A New Germacrane Sesquiterpene from the Brazilian Endemic Gorgonian Phyllogorgia dilatata Esper

Abstracts

A bioassay-guided re-investigation of natural products from the Brazilian endemic octocoral Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae), collected in Rio de Janeiro State, has revealed three further compounds from this species: the dinoflagellate pigment peridinin (1), the icthyodeterrent diterpene 11beta,12beta-epoxypukalide (2), both previously reported from other octocoral species, and the new natural sesquiterpene (E) germacra-1(10),4(15),7(11)-trien-5-ol-8-one (3). Spectroscopic analysis and comparison with literature data allowed the structure elucidation of 1-3.

Phyllogorgia dilatata; peridinin; germacrane; epoxypukalide

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 11beta,12beta-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).

Article

A New Germacrane Sesquiterpene from the Brazilian Endemic Gorgonian Phyllogorgia dilatata Esper

Danielle L. Martins, and Rosângela de A. Epifanio^*

Pós-Graduação em Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, 24020-150 Niterói - RJ, Brazil

Received: July 23, 1998

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 11b,12b-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).

A bioassay-guided re-investigation of natural products from the Brazilian endemic octocoral Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae), collected in Rio de Janeiro State, has revealed three further compounds from this species: the dinoflagellate pigment peridinin (1), the icthyodeterrent diterpene 11b,12b-epoxypukalide (2), both previously reported from other octocoral species, and the new natural sesquiterpene (E) germacra-1(10),4(15),7(11)-trien-5-ol-8-one (3). Spectroscopic analysis and comparison with literature data allowed the structure elucidation of 1-3.

Keywords: Phyllogorgia dilatata, peridinin, germacrane, epoxypukalide

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^3-5. 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^6-8.

The extensive Brazilian coast (more than 7,500 km) is rich in marine coral species⁹, but assessments of their natural products have been rare and restricted to very few publications¹⁰. 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, Brazil⁹. Previous studies of its hexane extract reported the isolation of one sterol¹¹ 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 fishes¹⁴. 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 11b,12b-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 concentration¹⁴. The presence of the known P. dilatata sesquiterpenes in the nonpolar fraction was deduced by the ¹H-NMR analysis of the total fraction as well as of semi-purified compounds¹⁴. On the other hand, the crude extract and the ethyl acetate fraction significantly inhibited the consumption of treated food pellets in comparison with controls¹⁴. 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, ¹H and ¹³C-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 octocorals¹⁶. The presence of 1 in the crude extract reveals unambiguously for the first time the presence of a dinoflagellate in P. dilatata tissues.

Compound 2 was obtained as white fine needles (4.7 x 10^-3% of dry weight gorgonian), mp 211-213 °C. LREIMS showed the molecular ion [M]^+· at m/z 388 consistent with the molecular formula C₂₁H₂₄O₇. The IR absorptions at n_max 1765 and 1710 cm^-1 indicated the presence of at least two carbonyl functions in the molecule. Analysis of 1D and 2D NMR spectra, including ¹H-¹H-COSY, ¹H-¹³C-HETCOR and n.O.e. experiments, led to the conclusion that compound 2 was 11b,12b-epoxypukalide, previously isolated from Leptogorgia setacea¹⁸. With the exception of the assignments for CH₂-13 (d_C 23.8; d_H 1.48, m, and 2.57, dd, J = 10.5 and 15.0 Hz) and CH₂-14 (d_C 26.6; d_H 1.43, m, and 1.66, m), all the carbon and hydrogen NMR spectral data obtained are identical to those proposed by Ksebati et. al.¹⁷ The ¹H-¹H and ¹H-¹³C long range correlation spectra as well as nuclear Overhauser enhancements experiments, allowed us to unambiguously assign the chemical shifts for all carbons and hydrogens for 2. The cross peaks observed between C-14 (d_C 26.6)/H-2 (d_H 3.00, dd, J = 5.1 and 18.4 Hz; and d_H 3.02, dd, J = 10.0 and 18.4 Hz), C-15 (d_C 145.3)/H-14 (d_H 1.43, m), and C-12 (d_C 62.4)/H-13 (d_H 2.57, dd, J = 10.5 and 15.0 Hz) in the 2D ¹H-¹³C long range correlation and between H-1 (d_H 3.48, ddt, J = 2.1, 5.1 and 10.0 Hz)/H-14 (d_H 1.66, m) in the 2D ¹H-¹H COSY spectrum showed that the carbon chemical shifts previously proposed¹⁸ for CH₂-13 and CH₂-14 should be exchanged.

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.

