A New Germacrane Sesquiterpene from the Brazilian Endemic Gorg

Visando a caracterização de substâncias de defesa contra predadores, a reinvestigação dos produtos naturais do octocoral Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae) revelou a presença de novos metabólitos para esta espécie. Neste trabalho descreve-se o isolamento e a elucidação estrutural de três substâncias: o carotenóide peridinina (1), pigmento característico de dinoflagelados, caracterizando a presença de simbiontes nos tecidos dessa gorgônia; o diterpeno fagorrepelente 11β,12β-epoxipukalídeo (2), previamente descrito para uma outra espécie de octocoral; e um novo sesquiterpeno natural, (E) germacra-1(10),4(15),7(11)-trien-5-ol-8-ona (3).


Introduction
Marine octocorals of the order Gorgonacea are recognized to be a rich source of structurally unique and biologically active terpenoids 1,2 that can play important roles in ecological interactions.Most of the gorgonians that inhabit shallow waters are known to live in symbiotic relationship with intracellular algae, called zooxanthellae, important for the nutrition of the colony, carrying out photosynthesis and transferring organic compounds to the host [3][4][5] .Compounds such as peridinin, gorgosterol and 4-methyl sterols already isolated from octocorals are known to be biosynthesized by symbionts, but little is known about whether zooxanthellae or the host are involved in the production of other metabolites [6][7][8] .
The extensive Brazilian coast (more than 7,500 km) is rich in marine coral species 9 , but assessments of their natural products have been rare and restricted to very few publications 10 .One of the rare examples of Brazilian marine invertebrates already studied is the gorgonian Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae) an endemic organism, distributed along the southwest Atlantic from the Ceará until to the state of Rio de Janeiro, Brazil 9 .Previous studies of its hexane extract reported the isolation of one sterol 11 and two nardosinane sesquiterpenes 12,13 .
During our studies searching for secondary metabolites from Brazilian marine invertebrates involved in chemical defense against natural predators, we found that the crude organic extract of P. dilatata significantly reduced the consumption of bites by co-occurring fishes 14 .In this paper we wish to report the isolation and structure elucidation of a new germacrane sesquiterpene (3) from P. dilatata, along with two already known compounds, peridinin (1) and 11β,12β-epoxypukalide (2).

