A New Bioactive Metabolite Isolated from the Red Sea Marine Sponge Hyrtios erectus

Chemical investigation of the lipophilic fraction of Hyrtios erectus, a Red Sea sponge, yielded a new pentacyclic nitrogen-containing scalarane; 24-methoxypetrosaspongia C (1), together with the previously reported scalaranes sesterstatin 3 (2), 12-deacetyl-12-epi-scalaradial (3) and 12-deacetyl-12,18-di-epi-scalaradial (4). The compounds were identified using HRESIMS, 1D and 2D NMR experiments. The isolated compounds showed growth inhibitory activity against hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116) and breast adenocarcinoma cells (MCF-7).


Structure Elucidation of Compound 1-4
Compound 1 (Figure 1) was isolated and purified as an amorphous solid.The molecular formula C 29 H 45 NO 5 was established from the positive HRESIMS (high-resolution electrospray ionization mass spectrometry) pseudomolecular ion peak at m/z 488.3374 [M + H] + .The 1 H-NMR spectrum of compound 1 (Table 1) displayed resonances for 45 protons, including five singlets belonging to five methyl groups (δ H 0.80, 0.82, 0.84, 0.91 and 1.20), two methoxyls, (δ H 3.20 and 3.39), one acetyl methyl (δ H 2.12) seven methylenes, six aliphatic methines, and an exchangeable broad signal at δ H 5.78 for a NH moiety (Supplementary Materials, Figure S1).The 13 C-NMR spectrum (Table 1) showed signals for 29 carbons, including eight methyls, seven methylenes, six methines , and eight quaternary carbons (Supplementary Materials, Figure S2).Analysis of the 1 H, 1 H-COSY and the HSQC NMR experiments led to the assembly of the following structural fragments: C-1 to C-3; C-5 to C-7; C-9 to C-12 with an acetoxy group at C-12; C-14 to C-16 with methoxy group at C-16 and C-16 to C-24 with methoxy group at C-24.These fragments allowed identifying a 12-acetoxy-16-methoxyscalarane skeleton (Figure 2) based on the correlations of H-12 and H-16 with neighboring protons and carbons in the COSY and HMBC (Supplementary Materials, Figures S3-S5).The C-17/C-18 double bond was inferred by heteronuclear long range correlations between H 3 -23 at δ H 1.20 and the quaternary olefinic carbon at δ C 144.7 (C-18) and between H-16 at δ H 3.85 and the olefinic carbon at δ C 151.1 (C-17).Furthermore, the 13 C chemical shifts of C-17 and C-18 indicated the location of the amide carbonyl at C-25 [26].

Biological Activity of the Isolated Compounds
The growth inhibitory effects of compounds 1-4 (Table 2) against breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2) and colorectal carcinoma cells (HCT-116) were evaluated using a logarithmic best fit equation (Emax model Equation).Compound 4 was the most potent against all tested cell lines with IC50 3.3, 1.7 and 3.4 µM in MCF-7, HepG2, and HCT-116 cell lines, respectively.Both compounds 1 and 3 showed intermediate activities with IC50 55.4, 25.4 and 26.5 µM for compound 1 and 36.0,23.4 and 27.1 µM for compound 3 against MCF-7, HepG2, and HCT-116 cell lines, respectively.On other hand, compound 2 showed no growth inhibitory effects against all tested cell lines.Furthermore, the morphological changes induced by compounds 1, 3 and 4 were carried out against HCT-116 cells using computer-assisted phase-contrast microscopy [31][32][33].In addition, the effect of compounds 1, 3 and 4 on cell membrane integrity was quantified using a lactate dehydrogenase leakage assay (LDH leakage assay).
The results illustrated in Figure 4 reveal that among the three active compounds against HCT-116 cells (Table 2), compound 4 induced apparent morphological changes suggesting a cytotoxic effect (cell killing).These morphological abnormalities attributed to cell exposure to compound 4 were not observed after treatment with the other two compounds.Moreover, LDH leakage assay was used to confirm the ell killing effect of compound 4 against HCT-116 cells.Treatment with compound 4 (10 µM) for 72 h significantly increased LDH leakage from HCT-116 cells by 2-fold compared to the control Compound 1 represents a further example of scalaranes containing nitrogen, a group which which includes petrosaspongiolactams A-C [26], hyatelactam [27], and the pyrrole-terpenes molliorins A [28], molliorins B [29], and molliorins C [30].

