Didemnaketals F and G, New Bioactive Spiroketals from a Red Sea Ascidian Didemnum Species

In continuation of our ongoing efforts to identify bioactive compounds from Red Sea marine organisms, a new collection of the ascidian Didemnum species was investigated. Chromatographic fractionation and HPLC purification of the CH2Cl2 fraction of an organic extract of the ascidian resulted in the identification of two new spiroketals, didemnaketals F (1) and G (2). The structure determination of the compounds was completed by extensive study of 1D (1H, 13C, and DEPT) and 2D (COSY, HSQC, and HMBC) NMR experiments in addition to high-resolution mass spectral data. Didemnaketal F (1) and G (2) differ from the previously reported compounds of this class by the lack the terminal methyl ester at C-1 and the methyl functionality at C-2. Instead, 1 and 2 possess a methyl ketone moiety instead of the terminal ester. Furthermore, didemnaketal F possesses a disubstituted double bond between C-2 and C-3, while the double bond was replaced by a secondary alcohol at C-3 in didemnaketal G. In addition, they possess the unique spiroketal/hemiketal functionality which was previously reported in didemnaketal E. Didemnaketals F (1) and G (2) displayed moderate activity against HeLa cells with of IC50s of 49.9 and 14.0 µM, respectively. In addition, didemnaketal F (1) displayed potent antimicrobial activity against E. coli and C. albicans. These findings provide further insight into the biosynthetic capabilities of this ascidian and the chemical diversity as well as the biological activity of this class of compounds.


Introduction
Natural products and their derivatives contribute more than half of all clinically administered drugs [1]. Less than 1% of the isolated marine-derived natural products have been screened for their bioactivities [2]. Many marine invertebrates such as sponges and ascidians (tunicates) are sessile (attached to the ocean floor) and use highly evolved chemical compounds to attract food, block the growth of intruding neighbors or repel predators. Biologists believe that these survival demands triggered the evolution of a particularly abundant mixture of bioactive compounds. Marine invertebrates, including sponges and ascidians, contribute a major proportion of novel marine-derived bioactive compounds [3]. The search for the bioactive compounds from marine ascidians is relatively young, but it is worthwhile mentioning that didemnin B, an ascidian metabolite, was the first marine natural product to be evaluated in a human clinical trial [4]. Most ascidians are consumed as food in some countries. They produce antifouling natural products and this can be considered as a kind of autogenic protection [5]. The genus Didemnum is well-known for its diverse and bioactive secondary metabolites including cyclic peptides [6,7], alkaloids [8][9][10][11][12], macrolides [13][14][15], and N,N′-diphenethylurea [16]. These metabolites were found to possess different biological activities such as antiplasmodial [17], antiviral [18,19], cytotoxic [20], and protein kinase inhibitors [13][14][15]. It is well-known that the first marketed marine-derived anticancer agent, trabectedin (ET-743, Yondelis), is a product of the tunicate Ecteinascidia turbinata [21]. It is commercialized by PharmaMar and is approved for the treatment of soft tissue sarcomas in Europe and other countries. It is also undergoing clinical trials for the treatment of locally advanced or metastatic ovarian cancer, liposarcoma, leiomyosarcoma or pleural mesothelioma [22].
To date, five spiroketals, including didemnaketals A-E, were isolated from the marine ascidian Didemnum species collected from different geographical locations including the Red Sea [13] and Palau [14,15]. Two of these, didemnaketals A and B, were reported as a result of oxidation and methanolysis of didemnaketal C during long storage of the ascidian sample in MeOH [14]. In this work, we describe the isolation, structure determination, and biological activities of two didemnaketal congeners, didemnaketal F (1) and G (2) (Figure 1) from a new collection of the Red Sea Didemnum species. The new compounds differ from the previously reported ones in which they lack the methyl functionality at C-2 and the terminal methyl ester moiety of the spiroketal skeleton. Instead of the terminal methyl ester, they possess a terminal methyl ketone moiety at the left part of the molecule (as in 1 and 2). Additionally, compound 1 possesses a disubstituted double bond at C-2/C-3, while 2 possesses a secondary alcohol at C-3. As reported before in didemnaketal E (Figure 1) [13], both 1 and 2 possess a spiroketal/hemiketal functionality at the right part of the molecule.

