Anti-Helicobacter, Antitubercular and Cytotoxic Activities of Scalaranes from the Red Sea Sponge Hyrtios erectus

The Red Sea specimen of the marine sponge Hyrtios erectus (order Dictyoceratida) was found to contain scalarane-type sesterterpenes. 12-O-deacetyl-12,19-di-epi-scalarin (14), a new scalarane sesterterpenoid, along with fourteen previously-reported scalarane-type sesterterpenes (1–13 and 15) have been isolated. The chemical structures of the isolated compounds were elucidated on the basis of detailed 1D and 2D NMR spectral data and mass spectroscopy, as well as by comparison with reported data. The anti-Helicobacter pylori, antitubercular and cytotoxic activities of all fifteen compounds were evaluated to reveal the potency of Compounds 1, 2, 3, 4, 6, 7 and 10. Amongst these, Compounds 1, 3, 4, 6 and 10 displayed a promising bioactivity profile, possessing potent activities in the antitubercular and anti-H. pylori bioassay. Compounds 2 and 7 showed the most promising cytotoxic profile, while Compounds 1 and 10 showed a moderate cytotoxic profile against MCF-7, HCT-116 and HepG2 cell lines.


Purification of Compounds 1-15
Successive fractionation of the lipophilic fraction obtained from the methanolic extract of the sponge using silica gel column chromatography followed by final purification on semipreparative reversed phase HPLC column afforded fifteen pure isolated compounds (1-15) containing a scalarane-type framework, of which Compound 14 was assigned as a new scalarane sesterterpenoid.

Structural Elucidation of Compounds 1-15
Compound 14 (Figure 2) was isolated and purified as an amorphous powder. The molecular formula, C 25 H 38 O 4 , was established from the positive ESIMS, as well as from 13 C-NMR data. In the mass spectrum of Compound 14, the molecular ion peak was of low abundance or absent, but strong peaks corresponding to losses of water were observed at m/z 385. 3 Figure S1). The 1 H and 13 C-NMR spectra measured in CDCl 3 (Table 1), as well as correlations in the HMBC (Table 1, Figure 3) suggested that Compound 14 was also a scalarane-type sesterterpenoid. Thus, five singlets at δ H 0.81, 0.83, 0.85, 0.86 and 0.93 were assigned to the five methyl groups of a scalarane skeleton. In the 13 C-NMR spectrum of 14 (Table 1), the carbon signals in the low-field region at δ C 98.5 (C-19), 127.3 (C-17), 136.3 (C-16) and 166.9 (C-20) were reminiscent of those of scalarin [56] (Figure 2).    The NMR spectra of Compound 14 (Supplementary Materials, Figures S2-S5) were quite similar to those of scalarin [56] (Figure 2). However, several diagnostic differences were observed. The most evident were the absence of the signals due to the acetyl group, the upfield shift of H-12 at δ H 3.57, the chemical shift of CH-18 at δ C 58.7, δ H 2.54/(δ C 50.8, δ H 3.14, in scalarin), and the 13 C chemical shift of CH-19 at δ C 98.5/(δ C 98.9 in scalarin).
The relative configuration of H-12 and H-19 was detected by coupling constants (for H-12) and confirmed by interpreting the NOESY spectrum (Supplementary Materials, Figure S6). The α-configuration of H-12 was deduced on the basis of the diaxial coupling of H-12 (δ H 3.57; dd, J = 11.05 and 4.25 Hz) with H-11 and cross-peaks with α-oriented H-9 and H-14 in NOESY ( Figure 4). Finally, the signal assigned to H-19 was correlated in the NOESY spectrum with H-18, which in turn was correlated with H-14, and H-12 indicated the α orientation of H-19 ( Figure 4); therefore, the configuration at the C-12/C-19 carbons was opposite to that of scalarin ( Figure 2). To the best of our knowledge, Compound 14 has not been reported before in the literature; therefore, it is considered as a new scalarane sesterterpene analogue. The name 12-O-deacetyl-12, 19-di-epi-scalarin was assigned for this compound. The other known Compounds 1-13 and 15 ( Figure 2) were identified by extensive study of their spectral data, including ESIMS, 1D and 2D NMR data, as well as by comparison with the published data.

Biological Activities of the Isolated Compounds 1-15
The isolated Compounds 1-15 were evaluated for their antitubercular, anti-H. pylori and cytotoxic activities ( Table 2). Anti-H. pylori compounds are reported herein for the first time for scalarane sesterterpene analogues. Among these, Compounds 1, 3, 4, 6 and 10 displayed a promising bioactivity profile, possessing potent activities in the antitubercular and anti-H. pylori bioassays. Compound 7 displayed an interesting bioactivity profile, possessing potent antitubercular and cytotoxic activities and being practically slightly active in the anti-H. pylori bioassay.  In the cytotoxic assay, the compounds tested against various cell lines (MCF-7, HCT-116 and HepG2) showed variable cytotoxic activity. Amongst these, Compounds 2 and 7 showed the most promising cytotoxic profile with IC 50 values ranging from 0.4 ± 0.1-1.2 ± 0.1 µM and from 1.4 ± 0.05-1.6 ± 0.1 µM against the cell lines under investigation, respectively. Compounds 1, 5 and 10 showed a moderate cytotoxic profile with IC 50 values less than, or approximately, 20 µM against all cell lines under investigation. Other compounds showed weak to no activity against the cell lines under investigation (Table 2).

