Pure & AppL Chem., Vol. 48, pp. 7—23. Pergamon Press, 1976. Printed in Great Britain.

NATURAL PRODUCT CHEMISTRYOF THE MARINE SPONGES

L. MINALELaboratorio per la Chimica di Molecole di Interesse Biologico del C.N.R.—Via Toiano n.2, Arco Felice, Napoli, Italy

Abstract—A systematic search for constituents of marine sponges has yielded over one hundred new compounds,most of them with unique structural features. A broad survey of the field is presented and certain topics, particularlythose closely related to recent work done in our own laboratory on sesquiterpenoids, are discussed in more detail.

INTRODUCTION

In the context of the recent increased interest in thechemistry of marine organisms, the sponges, very primi-tive multicellular animals, have also received attentionleading to the discovery of many novel molecules. SinceBergmann's pioneering work' on the fatty acids andsterols in sponges over a hundred different compoundshave been isolated, mostly in the last 5—6 yr.

When I started to prepare this lecture I had the choiceof either presenting a summary review, or selecting onlycertain topics. It seemed to me best to choose the former.This would give a general picture of the sponge-derivednatural products and should help to focus attention on thestructural relationship between compounds isolated fromdifferent species, and allow us to see if metabolites so farisolated from sponges also occur in other marine phylaand/or terrestrial organisms.

With your permission, I will attempt to discuss in moredetail the very recent results from our own Laboratory,particularly those concerning sesquiterpenoids.

In this lecture, the known natural products fromsponges have been grouped in accordance with theirprobable biosynthetic origins, and I will discuss bromo-compounds, terpenes, compounds of mixed biogenesis,and sterols in that order. A brief mention of somemiscellaneous compounds will be also made. Fatty acidsand pigments are excluded as very little has been pub-lished on these topics since they were last reviewed in1%8.23

BROMO-COMPOUNDS

About a hundred naturally occurring organobromo-compounds have been so far described, and only one ofthese was not isolated from a marine organism.4 Thusthese compounds, which belong to such diverse chemicalclasses as phenols, pyrroles, indoles, sesquiterpenes, di-terpenes, and polynuclear heterocycles, appear to becharacteristic of the marine environment. They have beenfound especially in algae.5'6 Several brominated monoter-penes, sesquiterpenes, and diterpenes have also beenextracted from the digestive gland of molluscs of thegenus Aplysia, but experiments revealed that the chemicalconstituents of the digestive gland depended on the algaldiet of the individual Aplysia,7 and there now seems noreason to doubt that the brominated terpenes from Ap -lysia are derived from red algae such Laurencia, acommon food of the sea hare. So, besides the algae, therichest source of bromo-compounds appears to be thesponges.

Sponges of the family Verongidae have provided aseries of antibiotics and other closely related compounds

which may be considered as metabolites of 3,5-dibromotyrosine. Figure 1 lists their structures. The firsttwo members of the series were isolated from themethanolic extracts of Verongia fistularis and V. caulifor-mis by Sharma and Burkholder.'° The failure to convert Iinto II by reacting with methanol under various conditionsallowed the authors to assume that the ketal II was agenuine natural product and not an artifact generatedduring the extraction. Recently Andersen and Faulkner"have isolated from the ethanolic extracts of an unde-scribed species of Verongia the dienone I and the mixedketal III, which latter was revealed to be a mixture ofdiastereoisomers (two methoxy signals in the 220 MHzNMR). This suggested that the ketal was not a naturalproduct and led the authors to propose that the dienone I,the dimethoxyketal II and the mixed ketal III may all bederived from a single intermediate, such as an arene oxide(XI), by 1,4 addition of water, methanol, or ethanol duringthe extraction process. The recent work of Kasperek etal.,'2 showing that acid-catalyzbd addition of methanol to1,4-dimethylbenzene oxide give 4 - methoxy - 1,4 - di-methyl -2,5 -cyclohexadienol, was quoted by the authorsin support of their arguments.

7

Br,BrHO CH2

HsCO OCH3

Br..>-BrHO CH2

H3CO OC2HS

HO CH2

CONH CONH CONH2

(Sharma and Burkholder,1967) (Andersen and Fau(kner,1973(

B OHHBr 0 Hr

H2C Br Br HBrNC OH v;aeroplysrnlnl(+( V1 aerop(ysin(n -i- v(1aerop(ysin;n 2

(Fulmor etal.1970(

CH2=O

BrN,..Bro°°° NH

V(l; n=V aerothionin (XVl((; fl 5 homoaerothionin ( Borders eta( 1974

BrT.L(BrC H2 CON H2

OHo (Arejoarek eta(.1975

Fig. 1. Tyrosine-derived bromo-compounds.

8 L. MINALE

OCH3

Br I Br

CONH2

xl

The compound IX listed in Fig. I has been isolated fromVerongia lacunosa22 and it is unique in that it appears tobe the first bromo-compound containing 2-oxalidone ringsisolated from a sponge. The latest addition to this group isX isolated from V. aurea by the Rinehart's group(Illinois).23 It represents a major departure from the pre-viously reported Verongia brominated metabolites inwhich the aliphatic side chain remains in the paraposition relative to the hydroxyl group flanked by bromineatoms. An analogy for such a rearrangement of thetyrosine skeleton is available, however, in the conver-sion of 4-hydroxyphenylpyruvic acid into 2,5-dihydroxyphenylacetic (homogentisic acid), catalyzed byan enzyme classified as a mono-oxygenase.24 Interestinglya nonenzymic pathway shown in Fig. 3 for the conversionof 4-hydroxyphenylpyruvic into homogentisic acid viathe quinol XIVa, itself formed most probably via a cyclicperoxide, has been very recently described.25 This couldalso offer an alternative plausible explanation for hebiogenetic formation of the dienone I (Fig. 1).

Beyond these speculations, all the Verongia bromi-nated metabolites seem fairly obviously biosynthesizedfrom 3,5-dibromotyrosine, itself found in sponge pro-teins,26 and presumably the central C4N2 and C5N2 chainsof aerothionin (VII) and homoaerothionin (VIII) are de-rived from ornithine and lysine, respectively.

Quite surprisingly V. aerophoba failed to incorporateradioactivity from {U-'4C}-L-tyrosine into aerothionin(VII), aeroplysinin-l (IV) and the dienone (I); inactiveaerothionin (VII) was also isolated when the animals werefed with {U-'4C}-L-ornithine and {CH3-'4C} methionine.27However the sponge utilized these aminoacids for thesynthesis of fatty acids. A very slow rate of biosynthesis.might account for these results. A dietary origin for thesecompounds can be also suspected and in this connectionthe recent report of the isolation of the brominated estersXV and XVa (after methylation) from hydrolyzed ex-tracts of the red alga Halopytis incurvus28 seems relevant.So it appears possible that the brominated compoundsisolated from sponges, like the bromo-terpenes frommolluscs, were originally fabricated by algae. In addition Imust emphasize that all the Verongidae, like many othersponges, have large amounts of symbiontic bacteria andblue-green algae in their tissues.29

0

HO——CH—C—CO2H---' OXOOCCOH

C H2-CO2H

icPi.XCH2CO2H

Fig. 3. Nonenzymic pathway for the conversion of 4-hydroxyphenylpyruvic acid to homogentisic acid (I. Saito et a!.,

1975).

