fattorusso - modern alkaloids - structure, isolation, synthesis and biology (wiley, 2008)

XVII Modern Alkaloids: Structure, Isolation, Synthesis and Biology... BerlinckUniversity of Sao Paulo CP 780, CEP 13560-970 3566590 - Sao Carlos, SPBrazil Stefan BieriOfficial Food Contro

Modern Alkaloids

Edited by

Ernesto Fattorusso andOrazio Taglialatela-Scafati

Tietze, Lutz F / Eicher, Theophil / Diederichsen, Ulf / Speicher, AndreasReactions and Syntheses

in the Organic Chemistry Laboratory

2007

ISBN: 978-3-527-31223-8

Hudlicky, Tomas / Reed, Josephine W

The Way of Synthesis

Evolution of Design and Methods for Natural Products

2007

ISBN: 978-3-527-31444-7

Sarker, Satyajit / Nahar, Lutfun

Chemistry for Pharmacy Students

2007

ISBN: 978-0-470-01780-7

Kayser, Oliver / Quax, Wim J (eds.)

Medicinal Plant Biotechnology

From Basic Research to Industrial Applications

2006

ISBN: 978-3-527-31443-0

Eicher, Theophil / Hauptmann, Siegfried

The Chemistry of Heterocycles

Structure, Reactions, Syntheses, and Applications

Prof Ernesto Fattorusso

Univ Federico II Dipto di

Chimica delle Sost Naturali

Via D Montesano 49

80131 Napoli

Italien

Prof O Taglialatela-Scafati

Univ Federico II, Dipto di

Chimica delle Sost Naturali

applied for British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek Die Deutsche Nationalbibliothek lists this publica- tion in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de.

# 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

All rights reserved (including those of translation into other languages) No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Typesetting Thomson Digital, Noida Printing betz-druck GmbH, Darmstadt Binding Litges & Dopf GmbH, Heppenheim Cover Design Grafik-Design Schulz, Fußgo¨nheim

Printed in the Federal Republic of Germany Printed on acid-free paper

ISBN: 978-3-527-31521-5

Michael Wink

Muriel Cuendet, John M Pezzuto

Angela Bassoli, Gigliola Borgonovo, Gilberto Busnelli

4.9 Molecular Gastronomy of Hot Food 98

5.3.2.1 Substrate Reduction Therapy 130

Rashel V Grindberg, Cynthia F Shuman, Carla M Sorrels, Josh Wingerd,William H Gerwick

Je´roˆme Kluza, Philippe Marchetti, Christian Bailly

8.3.1 Source of Manzamine Alkaloids 202

Ana R Diaz-Marrero, Christopher A Gray, Lianne McHardy, Kaoru Warabi,Michel Roberge, Raymond J Andersen

Anna Aiello, Ernesto Fattorusso, Marialuisa Menna,

Orazio Taglialatela-Scafati

Contents IX

Roberto G.S Berlinck, Miriam H Kossuga

Philippe Christen, Stefan Bieri, Jean-Luc Veuthey

Steven M Colegate, Dale R Gardner

13.2.1 Optimization 370

14 Applications of15N NMR Spectroscopy in Alkaloid Chemistry 409

Gary E Martin, Marina Solntseva, Antony J Williams

Hans-Joachim Kno¨lker

15.1.2 Synthesis of the Indolizidino[8,7-b]indole Alkaloid

Sabrina Dallavalle, Lucio Merlini

Michael Prakesch, Prabhat Arya, Marwen Naim, Traian Sulea,

Enrico Purisima, Aleksey Yu Denisov, Kalle Gehring, Trina L Foster,

Contents XIII

18 Daphniphyllum alkaloids: Structures, Biogenesis, and Activities 541

Hiroshi Morita, Jun’ichi Kobayashi

19.2.1 Indoles 591

Ce´sar Sa´nchez, Carmen Me´ndez, Jose´ A Salas

Contents XV

Alkaloids constitute one of the widest classes of natural products, being synthesizedpractically by all phyla of both marine and terrestrial organisms, at any evolutionarylevel The extraordinary variety (and often complexity) of alkaloid structures andbiological properties have long intrigued natural product chemists (for structuredetermination and biosynthetic studies), analytical chemists, and synthetic organicchemists Toxicologists, pharmacologists and pharmaceutical companies have usedand will certainly continue to use alkaloids as biological tools and/or as leadcompounds for development of new drugs

