Cymbastela hooperi and Amphimedon terpenensis: where do they really belong?

8 Pages·1999·2.5 MB·

by  G M Konig

CYMBASTELA HOOPERIANDAMPHIMEDON TERPENENSIS: WHERE DO THEY
REALLY BELONG?
GABRIELE M. KONIG AND ANTHONY D. WRIGHT
Konig, G.M. & Wright, A.D. 1999 06 30: Cymbastela hooperi and Amphimedon
terpenemis:wheredotheyreallybelong?MemoirsoftheQueenslandMuseum44:281-288.
Brisbane. ISSN 0079-8835.
A sponge sample identified as Cymbastela hooperi collected from Kelso Reef, the Great
Barrier Reef, Australia, yielded a series ofnatural products, mainly diterpene isonitriles,
whichdemonstratedsignificantinvitroantimalarialactivity.Asaresultofthesecompounds
beingconsumed inanumberofbioassays itwasconsidereddesirabletohave moreofthem
so astoenable furtherandmore detailedbiological testingtobe undertaken. Subsequently
C
three Cymbastela samples (two ofC. concentrica and one of coralliophila) and two of
Amphimedon{Cymbastela)terpenemisweretestedforantimalarialactivityandinvestigated
for their natural product content. The results of these investigations provided further
evidence that either, Amphimedon terpenemis is more appropriately Cymbastela
terpenensis, orthatboth C. hooperiandA. terpenemisbelongtoan asyetundefined genus
and may perhaps be the same species. GPorifera, Cymbastela, Amphimedon, Acanthella,
biological testing, malaria, cytotoxicity, taxonomy, Great Barrier Reef, diterpene
isonitriles, marinenaturalproducts.
Gabriele M. Konig & Anthony D, Wright (email: a.wright@_tu~bs.de). Institute for
D
Pharmaceutical Biology, Technical University Braunschweig, Mendelssohnstrasse I,
38106Braunschweig, Germany; 2March 1999.
SincethediscoverybyAngerhoferetal.(1992a) spectrometry; 'H NMR, proton detected nuclear
ofthe antimalarial activity ofaxisonitrile-3 (Fig.
magnetic resonance spectroscopy.
1A)isolatedfromthespongeAcanthellaklethra,
much of our research activity has focused on
MATERIAL. All sponges were collected using
bfiionldoignigcfaulrtachteirvmitayr.iTnheensaetuerfaflorptrsodruecstulstweidtihntthhies SCUBAfromtheGreatBarrierReef,inthevicin-
identification of other marine derived natural ity ofLizard Island between ll-30m depth. All
specimens were frozen, then freeze dried. Five
products having selective antimalarial activity
samples of 3 sponge species were: Cymbastela
(Konig et al., 1998, Wright et al., 1996), par- coralliophilaHooper& Bergquist, 1992 (Demo-
ticularlythe compounds isolatedfrom Cymbastela
hooperi(Konig&Wright, 1995, 1997a;Koniget spongiae, Halichondrida, Axinellidae)
(specimen CTA); C, coneentrica, Lendenfeld,
al., 1996; Lindeneta]., 1996; Wrightetal., 1996). 1887 (Demospongiae, Halichondrida, Axinelli-
pInroodrudcetrstoiotbtwaaisnfduretchiedreadmotuontisnvoefsttihgeasteenastuormael tdaeer)pe(nsepmeicsi,meFnrsomCoTnDt,an1d99C3TE()D;emAomspphoinmgeidaoen,
scpoomnpgoeunsda.mpIlnesthleikperleysetnotcpoanptearinwtehisprcolvaisdseoaf Haplosclerida,Niphatidae) (specimens CTB and
CTC).
discussion of these biologically-guided isolat-
tiohnes,pothsesirbelseulttsaxoofncohmeimcicailmpalniaclaytsieosn,sasofwetlhlesaes EXTRACTION AND ISOLATION. Initially, a
smallpiece(~5goffreezedriedtissue)fromeach
findings. DCM
sample was exhaustively extracted with
MATERIALS AND METHODS followed by MeOH. A portion of the resultant
extracts was then sent for antimalarial and cyto-
General methodology follows Wright et al. toxicity testing (~2mg). ]H NMR and TLC
(1996). Abbreviations: DCM, dichloromethane; investigations ofthese extracts were also made.
