_FORSCHUNQ6ZENTRVNTJÜLICH GmbH-.

z. Brasilianisch-DeutschesSymposium für Natursfoffchemie

Hannover28 . Juli --10. August 1991

(KFA)~~

herausgegeben vonG . Habermeh!

Bilateral Seminars of the International Bureau

Volume 6

Forschungszentrum Jülich GmbHBilateral Seminars of the International Bureau

z. Brasilianisch-Deutsches Symposium für Naturstoffchemie

2. Slmposio Brasileiro-Alemao de Produtos Naturais

Hannover28. Juli -10. August 1991

herausgegeben vonG. Habermehl

Chemisches Institut der Tierärztlichen Hochschule HannoverW-3000 Hannover - Bischofsholer Damm 15

Deutsch-Brasilianische Zusammenarbeitin wissenschaftlicher Forschung und technologischer Entwicklung

Herausgeber

Forschungszentrum Jülich GmbHund Vertrieb :

ZENTRALBIBLIOTHEKPostfach 1913 - D-5170 MichTelefon (02461) 61-5368 " Telefax (02461) 61-6103

Druck:

Forschungszentrum Jülich, Graphische BetriebeCopyright:

Forschungszentrum Jülich 1991

Bilateral Seminars of the international Bureau, Volume 6ISSN 0938-7668

ISBN 3-89336-0743

Contents

Brown , Trigo, Klitzke, dos

Chemical ecology and coevolu-Santos, Francini, Henriques

tion at the insect/plantet Cardoso

interface

2

Gottlieb

Evolution of plant metabo-lism : the redox theory

6Bringmann

Naphthyl isoquinoline alka-loids-stereochemically, bio-synthetically, and pharmaco-logically unusual biaryls fromtropical medicinal plants

eStdckigt

The biosynthesis of monoter-penoid indole alkaloids

10Boland

Oxidative bond clavage ; anubiquitous biosyntheticroute to olefines

il

Tonholo, Goulart, Sant'Ana ,

The electrochemistry of na-et Boodts

tural products : the reduc-tion of gossipol on mercury,in the acid pH region, in amixed water-acetone solvent

12

Brondani, Neto, Generino,

Synthesis and biological ac-Moraes e Souza, Lima et

tivity of 3-,5- and 6-alkyl-2-Bieber

methoxy-1,4-benzoquinones 15

Barata

Natural and synthetic bioac-tive neolignanes

18

Knölker

Applications of transitionmetal complexes in alkaloidsynthesis

22

Baader , Bartmann, Beck, Jen-

Synthesis of chiral lipiddral1a, Kesselar et Wass

lowering agents derivedfrom natural products

23

Wagner

New potent antiasthmaticconstituents form higherplants

24

Ddpke , Sewerin

Some investigations in theplant family Amaryllidacea

26

Kaplan , Pereira et Maia

Polyhydroxyiated pyrrolizi-dine and indolizidine alkaloids form Alexa grandiflora

28

Schatte

metabolism of plant protec-tion agents in higher plants

32

Adam

New aspects in synthesis ofgibberellins

33

Queiroga, Hayasida, Lancas,

New sesquiterpone alcoholde Meijere et Marsaioli

from vassoura oil

34

Reboucas et Marsaieli

A new aromadendrane alco-hol from vassoura essential oil

38

Sander , Tiemann et Sarz

Secondary products and eli-citor-induced reactions inphotomixotrophic cell suspension culture of Cicer arie-tum L.

40

Achenbach

studies of new natural pro-ducts-problems + experiences

42

Keiecom, Pereira and

Secondary metabolites fromTeixe ra

brazilian marine organsims .Chemical, ecological andchemotaxonomix studies

44

Ferreira , Henriques, Lucas

Biologically active factorsand Habermehl

isolated from scaptocosaraptoria venom

47

Pereira, Ruppelt, do Nasci-

An update on plants againstmento, Parente et Mors

. snakebite

48

Hammann et Grabley

Isolation and structure va-riation of secondary metabo-lites from microorganisms derived by a chemical screening 52

Sonfanti Eorin et Gottlieb

Steroids, taxonomic markers?

53

Gilbert

The industrial exploitationof natural products from thetropical rain forest-a research and development pro-gramme

55Schulz

Odor communication in tropi-cal lopidoptera

58

Spitzer, Marx, Pfeilsticker

Occurrence of very long chainet Maia

and conjugated fatty acids inthe seed oils of Amazonianchestnuts

60

Krohn

Isolation of biocide naturalsubstances from phytophotoge-nic fungi

fit

Vichnewski, Turco Nasi,D as Rodrigues, Takaha-shi, Lunardello et Mauro

Yoshida , ishige, Motidomeet Gottlieb

Kato, Yoshida et Gottlieb

Vanderlan da Silva Bolzani ,Trevisan et Young

Trigo , Barata, Witte, Hart-mann et Brown

The chemistry of HremanthusSpecies

64

Neolignans form Ocotea Catha-rinensis

67

Chemical compositions inflowers, fruits of severalphases and seedling of _Vi-rola venosa

71

Chemical constituents of Ru-biaceae from Brazilian cerrado regions

75

Capillary GC-MS analysis ofpyrrolizidine alkaloids inplants utilized by ithomiinaebutterflies (Lepidoptera : Nym-phalidae) in the southeast ofBrazil

79

F 0 R E W 0 R D

Cultural and scientific relations between Germany and South America,

especially Brazil, date back to the last century, to Alexander vonHumboldt times . Throughout the decades many scientists from Germany

came to Brazil and vice versa, and quite a number o£ BrazilianFaculties habe been built up from German Universities . From the Germanstandpoint, there is no region in the world with such close relations

like Brazil .

Five years ago a group of German scientists went to an information

trip to Brazil ; the trip had been sponsored by the German Academic

Exchange Service and it should serve the permanent connections between

former DAAD fellows and German scientists . One averting in Teresopolis

the idea came up, why not having a common symposium an natural

products chemistry, and three years ago our Brazilian colleagues,

under the leadership of Professor Dr . Walter Mors, started with the

First Brazilian-German Symposium

on

Natural Products Chemistry,

which was a full

success

in

every respect, and the German

participants remember this event still gratefully and with pleasure .

Now we are happy to extend a cordial welcome to our Brazilian friends .

The number of participants on both sides has been extended . So the

program will reflect a real scope of natural products chemistry in

both countries, even if not all representative scientists could follow

our invitation . This booklet contains the summaries of the papers as

submitted by the authors . We wish the conference to be a successful

one far further fruitful cooperation between institutes in Brazil and

in Germany .

On behalf of the Organizing Committee,

Gerhard G. Habermehl

CHEMICAL. ECOLOGY AND COEYOLUTION AT THE INSECT/PLANT INTERFACE :

METHODS, BIOASSAYS, RESULTS AND APPLICATIONS

Keith S . Brown 1r . , lose Aoberto Trigo, Clecio F . Klitzke, Paulo

tusebia R . dos Santos, Ranaldo B . Francini, Silvana Aparecida Henriques

e Härcio Zikän Cardoso

Laboratorto de Ecologia Qulmica, Departamento de Zoologia

Instituto de BIologia, Universidade Estadual de Campinas

C .P . 6109, Campinas, 5äo Paulo

13.081

Brasi1

a-S» 2 aüa aäa;aYaa

Natural product chemistry research programs have been traditionally directed

at easily isolated, often apolar compounds of a certain structural class useful in

taxonomic, chemical or pharmacological studies . More recently, some projects have

sought compounds or mixtures with a given biological activity (such as anticancer),guiding the fractionation by specific bioassays ; these have often led to minor com-

ponents with very novel structures, frequently polar or unstable .

The commercialapproach . i n contrast, has led to the isolation from natural sources of large quan-tities of certain structures that, while not necessarily showing useful activity,can be used as synthetic intermediates for compounds with established markets .

The relatively recently crystallized science of Chemical Ecology, while parti-cipating in and using the results of all these approaches, initially directs aquestion at natural systems : what chemicals are being used to carry informationfrom some. elements in the system to others? All living organisms possess some sortof sensitive receptors to environmental signals (physical or chemical) that permitthem tn,adapt to environments sufficiently to carry out their three principal func-tions of growth, survival and reproduction ; their efficiency in the first two per-mits fecundity in the third, guaranteeing that any adaptive genes involved in thisprocess will multiply in the population, leading to evolution (change in gene fre-quencies from one generation to the next), This very intensive adaptive and evolu-tionary process, often called natural selection, has led over long time to a tremen-dous diversity of genes and their expressions in nature, related to the many differ-ent environments with varying stability, dynamics, and resources .

Harry of these selected genes lead directly or indirectly to structural elements .primary biochemicals for metabolism, or even behavioral capacities of organisms .All are capable of directing enzyme and protein synthesis, which in turn favor somechemical transformations over others, fading to potential production of the speci-fic "secondary" compounds (episemntides) that are the objects of natural productschemical research, When such compounds produce greater efficiency in growth, sur-vival and reproduction, they are selected for, stabilized and accumulated until thebenefit/cost ratio of their synthesis is optimized,

Chemical ecologists ask anothertwo questions about these compounds : wh1 are they produced, selected and maintainedPrecisely how dg they aid the producing organism in its adaptation and eventual re-productive success (fitness in the OhNinian sense)? The answers to these questions

K.S . Brown, Jr et al .

require rather special approaches to phyto/zoochemistry : mild methods of extractionand fractionation, specific assays for ecological activities,,nd activity-directedisolation of compounds, all within a framework of appreciation for the significanceof their production and accumulation in the microcosm of each ecological interaction .

This, experimental work in Chemical Ecolopy starts with observation of an eco-logical interaction possibly involving chemical communication, isolation from theliving producer organism of a chemical essence (extract, fraction or compound), anddemonstration of its activity in the original interaction, through a carefullyplanned and constructed ecological assay with the receiver organism itself or atrustworthy surrogate or component part (such as an isolated antenna, single-cellreceptor or enzyme system) . All steps must be planned and guided by this ecologicalassay, and in such a way as to conserve in the isolated elements the basic chemicaland biological properties of the living systems, including structures, affinitiesand concentration of compounds, proportions of mixtures,

flow rates and sequen-ces, and even neurophysiological properties of receptors, without retaining all theother complications of the complete living organisms .

To obtain ecologically active compounds from plants or animals, it is necessaryto rapidly extract and disintegrate living or fresh material directly with polarsolvents or polar/apolar mixtures, inactivating enzymes to avoid production ofaglycones or artifacts .

In the Chemical Ecology Laboratory, Department of Zoologyof the State University of Campinas (URICAMP) , we have developed several generalmethods to do this, that minimize transformation of inherently labile, ecologicallyactive compounds to other (perhaps more easily isolated) inactive substances .

Appli-cation of these in standardized fashion, to a variety of olants and animals, hasoften revealed very high concentrations of labile active compounds . usually withstructures already known, but often previously isolated in far lower yield or astransformed artifacts by standard dry-grind-extract methods (Table 1) .

