New sesquiterpene hydroquinones from the Caribbean sponge Aka coralliphagum

Four new sulfated sesquiterpene hydroquinones siphonodictyals E1–E4 (1–4) and cyclosiphonodictyol A (5) were isolated from a sample of the Caribbean sponge Aka coralliphagum collected off the coast of San Salvador in the Bahamas. The structures of the new compounds were elucidated on the basis of mass spectrometric and NMR spectroscopic analysis. Compounds 1–4 are derivatives of siphonodictyal E (9). Siphonodictyal E4 (4) exhibited mild antiproliferation activity against L929 mouse fibroblast, KB-31 epidermoid carcinoma, and MCF-7 breast cancer cell lines, while siphondictyal E3 (3) and cyclosiphonodictyol A (5) showed moderate activity against Gram-positive bacteria.


Introduction
Aka coralliphagum (Siphonodictyon coralliphagum) is known to have four distinct morphological forms: forma typica, f. tubulosa, f. obruta, and f. incrustans [1]. This sponge has the ability to burrow into live coral heads, leaving only the oscular chimney protruding (typica) or the flat crusts (incrustans) exposed. The oscular chimneys or the flat crusts are encircled by a so-called "dead zone" which protects the sponge from overgrowth [2]. Sullivan and Faulkner have proposed that the coral polyps are killed by the viscous mucus containing the toxic secondary metabolites which are produced by the sponge tissue [3]. This ecological observation inspired the chemical investigation of this sponge by different research groups. Thus many secondary metabolites have been reported, such as the sesquiterpene phenolic aldehydes, siphonodictyals B, C, D, E, the monosulfated siphonodictyols G and H, the disulfated siphonodictyal B3, and siphonodictyoic acid [2,4,5]. The sponge under our investigation is of the growth form incrustans, which was collected off the coast of San Salvador in the Bahamas. Fractionation of the aqueous n-BuOH extract yielded the new compounds siphonodictyals E1-E4 (1-4) and Since the structure of siphonodictyal E4 (4) could not be unambiguously assigned, the two possible constitutional isomers (4a and 4b) are shown. cyclosiphonodictyol A (5). In this article, we describe the isolation, structural elucidation, and bioactivity of compounds 1-5 from the Aka growth form incrustans, as well as discuss the biosynthetic pathway of related compounds.

