihexosides [87]. These results reveal that phytoceramides in fungi can be modified to generate unconventional GSLs, which agrees with previous reports [88]. In summary, fungal cells are believed to construct two different pools of ceramides to be used for the synthesis of different sphingolipids
1051 1051
[38]. Ceramide backbones with C16 or C18 fatty acids linked to a 4,8diene-9-methyl-sphingobase, which were widely identified in several fungal species (Table 1), are thought to be exclusively used as precursors for glucosylceramide (GlcCer) synthesis. In contrast, ceramide backbones with relatively long chain C24 and C26 fatty acids bound to phytosphingosine were thought to be restricted to the synthesis of the inositol-containing phosphosphingolipids. In a recent investigation, however, Leipelt and colleagues [38] have identified and characterized novel glucosylceramide synthases from plants, animals and fungi, including M. grisea. Genetic approaches revealed that the expression of the GCS from M. grisea in a P. pastoris GCS null mutant resulted in the biosynthesis of GlcCer with the usual ceramide moieties comprising C16 and C18 fatty acids in an araidic linkage with 9-methyl-4,8-spbJngadienine, but also in the occurrence of GlcCer with phytosphingosine and mainly long-chain (C26) a-hydroxy fatty acids in amide hnkage. These results indicated that GCS could accept bom classes of ceramide as substrates to form GlcCer. These data also supported a previous report by Lester and co-workers [88], which described the occurrence of a ceramide tetrahexoside consisting of (GabGlc)-iV-hydroxytetracosonyl-hydroxysphinganine in Neurospora crassa. Additional functions of glycosphingolipids in yeast cells: lipid rafts Besides their relevance in as targets for antifungal drugs and role in fungal pathogenesis, GSLs act in several physiologic processes resulting in the control of cell functions. In this context, S. cerevisiae has been used as an adequate model for studying sphingolipid function in fungal and animal cells. Recent studies indicate that sphingolipids probably perform signaling functions during heat stress and normal growth conditions, which may be related with the presence of membrane domains referred to as lipid rafts. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and GSLs. They exist as distinct liquidordered regions of the membrane that are resistant to extraction with nonionic detergents. Rafts appear to be small in size, but may constitute a relatively large fraction of the plasma membrane [89]. While rafts have a distinctive protein and lipid composition, all rafts do not appear to be identical in terms of either the proteins or the lipids mat they contain. A variety of proteins, especially those involved in cell signaling, have been shown to partition into lipid rafts. As a result, Mpid rafts are thought to be involved in the regulation of signal transduction and membrane trafficking
1052
[90]. In S. cerevisae, lipid rafts are important for delivering specific proteins, including Gaslp, Pmap and Nce2p, to the plasma membrane [91], Lipid rafts also serve as sorting platforms for proteins destined to the cell surface in S. cerevisiae during mating. [92]. ABREVIATIONS CDH CMH GCS GIPC GSL IPC LCB MDPC PLM VLCFA
ceramide dihexoside ceramide monohexoside, cerebroside glucosylceramide synthase glycoinositolphosphoryl ceramide glycosphingolipid inositol phosphoceramide long chain base mannosylinositol phosphoceramide phospholipomannan very long chain fatty acid
ACKNOWLEDGEMENTS This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico (CNPq), Fundaelo de Amparo a Pesquisa no Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ), Fundaelo Universitaria Jos6 Bonifacio (FUJB), and CoordenacSo de Aperfeieoamento de Pessoal de Nivel Superior (CAPES). We thank Kildare R. Miranda and Anderson J. Franzen for the micrographs used in Figures 2 and 3.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©
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PHYTOCHEMICAL STUDIES AND PHARMACOLOGICAL ACTIVITIES OF PLANTS IN GENUS HEDYOTIS/ OLDENLANDIA NORDIN HJ. LAJIS3'* AND ROHAYA AHMADb "Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia Faculty of Applied Science, University Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia Tel: + 603-89468063; Fax: +603-89468080; E-mail:
[email protected] ABSTRACT: This paper attempts to present a review on the study of phytochemical and pharmacological activities of plants from the genus Hedyotis (Rubiaceae) in the last seven decades, which include our work on Malaysian Hedyotis species. The structure-activity relationships of compounds isolated from this genus are compiled and discussed. Finally, there is also a brief discussion on the biosynthesis of anthraquinones, iridoid glycosides and alkaloids, which are the common constituents of Hedyotis species.
INTRODUCTION Hedyotis Linn., a genus formerly grouped under Oldenlandia, is a group of erect decumbent or climbing herbs and somewhat shrubby plants of the family Rubiaceae. The genus consists of some 180 species and they usually grow well on dry and sandy soil, along rivers and coasts. There are 35 species recorded in Malaysia among which include H. capitellata Wall. syn. Oldenlandia recurca Miq., H. corymbosa Lam., H. dichotoma Hook., H. verticillata Linn., H. diffusa Willd., H. nudicaulis Roth., H. herbacea Linn., and H. pinifolia Wall. [1]. Among the most common medicinal uses of the plants in Malaysia are as tonic or febrifuge, stomachic or treatment for diarrhea and dysentery. The leaves and roots of most of the plants are also used as a poultice to treat bruises and wounds as well as to treat broken bones, rheumatism and lumbago. H. capitellata stems are taken for
1058
post-partum treatment and the leaves as an herb to treat kidney ailment [1]. hi the Chinese medicine, more than 20 species of the genus Hedyotis have been used [2]. The most popular among these are H. diffusa and H. corymhosa, which are regarded as the active ingredients in several Chinese herbal medicines, such as "Peh-huejuwa-chi-cao", "Jiedu Yanggan Gao" powder", "Xiao Wei Yan Powder" and "Feibao syrup". These herbal medicines are taken for cancer and other diseases [3]. H. diffusa is also extensively marketed as "Bai Hua She She Cao" (Figure 1), which is made into capsules called "Hedyotis capsules". This medicine is traditionally claimed to support the immune system and maintain body's natural balance and general well being. Other medicinal claims include treatment for ul ulcerations, swellings, snakebites, bronchitis, tonsillitis and hemorrhoids. It is also used in mixtures with other herbal preparations for cancer of the digestive tract and is said to be especially effective for cancer of the rectum [4,5].
«l v
X
Figure 1: "Bai Hua She She Cao" and Hedyotis Capsules Therefore, this paper attempts to review the phytochemical and pharmacological investigation of the species of this genus to date, and the structure-activity relationships on certain classes of compounds, as well as the previous studies on biogenesis.
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PHYTOCHEMICAL STUDIES ON PLANTS OF THE GENUS HEDYOTIS The first report on Hedyotis/ Oldenlandia was published in 1933 in India on the phytochemical investigation of the medicinal plant, H. auricularia syn Oldenlandia auricularia by Dey and Lakshminarayan [6]. Phytochemical studies on other species in the genus since then have yielded indole and /?-carboline alkaloids, anthraquinones, flavonoids and their glycosides, iridoids and their glycosides, lignans, triterpenoids, sterols, coumarins as well as saponins [6,722]. From these reports, it is quite apparent that the members of the Hedyotis genus have highly divergent structural differences. The diversity in this genus has also led to its selection in the study of the evolution of fruit development in the Rubiaceae family. This family has been chosen because it contains species with immense economic value such as Coffeea, Cinchona and Gardenia. This project named "The Hedyotis Genomics Project" undertaken by the New York Botanical Garden is focused on finding the genetic machinery responsible for fruit biodiversity among plants within the genus (such as H. centranthiodes and H. terminalis) and other flowering plants within the Rubiaceae family [23]. In the following discussion, we look into the structural diversity among the members of the genus based on the classes of compounds that have been isolated, namely the alkaloids, anthraquinones, flavonoids, iridoids, terpenoids, sterols, lignans, coumarins and other phenolics. ALKALOIDS A number of alkaloids have been isolated from only three Hedyotis species, which are H. auricularia, H. chrysotricha and H. capitellata. The bis-indole alkaloids, hedyotine and auricularine were both isolated from the roots of H. auricularia [6,24]. However, the molecular formula and the structure of auricularine was revised later [19]. Auricularine (1) belongs to a novel group of bis-indole alkaloids, borreverine (lb) and isoborreverine [25] whose structures were established by X-ray crystallography and HRMS. Spectroscopic experiments found auricularine to differ from borreverine in its molecular formula by the presence of only a
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methyl group. This was accounted for, by the presence of an extra iVjJV-dimethyl group in the form of an 7V-Me amino group. The /?-carboline alkaloid, chrysotricine, was first isolated from H. chrysotricha (Palib) Merr. [2]. The structure of chrysotricine was determined by X-ray diffraction analysis after H and C NMR data taken in CDC13 and CD3OD resulted in large changes in some signals, thus failing to give a definite structure. Further analysis showed that the spectra measured in CDCL were consistent with the crystal structure. The two additional CH signals in CD3OD and the disappearance of CH2 signals suggested that (2a) had converted into its tautomeric form (2b), as shown in Table 1. It is interesting to note that chrysotricine has been later reported in H. capitellata along with isochrysotricine and two other /?-carboline alkaloids, capitelline (3) and cyclocapitelline (4a) [9]. The tautomeric transformation as described by Peng et al. for chrysotricine to its tautomeric form was also observed in CD3OD [2]. Capitelline (3) and isocyclocapitelline (4b) from H. capitellata were reported as stereoisomers, and the constitution as well as relative configuration of (-)-isocyclocapitelline was subsequently confirmed by X-ray crystallography. Two /?-carboline alkaloids from H. capitellata var. mollis, hedyocapitelline (5) and hedyocapitine (6) have been reported later [9]. As in the case of chrysotricine, tautomerization was also observed for hedyocapitine upon standing in CD3OD. The preferential form of the equilibrium between (6a) and its tautomer (6b) is shown below.
(6a)
(6b)
1061 Table 1: Alkaloids from Hedyotis and structure of borreverine [11,19, 24,25] Species
Alkaloids
NHMe
H. auricularia
(1) Auricularine revised structure
(lb) Borreverine
(2a) Conformation in CDC13
(2b) Conformation in CD3OD
H. chrysotricha
(2) Chrysotricine
H. capitellata HO'
(4a) (3) Capiteiiine
(4b)
Cyclocapitelline R = CH3 Isocyclocapiteiline
1062 1062
.Me "Me H. capitellata var. mollis
(5) Hedyocapitelline
(6) Hedyocapitine
ANTHRAQUINONES Rubiaceaous plants are usually rich in anthraquinones. The first four anthraquinones, 2-methyl-3-methoxyanthraquinone (7), 2methyl-3-hydroxyanthraquinone (8), 2-methyl-3-hydroxy-4methoxyanthraquinone (9) and 2,3-dimethoxy-6-methylanthraquinone (10) reported in genus Hedyotis were isolated from H. diffusa [26]. All the compounds except for (10) are substituted only in ring C. The structure of (10) has been later confirmed by synthesis based on Diels-Alder reaction. This anthraquinone has been the only one reported from a natural source until today. Other anthraquinones possessing the same substitution pattern are synthetic products [27]. Our investigation on the roots of H. dichotoma [12] yielded two new anthraquinones, l,4-dihydroxy-2,3-dimethoxyanthraquinone (11) and 2,3-dimethoxy-9-hydroxy-l,4-anthraquinone (13). Compound (11) is the 2,3-dimethyl ether derivative of 1,2,3,4tetrahydroxyanthraquinone [28] but otherwise has not been reported. Interestingly, a study on H. herbacea [7] found both (11) and (13) along with l,4-dihydroxy-2-hydroxymethylanthraquinone, (12) and the new 2-hydroxymethyl-10-hydroxy-l,4-anthraquinone (14). Compounds (13) and (14) are 1,4-anthraquinones that occur rarely in nature.
1063 Table 2: Anthraquinones from Hedyotis [7,12,26] Species
Anthraquinones
H
O
H. diffusa H
H. dichotoma and
(7)
2-Methyl-3-methoxy anthraquinone R, = Me, R 2 =OMe, R 3 = R , = H
(8)
2-Methyl-3-hydroxy- anthraquinone Ri = Me, R 2 = OH, R 3 = R,= H
(9)
2-Methy1-3-hydroxy-4methoxyanthraquinone' R, = Me, R 2 = OH, R3 = OMe, R,= H
(10)
2,3-Dimethoxy-6-methyl anthraquinone R, = R 2 = OMe, R 3 = H, R,= Me
(11)
1,4-Dihydroxy-2,3-dimethoxyanthraquinone R,= R 2 =OMe
H
O
(12) l,4-Dihydroxy-2hydroxymethylanthraquinone Ri = CH 2 OH,R 2 =H
H. herbacea (13)
2,3-Dimethoxy-9-hydroxy-l ,4anthraquinone Ri = R 2 = OMe, R 3 = OH, R,= H
(14)
2-Hydroxymethyl-10-hydroxy -1,4-anthraquinone R, = CH2OH, R2 = R3 = H, R, = OH
FLAVONOIDS Flavonoids are another class of compounds found in abundance in Rubiaceae family that has also been found in some Hedyotis species. Lu et al. reported the isolation of a new acylated flavonol glycoside, kaempferol 3-0[2-0-(6-0-£-feraloyl)-i/?-Dgrucopyranosyl]-/?-D-galactopyranoside (15) from H. diffusa collected in Taiwan along with quercetin (20), quercetin 3-0glucopyranoside (21), quercetin 3-O-sambubioside (22), quercetin 3-0-sophoroside (23) and a number of iridoid glycosides [29]. A bioassay-guided search for neuroprotective compounds from H. diffusa collected in Korea yielded a number of flavonol glycosides
1064
[30]. The same flavonol glycoside (15) was reported together with a new quercetin 3-O[2-O(6-O-£'-feruloyl)-/?-D-glucopyranosyl]-/?-Dgalactopyranoside (16), as well as with quercetin 3-O[2-O-(6-O-Efemloyl)-/^D-glucopyranosyl]-/?-D-glucopyranoside (17), kaempferol 3-O(2-0-y#-D-glucopyranosyl)-/?-D-galactopyranoside (18), quercetin 3-0(2-O-/?-D-glucopyranosyl)-/?-D-galactopyranoside (19) and iridoid glycosides. Our investigation on Malaysian H. diffusa [31] as well as H. herbacea also yielded quercetin 3-O-glucopyranoside (21) together with quercetin 3-0-rutinoside (24). From H. herbacea, we have found kaempferol 3-Orutinoside (25), kaempferol 3-0-glucoside (26), kaempferol 3-O-arabinopyranoside (27) and quercetin 3-0galactoside (28) [7]. We have also reported kaempferitrin (29) from H. verticillata [32] and isovitexin (30) from the aerial parts of H. dichotoma [33]. Table 3: Flavonoids from Hedyotis [7,29-33] Flavonoids
Species
New flavonoids (15) R2 = gal -2— g l c - ^ - feruloyl (16) R, = OH R 2 =gal-^— glc -£- feruloyl (17) R! = OH R 2 = g l c ^ — glc - ^ feruloyl (18) Ri=H R 2 =gal — • g l c (19) R, = OH R 2 =gal ^ * g l c Ri = H
R
!
K
c
i
'(B1T
% Jr lf OH
0
H. diffusa 0
Feruloyl moiety for (15), (16) and (17)
Known flavonoids (20) Quercetin; R, = OH, R 2 =H (21) Quercetin 3-O-glucopyranoside R, = OH,R 2 =glc (22) Quercetin 3-O-sambubioside Ri = OH, R 2 =xyl - ^ - g l c (23) Quercetin 3-0-sophoroside Ri = O H , R 2 = g l c - ? - g l c (24) Quercetin 3-O-rutinoside Ri = OH, R2 = rutin .
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/y H. herbacea
TY OH
0H
OR2 Q
(25) Kaempferol 3-O-rutinoside Ri=H, R 2 =rutin; (26) Kaempferol 3-0-glucoside R 1 =H,R 2 =glc (27) Kaempferol 3-O-arabinopyranoside Ri = H, R2 = arabino (28) Quercetin 3-O-galactoside R, = OH, R 2 =gal
(29) Kaempferitrin
H. verticillata ::
V
^r* ^|^ O-Rha OH 0
(30) Isovitexin
H. dichotoma GIC-O-^TT OH
Q
IRIDOIDS Besides flavonoids, iridoids are also commonly found in a number of Hedyotis species. Nishihama et al. reported the isolation of the new E isomers of 6-(9-acylscandoside methyl esters [i.e. 6-0methoxycinnamoyl (31), -coumaroyl (33) and -feruloyl (35) along with asperuloside (48)] from H. diffusa collected in Hong Kong [18]. Following this the Z isomers of the acyl group of these scandoside methyl esters were found together with the previously reported E isomers (31-36) from the plant collected in China [15]. Recent investigation on H. diffusa from Taiwan also found (36) together with asperulosidic acid methyl ester (50) [29]. Kim et al. later have reported the isolation of only the E and Z isomers of methoxycinnamoyl and coumaroyl esters (31-34) from the plant collected in Korea [30]. In both studies, flavonoid glycosides were also isolated. The first six iridoid glucosides from H. corymbosa Lam. syn Oldenlandia corymbosa originated from China were reported by
1066
Takagi et al. [34]. Following this, nine iridoid glucosides from O. corymbosa L. collected in the Philippines were reported, four of which were the acylated derivatives of some of the known ones [35]. Among these were 10-0-benzoyldeacetylasperulosidic acid methyl ester (37), 10-0-benzoylscandoside methyl ester (38), 10-0p-hydroxybenzoylscandoside methyl ester (39), and 10-O-p-transand 10-0-p-czs-coumaroylscandoside methyl esters (33) and (34), respectively. The known asperuloside (48) and deacetylasperuloside (49) as well as asperulosidic acid, scandoside methyl ester and deacetylasperulosidic acid (51-53) were also isolated. A recent study on H. tenelliflora B.L or locally known as "Xiazicao" in Yunan, China had led to the isolation of two new iridoids, teneoside A (46) and teneoside B (47) along with deacetylasperuloside (49) and scandoside methyl ester (52) [36]. From the work on H. chrysotricha, Peng et al. reported a total of 10 iridoid glycosides including the new asperulosidic acid ethyl ester (40), 6'-acetyldeacetylasperuloside (41), 6'-acetylasperuloside (42) and hedyoside (43) [8,37]. Investigation on H. hedyotidea by the same group [38] found the new deacetylasperulosidic acid ethyl ester (44) and hedyotoside (45) besides asperulosidic acid (51), asperuloside (48) and deacetylasperuloside (49). Hedyotoside was reported to be the first iridoid glycoside with three conjugated double bonds. It is also interesting to note that from only five species, a total of 17 new iridoids have been isolated from this genus (Table 4).