0 C H3

B

rJ(BC H2-C02-CH3

Aeroplysinin-l (IV), the nitrile component, was firstisolated as the dextrorotatory isomer in our laboratoryfrom Verongia aerophoba,'3 which also contains thedienone I, the lactone VI,'4 and the more complex VII andVIII.'5 Fulmor et al.'6 isolated the laevorotatory antipodeof aeroplysinin-l from the closely related sponge lanthellaardis, for which they propose the absolute configurationas shown in V on the basis of combined chemical, c.d.and NMR data. The absolute structure of both antipodesas shown in IV and V have now been firmly establishedby two independent X-ray studies.'7"8

The occurrence of each enantiomorph in different gen-era of the same class of invertebrates is most unusual.

Aeroplysinin-1, which possesses, besides antibacterialproperties, antitumor activity, is the first example of anaturally occurring 1,2 - dihydroarene - 1,2 - diol and itcould be biosynthesized via an arene oxide'9 in agreementwith the stereochemistry.

The two more complex brominated metabolites (VIIand VIII) obtained from V. aerophoba were also isolatedby Moody and Thomson from V. thiona, and accordinglynamed aerothionin and homoaerothionin. Structural eluci-dation of these two spirocyclohexadienylisoxazoles wasthe result of a collaborative effort between ProfessorThomson's laboratory in Aberdeen and our own group inNaples.'5 I cannot specify in detail the arguments leadingto the structures VII and VIII but I must add that thestructure of aerothionin, the major component of bothsponges (ca. 10% in V. aerophoba), has now been con-firmed by X-ray crystallographic analysis (J. Clardy,personal communication), which also revealed the relativestereochemistry (0—H and 0—N trans). The spiro systemsin VIII and VIII could arise in various ways includingnucleophilic attack by an oxime function on an areneoxide as shown in XII (Fig. 2). Following suggestions thatnitriles may be derived in vivo from a-amino-acids byway of a-keto and a-oximino-acids,2° we speculated thatthe oxime (XII, R = OH) could be also a likely precursorof the nitrile aeroplysinin-1 (IV and V), as indicated in XIII.We now have obtained good support for this hypothesis byisolating from a marine sponge Hymeniacidon sanguineathe oximinopyruvic acid XIV.2'

OCH3

Br

0

OCH3

B

H3KHO-N. R0

XII

0,Th I IIHO N\JC

XIVa

OH

—I

CH2-C—CO2H

N-O H

Xv

OCH3

BrBrH300' CO2CH3

XVaxii I X IV

Fig. 4. Bromo-compounds (after methylation) from the hydrol-Fig. 2. Biogenetic hypothesis for the formation of the spiro yzed extracts of the red alga Halopytis incurvus (J. M. Chantrainesystem in aerothionin and of the nitrile function in aeroplysinin-1. et at., 1973).

Natural product chemistry of the marine sponges 9

Before leaving this subject I should like to show youthe list of sponges, collected in different seas and oceans,which have been reported to contain tyrosine-derivedbromo compounds (Table 1). So these compounds seem tobe confined to the Verongidae family and to the closelyrelated lanthella genus; this has received support from arecent survey for the presence of the above group ofcompounds covering 33 more species representative of 17families and 9 orders of the class Desmospongiae.3°

Table 1. Sponges in which dibromotyrosine-derived compounds have been reported3°

Sponges

Order DictioceratidãFamily Verongidae

V. aerophobaV. archedV. cauliformisV. fistularisV. thionaV. sp.V. lacunosaV. aurea

Family Disideidaelanthella ardislanthella sp.

Another group of bromocompounds are the biogeneti-cally related bromopyrroles isolated from two Agelas andthree Axinellida. Fig. 5 lists their structures. The simpledibromopyrroles (XVI—XVIII) have been isolated fromAgelas oroides,31 which also contains, in much largeramounts (2—3% of dry sponge), the more complex oroidin,to which the structure XIX has been definitively assignedafter the synthetic work of Garcia et al.32 Oroidin has alsobeen recently found in Axinella damicornis and A. verru -cosa.3° The polycyclic dibromophakellin (XX) and 4-bromophakellin (XXI) closely related to oroidin, andshowing broad spectrum antimicrobial activity, have beenisolated from Phakelliaflabellata (Axinellidae) by Sharmaand Burkholder.33 The complete structure of di-bromophakellin (XX), the major component of the sponge,was established by X-ray analysis of a single crystal of themonoacetyl derivative. The latest addition to this group isthe new antibiotic 4 - bromopyrrole - 2 - carbonyl-guanidine (XXII), isolated from an unidentified species ofAgelas.34 The occurrence of related bromopyrroles fromtwo Agelas (order Poecilosclerida) and three Axinellida

Br Br Br

Br N COOH Br N CN Br N CONH2H H H

XVII

H2N4,>3XIX,oroi din XX, Ri=RBr dibromophakellin

XXI, RLH; RLBr; 4-bromophakellin

(Sharma and Burkholder,1971 I

Br NH2H II0 NH

seems to indicate a relationship between Agelas and someAxinellida. In this connection it is important to note thatBergquist and Hartmann35 observed an anomalous amino-acid pattern in the Agelas genus with respect to those ofother Poecilosclerida and found it difficult to differentiateAgelas from typical Axihellidae.

In Fig. 6 the remaining bromo-compounds so far isolated from sponges are shown. The two brominatedphenoxyphenols, active against both gram-neg. and gram-pos. organisms, have been found in Disidea herbacea,36while the two new antibacterial bromoindole metaboliteshave been isolated from the Caribbean sponge Polyfibros-pongia maynardii.37

BrBr Br

B r__f_0 __$_ Br Br_-(_ o

XXIII XXIV

(Sharma and Vig ,1972)

::cçrN HXXV RCH3XXVI) R=H

Van Lear etal.,1973)

Fig. 6. Miscellaneous bromo-compounds from sponges.

TERPENES

Terpenes are amongst the most widespread groups ofnatural products. They are mainly of fungal and plantorigin, but they have also been isolated from insects andmarine animals. In marine organisms they have beenreported to date from algae, coelenterates, molluscs andsponges. The distribution among marine phyla may beeven smaller, as stated by Faulkner and Andersen in theirrecent excellent review which appeared in The Sea,38since there is evidence that algal symbionts are the truesources of terpenoid compounds isolated from coelenter-ates and that the terpenoids from molluscs are derivedfrom ingested algae.

Sponges have provided terpenes in large amounts, mostof them possessing unique structural features withoutparallel in terrestrial sources.

Furan rings occur frequently, although hitherto nearlyall known furanoterpenes were plant products. In thisgroup the linear furañoterpenes containing 21 carbonatoms are the most intriguing compounds from thebiogenetic point of view. More recently we have encoun-tered an interesting assortment of furanoid sesquiter-penes, and I would like to give you a brief account ofsome work on these compounds. Furthermore, sesterter-penes are relatively abundant in sponges in contrast withtheir very limited distribution elsewhere.39 Even moreunusual are terpenes bearing an isonitrile function, a veryrare feature in nature. At this time neither monoterpenesnot triterpenes have been reported from sponges sources,apart from squalene which was found in Irciniaspinosula4° and I. muscarum.41

SesquiterpenesNow let me discuss the sesquiterpenes. Disidea palles -

cens has provided ten new sesquiterpenes42 (Fig. 7).

XVI XVIII

XXII I Stempien etal,1972I

Fig. 5. Bromopyrroles from sponges.