When we started our project of a handbook on alkaloid science, we were facedwith an impressive number of papers describing the structures and activities ofalkaloids, and also with an intense review activity, published in excellent book series

or in single books covering specific classes of alkaloids Consequently, we decided toorganize our handbook to present the different aspects of alkaloid science (e.g thestructure and pharmacology of bioactive alkaloids; recent advances in isolation,synthesis, and biosynthesis) in a single volume, aiming to provide representativeexamples of more recent and promising results as well as of future prospects inalkaloid science Obviously, the present handbook cannot be regarded as a compre-hensive presentation of alkaloid research, but we feel that the diversity of topicstreated, ranging from bitterness to the anticancer activity of alkaloids, can provide agood idea of the variety of active research in this field

In particular, Section I describes the structures and biological activities of selectedclasses of alkaloids Almost half of the chapters focus their attention on terrestrialalkaloids (Chapters 1–5) The other half (Chapters 7–11) describe recent results inthe field of marine alkaloids, while Chapter 6 is focused on neurotoxic alkaloidsproduced by cyanobacteria, microorganisms living in both marine and terrestrialenvironments The particular emphasis on marine alkaloids undoubtedly reflectsour long-standing research activity on marine metabolites, but it is also a result ofthe impressive amount of work carried out in the last few decades on marine naturalproduct chemistry Section II (Chapters 12–15) gives an account of modern techni-ques used for the detection and structural elucidation of alkaloids, while Section III

is divided into two parts: different methodologies for the synthesis of alkaloids andaccounts of modern biosynthetic studies

XVII

Modern Alkaloids: Structure, Isolation, Synthesis and Biology Edited by E Fattorusso and O Taglialatela-Scafati Copyright ß 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

Finally, we should point out that even today the term alkaloid is ambiguous (adiscussion on the definition of alkaloid is presented in Chapter 4) The initialdefinition of Winterstein and Trier (1910) ("nitrogen-containing basic compounds

of plant or animal origin") has obviously been superseded The most recent tion of alkaloid can be attributed to S W Pelletier (1984): "compound containingnitrogen at a negative oxidation level characterized by a limited distribution inNature" In the preparation of this handbook we have decided to follow this lastdefinition and, thus, to include "borderline" compounds such as capsaicins and non-ribosomal polypeptides

defini-We cannot conclude without thanking all the authors who have made their expertcontributions to the realization of this volume, which we hope will stimulate furtherinterest in one of the most fascinating branches of natural product chemistry

Orazio Taglialatela-Scafati

List of Contributors

Anna Aiello

Universita` di Napoli ‘‘Federico II’’

Dipartimento di Chimica delle

National Research Council of Canada

Steacie Institute for Molecular Sciences

Place de Verdun

59045 LilleFrance

Angela BassoliUniversita` di MilanoDipartimento di Scienze MolecolariAgroalimentari

Via Celoria, 2

20133 MilanoItaly

Roberto G.S BerlinckUniversity of Sao Paulo

CP 780, CEP 13560-970

3566590 - Sao Carlos, SPBrazil

Stefan BieriOfficial Food ControlAuthority of Geneva

20, Quai Ernest-Ansermet

1211 Geneva 4Switzerland

Modern Alkaloids: Structure, Isolation, Synthesis and Biology Edited by E Fattorusso and O Taglialatela-Scafati Copyright ß 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

XIX

Via Celoria, 2

20133, MilanoItaly

Aleksej DansiovDepartment of BiochemistryMcGill University

3655 Promenade Sir William OslerMontreal, Quebec H3G IV6Canada

Ana R Diaz-MarreroInstituto de Productos Naturales yAgrobiologı´a del CSIC,

Avda Astrofisico F Sa´nchez 3Apdo 195

38206 La LagunaTenerife

Spain

Ernesto FattorussoUniversita` di Napoli ‘‘Federico II’’Dipartimento di Chimica delleSostanze Naturali