1067 Table 4: New Iridoids from Hedyotis [2,8,15,18,29,35,37,38] Structure and Name of Iridoid
Species
(31) R = (£)-6-O-p-Methoxycinnamoyl (32) R = (Z)-6-O-/7-Methoxycinnamoyl
COOMe
R
HOH2C
o-Glc
H. diffusa
(33)
Oldenlandoside I R = (£)-6-O-Coumaroyl (34) R = (Z)-6-O-j7-Coumaroyl
(35)
Oldenlandoside II R = (£)-6-OFeruloyl (36) R = (Z)-6-O- Feruloyl
Cinnamoyl moiety: Coumaroyl moiety: R, = OH, R 2 = H Feruloyl moiety: Ri = OH, R2 = OMe
(37) Ri = CH3, R2 = benzoyl (38) R, = benzoyl, R2 = H (39) Ri = hydroxybenzoyl, R2 = H
H. corymbosa R 2 OH 2 C
o-Glc
and (33), (34)
OH
COOEt (40) Asperulosidic acid ethyl ester
AcOH2C H. chrysotricha
O.G|C
0
o—y
CO ROH2L
0
(41)
6'-Acetyldeacetylasperuloside
(42)
6'-Acetylasperuloside R = Ac
1068
COOMe
H. chrysotricha
•r? Y° o-
7
(43)
Hedyoside
(44)
Deacetylasperulosidic acid ethyl ester R=Et Hedyotoside R = Me
O-Glc
COOR
OH
H. hedyotidea HOH2C
Y°
(45)
O-Glc
,O
ROH2C
(46)
Teneoside A R = rha
(47)
Teneoside B R = rha
O-Glc
H. tenelliflora OH
COOMe
1*
\I ROH 2 C
O-Glc
Table 5: Common Iridoids from Hedyotis [29,35,36,38]
Name oflridoid
Structure
O
o—[
(48) (49)
VT° ROH2C
Asperuloside R = Ac Deacetylasperuloside R=H
o_Glc
(50)
Asperulosidic acid methyl ester R, = Me, R 2 =Ac (51) Asperulosidic acid R, = H, R 2 =Ac,
1069
OH
(
:OOR,
vc
R2OH2C
(52) (53)
Scandoside methyl ester R, = Me,R 2 =H, Deacetylasperulosidic acid R,= R 2 =H
o-Glc
TRITERPENOIDS AND STEROLS More than 20 pentacyclic triterpenoids have been reported from this genus, mostly from H. lawsoniae and H. acutangula [20,39]. The structures of some compounds isolated from various Hedyotis species are shown in Table 6. The triterpenoids isoarborinol (54), arborinone (55) and their acetates as well as germanicol, taraxerol, erythrodiol and the new olean-12-ene-3p,28,29-triol (56) were reported from H. acutangula. Three new triterpene acids, 3/3,23dihydroxyurs-12-en-28-oic acid (59), 3/?,24-dihydrOxyurs-12-en28-oic acid (60), and 2a,3/?,24-trihydroxyurs-12-en-28-oic acid (61) together with sixteen known ones including ursolic acid, 3epiursolic acid, ursonic acid, asiatic acid, euschaphic acid, oleanolic acid, oleanonic acid, sumaresinolic acid, hederagenin, arjunolic acid, betulinic acid, betulin, 2oc-hydroxyursolic acid, 2oc-hydroxy-3epiursolic acid, 2a,3a,23-trihydroxyurs-12-en-28-oic acid and benthamic acid were reported from H. lawsoniae. The isolation of oleanolic acid (57) and ursolic acid (58) was also reported from H. corymbosa, H. auricularia and//, acutangula [22,26]. Three new triterpenoid saponins, nudicaucin A, -B and -C (62-64) have also been isolated from H. nudicaulis together with a known saponin, guaiacin D (65) [10]. The phytosterols, stigmasterol and sitosterol are frequently reported in most of the phytochemical work on this genus.
1070 Table 6: Some Triterpenoids from Hedyotis [10,20,22,39]
Species
Structure and Name of Triterpenoid
(54) Isoarborinol
\ (55) Arborinone H. acutangula
R =O
HOH2C, /
rrVcHOH
(56)
Olean-12-ene-3p,28, 29-triol
V H. lawsoniae, H. acutangula,
X X Jr COOH
(57) Oleanolic acid R = a-H j?-OH
H. corymbosa, and H. auricularia (58) Ursolicacid
f
|
PCOOH
1071 H. lawsoniae
COOH
(59)
3P,23-Dihydroxyurs12 - en-28-oic acid Ri = OH;R2=R 3 =H
(60)
3p, 24-Dihydroxyurs12 -en-28-oic acid R, = H;R2=OH, R3=H
(61)
2a,3P,24- Triihydroxy urs -12 -en-28-oic acid Ri=H, R 2 =R 3 =OH
(62) NudicaucinA Ri = gal,R2= (63) NudicaucinB Ri = glc, Ra = r
H, nudicaulis
(64) Nudicaucin C Ri = glc, R2 = rha (65) Guaiacin D R, = gal,R 2 =H
LIGNANS Only one specie, H. lawsoniae has been found to contain lignans. Novel sesquilignans named as hedyotol A, -B, -C and -D (66-69) [16] and their acetyl derivatives, (+)-pinoresinol, (+)-medioresinol, (+)-syringaresinol, (-)-dehydrodiconiferyl alcohol, (70-73) as well as the novel dilignans, hedyotisol A, -B and -C (74-76) have been isolated from this plant (Table 7) [17].
1072 1072 Table 7: Lignans and Sesquilignans from H. lawsoniae [16,17] Name
Structure
OMe HO (66) OH
(67)
Hedyotol A R=H Hedyotol B R = OMe
OMe
(68) OMe
Hedyotol C 7"=P-OH (69) Hedyotol D 7" = a-OH
OMe
(70) (71)
OMe OMe
(+) - Pinoresinol Ri = R 2 = H (+) - Medioresinol
(72)
(+) - Syringaresinol R[ = R2 = OMe
(73)
(-)-DehydrodiconifetyI alcohol
1073
<j>H
OMe
J I iL
|<*!X-J»(e0'^^\|/0 N
,J*yJ
OH
OMe
0Me
f^Y'0H
H _J 4—H
^0
OMe
(74) Hedyotisol A: 7", 8"-, 7 " \ 8 " '- erythro (75) Hedyotisol B: 7", 8"- erythro, 7 ' " , 8"'-threo (76) Hedyotisol C: 7", 8"-, 7"', 8 " '-threo
OH
OTHER COMPOUNDS Other secondary metabolites isolated from Hedyotis (Table 8) include coumarins, scopoletin and fraxin (77-78) from H. dichotoma [33] and arbutin (79), a hydroquinone monoglucoside, from//, herbacea [7]. Table 8: Other Compounds in Hedyotis [7,33] Species
Structure and Name of Compound
MeQ.
H. dichotoma
(77) Scopoletin 1
(78) Fraxin
O-Glc
H. herbacea
H0H2C
HO
T\__/0Ii
(79) Arbutin .
T OH
PHARMACOLOGICAL AND OTHER STUDIES OF CRUDE EXTRACTS
BIOACTIVITY
Most of the reports on pharmacological studies of Hedyotis species have mainly been on those used in Chinese herbal medicines (//. diffusa and //. corymbosa) as co-constituents. These studies include
1074
the effects of the medicinal extracts in reversal respiratory tract infection in children, intestinal metaplasia, atypical hyperplasia, anti-inflammatory as well as hepatoprotective activities. Bioactivity studies conducted on pure compounds isolated from a few Hedyotis species have also been reported but are quite scanty. Among these are brine shrimp lethality, antimicrobial and cytotoxicity assays, hi our own laboratory, selected bioactivity studies have also been carried out on some methanolic extracts of seven Malaysian Hedyotis species [40]. hi the early 90's, a clinical trial on "Feibao syrup", which contains H. diffusa together with Radix astragali and other medicinal plant extracts, was reported, hi this study, "Feibao syrup" was used to treat reversal respiratory tract infection in children. The clinical research proved that after taking the medicine, the general condition was improved and if the disease occurred, the symptoms were mild and the disease course was short. The efficacy of the medicine was 92.5%. Furthermore, experiment on mice indicated that the medicine could enhance the macrophage phagocytic activity and lymphocyte transformation rate [41]. Another Chinese herbal medicine, "Xiao Wei Yan Powder" (XWYP), which contains among others, H. diffusa, Smilax glabrae and Glycrrhiza uralensisis, was reported to be used to treat 138 cases of intestinal metaplasia (EVI) and 104 cases of atypical hyperplasia (AH) of the gastric mucosa of chronic gastritis. The results showed that in the treated group, the total effective rate of EVI was 91.3% and that of the AH was 92.2%, while in the control group, they were 21.3% and 14.5%, respectively, indicating that XWYP had marked therapeutic effects for both IM and AH [42]. "Peh-hue-juwa-chi-cao" is the common commercial name for the herbal extract of either of H. diffusa, H. corymbosa or Molluga penthaphylla. hi a recent investigation on the anti-inflammatory and hepatoprotective effects of these three extracts in rats, the results indicated that all three extracts possess anti-inflammatory activity and that they significantly reduced the acute elevation of serum glutamate oxalate transaminase and serum glutamate pyruvate transaminase concentrations. It also alleviated the degree of liver damage after the intraperitoneal administration of hepatotoxins [43].
1075
It is in view of these pharmacological activities, as well as the structural diversity of the compounds isolated, that we have investigated the antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of seven Malaysian Hedyotis species [40]. The species tested were H. verticillata, H. dichotoma, H. capitellata, H. nudicaulis, H. corymhosa, H. herhacea, and H. pinifolia. In some cases, different parts of the plant extracts were tested to compare the activities. The antioxidant activities were evaluated by ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods while the radical-scavenging activities were measured by the 1,1-diphenylpicrylhydrazyl (DPPH) assay. As shown in Table 9, all tested extracts were found to exhibit very strong antioxidant properties when compared to Vitamin E (atocopherol) with percent inhibition of 89-98% in the FTC and 6095% in the TBA assays. In the DPPH assay, we found that none of the extracts was a good radical scavenger. Only H. herhacea exhibited some radical scavenging activity, with an IC50 value of 32 Table 9: Comparison of absorbance values and percent inhibition of linoleic acid peroxidation as measured by the FTC and TBA antioxidant assays. Sample (0.02%, w/v)
Absorbance* (FTC)
Percent Inhibition
Absorbance* (TBA)
Percent Inhibition
H. capitellata (leaves)
0.039
98.2
0.043
97.5
H. verticillata (stems)
0.042
98.0
0.123
96.5
H .dichotoma (aerial)
0.044
98.0
0.136
95.7
H. capitellata (stems)
0.047
97.8
0.074
95.3
H. verticillata (leaves)
0.052
97.6
0.031
94.2
H. pinifolia (aerial)
0.062
97.1
0.071
93.9
H. dichotoma (roots)
0.062
97.1
0.053
92.4
H. corymbosa (aerial)
0.070
96.7
0.058
89.9
H. nudicaulis (aerial)
0.104
95.2
0.092
88.8
H. herbacea (aerial)
0.233
89.2
0.485
59.9
Vitamin E
0.500
76.7
0.550
54.5
Control
2.147
0
1.21
0
*Absorbanee reading on the 6* day (one day after control reached maximum)
1076
An antibacterial assay using the disc-diffusion method found that the stems and the roots of H. capitellata showed weak to moderate activities against the tested bacteria, Bacillus subtilis B28, Bacillus subtilis B29, Pseudomonas aeruginosa UI 60690 and methicillin resistant Staphylococcus aureus, (MRSA) while the leaves showed weak activity toward Bacillus subtilis B28, MRSA and P. aeruginosa only. However, the roots of H. dichotoma showed moderate antibacterial properties against all four bacteria. All other extracts did not exhibit any antibacterial properties. Table 10 shows the inhibition zones of the methanolic extracts of some Hedyotis species as measured by the disc-diffusion method. Table 10: Inhibition zones (mm) of methanolic extracts of Hedyotis species as measured by the disc-diffusion method. B28
B29
MRSA
UI 60690
H, capitellata (stems)
9
6.5
g
10
H. capitellata (leaves)
g
-
g
s
H. capitellata (roots)
10
7
9
11
H. dichotoma (roots)
15
12
11
13
Streptomycin (Control)
17
15
19
20
Species*
*Other extracts did not show any inhibition, (criteria used for activity: >15, strong; 10-15, moderate, <10, weak) B28: Bacillus subtilis (mutant) B29: Bacillus subtilis (wild type) MRSA: mcthicillin-resistant Staphylococcus aureus IU 60690: Pseudomonas aeruginosa
Through the MTT assay, all tested extracts were found to exhibit moderate to high cytotoxic properties against CEM-SS cell line (human T-lymphoblastic leukemia) giving CD50 values in the range of 21-45 (ig/ml. In the Griess assay to measure the potential antiinflammatory activity related to NO inhibition, only H, nudicaulis and H. capitellata (stems) were found to exhibit moderate inhibition (about 40%) at 100 u-g/ml. All other extracts did not exhibit any inhibition indicating that the constituents of the tested extracts were weak inhibitors of NO synthase.
1077
PHARMACOLOGICAL AND OTHER STUDIES ON ISOLATED COMPOUNDS
BIOACTIVITY
Up to 1990's, most works on Hedyotis were phytoehemieal in nature. Thus, not much of pharmacological data on the pure compounds isolated from Hedyotis species were reported. Hitherto, there has been a widening interest in the bioactivity of pure compounds as reported for H. chrysotricha, H. diffusa and H. nudicaulis. ANTIOXIDANT ACTIVITY In a study of the active constituents of H. diffusa [29], a new acylated flavonol glycoside, quercetin 3-0[2-0(6-0-2?-feruloyl)~/?D-glucopyranosyl]-/?-D-galactopyranoside (16), which was hydrolyzed to produce kaempferol 3-0(2-0-P-D-glucopyranosyl)^-D-galactopyranoside (18), along with three other flavonol glycosides and three known iridoid glycosides were tested for antioxidant effect using xanthine-oxidase inhibition, xanthinexanthine oxidase cytochrome c and TBA-MDA systems. Among the constituents tested, compound (18) and asperuloside (48) showed only minor anti-lipid peroxidation effect with no activities observed for the iridoid glycosides (31), (33) and (35) as well as the quercetin glycosides (22) and (23). However, (22) and (23) were found to be superoxide anion scavengers, whereas no activities were detected for the other four compounds. Recently, we have also found that quercetin monoglycosides, (20) and (23), at 100 ng/ml equivalent to 215 and 164 uM, respectively, were good inhibitors of linoleic acid peroxidation in the ferric thiocyanate assay. Both compounds were also found to be free radical scavengers in the DPPH assay as compared to vitamin C (100 p,g/ml). In our own study on H. diffusa using chemical-based assays such as the ferric thiocyanate (FTC), thiobarbituric acid (TBA), and diphenylpicrylhydrazyl (DPPH) radical-scavenging methods [31], we found that both quercetin 3-0-/?-glucopyranoside (21) and quercetin 3-O-/?-rutinoside (22) inhibited linoleic acid peroxidation and also exhibited good DPPH radical-scavenging properties (8788%) which was comparable to vitamin C (93%). We also found asperuloside to be inactive in both assays.
1078
NEUROPROTECTIVE ACTIVITY In a separate study of the active constituents of H. diffusa [30], the flavonol glycosides and iridoid glycosides were tested for their neuroprotective activity using primary cultures of rat cortical cells. The activities were evaluated by assessing the viability of cortical cells after treatment with glutamate. It was found that all the nine compounds isolated [flavonol glycosides (15-19) and iridoid glycosides (31-34)] exhibited significant neuroprotective activity in primary culture of rat cortical cells damaged by L-glutamate at 0.1 10 |JM dosages. CYTOTOXIC AND OTHER BIO ACTIVITIES One of the earliest pharmacological data reported on the constituents of H. diffusa and H. corymbosa. Asperuloside (48) and deacetylasperuloside (49) from H. diffusa and oldenlandoside (33) from H. corymbosa were reported to show various degrees of antineoplastic or antileukemic activities [44]. Chrysotricine, a J3carboline alkaloid isolated from H. chrysotricha, was found to inhibit the growth of HL-60 cells in vitro. The inhibition rates were reported to be 63% at 10 \iM [2]. However, there was no report on the other components of this plant. hi a recent study, Hsu and Tsai have studied the effect of two constituents from the extracts of "Peh-hue-juwa-chi-cao", namely oleanolic acid (OA) and ursolic acid (UA) on the apoptosis of HL60 cells in combination with a high dose of ionizing radiation. They found that the surviving HL-60 cells were decreased significantly at 12 hrs after OA or UA treatment at a dosage of more than 50 (j.g/ml. However, combining OA or UA treatment with radiation did not accelerate the death, but increased the survival of cells instead [45]. Earlier, we have reported the bioactivity of kaempferitrin and ursolic acid from H. verticillata against brine shrimp larvae. Both compounds were found to be toxic with LC„ = 21.9 ppm and 29.8 ppm, respectively [32]. The brine shrimp lethality test has been reported to be useful in predicting biological activities such as cytotoxicity, phototoxicity and pesticidal activities [46].
1079
Three new saponins, nudicaucin A, -B, and -C isolated from H. nudicaulis, showed only weak antibacterial activity against Bacillus subtilis M45 and HI 7 [10]. STRUCTURE-ACTIVITY RELATIONSHIPS (SAR) OF THE BIOACTIVE COMPOUNDS IN GENUS HEDYOTIS There have not been many reports on the structure-activity relationship (SAR) studies on the bioactive compounds from Hedyotis. However, some SAR studies on the antioxidant and the neuroprotective constituents from H. diffiisa have been reported recently. We include in this review some of our observations on the SAR studies of some active flavonoids. ANTIOXIDANT ACTIVITY In a study on the antioxidant effects of the components from H. diffusa on xanthine-xanthine oxidase cytochrome c, Lu et al. found that quercetin 3-0-sambubioside (22) and quercetin 3-0sophoroside (23) are superoxide radical scavengers. The kaempferol glycoside (18) and the iridoid glycosides having cinnamoyl or feruloyl substituents on the glucose units [(31) and (33)], as well as asperuloside (48), were found to be inactive in this assay [29]. However, in this study of the inhibitory effects of the constituents on FeCl2-ascorbic acid induced lipid peroxidation in rat liver homogenate in vitro (using TBA-MDA systems), the quercetin glycosides, (22) and (23), (at 125 jiM) were found to be inactive, while the kaempferol glycoside (18) and asperuloside at the same concentration were found to exhibit only minor activities as compared to Trolox (Vitamin E). In a related study on antioxidant activity of kaempferol methyl ether derivatives, we found that the presence of a free 3-OH in ring C is important in enhancing antioxidant activity [47]. It is, however, interesting to note that asperuloside (at 241 uM) was found to be inactive in our chemicalbased ferric tbiocyanate assay [31], but was found mildly active at 125 uM in the in vitro assay conducted by Lu et al. [29]. In this study, it was also found that the iridoid glycosides of H, diffusa containing the 2?-cinnamoyl and the iJ-coumaroyl moieties were inactive in their antioxidant assays.