MeOH, methanol; EtOAc, ethyl acetate; VLC, On the basis of the results obtained from the
vacuum liquid chromatography; HPLC, high biological testing and the !H NMR and TLC
performance liquid chromatography; TLC, thin investigations, specimens CTA, CTC and CTD
layerchromatography;GC,gaschromatography; were subsequently selected for bulk extraction
GC-MS, gas chromatography coupled mass and fractionation.
282 MEMOIRS OF THE QUEENSLAND MUSEUM
TABLE 1. Antimalarial activity, towards clones D6 and W2 ofPlasmodiumfalciparum, of the
dichloromethane(D)andmethanol (M)extractsfrom 5 spongesamples. SI=dieratiooftheKBcell
cytotoxicity to the Plasmodiumflaciparum toxicity. * Extract was non-toxic only towards KB cells, in
othercell lines itwas at least 100times moretoxic.
IC50(ng/ml) CloneD6 CloneW2
Sample Species
KBcells ICso(ng/ml) SI IC50(ng/ml) SI
CTA(D) C, coraUiophila >20,000 >10,000 - >10,000 _
CTA(M) C eontlliophiht >20.000 5150 >3.9 6380 >3.1
CTB(D) A. terpenensis >2OsO0Q 4820 >4.1 >10,000 _
CTB(M) A. terpenensis >20,000 3240 >6.2 9680 2.1
CTC{D)* A. terpenensis >20,000 <41 >490 <41 >490
CTC(M) A. terpenensis >20,000 540 >37 5250 >3.8
CTD(D) C. concentiiea >20,000 1360 >14.7 >10,000 .
CTD(M) C. concemrica >20,000 2730 >7.3 1360 >14,7
CTE(Df C. concentrica >20,000 >10,000 - >10,000 _
CTE(M) C. concemriea >20,000 8470 >2.4 5700 >3.5
1) Specimen CTA: Freeze-dried tissue (108.4g) the major components of fractions 8-12 was a
was exhaustively extracted with DCM (2L) and sterol ofthe type represented by the compound
MeOH (2L) to yield 15.9g (14.7%) of DCM shown in Figure 2A.
soluble material, and Tl.3g (10,4%) of
MeOH/H solubles. VLC separation of the GC SEPARATIONS. GC analysesweredoneac-
DCM sol2ubles over silica, employing gradient cording to methods previously described (Witte
elution fromhexanetoacetonetoMeOH,yielded etal., 1993). FromeacDhCofMVLCfractions 1 and2,
15 fractions each of approximately 90ml. obtained from the extract of sponge
Fractions 3-14 were predominantly compounds specimen CTC approximately lmg of material
depictedinFigure C-D.Theremainingfractions was taken and analysedmy GC-MS. The results
and the MeOH/H1 solubles were ubiquitous of these analyses indicated VLC fraction 1 to
lipids and a numbe2r ofother sterols. containthecompoundsdepictedinFigures 1E-H,
and VLC fraction 2 to contain the compounds
2) Specimen CTC: Freeze-dried tissue (38.8g) depicted in Figures 1I-Q.
wasexhaustivelyextractedwithDCM(1.5L)and
MeOH(2L)toyield3.0g(7.7%)ofDCMsoluble BIOLOGICALTESTING.Theantimalarial(anti-
material. VLC separation of the DCM solubles malarial activity is defined as the ability ofsome
over silica, employing gradient elution from substance, pure or mixture, to inhibit the growth
hexane to EtOAc to MeOH, yielded 12 fractions of, or be lethal to, one or other strains of
eachofapproximately 100ml. Fractions 1-5were Plasmodiumfalciparum)andcytotoxicitytesting
foundby GC-MS analysisto contain compounds was undertaken as previously described (Anger-
depicted in Figure 1E-Q. Fractions 6 and 7 were hoferetal., 1992b,Likhitwitayawuidetal., 1993).
essentially the pure compound shown in Figure
IB. Fractions 1 and 1 1 werefoundtocontainthe RESULTS
TchoemporuemnadisnidnegpicftraecdtiionnsFiagnurdesth1eRM-eXOaHn/dF2LAO. A smallpiece(~5gofdrytissue)fromeachof5
solubles were ubiquitous lipids and a number of sponge samples thought likely to contain di-
terpene isonitriles of the type represented by
sterols.