Purification of Compounds 1 and 2
Investigation of the CH2Cl2 soluble fraction of an organic extract of a new collection of the Red Sea ascidian Didemnum species using a combination of chromatographic techniques, including normal SiO2, Sephadex LH-20, and C18 HPLC, afforded two new spiroketals; didemnaketals F (1) and G (2) (Figure 1). The isolated compounds were evaluated for their antiproliferative, cytotoxic, and antimicrobial activities.

Structure Elucidation of Compound 1
Compound 1 possesses a molecular formula C45H72O15 as deduced from the HRESIMS molecular ion peak at m/z 853.4950 [M + H] + , suggesting 10 degrees of unsaturation. Compound 1 is 30 mass units (CH2O) less than didemnaketal E ( Figure 1) [13]. The IR spectrum of 1 displayed characteristic absorption bands for the functional groups in the molecule at 3460 (OH), 1735 (ester), 1720 (ketone), 1675 (α,β-unsaturated ketone) and 1650 (C=C), 1070 (C-O) cm −1 . The 13 C NMR, DEPT, and HSQC spectra revealed the presence of 45 carbon resonances: 12 methyls, 11 methylenes, 13 methines, five of them were assigned to oxymethines and two for disubstituted olefinic double bond, and 9 quaternary carbons including five ester and two ketone carbonyls. The presence of the unique spiroketal/hemiketal moiety was evident from carbon signals at δC 98.7 (C-16) and 99.8 (C-20) [13]. The 1 H-1 H COSY spectrum revealed the presence of several spin systems (Table 1, Figure 2). These spin systems were confirmed by the HSQC and HMBC cross peaks. The 1 H and 13 C NMR data of 1 were comparable to those of didemnaketal E in the C4C23 region [13]. The differences in the NMR spectra of 1 were the absence of the signals associated with the methyl group at C-2 and the terminal methyl ester moiety in didemnaketal E ( Figure 1) [13]. Instead, the 1 H and 13 C NMR spectra of 1 showed signals at δH 6.11 (d, J = 16.5 Hz, H-2)/δC 133.0 (C-2) and 6.75 (dt, J = 16.5, 6.8 Hz, H-3)/δC 145.8 (C-3). These signals were assigned for a disubstituted double bond and confirmed by the observed COSY correlations between H-2 and H-3 and between H-3 and H2-4 (δH 1.97, m). The E-configured double bond was confirmed by a large 3 JH,H value ( 3 JH-2,H-3 = 16.5 Hz) [23]. The position of the double bond at C-2 and C-3 was established by the COSY correlation between H-3 and H2-4 (δH 1.97, m) and further secured by the 3 J HMBC correlation between H2-4 and C-2. Moreover, signals for a terminal methyl ketone at δH 2.24 (s, H3-24)/δC 27.1 (C-24) and δC 198.5 (C-1) were observed. This was confirmed by the 2 J HMBC cross peak between H3-24 and C-1 (δC 198.5). The upfield shift of the ketone moiety at C-1 (δC 198.5) supported the α,β-unsaturation at C-2/C-3. The 13 C chemical shift values of C-2 and C-3 and the HMBC cross peak of H-3 with C-1 confirmed the connectivity of methyl ketone moiety at C-2. Furthermore, the 1 H and 13 C NMR spectra showed the presence of one acetate at δH 2.05 (H3-33)/δC 21.0 (C-33) and 170.  Table 1). The absolute configuration of 1 was assigned by comparison of 1 H and 13 C NMR data as well as coupling constants with those of previously reported didemnaketals [13][14][15][24][25][26]. From the above discussion and the results of the COSY, HSQC, and HMBC (Table 1, Figure 2) spectra, the structure of 1 was unambiguously assigned as shown and named didemnaketal F.    Instead, signals for a methoxy group at δH 3.64 (H3-44)/δC 51.8 (C-44) were observed in 2. The placement of methoxy group at C-25 in 2 was confirmed by the HMBC correlation between H3-44 and C-25 ( Figure 2). The assignment of all quaternary carbons were unambiguously assigned from HMBC correlation ( Table 2 and Figure 2) completing the assignment of 2 and was named as didemnaketal G.
The absolute configuration of the previously reported didemnaketals from the genus Didemnum was assigned on the basis of degradation/derivatization experiments, chiral shift methods, and chemical synthesis of the C9-C28 subunit [24][25][26]. Recently, the absolute configuration of the C10-C20 subunit was revised by Fuwa et al. [27]. Thus, the absolute configuration of didemnaketals F (1) and G (2) was assigned as 5S, 6S, 7R, 8R, 10S, 11S, 12S, 14R, 16S, 18R based on extensive spectroscopic studies, in addition to comparison with the previously reported didemnaketals.  Didemnaketals F (1) and G (2) were evaluated for their antiproliferative and cytotoxic activities against HeLa cells [28][29][30] as well as antimicrobial activity against three pathogens including a Gram-positive bacterium (Staphylococcus aureus ATCC 25923), a Gram-negative bacterium (Escherichia coli ATCC 25922) and yeast (Candida albicans ATCC 14053). Didemnaketals F (1) and G (2) displayed moderate antiproliferative activity against HeLa cells with IC50s of 49.9 and 14.0 µM, respectively (Figure 3). Didemnaketal G (2) also caused cytotoxicity as indicated by the dose response curve dropping below the dashed line ( Figure 3). In contrast, cytotoxic effects were not observed with didemnaketal F (1) at concentrations up to 50 μM ( Figure 3). Paclitaxel was used as a positive control with an IC50 of 0.0017 µM in this assay.
In the antimicrobial screening, didemnaketal F (1) showed strong antimicrobial activity against E. coli and C. albicans with inhibition zones of 20 and 24 mm at a concentration of 100 µg/disc, while didemnaketal G (2) showed moderate activity against E. coli and C. albicans with inhibition zones of 7 and 17 mm at the same concentration ( Figure 4).

