Spongenolactones A–C, Bioactive 5,5,6,6,5-Pentacyclic Spongian Diterpenes from the Red Sea Sponge Spongia sp.

Three new 5,5,6,6,5-pentacyclic spongian diterpenes, spongenolactones A–C (1–3), were isolated from a Red Sea sponge Spongia sp. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and the absolute configurations of 1–3 were determined on the basis of comparison of the experimental circular dichroism (CD) and calculated electronic circular dichroism (ECD) spectra. Compounds 1–3 are the first 5,5,6,6,5-pentacyclic spongian diterpenes bearing an β-hydroxy group at C-1. These metabolites were assayed for their cytotoxic, antibacterial, and anti-inflammatory activities. All three compounds were found to exert inhibitory activity against superoxide anion generation in fMLF/CB-stimulated human neutrophils. Furthermore, 1 showed a higher activity against the growth of Staphylococcus aureus in comparison to 2.


Introduction
Sponges of the genus Spongia have been proven to be rich sources of structurally diversified secondary metabolites [1,2]. A series of previous studies for the discovery of versatile molecular structures and bioactivities of compounds from sponges of the genus Spongia have been reported [3][4][5][6][7][8][9][10][11]. Our recent investigation on the secondary metabolites of a Red Sea sponge Spongia sp. has led to the isolation of a series of diverse new natural products, including a 5,5,6,6,5-pentacyclic diterpenoid, two furanotrinorsesquiterpenoid acids, a furanyl trinorsesterpenoid, a halogenated and oxygenated labdane, a highly oxygenated steroid, a steroid with the rare seven-membered lactone B ring, and an α,β-unsaturated fatty acid [12,13]. In our continuing effort to discover new metabolites from this sponge Spongia sp., we have further discovered three new diterpenes with 5,5,6,6,5-pentacyclic structures (5,6,6-tricarbocyclic ones with two five-membered lactones), spongenolactones A-C (1-3) (Figure 1). The molecular structures of 1-3 were established by detailed analysis of MS, IR, and NMR spectra (Supplementary Figures S1-S26), and by comparison of their NMR spectral data with those of structurally related known compounds. Further, the absolute configurations of 1-3 were determined by comparison of the experimental CD and calculated ECD spectra. Moreover, the cytotoxic activity of compounds 1-3 toward human hepatocellular carcinoma (HCC) Huh 7 cell line, their antibacterial activity against the growth of Staphylococcus aureus, and their anti-inflammatory activity toward the inhibition of the superoxide anion generation and elastase release in N-formylmethionyl-leucyl phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils, were also evaluated.

Results and Discussion
The sample of Spongia sp., collected off the Red Sea coast of Jeddah, Saudi Arabia, in 2016, was freeze-dried. The lyophilized sample (550 g) was chopped and extracted with EtOAc/MeOH/CH 2 Cl 2 . The crude extract was partitioned in water with CH 2 Cl 2 to obtain the CH 2 Cl 2 fraction (18.47 g), which was subjected to repeated column chromatography and high performance liquid chromatography (HPLC) to afford compounds 1 (2.4 mg), 2 (2.0 mg), and 3 (3.5 mg) ( Figure 1).

DFT and TD-DFT Calculations
The DFT approach at the B3LYP/6-31G (d,p) level of theory was applied to simulate the preliminary geometry optimization of conformers [16]. Then, the time-dependent DFT (TD-DFT) approach at the CAM-B3LYP/6-311+G(d,p) level of theory was used to simulate ECD spectra [16]. The integral equation formalism polarizable continuum (IEFPCM) solvent model for MeOH was used for the bulk solvent effect, with all programs calculated by the Gaussian 09 program [17]. The final ECD curves were transformed by GaussSum 2.2.5 and illustrated by Microsoft Excel.

Cytotoxicity Assay
The cytotoxicity assay was performed using the methods described previously [18,19]. Huh7 cells were used in resazurin assay (Cayman Chemical) and treated with indicated concentrations (12.5, 50.0, and 200.0 µM) of compounds for 72 h. The DMSO control was assigned 100% of relative cell viability. The positive control, Sorafenib, inhibited the 52% growth of Huh7 cells at 12.5 µM.

Antibacterial Assay
The antibacterial assay was performed using the methods described previously [23]. The bacteria S. aureus were cultured in LB (Lysogeny broth) medium in the shaker incubator at 37 • C for 24 h. The bacterial culture was diluted to an absorbance of 0.04 at 600 nm using a sterile LB medium. Tested compounds (cpd) were then added to bacteria aliquots (100 µL/well of 96-well) with the concentrations at 50 µM, 100 µM, and 200 µM, respectively. Background control (1% DMSO in LB solution), positive control (1% DMSO in the diluted bacteria solution), and known drug control (tetracyclin; concentration is 0.5 µg/mL) were run on the same plate. The absorbance at 600 nm (A) was measured right after the testing compounds were added for the basal absorbance and after 16 h incubation at 37 • C. The percentage bacterial growth was calculated as follows: [(Acpd − Acpd_basal) − Abackground control]/[(Apositive control − Apositive control_basal) − Abackground control] × 100.

Anti-Inflammatory Activity
The dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic lysis were used to enrich the neutrophils which were isolated from the blood of healthy adult volunteers, and these methods were described in a previous paper [22]. The neutrophils were incubated in Ca 2+ -free HBSS buffer (pH 7.4, ice-cold).

Conclusions
Three new 5,5,6,6,5-pentacyclic spongian diterpenes, spongenolactones A-C (1-3), were isolated from a Red Sea sponge, Spongia sp. These metabolites are the first 5,5,6,6,5pentacyclic spongian diterpenes bearing a β-hydroxy group at C-1. In our previous chem-ical study of the same organism, we also discovered a compound of the same skeleton, 17-dehydroxysponalactone, which was found to potently reduce the superoxide anion generation and elastase release [12]. Compounds 1-3 exhibited inhibitory activity against the generation of superoxide anion, and 2 also displayed inhibitory activity against elastase release. Furthermore, 1 showed significant inhibition on the growth of S. aureus.