diisocyanoadociane (Fig. IB), was exhaustively
3) Specimen CTD: Freeze-dried tissue (57.5g) extracted with DCM, followed by MeOH, and
was exhaustively extracted with DCM (2L) and theresultantextractssentforantimalarialactivity
MeOH (2L) to yield 600mg (1.1%) of DCM assessment. Out ofthe 10 extracts only 2 were
soluble material. VLC separation of the DCM found to have significant activity, the DCM and
solubles over silica, employing gradient elution MeOH extracts ofsample CTC (A. terpenensis)
from hexane to ethyl acetate (EtOAc) to MeOH, (Table 1). The only other extracts to show some
yielded 13 fractions, each ofapproximately 80ml. promise interms oftheiractivity and selectivity
All fractions and the MeOH/H2 solubles were were the DCM and MeOH extracts of sample
ubiquitous lipidsandanumberofsterols. One of CTD (C. concentrica), and to a lesser extent the
CYMBASTELA HOOPERIANDAMPHIMEDON TERPENENSIS 283
HO^
CH
FIG. 1. A-X, chemical structures of secondary metabolites derived mainly from sponges of the genera
Cymbastela andAmphimedon (refertotext forfurther information).
284 MEMOIRS OF THE QUEENSLAND MUSEUM
DCM and MeOH extracts of sample CTB (A. from C. hooperi (Konig et al., 1996; Konis &
terpenensis) (Table I). On the basis of results Wright, 1997b; Wright et al., 1996) showecfthe
presented in Table 1, TLC and 'H NMR ex- two VLC fractions to be almost identical. Pur-
aminationsoftheextracts,detailedinvestigations ificationofthemain components from bothVLC
were made of samples CTA (C caraUiophila\ fraction 1 Ishasresulted inthe identification of7
CTC (A. terpenensis)and CTD(C. concenJrica). diterpene formamide derivatives (Fig. 1R-X), a
For this purpose DCM and MeOH extracts were numberofwhich are newnatural products, anda
prepared from bulk material of each of the 3 mixtureofperoxidecontainingsterolsofthetype
samplestoidentifythemajorcomponentspresent. represented by Figure 2A; the detailedresults of
this investigation will be presented elsewhere
SAMPLE CTA (C CORALLIOPHILA). The (Konig et al., in preparation).
DCM extract of this sample (CTA, C. coral-
liophila) was found to contain predominantly SAMPLE CTD (C. CONCENTRICA). Both the
lipids and 2 sterols (Fig. 1C-D), previously iso- DCM and MeOH extracts ofthis sample (CTD,
lated from Pseudaxinyssa sp. The latter sample C. eoncentrica) were found to be complex mix-
wascollected from several mid-shelfreefsonthe tures of ubiquitous lipids and sterols. In VLC
Great Barrier Reef, by Hofheinz & Oesterhelt fractions 8-12, made from the DCM solubles of
(1979). These 2 sterols have also been isolated CTD, sterols ofthe type represented by Figure
from anotherPseudaxinyssa sp. collected from a 2A were abundant.
reeffringing Pelorus Island on the Great Barrier TLC and 'H NMR ofthe extracts ofthe two
Reef(Konig,G. M.& Wright,A. D.,unpublished remaining sponge samples, CTB (A. terpenen-
data). An interesting observation concerning sis), and CTE (C. eoncentrica), clearly showed
thesecompounds(Fig. 1C-D)is,thattheyalways thatspecimen CTB is very similar in allrespects
seem to occur as a 1:1 mixture which is essent- to CTC (A. terpenensis) and that sample CTE
ially inseparable, even by GC (Bergquist et al., showsthegreatestsimilaritytosamplesCTAand
1980). The MeOH extract was composed of CTD, particularly with respect to their 'HNMR
DCM
ubiquitous lipids and a number ofother sterols spectra. The reduced activity of the and
(Bergquist et ah, 1980). MeOH extracts ofCTB when compared to the
activity of the equivalent extracts of CTC, ap-
SAMPLE CTC {A. TERPENENSIS). Chromat- pearstobe due totherelatively large amounts of
ographic and !H NMR analyses indicated the lipids present inthe extracts ofCTB.
