Two new polyacetylene derivatives from the Red Sea sponge Xestospongia sp.

1 Introduction

Barrel sponges, or the species of the genus Xestospongia (class Demospongia, order Haplosclerida, family Petrosiidae) might be considered as exclusive producers of some interesting secondary metabolites. These include the halogenated polyacetylenic lipids, which have been shown to possess a broad spectrum of biological activities such as antimicrobial, HIV-integrase inhibition, and Na⁺/K⁺ ATPase inhibition [1–4]. The genus Xestospongia contains long-chain polyacetylenic alcohols with chemotaxonomic markers [5], and polyacetylenes with antimicrobial, cytotoxic, antitumor, antiviral, and immunosuppressant bioactivities, and also as enzyme inhibitors [6, 7]. Search for bioactive metabolites from Xestospongia led to the identification of two new polyacetylenes, viz. acylglycerolipid and xestospongiamide. Similarly, the extract from Xestospongia sp., collected from Pohnpei, Federated States of Micronesia, exhibited antimicrobial activity against Pseudomonas aeruginosa and Mycobacterium intracellulare with IC₅₀ values of 1.03–2.07 μg/mL [8]. Here we present the isolation and identification of two further new polyacetylenes from this sponge.

2 Materials and methods

3 Results and conclusion

In continuation of our research program on compounds from marine organisms, the sponge material was collected from Saudi territorial waters. Extensive fractionation of the organic extract employing NP-Silica gel, preparative thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) yielded four metabolites (1–4). Two were new acetylenic derivatives: an acylglycerolipid,2′-O-(7E,17E-18-bromo-octaoctadeca-7,17-dien-5,15-diynoyl)-3′-O-(9E,13E,17E)-18-bromo-octaoctadeca-9,13,17-dien-7,15-diynoyl-1′-O-β-D-galactopyranosyl glycerol (1), and xestospongiamide (2). The other two metabolites had been previously identified: known 26,27-Dimethylergosta-5,24(28)-dien-3β-ol (xestosterol) (3) and the xestosterol ester of 18-bromooctadeca-(9E,17E)-diene- 7,15-diynoic acid (4; Figure 1).

Figure 1:

Structures of compounds 1–4.

Compound 1 was isolated as a white powder. The molecular formula, C₄₅H₅₈O₁₀Br₂, was determined by HRESIMS at m/z 916.2384 [M]⁺. Liquid chromatography mass spectrometry (LCMS; positive-ion-mode) at m/z 916 [M]⁺ indicated 16 degrees of unsaturation. The 1:2:1 isotopic distribution at m/z 916, 918 and 920 indicated the presence of two bromine atoms. Moreover, the peak at m/z 179 could be assigned to a hexose sugar unit (Figure 2). The ¹³C NMR spectrum indicated the presence of 45 signals, categorized by an DEPT NMR experiment into two carbonyl δ_C (174.3 and 175.0 ppm), eight quaternary (93.7, 91.3, 89.1, 88.5, 85.6, 80.8, 80.3, and 78.3), 10 olefinic (145.7, 143.5, 142.8, 112.0, 119.1, 118.9, 118.7, 118.0, 111.6 and 111.1), 16 methylene (34.8, 34.0, 33.5, 33.3, 33.0, 29.7, 29.4, 29.1, 28.9, 28.9, 28.9, 25.5, 25.2, 19.8, 19.8 and 19.3), an acetal (104.7) and three oxygenated methylene (68.7, 63.9 and 62.7) carbons, respectively. The ¹H NMR spectrum comprised signals due to a sugar structure, such as oxymethines (δ_H 4.28, 3.33, 3.32, 3.27 and 3.17), and ¹³C NMR revealed the signal of a typical acetal carbon δ_C 104.7. ¹³C, ¹H and H-H COESY NMR spectral data indicated the presence of a glycerol moiety, based on the assignment of the three vicinal oxygenated carbons δ_C 68.7, 71.6 and 63.9. The downfield chemical shift indicated a substituted glycerol. Two esterified carbonyl functions (δ_C 174.3 and 175.0) and the IR absorption band at 1736 cm^-1 assigned a diglyceride skeleton. The stereochemistry of the sugar moiety was revealed to be that of a glucose derivative based on the large coupling constants (9 Hz) between H₂-H₃, H₃-H₄, H₄-H₅. The β-anomeric structure was assigned from the coupling constant (J_1–2 = 7.8 Hz) and NOESY correlations of H₁/H₃ and H₁/H₅. Hence, 1 is a glucopyranosyl diacylglycerol. The acid hydrolysis and methanolysis method of Hirsh et al. [13] yielded two UV₂₅₄ absorbing substances after purification employing column and PTL chromatography (cf. exp.). The two substances were assigned as R₁ and R₂ methyl esters (R1 and R2, respectively).