Compound 3 was obtained as a pale yellow oil (3.6 x 10^-3% of dry weight gorgonian) The LREIMS of this compound showed an [M]^+· ion at m/z 234 consistent with the molecular formula C₁₅H₂₂O₂. The mass spectra also exhibited a diagnostic fragment at m/z 216, suggesting the loss of H₂O from the molecular ion [M]^+·. The IR absorption bands at n_max 3432 and 1724 cm^-1 indicated the presence of hydroxyl and ketone funtionalities. Based on HBBD and DEPT ¹³C-NMR experiments, it was deduced that one carbonyl, four quaternary carbons, two methines, five methylenes and three methyl groups were present in the structure. The ¹H and ¹³C (HBBD and DEPT) NMR spectra confirmed the presence of one secondary hydroxyl group by the signals at d_C 74.8 (CH-5) and d_H 3.86 (1H, t, J = 7.0 Hz, H-5). The ¹³C-NMR spectra also revealed seven sp² carbon signals at d_C 112.3 (CH₂-15), 127.8 (CH-1), 130.3 (C-10), 133.9 (C-7), 134.4 (C-11), 151.5 (C-4) and 206.0 (C-8). The presence of an exomethylidene was deduced, by the singlets at d_H 4.97 and 5.12 (1H each, H-15ab). These data were in accordance with a decacyclic sesquiterpene skeleton for 3, which were confirmed by two dimensional NMR experiments [homonuclear ¹H-¹H and heteronuclear ¹H-¹³C (¹J_CH and ^2,3J_CH) correlation spectroscopy].

All the ¹H-NMR resonances of 3 were assigned to their corresponding carbon partner signals by direct 2D ¹H-¹³C spectra (¹J_CH) analysis (Table). Homonuclear ¹H-¹H and long range (^2,3J_CH) ¹H-¹³C 2D spectra allowed us to build partial structures for 3. The ¹H-NMR signal at d_H 5.33 (bt, J = 8.0 Hz, H-1) exhibited an allylic coupling with the hydrogens at d_H 1.61 (d, J = 1.2 Hz, 3H-14) and a vicinal coupling with the hydrogens at d_H 2.20 (m, 2H-2), which in turn was coupled with the signal at d_H 2.25 (m, 2H-3). ²J and ³J ¹H-¹³C couplings between the methylene hydrogens (d_H 3.17, d, J = 15.9 Hz, H-9a, and 3.22, d, J = 15.9 Hz, H-9b) and the carbons at d_C 127.8 (C-1), 130.3 (C-10) and 206.0 (C-8) allowed us to build the partial structure A (Fig. 1). In addition, the cross peaks, observed in the ¹H-¹H COSY spectra, between the hydrogens at d_H 2.69 (d, J = 7.0 Hz, 2H-6) with those at d_H 3.86 (t, J = 7.0 Hz, H-5) and 1.79 (s, 3H-12), together with the long range couplings observed in the 2D ¹H-¹³C spectra between the carbons at d_C 133.9 (C-7) and 134.4 (C-11) and the methyl hydrogens at d_H 1.79 (s, 3H-12) and 1.83 (s, 3H-13) led us to build the partial structure B (Figure). The E geometry of the double bond in A was deduced by the nuclear Overhauser enhancements observed for the hydrogens at d_H 3.17 and 3.22 (d, J = 15.9 Hz, H-9a and H-9b) when the signal at d_H 5.33 (bt, J = 8.0 Hz, H-1) was irradiated. This geometry is also consistent with the chemical shift observed for the methyl group (d_C 17.4, C-14) and confirmed by literature data for other similar germacrane derivatives^19,20.

The extensive NMR measurements and the UV absorption for the chromophore in 3 at l_max 249.5 nm (log e 3.17, calcd 250 nm) provided the data, which fully defined the structure shown in 3.

In order to confirm the proposed structure, compound 3 was acetylated by treatment with acetic anhydride in presence of pyridine. The incorporation of the acyl group deshielded the H-5 hydrogen (+ 1.13 ppm) and the carbons C-3 (+ 1.6 ppm), C-5 (+ 1.9 ppm), C-7 (+ 1.2 ppm) and C-15 (+ 2.7 ppm) and shielded C-4 (- 2.6 ppm) and C-6 (- 3.4 ppm), confirming the proposed structure (Table 1).

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 structure²¹.

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 pressure²². 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 controls¹⁴.

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 11b,12b-epoxypukalide (2), probably produced by the gorgonian and not by the symbiont, is a potent feeding deterrent compound against co-occurring fishes¹⁴. 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.

Experimental

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. ¹H and ¹³C-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 mm) 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 thin-layer chromatography (tlc) on pre-coated silica gel plates (Merck, Kieselgel 60 F-254).