Biological Activity of the Isolated Compounds
The growth inhibitory effects of compounds 1-4 (Table 2) against breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2) and colorectal carcinoma cells (HCT-116) were evaluated using a logarithmic best fit equation (E max model Equation).Compound 4 was the most potent against all tested cell lines with IC 50 3.3, 1.7 and 3.4 µM in MCF-7, HepG2, and HCT-116 cell lines, respectively.Both compounds 1 and 3 showed intermediate activities with IC 50 55.4,25.4 and 26.5 µM for compound 1 and 36.0,23.4 and 27.1 µM for compound 3 against MCF-7, HepG2, and HCT-116 cell lines, respectively.On other hand, compound 2 showed no growth inhibitory effects against all tested cell lines.Furthermore, the morphological changes induced by compounds 1, 3 and 4 were carried out against HCT-116 cells using computer-assisted phase-contrast microscopy [31][32][33].In addition, the effect of compounds 1, 3 and 4 on cell membrane integrity was quantified using a lactate dehydrogenase leakage assay (LDH leakage assay).
The results illustrated in Figure 4 reveal that among the three active compounds against HCT-116 cells (Table 2), compound 4 induced apparent morphological changes suggesting a cytotoxic effect (cell killing).These morphological abnormalities attributed to cell exposure to compound 4 were not observed after treatment with the other two compounds.Moreover, LDH leakage assay was used to confirm the ell killing effect of compound 4 against HCT-116 cells.Treatment with compound 4 (10 µM) for 72 h significantly increased LDH leakage from HCT-116 cells by 2-fold compared to the control cells (Figure 5).On the other hand, compounds 1 and 3 did not induce any significant cell membrane damage in HCT-116 cells at 10 µM concentration after 72 h exposure.Thus, compound 4 possesses cytotoxic properties while compounds 1 and 3 possess antiproleferative effects, which could be attributed to cytostatic effects.However, further biochemical and molecular biology-related experiments are currently underway to define the mechanism of action of compounds 1, 3 and 4 as cytostatic or cytotoxic agents.
Molecules 2016, 21, 82 5 of 9 cells (Figure 5).On the other hand, compounds 1 and 3 did not induce any significant cell membrane damage in HCT-116 cells at 10 µM concentration after 72 h exposure.Thus, compound 4 possesses cytotoxic properties while compounds 1 and 3 possess antiproleferative effects, which could be attributed to cytostatic effects.However, further biochemical and molecular biology-related experiments are currently underway to define the mechanism of action of compounds 1, 3 and 4 as cytostatic or cytotoxic agents.

Biological Materials
The marine sponge, Hyrtios erectus (Keller, 1889) (Figure 6) was collected off Sharm el-Sheikh, Red Sea, Egypt, using scuba diving at a depth of 11 m.

Biological Materials
The marine sponge, Hyrtios erectus (Keller, 1889) (Figure 6) was collected off Sharm el-Sheikh, Red Sea, Egypt, using scuba diving at a depth of 11 m.The collected material was immediately frozen and kept at −15 °C until investigation.The sponge was identified by Dr. R. van Soest (Institute of Systematic Population Biology, Amsterdam University, Amsterdam, The Netherlands) to be Hyrtios erectus (class Demospongiae, order Dictyoceratida, family Thorectidae) under the number ZMAPOR19761.A voucher specimen has been deposited in the Red Sea invertebrate's collection at the Faculty of Pharmacy, Suez Canal University, under the registration number SAA-59.

Cell Culture
Breast adenocarcinoma cells (MCF-7), hepatocellular carcinoma cells (HepG2) and colorectal carcinoma cells (HCT-116) were from the National Cancer Institute of Egypt (Giza, Egypt).Cells were maintained in RPMI-1640 supplemented with 100 mg/mL streptomycin, 100 units/mL penicillin and 10% heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO 2 atmosphere at 37 ˝C.Exponentially growing cells were collected using 0.25% Trypsin-EDTA and plated in 96-well plates at 1000-2000 cells/well.Cells were exposed to serial concentrations of test compounds for 72 h and subsequently fixed with TCA (10%) for 1 h at 4 ˝C.After several washings, cells were exposed to 0.4% SRB solution for 10 min in dark place and subsequently washed with 1% glacial acetic acid.After drying overnight, Tris-HCl was used to dissolve the SRB-stained cells and color intensity was measured at 540 nm.Doxorubicin was used as a positive control.The dose response curve of compounds was analyzed using a logarithmic best fit equation (E max model Equation).% Cell viability " p100 ´Rq ˆˆ1 ´rDs m K d m `rDs m ˙`R where (R) is the residual unaffected fraction (the resistance fraction), (D) is the drug concentration used, (K d ) is the drug concentration that produces a 50% reduction of the maximum inhibition rate and m is a Hill-type coefficient.IC 50 was defined as the drug concentration required to reduce absorbance to 50% of that of the control (i.e., K d = IC 50 when R = 0 and E max = 100 ´R) [35].

Cell Membrane Integrity Assessment
The influence of compounds 1, 3 and 4 on cell membrane integrity was assessed in colorectal adenocarcinoma cells (HCT-116) by LDH leakage assay [36,37].Briefly, exponentially growing cells were plated in 24-well plates (1 ˆ10 4 cells/well).Cells were exposed to 10 µM of tested compounds and compared to untreated cells (control) for 72 h.LDH concentrations were determined in each well using a colorimetric assay (Biosystems, Barcelona, Spain).

Conclusions
The investigation of the antiproliferative lipophilic extract of the Red Sea sponge H. erectus yielded the new metabolite 24-methoxypetrosaspongia C (1), along with sesterstatin 3 (2), 12-deacetyl-12-epi-scalaradial (3) and 12-deacetyl-12,18-di-epi-scalaradial (4).The structures of the isolated compounds were determined by HRESIMS, 1D and 2D NMR data, as well as by comparison with the available data in the literature.The compounds displayed variable growth inhibitory activity
(br s) C-17, C-18 a HMBC correlations are from proton(s) stated to the indicated carbons.

Figure 6 .
Figure 6.Underwater photograph of the Red Sea sponge Hyrtios erectus.

Figure 6 .
Figure 6.Underwater photograph of the Red Sea sponge Hyrtios erectus.
a positive cytotoxic control.
a positive cytotoxic control.