Via D Montesano, 49

80131 NapoliItaly

Trina L FosterApoptosis Research CentreChildren’s Hospital of Eastern Ontario(CHEO)

401 Smyth RoadOttawa K1H 8L1Canada

Dale R Gardner

Poisonous Plant Research Lab

USDA, Agricultural Research Service

University of California at San Diego

Scripps Institution of Oceanography

9500 Gilman Drive

La Jolla, CA 92093-0210

USA

Christopher A Gray

University of British Columbia

Chemistry of Earth and Ocean

Scripps Institution of Oceanographyand The Skaggs School of Pharmacyand Pharmaceutical Sciences,

La Jolla, California 92093USA

Mark T HamannUniversity of MississippiDepartment of PharmacognosyMississippi, MS 38677USA

Jerome KluzaINSERM U-524, Centre OscarLambret

Place de Verdun

59045 LilleFrance

Hans-Joachim Kno¨lkerUniversity of DresdenInstitut fu¨r Organische ChemieBergstrasse 66

01069 DresdenGermany

Jun’ichi KobayashiHokkaido UniversityGraduate School of PharmaceuticalSciences

Sapporo 060-0812Japan

Robert G KornelukNational Research Council of CanadaSteacie Institute for Molecular Sciences

100 Sussex Drive,Ottawa, Ontario, K1A 0R6,Canada

List of Contributors XXI

Miriam H Kossuga

Instituto de Quı´mica de Sa˜o Carlos

Universidade de Sa˜o Paulo

Universita` di Napoli ‘‘Federico II’’

Dipartimento di Chimica delle Sostanze

Via Celoria, 2

20133, MilanoItaly

Hiroshi MoritaHokkaido UniversityGraduate School of PharmaceuticalSciences

Sapporo 060-0812Japan

Mohammed NaimBiotechnology Research InstituteNational Research Council of Canada

6100 Royalmount AvenueMontre´al, Quebec, H4P 2R2Canada

John M PezzutoUniversity of HawaiiHilo College of Pharmacy

60 Nowelo St., SuiteHilo, Hawaii 96720USA

Michael PrakeschNational Research Council of CanadaSteacie Institute for Molecular Sciences

100 Sussex Drive,Ottawa, Ontario, K1A 0R6,Canada

Jangnan PengUniversity of MississippiDepartment of PharmacognosyMississippi, MS 38677USA

University of British Columbia

2146 Health Sciences Mall

University of California, San Diego

Center for Marine Biotechnology and

Biomedicine

Scripps Institution of Oceanography

and The Skaggs School of Pharmacy and

Scripps Institution of Oceanographyand The Skaggs School of Pharmacyand Pharmaceutical Sciences,

La Jolla, California 92093USA

Traian SuleaBiotechnology Research InstituteNational Research Council of Canada

6100 Royalmount AvenueMontre´al, Quebec, H4P 2R2Canada

Orazio Taglialatela-ScafatiUniversita` di Napoli ‘‘Federico II’’

Dipartimento di Chimica delleSostanze Naturali

Via D Montesano, 49

80131 NapoliItaly

Jean-Luc VeutheyUniversity of GeneveFaculty of Sciences

20, Bd d’Yvoy

1211 Gene`va 4Switzerland

Kaoru WarabiUniversity of British ColumbiaChemistry and Earth and OceanSciences

2146 Health Sciences MallVancouver

British Columbia V6T1Z1Canada

List of Contributors XXIII

University of California, San Diego

Center for Marine Biotechnology and

Biomedicine

Scripps Institution of Oceanography

and The Skaggs School of Pharmacy

and Pharmaceutical Sciences,

La Jolla, California 92093

USA

Michael WinkUniversity of Heidelberg,Institute of Pharmacy and MolecularBiotechnology

Im Neuenheimer Feld 364

69120 HeidelbergGermany

Bioactive Alkaloids: Structure and Biology

Modern Alkaloids: Structure, Isolation, Synthesis and Biology Edited by E Fattorusso and O Taglialatela-Scafati Copyright ß 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