1080
The findings from the structure-activity relationship studies reported thus far support the conclusion by Pieta on the role of flavonoids as antioxidants [48]. He concluded that the presence of a catechol moiety in ring B is a major determinant in radicalscavenging capability making quercetin derivatives good antioxidants. He also reported that the presence of the free 3-OH group in ring c (as in flavonols) increases their radical-scavenging activity. Thus, we can deduce that the diglycosylation at the 3position (ring c) in these compounds contributed to the reduction of their antioxidant activity. NEUROPROTECTIVE ACTIVITY In their study on neuroprotective activities using primary cultures of rat cortical cells, Kim et al. reported that the flavonoid glycosides possessing both the dihydroxyl functionality on ring B (a catechol moiety) and the acyl substituent (feruloyl group) in the sugar moiety as in structures (16) and (17) showed the strongest activity [30]. Furthermore, it was suggested that the neuroprotective activities were reduced by the loss of either feature as exemplified by structures (15) and (19). The weakest activity was displayed by compounds with neither feature as illustrated in structure (18). A catechol moiety in ring B of flavonoids (Ri=OH) as found in quercetin derivatives has been known to play a major role in enhancing antioxidant activity, thus it could possibly play a similar role in neuroprotection. R,
.OH
Flavonoid
The four iridoid glycosides isolated from H. diffusa as shown in structures (31-34) possess acyl moieties (Ri= cinnamoyl, feruloyl or coumaroyl) but they differ in the stereochemistry of the double bond as well as the presence of a p-methoxy substituent in the aromatic ring [30]. All four acylated iridoid glycosides showed significant neuroprotective activities. However, from the structureactivity study, it was suggested that the presence of a p-methoxy
1081 1081
group in the aromatic ring and a frvmy-double bond (E configuration) in the acyl moiety played a more significant neuroprotective role against glutamate-induced damage. The cinnamoyl and coumaroyl functionalities have been known to exhibit antioxidant properties in chalcones and flavanones [49]. It is therefore reasonable to suggest that the presence of cinnamoyl, coumaroyl and the feruloyl functionalities in these iridoid glycosides are the responsible moieties for the significant neuroprotective activities observed. The second structural feature corresponding to the presence of a/>-methoxy group in the aromatic ring has also been previously suggested [50]. In the work on Scrophularia buergeriana roots, the same authors had found that four phenylpropanoid esters of rhamnose isolated from this plant, all possessing/?-methoxycinnamoyl moieties, attenuated glutamateinduced neurotoxicity in a similar experiment. The third structural feature with regards to the ^-configuration in the cinnamoyl moiety was not emphasized in this study as a major factor in neuroprotection. However, this feature was also seen to have an effect on the neuroprotective activities exhibited for the isomers of 2-O-acetyl-3-O-/7-methoxycinnamoyl-a-L-rhamnopyranoside. An enhanced activity for the £-isomer was observed as compared to the Z-isomer. Thus, based on this work, the presence of the /?-methoxy group and the .^-configuration in cinnamoylated iridoid glycosides is suggested to be the two structural features responsible for enhancing neuroprotective activities. COMe (31) R = (£)-6-O-p-Methoxycinnamoyl (32) R = (Z)-6-O-p-Methoxycimamoyl HOH 2 C
Iridoid Glycoside BIOGENESIS OF ANTHRAQUINONES, IRIDOIDS AND INDOLE ALKALOIDS FOUND IN HEDYOTIS Biosynthesis of Anthraquinones There are two main biosynthetic pathways leading to anthraquinones in higher plants, the polyketide and the
1082
chorismate/o-succinylbenzoic acid pathways [51]. In the polyketide pathway, anthraquinones are biosynthesized from acetyl-CoA and malonyl-CoA via an octaketide chain. These types of anthraquinones such as emodin and chrysophanol occurring in the families Rhamnaceae, Polygonaceae and Leguminoceae are substituted in both rings A and C [52]. However, the anthraquinones in Rubiaceae are considered to be formed via the chorismate/o-succinylbenzoic acid pathway, thus are substituted only on ring C. In this type of anthraquinones, rings A and B are derived from mixed shikimate and a-ketoglutarate pathways via osuccinylbenzoate, whereas ring C is derived from mevalonic acid (MVA) via isoprene units. Exceptions are however found, especially in other genera such as Cinchona and to a lesser extent in Galium, Morinda and Coprosma [52]. Recently, it has been found that a non-MVA pathway termed as the 2-C-methyl-D-erythritol-4phosphate or MEP pathway is involved in the formation of isoprenoid unit in ring C of anthraquinones in the cell cultures of the family Rubiaceae [51]. As shown in Table 2, all 9,10anthraquinones reported for Hedyotis except for (10) are substituted only on ring C, thus typically follow the shikimate and mevalonate pathway of the rubiaceaous anthraquinones (Figure 2). The other anthraquinones are 1,4-anthraquinones which may be derived from 9,10-anthraquinone through oxidation at C-4 followed by tautomerization.
1083 Terpenoid Pathway I Shikimate Pathway
III MVA Pathway
PEP + E - 4 - P - * — -•
IV MEP Pathway
glucose
COOH
h
Shikimic acid
pyruvate
CHO OH glyceraldehyde
+ TPP
chorismic acid
CO 2 , PEP OH O
COOH + o.
SCoA
O.
OP
COOH a-ketoglutaric acid
isochorismic acid + TPP
3-hydroxy-3-methylglutaryl-CoA
1-deoxy-Dxylulose-5-P
CO 2 , PEP HQ OP
COOH
o-succinylbenzoic acid
COOH
OH
mevalonic acid
2-C-methyl-Derythritol 4-P
I,
OH
\
OH
\ COOH isopentyldiphosphate
OH 1,4-dihydroxy-2-naphtoic acid
OPP
OPP
dimethylallylphosphate
anthraquinones
Figure 2: Biosynthetic pathways leading to anthraquinones in the Rubiaceae (E-4-P: erythrose 4-phosphate; P: phosphate residue; PEP: phosphoenolpyruvate; TCA: tricarboxylic acid; TPP: thiamine diphosphate [51]).
1084
BIOSYNTHESIS OF IRIDOID GLYCOSIDES Mdoids and their glycoside derivatives are commonly found in a number of species of this genus including H, diffusa, H. hedyotidea, H. chrysotricha and H. corymbosa. The studies on iridoid glycolsides from the Rubiaceae and their distribution in the family can be traced back to the 60's. However, Inouye et al. suggested further that rubiacaeous plants could be classified into three groups or subfamilies based on his own chemotaxonomic study and others [53]. These subfamilies are: i) Ixoroideae, members of which contain gardenoside, gemposide and ixoroside; ii) Rubioideae, all of which contain asperuloside and/or deacetylasperulosidic acid; and iii) Cinchonoideae and Antirheoideae which contain loganin, secoiridoids and/or indole alkaloids biosynthesized via the latter two glucosides. In this classification, the iridoids from Hedyotis fall under the Rubioideae family, in the tribe Hedyotideae. hi their study, either by GC/GC-MS analysis or isolation of pure constituents, on thirty-five species of rubiacaeous plants, they found that asperuloside (48) and/or deacetylasperulosidic acid (53) was uniformly distributed in the subfamily Rubioideae [53], Recent work on Hedyotis plants, supported this generalization and revealed the presence of other iridoid congeners. For this subfamily, the iridoid glycosides are biosynthesized via the iridodial pathway (pathway I in Figure 3). However, for the subfamily Ixoroideae, the biosynthetic pathway is believed to be via the epi-iridodial pathway (pathway II) [54]. In addition, in this pathway, it is known that loganic acid or loganin is at the branch pointing to secologanin and indole alkaloids, on one hand, and asperuloside on the other. Based on these data, Inouye et al. depicted a biosynthetic pathway for iridoids of Rubiaceae (Figure 3) of which pathway I is suggested to be the main pathway for Hedyotideae and other tribes are in the subfamily Rubioideae.
1085 Me MVA
R= Me; loganin R= H; loganic acid
CHO
CHO COOMe
secologanin
COOH
AcO asperuloside
indole alkaloids
gardenoside
Figure 3: Biosynthetic pathway of rubiaceaous iridoid glycosides
BIOSYNTHESIS OF INDOLE ALKALOIDS The general biosynthetic pathway of indole alkaloids involves condensation of tryptamine with secologanin to afford strictosidine
1086
and vincoside [55]. Secologanin as seen in Fig. 3 was produced from loganin in the biosynthesis of rubiaceaous iridoid glycosides. However, in the case of chrysotricine isolated from H. chrysotricha, its structure suggested the union of tryptamine and linalyl oxide, a monoterpene widely distributed in plants (Figure 4), providing a viable biogenesis of indole alkaloids [2]. Similarly, isocyclocapitelline and cyclocapitelline isolated from H. capitellata can be formed in the same way [11]. Alternatively, these two compounds can be produced by a ring closure of capitelline via C2' and the hydroxyl function at C-5'.
N-R
"—-CH,
(a)
Isocyclocapitelline: R = H Isochrysotricine: R = Me
Figure 4: Proposed biosynthetic pathway for some indole alkaloids from H. capitellata (a) Putative biogenetic pathway of chrysotricine (b) Ring closure of capitelline to form isocyclocapitelline and cyclocapitelline.
CONCLUSION Until recently, more than forty new compounds have been isolated from only 13 species of the genus Hedyotis. hi this review, which is the first for the genus, we have attempted to show the highly divergent structural features found from the members of this genus as well as the biological significance of these compounds. Our results and data from others have shown that Hedyotis plants contain potent antioxidant compounds. The use of some of the
1087
plants from this genus as important constituents in Chinese herbal medicines could be attributed to these antioxidant properties which in turn could be related to the anti-inflammatory, cytotoxic and neuroprotective effects. We have also noticed that separate phytochemical studies on the methanolic fraction of H. diffusa from various parts of the world including Korea, China and Taiwan and also Malaysia yielded the same type of compounds showing, in this case, that climatic conditions did not influence the phytochemical constituents of the plant. From the information on the activities of crude extracts and pure constituents, we may deduce that the constituents of some Hedyotis species possess antioxidant, neuroprotective and cytotoxic properties. The antioxidative constituents include the quercetin derivatives which were found to be superoxide radical scavengers. The neuroprotective constituents in the genus include the active acylated iridoid glycosides (in the presence of cinnamoyl, coumaroyl and feruloyl groups) and the flavonol glycosides. Among the triterpenes, ursolic and oleanolic acids are the two most common constituents of Hedyotis and both were found to possess cytotoxic properties. Although bioactivities on the anthraquinones isolated specifically from this genus have not been reported, the antileukemic, cytotoxic, antiviral and antimicrobial properties of some anthraquinones from the same family are well documented [56-60]. It remains a question as to whether these bioactive constituents work as merely single entities or synergistically with others. The biogenetic studies on anthraquinones, iridoids and alkaloids discussed here mostly represent a general biosynthetic pathway for the Rubiaceae family. Despite the relatively large number of new compounds reported from only 13 species, the biosynthetic pathways for the new compounds of this genus are seldom elaborated. We believe that the genus Hedyotis will continue to be a source of novel bioactive compounds. REFERENCES [1]
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1090 [53]
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1091 SUBJECT INDEX A-105972 cytotoxic activity towards human cancer cell lines 726 A-289099 726 preclinical development of 726 ABT-751 731 Acetic acid 427 Acetic anhydride 427 Acetogenins 983 class of 983 8a-Acetoxydrimane-ll-oic acid 425 synthesis of 425 Acetylation 215,231,418 Ache la domesticus
115
Achiwa's methodology 558 Acid hydrolysis 220 Acrocynine 165,166 from Acronychia baueri 165 structure of 166 Acronychia baueri 165 acrocynine from 165, Acutiaporberine 165 from Thalictrum acutifolium 165 Adenosine 972 Adhesion 157 Adipokinetic effect 79 Adipokinetic hormones 81 biosynthesis of 81 from Schistocerca gregaria 81 Aedes aegypti 115 Aequorea GFP 17-21,31,32 chromophores from 5 derivatives of 31 isoforms of 18,32 properties of 21 Aequorea victoria 4,20 green-fluorescent protein (GFP)from 4 Aequorea victoria GFP 9 from Discosoma sp. 9 Aequorin 77 Agaricus bisporus 1042 Agastache rugosa 181,543 agastinol from 181 analytical methods/
identification of 543 isolation of compounds 543 Agastinol 181 from Agastache rugosa 181 Ageing 881 olive oil in 881 Aglaia cordata 543 Ailanthus altissima 442,443 against Epstein-Barr virus 442 against HIV replication 466 herbicidal effect of 471 shinjulactone L from 443 shinjulactone M from 443 shinjulactone N from 443 Ailanthus malubarica 440 in treatment of bronchitis 440 in treatment of dysentery 440 in treatment of dyspepsia 440 in treatment of opthalmia 440 in treatment of snake bites 440 AilantinolG 441 against Epstein-Barr virus 441 antitumor effects of 441 from A ilanthus altissima 441 Ajmalicine 831 AKH/RPCH families 78-80 members of 80 of neuropeptides 78 ofpeptides 80 primary structure of 80 Albicanol 408 from liverworts 408 Alcohol 213 ozonization of 213 Alder reaction 195 of 3,6-dihydro-3,5-dimethyl anisole 195 Aldose reductase inhibition 302 Alkaloids 163,180 apoptosis inhibitors of 180 Alkylant sesquiterpene lactones 322 effect of tripeptide glutathione (gly-cys-glu, GSH) 322 structure-reactivity relationships of 322 Alkylation 222
1092 1092 Allatostatins (ASTs) 105 superfamily of 104 Allocolchicine 723 tubulin binding affinity of 723 inhibitory activities against tumour cell lines of 723 Alpinia oxyphylla 169,791 yakuchinone A from 791 yakuchinone B from 791 Alzheimer's disease (AD) 160 3-Aminobenzophenone compounds 726 SAR of 726 Aminoethylphenantherene derivatives 969 Ampelopsin 604 Ampelopsin A 604 Ampelopsin E 628 antihepatotoxic activity of 628 from Ampelopsis brevipedunculata var. hancei 628 Analgesics 163 alkaloids as 163 Analgesic activity 670 of phenylethanoid glycosides 670 Andalasin A 610 from Morus macroua 610 Andalusol 532 immunomodulating properties of 532 inhibition of lymphocyte proliferation by 532 Angiogenesis 729 in chick embryo chorioallantoic membrane (CAM) assay 729 AngorosideC 701 effect on PGF2 701 Anhydrovinblastine 828 (-)-Anisatin 371 stereochemistry of 371 AnnocherineA 961 AnnocherineB 961 Annomosin A 1009 isolated from annonaceae plants 1009 Annomurilin 992 cytotoxic values (IC50) of 992
Annonaceae 957 as pantropic family 957 acetogenins from 957 diterpenoids from 957 phytochemistry research on 957 styrylpyrones from 957 Annonaceous acetogenins 975,977,978, 980,982,990-992 bullatalicin as 990 classification of 977 mechanism of action of 992 molvizarin as 990 mono-tetrahydrofuran ring in (THF) 978 mono-THF compound of 978 role of spacer moiety linking two rings in 991 stereocenters of 977 stereochemistries of ring functions of 977 stereochemistry of 991 structural features of 975 THF ring moiety of 990 with adjacent bis-THF rings 980 with non-adjacent bis-THF rings 980,982 Annonaceous alkaloids 959,973-975 Annona from 977 benzylisoquinoline as 959 bioactivities of 973 cytotoxicity of 973 Rollinia from 977 Annonebinide A 1009 from stems of Annona glabra 1009 Anopheles gambiae 76 Anthocyanin biosynthesis 835 vacuolar compartmentalization of 836 Anthraquinones 175,1062,1063,1081 1083 as chrysophanol 1082 as emodin 1082 biogenesis of 1081-1083 by synthesis based on Diels-Alder reaction 1062 from acetyl-CoA 1082
1093 1093 from Hedyotis diffusa 1062 from mevalonic acid 1082 from Rheum palmatum 175 Anti-AIDS agents 243 Antiallergic activity 670 ofphenylethanoidglycosides 670 Anti-apoptotic Bcl-2 family proteins 148 Antiapoptotic factor 161 NF-KB
161