Figure 2:

Fragmentation of LCMS spectrum of compound 1 and prominent mass spectral fragment peaks (m/z) of R1 and R2.

R1 was obtained as yellow oil with a molecular formula of C₁₉H₂₅O₂Br, as established by high resolution fast-atom bombardment mass spectrometry (HRFABMS).

Gas chromatography-mass spectrometry (GCMS) revealed the presence of one bromide atom (two mass unit isotopic peaks in 1:1 ratio). Combination of ¹³C NMR spectrometry with a heteronuclear single quantum correlation (HSQC) experiment revealed the presence of four singlet acetylenic carbon atoms in R1, four protonated olefinic carbon atoms, along with signals for nine paraffinic methylene carbon atoms (Table 1).

The ¹H NMR spectrum featured the following: a brominated vinyl group [δ_H 6.23 (dt, 13.8, 1.8 Hz, H-17″ and 6.69 (d, 13.8, Hz, H-18″)] and a trans-disubstituted double bond [δ_H 5.46 (dt, 15.6, 1.8 Hz, H-7″ and 5.98 (dt, 15.6, 6.6 Hz, H-8″)], i.e. H-7″. The COESY and HSQC experiments allowed the assignment of the subunits C-2″/ C-4″, C-7″/C-8″ and C-17″/C-18″ within R1, which were substantiated by heteronuclear multiple bond correlation (HMBC) data. HMBC correlations of H₂-4″/C-5″, H-7″/C-5″, and H-17″/C-16 established the unambiguous placement of the conjugated diyne moieties at C-8″/C-5″ and C-15″/ C-18″. The structure elucidation of R1 based on NMR data was further confirmed by mass spectral analysis. Electron impact mass spectrometry (IMS) data of R1 exhibited several conspicuous fragments containing linear alkyl chains [14]. Fragments observed at m/z 263, 199, 129, 101, and 64 supported the structure assigned for R1 (Figure 2). Accordingly, the moiety R₁ was assigned the structure 7E, 17E-18-bromo-octaoctadeca-7,17-dien-5,15-diynoyl.

R2 was obtained as yellow oil with a molecular formula of C₁₉H₂₃O₂Br, as established by HRFABMS. GCMS revealed the presence of one bromide atom (two mass units’ isotopic peaks in 1:1 ratio). By a similar treatment of both NMR and EIMS data, the connectivities, the geometry, and positions of double and triple bonds were indicated (Table 1 and Figure 2). Accordingly, the moiety R₂ was assigned the structure 9E,13E,17E-18-bromo-octaoctadeca-9,13,17-dien-7,15-diynoyl. Taking into consideration that R₁ and R₂ may be interchangeable, compound 1 is a new acylglycerolipid; 2′-O-(7E,17E-18-bromo-octaoctadeca-7,17-dien-5,15-diynoyl)-3′-O-(9E,13E,17E)-18-bromo-octaoctadeca-9,13,17-dien-7,15-diynoyl-1′-O-β-D-glucopyranosyl glycerol (Figure 2).

Compound 2 was isolated as pale yellow oil. The molecular formula, C₂₀H₂₈BrClN₂O, was deduced by LCMS at m/z 426:428:430 (2:3:1) [M]⁺, indicating that 2 has two halogen atoms (one Br and one Cl), with seven degrees of unsaturation. The infrared (IR) spectrum showed the presence of a carbon–carbon triple bond (2215 cm^–1), a carbonyl ester group C=O (1741 cm^-1), amide and amine function bands (3120–3434 cm^–1). The ¹³C and DEPT NMR measurements indicated 20 signals, categorized into 1) a quaternary carbonyl δ_C (173.1 ppm), 2) four quaternary acetylenic carbons (92.7, 88.5, 79.2 and 77.5), 3) four olefinic methine, (142.6, 118.0, 117.1 and 110.2), 4) a methine (68.9), 5) a halogenated methylene (62.1), and 6) nine methylene δc (33.9, 32.3, 28.4, 28.3, 27.9, 27.7, 24.4, 19.2 and 19.1) carbons, respectivekly (Table 1). So, compound 2 must be acyclic.

