Bisdioxycalamenene: A Bis-Sesquiterpene from the Soft Coral Rhytisma fulvum fulvum

A dichloromethane extract of the soft coral Rhytisma fulvum fulvum collected in Madagascar afforded a novel compound possessing an unprecedented pentacyclic skeleton, bisdioxycalamenene (1), as well as seven known sesquiterpenes. The structures of the compounds were elucidated using 1D and 2D NMR techniques, as well as high-resolution mass spectrometry. The absolute configuration of 1 was determined using X-ray diffraction analysis and anomalous dispersion effects. The structure elucidation and a possible biogenesis of the compound are discussed.


Introduction
The soft coral Rhytisma fulvum fulvum (Forskål) (R. fulvum fulvum) (previously Parerythropodium), family Alcyoniidae, was originally described from the Red Sea [1,2]. Its present zoogeographical distribution extends south to the reefs of Madagascar and eastward to Indonesia [3,4]. The Genera Rhytisma is a rich source of sesquiterpenoids and norsesquiterpenoids containing a variety of skeletons as well as sterols [5][6][7][8][9]. Dozens of compounds have thus far been reported with different ring systems [5][6][7][8][9]. In previous research, our group compared the yellow and gray morphs that inhabit the reefs of the gulf of Eilat and found differences in the compounds they produce [6]. Inter alia, we isolated a volatile yellow pigment, fulfulvene, from the yellow morph. This pigment is responsible for the soft coral's yellow color [6]. The pigment and other volatile compounds, mainly sesquiterpenes, are lost during freeze drying and/or evaporation [6].

Results and Discussion
Bisdioxycalamenene (1) was isolated from the crude extract using solvent partition, followed by separations on Sephadex LH-20 and Silica gel-H. The positive atmospheric pressure photoionization high-resolution mass spectrometry (APPI HRMS) of 1 exhibited a molecular ion [M] + at m/z 478.3080 consistent with the molecular formula C 31 H 42 O 4 and eleven double bond equivalents. The structure elucidation of 1 was based on its mass-spectrometric and NMR data (Table ??, CDCl 3 ). The 1 H NMR exhibited two singlet signals in the lower and mid-field of the spectrum (δ H 6.36 and 4.39), a methoxyl (δ H 3.74), six protons resonating between 3.40 and 2.50 ppm, ten sp 3 protons resonating between 2.00 and 1.40 ppm, a singlet methyl (δ H 1.33) and six doublet methyl signals resonating between 1.10 and 0.75 ppm. The 13 C NMR presented two ketone carbonyls (δ C 199.  The C-H correlations from a heteronuclear single quantum coherence (HSQC) experiment established the one bond connectivity between the carbons and protons (Table ??  Using long-range C-H correlations from a heteronuclear multiple bond correlation (HMBC) experiment enabled the connection of all the proton and carbon signals to the final gross planar structure of 1 (Figure 3). H-1 and -4 exhibited correlations with C-8 and -5, respectively, and both with C-9 and -10, extending fragment a to a 1-isopropyl-4-methyl-2-cyclohexene moiety substituted at positions 2 and 3 by two ketone groups. Oxymethine-7 presented HMBC correlations with C-5, -6, -8 and -9, while methyl-15-protons exhibited correlations with C-5, -6, and -7, closing a second six-membered ring. H-1 1 and -4 1 demonstrated a similar pattern of correlations (Figure 3), extending fragment b to a 1,4-disubstituted cyclohexene ring. H-7 1 exhibited HMBC correlations with C-1 1 , -5 1 , -6 1 , -8 1 , -9 1 and -15 1 , while methylene-15 1 -protons presented correlations with C-5 1 , -6 1 , -7 1 and -8 1 , and the methoxy-protons exhibited correlations with C-8 1 , supporting a substituted 15-methylene-8-methoxy-5-oxy-calamenene substructure. The HMBC correlations of H-7 with C-5 1 and -15 1 , of methyl-15-protons with C-15 1 and of both protons of methylene-15 1 with C-5, -6 and -7 suggested the connection of C-6 with C-15 1 and C-7 through the oxygen to C-5 1 , establishing the planar structure of 1. The relative trans relationships of the 1,4-and 1 1 ,4 1 -substituents of the two cyclohexene rings were suggested when comparing the chemical shifts of the corresponding protons and carbons with those of the known calamenenes (2)(3)(4). The cis 6-7 junction was established based on the NOE between H-7 and methyl-15. The complete structure including absolute configuration was confirmed by the X-ray diffraction analysis.
Crystallization of bisdioxycalamenene (1) from CHCl 3 /MeOH solution furnished suitable crystals for an X-ray diffraction analysis. Bisdioxycalamenene (1) comprises of a pentacyclic structure derived from coupling of two sesquiterpenes. The asymmetric unit contains assemblages of two molecules of 1, thus in one monoclinic cell there are four molecules of 1. Anomalous dispersion effects and relation of the two enantiomeric forms of the structural model to the diffracted intensities established the absolute configuration. The preferred fit is indicated by the Flack and Parsons parameter (Flack parameter, x = 0.01 (9)) [13]. The X-ray diffraction analysis established the complete structure of 1 including the absolute configuration of the six chiral centers (1S,1 1 S,4R,4 1 R,6S,7R) shown in Figure 4. To the best of our knowledge, there are no earlier reports of such a skeleton. A possible biogenetic route to 1 is a hetero Diels Alder condensation of the para-quinone c with the ortho-quinonemethide, d [14,15] (Figure 5). Compound 1 was evaluated for lethal toxicity in a brine shrimp toxicity assay (Artemia salina) [16] and displayed mild toxicity (LD 50 15 µg/mL). Compound 1 was assayed for antibacterial activity against Pseudomonas aeruginosa and Escherichia coli and found to be inactive at 10 µg/mL.