MeOHextractofCTC (A. terpenensis)tocontain
many ofthecomponentstobe found inthe DCM DISCUSSION
extract. The MeOH extract was therefore part-
itionedbetweenwaterandDCMandtheresulting Of the 3 species of Cymhastela investigated
DCM solubles combined with the DCM extract. nonewere showntocontainterpenoidssubstitut-
These DCM solubles were fractionated as ed with isonitrilebased functionalities. This is in
aonuatllyisniesd oinftthhee erxepseurlitamnetntfarlacsteicotnison.ind!HicaNteMdRa d(iKroenciteco&ntraWsrtigthotr,esu1l9t9s5,obt1a9i9n7ead;fKoronC.ighoeotpealr.i,
stihmeilfarraicttyioinnatcioonmpoofsitthieoDnCtoMthsoosleubplersodoubcteadinbeyd t1h9i9s6r;esLpiencdteint iestaolf.,in1t9e9re6s;tWtoringohtteetthaalt.,C1h99o6o)p.erIin
from the previously investigated C, hooperi isalsomorphologically distinguished from other
(Konig et al., 1996; Konig & Wright, 1997b). Cymhastelaspecies (Van Soestet al., 1996). The
Basedonthis observationGC-MS investigations investigationofthetwosamplesofA. terpenensis
of selected VLC fractions were undertaken. (CTB and CTC), however, led to the ident-
These analyses indicated the sample to contain ification of secondary metabolites identical, or
compounds shown in Figures 1E-Q, and thus, to closelyrelated,tothoseobtainedfromC. hooperi.
be almost identical in secondary metabolite Literaturerelatingtothe secondary metabolite
content to C. hooperi (Konig et al., 1996; Konig chemistry of sponges from Amphimedon and
8c Wright, 1997b). This finding also explained Cymhastela shows sponges from the former to
the observed antimalarial activity of its DCM have received the most attention. In the 40 or so
extract. As a result ofthese studies it was also publications on Amphimedon the compounds
observedthat VLC fraction 11 contained anum- whicharetypicallyreportedare:variousclassesof
berofresonances inthe 8.0-8.3ppm region ofthe alkaloids (e.g. Fig. 2B-F; Chehade et al., 1997;
NMR
proton spectrum. Comparison of this 'H Kobayashietal., 1994a;Kobayashietal., 1994b;
NMR spectrum with an equivalentVLC fraction Schmitz et al., 1983; Tsuda et al., 1994), long
CYMBASTELA HOOPERIANDAMPHIMEDONTERPENENSIS 285
B
n
19 39
C-mnH
14 29
H
OX
NH NH
2
HO
OCOCH3
K
OH
N O
FIG. 2. A-O, chemical structures of secondary metabolites derived mainly from sponges of the genera
CymbastelaandAmphimedon (refertotextforfurther information).
chain fatty acid derivatives (e.g. Fig. 2G-H; diterpenes(e.g.Fig. IB,J,K;Fookesetal., 1988;
Carballeira& Lopez, 1989; Garsonetal., 1994), Kazlauskas etal, 1980; Konig & Wright, 1995).
glycosphingolipids (e.g. Fig. 21; Hirsh & As there have only been about 11 reports on the
Kashman, 1989) and some isonitrile containing secondarymetabolite chemistry ofsponges from
286 MEMOIRS OF THE QUEENSLAND MUSEUM
Cymhastela it is possible to show most of the classification. I ) A. terpenensis belongs to
isolates in this contribution. From C. corallio- Cymhastela, as proposed by Van Soest et al.
philasteroidsofthetyperepresentedby Figure2J (1996); 2) both A. terpenensis and C. hooperi
were isolated (Makarieva et al., 1995). Pyraxi- belongtoanother, possiblynewgenus located in
nine(Fig.2K),anovelalkaloidwasisolatedfrom the family Axinellidae; 3) they are the same
C. cantharella (Mourabit et al., 1997), while species with hooperi representing an unusual
from twounidentifiedspeciesofCymhastelatwo morphotype of terpenensis. The results of the
peptides (Fig. 2L-M; Coleman et al., 1995) and current work indicate that the taxonomic clas-
two pentacyclic bromopyrroles (agelastatins C sification ofC. hooperiandA. terpenensis needs
and D; Fig. 2N-0, respectively; Hong et al., to be clarified, particularly since sponges be-
1998) were obtained. The present authors have longingtobothofthesespecies producesomany
also published a numberofworks about second- interesting and biologically active compounds.
