Nepheliosyne B, a New Polyacetylenic Acid from the New Caledonian Marine Sponge Niphates sp

A new C47 polyoxygenated acetylenic acid, nepheliosyne B (2), along with the previously described nepheliosyne A (1), have been isolated from the New Caledonian marine sponge Niphates sp. Their structures have been elucidated on the basis of extensive spectroscopic analyses. These metabolites exhibited a moderate cytotoxicity against K562, U266, SKM1, and Kasumi cancer cell lines.

In the course of our search for bioactive marine natural products, we have investigated the New Caledonian marine sponge Niphates sp. (Haplosclerida, Niphatidae). In this paper, we report the isolation and structural elucidation of new polyhydroxylated acetylene, nepheliosyne B (2), along with nepheliosyne A (1), previously isolated from the marine sponge Xestospongia sp., whose structural elucidation is herein completed. We also report their cytotoxic properties against K562, U266, SKM1, and Kasumi cancer cell lines.

Results and Discussion
The CH 2 Cl 2 /MeOH (1:1, v/v) crude extract of Niphates sp. was fractionated by Flash Vacuum Liquid Chromatography, eluting with a gradient of decreasing polarity from H 2 O to MeOH. The subsequent MeOH fraction was purified by semi-preparative reverse-phase HPLC (Phenomenex Luna C18, 250 × 10 mm id, 5 μm, gradient H 2 O/MeCN/Formic Acid 50:50:0.1 to 0:100:0.1) to afford pure nepheliosyne A (1) (8.7 mg) and nepheliosyne B (2) (6.7 mg) ( Figure 1).    The IR spectrum showed bands at 3450, 3300, 2250, 2100, and 1705 cm −1  suggesting the presence of double bonds, triple bonds, and hydroxyl groups. A preliminary NMR spectral analysis showed similarities and strongly supported the presence of a polyhydroxylated acetylenic skeleton. The 1 H and 13 C NMR spectra of compound 1 (see Supporting Information and Table 1) 8-30.4). In a similar way than fragment a in compound 1, HMBC correlations were observed between, both H-4 (δ H 4.26) and H-5 (δ H 3.58) and C-3 (δ C 85. 3), and between H-4 and C-2 (δ C 80.1) thus assigning the carbon resonances of the triple bond in fragment a′ ( Table 2). The down-shifted proton value of H-4 in fragments a and a′ suggested its connection to the α-yne carboxylic acid moiety that had to be the first terminal part of the chain.   The connectivities between these partial structures a-e for 1 and a′-e for 2, as well as the number of the linking methylene groups, were established on the basis of the 1 H-13 C HMBC, 1 H-1 H COSY/TOCSY correlations, and MS data. The correlation observed between H 2 -23 and H 2 -25 offered the connection between fragments b and c. In a similar way, the correlation between H 2 -28 and H 2 -30 provided the connection between the partial structures c and d. Finally, the correlation between H-37 and H-39 offered the connection between the partial structures d and e. The combinations a + b + c + d + e and a′ + b + c + d + e represented 670 m.u. whereas the molecular structure weight was 810 m.u. The difference corresponding to 10 methylene groups determined the length of the complementary alkyl chain between a (or a′) and b. All the spectral data of 1 (1D and 2D NMR, MS, and optical properties) led to its identification as nepheliosyne A, in accordance with previous published data [3]. Thus, nepheliosyne B (2) is a new metabolite defined as the 5-hydroxy-6-dehydroxy derivative of nepheliosyne A (1).
The geometry of the double bond Δ 43,44 was easily assigned as E by analysis of the coupling constant of the olefinic protons (J 43,44 = 15.5 Hz). The geometries of the double bond Δ 26,27 and Δ 39,40 were assigned as Z and E, respectively, based on the 13 C chemical shifts of the allylic methylenes δ C 28.0 (C-25) and 27.8 (C-28) for Δ 26,27 and δ C 36.8 (C-38) and 36.3 (C-41) for Δ 39,40 .
The relative and absolute configurations of the stereogenic centers of nepheliosyne A (1) and B (2) remained unassigned. They were observed also to degrade rapidly under different reaction conditions [14][15][16]. Like fulvynes [12], any attempts to obtain suitable derivatives for a stereochemical analysis were unsuccessful.
To date, several polyhydroxylated acetylenic metabolites of marine sponges with a diacetylenic carbinol and a α-yne carboxylic group have been reported: Nepheliosyne A from Xestospongia sp. [3], petrosolic acid from Petrosia sp. [6], osirisynes [10] and haliclonyne [11] from Haliclona sp., and fulvynes A-I from Haliclona fulva [12]. Nepheliosyne B (2) is, with nepheliosyne A and petrosolic acid, the third example of linear acetylene with diacetylenic carbinol, α-hydroxyketone, and α-yne carboxylic groups. To the best of our knowledge this is the first report on the isolation and structure identification of oxygenated polyacetylenic metabolites from Niphates sp. All these data suggest that from a chemotaxonomic point of view polyhydroxylated acetylenic metabolites could constitute potential markers of Haplosclerida species.
The cytotoxicity of the nepheliosynes A (1) and B (2) was evaluated against K562, U266, SKM1, and Kasumi cancer cell lines which are widely used for cytotoxicity assays, and the IC 50 values in μM (XTT assay) are indicated in Table 3. Compounds 1 and 2 were equally efficient with IC 50 values around 150-200 μM. Interestingly, their significant specificity against tumor cells were highlighted thanks to additional assays showing that, compared to the K562 cells, the peripheral blood mononuclear cells (PBMC) viability is not affected by compounds 1 and 2 (see Supporting Information).