1

Ecological Roles of Alkaloids

Michael Wink

1.1

Introduction: Defense Strategies in Plants

Plants are autotrophic organisms and serve as both a major and the ultimate source offood for animals and microorganisms Plants cannot run away or fight back whenattacked by a herbivore, nor do they have an immune system to protect them againstpathogenic bacteria, fungi, viruses, or parasites Plants struggle for life, as do otherorganisms, and have evolved several strategies against herbivorous animals, para-sites, microorganisms, and viruses Plants also compete with neighboring plants forspace, light, water, and nutrients [1–8]

Apparently plants have evolved both physical and chemical defense measures,similar to the situation of sessile or slow moving animals Among physical defensestrategies we find [8]

formation of indigestible cell walls containing cellulose, lignin,

or callose;

presence of a hydrophobic cuticle as a penetration barrier

for microbes and against desiccation;

formation of a thick bark in roots and stems against water loss,

microbes, and herbivores;

development of spines, thorns, hooks, trichomes, and

glandular and stinging hairs (often filled with noxious

chemicals) against herbivores;

formation of laticifers and resin ducts (filled with gluey and

noxious fluids);

a high capacity for regeneration so that parts that have been

browsed or damaged by infection can be readily replaced

(so-called open growth)

Secondly, plants are masters of chemical defense, with a fascinating ability toproduce a high diversity of chemical defense compounds, also known as secondarymetabolites or allelochemicals [1–17] Chemical defense involves macromolecularcompounds, such as diverse defense proteins (including chitinase [against fungal cell

Modern Alkaloids: Structure, Isolation, Synthesis and Biology Edited by E Fattorusso and O Taglialatela-Scafati Copyright ß 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

3

walls],b-1,3-glucanases [against bacteria], peroxidase, and phenolase, lectins, proteaseinhibitors, toxalbumins, and other animal-toxic peptides), polysaccharides, and poly-terpenes More diverse and more prominent are low molecular weight secondarymetabolites, of which more than 100 000 have been identified in plants (Figure 1.1).Among the secondary metabolites that are produced by plants, alkaloids figure as avery prominent class of defense compounds Over 21 000 alkaloids have beenidentified, which thus constitute the largest group among the nitrogen-containingsecondary metabolites (besides 700 nonprotein amino acids, 100 amines, 60 cyano-genic glycosides, 100 glucosinolates, and 150 alkylamides) [2,3,18,19] However, theclass of secondary metabolites without nitrogen is even larger, with more than 25 000terpenoids, 7000 phenolics and polyphenols, 1500 polyacetylenes, fatty acids, waxes,and 200 carbohydrates.

1.2

Ecological Roles of Alkaloids

Alkaloids are widely distributed in the plant kingdom, especially among angiosperms(more than 20 % of all species produce alkaloids) Alkaloids are less common butpresent in gymnosperms, club mosses (Lycopodium), horsetails (Equisetum), mosses,and algae [1–5,17] Alkaloids also occur in bacteria (often termed antibiotics), fungi,many marine animals (sponges, slugs, worms, bryozoa), arthropods, amphibians(toads, frogs, salamanders), and also in a few birds, and mammals [1–5,13,17,20].Alkaloids are apparently important for the well-being of the organism that pro-duces them (Figures 1.1–1.3) One of the main functions is that of chemical defenseagainst herbivores or predators [2,3,8,18] Some alkaloids are antibacterial, anti-fungal, and antiviral; and these properties may extend to toxicity towards animals.Alkaloids can also be used by plants as herbicides against competing plants [1,3,8,18].The importance of alkaloids can be demonstrated in lupins which – as wildplants – produce quinolizidine alkaloids (‘‘bitter lupins’’), that are strong neurotoxins(Table 1.1) [21,22] Since lupin seeds are rich in protein, farmers were interested inusing the seeds for animal nutrition This was only possible after the alkaloids (seedcontent 2–6 %) had been eliminated Plant breeders created so-called sweet lupinswith alkaloid levels below 0.02 % If bitter and sweet lupins are grown together in thefield it is possible to study the importance of alkaloids for defense For example,Figure 1.3 shows that rabbits strongly discriminate between sweet and bitter lupinsand prefer the former This is also true for insects, as aphids and mining flies alwaysfavor sweet lupins In the wild, sweet lupins would not survive because of the lack of

an appropriate chemical defense [8,21]