Ecological assays developed range from predators in nature (such as visuallyoriented bird or lizard species, or the chemically oriented giant tropical orb spi-der NMhila. cl avipes , a very sensitive species to some alkaloids which are also respected by other spiders) and behaviorally complex adult butterflies (for pheromoneprecursors and analogs), through seeds of plants (noting germination and growth)and specialist caterpillars (acceptance, growth rate, metamorphosis) to strictlylaboratory-restricted receptors like domestic chicks, down to single antennal sen-silla cells .

Each system and each activity must have its own special bioassay, mi-micking the ecological milieu of the activity of earth compound or mixture . each typeof compound should have its own special extraction, fractionation and purificationmethod, once the interest becomes focussed only, on it among the many others present.While these restrictions do not help broad nharmacoloaical- type screening, they cangive in-depth knowledge of the basis of known activiths, their mechanisms, thestructure and function of active compounds, and eventually the raisond'et_re ofthese "secondary" compounds in natural systems - exactly the information most useful

TABLE 1 . Some chemical classes or compounds (ecologically active principles)

obtained by the authors in high concentrations from Brazilian plants and insects

04P0i1NDSJCLASSES STRUCTURES

2-sorbotriglycerides,°~caW,C~t-cu'

'J 43ra,_wwKArA

lieriaphin

,a

"

RD

Sesquiterpenehydrocarbons

Prenylatedhydroxyben-zoi c acids Lderi va ti ves

Dehydropyrrolf-zidine alkaloldmonoesters (fiteisomers) 1. ff-oxides

Linamarin

+ many isomers

Labdanoic acids

Leaves of bi-

Defense

8-16

B2 Isolated in(bicyplit

c'm434

labiate Arts- against

0.1-1% yieldditerpenes)

`Otl`

tolochia ,esp, herbivores

in previous

wlät1

A. esperanzae

work

cpotl

K.S . Brown, Jr . et al .

Leaves of

Defense

10

82 Hvch lowerpiperMlum

against

yield fromherbivores

dried leaves0

öri°u Flli Hectarfes of

Pollinator 2-4

C

Isolated inEupatorium spp,

attraction

lower yield or

U f "w3 Heliotrwium leaves

Defense A-10

C

not reportedto previous

3

Ithomifnaeadults Defense and 5-

C

studies withpheromones 20

dry material

CN3-C.-0431"a

All stages of

Defense

1-5

0

Yield falls tocrt

Acraeinae

0 .5% in driedmaterial

a Extraction of fresh material with polar solvents and , raptd fractionation at room tom,The indicated compounds represent 60-901 of the crude fractions obtained by each method :A a acetone-water, then ELOAc extraction, FlorSs'iTelorömaU-qraphy of whole extract.6 = HeCH-HZO-CHC13-hexane-1X HCl extraction, then HCOj and CO

removal of acids (B2)and flash rhramatography of remaining neutral material (RI), CHC13 fraction,

C - M0011-1110 extraction, then CHC13, H2SOq~~to aq . layer, Zn, then NH40H, C11Cl3-MeiiH-0 - Acetone-1120 extraction, evaporation, QiCl3, then CHC13-MeOH .

araanaaaarawsaawwawuarrw~arrarrreraaewwr :wwrsrrwwrwwawrraaawrrrawarwrawaaatarwa~a~ararraa

We have chosen as a model rfremico-ecological system, the interface between toxicplants and theirinsect herbivores . The latter must deal with the plant toxins in someway, and often wind up not only tolerating them but also transforming, storing, andusing some as effective defences against their own epemies (competitors, parasites,parasitoids, predators and pathogens) . Depending upon 'the strength of natural selec-tion (very intense in the tropics] and the activity spectrum and levels of these de-fensive compounds, useful to both plants and insects, their quantities may reach 5-i5%of the dry weight of some parts of the producing or storing organisms (in exceptionalcases, up to 2071 of the whole organism ; Table 1) .

In plants, they are often synthe-si2ed in the roots and selectively mobilired to plant parts in resnanse to environmental

Sources Activities '4 of dryweightExraractionedurea} NOTES

A~his nerii Fungtcida, Z3 Extraction of(wlth U1ric!r fense A dried materialgives ouch re-Weiss, HiH) g 4-olu ration duced yields

Leaves of Herbivore 434-2? 111 Steam distillat-many plant regulation ion or hexanefamilies extraction

gives

K.S . Brown Jr . et al .

challenges or communication needs . In herbivores, they may be obtained from the lar-val or adult foodplants or both, or biosynthesized from plant-derived orecursors orentirely de novo from simple metabolic units ; it is not safe to predict which of thesesources will predominate in a given animal, nor can one generalize the patterns fromone group of herbivores to another.

We have discovered, for example, that the more primitive aposematic Lepidoptera(brightly colored to advertise strong chemical defenses) often obtain their defensivecompounds from the larval foodplants, usually selectively storing or transforming onlya part of the plant's toxins (the "classical" case, often generalized to all interact-ions) . More advanced species, however, may obtain much larger amounts of compoundsfrom adult food sources, and the most evolved groups completely reject (excrete ormetabolize) plant toxins and biosynthesize their own defensive compounds, sometimeseven the exact same compounds present in the~pI

s j ,`,but notstored by the herbivores .In contrast, primitive phytophagous Chrysomelid beetles on toxic hosts do not storethe plant toxins, but show de novo synthesis of a wide variety of endogenous defensecompounds, only occasionally supplemented by plant-derived substances in a few advancedgenera (Pasteels E Rowell-Rahier) . Other insect groups, not yet fully enough studiedto be able to detect such patterns,

show both storage of hostolant chemical andde novo synthesis of defense compounds, as well as further syndromes .

In the realm of broad theory, the specific assays and also the microanalyticalmethods for different compound classes have permitted detailed tests of coevolution-ary hypotheses at the plant/herbivore interface - usually revealed to be unsustainable,

ith evidence favoring the colonization of progressively more effectively de-fended, more primitive hosts by advancing, gradually more preadapted insect lines -and also have cast light on the precise flow of specific ecologically active compoundsin natural systems . thus, chemistry can fulfil a unique role in understanding ecolo-gical interactions, investigating evolutionary progression, testing biological theo-ries and studying the dynamics of natural systems - all useful in aiding and educatinghuman usage of natural resources, and adaptation to the chemically complex environ-ments encountered, constructed, or produced by human populations .

Acknowledgements :' We are deeply grateful to the Brazilian CNPq, CAPES, FAPESP, FAP-UNICAMP and BNDE for research grants and fellowships, to the Institute de Quimica -UNICAMP for use of instruments, to the Uepartamento de Fisiologia vegetal for use ofa colorimeter, and to Dr . Miriam Rothsthild for continuous stimulation of this work .

EVOLUTION OF PLANT METABOLISM : 1HE REDOX THEORY

OT10 R, (3011LIEB

I1lstituto de Oulniica .

kWivf'F F- idcSCi4`

Säo Paulo, EP, Era:il

it) the late Silurian, when olaiste iritt.iat.ed

coicmiization of terrestrial enviroisment " ,

tlle,y c-lic°misrtered

as major stress factor the oxygen pressure of atmospheric

air . ill plants, oxygen is partially reduced to suneroxide

and other reactive free radicals, apt Lo destroy 1:ey

biomolecules . Since already the Carboniferous was

characterized by arborescent lycopods, xr-caumaryl alcohol,

witiferyl alcohol and sinapyl alcohol are not only lignin

precursors, but also the most likely candidates for early

radical scavengers or antioxidants . Guaiacyl lignins,

vredominating hugely in Paleozoic pteridrohvte ai)d Mesozoic

glmnosoerin forests, were supplanted by

evrirrgvl l igiiiiis in

Snnots~ic; yrigiti;~i~etrnl forests . 11i1s pv0ltri i0nar v Sf"rlllwnce runts

parallel with increasing antioxidant activity f:t ijle

rnorroligrroids arid decreasing strength c%f their r,,,, id'ition

products, tile l ignites,

Flavoiloid tarlrlins are present, ill roltsider able

dssantities in pteridophytes and gyrnllosrierlns, lis allgiospernis

the trend towards evolutionary develol .rssent tii tlre-~,

nroarithocyanidins continues with their 5ttr11ir, acid eslers,f1%11owod by

gaIIotannins aiir! 1113git,arsrl ills,

'lariniric are

rilsoi,rliical scavengers and agmiii the evnlur.ioriarv se4tfiance runs

parallel

with

iItcreasing

ailti OYidarit pcng±r

mild

der,tP. ;i=1nq

bioactivity .

From the Ctetaceou*i le rthir y

bolirsdilt'y

wrtrr'tld

a

c

Naphthyl lsogWnoüneAlkaloids -Stereochemicaliy, ßiosynthetiraily, and PharnacoiogicallyUnusual

ßiatyls From Tropical Medicinal Plants

Gerhard BrlagummInstitut für Organische Chemle derürtiversität Würzburg

Am Hubland, L]-8700 WCuzbur& FRG

The families of the I?iancophyllaceae and the Ancistrodadaceae, botanically trop-ical lianas from Asian and African rain forests, are widely used in folk medicine(e.g. against malaria, dysentery, and elephantiasis).

In the lecture, the isolation and structure elucidation (resp. revision) of naphthyiisequinoline alkaloids]), first of all from the Westafrican plant Triphyaphyllumpehatum2,3l is reported. Exemplarily for the main alkaloid dioncophylline A4sl(previously "triphyophylline) (!), the spectrum of conventional and new meth-ods for the establishment of the complete stereostxucture of 1 is presented, In-cluding the "normal" spectroseopical and diiroptical methods and novel cheml-cal procedures for theelucidation of configuration at the axis and chemical degm.dation procedures, the stereoselertive total synthesis, and finally the c+anfiMs.tionby X-ray crystallography.

Completely different from the synthetically "disciplined" plant T. pdtafum is thebehaviour of the liana A. abbrrdaftOM : It produces a broad spectrum of partiallyvery similar al)caloids (e.g. 2 and 3) that nearly always occur as pairs of stereo,ismas (with varying central and axial configurations) and Involve several new

regioisomeric coupling types - which may be interpreted, from a human point ofview, either as an overwhelming structural variety or (rather) as a chaoticdeficiency in synthetic stereo- and regiocontrol.

Besides structural, especially stereochemical aspects, these naphthyl Isoquinolinealkaloids are interesting also from pharmacological and from biosynthetic pointsof view.