Results and Discussion
The aqueous n-BuOH extract from Aka coralliphagum was subjected to solvent partitioning, followed by gel chromatography and reversed phase (C18) HPLC to yield siphonodictyals E1-E4 (1-4) and cyclosiphonodictyol A (5) (Figure 1). The structure elucidation was based on 1D and 2D NMR as well as HRMS-ESI(−) experiments. The 1 H and 13 C NMR chemical shifts for the five new compounds are given in Table 1.
Examination of the NMR data for siphonodictyal E2 (2) revealed a high degree of similarity with the data reported for siphonodictyal E (9). However, the molecular ion peak of compound 2 was shown at m/z 469.1534 ([M − H] − C 22 H 29 O 9 S), which has 80 amu more than that of 9, indicating an additional sulfate ester group in compound 2. The 1 H, 13 C-HMBC correlations from 18-OH to C-17 (δ 122.8), C-18 (δ 141.8), and C-19 (δ 147.8) as well as from 19-OH to C-18 (δ 141.8), C-19 (δ 147.8), and C-20 (δ 115.5) indicated that C-18 and C-19 are    (6), siphonodictyal B2 (7), siphonodictyal B3 (8), and siphonodictyal E (9) from the sponge Aka coralliphagum. connected to hydroxy groups rather than sulfate ester groups ( Figure 4). Thus, the sulfate ester group should be at position C-21. Furthermore, the 13 C NMR chemical shifts of the three constitutional isomers 2-I to 2-III were calculated using an increment system (Table 2) with additional values for sulfate esters published by Ragan [8]. These calculations (Table 3) allowed us to confirm the position of the sulfate moiety (2-I, Figure 5). The comparison of the NMR data of 2 and siphonodictyal B2 (7, Figure 2) [5] further confirmed the identity of the aromatic moiety of both compounds.  , which suggested a sesquiterpene side chain containing three trisubstituted olefinic bonds. The two sulfate ester groups were assigned to attach to C-18 and C-21 after comparison of the NMR data of the aromatic moieties of 3 with those of siphonodictyal B3 (8, Figure 2). Nevertheless, the results of the increment calculations using the values for sulfate esters from Ragan [8] clearly favours the isomer 3-I ( Figure 5), which further supports our proposed structure.
The HRMS-ESI(−) spectrum for siphonodictyal E4 (4)    2) confirmed the position of the unsubstituted CH-17 as shown in the proposed structure ( Figure 6). Since the very weak HMBC correlation from H-1 to C-19 or C-21 could not be unambiguously assigned, the constitutional isomers with respect to the position of the oxygen bridge 4a and 4b (Figure 1) are in accordance with the NMR data. This is the first compound  isolated from this sponge which contains a macrocycle. Moreover, this macrocycle could probably be formed by an aldol addition from a precursor with an acyclic sesquiterpene side chain (Figure 7). The aromatic ring system of proposed precursor 3-ox in Figure 7 also appeared in the known compound siphonodictyal B3 (8) [5] isolated from the same sponge source as well as the newly identified compound 3. There are no similar compounds known which contained the aromatic ring system with two adjacent sulfate ester groups. Therefore, we assumed that the constitutional isomer 4a is more likely. The δ values of methyl carbons C-14 and C-15 below 20 ppm indicated the E configuration of the double bonds C-6/C-7 and C-10/C-11 of the isoprenoid chain [6,7]. Additionally, in the NOESY spectrum we could observe a weak correlation    H-14 and H-15 suggested that the three methyl groups as well as CH 2 -15 are axial. Examination of the NMR data for 5 revealed a high degree of similarity with data reported for bis(sulfato)cyclosiphonodictyol A [10]. The difference concerned the absence of the two sulfate ester groups. Compound 5 could be the precursor of bis(sulfato)cyclosiphonodictyol A. Another possibility is that bis(sulfato)cyclosiphonodictyol A lost its sulfate ester group during the isolation procedure.
The new compounds were tested for antimicrobial activity against different Gram-positive and Gram-negative bacteria, fungi, and for antiproliferative activity. The results given in Table 4 show that siphonodictyal E3 (3) and cyclosiphonodictyol A (5) exhibited mild activity against the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, while siphonodictyal E4 (4) show cytotoxic activity against the L929 mouse fibroblasts, KB-31 epidermoid carcinoma, and the breast cancer cell line MCF7, although none of them showed activity in the antimicrobial assays against Gram-negative bacteria and the fungus Candida albicans.
Many of the isolated compounds from Aka coralliphagum are bicyclic sesquiterpene hydroquinones. However, in the course of our investigation, three linear sesquiterpenes were isolated. From a biosynthetical point of view [11], we propose that the linear sesquiterpenes are the precursors of the bicyclic sesquiterpenes ( Figure 8). Therefore, siphonodictyals B1-B3 (6-8) could be derived from siphonodictyals E1-E3 (1-3), respectively.
Aromatic sulfates occur in a wide range of higher plants and animals, particularly those growing in marine, estuarine, or freshwater environments. This is probably due to the high content of inorganic sulfate in aquatic environments (both limnic and marine environments -seawater contains an average of 28.7 mM inorganic sulfate [12]). Jensen and Ragan have demonstrated the presence of the natural product 1,2,3,5tetrahydroxybenzene-2,5-disulfate in the marine brown alga Ascophyllum nodosum [13]. Kobayashi et al. isolated halenaquinol sulfate from the marine sponge Xestospongia sapra [14].
In the course of our investigation on the sponge Aka coralliphagum, we isolated a number of compounds containing sulfated phenols. These sulfated metabolites are labile [5], and easily loose the sulfate ester groups by hydrolysis in water [15]. Moreover, the influence of the sulfate esters on the bioactivity has been proven by activity tests: the desulfated compounds tend to be more active [5]. Therefore, we propose that the sulfate esters act as hydroxy protecting groups. The sponges excrete the sesquiterpene hydroquinones primarily in the sulfated form and their activities get increased by hydrolysis of these labile groups. This would lead to a prolonged bioactivity of these metabolites and therefore to a more efficient defense against predators or pathogens.

Antimicrobial assay
Determination of antimicrobial activities was carried out by the Helmholtz Center for Infection Research (HZI), and was based on the microdilution test, using 96-well micro titration plates. The MIC was defined as 50% growth inhibition after 24 hour incubation compared to that in the growth control well.

Cytotoxic assay
The cytotoxicity was determined using WST-1 assays, targeting cell lines L929 mouse fibroblasts, KB-31 epidermoid carcinoma, MCF-7 breast cancer, and FS4-LTM conditional immortalization human fibroblasts, respectively. The FS4-LTM cell line was incubated for 24 hours with the tested substances. The other cell lines were incubated for 5 days with the tested substances.

Supporting Information
Supporting Information File 1 1D and 2D NMR spectra of the isolated compounds.