Biological Materials
The marine sponge specimens used in this study ( Figure 1) were collected from the Red Sea, Egypt, by scuba diving. The sponge material was immediately frozen after collection and kept at −20 • C until investigation. The sponge specimen was later identified to be Hyrtios erectus (class: Demospongiae, order: Dictyoceratida, family: Thorectidae) by Dr. Rob van Soest (Institute of Systematic Population Biology, Amsterdam University, The Netherlands). A voucher specimen was preserved at the Zoological Museum of the University of Amsterdam, under Registration Number ZMAPOR19761.

Purification of Compounds 1-15
The collected sponge specimen (0.90 kg, wet wt.) was cut into small pieces and was macerated exhaustively at room temperature in MeOH. The combined extracts were concentrated under reduced pressure to yield the organic crude extract (85 g). The total crude extract was subjected to silica gel column using VLC (vacuum liquid chromatography) gradient elution (n-hexane-CHCl 3 -MeOH) to afford Fractions 1-9.

Anti-Helicobacter pylori Activity Assessment
The anti-H. pylori activity was assessed against a strain of Helicobacter pylori (American Type Culture Collection, H.b., ATCC 700392) using a micro-well dilution method as previously described [57]. Clarithromycin was used as a positive control and exhibited an MIC of 1.31 µM.

Antitubercular Activity Assessment
A nonvirulent strain Mycobacterium tuberculosis (ATCC 25177, H37Ra) was obtained from the American Type Culture Collection. The antitubercular activity was assessed according to the protocol described by Franzblau [58,59] using the microplate Alamar blue assay (MABA). Isoniazid was used as positive control and exhibited an MIC of 0.87 µM.

Trypan-Blue Exclusion Assay
Cell viability was measured prior to seeding using the trypan-blue exclusion method. Briefly, exponentially-growing cells were trypsinized by 0.25% trypsin-EDTA solution. Aliquots of cell suspensions were mixed with trypan blue solution (0.4% w/v), and blue color (positive cells) was determined. Cells were not seeded unless viability was greater than 95%.

Cytotoxic Assessment
The cytotoxic effects of Compounds 1-15 on breast (MCF-7), colorectal (HCT-116) and liver cancer cells (HepG2) were evaluated using the sulforhodamine B (SRB) method as previously described [60]. Briefly, mid-exponentially-proliferating cells were trypsinized by trypsin-EDTA (0.25% w/v) and seeded in 96-well plates (1000-2000 cells/well). Cells were treated with serial concentrations of the compounds under investigation for 72 h and subsequently fixed with TCA (10% w/v) for 1 h at 4 • C. After washing three times, cells were stained with 0.4% SRB solution for 10 min in a dark place and then washed with glacial acetic acid (1% v/v). After drying, Tris-HCl (50 mM, pH 7.4) was used to dissolve the SRB-stained cells, and color intensity was measured at 540 nm. The dose response relationship of test compounds was analyzed using the E max model (Equation (1)).
where (R) is the residual unkilled fraction (the resistance fraction), (D) is the drug concentration used, (K d ) or IC 50 is the drug concentration needed to produce 50% viability reduction and (m) is the Hill-type coefficient. IC 50 is defined as the drug concentration required to reduce maximum absorbance by 50% compared to untreated control cells [61].

Statistical Analysis
Data are presented as the mean ± SEM using GraphPad prism™ software Version 5.00 (GraphPad software Inc., La Jolla, CA, USA) for Windows Version 10.00. Analysis of variance (ANOVA) with the Tukey-Kremer post hoc test was used to calculate the significance using SPSS ® for Windows, Version 17.0.0. p < 0.05 was taken as a cut-off value for significance.

Conclusions
Chemical investigation of the bioactive extract of the sponge Hyrtios erectus, collected in the Red Sea, Egypt, yielded fifteen compounds of scalarane-type sesterterpenes. Compounds 1-15 including a new one (14) were purified, and their chemical structures were characterized using spectroscopic studies. The isolated compounds belong to the class of scalarane sesterterpenes and displayed diverse biological activities including anti-H. pylori, antitubercular and cytotoxic. Anti-H. pylori compounds are reported herein for the first time for scalarane sesterterpene analogues. Among these, Compounds 1, 3, 4, 6 and 10 displayed a promising bioactivity profile, possessing potent activities in the antitubercular and anti-H. pylori bioassays. Compound 7 displayed an interesting bioactivity profile, possessing potent antitubercular and cytotoxic activities and being practically slightly active in the anti-H. pylori bioassay. Moreover, Compounds 2 and 7 showed the most promising cytotoxic profile, while Compounds 1, 5 and 10 showed a moderate cytotoxic profile against MCF-7, HCT-116 and HepG2 cell lines. Other compounds showed weak to no activity against the cell lines under investigation.
Supplementary Materials: The supplementary materials are available online.