Literature:

1)

2)

3)

4)

5)

6)

review: G. Bringmann (The Naphthyl Isoquinoline Alkaloids) in The Alkal-oids (A. Brossi, ed .) Vol. 2.9, p.141, Academic Press, NewYork, 1986.G. Bringmann, M. Rübenacker, J.R. Jansen, T. Geuder, Plants Med. 56 (1990)495.G. Bringmann, M. Rübenacker, P. Vogt, H. Busse L AM Assi, K. Peters, H.G .v. Schnering, Phytochem . 30 (1991) 1691 .G. Bringmann, M. Rübenacker, J.R. Jansen, D. Scheutzow, L. Aki Assi,Tetrahedron Lett . 31. (1990) 639.G. Bringmann, J.R Jansen, H. Reuscher, M. Rübenacker, K. Peters, H.G . v.Schnering, Tetrahedron Lett. 31 (1990) 643.G. Bringmann, D. Lisch, H. Reuscher, L. Aki Assi, K. Günther, Phytochern .30 (1991) 1307.G, Bringmann, F. Pokorny, H. Reuscher, D. Lisch, L. Akts Ass!, Planta Med.56 (1990) 496.

Joachim H.H.StäckigtLehrstuhl für Pharmazeutische Biologie, Institut für Pharrnazie, JohannesGutenberg-Universität Mainz, Gerntiany

The Bias nthesis of Monoter enaid indole Alkaloids

During recent years much progress bas' been made to understand the

biosynthetic mechanisms leading to monoterpenoid indole alkaloids of the

Apocynaceae plant Rauwolffa serpentine Benth . This progress was exclusively

based on one hand an the use of Rauwolfa cell cultures producing substancial

amounts of ajrnallne and its derivatives - on the other hand on the isolation and

identification of the enzymes responsible for alkaloid formation . These in vitro

studies provided for the first time a detailed insight into the biosynthesis of the

structural complex and pharmaceutical interesting Rauwolffa alkaloids belonging

to the sarpagan and ajmalan group. In contrast, classical in vivo feeding

techniquesapplied in the past in biosynthetic research did not result in a detailed

description of the appropriate biosynthetic routes .

At the p;eserit time the identification of a whole series of enzymes and the

isolation of the pathway intermediates allows to summarize and to connect thebiosynthesis of the three typical Rauwolfs constituents ajmalicine (I), sarpagine(1I) and ajmaline (II1).

10

Ajma1scine (1)Serpagine(11)

Ajmaline (111)

Oxidative Bond Clavage; an Ubiquitous Biosynthetic Route to OlefInes .

Wilhelm Boland .

Institut flir Organische Chemle der Universität Karlsruhe.Richard-Willstätter-Allee 2, 0-7500 Karlsruhe, FRG

Olefinic, Irregular carbon backbones arewidely distributed ¬n nature . These Include

among many others Irregular terpenes, odd numbered 1-alkenes or terminally un-

saturated polyacetylenes derived from fatty acids, furanocoumarins and certain

C11 hydrocarbons from terrestrial and marine plants . Without exception, the bio-

synthesis ofthe aforementioned compound classes has at least one oxidative bond

cleavage reaction in common which introduces a double bond with simultaneous

loss of one or more carbon atoms the originai backbone . Two ofsuch bond clea-

vage reactions will be discussed In detail . These are: 1 . Oxidative decarboxylation

of fatty acids leading to 1-alkenes In higher plants and insects . 2. Oxidative clea-

vage of the terpene alcohol nerolldol In flower heads of many terrestrial plants to

give the homoterpene 4,8-dlmethylnona-1,3,7-trlene.

2) +

2CH3000H

In each case mechanistic Informations are gained from administration of enantio-

specifically labelled ( 2M)precursors and subsequentspectroscopic analysis ofthe

resulting metabolites which are usually obtained at the ng --> lower fig level . The

results are consistent with a direct conversion offatty acids into 1-alkenes, while

the terpene alcohol nerolldol Is cleaved via a sequence of reactions Into two C2

fragments and the homoterpene. The latterconversion shows striking similarities to

steroid demethylatlons .

THE MGCTROCIl1SMISTRY Oil NATURAL PRODUCTS- THE REDUCTION OF COSSIPOL ONMERCURY,

1N TIM ACID pli RECION, IN A MIXED W001-AMONE SOLVENT .

'J .Tonltolo *, s't .O.r.Goulart **, A .L .G .Sant'AnaA* attd J.f.C .Boodts* .

.* Departuweuto de quiutica-raculdade de Filosofia, Cicucias'c Letras .ac Ri-

beirao freto/USP . Av .üandeiratites, 3900 -- 14 .049 Ribeirao 1't'eGo,Sl',Btca-

sil .

* *Departautento do quinica/CCEN-Univcrsidade federal de Alagoas/UFA1 . 57 .000

fläcelö, Alagoas, Brasil .

1 . INTRODUCTION

Cossypol, a polypltcuolic binaphLEtyl, is a male antifertility agent

of

considerable current interest . An excellent review of it's biological. acti

vity has been recently published by Walter at a1ii (1) .

'

. Few 'clectrochemical investigations of-£ossypol are available in tike lit

terature, tuost of thatu focusing on analytical aspects (2-4) . 13rauik (5) pro-

posed a two electron/two proton clectrode mechanism on taercury and wethattol

ups solvent . The polarograpikic wave was ill-defined because of kite proxiutity

of tike discharge of kite supporting electrolyte, S .L .

' 2 . EXPCltxau:NTAt

. Tike alcctrocltetuical behavior 01, gossypol on mercury, in 11 20 : ., acetone

v/v,Nas investigated by d .c . and a .c . polarography and

coulontatry .D.C. and a .c . host poiarograms were recorded on a Polarecord f:505

-r 8506

cquipuicnt (lletroluu, Suisse) . folarographic worlci.ng' solutions contained -ltng

gossypol%l0 tut

-414) . Ionic strength wäs maintained constant at 0 .514trouglt ,the addition of NaCl and the p1P, adjusted by Rellvaine buffers

01:

11C1 . ALL polarogratus were recorded at 2:'i C and 1 .4s

dropl:i.me . Tho intluen--cc of plhc, concentration, droptime, lieight of tire mercury column and tempe-rature were examined . '

The controlled potential coulometric iovostigatiou was executed ail

aVAR 173 potsutiastat equipad with a model 1'AR 179 digital coulontetcr .

Theinfluence of the potential and concentration (under way) oil Cite

apparentnumber or transferred alectrone, nap , have been examined .

1 2

3 . RCSULTS

The d .c . polarograms, showing a single wave'overlapping with the dis-

charge of the S .C ., were analyzed by the Gauss-Newton algorithm (6) . .A 65

5mV slope of the 1ogaritlunic analysis was obtained . The influence of all* on

E1/2 is shown in fig . 1 . A linear relationship between'iI and l:l/6 was

ob-

tained. The same behavior was observed between E1/2 and log t, the

wavebeing displaced

anodically

by 30 suV In the 34-80 cm range,

depen

ded linearly on thf1g2. An approximatly logarithmic relationship bctweeu

iland the gossypol concentration was observed in the P .01-0.4 n11 interval,thelimiting current tending to a-constant value for

higher

couceArations .

131/2 is independent of the concentration . Temperature

coefficients found

-for the limiting current were 3 .9 (pA* 1 .1) and 1.6ZOC1 (alt* 2 .8) .Two peaks were observed in the a .c, polarograms . The principal

peak,

coinciding with the d.c . wave, is displaced to more negative potentials siLth

increasing pli* . The second peak, having no counterpart in the d .c . and over

lapping at the lower all* values with the principal peak, has a peak poten-

tial (-0 .80V vs AS/AgCl, HaCl sat) independent of pit* .

.

The influence of Eap an nap is shown in fig .2.

4 . CONCLUSIONS

Considering that the gossypol molecule has two identical parts,

eacli

containing one aldehyde group, the C .F .C . results indicate a two

electron

transfer per aldehyda . Having tire first linear'segonant (all* 3 .2)

of ' .-'the ,

El/2 vs all* grapha 70 mV slope, one can conclude that twolwotonsparticipate

in the electrode mechanism . For pH* 3 .2 the participation of no protons are

observed polarographically % The behavior of nap vs Eap is clear

evidence

for ,the participation of a chemical reaction (probably dimerization ) in the

electrode mechanism .

5 . RCIERENCES

(1) D.P.Waller, L .J.D .Zanoveld and N .at.farnsworth, Economic

and fedicitial

plant Research, Chap . . 3, Academic fress'Inc., London, England (1985) .

(2) L.Lacombe, A.Valkafouki and H .Kunesch, J.Nat.Prod ., 1987, 50, 227 .

(3) A.L .1larkman and N .Koiesov, Zhur Priklad, Khim, .1956, 29, .242.Xbid,, 1956,

z9, 424'.

13

(4) Y .Jiang and T .Zhou, Penxi 1[uaxuc, 1984', 12, 338 . Ibid, * 1985,13,536 .Ibid .Yaoxue Xuebao, 1984, _19, 195 .

(5) It .D.Drauu, Iaectrochimica Acta, 1987, _32, 459 .(G) O .L .AoLtecchia, L.Degreve and J.P.C .Doodts, J .Dlectroaual . Chem .,, 1990,

in press .

G . ACKNOWLEDGMENTS

Tlte authors wish to acknowledge the financial support received fromIe'M'INI', CNINI ;Mud FINEI'/PAUCT .

SY3ülü'%SiS AIiD HIßLOGICAL ..Li1YITy ui~ 3-, ;- and a-AL1CY1.~~2

~~rHOx~c-1, 4-BExzflQ«xr+ßt~~s .

pale J . Brvndani, Pedro Rolim Neto, Eegina M. Generina, 6~ariaA. de SToraes e Sauza, Rosälia H. ß. C . Lima and Lothar W . Bieber,

Bepartamento de Antibiöticos, Universidade Federei de Pernembuoo,

50.739 Recife .. Pernambncv, Brazü .

Several simple n-alkyl-s++bstit++ted 2-tnethoxybenzaauinones have

been fo++nd in nnt++rC . Part of them are prod+eced by f++ngi 1-3 .

one is fownd in higher plants 4,5 and another in arthropods 6 .

They all shoe interesting biological properties, s++ch as aller-

genie and antifeedant activity, antimicrobial, trypanocidal and

vytotoxic action ar even antic++mrn+r activity . HvRever, no syste-

matie pharc+ecvlvgical study eas nndertnken except far primin

{1, R 4 n-pentyl) which has been used clinically against severe}.

types of skin cancer 7. This may be dne in part tv the problem

of providing s++ffioient q++antities of the grad++cts from natr+ral

sanrces . Qn the other hand, all kno+rn synthetic approaches descri-

bed in the litnrat++re are loa yielding multistep groced++rea or

proceed non-regioaelectively. In order to enable strict++re/acti-

city st++dies of the nat+rrai products and their ,+nnetural homolo-

goes, ne developed nee short and regivselective syntheses for

the three isomeric series of type 1, 2 and ~, .

xhe 6-s++bath,+ted compmsnds 1 are best prepared by catalytic oxy-

dation of the corresponding phenols A which can be obtained regio-

selectively by üthiation and alkylation of the protected g++aiacvl

The isomeric 5-fslkyl-2~othoxy-l,4-benzogninoncs 2 +rere syn-

thesized hy additfvn of trialfsylboranes tv aethaz;~benzvquinvne ö

15

twined by regioselective lithiation and alkylation of wiitable

protected methoxyhydrog1+inone 7 to 8, followed by hydrolysis

and oxydation . Most interestingly, by proper choice of the reac-

tion conditions of this sequence, the alkylation can also be di-

rected selectively to the 5- or 6-position. This opens a direct

synthetic access to all three isomers of the desired prodrncte

from the same starting material .

