Dragmacidoside: a new nucleoside from the Red Sea sponge Dragmacidon coccinea

Chemical investigation of the Red Sea sponge Dragmacidon coccinea led to the isolation of a new nucleoside, dragmacidoside (1), along with eight known compounds: adenosine (2), inosine (3), deoxycytidine (4), methyl-α-d-glucopyranoside (5), clionasterol (6), stigmasterol (7), campesterol (8) and brassicasterol (9). The compounds were isolated from chloroform and ethyl acetate fractions of the methanolic extract of the sponge, and their structures were established based on various spectroscopic data including MS, 1D and 2D NMR (COSY, HSQC and HMBC). Biological testing revealed that the chloroform fraction possesses significant anti-inflammatory activity in the carrageenan-induced hind paw oedema in rats.


Introduction
Marine sponges have gained a significant attention with respect to the diversity of their secondary metabolites. The biological activities of new metabolites from sponges have been reported in hundreds of scientific papers. Sponges have the potential to provide future drugs against important diseases such as cancer (Bai et al. 1993;Blackburn et al. 1999;Hood et al. 2002;Molinski et al. 2009), inflammation (Tan et al. 1997;Pope et al. 1999;Festaa et al. 2012), cardiovascular (Maryanoff et al. 1993;Shuman et al. 1993;Chackalamannil 2001), viral (Müller et al. 1987;Ford et al. 1999;Wellington et al. 2000), plasmodial (Ang et al. 2001) and bacterial (D_Ambrosio et al. 1996). Previous work on another species of the genus Dragmacidon collected from Andaman Sea in Thailand resulted in the isolation of a number of b-carboline alkaloids; some of them are characterised by possessing potent anti-inflammatory and antitumour activities (Pedpradab et al. 2004). In this study, chloroform and ethyl acetate fractions of the methanolic extract of the sponge Dragmacidon coccinea collected from the Red Sea, Hurghada, Egypt were examined. This resulted in the isolation and identification of a new nucleoside (1) along with eight known compounds (2 -9) (Figure 1). In addition, the antiinflammatory activity of the fractions using rat paw oedema test was evaluated.

Results and discussion
Compound 1 was isolated as a white precipitate. Combined spectral data of 1 including 1D ( 1 H, 13 C) and 2D ( 1 H-1 H COSY, HSQC, HMBC) NMR as well as ESI-MS data suggested the molecular formula C 10 H 13 N 5 O 5 requiring seven degrees of unsaturation. 13 C NMR spectrum revealed resonances for ten carbons of which five were detected in the range from 52.9 to 94.8 ppm indicating the presence of a sugar moiety. Comparison of the 13 C NMR data with those reported for sugars (Agrawal 1989) confirmed the identity of the sugar part as a-D-ribofuranose. 13 C signal at d C 52.9 ppm was assigned to C-5 0 and the relatively low chemical shift (around 10 ppm) suggested an attachment to a nitrogen atom. Five signals in 13 C NMR spectrum indicated the presence of an amide group (d C 162.8; C-6), one protonated sp 2 carbon (d C 136.0; C-8) and three non-protonated sp 2 carbons (d C 119.1, 142.3 and 152.7; C-5, C-4 and C-2, respectively), suggesting the existence of a guanine nucleotide. Apart from the signals of the sugar moiety, only one proton singlet resonating at d H 7.79 ppm (H-8) was detected in the 1 H NMR spectrum. HMBC experiment indicated cross-peaks from H-8 (d H 7.79) to each of C-4 and C-5 detected at d C 142.3 and 119.1 ppm, respectively. This provided a further confirmation for the presence of a guanine moiety. The link between the nucleotide and the sugar moiety through C-5 0 was assigned by HMBC experiment: correlations of H-5 0 a (d H 4.73) to C-4 (d C 142.3) and C-8 (d C 136.0) were detected. The linkage was confirmed by the chemical shift value of C-5 0 (52.9 ppm). Moreover, cross-peaks from H-1 0 (d H 6.13) to C-3 0 (d C 71.8) and C-4 0 (d C 85.6), from H-2 0 (d C 4.02) to C-1 0 (d C 94.8) and from H-3 0 (d H 4.32) and H-4 0 (d H 4.65) to C-5 0 (d C 52.9) were displayed by HMBC experiment. A further COSY correlation of a non-interrupted spinspin coupling from H-1 0 to H 2 -5 0 supported and secured the assignment of the signals of the a-Dribofuranose moiety. Compound 1 was reported here for the first time from a natural source and was named, dragmacidoside ( Figure 2).
Compounds 2 and 3 were identified as adenosine and inosine, respectively, by comparing 1 H NMR data and TLC results with those of reference compounds previously isolated by the authors from Eudistoma laysani (Abou-Hussein et al. 2007).
13 C NMR spectrum of compound 4 revealed resonances for nine carbons of which five were detected in the range 62.8 -88.8 ppm, indicating four oxygenated carbons of a sugar moiety and one deoxygenated carbon that resonated relatively upfield at 41.0 ppm. 1 H NMR spectrum revealed signals in the range of 2.2 -4.38 ppm besides a triplet detected at 6.27 ppm confirming the suggestion of the sugar moiety. HSQC experiment correlated each proton to its corresponding carbon. Comparing 13 C NMR data with those reported for sugars (Agrawal 1989), the sugar moiety could be identified as 2 0 -deoxyribose. The other four signals detected in the 13 C . These data confirmed the structure of the nucleobase as cytosine. Accordingly, compound 4 was identified as 2 0 -deoxycytidine. It was previously detected in marine sediments (Dell'Anno et al. 2002). 1D and 2D NMR data of compound 5 were found to be identical to those reported for methyla-D-glucopyranoside (Agrawal 1989). Methyl-a-D-glucopyranoside was previously isolated from Tulbaghia violacea extract (Lyantagaye 2013).
Biological evaluation indicated the ability of the chloroform fraction to reduce the thickness of the oedema induced by carrageenan in rats. The results of anti-inflammatory activity (Table 1) revealed that a gradual decrease in the thickness of the induced oedema was clearly detected over time. After 24 h of dose administration, the chloroform fraction produced 45.1% of change from the untreated inflamed group of rats thus performing 87% of indomethacin (reference drug) potency. On the other hand, the ethyl acetate fraction displayed only 20.0% of change from the control group, exhibiting 39% of indomethacin activity. Sterols were verified to possess antiinflammatory activity (García et al. 1999) and the capability of reducing the secretion of proinflammatory cytokines and tumour necrosis factor-a (Gupta et al. 1980;Bouic et al. 1996). The significant anti-inflammatory activity produced by the chloroform fraction may then be attributed to its sterol' contents (compounds 6 -9).

Biological material
The sponge was collected in July 2009 from the Red Sea, Hurghada, Egypt at a depth of 5 -9 m and identified by Prof. Rob van Soest. A voucher specimen, measuring 3.5 cm, has been incorporated in the collections of the Zoological Museum of the University of Amsterdam under registration number ZMAPOR 17592. Another voucher specimen has been deposited in the Red

Extraction and isolation
The fresh sponge (1.5 kg) was cut into very small pieces and macerated in methanol (3 £ 4000 mL) at room temperature. The combined methanolic extracts were dried under vacuum (7 g), dissolved in aqueous methanol and extracted with ethyl acetate (3 £ 500 mL). The organic layer was dried under vacuum to afford 1.5 g. The residue left was extracted with chloroform (3 £ 2000 mL) and dried to yield 5 g.