10 L. MINALE

Fig. 7. Furan sesquiterpenes from the sponge Disidea pallescens.

These include three of a mono-cyclofaresane type,pallescensin-1 (XXVII), —2 (XXVIII) and —3 (XXIX), andseven, pallescensins A—G (XXX—XXXVI), closely re-lated, having a 2,3-disubstituted furan ring and two addi-tional cycles. Lack of material and the instability of mostof them prevented extensive chemical investigation andthe structural assignments are mainly based on spectralgrounds, biogenetic considerations, and interrelation be-tween them.

U.V. and NMR spectra (Fig. 8) clearly revealed the

OH

0'•XXIX, paltescensin-3

5.94d.d(J,1O,2)/,70bs

\( {486bs55cm H2\ MS

XXVIII, pallescensin-2Xmax 230 nm(E,22,000)

XX VII, paLlescensin-1 m/e 162

Fig. 8. Structures of pallescensin-1, -2 and -3. Chemical shifts arein ppm from TMS and coupling constants in Hz.

presence in pallescensin-2 of a f3-substituted furan ringisolated from a conjugated diene system. The NMRpattern due to the olefinic protons was readily assigned toa 1,3-disubstituted conjugated butadiene system. Thedownfield vinyl-H ( 5.94) is clearly an internal hydrogenof the conjugated system and the 10Hz coupling indicatesa cis double bond. Hydrogenation yielded two dihydro-derivatives and the major one is the 1—4 addition product(XXVII); in the mass spectrum a significant m/e 162fragment corresponding to the elimination of isobutene bythe retro-Diels—Alder process, supported the presence inits structure of a 4,4 - dimethylcyclohex - 1 - ene ring. Thestructure XXVIII, proposed for pailescensin-2, fits withall data and its mass spectrum, which is marked by intense"McLafferty type" fragments at m/e 122 and 94 (Fig. 8),added confirmatory evidence. The structures ofpallescensin-1 (XXVII) and pallescensin-3 (XXIX) inwhich latter the furan ring is modified as a y - hydroxy -

a,/3 - butenolide, were determined by spectral data andinterrelations with pallescensin-2 (XXVIII) shown in Fig.8.

Pallescensin A (XXX), which showed NMR charac-teristics indicative of three tert-methyl groups and a2,3-substituted furan ring (Fig. 9), was also interrelatedwith pailescensin-1 (XXVII). The latter, on treatment withBF3—etherate, yielded a tricyclic compound identical withXXX in GLC, Si02—AgNO3 TLC, and mass spectrometry.

The cage like structures of pailescensins B—D (XXXI—XXXIII) were essentially based on a detailed analysis ofthe NMR data summarized in Figs. 10 and lOa, togetherwith decoupling experiments.

.17'H 7.02d(J=2)

ddeubLet0.93'0.91'

XXX, pallescersin 4 ['1, 97'

Fig. 9. Conversion of pallescensin-1(XXVII) to pallescensin-A(XXX).

Xxxi pailescensin 9, [a] +

Fig. 10. Proton interactions in pallescensin B detected bydecoupling; coupling constants in Hz.

In the mass spectrum of pailescensin B (Fig. 10) thesignificant m/e 160 fragment was interpreted as originat-ing by elimination of isobutene by the retro-Diels—Alderprocess and suggested the presence of a dimethyl-cyclohexene ring. The sequence of protons in the six-membered ring was determined by decoupling, and thedetected proton interactions are indicated in Fig. 10.Irradiation at ô 3.40 (H-2) sharpened both the furanoidprotons, reduced the olefinic broad doublet at t5 5.83 into abroad singlet and also the signal at 5 1.60 (2H; H2 at C-3)

XXVII, pallescensin-l XXVIII, pallescensin-2

QJOHXXIX , pallescensin-3 XXX , pallescensin A

H HH

XXXI, pallescensin B XXXII, pallescensin C

0

XXX II, pallescensin 0

XXXV , pallescensin F

XXX IV, pallescensin E

XXXVI,pallescensin S

XXVII paiiescensin-l

AH

[]+ []4m/e122 11* 94

MS

+.

0

m/e 160

H2, Pd-C

Natural product chemistry of the marine sponges 11

607 d (J1 0)

6.g8dIJ=2) H S.gSd(J2)

XXXII, pallescensin C; No + 42.6*

6.ggd

IJ=2),\5952) { ;:I J=4,1.5I H1'kH\

5dd)J=g,4)H IIIçIII

12 L. MINALE

MeMeHX HB HA

H JAXJAY =53 5\ Hy JBX6HH / / JBY9

Fig. 12. Absolute configuration of pallescensin G (XXXV17; c.d.O266 18,200.

axial orientation is required for H-S (in the case of aquasi-equatorial orientation, H-S should be in a W rela-tionship with H-3 and one should expect couplings, evensmall, between them). On this basis, we tentatively pro-pose for pallescensin G the absolute configuration shownwith R-chirality at the sole asymmetric center C-S. Furth-ermore from inspection of the Dreiding models of the twopossible conformers, one is unreal because of the sterichindrance between one tert-Me and H2C-7, and for themore favourable one the coupling constants HA—HX, HA—Ify, HB—HX and HB—HY calculated from the correspondingdihedral angles by applying the Karplus equation accordwith the observed values.

Figure 12a summarizes the spectral data of pallescensinE, the benzenoid component of this group. An accurateanalysis of the small long-range couplings, detected bydecoupling, was also in this case the key argument whichfavoured the structure XXXIV for pallescensin E.

,- H 709d(32)

594d (J2)

Fig. 12(a). Chemical shifts (jipm from TMS) of pallescensin E(C6D6);————longrangeprotoninteractiondetectedbydecoupling.

Pallescensins A—G represent new skeletal typesamongst the sesquiterpenoids, and their structures canalso be rationalized biogenetically when a furanoid mono-cyclofarnesane intermediate is submitted to C—C cycliza-tions, as shown in Fig. 13, and further oxidations.

The co-occurrence of furanoid sesquiterpenes of themonocyclofarnesane type provides good support for thissuggestion.

A further group of furan sesuqiterpenes have beenextracted from the sponge Pleraplysilla spinifera. Amarked difference was found between the components oftwo samples collected in the Bay of Naples. However,according to the expert opinion of an authoritative zoolog-ist, both samples were identical from a spicule analysisstandpoint.

This, we believe, is an interesting finding. The twosamples of the sponge show only slight morphologicaldifferences, but enough to be differentiated. I must em-phasize that sponges are exceptionally difficult to classifyand possibly the two samples belong to different species.

Figure 14 lists the structures of the constituents of thefirst sample.43'66

XLI, pleraplysillin-l ,MYe 216 XLII

IMS /+ COH[]

Fig. 14. Furan sesquiterpenes from Pleraplysilla spinifera (firstsample).

Dehydrodendrolasin (XL), the major component (5% ofdry sponge), is closely related to dendrolasin, the odour-substance of the ant Dendrolasius fuliginosus ; the secondcomponent, pleraplysillin-1 (XLI), is a new type of ses-quiterpene. The formation of the six-membered ring inpleraplysillin-1 is of biosynthetic interest, as it seems toarise by a C—C cyclization involving a lateral methylgroup.