Preliminary biological at+dies indicate that all compounds of

the three series have similar antimicrobial and cytotoxic proper-

ties . The allergenic activity is most pronounced in the 6-s+lb-

stinted series with a maximim for the natrral product primin .

Only 3- and 6-s+ibstit,oted compounds were active against trans-

plantable tumours in mice and best inhibition rates were obser-

ved for 1 ($ - n-propyl) . Based on these preliminary rewilts,

it seems possible to dissociate allergenic and antitomour action,

opening a new chance for a primin analogee in the treatment of

skin cancer.

REFERENCES :

1 . R,5. SOOd, K. Boy, G.C.S . Reddy, x. Reden, B .M . Cang+jli, 3,

Antib. 35, 985 (1982) .

2, I . Volc, P . Sedmera, K. Hoy t V . Yasek, I . Vokoun, Collect .

Czech. Chem. Comm. 42, 2957 (1977).

3, M. Anchel, A . Henvey, F. Favanagh, J . Polatwich, W,J. Robbins,Proc . Irnt . Ac~-ad . 5

, ci, U.S,

4, 498 (19$$) .

4 . ' H . Schild]anecht, I. Bayer, H. Schmidt, Z. Nat�rforsch . B 22,36 (1967) .

1 6

5 . O.G . de Lima, G.B . ICarini-Bettölo, F. Delle Monache, J.S.B .

Coelho, I .L . DIAlb+iq++erolre, G .M . Mactel, A .L . Lacerda, U.G .

Martins, Rev . Inst . Antib . (Recife) 10, 29 (1970) .

.6 . R.H . Thomson, Yabirally Occurring Qsvinonea, 2nd ed ., London,

Academic Press, 1971.

7 . A.M . Melo, M.L . Jardim, C.P. do Santana, O.G . de Lima, I .L .

D'Albnignerque, Rev . Inst . Antib . (Recife) 14, 9 (1974) .

8 . L .71 . Bieber, A .A . Chiappeta, M .% .M . Souza, R.M . Generino,

P. Rolim Heto, J . Nat . Prod . 53, 7C6 (1990) .

OTHP

,THP

OCH3

Q,b $c

at n"BuL1

c: H30+e : fi304/Fe3{

b : RX

d : 02/salcomine

f: R3B

R m n-allcyl

17

IH

'- I :!-I ,

i~iQCF13

L

QTHP

0

. OCH3+~OCH3

e

1NTRODUC1x014

NAT(1RA1. AND SYNTHETIC l3IOACTIVE NEOLIGNANF.S

prol, Ar . LALtpa C . 10- PAAATA10-VNSCAMA campinn6 . Sp

13ac11gnanx [13 are dimdrs from oxidative coupling or ally) andprapanyl phonois, that occur in Oho Myristicaceao and ot.horprimitive plant families. Of the five general of MyrisLicAceaeoccurring throughout, Lho Americas where Virola is thw mostrepresenCa6lvo, with the largest number of species 12 .37 .Virol surinamonsis C12o1 .) Warb represents Lho principalBrazilian wood cut and exported to tho PYrst World as venovrand plywoods [3i .

In 3970 . initial studies of loaves of VS toia 143 loci toisolation ofthe first 8,0.4' neolignan of this genus .'fho crude hexane extract of V. uri amensic loaves Crejectvci in thesawmill) showed high efficacy in the in viva blockage texts ofponotration of cercaria worms of 5ehistosoma man2ani 14-Ci3 . in mice .Tiw autivw substances resporssiplo for protection were isolated andAuan16.111oa as naLuras tj .v.~j'- uoltyi~ans"y3'1'Otxn',.'I) aril$ '

-

`surinamansin (Z) 1ß.t31 .

Recently the absolute configuration ofC-f)-surinamensin was datormined using diastorvomoric C1-methyl mandQlJCosiers . {7, Additionally we have isolated voraguonsin 3 . a noolignanwhich was recently show,% to gave pront.unzod anti -YAFC pial.olaL activating £actor$ *&Lion 183 .

ko-working of V. surinamonsisleaves . gave further THE noolignans and also flavones, lsolatwd forLho first time from this plant family .

In order Lo obtain noolignans from leaves o!' y.

surinamon, ,i

,yav n[s CA.MC.)Smith. V_t oloifora CSchotL?A.C . Smith and

Qsteo hiaauzn las ysparmum . a straightforward separationmel.hod woes developed to eliminate chlorophylls and coloring matter .which also concontratw bioacLivo neolignans . ready for biologicalassays and spocLroscopic data (1-J) .

Ph)ftr.~hcmieal study of O~pjAL%Mr;r&untpenes and ditorpones, but no noolignans .

The extract. and puluz~iitist.~.nco_ from thin plant, nhowod good rosultm against. tur.yi andbactaria C E.coll and S

au our) .(10) .From V, oavoas loaves we have isolated, among others. a T%:'

noolignan C4) .

which had significant anti-PAP acLiviLy in the mousevascular pormeabilityiassay (113 and a new aryl-propanuid C5) .

In research aimed at, development of new drugs .

thebiological act1vi6y was dwi.orminAd for thoso of ours nat.uralnoolignans and ca . 50 now synthetic noolignans . derivatives andanalogs carrying orvgon CType I], thiLroaor. CTypo I3) "lid yulfurCType IIID in Lho C "-8 bridge of the dimor. A71 empirical correlationstudy is being undorLaken botwogn biological activity and chemicalstructure of those natural and syntio®tics subrt,"ncos . III close,collaboration with skientists irs Euronn .

ti fi

anri Mra " ri t

Tsr"target. is the chamotherapeutic contra! of certain tropicaldlsoases (Schistosomiasis and lVishmaniasis), cancer, cartainbacteria and fungi . as well as discovery of ant,$-PAP synLhPM r.cubstancac .

18

Thu chlorophyll -free fraction of Lhn hrx:ino ovlrs.a l. mfV.

s..lrin rags©nets, laavos . rn wol l us . a koLolur ddriv:.Ll v .r c31'

'vlrollno C1) have shown anti -cantor activity In oxporimontal loukomiaP-388 1121 . About 30 synthotsc naolignan analogues . carryingoxygen (Typo ID in Lho G-8 bridge were tasted and have shown importantactivi l .,y i n the i n vi vo Lost of cercari a . worm ponotrcnl x on . -

tldol i gnananalogues carrying oxygen CTypo I3 or nlLrogon CT,ypw lI'l .in Lllo G-8bridgo are equally active to bacteria and fungi 11'3,14] . Sulfurdoriva%A vos CTypq 111) are 1nacLive .

In order to expand Oho spectrum of biological activity .c;mmpounds Type I and III warc 4ostvd acainst . 1 Pi ;hmani act c j n .,1 1 rte.Cmacroph.,ges Mfo`-tod wltli alnasCigotos df L:oishmaniA donovani g .H1 ghoct acts vi Ly was found in uumpuuntim of Typo I , with nosubsti.Luents .

Thiol dorlvatives°CType III :), showed good' Ln, vitroactivity similar to the first choice drug sodium &tibogluconaLe .Unfortunately . Ln vi vo C 8alb nd co] t etl .c. dicL not_ show- t hw %ame -performance .

Recwr.tly . a sulfur neolignan analog CTypo III :) coded LS-91 -wastested as a PAP antagonis .L.This compound . inhibits dose-doporl-dontly the PAP-induced increase of vascular pormoability in rats . butfails to affdct the same phonomana produced by bradykinin, histamine,sorotonin or carragoonin . PAP-acoLhor induced aggrogaLtion or rabbitplatolots in plasma is effoctivoly inhibited by LS-Sl . whoroas Lhoaggregation Lriggored by collagen or ADP is no4 modified .

Thoseresults suggest that LS-S1, could be a spwcifir. AnLagonick of PAF-aooLhor" . " ao pel.an6 - .rl.llml - &;ululxrujIdU"Ail r0adyvvz'crItwo l1Yhli ?U131f MW9N52021 .) .

o rk-

STRUCTURES OF DIOACTIVE N£OLIGNANS

R.

i

Z

3

oll

.%OMC

F

1 9

oxt

ACK?4OIRLEDGLZHENTSf " 'i3t,o uuthor-would like La Lhatsk-to ärs .Padro_H .

Farr1, L^ttrivaldc% ft Silva Santos . Marcai clu ouoiroz1'w�1,:, fc,r

aSonLific aunport of this work, Lhu Labraratorio du lo,:r,oleyiaF'i,r

,nacöuLic .a - UFRh . fnr. mLcrobial Lasts, Profos%or Rilph Hwal 5--:of tho l.orsdon School of HygienQ and Tropical Modicino for3aishmzniatla torts . Profossor Ronato B, CovdrtEra . of FIOCRUZ -Rio do Janoiro for anti-PAF torts, and rr, Mvrlan C. Ivrc)an oftho UFRJ - Rio do Janeiro for schisLosomiasis Lasts,

REFERENCPS

1 . O.p . CoLtiiob . Ro*Lignane Fnrtcrhr . Chnat . nrp . Nalurxt . . 351C1078)

2 .

1l . F .

Paul i no l""° . t

Gcol ogla quimE ca da C ami l i a Myt i s1.i r, ac oaw,PhD ihoais, TO-1t4F . Säa Paulo . Brazil CIOSG)

3. W,A. Rodriguos : Rfrvisäo googrhi'Lca das o_p3cEpc do Vi .rolaAublaL CMyriotichcoaa) 'do Bracll . Acta An-z+anica CSupl -? 10i 27C1080)

4 . L.E.S . Baraa. o P .M . Baker . kuimiaprofilaxia da osquistosso-moso . aLividado do V . *; urCttamvrtsit . Ciarre, n Cult., 25, 100C 1a7;4')

G . L.E.T . 8arala . P.M. Sakar, O.ZCottliab o E.I~ Ruvoda:- "" .Hoolignans of V . surinamanstir . Phytochvmiztry, 17, 783CIO78]

D . L.£.5. Barata : I sol amonto o si rtLoso da ntaol i ananax do V.i re t a .nuri nurn,rnr: i L C RUi , 7

har ti. - Ohfs,

I'},osi s .

20 UNi CAIIP .

C.~rnF.l n:~s ""SPC 107ß.)

7 . L.S . Santos v L.F,S. Barata : Dof.orminAtion absoluteConflyuraLlon of C +] -surinarnonzin's XIa diastaraomoricO-MoLhyl mandollc ostors . J . 8raz . Chom . Soc., 1, 47CIO40)

O . 1N . OraquaL, nnd J,J . Cddft+oid% Con!'nrwational propc,rtioc c%rLha PAP-acather t"oeaptor in piatalots barod on =it uctuvo --Acti+vit.y studios, Sn Platalot ActravatLng Faot.or' . F . S�vdor Ed . -f1gnum Proms CIOSEY.)