Collection

Phyllogorgia dilatata Esper (Octocorallia, Gorgonacea, Gorgoniidae) was collected at Tartaruga beach, Armação dos Búzios, Rio de Janeiro State, Brazil (22° 45’ S; 41° 51’ W) in December 1995 by free diving between 3 and 5 meter depths. Colonies were air dried for ca 2 h and kept frozen until laboratory analyses. A voucher specimen (PD1295) was deposited at IQ-UFF.

Extraction and isolation of compounds 1 - 3

P. dilatata (3.44 cm³, 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.

Silica gel (tlc grade) vacuum chromatography of the ethyl acetate fraction employing a gradient of 0-100% of ethyl acetate in hexane, followed by methanol afforded 13 fractions. Purification of fractions 5 (1,184.6 mg) and 6 (749.3 mg) (1:1 and 3:7 of hexane:ethyl acetate, respectively) yielded compounds 1, 2 and 3.

Reversed phase chromatography of 700 mg of fraction 6 using water:methanol (2:8) followed by silica Sep Pack (10 g) filtration with hexane:ethyl acetate (1:1) yielded 25.4 mg of an orange-red oil, [a]²⁰_D - 2.94 (c 0.0034, MeOH). UV, IR and ¹H and ¹³C-NMR (CDCl₃, 300 MHz) data of 1 showed it to be identical to peridinin (1)^6,15.

Fraction 5 (1,184.6 mg) was recrystallized from methanol yielding 156.8 mg of white needles, mp 211-213 °C, identified as 2¹⁷.

11b,12b-Epoxypukalide (2): [a]²⁰_D - 3.47 (c 0.0270, CH₂Cl₂); UV (CHCl₃) l_max (log e) 251,5 (3.75) nm; IR (film) n_max: 3090, 2980, 2940, 1765, 1710, 1640, 1620, 1575, 1440, 1380, 1350, 1275, 1255, 1245, 1220, 1200, 1090, 1070, 880, 870, 770 cm^-1. ¹H and ¹³C-NMR (CDCl₃, 300 MHz): with the exception of the data shown in "Results and Discussion" all the chemical shifts are identical to those published¹⁷. LREIMS (70 eV) m/z (rel. int.): [M]^+· 388 (4), 373 (2), 356 (5), 328 (3), 313 (2), 208 (11,4), 179 (14), 173 (11), 168 (20), 165 (14), 159 (10), 152 (14), 137 (22), 110 (16), 91 (20), 83 (32), 79 (32), 69 (30), 67 (29), 55 (48), 43 (100).

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).

(E) Germacra-1(10),4(15),7(11)-trien-5-ol-8-one (3): [a]²⁰_D - 8,78 (c 0.0260, MeOH); UV (CHCl₃) l_max (log e) 249.5 (3.17); IR (KBr) n_max: 3433, 3076, 2925, 2854, 1725, 1670, 1448, 1376, 1286, 1263, 1193, 1137, 1079, 1040, 904, 755 cm^-1; ¹H and ¹³C-NMR (CDCl₃, 300 MHz): See Table and "Results and Discussion". LREIMS (70 eV) m/z (rel. int.): [M]^+·234 (98), 220 (30), 216 (15), 201 (55), 191 (15), 177 (35), 175 (94), 173 (38), 163 (45), 162 (28), 159 (30), 150 (32), 149 (100), 136 (55), 124 (60), 123 (70), 122 (56), 121 (80), 119 (46), 109 (80), 107 (65), 105 (48), 93 (65), 91 (70), 79 (50), 67 (45), 53 (35), 41 (60).

(E) 5-Acetyl-germacra-1(10),4(15),7(11)-trien-8-one (4): A solution of 10 mg of 3 in 5 mL of dry acetic anhydride and 2 drops of pyridine was left at room temperature for two hours. After the usual work-up and purification of the resulting oil by silica gel chromatography with 9.5:0.5 hexane:ethyl acetate, 3.5 mg (30%) of pure 4 was obtained as a colorless oil. ¹H and ¹³C-NMR (CDCl₃, 300 MHz): See Table.

Acknowledgments

We wish to express our gratitude to Prof. Angelo C. Pinto (IQ-UFRJ) and Prof. William Fenical (SIO-UCSD) for the work opportunities and for always stimulating our research interests. This research was financially supported by the National Brazilian Research Council (CNPq and PADCT-CNPq, Brazil) and International Foundation for Science (IFS, Sweden). DLM gratefully acknowledges CAPES for providing a M.S. Fellowship.

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Publication Dates

• Publication in this collection
  25 Oct 2002
• Date of issue
  Dec 1998

History

• Received
  23 July 1998