General
All organic solvents used for material extraction were of analytical grade and purchased from Merck (Darmstadt, Germany). Acetonitrile used for HPLC was of HPLC-grade and purchased from Fisher Scientific, USA. Formic acid of HPLC grade was purchased from Acros, USA. 2,5-Dihydroxybenzoic acid (DHB), used as the matrix for MALDI-TOF experiments, was of the highest grade available and used without further purification was purchased from Sigma Aldrich Co, France. The Chromabond C18 preparative column used for flash chromatography was obtained from Merck, USA. UV measurements were performed on a Varian Cary 300 Scan UV-visible spectrometer. IR spectra were obtained with a Perkin-Elmer Spectrum 100 series FT-IR spectrometer equipped with an universal attenuated total reflectance sampling accessory (ATR). Optical rotations were measured on a Jasco P-1010 polarimeter. Flash chromatography was performed on an Armen Instrument Spot Liquid Chromatography system, the detection wavelength was set at 254 nm. HPLC purifications were carried out on a Waters 600 system equipped with a Waters 717 plus autosampler, a Waters 996 photodiode array detector, and a Sedex 55 evaporative light-scattering detector (SEDERE, Alfortville, France). Detection wavelengths were set at 214, 254 and 280 nm. 1 H and 13 C NMR spectra were recorded with 500 MHz Bruker Avance NMR spectrometers. Chemical shifts (δ) are recorded in ppm with CD 3 OD (δ H 3.31 ppm and δ C 49.0 ppm) as internal standard with multiplicity (s singlet, d doublet, t triplet, m multiplet). High resolution mass spectra (HRMS) were conducted on a Voyager DE-STR MALDI-TOF mass spectrometer (ABSciex, Les Ulis, France), equipped with a 337 nm pulsed nitrogen laser (20 Hz) and a Acquiris ® 2 GHz digitizer board, was used for all experiments. Mass spectra were obtained in reflectron positive ion mode with the following settings: Accelerating voltage 20 kV, grid voltage 62% of accelerating voltage, extraction delay time of 100 ns. The laser intensity was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise (S/N) ratio without significant peak broadening. All data were processed using the Data Explorer software package (ABSciex).

Sponge Material
We collected the sponge Niphates sp. (Schmidt, 1862) (Demospongiae, Haplosclerida, Niphatidae) in November 2008 using scuba at a depth of 22 m in the south-west lagoon of New Caledonia (Philippe Amade, Figure 3). The sponge was identified by J. Vacelet and a voucher specimen (MHNM 1646) was deposited at the Natural History Museum of Marseille (France) [17].