A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated.

In the course of our search for bioactive marine natural products, we have investigated the Mediterranean marine sponge Sarcotragus spinosulus (Dictyoceratida, Irciniidae) collected from Callejones, Ceuta. In this paper we report the isolation and structural elucidation of a new hydroxylated nonaprenylhydroquinone, along with two known polyprenylhydroquinones, hepta-and octaprenylhydroquinones. We also report the antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562.
The 1 H-and 13 C-NMR spectra of compound 3 were similar to those of compounds 1 and 2, except for the presence of signals at δ 4.06 (2H) and δ 60.0, respectively, in the 1 H and 13 C NMR spectra (Table 1), assigned to the primary alcohol group of the side chain, and small differences in the chemical shifts around the OH group. Further examinations of the 1 H-and 13 C-NMR data of 3 led to its identification as a hydroxylated polyprenylhydroquinone, close analogues of which have been previously isolated from S. spinosulus [5,6,20,21]  To the best of our knowledge this is the first report on the isolation and structure identification of a hydroxylated nonaprenylhydroquinone. Until now, four hydroxylated polyprenylhydroquinones have been reported: two heptaprenyl bearing the OH group on the first [20] and fifth prenyl moiety [6]; and two octaprenyl congeners bearing the OH group on the fourth [21] and fifth [5] isoprene unit.
From a chemotaxonomic point of view hydroxylated polyprenylhydroquinones could constitute potential markers for S. spinosulus species.
The metabolites 1-3 were evaluated for their potential antileukemic effect towards the chronic myelogenous leukemia (CML) cells line K562, which is widely used for cytotoxicity assays. The effect of compounds 1-3 was compared with that of Imatinib, the leading compound to treat patients suffering CML. This compound has proven very efficient in killing Bcr-Abl-positive cells in a caspase-dependent manner [25,26]. The IC 50 values for Imatinib and compounds 1-3 for loss of cell metabolism (XTT assay) and cell number are given in Table 2. Compounds 1 and 2 inhibited cell metabolism and cell number with very similar IC 50 values (around 10 µM). Compound 3 was less efficient that the two former compounds with IC 50 values of 193 and 191 µM, respectively. As a control, Imatinib was shown to inhibit cell metabolism and cell number with IC 50 values of 0.4 and 0.5 µM, respectively, in agreement with previous results [26]. As compounds 1 and 2 were also found to induce annexin V externalisation in K562 cells (not shown), it is likely that the main mechanism by which both compounds inhibit cells metabolism and cells number is by inducing apoptosis.

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, 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) and Silver trifluoroacetate (AgTFA, used as the cationizing agent) were purchased from Sigma Aldrich Co. The Chromabond C18 preparative column used for flash chromatography was obtained from Merck, USA. Imatinib was kindly provided by Novartis Pharma. UV measurements were performed on a Varian Cary 300 Scan UV-visible spectrometer. IR spectra were obtained with a Perkin-Elmer Paragon 1000 FT-IR spectrometer. 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, 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 standards with multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet). High resolution mass spectra (HRMALDITOFMS) were conducted on a Perseptive Voyager DE-STR MALDI-TOF mass spectrometer (Perseptive Biosystems, Framingham, MA, USA), equipped with a 337 nm pulsed nitrogen laser (20 Hz) and a Acqiris ® 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 (Applied Biosystems).

Sponge Material
A sponge sample of Sarcotragus spinosulus (Schmidt, 1862) ( Figure 2) (Demospongiae, Dictyoceratida, Irciniidae) was collected by hand using scuba at a depth of about 10 m from Callejones, Ceuta in June 2009. Taxonomic examination of the sponge was made by the authors (C.A. and J.V). The sponge shape is massive and subspherical. The color in situ is dark grey exterior, white to beige interior, and in EtOH the flesh color turned slightly to reddish. The texture is difficult to tear and has a firm and compressible consistency. The ectosome is unarmoured, thick with conules (1 to 2 mm height) irregularly scattered over the entire surface. The choanosome is cavernous. Its skeleton consists of laminated primary and secondary fibers and comprises very dense spongin filaments. Primary fibers (90 to 180 µm diameter) are pithed and clear of foreign detritus. Secondary fibers (50 to 100 µm diameter) are uncored, and the spongin filaments are long with a very thin diameter of 0.7 to 2 µm . A sponge sample (090618Ce7-05) is kept in the sponge collection of the Centre d'Océanologie de Marseille (Station Marine d'Endoume, France). The sponge was kept frozen until the extraction process.

Biological Activity
Cell Line: The human CML K562 cell line was provided by ATCC and were grown at 37 °C under 10% CO 2 in RPMI 1640 medium (Gibco BRL, Paisley, UK) supplemented with 5% FCS (Gibco BRL, Paisley, UK) completed with 50 units/mL penicillin, 50 mg/mL streptomycin and 1 mM sodium pyruvate.
Cell death measurement: After the indicated treatment, cells were harvested and percentage of viability was measured by propidium iodide (PI) staining (0.5 Ag/mL) and flow cytometry analysis in FL-3.

Conclusions
A new hydroxylated nonaprenylhydroquinone, 2′-[38-Hydroxy]nonaprenyl-1′,4′-hydroquinone, along with two known metabolites, hepta-and octaprenyl-1′,4′-hydroquinones, have been isolated from the Mediterranean marine sponge Sarcotragus spinosulus. These compounds exhibited a good activity against K562 cells which will warrant further analysis at the molecular level and offer promising opportunities for the development of new antitumor agents.