Results and Discussion
Phyllogorgia dilatata Esper (Octocorallia, Gorgonacea, Gorgoniidae) was collected at Tartaruga beach, Armação dos Búzios, Rio de Janeiro State in December 1995 by snorkel diving.The gorgonian colonies were air dried for ca 2 h and kept frozen until extraction.
The crude organic extract of the gorgonian was partitioned between different solvents furnishing non-polar (hexane), medium polarity (ethyl acetate) and polar fractions (water).The hexane and the polar fractions did not show any deterrent activity against generalist fishes, when incorporated into artificial food pellets in their natural volumetric concentration 14 .The presence of the known P. dilatata sesquiterpenes in the nonpolar fraction was deduced by the 1 H-NMR analysis of the total fraction as well as of semi-purified compounds 14 .On the other hand, the crude extract and the ethyl acetate fraction significantly inhibited the consumption of treated food pellets in comparison with controls 14 .These results indicated that the bioactive metabolites were not those previously isolated by Kelecom et.al. from the apolar extract of P. dilatata 12,13 and prompted us to investigate the medium polarity fraction in order to identify the icthyodeterrent compounds.Purification of the ethyl acetate fraction by a series of chromathographic steps yielded three pure compounds.
Compound 1 was obtained as an orange-red oil (7.6 x 10 -4 % of dry weight gorgonian).Spectroscopic analysis of 1 (UV, IR, 1 H and 13 C-NMR) revealed that it was the known pigment called peridinin 6,15 , a carotenoid unique to dinoflagellates, found in more than 75% of the investigated species, and proposed as an important taxonomic marker for symbiont determination in octocorals 16 .The presence of 1 in the crude extract reveals unambiguously for the first time the presence of a dinoflagellate in P. dilatata tissues.
Although cembranolide diterpenes are common in gorgonians possessing symbionts, there is evidence that they are produced by the host.In fact, Leptogorgia setacea (Gorgonacea, Gorgoniidae), from which 2 was previously isolated, lack zooxanthellae as do other octocorals that yield cembranolides 17,18 , suggesting that both P. dilatata and L. setacea can biosynthesize this diterpene.
The extensive NMR measurements and the UV absorption for the chromophore in 3 at λmax 249.5 nm (log ε 3.17, calcd 250 nm) provided the data, which fully defined the structure shown in 3.
A literature search about germacrane sesquiterpenes revealed that this is the first time 3 has been isolated from natural sources.Although this compound has been once proposed as a product from the photosensitized oxygenation of germacrone, the published spectral data are inconclusive for its structure 21 .
Germacrane sesquiterpenes are common in terrestrial plants and algae, and a few of them have been also obtained from octocorals.In addition to 3 from P. dilatata, the majority of these sesquiterpenes have been found in the families Nephtheidae, Gorgoniidae, Xeniidae, and Plexauridae 1,2 .Furanocembranolides, on the other hand, as a particular class of cembranes, are exclusive to octocorals.They have been found only in the families Gorgoniidae and Alcyonidae 1,2 .
Whereas octocoral secondary metabolites have been extensively studied for their medical potential, their ecological functions began to be investigated only a decade ago, showing that ancepsenolides, sesquiterpenes and diterpenes can be important for survival of the species in areas of high levels of predation pressure 22 .In a parallel study about feeding deterrent properties of Brazilian octocoral species, we found that among P. dilatata lipophilic metabolites, only the diterpene 2 reduced the consumption of artificial diets by co-occurring fishes relative to controls 14 .
This study led to the isolation of three compounds not previously reported for Phyllogorgia dilatata.One of them, the pigment peridinin (1), confirmed the presence of zooxanthellae in symbiosis with this octocoral.The diterpene 11β,12β-epoxypukalide (2), probably produced by the gorgonian and not by the symbiont, is a potent feeding deterrent compound against co-occurring fishes 14 .During this investigation, we also identified a new natural sesquiterpene, (E) germacra-1(10),4(15), 7(11)-trien-5-ol-8-one (3), not previously reported from natural sources and for the first time structurally assigned by spectroscopic methods.

General experimental procedures
The melting points were determined using a Fisher Johns melting point apparatus and are uncorrected.Optical rotations were measured on a Perkin Elmer 243 B Polarimeter.UV spectra were obtained on a Shimadzu UV-160A spectrophotometer. 1 H and 13 C-NMR spectra were recorded in a Varian-Unity Plus 300 NMR spectrometer.LREIMS spectra were obtained through HRGC-MS analysis (SE-54 glass capillary column, 24 m x 30 mm, df = 0.25 µm) on a Hewlett Packard 5890 A, using linear scanning (50-500 DA, 1.87 s dec -1 ) and EI (70 eV) ionization.HPLC was carried out on a semi-preparative column using a Waters 510 pump, a 410 refractometer and a 486 UV. Isolation procedures were monitored by employing thinlayer chromatography (tlc) on pre-coated silica gel plates (Merck, Kieselgel 60 F-254).
Extraction and isolation of compounds 1 -3 P. dilatata (3.44 cm 3 , 3326 g of dry weight gorgonian) was cut into small pieces and extracted with a mixture of methanol:dichloromethane 1:1 (once) and dichloromethane (twice).The extracts were combined and evaporated under reduced pressure yielding 368 g of a brownish gum (11.06% of dry weight gorgonian).Partition of the crude extract between hexane/methanol and ethyl acetate/water, afforded non-polar (hexane, 260 g), medium polarity (ethyl acetate, 11 g) and polar (aqueous, 90 g) fractions.
The remaining fraction 5 was purified by silica gel flash chromatography and normal phase semi-preparative HPLC, using 4:6 hexane:ethyl acetate to furnish 120 mg of a pale yellow oil (3).

Figure 1 .
Figure 1.A and B = partial structures for 3, deduced by 2D NMR experiments.
a) Number of H bonded to C deduced by DEPT experiments; b) Assignments aided by direct 1 H-13 C HETCOR; *Values may be interchanged.