0 .

P. H.

Perri .

A. 14 . A, P .

Fornandfir and L . E . S.

Barata "- A st raightrorward saparatJvo rnothod of' rtoclignans rrom lc,:rvos,1?1 atsta Modi ca C bui ng submi LL od)

10. M,O, PAU10: f4tudo f1tPquimico dar. fcolhas . do Viroln rrurinrmtarutso Ostoophlooua plalyFpormum . Me, Thocir, IQ/UNICAMP Can+Pinas-SFC1983>

13 . P . H . Parri : Nool I gnanas v ari l propanaI da da:: rol hac doV . pauonea C A, DC. 3 A. C, ymi th . MSo . Thosi s . Campinas-SPI CIrUNX CAKP C 1 UE1fii~

12,

M. M. Ol i val r'~ .o MrR . "p . 'Saa,pal os

Arr~Y, da- 11-drsarr:e vl r-ol l-na vdarlvado nm loucomia, oxparimuntal . Ci6nc. o Cult.., 32,104C1979)12 . L.£.S . Barratat svnthorle Ahd ahLxtosomjcldal, uhLlbiotic andr-ngw4cw ytlyil-las or 8.0.4' nvolinan xncl~.

ncl analoci+ps.~ vcon~airt~ng

rrtt-

8tt'3nd'sirllitr ADULFa..Ls S1

al .t : 'K-ä .' CnrtgrwsFon NaLuval Products ASP . Norti, Cavvlit%a . Chaptrll MillP . I IQ C1485)

J4 .

E. P.

Llma .

R, f,

Mal A .

R. C . S, P . C.

8arhosa .

L. Xavi sr

Pi I ho,

M. Ca.Paulo,

L. S.

Santos w L, E, S,

Barata :

ati vi d:tde anLI ml crobi anadP nool 1 grnanac 8,0,4* o Dori vados Sl nL6ti aas-l Ciärrc . a Cult .Saüde, v, r33C 1089)

2 0

1G .

I .H.

Florra .

L.E.S.

BaraLa,

L. S.

Santos .

H. N.

Cruz, P .M.R .Rodri guos o Si l va .

M.C.R.

Lima.

M. A.

Martins .

R .S.B .

Cordoff roiLS-S] . a synthotic dorlvad nuoliui,an . a poLont PW-ACulhorantagonist compound . Abstracts of Lho Brazilian - Sino Symposiumon Chemistry and pharmacology of Natural Products, R]o doJanoiro . p.11 .4 C1480)

Applications of Transition Metal Complexes in Alkaloid Synthesis

Abstract

Priv.Doz. Dr. Hans-Joachim Knälker

Institut frir Organische Chemie der Universität Hannover

Schneiderberg lB, 3000 Hannover 1, Germany

We developed methodologies involving consecutive ironinduced C-C and C-N bond formations

for the synthesis of nitrogen heterocyclic compounds. Some applications of this strategy to alkaloidsynthesis are presented. Using a sequence of eiectrophilic aromatic substitution of an arylamine

with an iron-complexed r15-cyclohexadienylium cation and subsequent oxidative cyclization thetotal synthesis of several pharmacologically active carbazole alkaloids has been achieved . Theoxidative cyclization can be performed directly as an iron-mediated ary[amine cyclization . Thismethod has been used for the syntheses of the antibiotic carbazomycin B, carbazomycinal, andsome 1-methoxycarbazole alkaloids including the cytotoxic koenoline. The iron-mediatediminoquinone cyclization and subsequent dernetalationlaromatization provides an improved route tothe 3-methoxycarbazole alkaloids, which has been applied to the total synthesis of 4-deoxycarbazomycin B and carbazomycin A.

The iron-mediated iminoquinone cyclization proceeds in a diastereoselective fashion and istherefore exploited for synthetic approaches to other indole alkaloids . Ahighly chemo-, regio-, andstereoselective Sakurai reaction of the iron-complexed 1-methyl-4b,Ba-dihydrocarbazol-3 -oneprovides a promising precursor ofaspidospernma alkaloids.

The combination of electrophilic substitution of an arylamine and cyclization vianucleophilic substitution of a leaving group by using an appropriately substituted iron-complexedcation results in a diastereoselective access to spiroquinoline derivatives. This reaction representsthe key step of our projected total synthesis of the discorhabdin and prianosin alkaloids.

Synthesis of chiral lipid lowering agents derivedfrom natural products

E . Baader, W. Hartmann, G . Beck, H . Jesdralla,K . Kesseler, G. Wess

Hoechst AG, 623o Frankfurt 8o, Germany

A variety of mevalonolactone derivatives have beenprepared from either chiral pool intermediates orthrough enantioselective total synthesis . Systema-tic exploration of structure-activity relationshipsof these compounds with respect to lactose ring anddifferent heteroaromatic and acyclic lipophilic moi-ties was performed . The compounds of these studieswere evaluated for inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase in vitro . In severalanimal species the pyridine analog HR 780 exceedsthe biological activity of mevinolin and exhibitsa longer biological half-life and a more favorableorgan selectivity .

New Potent Antrasthmatle Constituents from Higher Plants

H. Wagner

Institute of Pharmaceutical Biology

University of Munich

IGarlstrasse 29, 1a-8000 München 2, FRG

Theestablished andasthmaticpharmaceutical products available today, for themost part incur

considerable side effects, and, with theexception of corticoids, have little or no effect on the

late phase bmnchoconstriction . Thm disadvantages, and the fact that most present day

antiasthmatics are derived from natural products, prompted us to search for new potentially

andasthmatic plantconstituents . in contrastto previous screening investigations we used only

ethnopharmacologically preselected drugs, and inidAy an in vivo model system for testing

extractsand fractions. This non-invasive method (plethysmography) using guinea pigs, allows

to measure the antagonistic effect upon artificially induced bronchial constriction (PAF,ovalbumin, acetylcholin or histamin). As a measure of the obstruction level compressed air

was used . Inaddition theextract fractions and isolated compounds were tested in enzymeand

primary cell culture assays for inhibition of cyclooxygenase, 5-lipoxygenase, thromboxaneand leueotriene biosynthesis, and platelet aggregation as well as the effect on chemotaxis ofleucocytes and release of histamine from mast cells . From this plant screening four drugswere chosen for detailed studies.AljiUm CM (onion)

From the chloroform extract antiasthmatieactive thiesulfinates and sulfrnvidisultdes (cepae-nes) of as yet unknown structure were isolated, Its functional profile indicated that themechanism ofaction is different from that of ll-sympathomimetics and andcholinergic agentsas well as corticoids .

The andu3sthmatic active principle of this plant which is indigenous to South and MiddleAmerica and which is used in Germany as a homeopathic remedy, consists of gallic acid-Me-ester and tetragailoyl-duinic acid . The flavonoides only marginally contribute to thiseffect .

24

Adhatoda vesica

From this drug which is used as an expectorant in India, we succcded in isolating a vasicin-and vasicinon-free alkaloid fraction which in the in vivo test surpassed the effect of the mainalkaloids. The alkaloid which is responsible for the antiasthmatc effect is suggested to be anoxy-derivative ofvasicin with an as yet unknown structure.ficrorhiza currooa

The antasthmatic property of this drug was localized to the phenol fraction from which theantiasthmatic effective acetophenone glucoside Androsin and its aglykone Bwgxnin wereisolated. Thecucurbitacin and iridoide glycosides which were found in another fraction werenot directly involved in the effect. Synthetic modification of these phenol derivatives resultedin some compounds with an increased antiasthmatic effect,

A dose response relationship was established for all compounds isolated from these drugs.These results are compared with those of other compounds meanwhile detected in plants ofThe Traditional Medicin ofAsia.

Same investigations in the plant family Amaryllidacea

Werner nöpke und Edda SewerinFachbereich Chemie der Oumboldt Universität zu Berlin

Amaryllidaceas are, apart from a few exceptions, bulbous

plants and have their main field of distribution in the

steppes of the tropics and semitropics, preferably in the

southern hemisphere . Several tropical genera and species,

however,are cultivated as ornamental plants in Central Europe

making them available rather easy . The best known represen=

tatives to be mentioned here are amaryllis,cliv is, crinum,

hippeastrum,haemanthus,vellote and nerine .

The chemical investigations of the amaryllidaceas until

recent years was confined, an the whole, to the isolation

of lycorin, which due to its small solubility is easy ob=

tainable .When our team in Berlin began to deal with this

field,only about ten alkaloids of amaryllidaceas where known

to be considered as uniform . It was found that in the case

of amaryllidaceas we are concerned with a plant family, un=usually rich of alkaloids . In about 150 different species

and verietis analysed in our laboratory in recent years,

more then one hundret thirty alkaloids where found so farand this number is expected even to grow if the investiga=tion are extended to species,ths access to which is moredifficult . Two remarkable findings have been made ; first,none of this alkaloids occurs at another place in thevegetable kongdom or has been discovered so far and second,the structural types apparently do not occur with alkaloidsof other plant families . Nor are there any indications thatthe alkaloids of the amaryllidaceas Bra structure relatedin any way with those of other plant families, especiallynot with the alkaloids from the botanically relatedfamily of the liliacee,some species of which are formingsteroidbases,such as veratrum and fritilaria, whereaswith others,e,g .colch icum colchicine and related compoundsoccur .

26

On the basis of their structural types, the alkaloidsknown so far can be subdivided into six groups which arereferred in accordance with the best known representatives

In the lecture T should like to deal with the lac=tone alkaloids exclusively, since this group offers anumber of chemical and stereochemically interesting as=pacts .

POLYHYDROXYLAIED PYRROL371DI14E A14D INC10LIZIDINE ALKALOIDS

FROM ALEX GRAND F

Q

MARIA AUK]LIADORA C . KAPLAN,

ANA CPI Sl I14A DE

S .

PEREIRA*

and JOSE

GUI LHERI.E S .

MAI a)"

Pq .duesas de Produtos Naturais, UniversidadeFederal do Rio de Janeiro . 21441 Rio de Janviro . PJ,

x" Mugc- v' Faraense Emilio Goeld" . 6600 . 50ki . FA

Trte tetrahydroxy indc"lizidine castanosclermlne + 1 ! hss

potent and r-glucosidase inhibitory activity . The

alkaloid has been located initially in Castanasaermum

australe A . Cunn . from northeastern Australia and later also

in a series of Amazonian Aletia soeaies . incloding A,

aran¢iflor Dude (1] . Only a herbarium spec}nier, of the

latter plant having been analysed, it was deemed of interest

to examine also freshly collected mateeial cvch as leaves .

branches, trunk wood and seed:,

2 8

The major component of the ethanol ic extract of the

leaves was found to be the novel 3-carboky-1,2 .7-trihydroxy

p~-rrolizidine alkaloid alexaflorine (2) [2) . In the polar

fraction saponins and aminoacids were located . The

chloroform extract of the leaves contained the triterpene

lupenone (3) and the steroids 2,5(27)-dihydroporlferasterol

14 r a , ,d cleroeterol IF) . The latter com;-:-uno was i ;~ " lat4" o

additionally from the chloroform extract of the truni: wooo

samöle .