Its structure was deduced from spectral and degrada-tive data: UV and NMR spectra pointed out the presenceof a /3-substituted furan ring isolated by a methylene fromthe 1,3-diene system; oxidative ozonolysis gave 3,3'-dimethyl adipic acid and the position of the double bondin the cycloliexene ring was confirmed by a strong peak atm /e 160 in the mass spectrum corresponding to elimina-tion of isobutene by the retro-Diels—Alder process. Thethird component in P. spinifera is the ester pleraplysillin-2(XLIII).

The second sample of Pleraplysilla spinifera containslongifolin (XLIV), previously found in a terrestrial plant,45and two more cyclic furan sesquiterpenes, namedspiniferin-1 and -2, for which we propose the alternativestructures (XLVa)—(XLVb) and (XLVIa)—(XLVIb), re-spectively (Fig. 15), with carbon skeletons of a newstructural type.66

XL, dehydrod endrolasin

211

210s XXX IV

m/e 160

XLIII, pleraplysi IIin-2

ccpallescensinsF-G

skeleton \12,lsin kaBe"\\

pallescensrn Eskeleton

nkagepaUescensin C

skeleton

skeleton

pallescensin 0skeleton

pallescensin Bskeleton

Fig. 13. Possible biogenetic scheme for the formation ofpallescensins A—G.

Natural product chemistry of the marine sponges 13

XLVIa XLVI bspiniferin -2

Spiniferin-2 is an optically inactive oil showing UVabsorptions (Fig. 16) consistent with the presence of furanand benzene rings. In the NMR spectrum the presence ofa 2,3-disubstituted furan is indicated by doublets at 6 7.03and 5.%. The benzenoid protons are seen as a singlet at6 6.82 and two aromatic methyl groups resonate at 6 2.21and 2.25. An isolated methylene group between thearomatic and furan rings is indicated by a low field broadsinglet at 6 4.02, while a C2 saturated chain is suggested byan A2B2 system with line positions at 6 2.61 and 2.92 ppm.An accurate analysis of the small long-range couplings,detected by decoupling, established that the isolatedmethylene is proximate to the methyl resonating at 6 2.25(homobenzylic coupling), while the multiplet at 6 2.92couples to the benzenoid protons (benzylic coupling).Decoupling experiments also revealed benzylic couplingbetween the C-6 methylene protons and both the furanoidprotons and established the existence of "homobenzylic"couplings between the C-6 and C-9 methylene protons(.16,9 1 Hz). Smaller but observable couplings of the C-6protons with the C-lO protons were also noted.

Oxidative ozonolysis, followed by methylation withdiazomethane, afforded a dicarboxylic acid methyl ester,whose spectral properties fully agreed with the structureXLVII: the two aromatic protons now appear as an ABquartet with J =8Hz indicating an ortho -relationship.

Now only two alternative structures (XLVIa and

4282_system6.82—I'-../1o 9\

I H 5.96d(J=2)-... 6 /a OR

( H 7.03d (J=2)4.02'\, 2.25

2.2 is

spiniferin-2 Xmax 222,224 and 264 nm(E,iO,200 i3,200790)H

O2C

XLVII £Ar-H2 6.861 ABq; 1=8Hz)

--H H F-I,Ii H.. /, -I

51j .---.,. H - _- --- H 725d(j2)

075d(310) 362d(y.10)

XLVa ,k)0—4-2; ''Xmas 240302 nm C

XLVIII ;

14 L. MINALE

Both pairs XLVa-XLVIa and XLVb—XLVIb appearbiogenetically reasonable and they are based on carbonskeletons so far unique amongst sesquiterpenoids. Figure18 shows a possible biogenetic scheme for the formationof both pairs starting from a cis -farnesyl precursor. Theformation of the dimethylcyclohexane ring in XLIXthrough aC—C cyclization involving a lateral methyl groupof the polyisoprene chain also occurs in pleraplysilin-1(XLI), isolated from the first sample of Pleraplysillaspinifera.

A series of four more furano sesquiterpenes have nowbeen obtained from the sponge Microciota toxystila andtheir structures are listed in Fig. 19. Microcionin-3 hasbeen formulated as LII on spectral data and formation, onozonolysis, of 2,2,6-trimethylcyclohexanone.

OR XLVa-XLVIa

biogenetic scheme for the formation ofspiniferin-1 and -2.

L, microcionin-1 LI, microciornn-2

LII, microcionin-3 LIII ,microcion,n-4 LIV

Fig. 19. Furan sesquiterpenes from Microciona toxystila.

The two double bond isomers, LI and LIII, wereinterrelated by showing the identity of their dihydroderivatives, while the relationship between LI and tricyc-lic component L has been established by converting theformer to the latter, using BF3—etherate. The structure ofmicrocionin-2 (LI) has been deduced from NMR charac-teristics indicative of a a-substituted furan ring, an olefi-nic hydrogen and of three methyl groups, one vinyl on atrisubstituted double bond, one tertiary and one secon-dary,. together with chemical transformations and relatedspectroscopic properties. Microcionin-2 has been con-verted into the enone LV (Fig. 20). In the NMR spectrumof this compound, recorded in the presence of Eu(fod-d9)3, the resonances of the methylene a to the ketonewere resolved giving rise to an 8-line multiplet clearlyconstituting the AB part of an ABX system, thus confirm-ing that C-6 is tertiary and C-S quaternary. An indicationof the relative stereochemistry at C-S and C-6 comes froma study of Eu(fod-d9)3 induced shifts of the methylresonances of the two diastereoisomeric epoxides LVIand LVII. The normalized ratios of 10:6.14:5.65 and10:8.41:3.73 for the induced shifts of the C-4, C-S and C-6methyl groups in LVI and LVII, respectively, are consis-tent with the relative stereochemistry as indicated. Inaccordance the shift of the H-6 signal in the spectrum of

LVII is approximately four times greater in magnitudethan that of the same signal in the spectrum of LVI.Reinforcing evidence for the structure assigned tomicrocionin-2 (LI) came from the mass fragmentationpatterns of the two epoxides. The mass spectrum of LVIis marked by peaks at m/e 140 and 94 resulting from a"McLafferty type" rearrangement, in agreement with thesyn-relationship between the epoxide ring and the alkylchain at C-5, while LVII breaks down without hydrogentransfer giving intense ions at m/e 139 and 95.

The co-occurrence of microcionins-l, -2 and -4, havinga rearranged skeleton, along with microcionin-3 suggeststhat the whole group is derived from a commonbiosynthetic precursor, the ion LIV (Fig. 19).

As pointed out before the isonitrile function is a veryrare feature in nature. Until 1973, only xanthocillin,isolated from Penicillium notatum, had been described asa natural isonitrile.47 The isonitrile function has now beenfound in five sponge sesquiterpenes and one diterpene.The structures of the sesquiterpenes are shown in Fig. 21.Fattorusso and his co-workers48'49 obtained the first two,accompanied by the corresponding formamides andisothiocyanates,5° from Axinella cannabina. Interestingly,

LVIII, axisonitrile-1 LIX axisonitrile-2

IFattorusso etal.,197374I

I H,,L,,

LX,acanthellin-1(Burreson etal.,1975)

LXII, axisonitrile-3I Di Blasio etal.,1975I

Fig. 21. Isomtrile sesquterpenQiØs from sponges.

123 95

LI, microcionin-2

1 6

LV, \Imax 1670 cm

acid

'H

139 95LVI

1M9

LVII

+ +

LH0m/e 140 m/e 94

Fig.20. Structure microcionin-2.