Anti-apoptotic mechanism 155 Anti-apoptotic proteins 148,171 Mcl-1 171 Bcl-2 171 Anti-AR reagents 802 expression of 806 Anti-asthmatic drug 646 Plantago as 646 Antibacterial activities 668 against Staphylococcus aureus 668 Antibacterial activity 394 ofdrimanes 394 Antibiotic 820,959 formosan annonaceous plants as 959 terpenoid indole alkaloids (TIAs) as 820 Anticancer agent 162,163,739 alkaloids as 163 taxolusedas 739 Anticancer activity 173 oftangeretin 173 Anticancer drug 243 Tripterygium wilfordii use as 243 Anti-carcinogenic 167,891 capsaicinas 167 medicinal plants as 891 Anti-carcinogenic activity 167,623 of oligostilbenes 601 of resveratrol 167 Anti-CMH antibodies 1033 fungal growth by 1033 Anticomplemental activity 394 ofdrimanes 394 Antidote 646 Forsvthia as 646
Antifeedant activity 240.302,394,820, 1013 of clerodane hydroxybutenolides as 1013 ofdrimanes 394 of pristimerin 302 of sesquiterpenes 240 of triterpenes 240 of triterpenoid quinonemethides 240 of terpenoid indole alkaloids (TIAs) 820 Antifungal activity 206,219, 228,230, 394.771 ofcapsidiol 228 ofdrimanes 394 of luttucenin A 230 of mansonone F 219 ofphytuberin 206 of viridiofungin A 771 of viridiofungin B 771 Antifungal drugs 1045 development of 1045 Antifungal norsesquiterpene 200 rishitin as 200 Antifungal properties 224 of sesquiterpenes quinones 224 Antifungal sesquiterpene 193,218 glutinosone as 193 mansonone Fas 218 from ulmus Hollandice 218 Anti-genotoxic 167 capsaicinas 167 Antihepatotoxic activity 628 of oligostilbenes 601 Anti-HIV agent 466 Anti-HIV principle 243 Antihyperlipidemic activity 777 of descarestrictines E 777 of pangamic acid as 909 Antihypertensive agents 892 reduction of atherosclerotic complications by 892 Anti-inflammation actions 623 of oligostilbenes 601 Anti-inflammatory activity 177, 646,700, 788,891,1001
1094 1094 ofcurcumin 788 of curcumin analogs 788 of Medicinal plants as 891 ofparthenolide 177 of Plantago 646 ofPPGs 700 of Stevia rebaudiana 1001 Anti-inflammatory agent 162,176,646 Forsythia as 646 in vivo 176 in vitro 176 of phytochemicals 162 triterpenes as 176 Anti-inflammatory compounds 701 angoroside A 701 angoroside C 701 angoroside D 701 Anti-inflammatory cytokines 158 IL-10 158 TFG-P 158 Anti-inflammatory drug 155 anti-oxidant agents 155 glucocorticoids 155 NSAIDs 155 Anti-inflammatory effects 155,178 implication in 155 ofcorticoids 155 of glucocorticoids 155 oftriptolide 178 Anti-inflammatory property 162,167,170 in vivo 167 ofcapsaicin 167 of natural products 162 of yakuchinone A 170 ofyakuchinoneB 170 structure of 170 Anti-inflammatory therapies 162 antioxidants in 162 Anti-leishmanial activity 924 Antileukemic activity 473 in vivo 473 Antimalarial activity 240,462 of quassinoids 462 of sesquiterpenes 240 of triterpenes 240 of triterpenoid quinonemethides 240
Antimicrobial 878,1001,1013 clerodane hydroxybutenolides as 1013 Olea europaea as 878 Stevia rebaudiana as 1001 Antimicrobial activity 240,352.704 ofPPGs 704 ' of sesquiterpenes 240 of sesquiterpene lactones 352 of triterpenes 240,298 of triterpenoid quinonemethides 240 Antimicrobial compounds 193 against bacteria 193 against fungi 193 phytoalexins as 193 Antimicrobial drugs 1026 for fungal infections 1026 Antimicrobial property 226,546,962 ofberberine 962 of 7-hydroxycalamenene 226 of lignans Antimitotic activity 544,813 of lignans 544 of vinca alkaloids 813 Antimitotic agents 725 combretastatin A-4 725 Antioxidant 698,882 mechanism of action of 698 olive oil as 882 PPGsas 698 Anti-oxidant activity 698,791.872 ofcurcumin 791 of curcumin analogs 791 ofPPGs 698 ofverbascoside 872 Antioxidant compounds 162 andapoptosis 162 Antioxidative 878 Olea europaea as 878 Antioxidative activity 171 ofhonokiol 171 ofhumulone 171 Anti-proliferative activity 724,731,840 of 2-methoxyestradiol/NSC659853 724
1095 1095 of sulphonamides 731 of vinca alkaloids 840 Anti-rheumatic drags 165 mechanism of apoptosis induction by 165 Anti-stress 909 pangamic acid as 909 Antistress effect 670 of phenylethanoid glycosides 670 Antitrypanosomal activity 353 of sesquiterpene lactones 353 Anti-tumor 959 formosan annonaceous plants as 959 Antitumor activity 294,544,743 ofBMS-247550 743 oflignans 544 of nor-triterpene quinonemethides 294 of triterpenes 294 Antitumor agent 483 bruceantinas 483 Antitumoral effect 167,178 ofcapsaicin 167 oftriptolide 178 Anti-tumour-promoting activities 300 of triterpenes 300 Antitusive 646 Plantago asiatica as 646 Antiviral activity 678 of PPG 678 Apigenin 172 apoptotic effect of 172 Apoptogenic effects 157 of glucocorticoids 157 Apoptogenic factors 796 apoptosis by 796 Apoptosis 141-144,147,150,151,163,993 activation of 709 and inflammation 150 characteristics of 142 defective regulation in 151 disease associated with 151 induction by ultraviolet (UV) light 150 mechanism of 144,147 modulation by natural products
162 modulators of 141 natural product as activators of 163 pro-apoptotic proteins in 143 role in inflammation 141 Apoptosis proteins (IAPs) 145 in cancers 145 Apoptosis-inducing genes 144 Ced-3 144 Ced-4 144 Apoptotic cells 158 phagocytosis of 158 Apoptotic effects 162 of natural products 162 Aporphines 964,965 structure of 964,965 Arabidopsis ABC transporter gene 836 Arachidonic acid 167 from phospholipase A2 (PLA2) 167 Arachidonic acid metabolites 159 eicosatetraenoic acid (12-HETE) 159 5-HETE 159 in regulation of apoptosis 159 PGE2 159 Arachis hypogea 928 Arachis paraguariensis 928 Argentinine 969 Aristolactams 970 Aristolochic acid 970 biosynthetic pathway of 970 Aromatic hydroxylation 547 oflignans 547 2-Aroylindoles 729 against carcinoma cell lines 729 in chick embryo chorioallantoic membrane (CAM) assay 729 Artabotrys uncinatus 358 Artemisininoids 355,361 mechanism of action of 355 QSARof 361 Artherospermidine 966 from Annona cherimola 966 Artherospermidine A 966 from Fissistigma glaucescens 966
1096 Arthropod neuropeptides 69 function of 69 mode of action of 69 structure of 69 Artogomezianol 627 from Artocarpus gomezianus 627 inhibition of tyrosinase activity by 627 Arylbenzofuran 921,948 biosynthesis of 923 by palladium-catalyzed coupling of 948 cicerfuran as 923 isoflav-3-enes sub-goup of 921 role in natural resistance of 923 Aschochyta blight pathogen 919 Aschochyta rabiei 906,915,917,919,935 as phytoalexins 915 spores of 919 Ascorbic acid 883 Asiminia 957 Aspergillus niger 924 as mammalian pathogens 924 A spergillus oryzae 410 Aspergillus terreus 760 dihydromevinolin from 760 Asperuloside 1065 from Hedyotis diffusa 1065 Asperulosidic acid methyl ester 1065 Astacideans 91 crayfish as 91 Asthma 156,700 role of leukotrienes in 700 role of neutrophils in 156 Astisane diterpenoids 533 from Sideritispusilla 533 Asymmetric aldol reaction 572 Asymmetric cycloaddition reactions 577 Asymmetric Strecker reaction 567 Atherosclerosis 753 low-density lipoprotein (LDL) in 752 Atorvastatin 762 to lower total cholesterol levels 762 ATP molecules 754 phosphorylations with 754
ATP-binding-cassette (ABC) transporter 836 Auricularine 1059 from Hedyotis auricularia 1059 Autoimmune lymphoproliferative syndrome 149 Azafluorenes 971 in Polyalthia 971
Baicalein's 172 mechanism of action of 172 structure of 172 Balanocarpus heimii 602 hopeaphenol from 602 resveratrol from 602 Basophils 157 in allergic inflammations 157 Bcl-2 family proteins 148 Belamcanda chinensis 173 tectoridin from 173 tectorigenin from 173 Benzodiazepine 711 Benzoyl chloride 945 condensation of 945 Benzylation 222 Berberine 962 antimicrobial properties of 962 inhibition of reverse transcriptase by 962 Beta-carbonyl acid 875 decarboxylation of 875 Betulinaldehyde-3p-yl-caffeate 250 from Celastrus stephanotifolius 250 Betulinic acid 176 structure of 176 Betulinic aicd methyl ester 176 structure of 176 Bioactive triterpenes 239 from celastraceae 239 Biochanin A 935 production of 935 Biological activity 293 of triterpenes 293 Biological assays 73 in neuropeptide research 73
1097 1097 Biological processes 879 leukocyte/eicosanoid production 879 lipoprotein oxidation 879 platelet aggregation 879 platelet production 879 Biosynthesis 813 of vinca alkaloids 813 Bisabosqual A 772 from Stachybotrys sp. RF-7260 772 inhibitory activity against microsomal squalene synthases 772 Bisdeoxy-asperuloside 864 Blattella germanica 109 Blood (plasma) lipoproteins 752 chylomicrons as 752 families of 752 high-density lipoproteins as (HDLs) 752 low-density lipoproteins (LDLs) as 752 very low-density lipoproteins (VLDLs) as 752 Blue fluorescent proteins (BFP) 7,31 aggregation of 34 emission spectra of 33 excitation of 33 GFP derived 32 isoforms of 32 isomerization of 34 oligomerization of 34 pH of 34 photobleaching of 34 salt of 34 spectra of 32 BMS-184476 739 comparative preclinical study of 739 in L2987 lung carcinoma 739 in paclitaxel-resistant colon carcinoma 739 BMS-188797 739 comparative preclinical study of 739 in L2987 lung carcinoma 739
in paclitaxel-resistant colon carcinoma 739 BMS-247550 741,743 anti-tumour activity of 743 Bombyx mori 85 Borreverine 1061,1062 anthraquinones from 1062 structure of 1061 Botrytis cinerea 626,906,921,924 as fungicides 921 cytotoxicity against human lymphoblastoid cell (CEM) 626 germination of spores of 921 in BGM resistance 921 Botrytis cinerea 924 Botrytis grey mould (BGM) 917 Bovine aortic endothelial cells (BAECs) 806 Breast cancer 626 vatdiospyrodol C against 626 Bronchial asthma 164 Bronchial inflammation 156 eosinophils role in 156 Bronchitis 440 Ailanthus malubarica in 440 Bruccosides C 448 against human epidermoid carcinoma 448 from Brucea javanica 448 Brucea antidysenterica 466 against HIV replication 466 Brucea javanica 439,446,466 in cancer 439 in dysentery 439 in malaria 439 inhibitory activity of 466 Bruceanic acids B 445 from Brucea antidysenterica 445 Bruceanols A-H 445 from Brucea antidysenterica 445 Bruceantin 445,483 antileukemic compound 445 as antitumor agent 483 Bruceantinoside C 467 in vitro 467 Brunus grayana 867
1098 Buddleja 675 as poultice/lotion 67 in healing wounds 675 in ulcers 675 Bullatacin-induced cell death 993 Butylated hydroxytoluene (BHT) 883 Butyrolactones 549 synthesis of 549 y-Butyrolactones 554 synthesis of 554
C19 Quassinoids 438 structure of 438 C2o Quassinoids 442 structure of 442 C22 Quassinoids 461 structure of 461 Caffeic acid phenethyl ester 175 from propolis 175 Callinectes sapidus 96 Calliphora vomitoria 105 cloning from Drossophila melanogaster 108 protein-coupled receptor 108 cAMP-increasing agents 164 effects on granulocyte apoptosis 164 Canavalia brasiliensis 180 lectins from 179 Cancer 145,439.444,720,881 apoptosis proteins (IAPs) in 145 Brucea javanica in 439 cyclin-dependent protein kinases (CDKs)in 720 dynamics of tubulin polymerization in 720 hallmark of 720 olive oil in 881 Cancer borealis 82 Cancer chemotherapy 735 vinca site binding agents in 735 Cancer pagurus 75 use in enzyme-linked immunoassay (ELISA) 75
Cancer therapy 814 vinblastine in 814 vincristine in 814 Candida albicans 1038 as fungal pathogen 1038 Capitelline 1060 from Hedyotis capitellata 1060 Capsaicin 167 anti-tumoral effects of 167 Capsidiol 227 antifungal activity of 227 biological activity of 227 isolation of 227 Caragana chamlague 629 inhibitory activity on acetylcholinesterase (ACliE) 629 Caraphenols B 605 from Caragana sinica 605 Carausius morosus 81 Carex humilis 624 inhibition of COX by 624 Carnoside 876 chemical modification of 876 Carrageenan-induced inflammation 167 (-)Carvone 210 alkylation of 210 Carvone 211 transformation of 211 Caspases 156 in eosmophil apoptosis 156 Caspase-8 145 Caspase-9 145 Caspase-induced apoptosis 145 of cancer cells 145 Cassine 240 Cat's claw 181 an herbal medicine 181 from Amazon 181 to treat inflammatory disorders 181 Catalytic asymmetric method 550 Catharanthine 824 biosynthesis of 824 Catharanthus alkaloids 837 cytotoxic to cells 837 effect on microtubule-dependent processes 837
1099 mechanism of action of 837 mitosis arrest by 837 Ced-9 144 as antagonist 144 Celasdin-A 247,248 from Celastrus hindsii 247 structures of 248 Celasdin-B 247,248 from Celastrus hindsii 247 structure of 248 Celasdin-C 247,248 from Celastrus hindsii 247 structure of 248 Celastolide 244 from Tripterygium hypoglaucum 244 structure of 244 Celastraceae family 240 chemotaxonomy of 240 Celastrol 268 from Pristimeria indica 268 from Celastrus scandens 268 from Tripterygium wilfordii 268 structures of 268 Celastrus 240 Cell differentiation 708 induction of 708 Cell growth 883 in breast 883 in colon 883 in skin cancer 883 Cellular kinase complex 160 as IKK 160 Central neuromodulators 120 sulfakinins as 120 Cepharanthine 165 matrix metalloproteinase 9 production on 165 Ceramide monohexosides (CMHs) 10261033 analysis of 1028 as bioactive components from fungal cells 1030 cellular distribution of 1033 from Cryptococcus neoformans 1027 from Histoplasma capsulatum
1027 from Magnaporthe grisea 1027 from Paracoccidiodes brasiliensis 1027 from Pseudallesheria boydii 1027 in aspergillosis 1032 in fungal cells 1026 induced accumulation of antimicrobial compounds 1030 purification by chromatographic methods 1028 serological reactivity of 1032 thin layer chromatography 1029 Ceratocystis ulmi 225 against Cladosporium cucumerinum 225 Cerebrosides 1026 as neutral glycosphingolipids 1026 ceramide monohexosides (CMHs) as 1026 in Saccharomyces cerevisiae 1026 Cerivastatin 762 to lower total cholesterol levels 762 Chalconaringenin 935 Chalcones 944 conversion of 944 Chamissonolide 343 on gene transcription profiles 343 Chang liver cells 778 for screening of cholesterol biosynthesis inhibitors 778 Chaparrin 474 antineoplasic activity of 474 from Castela species 474 Chaparrinone 464,479 against Plasmodium falciparum 464 from Hannoa klaineana 479 Chelidonium majus 833 chelidonic acid from 833 Chemoprevention 800 Chemopreventive activity 624,800 ofcurcumin 800 of curcumin analogs 800 of natural products 624
1100 Chemopreventive agent 800 epigallocatechin gallate as 800 Chemopreventive potency 802 of dibenzoylmethane 802 Chemotaxis 157 Chemotherapy-induced apoptosis 169 curcumin inhibitory effect on 169 Cherinonaine 971 cHH peptide family 103 sub-grouping of 103 cHH peptide family 90 Chickpea kairomone 929 Chilean propolis 543 Chiral butyrolactones 549 diastereoselective alkylation of 549 Chiral oxazolidinones 570 uses of 570 Chloramphenicol acetyl transferase 63 Cholecystokinin 119 Cholesterol 751 in central/peripheral nervous systems 752 Cholestesterol biosynthesis 757 against hypercholesterolemia 757 effect of hydroxymethylglutarylCoA (HMG-CoA) reductase inhibitors 757 inhibitors of 757 Cholesterol biosynthesis inhibitors 751 bile acids as 752 biosynthetic pathway of 753-756 microbial origin of 751 screening by using cell lines 776 steroid hormones as 752 structure of 752 vitamin D as 752 Cholesterol esters 893 Chondrocyte apoptosis 153,154 Chromic anhydride oxidation 417 diacetate 417 Chromophore maturation 9 Chromophores 5 from A equorea GFP 5
from RenillaGY? 5 Chromoproteins 8 nomenclature for 8
Chronic inflammations 154 in atopic dermatitis 154 in psoriasis 154 Chronic inflammatory diseases 157 neutrophils in 157 Chronic inflammatory synovitis 154 Chronic tissue eosinophilia 156 Cicer arietimum 907,913,928-935 acetaldehyde in 929 aliphatic hydrocarbons in 928 analysis of 907 butanal in 929 cadaverine from 932 citric acid from 907 coumarins from 933 deadienal in 929 decenal in 929 formaldehyde in 929 GC-MS analysis of 928 germinated cotyledons of 935 malic acid from 907 malonic acid from 907 nonenal in 929 octanal in 929 octanone in 929 octenal in 929 oxalic acid from 907 oxaloacetic acid from 907 fumaric acid from 907 pentanal in 929 polyamines from 932 saccharides from 932 scopoletin from 933 spermidine from 932 spermine from 932 succinic acid from 907 umbelliferone from 933 volatile component of 928.930 Cicer bijugum 920 effect of Botrytis cincerea 920 resistance to Botrytis grey mould (BGM) 920 Cicer flavonoids 934 biosynthesis of 934 Cicer pinnatifidum 940 2-arylbenzofuran from 940
1101 Cichoralexin 231 biological activity of 231 from Cichorium intybus 231 isolation of 231 Cichorium intybus 231 cichoralexin from 231 Cinnamic acids 909,911 benzoic acid from 911 by phenylalanine ammonia lyase (PAL) 909 derivatives of 909 gallic acid from 911 gentisic acid from 911 a-resorcyclic acid from 911 with caffeic add 909 Cistanche deserticola Y.C. 646 in cold sensation 646 in female sterility 646 in male impotentcy 646 used for staminal tonic 646 Cistanche plants 650 HPLC chromatogram of 650 Cistanche salsa 646 CJ-13,981 774 as squalene synthase inhibitor 774 structure of 774 CJ-13,982 as squalene synthase inhibitor 774 structure of 774 CJ-15,183 773 antifungal activities against filamentous fungi/yeast 773 from Aspergillus acuhatus 773 structure of 773 Cladosporium cucumerinum 225 ceratocystis ulmi against 225 Clerodane 1010 isolation from Polyalthia sp, 1010 Clerodane hydroxybutenolides 1013 as antifeedant 1013 as antimicrobial 1013 Cloned CFPs 38 alignment of 38 amino-acid sequences of 38 Cloned green fluorescent proteins 20