General Experimental Procedures
Optical rotations were measured on a JASCO DIP-370 digital polarimeter at 25 °C at the sodium D line (589 nm). UV spectrum was recorded on a Hitachi 300 spectrometer. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). NMR spectra were determined on BRUKER AVANCE III 600 instruments (600 MHz for 1 H and 150 MHz for 13

Biological Materials
The marine ascidian Didemnum species was collected in the Mangrove located in Nabq/Sharm El-Sheikh on the Egyptian Red Sea coast at depth (~1 to 2 m) during July 2013. A complete description of ascidian as previously mentioned [13]. The ascidian forms thin (2-3 mm) deep orange colored sheets around the pneumatophores of the mangrove plant Avicennia marina. The color of the ascidian fades on preservation in formalin. Didemnum species share many characteristics common among colonial tunicate species. Characteristics such as colony shape and color, where the colony grows, zooid structure, and spicule shape have been used previously to separate and identify different Didemnum species. Didemnum species are characterized by: many small zooids 1-2 mm in length, embedded in a sheet-like, gelatinous matrix called a tunic or test. White, calcareous, stellate spicules are embedded within the tunic's surface among zooids that contain individual oral siphons while atrial siphons discharge into a common cloacal aperature maintained in deep crevices within the colony [31,32]. Spicules measure 40 μm in diameter on average, but can reach 100 μm. This combination of spicules and zooids give the tunic's surface an overall appearance described as "small, white dots and pinhole-sized pores". Descriptions of Didemnum sp. colony shape include long, ropey or beard-like colonies; low, undulating mats with short appendages that encrust or drape; sponge-like colonies. However, it should be noted that Didemnum sp. colony shape changes with age. Young Didemnum sp. colonies usually present as thin mats but as the colony matures, irregular lobes are formed, thereby greatly increasing surface complexity. A voucher specimen, measuring 2.5 × 3.0 cm was deposited in the Red Sea Invertebrates collection of the Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University under the registrationcode 13DY31.

Evaluation of Antiproliferaive and Cytotoxic Activities
The effects of the compounds 1 and 2 on HeLa cell proliferation and cytotoxicity were evaluated using the sulforhodamine B (SRB) assay [28][29][30]. HeLa cells were grown in Basal Medium Eagle (BME) containing Earle's salts, 10% FBS and 50 µg/mL gentamycin sulfate. Cells were plated at a density of 2500 cells per well in a 96-well plate and allowed to adhere and grow for 24 h before compounds were added. The compounds were solubilized in DMSO and added to a final DMSO concentration of 1% in both test wells and vehicle controls. The cells were incubated with compounds or vehicle for an additional 48 h. The IC50s, the concentrations required to cause a 50% inhibition of cell proliferation, were calculated from the log dose response curves. The values represent the average of 3-4 independent experiments, each conducted in triplicate ± SEM. Cytotoxicity was determined by a cell density lower than that measured at the time of drug addition (dashed line in Figure 3). Paclitaxel was used as a positive control.