ary metabolites from C. hooper/ (Konig & This study also serves to further highlight the
Wright, 1995, 1997a; Konig et al., 1996; Linden significance of secondary metabolite chemistry
et ah, 1996; Wright et al., 1996) and the typical as an important taxonomic tool. It is also hoped
metabolites described are mainly isonitrile con- that continued investigations into the biologic-
taining diterpenes (e.g. Fig. IB, l-Q). ally active secondary metabolites produced by
When the literature is considered for Cym- both ofthese sponge specieswill eventually lead
hastelaandAmphimedonitisevidentthatthereis to the development ofan agent suitable for the
currently no class of secondary metabolite one treatment ofmalaria and/or some other disease.
might designate as being 'characteristic' for one ACKNOWLEDGEMENTS
or the other of these genera. What is evident,
however, is that in both genera only one species WethankJ.N.AHooper,QueenslandMuseum,
C
produces isonitrile containing diterpenes; Brisbane, for identification ofall sponges. C.K.
hooperi and A. terpenensis. Angerhofer,DepartmentofMedicinal Chemistry
Two observations can be made: A. terpenensis and Pharmacognosy, University of Illinois at
Chicago, and N. Lang-Unnasch, Division of
is positioned in an order (Haplosclerida) and
family (Niphatidae) where no other sponges are Geographic Medicine, University ofAlabama at
known to produce secondary metabolites that Birmingham, provided the antimalarial and
have isonitrile or similar functionalities, and C. cytotoxicity data associated with this study. L.
hooperi is located in an order (Axinellida or Witte, Institute for Pharmaceutical Biology,
Ilalichondrida; see Van Soest, 1996) and family Technical University Braunschweig, Germany,
(thAaxtinperloldiudcaee)istohnaittrairleekconnotwainnitongcsoentcaoinndasrpyonmgeets- psearmfpolremeCdTCth.e GACl-lJVNIMS Ranalmyesaissuarsesmoecniattsedmwaidteh
abolites. It is clear that "A. terpenensis* does not were undertaken by V. Wray and his team,
(fiBterginquiAstm,phFriommeondto,nHoaospecru,rrVeanntlSyoesdte,fipenresd. Gmebslelll(sGcBhaFf)i, fBurrauBnisocthewcehingo.loWgeiscahreeiFnodresbtcehduntgo
comm.), nor is it clearly a haplosclerid, it is J. Fromont, WesternAustralia Museum, Perth, P.
possibly an axinellid close to Cymhastela, as Bergquist, University of Auckland and R.
suggested by Van Soest et at. (1996), but it's life Desqueyroux-Faundez, Museum d'Histoire
characteristicsdonotconformwell withtheother Naturelle, Geneva, for their constructive
speciesofCymhastela(e.g.growth form,texture, comments concerning the possible taxonomic
mucus production, surface features and amount significance ofour findings.
of spongin to spicule ratio). Cymhastela as
definedby Hooper& Bergquist is a fairly homo- LITERATURE CITED
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: iM,, ! :, PT, RICCWl, C. & IIART- DC.EKS..QUGERYEIROU[XI -FA.LPJ.,NDLETZND,1 R. 199(5
i\NNk I i i ti i Antimalarial activity: The search for marine
pyrroli/idine alkaloids from natural sources b> derived natural products which demonstrate
chromatography -ma- | ClrOIT) otivi Journal ofNatural
. Products59; 7IU-7I6.