Cloned naturally occurring GFPs 23,24 amino-acid sequences of 23,24 Cloned red fluorescent proteins (RFPs) 48,52 amino-acid sequences of 50 emission spectra of 52 typical excitation 52 Clover 916 in nodule primordial 916 on colonization by vesiculararbusucular mycorrhizal (VAM) 916 CNS cancers 728 diarylindoles against 728 CNS-active peptides 892 atrial natriuretic factor as 892 bradykininas 892 enkephalins as 892 substance P as 892 CoASH 328 in primary cell metabolism 328 COBRA-0 733 COBRA-1 733 Coenzyme A (CoASH) 326 Colchicine 721 analogues of 721 chemical structure of 721 Colchicine binding site 733 miscellaneous compounds affecting 733 Colchicine-site binding tubulin inhibitors 722 family of 722 Colchicum autumnale 722 colchicinoides in 722 colchicine from 722 Coleus forskohlii 424 offorskolin 424 Colletotrichum lindemuthianum 920 infection with 920 Colon cancer 626 vatdiospyrodol C against 626 Colonocyte apoptosis 160 TNF-a effect on 160 via caspase-3-independent mechanism 160
1102 Combretastatins 722,727 structures of 727 Combretastatin A-4 725 activity towards multi-drugresistant (MDR) cell lines 725 as antimitotic agent 725 as tubulin inhibitor 725 synthesis of 725 Compactin 758 from Penicillium brevicompactum 758 Convulsant sesquiterpene lactones 373 structure-activity relationships of 373
Coptisjaponica 836 Cornigerine 722 from Colchicum comigerum 722 Cornoside 861,865 enzymatic hydrolysis of 866 from Forsythia genus 866 Cornoside rearrangements 876 Corpora cardiaca 79 Locust adipokinetic hormone I (Locmi-AKH-I) from 79 Corticoids 155 anti-inflammatory effects of 155 Coumestan 944 from Cicer anatolicum 922 from Cicer canariensis 922 from Cicer chorassanicum 922 from Cicer nuristanicum 922 from Cicer oxyodon 922 from Cicer pungens 922 from Cicer reticulatum 922 from Cicer yamashilaz 922 intramolecular Heck reaction of 944 isoflav-3-enes sub-goup of 921 palladium catalyzed cyclization of 944 Coumestrol 944,945 intramolecular Heck reaction of 944 palladium catalyzed cyclization of 944 synthesis of 945
COX-2 169 TPA-induced expression of 169 COX-2 expression 156 role in pathological processes 156 COX-2 inhibitors 159.701 arenarioside as 701 forsythoside as 701 PGE2 production in 159 verbascoside as 701 Croton oil-induced mouse ear edema 167 Crustacean cardioactive peptide 111 Crustacean hyperglycaemic hormone (cHH) 75 90 Crustacean neuropeptides 77 Cryptococcus neoformans 1041 ceramide-(phosphorylinositol)2mannose in 1041 ceramide-phosphorylinositol mannose in 1041 tandem mass spectrometry of 1041 Cryptolepine 165 from Cryptolepis sanguinolenta 165 Cryptolepis sanguinolenta 165 cryptolepine from 165 neocryptolepine from 165 Cryptophycins 736 as macrolide antimitotic agents 736 Cryptophycin A 736 block cells at mitosis 736 Culex salinarius 115 Culture filtrate proteins (CFPs) 7 from Mycobacterium tuberculosis 1 Curcuma longa 168 anti-inflammatory activity of 788 anti-oxidant activity of 791 antioxidant mechanism of 796 as anti-prostate cancer 802 biological activities of 785,787 biology of 787 chemopreventive activity of 800 Curcumin 785 cyclovalone from 790 effect on mitochondrial
1103 permeability transition pore (FTP) 796 from Curcuma longa 785 mechanisms of action of 787 natural analogs of 788 sites of action of 788 structure activity relationships of 785,787
structures of 786 synthetic routes to 787 therapeutic effects of 786 Curcumin analogs 785 activity in carrageenin-induced rat paw edema 789 activity in cotton pellet granuloma assays 789 anti-inflammatory activity of 788 anti-oxidant activity of 791 as anti-prostate cancer 802 biological activities of 785,787 bisdemethoxycurcumin as 785 chemopreventive activity of 800 demethoxycurcumin as 785 for treatment of inflammation/ sprain 785 from Curcuma longa 785 inbioassays 793 inhibition of lipid peroxidation by 794 structure activity relationships of 785 structures of 786,796,798,799 synthesized for anti-AR assay 803 synthetic routes to 787 therapeutic effects of 786 Curcuniin-induced cell apoptosis 169 byROS 169 Cyan fluorescent proteins (CFPs) 36,41,63 GFP-derived 36 spectra of 36 Cyanation reaction 198 Cyclic AMP phosphodiesterase 669 phenylethanoid glycosides effects on 669
Cyclin-dependent protein kinases (CDKs) 720 in cancer 720 Cyclin-kinase inhibitors 177 Cyclization 13,14,22 Cyclocapitelline 1060 from Hedyotis capitellata 1060 Cyclohexanone 790 Cyclooxygenase-2 156 glucocorticoids effects on 156 Cyclopentanone 791 Cyclovalone 791 analogs of 791 Cydia pamonella 109 a-Cyperone 207 oxygenation of 207 Cyphostemma bainesii 619 oligostHbenes from 601 Cysteinyl leukotrienes 157 inflammatory mediator from 157 Cytochrome P450 800 oxidation cycle of 884 oxidative reactions of 885 Cytokines 157 glucocorticoids effects on 158 inflammatory mediator from 157 Cytotoxic activity 240,394 ofdrimanes 394 of sesquiterpenes 240 of triterpenes 240 of triterpenoid quinonemethides 240 Cytotoxic anti-tumor activity 626 ofoligostilbenes 601 Cytotoxic cardenolides 264 against KB cell line 264 from Crossopetalum gaumeri 264 Cytotoxicity 710 ofverbascoside 710
D-24851 729 against ASPC-1 pancreatic cancer cells 729 against human tumour cell lines 729 against SKOV3 ovarian cancer
1104 1104 729 against U87 glioblastoma 729 Daidzein 914 Dammarane triterpenes 261,263 from Maytenus macrocarpa 261,263 Dammaranes 261-263 chemistry of 261-263 Deamination 547,959 oflignans 547 Decarboxylation 195,196 Decarestrictines 776 Decarestrictine A 776 inhibition effects of 776 Decarestrictine B 776 inhibition effects of 776 Decarestrictine C 776 inhibition effects of 776 Decarestrictine D 776 hypolipidemic activity of 776 inhibition effects of 776 Dehydration 13,14,215,863,971 5,6-Dehydro-7-oxoisodrimenin 421,422 from Porella cordeana 421,422 6a,7-Dehydroaporphines 967 Dehydrogenation 12 ofGln66 12 Demethyloleuropein aglycon 871 Deoxygenation 220 Descarestrictines E 777 antihyperlipidemic activities of 777 purification of 777 structure elucidation of 777 Descarestrictines M 777 antihyperlipidemic activities of 777 purification of 777 structure elucidation of 777 Desilylation 218 Destabilisation 879 of lipid mosaic 879 Diabetes 302,418 oxidation of 418 Salacia oblonga in 302 Diarylindoles 728 against CNS cancers 728
against non-small cell lung 728 as heterocombretastatins 728 cytotoxicity towards leukaemia 728 Diastereoselective conjugate addition 563 to 2(5/f)-furanones 563 Diazotization 220 Dibenzocyclooctadiene lignans 546 schinzandrin B as 546 Dibenzoylmethane 801 chemopreventive potency of 801 Dibenzyl-butan(diol)es 542 structure of 542 Dibenzylbutyrolactones 570 asymmetric synthesis of 570 Diels-Alder reaction 219,265 Dienes 197 from aldehydes 197 Dienone 198 reduction of 198 Diglycosylation 913 Dihydroartemisinin 358 structure-activity relationships of 358 Dihydrocompactin 759 Dihydromevinolin 759,760 isolation from Aspergillus terreus 760 Dihydromonacolin L 759 6',7"-Dihydro-scutionin aB 290 structure of 290 (+)-14f3,15p-Dihydroxyklaineanone 484 total synthesis of 484 Dimeric e«f-kaurane diterpenoids 1009 from bulbs of Fhtillaria thunbergii 1009 (-)-a-Dimethylretrodendrin 578 Dioclea grandiflora 180 Dioclea violacea 180 1,1-Diphenylpicrylhdrazyl (DPPH) 1075 (+)-Discodermolide 742 from Discodermia dissoluta 242 Dispermoquinone 277 structure of 277 Diterpenoids 494 candicandiol as 494
1105 1105 candidiol as 494 dehydroabietane as 494 discovery of 494 epicandicandiol as 494 epoxysideritriol as 494 epoxysiderol as 494 isofoliolas 495 isosidol as 495 isolinearol as 495 linearol as 495 sideripol as 494 sideritriol as 494 siderone as 494 sideroxol as 494 Ditylechnus dipsaci 916 Diuretic 646 Forsythia as 646 Plantago asiatica as 646 Diuretic actions 891 of medicinal plants 891 DMPC liposomes 879 interaction with oleuropein 879 Doastatins 737 as cytotoxic cyclic pentapeptides 737 from Dolabella auricularia 737 in advanced solid tumours 737 Dopamine 959 Doxetaxel 736 DPE counteraction of cytotoxicity 886 drFP583 9 from Discosoma sp. 9 Drim-5,8-dien-7-one 414-,415 synthesis of 414,415 (+)-Drim-8-en-ll-oicacid 424 synthesis of 424 Drim-8-en-7-one 414-,415 synthesis of 414,415 Drim-9(ll)-en-8a-ol 410-413 synthesis of 410-413 Drimane sesquiterpenoids 393 synthetic investigations in field 393 Drimane-8a,ll-diol 406-410 synthesis of 406-410 Drimanes 394 antibacterial activity of 394
anticonTplemental activity of 394 antifeedant activity of 394 antifungal activity of 394 cytotoxic activity of 394 partial syntheses of 394 plant growth activity of 394 Drimanic dialdehyde 396 warburganal 396 Drimanic sesquiterpenoid 404 synthesis of 404 Drimenin 420 oxidation of 420 (-)-Drimenol 395 synthesis of 395 use of 395 Drimenol synthesis 403 from larixol 403 Drimys winferi 395-405 drimenol from 395-405 (-)-drimenol from 393 Drosophila melanogaster 76,85,88,380 sequence of Rdl protein from 380 DsRED 11,51,55,56 aggregation of 55 folding/maturation of 51 oligomerization of 55 pHof 56 salt of 56 structure of 11 temperature of 56 DsRED chromophore formation 14 mechanism for 14 DsRED fluorescence 57 pH-dependence of 57 stability of 57 DsRED maturation 53,54 pH-dependence of 54 temperature dependence of 53 DsRED protein 55 monomeric form of 55 Dysentery 440 Ailanthus malubarica in 440 Bruceajavanica in 439 Dyspepsia 440 Ailanthus malubarica in 440
1106 1106 E7010 731 E7070 723 as antimitotic agent 723 tumour regression by 732 Ecdysteroids 629 as insect steroid hormones 629 in moulding/metamorphosis 629 Eicosanoids 158,700 role of 158 Electrospray ioruzation (ESI) mass spectrometry 80 Elemanolides 317 Eleutherobin 741,743 from Eleutherobia sp. 743 from Sarcodictyan roseum 743 Emodin 175 structure of 175 (+)-Enantiomer 225 from Eremophila drummondii 225 Endoelicitors 193 Endogenous alkaloids 834 Entacmaea quadricolor 1 Enterolactone 557 asymmetric synthesis of 557 enantiomers of 557 synthesis of 557 (-)-Enterolactone 576 asymmetric synthesis of 576 iM-Halimane diterpenoid 1010 isolation from Polyalthia sp. 1010 isolation from Polyalthia longifolia var. pendulla 1010 isW-Kaurane diterpenoid 1001 biological activities of 1001 from Annona species 1001 from Rollina species 1001 NMR spectrum of 1001 Enzyme-linked immunoassay (ELISA) 75 use in Cancer pagurus 75 Enzymes 815 transport mechanism of 815 clinical uses of 816 Eosmophil apoptosis 156 mechanism of 156 role of caspasesin 156
Epigallocatechin gallate 800 as chemopreventive agent 800 in colon cancer 800 phase I clinical trial of 800 structure of 175 EpothiloneB 741,743 from Sorungium cellulosum 743 Epoxidation 948 ofstilbenes 948 Eremophila drummondii 225 Eremophilanolides 317 Esters 197 oxidation of 197 treatment of 197 Etoposide 545 in testicular carcinoma 545 Eudesmanolides 317 (-)-Eudesmin 565 synthesis of 565 Euonymus sp. 240 Euonymus tingens 272 tingenone from 272 Euphorbia fischeriana 178 jolkinolide B from 178 Extra-virgin olive oils 877 phenolic antioxidants in 877 uses of 877
Famesol 179 proapoptotic effects of 179 Farnesyl pyrophosphate (FPP) 754 Far-red fluorescent proteins 50 spectra of 50 Fas signaling pathway 146 O-3 fatty acids 894 lowering of blood pressure by 894 improvement of lipids by 894 reduction of cardiovascular disease risk factors by 894 Ferula ceratophylla 395 drimenolfrom 395-405 Fevers 464 Honnaa klaineana against 464 Fictitious green fluorescent protein 9 nomenclature from 9 Fissicesine 969
1107 Fissistigama glaucescens 957 Flavones 731 structure of 731 Flavonoids 171,892,927,1063 anti-inflammatory property of 171 antioxidative properly of 171 biological activities of 927 inhibitory effect on NEP 892 Flavonols 925 flavonols kaempferol from 926 from glycosides 925 isorhamnetin from 926 phenylchroman skeleton in 925 FLRFamides 118 Fluorescent protein 3,8,16,57,59,61 basics of 9 biological function of 57 p-ean-structure of 11 ehromophore structures of 16 definitions of 9 focus on 3 from Vibrio fischeri (YFP) 7 genes encoding 3 historical overview of 3 limiting factors of 61 nomenclature for 7,8 problems of 61 properties of 9 usage of 59 Fluorescent timer 17 Fluvastatin 762 lowering of total cholesterol levels 762 FMRFamide-related peptide superfamily 117 FMRFamides 118 Food-derived compounds 886 Formal total synthesis 198 ofglutinosone 198 Formononetin 914,935 Formosan annonaceous plants 957 as anti-tumor agents 959 as antibiotic agents 959 as muscle relaxant agents 959 nitrogen-containing constituents of 959 proaporphines in 963
L-tyrosine from 959 research on 957 Forskolm-stimulated activity 123 inhibition of 123 Forsythia 646 anti-inflammatory agent from 646 as antidote 646 as diuretic 646 use as health tea for colds 646 Forsythia koreana 646 Forsythia suspensa 646 Forsythia viridissima 646 Friedelanes 242 chemistry of 242-249 from Celastraceae 242 Friedel-Craft acylation 223 Friedel-Craft-like cyclization 570 Friedelin 242 structure of 242 (+)-Fuegin 419 from Drimys winteri Forst 419 Fumaria capreolata 834 Fungal cells 1045-1047 biosynthesis of glycosphingolipids in 1045 Fungal cerebrosides 1030 as antigenic molecules 1030 in cell growth/differentiation 1030 Fungal CMHs 1033 membrane components of 1033 Fungal glycosphingolipids 1025 functional aspects of 1025 structural aspect of 1025 Fungal monohexosylceramide 1026 functional elucidation of 1026 Fungal sphingolipid synthesis 1045 inhibitors of 1045 Furofurans 542 structure of 542 2(5#)-Furanones 563 diastereoselective conjugate addition to 563 Fusarium oxysporum 906 Fusarium oxysporium f. sp. batata 933 Fusicoccum amygdati 1027 Fusrium oxysporium L. sp. cieeri 919
1108 1108 GABAA antagonistic convulsants 371 GABA-antagonistic sesquiterpene lactones 370 inhibiting action of yaminobutyric acid (GABA) 370 mechanism of action of 370 Galactose 1027 Gallic acid 174,175,884 antioxidative activity of 174 apoptotic effect of 175 in human liver microsomes 884 Gastrin 119 Genistein 173 as anti-inflammatory agent 173 inhibitory effect of 173 mechanism of action of 173 structure of 174 Genus deer L. 905 aliphatic acids from 907 chemistry of 905 exudaton of 907 flavonoids from 912 isoflavones from 913 isoflavanones from 913 isoflavonoid glycosides from 914 miscellaneous compounds from 932 phenylpropanoids from 909 polyamines from 932 Genus HedyotislOldenlandia 1057 alkaloids from 1059 anthraquinones from 1059 antioxidant activity of 1077 as febrifuge 1057 as poultice 1057 as stomachic 1057 as tonic 1057 bioactivity studies on compounds from 1073 borreverine from 1059 P-carboline alkaloids from 1059 coumarins from 1059 cytotoxic study of 1078 family Rubiaceae of 1057 flavonoids from 1059 glycosides from 1059 indolefrom 1059
iridoidsfrom 1059 isoborreverine from 1059 isolated compounds of 1077 lignansfrom 1059,1071 medicinal uses of 1057 MTT assay of 1076 neuroprotective activity-of 1078. 1080 pharmacological activities of 1057 phytochemical studies of 1057 plants in 1057 role against glutamate-induced damage 1081 saponinsfrom 1059 secondary metabolites from 1073 sterols from 1059 to treat broken bones 1057 to treat bruises/wounds 1057 to treat diarrhea 1057 to treat dysentery 1057 to treat lumbago 1057 to treat rheumatism 1057 triterpenoids from 1059 Genus Sideritis 493 chemical investigations on 493 chemistry of secondary metabolites from 494 diterpenoids from 493 essential oils from 493 flavonoids from 493 Geranyl pyrophosphate (GPP) 754 Germacranolides 317 GFP 10,11,18 from Aequorea victoria 18 from Renilla reniformis 18 maturation of 10 structure of 11 GFP chromophore formation 13 mechanism for 13 GFP-chromophore 17 GFPI-gene 8 from Aequorea victoria 8 GFP-like proteins 10 Gingerol 170 anti-inflammatory activity of 170 chemopreventive potential of 170 from Zingiber officinalis 170