Fig. 18. Possible

—'XLVb-XLVIb

XLIX

LXI

Natural product chemistry of the marine sponges 15

the new skeletal type of axisonitrile-1 also occurs inoppositol, a bromo-sesquiterpene recently isolated fromthe red alga Laurencia subopposita.5'

The third isonitrile sesquiterpene in Fig. 21, which has a4-epi-eudesmane skeleton, has been found in the closelyrelated species Acanthella acuta (Axinellidae),52 whilethe amorphane sesquiterpenoid LXI has been isolated,with its corresponding formamide and isothiocyan-ate, from an unrelated Halichondria sp. (Halichondridae)by the Scheur's group in Hawaii.53 Co-occurrence Ofterpenoid isonitrile—formamide pairs is strong evidencethat a formamide is the biogenetic precursor of the rareisonitrile function. The latest addition to this group isLXII, which has been isolated in the form of beautifullycrystalline substance from Axinella cannabina.54 This wassubmitted immediately to X-ray crystallographic analysiswhich established that it has the spiro structure LXII.

This represents the fourth type of spiro(4,5)decane-derived sesquiterpene, after the discovery of the acoranesand the sketally related enantiomeric alaskanes, and thespirovetivanes, all occurring in terrestrial sources, and thespirolaurenone, a bromine-containing sesquiterpene iso-lated from a marine source (Fig. 22).5556

a pirovetiva neske(eton

spirolaurenone sponge-derived(from Lauren cia granduliferal spirot45) decane

skeleton

Fig. 22. Spiro[4.5}decane carbon frameworks in sesquiterpenoids.

DiterpenesOnly two examples of true diterpenoid compounds

have so far been reported from sponges. One, isolatedfrom Spongia officinalis,°7 proved to be the first naturallyoccurring compound with the carbon skeleton ofisoagathic acid, the acid-catalyzed cyclization product ofagathic acid, and accordingly named isoagatholactone(LXIII). The second one, elaborated by a Halichondriasp.,58 is the isonitrile analogue of geranyl-linalool (LXIV),co-occurring with the corresponding formamide (LXV)and isothiocyanate (LXVI) (Fig. 23).

c0LXIII, isoagatholactone

LXIV; RNvCLXV R=NHCHO (Burreson and Scheuer,1974)

LXVI ;R=NCS

Fig: 23. Ditarpenoids from sponges.

The C21 furanoterpenes and sesterterpenesAmong the most unusual terpenes isolated from

sponges are the linear furanoterpenes containing 21 car-bon atoms occurring in the genus Spongia. All of thempossess the same carbon skeleton, LXVII and oxidationin the central chain accounts for all their differences. Theindividual structures are listed in Fig. 25. Spongia nitenscontain nitenin (LXVIII) and dihydronitenin (LXIX),59while Spongia officinalis and Hippospongia commu'iisboth yielded furospongin-1 (LXX) as the major terpenoidcompound6° and the related compounds (LXXI—LXXV)as minor components.61

LXVII

Fig. 24. Carbon skeleton of the sponge-derived C21 furanoter-penes.

XIII,isofurosin-2

XIVzdihydrofurospongin2

LXXV tetrahydrofurospongin-2

Fig. 25. C21 linear difuranoterpenes from sponges.

At present the biogenetic origin of these unique C-21compounds is a matter of speculation. Radio labellingexperiments using 1-14C acetate in the sponge Spongianitens resulted only in non-radioactive nitenin and dihyd-ronitenin.62 This might suggest that the sponges, like theMolluscs and Coelenterata, are unable to synthesize denovo terpenoids, which may be derived from the diet ormay be synthesized by algae symbiotically associatedwith the animals.

While evidence on the biogenetic origin of these C-21compounds is lacking, in view of the occurrence in relatedsponges (genus Ircinia) of several furanoid sesterter-penes, we prefer the idea that they are derived bydegradation of sesterterpenoids to the possibility ofbiosynthesis by addition of a C-i unit to a diterpenoidprecursor. Figure 26 lists the structures of the linearsesterterpenes isolated from sponges. The isomericircinin-i (LXXVI) and ircinin-2 (LXXVII) have been

acorane -alaskaneskeleton

Br)Ifl

.J LXVIII nitenin \__0

dihydronitenin

X,furospongin1"(�anhydrofurospongin-l \—

0

furospongin-2

16 L. MINALE

LXXVI; b12'13, ircinin-10

LXX VII;L3'15, ircinin-2

LXXVIII fasciculatin

LXXIX variabilin (Faulkner,1973)

C02 C H3

LXXX R=C02H R=CH3 furospongin-3LXXXI R=CH3 R=CO2H furospongin-4

Fig. 26. Linear furanoid sesterpenes in sponges. *Stereochemisftyof olefin unknown.

isolated from Ircinia oros,63 while the closely relatedmonofurano derivatives fasciculatin (LXXVIII) and var-iabilin (LXXIX) have been found in I. fasciculataTM andI.variabilis,65 respectively. Significantly, furospongin-3(LXXX) and furospongin-4 (LXXXI), the less elaboratecomponents of this interesting group, have been isolatedfrom Spongia officinalis, which also contains the C-21furanoterpenes. Better support for our biogenetic conjec-ture comes from the discovery of two isomeric C-21furanoterpenes, ircinin-3 (LXXXII) and -4 (LXXXIII)(Fig. 27) co-occurring in the sponge Ircinia oros66 with thesesterterpenes, ircinin-1 (LXXVI) and ircinin-2(LXXVII).

C 02H

LXXXII, ircinin-3

C 02 H

LXXXIII, ircinin-4

Fig. 27. C-21 furanoterpenes in Ircinia oros.

The two isomeric pairs (C21 and C25) have structuresvery closely related even in the position and stereo-chemistry of the central double bonds. In addition, theisolation from Ircinia spinosula4° of a C35 linear furanoter-pene, LXXXIV (n =6) along with the C-31 difuranoter-pene (LXXXV) (Fig. 28), which are in the same biogeneticrelationship as the above C25—C21 compounds, lendsfurther substantial support to this suggestion, since it isdifficult to accept the increasing co-occurrence of suchclosely related structures as coincidence.

In Table 2 there is the list of sponges which have beenreported to contain furanoterpenes, and you will see thatthese compounds• have been found in three families

LXXXIV; n,L,5,6

LXX XV

Fig. 28. Furanoid terpenes in Ircinia spinosula.

Table 2. Sponges in which furanoid terpenes havebeen reported3°

Sponges

Family AplysillidaePleraplysilla spinifera sample 1Pleraplysilla spinifera sample 2

Family DisideidaeDisidea pallescens

Order PoeciloscleridaFamily Clathriidae

Microciona toxystila

belonging to the order Dictioceratides and in the unrelatedMicrociona toxystila.

Another impOrtant group of sesterterpenes with a tet-racarbocyclic skeleton have been found in sponges of thegenus Cacospongia, and in the taxonomically relatedSpongia. Their structures are shown in Fig. 29.

Scalarin (LXXXVI) from Cacospongia scalaris was thefirst compound of this group to be isolated and itsstructure determination was reported by Fattorussoet aL67 Deoxoscalarin (LXXXVII) and scalaradial(LXXXVIII) have subsequently been isolated from Spon-gia officinalis68 and Cacospongia mollior,69 respectively,and their structures assigned on chemical interrelationwith scalarin.