Compounds 3[ :), 4(4] and 5[51 were identified b~

careful comparison chiefly of MAR spectra wilh analogous.

cata reported in the literature . .

The structure determination of alexaflor ;ne (2) relied

orr, elementary analysis and mass spectrometry for the

determination of the molecular formula C ` Ht3 NC5, infrared

spectrometry for the identification of a carboyylate group .

a s well as 'H and "C NMR spectrometry for the estat-lishmeno

of the constitutional formula 2 . Analysis of the JH- '3C

heteronuclear correlation spectrum resulted in the complete

assignment of all proton resonances to the respective carbon

signals . Furthermore the pyrrolizidine constitution for

alexaflorine was consubstantiated by all possible decoupling

2 9

HO

experiments and the MS fragmentation pattern . Analysis of

the proton coupling constants led to the relative

configuration as shown in the for muls ; .

Detailed pharmacological study of alexaf lorine and of

the other products from Alexa arandiflora will dE reported

sevaratelv .

30

OH

REFERENCES

[11 Ilash . R .J . . Fellows . L .E ., Dring . J .V . Stirton . C .H .,Carter . D . . Heoarty . M .E, . . anc Bell . E .A, I1~E.F1Phytecheml :try 27 . 14(i :. .

[~] Pereira, A.C . de S . . Kac+lan, K.A .C . . Mala . .I .G .S . andGottlieb . O.R . (19901 Anais Acad . brasil . C1enc . . inprint .

[~1 henFert,E . . Bacdelep . G .V ., BlirI11 . I R . and Moren .n . L,N . (19781 Org . Magn . Res . 11 . 2S7,

(4)

Garo .

V .F, .

and

Ne; .

W .re .

I1Gk41

PitvI( "cher+i :trv

: : .2925 .

[ :.] Matsumoto, T ., Shiaemote, T . and Itih . T . i1~4:1Phytochemistry 22, 2622 .

Metabolism of Plant Protection Agents in Higher Plants

Horst-Robert SchütteInstitut für Biochemie der PflanzenWeinberg 3, 0-4050 Halle/Saale

in living systems plant protection agents - like other xeno-biotics - are subject to a more or less strong metabolism .This is of great importance concerning the ecological and to-xicological situation of these compounds in plants and soil .Often different metabolism of herbicides in culture plantsand weeds is responsible for the selective effects of the lat-ter . In the process of metabolism mostly hydrophile compoundsare produced that can be stored in vacuoles in the plants .Transformation reactions take place by p.e . hydroxylations,oxidations, reductions, hydrolyses, acylations, alkylations,desalkylations and conjugations . The principles are discussedwith some examples like different phenoxyalkane acids, phenyl-urea herbicides, carbamat herbicides, diphenylether herbicides .

For example the metabolism of 3,5-dibrome-4-hydroxy-benzonitrile(Bromoxynil) was investigated in plants that have a variable sen-stivenesa concerning this active component . on this occasion agradation of the metabolism rate was found depending on the sen-sitiveness of the respective plants . After 48 hrs we traced only19% of the incorporated 14-C-Bromoxynil in the resistent Stella-ria _media . 678 of the radioactivity were incorporated in polarconjugates and 14% in bound residues . After the same test periodwe found in Chrysanthemum seaetum only 568 radioactive Bromoxynilas well as 32$ conjugates and 12% in bound residues, on the otherhand Galium a arine which is very sensitive to Bromoxynil was notfound to be able to decompose a larger quantity of this activecomponent, even when the latter was used in a concentration. .50 fold lower . After 48 hrs we still detected 948 unchanged Bro-moxynil .

NEW ASPECTS IN SYNTHESIS OF GIBBERELLINS

G . Adam

Institute of Plant Biochemistry, Weinberg 3, 0-4050 Halle/Saale, F . R . G .

The gibberellins represent an important class of diterpenoid phyto-hormones o£ ubiquitous occurrence in higher plants . From the known79 native members detected hitherto in higher plants or fungi onlygibberellic acid (GA3 ) is available in large scale by fermentationof Gibberella fujikuroi and serves as starting material for furtherchemical and biological studies .

After a brief discussion of bioactivity and application of gibberellinsrecent progress towards the regio- and stereoselective synthesis o£new gibberellin structure types starting from the polyfunctionalizedand highly reactive GA3 will be reported . Our studies expecially in-volve

* synthetic pathways to scarce native memberssuch as GA4 , GA S , GA55, GA57 and GA60' ,

* reaction sequences of general applicability forspecific labelling of gibberellins

* the synthesis of new halogen-, nitrogen- andsulphur-containing analogs for structure-activityrelationships .

For the structural determination X-ray analysis, MS and NMR-studies

including 2D-NMR techniques were of special importance and will be

also discussed .

l4EW SSESQUITERPENE ALCOIIOL FROM VASSOURA OIL

Carmen Lucia Qveiroga" Leonardo J. üayasidab . F'e

~rriarido N . LancasArmin de Maijere° and Anita J . Marsaioii

ux

° Instituto de Quimica, Universibdade Estadual de Can,Pinas . GP6164 Gampinas 23OB1 , 5P, Brazil. Univer si dade Estadual eje S.Paulo , Departamento de Quimica e Fisica 1Solpcular CP 3159 S.Carlos 131580, 5. Paul o Brazil-.u I nst i tüt für C~r gani sr.F~es Chemie derGeorg-August-Universität . Tammarnnstrase-e 2, 34c)Q (.'-~5ttirngen,

Ger many

The genus 8accharis with very few exceptions occurs Only

in the new world and the largest concentration of species is

located in Brazil and the Andes.

We have therefore dedicated partof our research programm to the phytochemical study of BrazilianSacc har tis focusing the essential oil producing species

beginningwith

Vassoura

oil

CBroom oil)

C

8.

dracuncuitfoLia

D.C. ,

Compositae) . This particular name has its origin& in older days

when a rudimental broom was made from its aerial parts and used to

sweep hot embers from clay ovens leaving I-&hind a fresh herbalfragrance. Nowadays this oil is produced in Brazil by steam

distillation and used in perfumery industries .An earlier report i in the literature has shown that

this light yellow oil contains nerolidol

a ,

spathlilenol

2 andseveral

raonoterpenes .

We

have

reinvestigatedI

this

oil

anddetected 43 constituents by CC-MS, and/or PIAR . CHR, IP andcomparison with model compounds containing thetricyclo/8,3,0,02,4-undecane skeleton .

It was obvious from this study that we were dealing witha complex. mixture of sesquiterpene alcohols possessing thearomadendrane and cadinane skeletons, many of which could not be

34

identified due to the lack of data .This paper deals with the detection, isolation and the

structure elucidation of dracunculifoliol .a Sesquiterpene alcoholwhich is a minor component of Vassoura oil .

3

7

Compound 3 was first detected by GC/MS and identified asd-cadinol 3, by comparison of its mass spectrum with -those in theNSS-REVEL C38791 MS) and Nature L. C545 MS) data system library.Several chromatographic techniques CCC, TLC, silver nitrate/silica TLO led to the isolation of dracunculifoliol which showedspectroscopic data different from those of 3 and other reportedcads nols.

Taking into consideration that the proposed skeleton wascorrect we had to locate the functional groups (trissubstituteddouble

bond,

a

quaternary

hydroxyl

,

a

mothyi

link-ed

to

acarbinolic carbon and a 2-propyl] and to determine the relativeand absolute configuration. We had therefore to seleat among 46possible isomers Mg I .) and from a brief survey of the literatureon a spectroscopic method to distinguish Chp 24 diastereomers itwas obvious that mass spectrometry would be of little help due tothe rather small differences in fragmentation patterns and NMRdata were incomplete Cno carbon-13 NMR) for the few reportedisomerc.

Figure ie PoccLbta sirucLUroa for dracuneulifaliol

CLASS 1 132 isomers)

GLASS 2 116 Isomers)

We finally came to the conclusion that syntheses of

Selected isomers might be the clue to this problem, as X-raycrystallography was not accessible for this specific problem.

Taking into consideration the various spectroscopic

characteristic of dracunculifoliol our target compounds belongedeither to class 1 or 2 C Fig 1 .) .

Compounds 4, 5, 6 and 7 were easily accessible from themethylation of the octalones 8 and 9`. From GC/MS and 114 N1412datawe could select structure 7 for dracunculifoliol, which is anew sesquiterpene alcohol possessing theC1e, t:Sam , 7S*~1--7-C2-propy17 -4,10-di mett

Acknowledgements

REFERENCES

Tetrahedron 37, 425 C1991D ;Tetrahedron 40, 3491 C1994D ;Rana, Indian J. Chem . 179MS. C: .

Nath

and

R. P.

Sbarma.

references cl ted therein.

THF,-10'C

MetlTHF

-IO' C

6

4 R~=OH RZ=CHy5 R l =CH3 R 2 = 0H

6 Rl= OH

R2=CH37 RI=CH3 Rz=OH

A.J . Marsaloli and C.L.Queiroga are indebted to DAAD and toFAPESP for support and scholarship.

1 . O. Motl, and A. Trka, Parfum . Kosmet . 84, 488 019933 .2. C.L . Queiroga, A. Fukai and A.J . Marsaioli, J_,. Brag . Chem 5oc019913 in press .3. A.I . Rezvukhin, K.A . Khan and Z.V. Dubovenko, Bull . Ar-ad . Sci .USSR 24,, 1208 019753 ; A.K.Borg--Karlson, T.Norin and A. Talvitie,

L . R. R. A . Franke,

M.

Wolf and V.

Wray,O. P. Vig, S. S. Pari ,

D.M.

Dua and S.S.552 C1979) ; M. Pordolof , V. S. 'Mink-la,Phytoghemist= 2L;, 2007 019893 and

4 .

F. Fr i nguel l l ,

F.

Pizzo,

A.

Tati cchl .Richelotti, B. Porter and E. Wenkert,(19953, and references cited therein.

V.

Ferreira .

E. L.gm. Chem . 50, 890

A NEW AROMADENDRANE ALCOHOL FROM VASSOURA ESSENTIAL OM

Lucia Rebocrgas and Anita j. Marsaiolfshistituto de Qulmica , Universidadr Cntadu~il do-* t1 .:nireirris ,C1 , 6154

Campinas 13081 5P Brazil

Sesquiterpenes coW.aining

thetr ,1cyc1o1G,3,U,Oz''2undecene skeleton Caromaderidvaijg tkeletan) .ar"R

widely distributed in nature and have been isolated from plantsand ardnraia of terrestrial acid marine origin1.