Secondary metabolites are not only mono- but usually multifunctional In manycases, even a single alkaloid can exhibit more than one biological function Duringevolution, the constitution of alkaloids (that are costly to produce) has been modu-lated so that they usually contain more than one active functional group, allowingthem to interact with several molecular targets and usually more than one group ofenemies [3,18,19,21–24] Many plants employ secondary metabolites (rarely alka-

Fig 1.1 Relationships between plants, their secondary

metabolites, and potential enemies (herbivores,

microorganisms, and viruses) Example: Lupins produce

quinolizidine alkaloids, isoflavonoids, and saponins as

main defense compounds.

1.2 Ecological Roles of Alkaloids 5

Function Secondary metabolites

•seed dispersing animals

•root nodule bacteria

•adapted organisms

(specialists)

UV- Protection N-storage

Fig 1.2 Overview of the ecological functions of secondary

Completely eaten lupins (%) Lupins with Agromyzidae (%)

Fig 1.3 Importance of quinolizidine alkaloids for lupins

against herbivores.In this experiment, lupins with or without

alkaloids were grown in the field When rabbits got into the

field, they preferentially consumed the sweet, alkaloid-free

lupins Also larvae of mining flies preferred sweet lupins.

Tab 1.1 Molecular targets of alkaloids in neuronal signal transduction [2,3,19].

Neuroreceptor

Muscarinic

acetylcholine

receptor

Hyoscyamine, scopolamine, and other tropane alkaloids (AA);

acetylheliosupine and some other pyrrolizidine alkaloids;

arecoline (A); berbamine, berberine, and other isoquinoline alkaloids; dicentrine and other aporphine alkaloids; strychnine, brucine; cryptolepine (AA); sparteine and other quinolizidine alkaloids (A); pilocarpine (A); emetine; himbacine and other piperidine alkaloids (A); imperialine (AA); muscarine (A) Nicotinic acetylcholine

receptors

Nicotine and related pyridine alkaloids (A); Ammodendrine (A); anabasine (A); arborine (AA); boldine and other aporphine alkaloids (AA); berberine and related protoberberine alkaloids;

C-toxiferine (AA); coniine and related piperidine alkaloids (A);

cytisine, lupanine, and other quinolizidine alkaloids (A);

tubocurarine (AA); codeine (A); erysodine and related Erythrina alkaloids (AA); histrionicotoxin (AA); lobeline (A);

methyllycaconitine (AA); pseudopelletierine (A) Adrenergic receptors Acetylheliosupine and related pyrrolizidine alkaloids; ajmalicine,

reserpine (AA); arecoline; berbamine, berberine, laudanosine, and other isoquinoline alkaloids (AA); boldine, glaucine, and other aporphine alkaloids (AA); cinchonidine and other quinoline alkaloids; corynanthine, yohimbine, and other indole alkaloids (AA); emetine; ephedrine; ergometrine, ergotamine, and related ergot alkaloids (A/AA); ephedrine and related phenylethylamines (A); higenamine (A); N-methyldopamine, octopamine (A) Dopamine receptor Agroclavine, ergocornine, and related ergot alkaloids (A);

bulbocapnine and related aporphine alkaloids (AA); anisocycline, stylopine, and related protoberberine alkaloids; salsolinol and related isoquinolines (A); tyramine and derivatives (A) GABA receptor Bicuculline (AA), cryptopine, hydrastine, corlumine, and related

isoquinoline alkaloids (AA); securinine; harmaline and related b-carboline alkaloids (A); muscimol (A); securinine (AA) Glycine receptor Corymine, strychnine, and related indole alkaloids (AA)

Glutamate receptor Histrionicotoxin and related piperidines (AA); ibogaine and related

indole alkaloids (AA); nuciferine and related aporphine alkaloids (AA)