General Experimental Procedures
Optical rotations were determined on a JASCO P-1010 polarimeter. UV spectra were recorded on an Agilent 8453 spectrophotometer. NMR spectra were recorded on a Bruker DMX-500 spectrometer at 500.13 MHz for 1 H and 125.76 MHz for 13 C and a Bruker Avance 400 spectrometer at 400.13 MHz for 1 H and 100.62 MHz for 13 C. DEPT, COSY-45, gTOCSY (mixing time 60 ms), gROESY (spinlock pulse 0.2 s), gHSQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. Mass spectra were recorded on a Synapt High Definition Mass Spectrometry (Waters Inc., Milford, MA, USA) instrument. For the GC MS analysis, separation of the crude samples was performed on an Agilent-7890-GC equipped with Agilent-5977A-MSD with an HP-5MS UI column (30 meterˆ0.25 mmˆ0.25 µm). The X-ray diffraction patterns were obtained with CuKα radiation from an Imus microsource, on an ApexDuo Bruker-AXS diffractometer.

Biological Material
A sample of the soft coral R fulvum fulvum (yellow morph, Phylum Cnideria, Class Anthozoa, Order Alcyonacea, Family Alcyoniidae) was collected in December 2012 at a depth of 15 m at Banc du Castor, Mitsios Archipelago, Madagascar. It was identified by Professor Yehuda Benayahu. A voucher specimen (MAD12-IM052) was deposited at the Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments (LCSNSA) at the University of Reunion Island, France. The soft coral sample was frozen immediately after collection and kept at´20˝C until processed.

X-Ray Crystallography
The structure of 1 was confirmed by single crystal X-ray diffraction analysis of a crystal obtained from a CHCl 3 /MeOH solution. The measurements were carried out on an ApexDuo (Bruker-AXS) diffractometer with CuKα radiation at low temperature in order to optimize the precision of the crystallographic determinations. Crystal

Conclusions
The soft coral R. fulvum fulvum is widely distributed throughout the Indopacific [1][2][3][4] and present several color morphs. The genus Rhytisma is very rich in sesquiterpenes, norsesquiterpenes and other metabolites such as fulfulven that gives the yellow morph of R. fulvum fulvum its color [1][2][3][4][5][6][7][8][9]. In the present study, we have isolated, from a specimen collected in Madagascar, a sesquiterpene dimer of new pentacyclic skeleton, bisdioxycalamenene (1), along with seven previously described metabolites of R. fulvum fulvum. Although 1 did not present significant biological activity in the set of bioassays, we believe that there should be a good reason for its biosynthesis and for the investment of considerable amount of energy in it. Such purpose might be, for example, involvement in the reproduction process of soft coral as demonstrated for alkyl acetylenes produced by the hard coral Montipora digitata and the cembrane type diterpenoids in the soft coral Lobophytum compactum [18].
Supplementary Materials: 1D ( 1 H, 13 C) and 2D NMR (HSQC, HMBC, COSY) spectra and HR MS data of compound 1, and photograph of the soft coral, are available. the measurements of the mass spectra; and Sigal Shefer from the Department of Zoology, for guidance with the brine shrimp toxicity assay.
Author Contributions: Y.J.T. contributed to the isolation and structure elucidation and writing of the manuscript with the supervision of S.C. and Y.K. M.A. and A.Gauvin.-B. contributed to the collection and extraction of the sample of the soft coral; Y.B. contributed to the identification the soft coral. S.C. and Y.K. contributed to the structure elucidation, writing and editing of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.