In our Croup wehave been particularly interested by the large amount of thesesesquiterpenes present in Vassoura all, Unfortunately only GC/MSdata were not enough to identify several components, we havetherefore" synthesized model compounds t, 2, 3, d, 5 and dz whichallowed the unambiguous detection of the presence of t, 2, 3 and 43n Va_rsoura oil.

f RI= OH ; R2-- CKRl=CH3 i R32 0H

38

3

5

R1 = OH f R2 = CH36

RI c CH3 ) 112 r OH

In this paper we amplify on our- earlier work byreporting the presence of 8 in Yassoura oil as a novelsesquiterpene alcohol belonging to the aromadendirene class ofterpenes . The coinjection of the essential oil with the modelcompounds 7 and 8, obtained by hydroboration of commercialaromadendrane,allowed the detection of 8 In the oil mixture.

AROMADENDRANE

e

?

Acknowledgements

The authors are indebted to FAP£SP for support and to CNPq forscholarship to L.R .. We are also grateful to Prof. Dr . F.N.Langgas and to L.j. Hayasida fox, GC/MS data.

REFERENCES

1%Ydrobor ( tlaa/H2o2

i."The Total Synthesis of Natural Products" Edited by j.ApSimonVol 5 (1983) John Wiry & Sons NY.;1).j . Faulkner Nat. Prod,Reports (1989) 619.2. C.L . Queiroga, A. Fukai and A.j . Marsaioli, L Bram. Chem . Soc(1991) in press.

SECONDARY PR0VUCfS and SLICITDR-TNDUCi01 RP.ACTIOHS in

PSM1OMiIODTAOPF1IC UZL SUSPSNSIOBI CMTUFE of c_icer Adgll= L.

PC SADDER, x TIF.KM, W BAZ

Institut für Biochemie und Biotechnalagie der Pflanzen,

WeatfäliSChe Wilhelms-Universtität Monster,Eindenburgplatz 55, A 4400 MÜNSTER - west Germany

Chickpea (Cicgr =, atinum L .) is an important seed legumecrop. In Brazil this legume is known by the trivial name "grao-de-bico".

The distribution arLd economic importance of this plant isover many countries of Asia, Africa, Europa and the Americas . In1985, the total world area for this legume production was approximately ten million hectares (PAO, 1985) . In South America, theproduction in the same period reached 25 thousands metric tons(FAO, 1985) .

This valuable crop plant suffers from several very destruc-tive diseases, among which the most serious one is the chickpea-Ascochyta-blight . The patogenic fungus - Asevchvta

i - causesbrown spots on the entire aerial surface of the plant, which may

lead to total loss of the plant.Earlier investigations in our institute used a considerable

number of experimental systems : i .e. whale plantlets, dissectedorgans, in vitro tissue and toll culture techniques, to demon-strate biochemical events in the interactions between the host andthe parasite .

Present investigations employ chickpea chlorophyllous cellsuspension cultures, growing photomixotrophically. Such a model.system, closely resembles the characteristic green aerial parts ofthe plant, which are predominantly susceptible to attack by thefungus .

In order to define some properties of the photomixotrophiccell culture system, essential. physiological parameters were Ana-lyzed. Thexfore fresh weight, dry weight, call number, chlorophyll- and protein-content of the cell mass, carbon dioxide

40

fixation rates, marker enzymes of the photossynthetical activity,as the Carbcxylases : 8ubisco and PEP, were measured during thegrowth cycle of the culture. Furthermore, the uptake of nutrientsfrom the medium was monitored. This included the consumption ofcarbohydrate, nitrogen "d phosphate sources and ionic entities,as well as pß-, conductivity- and osmotic pressure-profil of thegrowth medium.

C, , arietinvm possesses a pronounced secondary metabolism bysynthesizing many isoflnvonoids . The occurrence and the accumula-tion of the numerous different 3-phenylchromane derivatives duringa growth cycle of the cell suspension culture were determined .

In order to sir-alate the physiological response of the hostagainst a microbial pathogen, we applied cell wall glucan frag-ments of fungi (called elicitor), to the cell suspension cultures .This work aims at understanding how the plants defend themselveagainst the disease.

From the multitude of defence reactions produced by theplant system, we concentrated on the formation dud secretion, fromthe cells of pterocarpan phytonlexins, of oxidative enzymes (Po4),of hydroloses (i.e.chitinases and beta-1�3-glucanases) and certaincell wall bound phenolics.

In elicited cultures, the metabolic regulation of pteracar-pan phytealexins biosynthesis and of isoflavone conjugation reac-tions, was studied using marker enzymes as such phenylalanineammonia lyase, cinnamic acid-4-hydroxylaue, chalcone synthase,chalcone isomerase, mAlonyltransferase, isoflavone 2'- and 3'-hy-droxylases and pterocarpan synthase .

In order to elucidate another mechanism by which the patho-gen - 8., . rabies - possibly induces the pathogenic disease symptomsof chickpea, we also evaluate the role of sesquiterpenaid -liketoxins (i .e . solanapyrones) . These phytotoxins are secreted by themicroorganism into the plant cells.

Acknowledgement :

Financial support by Coordenapäo do Aperfeigoamento de Pes-scal do Ensino Superior - CAPES - Minietärio do Educapao to PCS isgreatly acknowledged.

41

STUDIES OF NEW NATURAL PRODUCTS -- PROHZEMS AND EXPRIENCES

R -C

H . Achenbech

University of Erlangen

bepartment of Pharmaceutical Chemistry,

D-8520 Erlangen, FRG

From two African Gochlos2ermum species and from the Middle

American Cochlospermum vitifoiium more than 10 novel naturalproducts were isolated among them the monocyclic aromaticcochlospermins (1) and the tricyclic cochlospermatins (2)(R = CnH2n+1 ; n = 11 or 13) .

r=n

R-II

C=OR

R

4 2

-R

All the isolated compounds - which mostly occur as mixturesof homologues - can well . be arranged into a biogenetic schemewith 3-ketotetradecanal (and 3-ketohexadecanal) as the keyprecursor .

Therefore, it needs special consideration whether the isolatedcompounds and particularly the monocyclic and tricyclic componentsare genuine natural products or whether they might be artefacts .

The galbonolides (3 : galbonolide A) constitute novel microbialmetabolites of high antifungal activity but only low chemicalstability . In addition, they are produced in very low concen-trations only .

We report on the isolation and structure elucidation of thegalbonolides .

3

srrcmannRY METABOLIT15S FROM ERAZILM MApllIE ORGANISMS.CHEMICAL, ECOLOGICAL AHD CREMOTAXOI10MIC STUIlJCS .

A]Chonse I:alecom, Renato Crespo Pereira ark-4 Ualdri3 Lv,ll., lai;keiraDepartment of General E ¬olog-z;, Uni-iersidade Federal Ftutoir,rrr, se,

C.P . 1CDAß3, 24.0010 ¬Iiter6i, 1ZJ, BRAZIL

Marine 11atural Products chemistry has received consid-gableattention in the past decades. The major goals urer-~ the obtenFion, fromurti~,onventional natural sources, of new valuable substances for use as

driv.ss, foods or for Industrial needs.In Crazil, a continuous program on

organisms has started ¬n the late seventies .exelusivetq with steroids, terpenes and moreOur most relevant results on gorgonian; and brownobject of this cotinotiunication .

Gorgonians are relatively abundant along the Brazilian coast. Theendemic species, Phyll,vgoryia arila'taea Esper, 3cß6 is a largeconspicuous benthic sea-fan that 1s almost never ?ncrusted bu otherorganisms, and when this happens, we observed that Its snia11 uras tiy'tiuchless active . Indeed, healthy gorsonians produce large a,v~ounts of ane=sential oil that may have antifoulins activitu. From this oil, ueeobtained four new Sesquiterpenes, two nardosinan+-as (1. and 2) and tVdgarmacranas Q and -:I). Their structures were ostabtished from sp_tbratanalysis, mainly 111 "18 . Hardosinanea were known previously froia softcorals

On

the

Other

hand,

our

phytc :heniical- studies

on

brown

algae

orthe genvs -Pictyata have resulted in the isolatior, of xeveralditerpenes from the dolastane JR (12 compounds),prenylated 9vaianes C (2) skeletons, Sevenstructures that were determined by spectralabsolute configurations of the dolastanes wereearrelativm

45

sacodolast ane J1 (5) andmetabolites had new

and cheMical data . Theestablished by chemical

Laboratory experiments showed that pachydictyol H «,), obtainedfrom ~ictyata &Ichatawa in 0.2-0.5'% dry weight., had, at its naturalabundance,

anti-herbivorous

activity against

the

amphipod

Parhc a.1,ehamai~nsis . The same compound is also present in trace ainounts inJ7ictsrota wcrten.sü and cannot therefore be responsible, by itself,for protection against predation. In this case,,dictyol-H «) was foundto play this ecological role.

oac

But are terpenes the only antifeedants in brownsystemAic study of a dozen of brown algae from theconcitod.2d that the algae that do not produce terpenesrich in steroids . Laboratory assays indicated that steroida are, at theirnatural concentrations, devoid of any anti-lierbivorous a--twjLy saainstP. ham..i~nsi~ .

algae? From aPhae "~t-Ioy:3, weare fregvently

Fhlorot.annins are ubiquitous in brolon algae and have been shoran to

play deterrent activity in temperate brown algae, the question arrosewhether, these compounds had the same activity in tropical regions,

Thus, we quantified tannins in a number of 17ra=ilian phaeophyka. and we

observed low a,nounts of phlorotannins (8.E to 2.F? dry weight) as

compared udth literature data on algae from Lzmperste wat?rs Cup to

15f) . Our preliminary results on herbivory showed that, at the

concentrations observed by us in tropical algae, phlorctannins are not

effective as antifeeding suxbs'tances . However, at the mean phlorotannins

cor,centraLion observed inphloroslucinol deters theindicate a differenttemperate and tropical regions.

temperate slgae (i.e . 15e. dry weight),

aMphipod P, hamaiensis . fill this IT'"functionality or Fhlorotar,nins ineca1aeical

Diterpenes from pictyotales were the object of a ch_motaxonoanicstudu. We showed that these compounds are excellent taxonotslic andFhylosenetic markers . Thus we were able to point wrong algalidentifications ;. to show the heterogeneity of collected algal materialfor che.tiaical studies . to corroborate the synonymy between llictyuta~~ntai~a and X. evertensii, and to propose that the- cosmopoliteh. cfichotoxua is in fact a complex of species and probably or genera.We showed that diterpenes are efficient to distinguish between ver9close genera Such as V ctyo4,*, A;IDphu.s' 61v.S .xophora andPachyd,ctsrosr, and we even suggested that the genus 39ictyoOadoes not produce any dolabellane diterpene, on the contrary to what isrelated in the literature . General biogeographic considerations Indicatedthat Dictyotales probably in4"aded the. Mediterranean Sea s~"ia the archaicThetys Sea and that Erazilian Dictuotales may have originated from thePacific Ocean by migration between both Americas before completeclosure of Central America, (CHPR, CANES, FAP9RJ, CINCH)

F

Aa.nt enledc ;! ii)enF,s

this

work

Is

part

of

the

MSc

thezesFernanden, Beatrix Grosse Fleury, Liane France fiitomboAnt6nic, da Silva Aloneida . Spectroscopy vies recoi-j-a-1 a`Unksersif.4 Libre de Eruxetles, Eelaium, bu Dr, Robert Ot :ing+:r,

4 6

of Lenize:,r~d w rgiati~e CIF,C11i

BIOLOGICALLY ACTIVE FACTORS ISOLATED FROM SCAPTOCOSA RAPTORIA VENOM

Luiza A .F.Ferreira, O .B.Henriques, S . Lucas, Instituto Butantan, Sao Pauloand Gerhard Habermehl, School of Veterinary Medicine, Hannover

In 1953 Lebez isolated polypeptides from Lycose tarantula venom showingactivities similar to that of bradykinin .