1109 1109 Ginsenosides 177 Ginsenoside Rg3 177 from Panax ginseng 177 structure of 177 Glaucarubolone 482 synthesis of 482 Gln66 12 dehydrogenation of 12 Glucocorticoid effects 156 on proliferation 156 Glucocorticoid receptor antagonist 156 mifepristone 156 Glucosamine 1034 Glucose 1027 3-0-Glucosides 919,927 occurrence of 919.927 Glucosylation 867 Glutathione reductase 917 Glutathione-S-transferase (GST) 326 cytotoxicity of 330 in formation of STL-GSH adducts 326 Glutinanes 256 chemistry of 256 Glutinosone 193,194 isolation of 193 biological activity of 193 formula of 194 chemistry of 194 (±)-Glutinosone 195 total synthesis of 195 Glutinosone 198 formal total synthesis of 198 Glycoinositolphosphoryl ceramides (GIPCs) 1035,1036,1040-1043 derived oligosaccharides 1041 ESI-MSof 1040 from higher fungi 1042 from Histoplasma capsulatum 1040 from mycelial forms of 1041 from pathogenic fungi 1036 from yeast 1041 in Amanita virosa 1043 in Lentinus edodes 1043 MS/CID of 1040
nuclear magnetic resonance spectroscopy (NMR) of 1040 Glycolipids 1026 Glycosphingolipids (GSLs) 1025. 1028.1051 amphipathic molecules of 1025 ceramide (jV-acylsphingosine) lipid moiety in 1025 from primitive class of phycomycetes 1025 in fungi 1025 in yeast cells 1051 Glycosylation 867 in natural products 867 Glycosylceramides 1050 phytosphingosine by 1050 Glycrrhiza uralensisis 1074 to treat atypical hyperplasia (AH) 1074 to treat intestinal metaplasia 1074 Glycyrrhizic acid 174 effect on LOX 174 effect on COX 174 Glyptopetolide 260,261 from Glyptopetalum sclerocarpum 260.261 Gneafricanin A 610 from Gnetum africanum 610 GnemonolA/C 609 from Gnetum gnemon 609 Gnemonoside B/K 607 GnetifolinC 608 oxidation of 608 GnetuhaininD 610 from Gnetum hainanense 610 GnetuhaininK 610 from Gnetum hainanense 611 Gnetuhainin R 608 from Gnetum hainanense 608 Gnetuhainin S 609 Gnetulin 608 Gnetum africanum 610 oligostilbenes from 601 Gnetum gnemon 609 oligostilbenes from 601 Gnetum hainanense 608 gnetuhainin R from 608
1110 1110 Gnetum parvifolium 609,623 oligostilbenes from 601,609 Gnetupendin D 607 from Gnetum species 607 Gold-labelled vitellogenin 99 endocytotic uptake of 99 Gomisin 558 asymmetric synthesis of 558 Gomisin A 559 synthesis of 559 metabolite D of 559 Goniothalamin 1013 from Goniothalamus 1013 Goniothalamin epoxide 1013 Goniothalamus amuyon 957,1013 GPI-anchors 1034 role of 1034 cleavage by PI-PLC 1034 Green fluoresent protein (GFP) 4,7,17. 28.29,31,40 aggregation of 29.40 excitation spectrum of 4 chemical structure of 4 from A equorea victoria 4 isomerization of 40 oligomerization of 29,40 pH of 30 pH-optima of 30 photoisomerization of 28,40 salt of 30 temperature of 31 Grignard reaction 195 Groundnuts 910 from Arachis paraguariensis 910 GSH addition 326 GST inhibitor 326 Guaianolides 317 Guaianolide type STLs 364 from Thopisa species 364 Gutolactone 459 from Simaba guianensis 459
Halleridone 861 Hannoa chlorantha 453,464 against malaria 464 used in traditional medicine 453
Heart ailment 881 olive oil in 881 Hedyocapitelline 1060 from Hedyotis capitellata var. moths 1060 Hedyocapitine 1060 from Hedyotis capitellata var. moths 1060 Hedyotis acutangula 1069 arborinone from 1069 germanicol from 1069 taraxerol from 1069 Hedyotis auricularia 1059 alkaloids from 1059 auricularine from 1059 Hedyotis capitellata 1057,1059.1076 activities against Bacillus subtilis B28 1076 activities against Bacillus subtilis B29 1076 activities against Pseudomonas aeruginosa UI 60690 1076 alkaloids from 1059 antibacterial assay of 1076 for post-partum treatment 1058 isochrysotricine from 1060 to treat kidney ailment 1058 Hedyotis chryostricha 1059,1060 alkaloids from 1059 asperulosidic acid ethyl ester from 1066 P-carboline alkaloid from 1060 chrysotricine from 1060 dataof'H/ 1 3 C-NMR 1060 structure of 1060 X-ray diffraction analysis of 1060 Hedyotis corymbosa 1058 for bronchitis 1058 for cancer 1058 for hemorrhoids 1058 for other diseases 1058 for rectal cancer 1058 for snake bites 1058 for swellings 1058 for tonsillitis 1058 for ulcerations 1058 iridoid glucosides from 1065.1066
1111 Hedyotis dichotoma 1062,1073,1076 antibacterial properties against bacteria 1076 fraxinfrom 1073 scopoletin from 1073 Hedyotis diffusa 1058,1079 antioxidant effects of 1079 capsules of 1058 flavonol glycosides from 1063 for bronchitis 1058 for cancer 1058 for hemorrhoids 1058 for rectal cancer 1058 for snake bites 1058 for swellings 1058 for tonsillitis 1058 for ulcerations 1058 inhibitory effect of 1079 neuroprotective compounds from 1063 role against glutamate-induced damage 1081 Hedyotis hedyotidea 1066 asperuloside from 1066 deacetylasperuloside from 1066 hedyotoside from 1066 investigation of 1066 Hedyotis herbacea 1062 Hedyotis lawsoniae 1069 arborinone from 1069 benthamic acid from 1069 germanicol from 1069 taraxerol from 1069 Hedyotis nudicaulis 1069 guaiacin D from 1069 nudicaucin A from 1069 nudicaucin B from 1069 nudicaucin C from 1069 Hedyotis tenelliflora 1066 teneoside A from 1066 teneoside B from 1066 Helicoverpa armigera 906 Heliothis virescens 924 Heliothiszea 928 maysin against 928 Helmchen 571 auxiliary of 571
Helminthosporium carbonum 918.919 Hemiquinone glucoside 861 HemsleyanolD 619 against methicillin-resistant Staphylococcus aureus 619 Hepatic microsomial cytochromes P450 884 Hepatoprotective activity 240 of sesquiterpenes 240 of triterpenes 240 of triterpenoid quinonemethides 240 Hepatoprotective lignans 543 from Saurus chinensis 543 sauchinone as 543 HepG2 778 Herbicidal compounds 471 ailanthone 471 Herbivores 241 triterpenes against 241 Heterocombretastatins 725 inhibition of tubulin polymerization by 725 Hexahydronaphthalene ring system 760 Hippocratea 240 Hippocratea volubilis 265 Histoplasma capsulatum 1036 histoplasmosis by 1036 inositolphosphosphingolipids from 1036 HIV infection 149 HIV replication 466 Brucea antidysenterica against 466 HIV-1 integrase 798 binding site of 798 Hodgkin's disease 816 vincristine in treatment of 816 Homarus vulgaris 110 Honnao klaineana 464 against fevers 464 Honokiol 171 antioxidative activity of 171 from Magnolia officinalis 171 structure of 171
1112 1112 Hopea odorata 602 hopeaphenol from 602 resveratrol from 602 Hopeaphenol 602 from Balanocarpus heimii 602 from Hopea odorata 602 Hordeum vulgara 836 Hormoconis resinae 778 inhibitory activity of 778 Human epithelial carcinoma 711 Human immunodeficiency virus (HIV) 465 causative agent debilitating disease 465 a-Humulene 179 structure of 179 Humulone 171 antioxidative activity of 171 from Humulus lupulus 171 structure of 171 Humulus lupulus 171 humulone from 171 (-)7-Hydoxycalamenene 225 biological activity of 225 isolation of 225 Hydrazinocurcuminoids 806 Hydrogen peroxide 711 Hydrolysis 195,214,412 7-Hydroxycalamenene 226 antimicrobial properties of 226 14-Hydroxychaparrinone 464 against P-388 cells 464 2'-Hydroxyformononetin 938 occurrence of 938 Hydroxyketone 214 from 7?-carvone 214 Hydroxyphenstatin 726 (+)-Hydroxysamin 574 asymmetric synthesis of 574 6ot-Hydroxytriptocalline A 245 Hydroxy-tyrosol 886 Hydroxy-tyrosol glucosides 866 Hypercholesterolemia 757,762 cholestesterol biosynthesis against 757 treatment of 762 Hyperchromatic effect 15
Hyperlipaemic 79 Hypertrehalosaemic hormone (HrTH)-l 81 Hypodiol 251 structure of 251 Hypoglycaemia 92 Hypolipidemic activity 776 ofdecarestrictinesD 776 Hypotensive activity 878
Oka europaea as 878 Hypoxanthine/xanthine oxidase model 882 to generate reactive oxygen species 882
IDN-5109 741 tumour regression by 741 Iguesterine 269,271 from Maytenus canariensis 271 structures of 269 IKK inhibition 161 IKK-a 160 IKK-P 160 Immune deficiency syndrome (AIDS) 465 Immune regulation 700 leukotrienes in 700 Immunocytochemistry 92 Immunodeficiency syndrome 149 Immunological skin diseases 153 keratinocyte apoptosis in 153 Immunomodulating properties 532 ofandalusol 532 Immunomodulatory activity 163 by xanthine theophylline 163 Indanone 232 Indoles 727 Indole alkaloid 972,1081 biosynthesis of 1085 cheritamine as 972 perlolidine as 972 Inducible transcription factors 160 in protecting cells 160 mediators of inflammatory
1113 response 160 NF-KB
160
Inflammation 141,150,156,700 and apoptosis 147,150 leukotrienes in 700 role of cells in 156 use of glucocorticoids in 151 Inflammation reaction 151 macrophages in 151 Inflammatory cells apoptosis 154 in rheumatoid arthritis 154 prevention of 154 Inflammatory cytokines 158,164 GM-CSF 164 EL-5 164 Inflammatory diseases 163 natural products in 163 Inflammatory disorders 181 use of cat's claw 181 Inflammatory response 155 apoptosis in 155 Inositolphosphoceramides (IPCs) 1036 classes of 1036 Inositolphosphoryl ceramide-type sphingolipids 1038 isolation from hyphal form of Candida albicans 1038 Insect NPF family 121 Insecticidal activity 240,301 of sesquiterpenes 240 oftriterpenes 240,301 of triterpenoid quinonemethides 240 Insecticides 630 Insoligosaccharide 1042 NMR spectroscopy of 1042 Interleukin-ip (IL-ip) converting enzyme 144-148 caspase activation mechanism of 144-148 I-Oleyltyrosol 873 structure of 873 spectroscopic methods for 873 Ion-transporting peptide 102 stimulation of reabsorption of ions/water 102 Ion-trap model 832
IPCI gene 1047 modulation of virulence traits of Candida neoformans 1047 Iridoids 1081 biosynthesis of 1084 Iris clarkei 630 action of 630 Isobrucein-B 468 from Brucea antidysenterica 468 Isochrysotricine 1060 from Hedyotis capitellata 1059 Isoflavones 946 by oxidative rearrangement of 946 single step synthesis of 946 Isoflavonids 913,944 palladium catalyzed cyclization of 944 intramolecular Heck reaction of 944 Isoiguesterine 269 structures of 269 Isolation 543,645 of Agastache rugosa 543 of phenylethanoid glycosides 645 Isomerization 46,54 of red-fluorescent protein drFP583 54 of yellow fluorescent proteins (YFPs) 42-47 Isopentenyl pyrophosphate (IPP) 754 Isoprenes 822 synthesis of 822 Isoquinolines 959 aporphines alkaloid as 960 aristolactam alkaloid as 960 benzylisoquinoline as 959 cherianoine as 961 doryphornine as 961 from oxidation of benzylisoquinolines 961 tebamine alkaloid as 960 phenanthrene alkaloid as 960 proaporhine alkaloid as 960 thalifoline as 961 Isovitexin 1064 from Hedyotis dichotoma 1064
1114 IKB
160 phosphorylation of 160
Jasus lalandii 75,91 JERE (jasmonate- and elicitor-responsive element) 830 JolkinolideB 178 from Euphorbia fischeriana 178 structure of 178 Judaicin 921,924 activity of 924 from Achillea pratensis 921 from Artemisia judaica 921 from Artemisia taurica 921 from A rtemisia vulgar is 921 from Cicer judaicum 921 larval feeding by Helicoverpa armigera 924
Kadsurin 558 asymmetric synthesis of 558 (-)-Kadsurin 560 synthesis of 560 Kaempferitin 1064 from Hedyotis verticillata 1064 Kaempferol 172,1063,1064 apoptotic effect of 172 isolation of 1063,1064 from Hedyotis dijfusa 1063 Keratinocyte apoptosis 153 in skin diseases 153 Ketal 195 Wittig reaction of 195-200 Kokoona zeylanica 246,278 celastrahydride from 246 zeylasterone from 278 Kotalagenin 16-acetate 249 structure of 249
Labdane diterpene 1010 from Polyalthia sp. 1010 Lactobacillus plantarum 897,898
Lactone 210,551 epoxidation of 210 saponification of 551 Lathyrus 926 flavonoids in 926 Lecithin-cholesterol acyltransferase (LCAT) 895 Lectins 179 from Canavalia brasiliensis 180 immunostimulatory properties of 179 use of 179 Lectin-treated Fusarium 1035 Leguminosae 905 sub-family Papilionoideae of 905 Lens esculenta 932 Leptinotarsa decemlineata 121 LettuceninA 229 antifungal activity of 229 biological activity of 229 isolation of 229 Leucophaea maderae 89 Leukoblasts 816 Leukotrienes 700 in asthma 700 in immune regulation 700 in inflammation 700 Leukotriene B 4 (LTB4) receptor 160 Leurocristine 816 Libinia emarginata 101 Lignans 541,542,548,881 asymmetric synthesis of 548 structure of 541 synthesis of 541 use as folk remedies 542 (+)-Lintetralin 569 synthesis of 569 Lipids 752 triglycerides by 752 Lipid peroxidation 671 inhibitory effect on 671 Lipoxin A4 (LXA4) 160 Liver cirrhosis 704 Liver diseases 628 Ampelopsis brevipedunculata in 628
1115 1115 Vitaceaeous in 628 Vitis coignetiae in 62S Liver function test 738 Liver injury 241,629 byCCl 4 629 by N-acetyl-P-aminophenol (APAP) 629 oleanolic acids against 241 ursolic against 241 Liver metastases 738 Locust adipokinetic hormone I (LocmiAKH-I) 79 from Corpora cardiaca 79 primary structures of 80 Locusta migratorla 79
LongusolA 621 from Cyperus longus 621 LongusolB 621 from Cyperus longus 621 Longusol C 621 from Cyperus longus 621 LongusoneA 621 from Cyperus longus 621 Lovastatin 758 inhibitory activities against HMGCoAreductase 758 structure of 758 Low-density lipoprotein (LDL) 752 in atherosclerosis 753 5-LOX 159,160 expression of 160 in vivo inhibitor of 159 inhibition of 160 12-LQX 159 in prostate cancer cells 159 5-LOX activity 159 LOX inhibitors 159,171 nordihydroguaiaretic acid (NDGA) 171 5-LOX inhibitor 172 caffeic acid as 172 12-LOX inhibitors 171 15-LOX-l 159 in prostate cancer cells 159 15-LOX-2 159 inhibitory role for 159
LOX-derived eicosanoids 160 lipoxins (LXs) 160 Lubimin 228 Luciferase 63 Lupane caffeoyl esters 250 from Celastrus stephanatifolius 250 from Hippocratea volubilis 250 Lupane triterpenes 249-250 chemistry of 249-250 Lupeol caffeate 250 from Hippocratea volubilis 250 Lupinus albus 932 Lupinus polyphyllus 834
LuttuceninA 230 against Bipolaris lecrsiae 230 antifungal activity of 230 synthesis of 230,231
Maackiain 924 activity of 924 larval feeding by Helicoverpa armigera 924 Macrobrachium potiuna 85 Macrocarpin A 296 structures of 296 Macrocarpin A acetate 296 structures of 296 Macrocarpin B 296 structures of 296 Macrocarpin C 296 structures of 296 Macrocarpin D 296 structures of 296 Macrolactins A/F 775 structures of 775 Macrolide polyether 734 Magnolia offwinalis 171 honokiol from 171 Malaria 355,439,464 Bruceajavanica in 439 by Plasmodium falciparum 355 Hannoa chlorantha against 464 MalibatolA/B 626 inhibit cytotoxicity to host cell (CEMSS) 626
1116 1116 Mandibular organ-inhibiting hormone 100 in brachyuran crabs 100 Manduca sexta 84 Mansonone A 221 Mansonone C 221 Mansonone D 221 Mansonone E 218 as phytoallexin sesquiterpene 218 Mansonone E 221 Mansonone F 218,219,221,223 antifungal activity of 219 biological acvitiy of 218 formal total synthesis of 221 isolation of 218 structure of 223 synthesis of 219 Mansonone G 221 Mast cells 157 inflammatory mediators from 157 Maturation 26,33 factors affecting 26,33 Mature protein 27,34,40 factors affecting 40 Maysin 928 against Heliothis zea 928 production of 928 Maytenfolone-A 247,248 strucutres of 248 Maytenus 240 Maytenus amazonica 295 in treatment of rheumatism 295 Maytenus aquifolium 242 Maytenus blepharodes 289 Maytenus chuchuhuasca 286 Maytenus macrocarpa 295 in folk medicine 295 Maytenus magellanica 289 Maytenus seutioides 297 Maytenus aquifolium 242 Medicagol 922 from Cicer anatolicum 922 from Cicer canariensis 922 from Cicer chorassanicum 922 from Cicer nuristanicum 922 from Cicer oxyodon 922 from Cicer pungens 922
from Cicer reticulatum 922 from Cicer yamashilae 922 Medicinal plants 891 as anti-carcinogenic 891 as anti-inflammatory 891 as spasmolytic 891 vessel diuretic actions of 891 stabilizing actions of 891 Melanin 627,1048 in pigmentation 627 Melanization/reddish colouration hormones (MRCHs) 123 Mesophyll cells 835 2-Methoxyestradiol/NSC-659853 724 anti-proliferative effects of 724 as cytotoxic human metabolite 724 hormone dependent/independent breast cancer cells 724 Methoxyfluoromethylphenylacetic acid (MPIA) ester (Mosher esters) 977 Methoxylation 702 (-)-Methylpiperitol 568 Mevastatin 758 inhibitory activities against HMGCoAreductase 758 structure of 758 Michael addition 564 of lithiated dithianes 564 Michael addition-aldol reaction 565 Microtubule associated proteins (MAPs) 720 Microtubule inhibitor 544 podophyllotoxin as 544 Mifepristone 156 as glucocorticoid receptor antagonist 156 Mitochondria 142 Mitsunobu reaction 217 Molecular biological techniques 76 Molecular electrostatic potentials (MEPs) 361 self-organizing maps 361 Molluga penthaphylla 1074 as anti-inflammatory 1074 hepatoprotective effects of 1074
1117 1117 to reduce serum glutamate pyruvate transaminase 1074 Monacolin J 759 MonacolinL 759 Monacolin M 759 Monacolin X 759 Monobutyrolactones 553,549 asymmetric synthesis of 553 deprotonation of 549 Monohexosylceramides 1027 Monosaccharides 1029 Mono-tetrahydrofuran (THF) ring 975 Monounsaturated fatty acids (MUFA) 893 Morphine 833 from Papaver latex vacuoles 833 Morus macroua 610 oligostilbenes from 601 Moult-inhibiting hormone 96 from cephalothoracic gland 96 Multidrug resistance (MRD) 843 Multidrug resistance protein 836 Multifloranes 256 chemistry of 256 Musca domestica 89 Muscle activity 110 by neuropeptide families 110 Muscle relaxant 959 formosan annonaceous plants as 959 Mutant isoforms 44 naturally occurring 44 Mutations 12 effects of 12 Mycoglycolipid 1036 Myoactive peptide family 113-115 primary structures of 113-115 Myokinin family 115 Myricetin 172 apoptotic effect of 172