Serotonine receptor Akuaminine and related indole alkaloids (A); annonaine, boldine,

liriodenine and related aporphine alkaloids (AA); berberine and related protoberberine alkaloids; ergotamine, ergometrine, and related ergot alkaloids (AA); psilocin, psilocybine (A); bufotenine, N,N-dimethyltryptamine, and related indoles (A); harmaline and related b-carboline alkaloids (A); kokusagine and related furoquinoline alkaloids (AA); mescaline (A); ibogaine and other monoterpene indole alkaloids (A); gramine;

N,N-dimethyltryptamine and derivates (AA) Adenosine receptor Caffeine, theobromine, and other purine alkaloids (AA)

Opiate receptor Morphine and related morphinan alkaloids (A); akuammine,

mitragynine (A), ibogaine and related indole alkaloids

(continued ) 1.2 Ecological Roles of Alkaloids 7

loids, mostly colored phenolics and fragrant terpenoids) to attract pollinating andseed-dispersing animals; the compounds involved are usually both attractant andfeeding deterrents Attracted animals are rewarded by nectar or fleshy fruit tissuesbut should leave seeds or flowers undamaged Hence, a multifunctional or pleio-tropic effect is a common theme in alkaloids and other secondary metabolites.

Acetylcholine esterase Galanthamine (AA); physostigmine and related indole alkaloids

(AA); berberine and related protoberberine alkaloids (AA);

vasicinol and related quinazolines (AA); huperzine (AA);

harmaline and related b-carboline alkaloids (AA); demissine and related steroidal alkaloids (AA)

Monoamine oxidase Harmaline and related b-carbolines (AA); carnegine, salsolidine,

O-methylcorypalline, and related isoquinolines (AA);

N,N-dimethyltryptamine and related indoles (AA);

Neurotransmitter

uptake (transporter)

Ephedrine and related phenylalkyl amines (AA); reserpine, ibogaine, and related indole alkaloids (AA); cocaine (AA); annonaine and related aporphine alkaloids (AA); arecaidine (AA); norharman and related b-carboline alkaloids (AA); salsolinol and related isoquinolines (AA)

Naþ, Kþchannels Aconitine and related diterpene alkaloids (A); veratridine,

zygadenine, and related steroidal alkaloids (A); ajmaline, vincamine, ervatamine, and other indole alkaloids (AA);

dicentrine and other aporphine alkaloids (AA); gonyautoxin (AA); paspalitrem and related indoles (AA); phalloidin (AA); quinidine and related quinoline alkaloids (AA); sparteine and related quinolizidine alkaloids (AA); saxitoxin (AA); strychnine (AA); tetrodotoxin (AA)

Ca2þchannels Ryanodine (A); tetrandrine, berbamine, antioquine, and related

bis-isoquinoline alkaloids (AA); boldine, glaucine, liriodenine, and other aporphine alkaloids (AA); caffeine and related purine alkaloids (A/AA); cocaine (AA); corlumidine, mitragynine, and other indole alkaloids (A/AA); bisnordehydrotoxiferine (AA) Adenylate cyclase Ergometrine and related ergot alkaloids (AA); nuciferine and related

aporphine alkaloids (AA) cAMP

phosphodiesterase

Caffeine and related purine alkaloids (AA); papaverine (AA); chelerythrine, sanguinarine, and related benzophenanthridine alkaloids (AA); colchicines (AA); infractine and related indole alkaloids (AA)

Protein kinase A (PKA) Ellipticine and related indole alkaloids (AA)

Protein kinase C (PKC) Cepheranthine and related bis-isoquinoline alkaloids (AA);

michellamine B and related isoquinoline alkaloids (AA);

chelerythrine and related benzophenanthridine alkaloids (AA); ellipticine and related indole alkaloids (AA)

Phospholipase (PLA 2 ) Aristolochic acid and related aporphine alkaloids (AA);

berbamine and related bis-isoquinoline alkaloids (AA)

A ¼ agonist; AA ¼ antagonist.

Tab 1.1 (Continued )

An alkaloid never occurs alone; alkaloids are usually present as a mixture of a fewmajor and several minor alkaloids of a particular biosynthetic unit, which differ infunctional groups Furthermore, an alkaloid-producing plant often concomitantlyaccumulates mixtures of other secondary metabolites, mostly those without nitrogen,such as terpenoids and polyphenols, allowing them to interfere with even moretargets in animals or microorganisms When considering the total benefits to a plantfrom secondary metabolites or the pharmacological activities of a drug, the potentialadditive or even synergistic effect of the different groups of secondary metabolitesshould be taken into account [10,25].