We have isolated four factors I, II, III, and IV from Scaptocosa reptorisvenom, by gel filtration on Sephadex G-25 M, followed by HPLC .

Factors I, II, and III showed contractions of the isolated guinea-pig ileumas well as on isolated rat uterus at equivalent amounts of that shown bybradykinin to cause the same effects on the respective organs . Factor Ishowed a biphasic effect on rat duodenum, whereas II only relaxes isolatedrat duodenum . When factors I, II, and III were injected separately into therat femural vein, and arterially, factors I and II showed biphasic effects,but factor III showed a small decrease of the rat arterial blood pressureonly . These three bellow factors increased also the capillary permeabilitywhen compared with the effect of bradykinin ; they also caused edema onguinea-pig foot, but they did not show hemorrhegic effects in mice .

Factor IV possesses an irreversible effect on muscle relaxation in insectsas well as in Scaptocosa raptorie itself .

AN UPDATE ON PLANTS AGAINST SNAKEBITE

Nano Alvares Pereira l , Bettina Monika Ruppeltl , Maria Cilia doNascimento2 , Jose Paz Parente2 and Walter B . Morst . 1 Departa-mento de Farmacologia Bäsica e Clinica, Instituto de Ciincias

Biomedicas, Centro de Ciencias da saEde; 2Nücleo de Pesquisasde Produtos Naturcis; Universidade Federal do Rio de Janeiro,21941 Rio de Janeiro, Brasil

Investigation of plants of popular use against enveno-mati.on by snakebite is still in its infancy . A review article,summarizing the scattered information which exidtd on the subject,was recently written by one of us (Mors, 1991) and is expected toappear this year . We now wish to report our more recent results,derived from assays conducted through ora administration of the ma-terials tested . By this approach, the experimentation comes closeto reproducing actual field conditions, since medication, even asa prophylactic precaution, is mostly performed by ingestion of theactive materials in the form of aqueous decoctions or alcoholicinfusions .

Materials hnd MethodsGroups of 5, 14 or 15 s4iss mice were used in the assays .

The materials to be tested - aqueous extracts or pure compoundswere introduced into the stomach by gastric sonde at the dosage of100 mg per kilo bodyweight for pure compounds as 1-2 g of plant me-terial in the form of aqueous decoction {"tea"} .

After one hour,5 mg/kg of Bothrops 'araraCa venom was injected subcutaneously,corresponding to twive t e lethal dose . Protection was assessedthrough the number of surviving animals, calculated as percentageof the total in the respective group, six, 24 and 4S hours afteradministration . Survival was 0% among the control animals .

Aqueous

Family_

extracts of the following plants were tested%

Species Popular name_ Part_ tested

COMPOSITAE C nary scoff alcachofra leavesEcl roserva-botäo~ ipts etrata aerial partElephantopus scaber lingua-de-vacs leavesMixania . l�mera~ta� guace . leavesVernonia condensata aluma leaves

CUCURBITACEAE Wilbrandiameata taiuiä rootsra_e cEUPHORBIACEA Phyllanthus klotzscianus - leavesFLACOURTIACEAE Casearia sylvestris erva-de-bugre barkLABIATAE Mars ianthes bola-caÄ serial partypto esLEGUMINOSAEPAPILIONOIDAE A uleia leiocarpa jutai

VERBENACEAE

Stachytar2heta

gerväo

aerial part'

dichotomy

The following pure compounds were also tested :

Wedelolactone and several other substances isolated fromEclipta prostrata ; bergapten and a novel furocoumarin from Dorsteniabrasiliensis, bredemeyeroside from _ered_emeyera floribunda . Alsoassayed, although from other sources, were giycyrrhizin, bromelinand silimarin. Several substances of wide distribution, such astrivial flavonoids, chlorogenic acid and triterpenes, some of whichhad shown highly interesting results in tge parenteral assaus (Mors

et al . , 1989) were also included in the test series .

Results and DiscussionAmong the extracts, best results were observed with Ph l-

lanthus klotzschinnus and Apuleia leiocar2a, which conferred 1008

protection after 6, 24 and 48 h afteradministration . The two furocoumarins isolated from the first were not near as active, showing

only 204 survival after the same time intervals ; but the hexane ex-

tract of Apuleia leiocar~a was only slightly less active than the

total crude.

Next in activity was the extract of Periandra pujalu , with

1004 protection after 6 h, decreasing to 804 after 24 h and to 608

after 48 h . P . mediterranea was somewhat less active (804 after 6 h),

having its activity matched by that of giycyrrhizin, the sweet con-

stituent of European licorice .

Total protection was also conferred by an aqueous extract

of artichoke leaves, 6 h after administrationt the activity, however,decreased markedly with time, being only 104 after 48 h. A hydroalco

holic extract of the same plant and an extract with standardized cy-

marin content 13 .6%) were considerably less active .

Periandra alcacuz-mediterranea da-terra roots

Periandra pujalu pujalu rootsMORACEAE Dorstenia carapii bulbras liensisPIPERACEAE Potomor2he meltata caapeba leavesPOLYGALACEAE Bredemeyeza pacari rootsfloribundaRUBIACEAE Chicocca brachiata cainca rootsSOLANACEAE Brunfelsia uniflora manaci leaves

Still remarkable were the results with the bark of Casearia

s ivy estxis, sustaining their e0^908 protection through the whole period

of observation . of the more common flavonoids, rutin gave 1008 pro-

tection after 6 h, decreasing rapidly afterwords ; less active was

quercetin . hesperidin showed almost no activity .

The remaining extracts still showed considerable activity

(70-808 after 6 h ; 20-50% after 48 h), although less than one would

expect from their popular reputation . Wedelolactone, which had shown

extraordinary activity by intraperitoneal injection (Mors et al ., 1989)

belogs into this group. One pure compound which exceeded the activity

of the respective drug wasthe saponin bredemeyeroside, being better

than pacari throughout the period of observation .

of course, this narrow range of test results does not allow

for definite conclusions . Even so, the results add significantly toearlier, isolated, findings reported in the literature and our own

preliminary observations, to extend the range of orally active anti-

snake compounds . The fact that they do not belong to a limited num-ber of chemical structural classes can explain the existence of plantsactive against snakebite in almost every corner of the plant kingdom.

What these substances have in common is that they are lowmolecular weight compounds with strong biodynamic activity . Theirtargets in living organisms are polypeptides and proteins - thekind of compounds present in toxins and enzymes which make up snakevenoms .

That the extracts, in most cases, are more active than iso-lated constituents is probably due to a number of factors .

The pos-sible synergism between compounds must be taken into consideration,particularly since the venoms themselves are not homogeneous entities .Also, the complexity of the crude mixtures allows for increased solu-bility of individual constituents . Similar behaviour has been repor-ted, for instance, in the case of the antihepatotoxic properties ofextracts of saccharis trimera and of the individual flavonoids iden-tified therein (Soicke and Lang-Peschlow, 1987) . Analogous observa-tions have been reported in the investigation of extracts of Schuman-niophyton mactnificum , active against the cardiotoxic effects of cobravenom (Houghton and Harvey, 1989) .

A marked parallelism between snake venom neutralizing andanalgesic and/or antiinflammatoxy activity has been observed (Ruppeltet al ., 1990) . Thus, of the plants mentioned in this report, four

so

(C nara acolymus , borstenia brasiliensis , Casearia sylvestr_is andVernonia condensata ) reduced the number of contorsions in the Whittle'test by better than 90% ; and five (Cynara s_colymus, , Chiococca braciataCasearia sylvestris , Apuleia leiocarpa and Mars ianthes chamaedr s)inhibited inflammation by better tah 508, measured by the diffusionof Evans blue after intravenous administration .

REFERENCES

Houghton, P .J . and A.L . Harvey, Planta Medica 55 : 273 (1989) .Mors, W.B ., M .C . do NaSCiment0, J .P . Parente, M .H . da Silva, P.A .

Melo and G. Suarez-Kurtz, Toxicon 27 : 1003 (1989) .Mors, W .e ., "Plants against snakebite", in H . Wagner, H . Hikino and

N .R . Farnsworth, eds ., "Economic and Medicinal Plant Re-search " Vol . 5, 1991 (in print) .

Ruppelt, B .M ., E.F.R . Pereira, L.C . Gongalves and N .A. Pereira,Rev. Bras . Farm. 71 : 54 (1990) .

Soicke, H. and E. Leng-Peschlow, Planta Medlca 53 : 37 (1587) .

ISOLATION AND STRUCTURE VARIATION OF SECONDARY METABOLITES

FROM MICROORGANISMS DERIVED BY A CHEMICAL. SCREENING

P. Hammann` and 5 . Grabley, ZF I, Hoechst AG, 6230 Frankfurt

The investigation in the field of microbial secondary metabolites is still a

challenging area in natural product chemistry . The coresponding culture broth,

containing the different secondary metabolites, is normally used in a biological

screening against different pathogens (e . g. bacteria, fungi, protozoa, hetminths,

viruses), as well as in pharmacological areas (e . g . immunomodulation, lipid

metabolism, carbohydrate metabolism, tumor therapy), with additional fields in

veterinary medicine and plant protection. Obviously, interesting compounds may

be overlooked, if the biological screening concentrates on a single focus.

Therefore, the possibility to supplement a distinct btologicat screening by achemical screening method was iinvestigated . 1 ) One of the advantages of thistype of screening is the possibility to select strains with sufficient production of,new or known, metabolites for further structure modification . The isolation andpurification of the desired products out of the large amount of water necessaryin fermentation requires a simple work-up procedure, which wilt be discussed forselected examples .Application perspectives resulting trom an intensive evaluation of the reactionpossibilities of multifunctional compounds will be discussed in the case of themacrodiolide ela¬ophylin (J), The antibacterial active _I can be transformed intoan antiviral, anfcoccidlal2) or cytotoxtc compound by simple chemical reactions .Furtherrnore, the chemical screening method allows the detection of naturalproducts Independent from their biological activity, which can be applicated asmeans of chiral synthons . Therefore, this type of screening extends the chiralpool . For example, streptenol A (2) can be transformed into all four possiblestereolsomers of 3-hydroxy-5-dec-S-enollde [(3$,5B)-is