Order DictioceratidaFamily Spongidae

Spongia nitensS. officinalisS. agaricinaHippospongia communisIrcinia orosI. fasciculata

I. variabilis

I. spinosula

LXXX VI, scalarin LXXXVII, deoxoscalarin

I Fattorusso etal,1972I

LXXXVIII, scalaradial

Fig. 29. Tetracarbocyclic sesterpenes in sponges.

Natural product chemistry of the marine sponges 17

These closely related cyclic C25 terpenes represent anew structure type in sesterterpenes,7° themselves a rela-tively rare group of compounds. Interestingly, the sponge-derived cyclic sesterterpenes have a terrestrial represen-tative in cheilanthatriol (LXXXIX), recently isolated froma fern Cheilanthes farinosa,7' and the whole group may bederived from a geranylfarnesyl precursor (XC) by acyclization initiated at the isopropylidene group, which istypical of triterpenes.

çtiir CLXXXIX cheilanthatriol

(Khan et aI,1971)

CHrOR

HLErXC

Compounds of. mixed biogenesis: mevolonate-benzenoidprecursor

Compounds of mixed biogenesis originating partly frommevalonate and partly from a benzenoid precursor arewidespread in nature. Recently two isomeric sesquiter-penoid hydroquinones, zonarol and isozonarol and thephenol taondiol with a cyclized diterpene chain have beenisolated from marine sources, the algae Dictyopterizonarioides72 and Taonia atomaris,73 respectively.

Sponges have also provided a series of compoundshaving isoprenoid skeletons linked to a benzoquinone orbenzoquinol ring.

Isoprenologous 2-polyprenyl benzoquinones (XCI, n =5, 6, 7), a novel group of terpenoid quinones, and thecorresponding quinols (XCII, n = 5, 6, 7), present in thesolvent extracts in much larger amounts, have beenisolated from Ircinia spinosula,4° which also contains thehydroxylated 2-octaprenyl quinol (XCIII) as minor

XCI n:3,5,6 0R7

rjhflOH

XCII, n:3,5,6 0R7

OH CH2OH

OH xc

CO2HXCIV

Fig. 30. Prenylated benzoquinones and quinols from Irciniaponges

metabolite. From another Ircinia species (I. muscarum),4- hydroxy -3 - tetraprenylbenzoicacid (XCIV) has beenisolated along with 2-tetraprenyl benzoquione (XCI, n =3) and the corresponding quinol41 (Fig. 30). This stronglysuggests that p-hydroxybenzoic acid is the ring precursoras in ubiquinone biogenesis.74

The acetone extracts of Halichondria panicea haveyielded75 five new compounds, panicein-A (XCV), -B1(XCVI), -B2 (XC VII), -B3 (XCVIII) and -C (XCIX), havinga sesquiterpenoid moiety linked to a benzoquinone or abenzoquinol residue, except for panicein-B2, which is thecorresponding chromenol of panicein-B3. The structuresof the paniceins are shown in Fig. 31. In the sesquiter-penoid moiety the paniceins have the uncommon featureof an aromatic ring which, notably, has been encounteredalready in renieratene (C) and isorenieratene (CI), aryl-carotenoids found in the sponge Reniera japonica76 (syn.Halichondria panicea), which also yielded renierapurpu-nfl (CII)77 and two further arylcarotenoids (CIII and C1Y)with the unique feature of an acetylenic bond.78 (Fig. 32).

A further sesquiterpenoid hydroquinone, avarol (CV),has been recently isolated from Disidea avara.79 It repre-sents the first "friedo" structure in sesquiterpenoids.

H3CO

XCV; panicein-A

OH

HOçOO=HC'-

XCVII; panicein-B2

OH

HOOH

O=HC15'!IH

XCIX panicein-C

XCVIII; panicein-B

Fig. 31. Paniceins from Halichondriapanicea.

The gross structure suggested for avarol was deducedfrom spectroscopic data, which were indicative for thepresence in the sesquiterpenoid moiety of one olefinicproton, four methyl groups, two tertiary, one secondaryand one vinyl, and a benzylic methylene linked to asaturated quaternary carbon, along with chemical trans-formations. The relevant chemical arguments are sum-marized in Fig. 33

Oxidation with Cr03-pyridine complex of avarol di-methyl ether gave the enone CVI, in the NMR spectrumof which H-b and the two C-i protons formed a cleanisolated AMX system, thus confirming that C-b is tertiaryand C-S and C- quaternary. The conversion of avaroldimethyl ether on treatment with acid into the tetrasubsti-tuted olefin CVIII and dehydrogenation of both the parentcompound and its acid-catalyzed rearranged product

XCVI; panicein-Bi

18 L. MINALE

Qr+CIX

Fig. 33. Structure of avarol(CV).

CVIII, affording 1,2,5,6 - tetramethylnaphthalene andl,2,5-trimethylnaphthalene along with a major amount oftetralin CIX, eventually confirmed the structure CV with-out stereochemical implications. The stereochemistry ofavarol remains for discussion and now let me make a briefmention of this work.

The magnitude of the coupling constants between H-1Oand H-lax and H-leq in the enone CVI indicated that thecompound has either the trans -AB ring fusion or thecis -fusion in the conformation with H-1O axial. Since thededuction of absolute configuration from the Cottoneffects of a,3-unsaturated ketones is known to be fraughtwith difficulties, c.d. measurements were also performed

• on the ketone CVII, prepared by hydroboration-oxidationof avarol dimethyl ether. On the assumption of a trans-

ring junction the strong negative Cotton effect would leadto the assignment of absolute configuration as indicated(5,l0a series). Even in the cis-clerodane diterpenoidsseries the enones corresponding to CVI are known toprefer a "steroid like" conformation with H-b equatorial,so a cis-AB ring fusion could not be excluded for avarol.An useful approach to solve this was the application ofthe nuclear magnetic resonance shift reagent Eu(fod-d9)3to the study of the diastereoisomeric epoxides CX andCXI, which also provided evidence for the stereo-chemistry at C-9.

For the purpose of comparison it was convenient tonormalize the induced shifts to give a value of 10.0 to thelowest field methyl signal and the results are shown in Fig.34. The comparison of changes of the chemical shifts of4-Me and 5-Me protons of both diasteroisomers clearlyrevealed the relative stereochemistry between the oxiranering and the angular methyl group; in the major compoundCXI, in which the oxirane ring and the angular methyl areanti each to the other, the H-b proton is more stronglyshifted towards lower field than the epoxide proton, andthis is only consistent with a syn-relationship to theepoxide ring, a- and f3-epoxides of friedel-3-ene producepatterns for H-lO and 4- and 5- methyl protons verysimilar to these produced by CX and CXI, respectively.Furthermore the "normalized" induced shifts of the ben-zylic methylene and 9-methyl protons are consistent withthe stereochemistry as indicated.

The observations that with boron trifluoride the a-epoxide (CXI) gives exclusively the A5"o olefin while thef3-epoxide (CX) furnishes the A510 (CXII)- and A5,6(CXIII)-olefins in approximatively equal amounts, pro-vide confirmatory evidence for the a-orientation of H-b.Trans-clerodane diterpenoid 3,4-epoxides are knownto behave similarly on BF3 exposure and McCrindle andNakamura8° offered a plausible explanation, which fol-lows. In the case of the a-epoxide (CXI), as the epoxidering opens, the C-3 oxygen function is suitably disposed toremove the C-b proton (CXV or related species) in anintramolecular process, forming the tetrasubstitutedolefin. In the case of the 3-epoxide (CX) either the C-l0 or

CXII CXIII CXIV

rxC, renieratene

CI, isorenieratene

CII, renierapurpurin

Fig. 32. Arylcarotenoids from Reniera japonica (syn. Halichon-dna panicea); (Yamaguchi, 1957, 1958, 1960; Hamasaki et a!.,

1973).