Na (sodium) 891 elimination of 891 in urinary flux 891 roleofANP 891
Natural curcuminoids 792 pro-oxidant effects of 792 Natural products 624 chemistry of 860 chemopreventive activities of 624 Naturally occurring CFPs 37 Nei's genetic distance analysis 939 Neochanin 914 Neocryptolepine 165 from Cryptolepis sanguinolenta 165 Nephrops norvegicus 99 Netzahaulcoyene 271 obtentionof 271 Neuroendocrine complexes 71.72 diagrams of 72 in decapod crustaceans 72 in insects 71 Neuroendocrine system 71,72 in crustaceans 72 in insects 71,72 Neuropeptide 75 in crustaceans 77 in insects 77 localization of 75 neurosecretion of 71 quantification of 75 Neuropeptide families 110 muscle activity by 110 Neuropeptide research 73 biological assays in 73 methods used in 73 Neuropeptide structure 74 elucidation of 74 Neuroprotective effect 671 phenylethanoid glycosides effect on 671 Neurosecretion 71 of neuropeptides 71 Neurospora crassa 1036 Neutral proteases 157 inflammatory mediator from 157 Neutropenia 738 Neutrophil activation 157 effect of glucocorticoids on 157 Neutrophil functions 157 Nezara viridula 81
1118 NF-KB
343
in regulation of cell homeostasis/ apoptosis 343 NF-KB activation 161
inhibition of 161 resveratrol inhibitor of 167 N F - K B effects 161
NH2-terminal kinase (INK) activity 157 in eosinophils 157 Nitric oxide (NO) 161,890 activity of 890 apoptosis induction 161 as cytotoxic molecule 161 effect on Leishmania major 890 effect on Plasmodium falciparum 890 effect on Schistosoma mansoni 890 in inflammatory conditions 161 inhibition of 703 mechanism of 161 role in apoptosis 161 NMRtube 875 enzymatic hydrolysis in 875 Non-steroidal anti-inflammatoiy drugs (NSAIDs) 158,161,162 anti-inflammatory mechanism of 158 effect on NO 161 role of 158 Non-competitive inhibitors 775 of rat liver squalene synthase 775 Non-cyclized glutamine (Gin1) residue 91 Norcoclaurine synthase 959 condensation of dopamine by 959 Novel arylbenzofuran analogues 949 i^-oxidation products 965 ofaporphines 965 NPY superfamily 121 in porcine brain 121
Oldenlanda corymhosa 1066 asperulosidic acid from 1066 iridoid glucosides from 1066
Oka europaea 859 as antimicrobial agent 878 as antioxidant agent 878 as hypotensive agent 878 as vasodilatator agent 878 biological activity of 877 chemistry of 859 clincial assay of 893 flavonoidsin 871 history of 860 hydroxy-tyrosol glucosides in 867 molecular composition of 862 molecular modification of 873 Oleuropeic acid from 871 Oleuropeic saccharose from 871 pharmacology of 877 phenolic esters from 873 phenolic fraction of 871 verbascoside from 872 Oleanes 250-255 chemistry of 250-255 Oleanolic acids 241 against liver injury 241 Oleuropein 862,863,880,896,897 against Enterococcusfaecalis 896 against Lactobacillus plantarum 896 against Lactobacillus plantarum 897 against Pseudomanasfragi 896 against Staphylococcus carnosus 896 antimycoplasmal activity of 897 as antioxidants against lipid peroxidation 880 as chemo-taxonomic marker 865 as secoiridoid 862 chemical modification of 874 free radical-scavenging capacity of 880
partial synthesis of 863 related compounds of 868-871 structure of 862 Oleuropein aglycon rearrangements 875 Oleuropein related compounds 868-871
1119 1119 Oligomerization 29,40 of GFP 29,40 Oligostilbenes 601 absolute configuration of 617 against methicillin-resistant Staphylococcus aureus 619 ampelopsis D as 605 amurensins B-F/J as 611 amuresin C 604 amuresinD 604 amuresin E 604 amuresin F 604 amuresin H 604 amuresin L 604 angiopreissin A as 604 antibacterial activities of 619 anti-carcinogenic activities of 623 antifungal activities of 610 antihepatotoxic activity of 628 anti-inflammation actions of 623 2-aryl-2,3-dihydrobenzofuran ring of 603 as group of polyphenolic compounds 601 biogenesis of 611-614 biological activities of 619-630 biomimetic pathways of 630 by polymerization 603 cassigarol A as 609 cassigarol D as 609 cassigarol E as 609 catalysis by metal 631 catalysis by oxidase 631 classification of 603-610 conformations of 611-614 containing benzo-oxygen heterocyclic ring 609 coupling reaction using silver oxide 634 coupling reaction with acid 636 cyclo [5,3,0] decase bicyclo [6,3,0] undecane ring system of 605 cytotoxic anti-tumor activity of 626 distribution of 611 from Artocarpus gomezianus 627 from Belamcanda chinensis 607
from Caragana sinica 624 from Cyphostemma bainesii 619 from Gnetum africanum 610 from Gnetum gnemon 609 from Gnetum gnemonoides 606 from Gnetum hainanense 607-611 from Gnetum pravifolium 609 from Morus macroua 610 from Polygonum cuspidatum 607 from resveratrol 601 from Shorea hemsleyana 619 from Vitis amurennsis 604 gnemonol L as 605 gnemonoside H/F as 607 gnetifolins C/D as 607 gnetuhainin R 608 groups of 602 hemsleyanoside F as 606 in MTT assay 625 inhibition of TNFa by 626 inhibition of tyrosinase activity by 627 inhibitory activities on LTC4/D4 enzymes 623 inhibitory activity on acetylcholinesterase (AChE) 629 IR characteristics of 617 maximol B as 605 mechanism of coupling reaction of 633 MS characteristics of 616 multi-faceted biological activities of 601 naturally occurring 601 13 C-NMR characteristics of 614616 2D-NMR spectroscopic methods tosudy 608 'H-NMRof 614-616 other biological activities of 629 oxa-cyclic rings in 603 oxygen heterocyclic ring in 609 photochemical transformation of 635 photooxidative coupling reaction using UV 635 polymerization by other
1120 monostilbene units 610 polymerization from piceatanol 608 polymerization from resveratrol monomers as 605 protective effects of 621 role of e-viniferin 601 screening by filter paper disc method (FPDM) 619 shegansu B as 607 SOD like activities of 622 spectral characters of 614 structure-activity relationship of 622 to cure apoptosis 621 to cure oxidative cell injuries 621 UV characteristics of 617 with benzofuran ring 604 X-ray crystallographic analysis of 618 Olive 861 fats in 861 fragmentation's phase of 874 glyceridesin 861 lipids in 861 phenols in 861 Olive components 889 effect on blood pressure control 889 effect on glycaemic state 889 Olive oil 881,882 as antioxidant 882 in ageing 881 in cancer 881 in heart ailment 881 to reduce oxidative stress 881 Opthalmia 440 Ailanthus malubarica in 440 ORCAs (octadecanoid responsive Catharanthus AP2-domain proteins) 830 accumulation of 831 Orconectes limosus 92 biological activity of 92 Organic acids 908 asdisruptors 908 Orophea enneandra 543
Orphan receptor 76 Osmanthus asiaticus 867 (+)-Osmundalactone 1015 Osteoarthritic cartilage 153 Osteoarthritis 154 Owenia cepiodora 262 Oxazolidinones 556 use of 556 Oxidation 13,14,197,204.222,271. 418,420,608 ofdiacetate 418 ofdrimenin 420 of esters 197 ofgnetifolinC 608 Oxidative burst 157 Oxidative stress-related diseases 160 Oxidosqualene cyclase 753 Oxindole 181 Oxoaporphines 967,971 structure of 967 Oxodicarboxylate 196 7-Oxoisodrimenin 420,422 dehydrogenation of 422 from Porella cordeana 420 7-Oxoquinonemethidedispermoquinone 276 from Maytenns dispermus 276 Oxylubimin 228 Ozonization 210 Ozonolysis 401,402
p38 MAPK activity 157 in eosinophils 157 regulation of 157 Paclitaxel 724 Paclitaxel prodrugs 741 Paclitaxel-induced apoptosis 177 of breast cancer cells 177 Palaemon adspersus 84 Panax ginseng \11 senoside Rg3 from 177 Panbo-RPCH 80 primary structures of 80 Pandalus borealis 79 Pandahis jordani 87
1121 1121 Pangamic acid 909 as anti-hyperlipidemic 909 as anti-stress 909 Panicum milliaceum 933 Panulirus interruptus 82 Papaver latex vacuoles 833 morphine from 833 Paracoccidioides brasiliensis 1040 analyses of 1040 Paradol 170 structure of 170 Parthenium hysterophorus 350 allergenic principle of 350 Parthenolide 177 anti-inflammatory activity of 177 from Tanacetum parthenium 111 structure of 178 Parvifol A 609 from Gnetum pravifolium 609 Pathogens 241 triterpenes against 241 PDH/DF family 86,88 members of 88 primary structure of 88 PDH/PDF families 78 of neuropeptides 78 PDHs 89 functions of 89 Pedicularis sp. 675 in collapse 675 in exhaustion 675 Penaeus japonicus 91 Penicillium citrinum 758 inhibitor of HMG-CoA reductase from 758 Pentacyclic alkaloids 181 Peperomins 575 asymmetric synthesis of 575 Peripheral oedema 738 Periplaneta americana 79 Peroxisomes 753 PGE 887 againt NSAIDs-induced gastric ulcer 887 effects on gastric mucosa 887 PGE2 production 159 inhibition of 159
PGF 2a 887 in synthesis of protective mucus 887 P-glycoprotein 843 in tumor 843 overexpression of 843 Phagocytosis 157 Phaseolus vulgaris L. 906,920 phytoalexin in 920 Phenolics 166,871 Phenolic triterpenoids 278 Phenstatin 726,727 inhibition of cancer cell growth by 726 Phenylethanoids 703 scavenging activity of 703 Phenylethanoid glycosides 645-654,659, 660,661,662,665.666.669.670 analgesic activity of 670 antiallergic activity of 670 antihypertensive activity of 669 antistress effect of 670 bioactivities of 645 echinacoside 647 cistanoside A-E 647 cistanoside G,H 647 crenatoside 647 from datanche plants 649 from Cistanche deserticola 640 from Cistanche phelypaea 640, 647 from Cistanche salsa 647 from Cistanche sinensis 647 from Cistanche tubulosa 640 from Cistanchis herba 649 from Forsythia plants 645,661, 663 from Forsythia suspense 661 from Forsythia viridissitna 661 from Plantago asiatica 665 from Plantago depressa 665 from Plantago lanceolata 665 from Plantago major 665 from Plantago ovata 665 from Plantago plants 663,665 from Plantago psyllium 666 HPLC analysis of 649
1122 1122 inhibitory effect on 5-lipoxygenase 669 isoationof 647,661,665 jionosideD 647 MS spectra of 661 13 C-NMR chemical shifts 660 sinensideA-B 647 spectroscopic analysis of 659 SSI-MS of 654 structures of 648 structure elucidation of 649,662,666 Phenylpropanoids 909,910 and crop resistance 910 from shikimic acid pathway 909 Phenylpropanoid glycosides 675,676 chemical structures of 676 pharmacological activities of 675 Pheromone biosynthesis-activating neuropeptides (PBANs) 123 Phosphodiesterase isoenzyme inhibition 163 by xanthine theophylline 163 Phospholipase C 1034 Phospholipids 893 Phospholipomannan (PLM) 1038 from Candida albicans 1038 role in pathogenesis 1038 structure of 1038 Phosphorylation 160 oflKB 160 Phosphorylinositol-containing sphingolipids 1035 in fungi 1035 in parasite nematode^scara suum 1035 in plants 1035 in protozoan 1035 Photobleaching 28,46,54 of yellow fluorescent proteins (YFPs) 42-47 of red-fluorescent protein drFP583 54
ofRenillaGFP 28 Photoisomerization 28,40 of GFP 28,40
Phylum arthropoda 69 subphyla of 69 Phytoalexins 193 Phytoalexin sesquiterpenes 228 lubimin 228 oxylubimin 228 Phytoceramide formation 1047 in fungi 1047 Phytoglycolipids 1036 in seeds of corn 1036 in soybean 1036 Phytuberin 205,207-218 biological activity of 205 isolation of 205 synthesis of 207 Phytuberin 214 from a-santonin 214 (-)-Phytuberin 209 synthesis of 209 Picrasma crenata 455 in diabetes mellitus 455 in gastric disturbance 455 in hypertension 455 use in traditional medicine 455 Picrocarpans medicarpin 935 production of 935 Picrodendranes 370 as toxic principles of Picrodendranes sp. 370 in inhibition of y-aminobutyric acid(GABA) 370 mechanism of action of 370 Picrotoxane 371 computer-assisted studies of 371 structure-activity relationships of 371 Picrotoxane type convulsants 371 Piperolactam A 970 Piperolactam C 970 Pisum sativum 906 Pitavastatin 762 to lower total cholesterol levels 762 Plant growth activity 394 of drimanes 394 Plant physiology 677 phenylpropanoids role in 677
1123 Plantago 646 as anti-asthmatic drag 646 as anti-inflammatory 646 as diuretic 646 Plantago asiatica 646 asantitusive 646 as diuretic 646 Plantago depressa 646 Plantago lanceolata L. 646 Plantago major L. 646 Plasmodium falciparum 355,464 chaparrinone against 464 malaria by 355 Plutella xylostella 934 lactonesfrom 934 mortality of diamondback moth 934 Pod borer 908 feeding behaviour of 908 Podophyllin 544 pharmacological activity of 544 (-)-Podophyllotoxm 581,591,592 synthesis of 581 total synthesis of 592 Polonovski reaction 828 5(i?)-/5 (S)-Polyandranes 480 from Castela polyandra 480 from Castela texana 480 synthesis of 480 Polyandranes 481 total synthesis of 481 Polygonum hydropiper 395 drimenol from 395-405 Polygonum hydropiper L 400 Polylongine 972 Polymerase chain reaction (PCR) 63 Polymerization 615 of resveratrol in dihydrobenzofuran moiety 615 Polymorphonuclear (PMN) leukocytes 152 Polyphenol 174 properties of 174 Polyunsaturated fatty acids (PUFA) 894 porcine brain 121 NPY superfamily in 121
Porella 395 drimenol from 395-405 PPG repairing DNA adducts 707 activity of 707 mechanism of action of 707 PPGs 698,700,704,705 against gram-positive bacteria 704 against gram-negative bacteria 704 anti-inflammatory activity of 700 antioxidant activity of 698 antimicrobial activity of 704 antitumoral-chemopreventive activities of 705 in vitro scavenging activity of 698 to repair of DNA adducts 705 Pratensein 914 Pravastatin 761,762 microbial transformation of 761 structure of 762 Pristimerin 268 from Celastrus scandens 268 from Pristimeria indica 268 from Tripterygium wilfordii 268 structures of 268 Pristimerin-related compounds 269 from Maytenus scutioides 269 Proangiogenic genes 806 of matrix metalloproteinase 806 Pro-apoptotic 148,161 NF-KB
161
Proapoptotic agents 165 mechanism of 165 Pro-apoptotic proteins 143,171 role in regulation of apoptosis 143 Procambarus bouvieri 97 Procambarus clarkii 97 Proctolin 110 Prostacyclins 700 Prostaglandins (PG) 700,887,888 biosynthesis of 888 effects on gastric mucosa 887 Prostate cancer 159,802 androgen receptor (AR) in 802 use of hydroxyflutamide 802
1124 Prostate cancer cells 159 12-LOXin 159 15-LOX-lin 159 Protease 798 KB (IKB) proteins 160 as cytoplasmic inhibitors 160 Protein folding 26,33,39,51 factors affecting 26 Protein G affinity chromatography 1031 Protein kinase C (PKC) 624 effect on 624 Protein kinases 150 Protein phosphatases 150 Prothoracicotropic hormone (PTTH) 104 synthesis in neurosecretory cells 104 Protoberberines 959 aporphines alkaloid as 960 aristolactam alkaloid as 960 benzylisoquinoline as 959 litebamine alkaloid as 960 proaporhine alkaloid as 960 phenanthrene alkaloid as 960 Pseudomonas cepacia 555 Pseudomonasfluorescens 555,912 Pseudomonas lachrymans 205 Pterocarpans 917 as maackianin 917 as phytoalexin 918 biosynthesis of 917 synthesis of 947 Pusillatriol 496 partial synthesis of 496 Pyridine 427 Pyrokinin family 123 Pyrolysis 220
Quassinoids 433-436,444.449-460,462 473-485 antimalarial activity of 462 biogenesis of 435,436 biological activity of 433,462 fromAilanthus vilmoriniana 444 from Caste la peninsularis 449 from Castela polyandra 451 from Castela texana 450
from Castela tortuosa 450 from Eurycoma harmandiana 452 from Eurycoma longifolia jack 452 from Hannoa chloraniha 453 from Hannoa klaineana 454 from Picrasma ailanthoides Planchon 454 from Picrasma crenata 455 from Picrasma javanica 456 from Quassia amara 457 from Quassia indica 458 from Simaba guianensis 459 from Simaba multiflora 458 from Simaba orinocencis 459 from Soulamea amara 460 from Soulamea fraxinifolia 460 general features of 434,435 structural diversity of 433 structure of 437 synthetic studies of 433.473 total synthesis of 475 Quassinoid glycosides 446,457 from Brucea javanica Merr 446 from Picrasma javanica 457 Quassinoid shinjulactone C 482 total synthesis of 482 Quercetin 172 apoptotic effect of 172 structure of 172 Quinazolones 731 structure of 731 Quinolines 722 Quinolones 730,731 cytotoxicity towards human tumour cell lines 730 in P-gp-expressing vincristine resistant HeLa/KB-VIN cell lines 730 in ovarian cancer IA9 730 structure of 731 Quinonemethide 242,301 biosynthesis of 242 insecticidal activity of 301 structure of 242
1125 1125 Radioreceptor assay 123 Radix astragali 1074 to treat respiratory tract infection 1074 Rat aortic smooth muscle cells 711 Rathepatoma 711 Recombinant DsRED isoforms 49 properties of 49 Recombinant isoforms 48 Red fluorescent protein (DsRed) 12,47 chromophore maturation of 12 DsRED member of 47 from Discosoma coral 12 Red-fluorescent protein drFP583 54 photobleaching of 54 isomerization of 54 Red pigment-concentrating hormone (RPCH) 75 Reduction 198 ofdienone 198,221,224 of tetralone Regelone 246,247 from Tripterygium wilfordii var. regelii 246 structure of 247 RegeolA 280 structure of 280 RegeolB 243,280 from Tripterygium wilfordii var. regelii 243 structure of 280 RegeolC 279,280 structure of 279,280 Renilla GFP 5,28 chromophores from 5 photobleaching of 28 Resveratrol 167,602 anti-carcinogenic activity of 167 from Balanocarpus heimii 602 from Hopea odorata 602 Reverse transcriptase 798 Rheum palmatum 175 anthraquinones from 175 Rheumatism 295 uses of Maytenus amazonica 295 Rheumatoid arthritis 154,243 inflammatory cells apoptosis