THE REPLACEMENT OF NATURAL HARD ascidf.fi rrtical than on horizontal surfaces. It has
SUBSTRATA BY ARTIFICIAL SUBSTRATA: ' whether these pattern^ exist in the
S EFFECTS ON SPONGES AND ASCIDIANS tempi
I l .'',.
a f\ii r/w W: 2-8&.,i . Studiesthathave examined rhe effectsol al and
i ; ,,, vortic.i ' (he distribution ofsponges and
Coim,mm,,onl,y; . thiese LaerceyQnfantuartaulrarloaenkdarneieflicfalsubstrata. thai cause the-se d.biasgtr,ielxup.tejir.imi e,ntal^ rested the factors
scawalkaitd pi pesofhard Hen | | :
differ in their structure. Most natural hard ib i that sponges and aseidvans are more ahundai1
consist bcu ntal surfaces; mosi ces on vertical thanhorizontalsurfaces in the sha h nbthtal
artificial hard substrata are vt [a ;ncrcforc. 50ne around '• i .,
. ; . ii. .. i !; Rcjj ' I] 11.11i\e experiments to determine which !fl!
is likely to ibstraia are.i.mp.or.ta.nt.in creating these distributions
Itom predoi i lOrizonlal ta mostly vertic iJ , .
understand and pre*
changes on the assembI lag cans it
rerrnine their distribution on Nathan
fooriz i d vertiical surfaces,.. h Centreon I
l_. . ti iviiesituodni horizontali an.idil vertnica"li i IJuneJm.
-.polled thai there are esand
i
CCOONNTVIENRUGUEMN:CE AIN TPHREEDTAITMOER-SPPRAECEY K.nv inddaktaatiwoerecoiimajor monalfrrjimeev.ents. No pc i dj I
IMNusreiulmtA44C:TI28O8N, 1999:- Community 1 .I al iambers r>f tl I I i ' i Iiihredatocrhiod
influenced b\ man; bioiic and a1 ctors, . .
atioj cturing rnechanisin fbi some 1996, In the spring ol
withosetrceonmgtmhunoiftiperesd.atPorreyreacbruuintdmaennctesandmapyersfilsutcetnucaet,e 15! A.i rmeucnrtueirtemyeenntsir.esulted inFarnomavMearaygetoJim peiiioneonf
'vheresomeoftheprey population hasa ooverol (i fhtrrnca declined ualestimatesol
i _.e in space or Time from predation. Consistent. 40% in May to 15% in July. L-: I 1^97, whenthe
predation levels on a predicabl) available 10 permanent quadrats and IG haphazard]}
reIsource should lead le community quadratsweremeasured,essentially nospongeco|uIldbe
structure with relatively small fluctuations in predator found at the study site. After July, ihe abundan U
andpreypopulationdensities.Conversely,rjre> species nudihranchs declined to 32 individuals commensurate
i' ge from predation are subject In major sponge reduction. By September, only one small
population fluctuationscommensurate -1 igthof \y and 7 predate ircfts were
Ltor reeniiTmeni and abundance. nt atrhe sue. IIven though // panlcen is abundant
rhe sponge Haiteftondrlci i is patently in theregionand potential recruitsshouldbenumerous,
dlKioascctarhticibmuotaneskd iiBnsatyht,eher5o0Usc1pkaytiCiiCannltfierdnominant. At oneorseiotef aisnnp;uriaeldoi-mimi1a1Dic19r9io8!,.tohepsiioteccounrcreindgo.minTahteedpbrvedHa.tpoarn-ipcfeeya
n 11- //.panieeahas relationship of I n eastsandI ris an
dominated themid-intertidal lor at least 10 years, v iih example of li chase through space and time with
low densities ofpotential molluscan predak,. sucha convergence resulting L] E :treme population
crisis, Kiithcrim umicata^ and rid an unstable canunurd 3 Forifsro.
primarysp.a,ce occu| peiseernst.atPtehercent cover estimatdefsroomY interaction cnoumdmjubnriatnychs,truicntnurer Alaska, recmiitm. >m
1109904..25tim.'ppaenrimcaenaenatveqruaagdrats established1.i9n%\cover A'm'I.'Knawlton (email ]}Luk<n:i:;;;.,:<h:> & Raymond C.
i 1996. I lories i kh ofFfsfia
wLe."r-eio i i'rs! to, th.e-6.s4i^t,e. and roipensprrioncgk,of261.9197% aBtnxtxfO"ceaf}S ewbank trUw/^/!fj ' \tasl fyiri .. '• PC).
wealed that lb colonies overwintered with
.