CVI;JAx=3Hz;JMx14Hz CVII1c.d.[O]287—7,666c.d.[G]32o 6035

tIC HSO-NaOH

2Cr 03-py

HO

c5,1.CCH3)2S04-NaOH

2 H'

MeO

L,.L,OMe

Q5,CV; avarol; rn p. 148-150 []6.1' CVIII

Pt-C270'

Pt-C270' Me

5.6O

CX CXI

JBF3-Et20 JBF3-Et20

MeO MeO MeO

e H OMe -'--" OMe

HO"' HOQ" HOcA

0 -®F3BO 'N'

CXV

Fig. 34. Normalized shifts ( = öcDcIS) induced by 'Eu'(fod-d9)3 in the spectra of 3f3,4- and 3a,4a-epoxides of avarol

dimethyl ether.

Natural product chemistry of the marine sponges 19

the C-6a-proton, both of which are trans-antiparallel tothe C-S methyl, is removed in an intermolecular reaction.Now the stereochemistry at C-8 remains to be assignedand this was established with the aid of '3C-NMR spec-troscopy, which also confirmed the stereochemistry atC-5, C-9 and C-1O. The '°C-NMR spectra of avaroldimethyl ether and its dihydro derivative were comparedwith those of some model compqunds of knownstereochemistry shown in Fig. 35.

Me 0

49.25 H: OMe0 s........38.O0

L4k6)£ \,3577

45.47

CXVII, dihyd roavarol dimethyl ether

HOH H tT- C H= 0

--

2.76C H3

CXX liner I dial, CXXI

Fig. 35. '°C-NMR data of avarol, dihydroavarol and some modelcompounds.

The compounds with a cis-AB-ring junction produced13C-NMR spectra clearly distinct from those produced bythe trans -models and particularly diagnostic, as expected,was the chemical shift of the angular methyl group whichin the former is at least 12 ppm further downfield than thatin the latter. Now the comparison of the '3C NMR spectraof dihydroavarol dimethyl ether and the clerodane diter-penoid CXIX revealed them to have nearly identicalsignals for the methyl and decalin carbons; particularlydiagnostic for the equatorial orientation of the 8-methylgroup are the shifts of the carbons C-6, C-8 and C-b,which are heavily dependent on the C-8 stereochemistry.The methine carbons could be easily distinguished by thestrong f3 effect of the OH substituent deshielding C-4 inCXVIII, and by considering the endocyclic homoallyliceffect exhibited by C-1O in CXVI. This leaves the doubletranging from 35.88 to 38.48 to the C-8 carbon. The C-6methylene carbon was assigned on a methyl-substituentparameter calculation and because its shift is nearlyinvariant in the spectra of all four compounds. As pointedout before avarol represents the first "friedo" structure insesquiterpenoids and it can be conceived as derivablefrom a farnesy precursor by cyclization to an inter-mediate cation involving a drimane skeleton, followed bya "friedo" rearrangement and finally deprotonation. As-suming the sequence of 1,2-shifts of methyl groups andhydrogen atoms be concerted, the stereochemistry of theintermediate cation should be as shown in CXXII (Fig.36). Interestingly the sponge Disidea pallescens, in addi-tion to being a rich source of furanoid sesquiterpenes(Fig. 7), has also yielded a chromansesquiterpenoid,entchroinazonarol,8' with the absoluteconfiguration CXX-Ilibiogeneticaily related to that of avarol. It is worthy ofmention that the brown alga Dyctyopteris undulata82

(from the sponge Disidea avara)

cpoch romazonaro(

(from the a(ga Dyctiopteris zonarioides;Fenica( eta(.1973,1975(

CXXIII; ent-chromazonaro((from the sponge Disidea pa((escens(

Fig. 36. Relationship between the sponge-derived avarol andent-chromazonarol and the algal zonarol and chromazonarol.

contains the antipodal isomer, chromazonarol, along withits phenolic isomer, zonarol.

The biosynthesis of antipodal terpenoids by differentorganisms is of considerable interest.

Disidea pallescens also contains in larger amountsdisidein, having a sesterterpenoid moiety linked to ahydroxyhydroquinone residue. It has been isolated as thesodium calcium salt of the disulphate. For the free phenolthe structure CXXIV, with a carbocyclic system of thescalarin type (see Fig. 29), has been proposed83 as themore probable one, which is also well explained from thestandpoint of its biogenesis. In fact, we may imagine that

CXXIV; disidein(from Oisidea pa((escens(

CXXV undergoes an essentially synchronous process forring formation if H is furnished at C-3.

STEROLS

Now let me consider briefly the recent results on thesterols from sponges. Since the extensive work of Berg-mann on the sterols of invertebrates, it has been recog-nized that, in the Animal Kingdom, the sponges containthe greatest variety of sterols. The elegant work ofBergmann and his co-workers resulted in the isolation of anumber of new sterols, but Bergmann himself was awarethat much of the data accumulated probably referred tosterol mixtures. Thus most of the earlier work nowrequires revision with the aid of more sophisticatedchromatographic techniques.

CXXII CV;avaro(

HO

zonarol

MeO

45.66 )1I10Me35.88

c7.O2CXVI, avarol dimethyl ether

4984

1b0c369CH20H 38.67

54.56

CX VIII

4874

it e

(' '367545.38

CXIX

)H

CXXV

20 L. MINALE

Since 1972 modern reinvestigations of the sterols ofsponges have began to appear, confirming the complexityof sterol composition in this phylum and announcing thediscovery of sterols of completely new types.

The reexamination of the sponges Cliona celata andHymeniacidon perleve by Erdman and ThomsonTM resultedin the isolation of three new marine stanols: 24 -norcholesta -22 - en - 33 - ol, 22-dehydrocholestanol and24-methylenecholestanol. This is the first occurrence insponges of a sterol with twenty-six carbon atoms (24-nor)which are widespread in marine invertebrates and to datehave been reported in five marine phyla, the Coelenterates,the Echinoderms, the Molluscs, the Tumcates and theSponges. An analysis of the sterols of Axinella cannabinaby the Fattorusso's group revealed the presence of fifteencomponents, C27 7,A89 and i5'7-sterols85 accom-panied by a mixture of 5,8-peroxides, from which the twomajor compounds, ergosterol peroxide and 5,8 -epidioxycholesta - 6,22 - dien - 3 - ol, have beenisolated.86 Sheikh and Djerassi87 in an examination of fivesponges for their steroid content, have also succeeded inisolating a series of 5a,8a -peroxides, listed in Fig. 37, fromTehya aurantia which also contains Z -24- propylidene -cholest -5- en -33 - ol (cxxvi), previously encounteredin the scallop Placopecten mage11anicus. The sameauthors have also found in Stelleta clarella a series ofz4-3-ketones, the first occasion on which any aj3-unsaturated steran-3-ones have been detected from marinesourceL

HO'

R = H2

R = 22R CH2 21d28)R = CH2CH3

HOcxxv'

Fig. 37. 5a,8a-Peroxides of sterols and the C-30 sterol from thesponge Tethya aurantia (Sheikh and Djerassi, 1974).