The ¹H and ¹³C NMR together with ¹H-¹H COSY spectral data indicated a disubstituted carbon-carbon double bond [δ_H 6.17 ppm (dt, 13.8, 1.8 Hz, H-17; δ_C 118.0, C-17) and 6.57 (d, 13.8 Hz, H-18; δ_C 117.1, C-18)], which was coupled on one side only to a two proton signal at δ_H 2.25 (td, 6.6, 1.8 Hz, H-14). The small coupling (1.8 Hz) originated from a long range effect between H-17 and H-14 through the acetylenic group. The large coupling constants between H-17 and H-18 (13.8 Hz) indicated the E geometry of the olefinic moiety. Investigation of the HMBC spectral data indicated correlations between H-18 and C-17, C-16 and C-15 and between H-17 and C-18 and C-15 (Figure 3). The presence of an ion peak at m/z 129[C₄H₂Br]⁺ in the LCMS supported this fragment (Figure 4), led to establishing fragment 1 (Figure 4).

Figure 3:

Selected HMBC of compound 2.

Figure 4:

Fragmentation of LCMS spectrum of compound 2 and its main fragments.

The ¹H-¹H COSY spectrum showed that chlorinated methylene protons (H₂-2′) at δ_H 4.28 (dd, 12.6, 4.2 Hz) and 4.13 (dd, 12.6, 6.6 Hz), are coupled to a low field methine proton H-1′ at δ_H 5.26 (dd, 6.6, 4.2 Hz) of an ABX system. The attachment of a primary amine and amide to C-1′ shifted the absorption of H-1′ to a more down field value (δ_H 5.26). Further investigation of the HMBC spectrum indicated a correlation between H-1′ and C-2′ with the amidic carbonyl between H-2′ and C-1′ and C-1 (Figure 3) establishing fragment 3 (Figure 4).

With the aid of the data in Table 1 and Figure 4, the structures of fragments 2, 4, and 5 can be deduced. The connections between the fragments were performed through interpretation of ¹H-¹H COSY and HMBC spectral data. A computer survey of different data bases, including SciFinder, indicates that 2 is a new xestospongia amide. Compounds 3 and 4 were identified as xestosterol and xestosterol (9E,17E)-18-bromooctadeca-9,17-dien-7,15-diynoic acid, respectively, by comparing their spectroscopic data with those in the literature [15, 16]. The antimicrobial activities of 1 and 2 were determined against some multidrug-resistant bacteria using an agar well diffusion assay. DMSO was used as negative control while ampicillin and amphotericin B were used as positive antibacterial and antifungal agents, respectively. The mean diameter of the inhibition zones was in the range of 12–23 mm (Table 2). The antifungal activity was recorded against the dermatophytes A. niger,C. albicans ATCC1023, C. tropicalis, C. neoformans. Product 1 showed no antifungal activity, while compound 2 exhibited excellent antifungal activity against A. niger, C. albicans, C. tropicalis, C. neoformans, Epidermophyton sp., M. gypseum, T. mentagrophytes, and T. rubrum (diameters of inhibition zones ranged from 14–22 mm). MICs were calculated for each product and ranged from 2.2 to 4.5 μM for A. baumannii, K. pneumonia and P. aeruginosa; those for C. albicans, A. niger, and Epidermophyton sp. were in the range of 2.2–2.5 μM (Table 3). Compounds 1 and 2 were not cytotoxic against Artemia salina, while compound 2 exhibited antitumor activity against Ehrlich ascites carcinoma and lymphocytic leukemia at LD₅₀ 5 μM (Table 4).

In conclusion, the results of this study confirm that product 2, obtained from the Xestospongia extract, can be considered a potential material for designing effective antibacterial, antifungal and antitumor agents for treatment of multidrug resistant bacteria, tumors, dermatophytosis, and other fungal infections with no cell toxicity.