The multiple functions that alkaloids can exhibit concomitantly include a fewphysiological tasks: sometimes, alkaloids also serve as toxic nitrogen storage andnitrogen transport molecules [3,8] Plants that produce few and large seeds, nearlyalways invest in toxic defense compounds (often alkaloids) that are stored togetherwith proteins, carbohydrates, or lipids [8] Since nitrogen is a limiting factor for plantgrowth, nitrogen apparently is a valuable asset for plants In many species that storenitrogen in proteins and/or secondary metabolites in seeds or tubers, a remobiliza-tion has been observed after germination or regrowth in spring [2] In plants that shedtheir leaves, alkaloids are usually exported to storage organs prior to leaf fall [2].Alkaloids are definitely not waste products as had previously been assumed

Aromatic and phenolic compounds can mediate UV-protecting activities, whichmight be favorable for plants living in UV-rich environments, such as highaltitudes [1] Alkaloids (such as isoquinoline, quinoline, and indole alkaloids) thatderive from aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan,may have UV-absorbing properties, besides antiherbivoral and antimicrobial activities.Only the defensive properties of alkaloids will be discussed in more detail in thischapter

1.3

Modes of Action

In order to deter, repel, or inhibit the diverse set of potential enemies, ranging fromarthropods and vertebrates to bacteria, fungi, viruses, and competing plants, alka-loids must be able to interfere with important cellular and molecular targets in theseorganisms A short overview of these potential targets is given in Figure 1.4a and b.The modulation of a molecular target will negatively influence its communicationwith other components of the cellular network, especially proteins (cross-talk ofproteins) or elements of signal transduction As a consequence, the metabolism andfunction of cells, tissues, organs, and eventually the whole organism will be affectedand an overall physiological or toxic effect achieved Although we know the structures

of many secondary metabolites, our knowledge of their molecular modes of action islargely fragmentary and incomplete Such knowledge is, however, important for anunderstanding of the functions of secondary metabolites in the producing organism,and for the rational utilization of secondary metabolites in medicine or plantprotection [10,25]

1.3 Modes of Action 9

DNA/RNA-polymerases; repair enzymes

•Intercalators: aromatic alkaloids; berberine

•Alkylants: PAs, aristolochic acids, cycasin

DNA/RNA polymerases; repair enzymes; topoisomerase I/II:

•Intercalators: berberine, indoles, isoquinolines

•Alkylants: PAs, aristolochic acids, cycasin

•Protein inhibitors: camptothecin

Fig 1.4 Molecular targets for secondary metabolites,

especially alkaloids (a) Targets in bacterial cells, (b) targets

in animal cells.

Whereas many secondary metabolites interact with multiple targets, and thus haveunspecific broad (pleiotropic) activities, others, especially alkaloids, are more specificand interact exclusively with a single particular target Secondary metabolites withbroad and nonspecific activities interact mainly with proteins, biomembranes, andDNA/RNA which are present in all organisms.

1.3.1

Unspecific Interactions

Among broadly active alkaloids, a distinction can be made between those that are able

to form covalent bonds with proteins and nucleic acids, and those that modulate theconformation of proteins and nucleic acids by noncovalent bonding

Covalent modifications are the result when the following functional groupsinteract with proteins [18,25]:

reaction of aldehyde groups with amino and sulfhydryl groups;

reaction of exocyclic methylene groups with SH groups;

reaction of epoxides with proteins and DNA (epoxides can be

generated in the liver as a detoxification reaction);

reaction of quinone structures with metal ions (Fe2þ/Fe3þ)

Noncovalent bonds are generated when the following groups interact with proteins[18,25]:

ionic bonds (alkaloids with phenolic hydroxyl groups, that can

dissociate as phenolate ions; alkaloid bases that are present as

protonated compounds under physiological conditions);

hydrogen bonds (alkaloids with hydroxyl groups, carbonyl, or

inter-OH groups may dissociate under physiological conditions to form phenate ions thatcan form ionic bonds with positively charged amino acid residues, such as those fromlysine, arginine, and histidine These OH groups are crucial for the biological activity

of phenolics [18,25]