in 154 Tripterygium wilfordii in 243 Rhizopus stolonifer 933 Ricciocarpus natans 867 Rishitin 200-202 biological activity of 200 isolation of 200 structure of 200,201 total synthesis of 202 Rosuvastatin 762 to lower total cholesterol levels 762 Rotunol 207 oxidation of 207 RPR-109881A 740 in phase III 740 RPR112378 733 as destabilizing tubulin inhibitor 733 from Ottelia alismoides 733 RPR115781 733 as destabilizing tubulin inhibitor 733 from Ottelia alismoides 733 Rubiaceae family 1059 Coffeea 1059 Cinchona 1059 Gardenia 1059
Saccharose 872 hydrolysis interglycosidic linkage of 872 structure of 872 Salacia 240 Salacia campestris 242 Salacia kraussii 271 against bilharziasis 299 against dysentery 299 Salacia madagascariensis 271 isoiguesterine from 271 Salacia oblonga 249,302 in Ayurvedic system 249 use in treatment of diabetes 302 Salidroside 868 isolation of 868
1126 1126 Samaderine A 437 from Samadera indica 437 (+)-Samin 583 asymmetric synthesis of 583 Samin type lignan 585 asymmetric synthesis of 585 Sanguinarine 164,165 antioxidant properties of 164 as anti-inflammatory agent 164 effect on human epidermoid carcinoma cells 164 structure of 165 Santonin 217 metal hydride reduction of 217 Scavenging activity 703 of phenylethanoids 703 Schisandra chinensis 543 Schistocerca gregaria 79,81 adipokinetic hormones from 81 Schizandra arisanensis 543 dibenzocyclooctadiene from 543 Schizandrin 558 asymmetric synthesis of 558 (+)-Schizandrin 560 synthesis of 560 Sclerotium rolfsii 909 as soil borne disease 909 Scrophularia buergeriana 1081 neuroprotective activities of 1081 Scrophularia genus 675 used in skin inflammatory ailments 675 Secoiridoids 881 Secologanin 816 Seco-prezizaanes 378 QSAR models for 378 Selective COX-2 inhibitors 161 Selective oxidation 215 (-)-Sesamin 568 (-)-Sesaminone 573 Sesquiterpenes 193 from potatoes 193 from tobacco 193 from tomato 193 Sesquiterpene lactones 309 ADME properties of 313
anti-microbial activity of 352 antitrypanosomal activity of 353 chemical diversity of 314 chemical/biological diversity of 311 cytotoxicity of 330 distribution of potentially reactive sites (PRS) of 316 effect on nuclear transcription factors 336 identification of 313 in energy storage/recovery 328 in inflammatory responses 343 inhibition of 5HT release by 344 inhibition of oxidative stress 328 mechanism of NF-KB inhibition 343 molecular targets of 334 monofunctional MGL-compounds of 320 PC2of 319 pharmacodynamic testing of 313 principal component analysis (PCA)of314 prinipal components (PCs) of 314 QSAR studies of 343 structural diversity of 311 structure activity relationships (SAR)of 312" Sesquiterpene phytoallexin 229 cichoralexin 231 lettuceinA 229 Sesquiterpenes classification 193 as phytoalexins 193 Shinjulactone C 482 Sideritis akmanii 505 Sideritis angustifolia 495 conchitriol from 495 jativatriol from 495 lagascatriol from 495 X-ray analysis of 495 Sideritis arborescens 496 e«/-kaurane siderol from 496 Sideritis arborescens 502 Sideritis biflora 498 diterpenoids from 498 Sideritis bolleana 503
1127 Sideritis canariensis 504 Sideritis candicans 504 Sideritis chamae dry folia 497 foliol from 497 sideridiol from 497 Sideritis cystosiphon 503 Sideritis diterpenoids 531 biological activities of 531 decoctions of 531 infusions of 531 Sideritis euboea 498 sideridiol from 498 siderol from 498 Sideritis ferrensis 504 diterpenoids from 504 Sideritisflavovirens 500 e«f-kauranes from 500 Sideritisfoetens 500 andalusol from 500 micropropagation of 500 Sideritis funkiana 499 Sideritis gomerae 498 e«/-labdanes from 498 Sideritis granatensis 501 diterpenoids from 501 Sideritis grandiflora 496 from sideridiol 496 Sideritis hirsuta 499 Sideritis huber-morathii 505 Sideritis incana subsp. virgata 499 Sideritis infernalis 503 Sideritis leucantha var. meridionalis 500 Sideritis leucantha var. tragoriganum 499 Sideritis macrostachya 503 Sideritis nutans 502 diterpenoids from 502 Sideritis paulii 498 epoxy-isofoliol from 498 Sideritis perfoliata 502 diterpenoids from 502 Sideritis pusilla 496 investigation of 496 Sideritis pusilla subsp. flavovirens 501 Sideritis reverchonii 497 derivatives of 497 Sideritis rubriflora 506
Sideritis scardica 499 Sideritis serrata 497 agascatriol from 497 Sideritis sipylea 506 Sideritis tragoriganum 499 diterpenoids from 499 ewf-atisanes from 500 Sideritis varoi subsp. oriensis 503 Simaba cedron 439 in vitro 439 Simalikalactone D 477 from Quassia africana 477 synthesis of 477 Simvastatin 760,761 microbial transformation of 761 structure of 760 Sinococuline 165 from Stephania sutchuenesis 165 Sireptomyces species 774 as squalene synthase inhibitors 774 macrolaetins A/F from 774 Skin diseases 153 keratinocyte apoptosis in 153 Skin inflammatory ailments 675 Scrophularia genus use in 675 Smilax glabrae 1074 to treat atypical hyperplasia (AH) 1074 to treat intestinal metaplasia 1074 Snakebites 440 Ailanthus malubarica in 440 Soil borne disease 909 Sclerotium rolfsii as 909 Solamargine 165 from Solanum incanum 165 Somatic mutation/recombination test (SMART) 841 of Drosophila 841 Spasmolytic 891 medicinal plants as 891 Spectral tuning 12 Sphingolipids 1045,1047 derivative of sphingoid bases 1045 with phytosphingosme-containing ceramides 1047
1128 1128 SPIKET 734 preclinical/clinical status of 734 SPIKET compounds 734 as novel synthetic spiroketal pyran 734 Spodoptera eridania 111 Spodoptera littoralis 924 Spondylitis 243 Tripterygium wilfordii in 243 Spongistatin 734 Sporothris schenckii 1040 as dimorphic fungus 1040 as human pathogen 1048 sporothricosis by 1040 Squalene epoxidase 753 Squalene oxide 242 structure of 242 Squalene synthase 753 Squalene synthase inhibitors 764 against hypocholesterolemics 764 Squalestatins I 764 from cultures of Phoma sp. 764 Squamolone 972 Statins 761 as microbial transformation products 761 (-)-Steganone 592,594,595 total synthesis of 592 asymmetric synthesis of 592 Stephania sutchuenesis 165 sinococuline from 165 Stevia rebaudiana 1001 as antimicrobial 1001 as anti-inflammatory 1001 biological activity of 1001 Stevioside 1001 as sweetener 1001 Stilbenes 948,949 epoxidation of 948 synthesis by Witting/Heck approaches 949 Stilbene monomers 602 isorhapontigenin as 602 oxyresveratrol as 602 Styrylpyrones 1013,1014 CD spectrum of 1013
cytotoxicity of 1013 cytotoxic activity against human tumors 1013 X-ray crystallographic data of 1014 Substance P 124 Sulfakinins 120 as central neuromodulators 120 Sulfakinin family 119 Sulphonamides 722,731 E 7010 as 731 inhibition of tubulin polymerization by 731 anti-proliferative activity of 731 Superstatins 762 Survival genes 155 Bcl-2 155 Bcl-X, 155 NF-KB
155
Synthesis 414,415,541,942-949 ofdrim-5,8-dien-7-one 414-.415 of drim-8-en-7-one 414-.415 of (+)-drim-8-en-11 -oic acid 424 ofdrim-9(ll)-en-8a-ol 410-413 of flavonoids of structural relevance to deer L. 942-949 oflignans 541,542 Synthetic small molecules 733 targeting bindng sites 733 Synthetic statins 763 strutures of 763
T lymphocytes 158 T138067 (Batabulin) 732 in phase III clinical trials 732 in therapy of liver cancer 723 Tabersonine 824 biosynthesis of 824 Tachykinin-related peptides 124 Tanacetum parthenium 111 parthenolide from 177 Tangeretin 173 anticancer activity of 173 structure of 173 Tannins 174 antioxidative activity of 174
1129 Taxol 721,739 chemical structures of 721 used as anti-cancer agents 739 Taxol resembling compounds 739 T-cells 157 in various inflammatory pathologies 157 Tectoridin 173 from Belamcanda chinensis 173 Tectorigenin 173 from Belamcanda chinensis 173 Telomerase inhibition 710 Terpenoid indole alkaloids (TIAs) 819,830 antifeedant activity of 820 as antibiotic 820 from Catharanthus roseus 820 regulation of biosynthesis of 830 role of tryptamine 821 role of tryptophan 821 role of shikimate pathway 822 Terpenoids 176 mechanism of action of 176 Terpenoid stress compound 205 phytuberin as 205 Tetrahydrocurcuminoids 792 Tetrahydropalmatine 962 discretamine from 962 Tetrahydroprotoberberine 961 Tetrahydropyran (THP) ring 975 Tetralone 221,224 reduction of 224 Tetrandrine 165,166 apoptotic effect of 165 apoptotic mechanism of 165 structure of 166 Thalicpureine 969 Thalictrum acutifolium 165 acutiaporberine from 165 Thapsigargin 364 analogues of 364 histamine liberation by 364 induced mast cell degranulation in 364 Theophylline 164 apoptosis in humans 164
Thromboxanes 700.888 biosynthesis of 888 Thymocytes 158 apoptosis of 158 Tingenone 269,272 from Euonymus tingens 272 structures of 269 TK-related peptides 125 TNFR family 149 death receptors induction 149 Tobacco mosaic virus (TMV) 193.205 Tobacco necrosis virus (TNV) 205.227 a-Tocopherol 883 Total synthesis 475 of quassinoids 475 Transcription factors 150 Transcription signals 155 modification of 155 Transmigration 157 7ra/7.?-resveratrol 167 anti-inflammatory properties of 167 structure of 167 7ra«s-tetrahydroactinidiolide 423 from drim-8-en-7-one 423 from tobacco 423 Trifolium repens 916 Trifolium subterraneum 916 Triol-styrylpyrones 1015 CD spectra of 1015 Tripterygium doianum 262 Tripterygium wilfordii 165,178,243 effect on T-cell 165 in rheumatoid arthritis 243 in spondylitis 243 triptolide from 178 use as anticancer drug 243 Triptocallic acid B 256 from Tripterygium wilfordii 256 Triptocallic acid C 256 from Tripterygium wilfordii 256 structure of 256 Triptocallic acid D 252 from Tripterygium wilfordii 252 Triptocalline A 244,245 from Tripterygium wilfordii 244 structures of 245
1130 TriptohypolA 281,282 from Tripterygium hypoglaucum 281 structures of 282 TriptohypolB 281,282 from Tripterygium hypoglaucum 281 structures of 282 TriptohypolC 281,282 from Tripterygium hypoglaucum 281 structures of 282 TriptohypolF 251,252 Triptolide 178 anti-inflammatory effects of 178 antitumoral effect of 178 from Tripterygium wilfordii 178 structure of 178 TriscutinA 291 structure of 291 TriscutinB 292 structure of 292 2,3,4-Trisubstitutedtetrahydrofuran lignan 562 synthesis of 562 Triterpene dimers/trimers 286 Triterpenes 176,241 against herbivores 241 against pathogens 241 apoptotic effects of 176 Triterpenoid quinonemethides 268 Trypsin 939 characteristics of 939 Tubulin inhibitors 719 as antimitotic agents for cancer therapy 719 development of 719 Tumors 843 P-glycoprotein in 843 Tumor formation 708 induction by carcinogens 708 Tumor necrosis factor (TNFa) 625 as pro-inflammatory cytokines 625 Two-dimension electrophoresis 826 TXB2 702 formation of 702
TXD-258 740 from 10-deacetylbaccatin 740 Tyrosol 866 Tyrosol related compounds 871
Ubiquitin/ATP-dependent proteolysis pathway 841 in regulation of cell cycle 841 Uca pugilator 86 Ulcers 675 buddlejain 675 Uncaria guianensis 181 anti-inflammatory activity of 181 Uncaria tomentosa 181 pharmacological properties of 181 Uncinine 972 Undine 972 Ursanes 257 chemistry of 257 from Tripterygium wilfordii 257 Ursolic 241 against liver injury 241 UvidinC 397 from Lactarius uvidus Fries 397
Valine 934 inhibition of biosynthesis of 934 Vanessa cardui 81 adipokinetic hormones from 81 Vanilloid receptor type I (VRI) 168 Vasodilatator 878 Olea europaea as 878 Vatdiospyrodol C 626 against breast cancer 626 against colon cancer 626 against oral epidermoid carcinoma 626 Vateriaphenol 605 from Vateria indica 605 Vaticanol 605 from genera of VitislVateria 605 Verbascoside 700,702.705,710.861.872 antioxidant activity of 872 carrageenan-induced rat paw 710
1131 1131 edema inhibition by 700 mode of action of 705 TX-synthase inhibition by 702 Vessel stabilizing actions 891 of medicinal plants 891 Vigna unguiculata 940 Vilmorinine A-F 444 fromAilanthus vilmoriniana 444 Vinblastine 814 from Catharanthus roseus 814 from Vinca rosea 814 hematological toxicity due to 817 in treatment of breast carcinoma/ choriocarcinoma 817 in treatment of Kaposi's sarcoma 817 in treatment of histiocytosis X 817 inhibition of taurocholate transport by 836 strucutreof 721,814 Vinca alkaloids 735,813 activity of 813 antimitotic activity of 813 antiproliferative activity of 840 as dimeric terpenoid indole alkaloids 813 biosynthesis in Catharanthus roseus 819 biosynthesis of 813 cellular targets of 840 clinical uses of 816 evolutionary link to 845 extrusion of 841 from Catharanthus roseus 735 from plants to animals 845 in animal cells 841 in cancer chemotherapy 735 mechanism of cell proliferation inhibition 839 metabolism in animal cells 813 monomeric precursors of 827 role of cytochrome P450 monooxygenase CYP3A4 844 semisynthetic process for 827 transport mechanism of 815 uptake of 841
Vinca site binding agents 735 in cancer chemotherapy 735 from Catharanthus roseus 735 Vincaleucoblastine 816 Vincaleucocristine 816 Vincristine 11,721,814 adverse effect of 818 chemical structure of 721 clinical uses of 816 discovery of 815 from Catharanthus roseus 814 horn. Vinca rosea 814 in treatment of acute leukemias 817 in treatment of Hodgkin"s disease 816,817 in treatment of lymphoma 816. 817 in treatment of other cancers 816 neurotoxicity of 818 structure of 814 transport mechanism of 815 with prednisone 817 Vindesine 818 in treatment of advanced nonsmall cell lung cancer 818 (+)-s-Vinferin 602 from infected grapevine leaves 602 a-Vinferin 602 from infected grapevine leaves 602 Vinflunine 735 clinical trials of 735 in advanced breast cancer 735 in treatment of NSCLC 735 Vinorelbine 818 in treatment of breast cancer 818 in treatment of non-small cell lung cancer 818 Virgin olive oil (VOO) 881 phenolic antioxidants of 881 Viridiofungins A 771 antifungal activities of 771 from Trichoderma viride 111
1132 isolation of 771 structure elucidation of 771 Viridiofungins B 771 antifungal activities of 771 from Trichoderma viride 771 isolation of 771 structure elucidation of 771 Viridiofungins C 771 antifungal activities of 771 from Trichoderma viride 111 isolation of 771 structure elucidation of 771 Vitamin E 160,1075 inhibitory effect on 160 in TBA assay 1075 Vitellogenesis-inhibiting hormone 99 Volubilide formation 267 mechanism of 267 structure of 267
(±)-Warburganal 421 total synthesis of 421 Warburgia 395 drimenol from 395-405 Wharton-Bollen rearrangement 416 Wheat germ agglutinin (WGA) 1034 Wild type Aequorea GFP 29 Wilforic acid B/C 243,244 structure of 243 Wittig reaction 195 of aldehydes 195 ofketal 195 Wodeshiol 587 asymmetric synthesis of 587
Xanthanolides 317 Xanthine 882 Xanthine theophylline 163 Xenobioties 831 detoxification of 831 regulation of 831 X-ray crystallographic analysis 977 Xylopianin 992 cytotoxic values (IC50) of 992
Yadanziosides F 447 against murine P-388 lymphocytic leukemia 447 antileukemic activity of 447 Yakuchinone A 791 structure of 791 Yakuchinone B 791 structure of 791 Yakuchinones A/B 169 (+)-Yatein 568 Yellow fluorescent proteins (YFPs) 7, 42-47 aggregation of 46 emission spectra of 45 from A equorea GFP 42 GFP derived 42 isoforms of 43 isomerization of 46 oligomerization of 46 pH of 47 photobleaching of 46 salt of 47 spectra of 42 temperature of 47
Zaragozic acid A 765,766,768 by microbial transformation using Streptomyces cyanus 768 from Sporormilla intermedia 765 inhibitory activity against cholesterol biosynthesis 765 Zaragozic acid B 765,766 from Sporormilla intermedia 765 inhibitory activity against cholesterol biosynthesis 765 structure of 766 Zaragozic acid C 765,766 from Sporormilla intermedia 765 inhibitory activity against cholesterol biosynthesis 765 structures of 766 Zaragozic acid D 768 as inhibitors of rat liver squalene synthase 768 from Amauroascus niger 768
1133 1133 Zaragozic acids D 2 768 as inhibitors of rat liver squalene synthase 768 from Amauroascus niger 768 Zaragozic acids F-10863 770 structures of 770 ZD6126 723 in phase II clinical trials 723 in solid tumours 723 Zea mays 928 flavonoidin 928 Zerumbone 178,179 from Zingiber zerumbet 178 structure 179 Zeylasterone 278,280 from Kokoona zeylanica 278 structures of 280 Zinc-induced tubulin sheets 733 crystallography of 733 Zingiber zerumbet 178 zerumbone from 178 Zingiberaceae family 785
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