Even more unexpected is the isolation by our researchgroup of aplysterol (CXXVII) and 24,28 - didehydro-aplysterol (CXXVIII), the first examples of 26-alkylationin steroid biosynthesis (Fig. 38). The structure of 26 -methyl - 24 - methylenecholesterol was suggested for24,28 - didehydroaplysterol from spectroscopic data alongwith degradative work and confirmed by synthesis,9°while the structure of 24,26 - dimethylcholesterol,suggested for aplysterol on spectral evidence and interre-lation with didehydroaplysterol,89 has been confirmed bysingle-crystal X-ray diffraction studies of its p-iodobenzoate, which also revealed the stereochemistry ofthe side-chain to be 24R, 25S as shown in CXXVII.9°

These unique sterols appear to be confined to theVerongia genus: in an examination of 25 sponges for theirsterol composition, using GLC and mass spectrometry we

CXXVII aplysterol

HOECXXVIII; 24,28-didehydroaplysterOl

Fig. 38. 26-Methyl sterolfrom Verongia genus.

have found that all Verongia species examined containedas principal components the two new sterols (Table 3),and they proved to be absent in all the remaining speciesexamined.9' This study also confirmed the complexity ofthe sterol pattern in this phylum; interestingly, the C26sterols have been found in very low yield in almost all thespecies examined.

A further remarkable side-chain alkylation pattern hasbeen found in calysterol, the principal sterol componentof the sponge Calyx nicaensis. The structure CXXIXassigned to this sterol which includes the unique featureof a cyclopropene ring in the side-chain, has been recentlyproposed on the basis of spectral data and chemicaldegradations by Fattorusso and co-workers.92 The samegroup has also discovered two further "unusual" sterols,CXXX and CXXXI, from Calyx nicaensis.92 The occurr-ence of CXXX, the first example of an acetylenic func-tionality in a steroid, might be an answer to the problem of,the biochemical precursor of the unique calysterol itselfand the gorgonian sterols having a 22,23-cyclopropanering. Figure 39 lists the structures of the Calyx sterols.

Table 3. Sponges in which aplysteroldidehydroaplysterol have been found9'

and 24,28-

% of thetotal sterol

Sponges Source content

Ordr DictioceratidaFamily Verongidae .

V. aerophoba Naples 70%V. archeri (hard) Jamaican N. Shore 60V. archeri (soft) British Virgin Islands 60V. fistularis Bermuda 67V. thiona La Jolla (California) 78

CXXIX calysterol

HOCXXVX CXXXI

Fig. 39 Sterols with Unusual side-chain alkylation patterns fromCalyx nicaensis; (Fattorusso et a!., 1974, 1975).

R

Natural product chemistry of the marine sponges 21

Modifications of the sterol nucleus has also been found insponges.

The total sterol content of Axinella polypoides is amixture of stanols having a 19-norcholestanol nucleuscarrying conventional saturated and mono-unsaturated C7(24-nor), C8, C9 and C10 side-chains93 (Fig. 40).

Axinella verrucosa contains a series of stanols with anew 3/3 - hydroxy - A - nor - 5a - cholestane nucleuscarrying conventional C8, C9 and C10 side-chains94 (Fig.40). Interestingly in this sponge the usual sterols are alsoabsent.

These examples indicate that the sponges may be asource of further new types of sterols and a carefulreinvestigation of marine sponges for their sterol contentis clearly warranted.

Whereas our knowledge about the sterols present insponges has rapidly increased in the last few years, ourinsight into the metabolism of sterols in these animals isstill meagre.

R = H, 24-norR= HR= MeR= EtR=HR= Me,R= Me, 2(28)R= Et ,

HOH2C_MeR= H 22R= Me,L22R= Et,i22

Fig. 40. Stanols with modified tetracyclic nuclei in sponges.

Recent results from our laboratory have indicated thatVerongia aerophoba failed to incorporate either 1-'4C-acetate or 2-'4C-mevalonate into aplysterol (CXXVII) and24(28)-didehydroaplysterol (CXXVIII). A radiolabellingexperiment using CH3—'4C methionine in the spongeV. aerophoba also resulted in nonradioactive CXXVII andCXXVIII.27

A similar situation arose with Axinella polypoides andA. verrucosa, the sponges containing the 19-nor-stanolsand the hydroxymethyl - A - nor - steranes, respectively,when they were fed with labelled acetate. However thetwo sponges have been shown capable of converting veryefficiently cholesterol to 19 - nor - cholestanol and 3/3 -hydroxymethyl - A -nor - cholestane,95 respectively. So, itappears that in A. polypoides and A. verrucosa, the sterolscannot originate from de novo biosynthesis but arise bymodification of dietary sterols. A similar conclusion ispossibly applicable to the sponge Verongia aerophoba.

Clearly, much work is required before any definiteconclusions can be drawn about sterol metabolism insponges.

MISCELLANEOUS COMPOUNDS

Finally, I would like to remind you of the extensivework of Ackermanñ on amines and, more generally, onproducts associated with amino-acid metabolism inmarine invertebrates including sponges?6

I am sorry to be unable, because of lack of time, to gothrough this excellent contribution. However, a briefsummary of nitrogen compounds in Porifera has appearedin 1970 from Ciereszko?6 I can just report here the latestadditions to this miscellaneous group (Fig. 41).

H CH2

CH3CH2 /N( NCOCH0

CXXXIV, n = 13 to 17

(Kashman etal.,1973)

C H3

H2NNC H(CCO2H

CXXXV

(Beryquist and Hartmann, CXXXVI1967)

C H2

t( JL. CH2CH3 CH2),c=CH2CH3N CH=0 N CH=OH H

CXXXVIII CXXXIX

C H3H2 —2c H2C H2=)

N CH=0

HCXX XX

Fig. 41. Some miscellaneous compounds recently isolated fromsponges.

A series of N - acylated - 2 - methylene - /3 - alaninemethyl esters (CXXXIV) has been found in Faciospongiacavernosa, in remarkably high concentrations, by Kash-man et al.98 It is interesting to note that the rare 2 - methyl-

/3- alanine (CXXXV) was found in several sponges by

Bergquist and Hartmann.3°The simple 2-aminoimidazole (CXXXVI), a metabolite

possibly associated with arginine metabolism, has beenobtained from Reniera cratera?6

A novel group of compounds characterized by satu-rated, mono and diunsaturated long alkyl chains linked atposition 3 of a pyrrole-2-aldehyde moiety (CXXXVII—CXXXX) has been isolated from Oscarella lobularis,which also yielded a series of corresponding pyrrole-2carboxylic acids and methyl esters.

Acknowledgements—I have the fortune of being associated with agroup of enthusiastic colleagues, Drs G. Cimino, S. De Stefano, R.Riccio and G. Sodano, whose ability, generous efforts andstimulating ideas have made this work possible. I would like toexpress my special thanks to Professor E. Fattorusso, who was'issociated with our group in the earlier phase of this project. I amalso indebted to Professors E. Lederer and R. H. Thomson foradvice and interest in this work. The cooperation of the ZoologicalStation (Napoli) in the collection of sponges is gratefully acknow-ledged.

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Natural product chemistry of the marine sponges