Molecules of nitrogen-containing compounds, such as alkaloids, amines, andpeptides, usually contain (under physiological conditions) positively charged N-atoms that can form ionic bonds with negatively charged amino acid residues ofglutamic and aspartic acid in proteins Both the covalent and the noncovalentinteractions will modulate the three-dimensional protein structure, that is, theconformation that is so important for the bioactivities of proteins (enzymes,

1.3 Modes of Action 11

receptors, transcription factors, transporters, ion channels, hormones, cytoskeleton).

A conformational change is usually associated with a loss or reduction in the activity

of a protein, leading to inhibition of enzyme or receptor activity or interference withthe very important protein–protein interactions [17,18,25]

Lipophilic compounds, such as the various terpenoids, tend to associate with otherhydrophobic molecules in a cell; these can be biomembranes or the hydrophobic core

of many proteins and of the DNA double helix [10,18,24,25] In proteins, suchhydrophobic and van der Waals interactions can also lead to conformational changes,and thus protein inactivation A major target for terpenoids, especially saponins, isthe biomembrane Saponins (and, among them, the steroid alkaloids) can change thefluidity of biomembranes, thus reducing their function as a permeation barrier.Saponins can even make cells leaky, and this immediately leads to cell death This caneasily be seen in erythrocytes; when they are attacked by saponins these cells burstand release hemoglobin (hemolysis) [1,6,17] Among alkaloids, steroidal alkaloids(from Solanaceae) and other terpenoids have these properties

These pleiotropic multitarget bioactivities are not specific, but are neverthelesseffective, and this is critical in an ecological context Compounds with pleiotropicproperties have the advantage that they can attack any enemy that is encountered by aplant, be it a herbivore or a bacterium, fungus, or virus These classes of compoundsare seldom unique constituents; quite often plants produce a mixture of secondarymetabolites, often both phenolics and terpenoids, and thus exhibit both covalent andnoncovalent interactions These activities are probably not only additive but syner-gistic [10,25]

1.3.2

Specific Interactions

Plants not only evolved allelochemicals with broad activities (see Section 1.3.1) butalso some that can interfere with a particular target [3,6,17–19,25] Targets that arepresent in animals but not in plants are nerve cells, neuronal signal transduction, andthe endocrinal hormone system Compounds that interfere with these targets areusually not toxic for the plants producing them Plants have had to develop specialprecautions (compartmentation: resin ducts, trichomes, laticifers) in order to storethe allelochemicals with broad activities that could also harm the producer.Many alkaloids fall into the class of specific modulators and have been modifiedduring evolution in such a way that they mimic endogenous ligands, hormones, orsubstrates [1,3,18,19] We have termed this selection process ‘‘evolutionary molecularmodeling’’ [12,13,19,23] Many alkaloids are strong neurotoxins that were selected fordefense against animals [2,3,19] Table 1.1 summarizes the potential neuronal targetsthat can be affected by alkaloids Extensive reviews on this topic have been published[2,3,19]

Neurotransmitters derive from amino acids; most of them are amines that becomeprotonated under physiological conditions Since alkaloids also derive from aminoacids (often the same ones as neurotransmitters) it is no surprise that severalalkaloids have structural similarities to neurotransmitters They can be considered

as neurotransmitter analogs (Figure 1.5a–c)

Fig 1.5 Agonistic or antagonistic modulation

of neuroreceptors by alkaloids that mimic

neurotransmitters (a) Interaction at

cholinergic neurotransmitters that bind

acetylcholine: nicotinic acetylcholine receptor

(nAChR) and muscarinic acetylcholine receptors (mAChR), (b) interaction at adrenergic receptors that bind noradrenaline and adrenaline, (c) interaction at serotonergic receptors that bind serotonin.

1.3 Modes of Action 13

Fig 1.5 (Continued )

Fig 1.5 (Continued )

1.3 Modes of Action 15