New 3,4-seco-3,19-Dinor- and Spongian-Based Diterpenoid Lactones from the Marine Sponge Spongia sp.

Continuing chemical investigation of the Red Sea sponge Spongia sp. led to the isolation of four new 3,4-seco-3,19-dinorspongian diterpenoid lactones, secodinorspongins A−D (1−4), along with a classical spongian diterpenoid lactone, sponginolide (5). The chemical structures, including the absolute configurations of these compounds, were elucidated using the extensive spectroscopic study composed of 1D and 2D NMR data analyses, and a comparison between calculated-electronic-circular-dichroism (ECD) and experimental-circular-dichroism (CD) spectra. A plausible biosynthetic pathway of 1−4 was also proposed. Furthermore, the cytotoxicity, antibacterial and anti-inflammatory activities of 1−5 were evaluated. Compound 1 was found to exhibit inhibitory activity against the growth of Staphylococcus aureus (S. aureus), and 4 and 5 exhibited suppression of superoxide-anion generation and elastase release in fMLF/CB-induced human neutrophils.


Introduction
Spongian diterpenoids and structurally related metabolites are a series of natural products with unique structures distributed in sponges of the genus Spongia, and the structural diversity and potential biological significance of these compounds are well known [1][2][3][4][5][6][7][8][9][10][11]. Recently, we have reported the discovery of new spongian diterpenes, including four rare 5,5,6,6,5-pentacyclic diterpenoids, from a Red Sea sponge, Spongia sp. [12,13]. Some of these compounds have been shown to display antibacterial and anti-inflammatory activities. In including four rare 5,5,6,6,5-pentacyclic diterpenoids, from a Red Sea sponge, Spongia sp. [12,13]. Some of these compounds have been shown to display antibacterial and anti-inflammatory activities. In our continuing study on the chemical constituents from this sponge, we further discovered four new 3,4-seco-3,19-dinorspongian diterpenoid lactones, secodinorspongins A−D (1−4), and one classical spongian diterpenoid lactone, sponginolide (5) (Figure 1). Compounds with the same carbon skeleton of 1−4 have been found only two times so far [1,2], and were discovered in Red Sea sponges for the first time. The relative structures and absolute configurations of these compounds were established by the analyses of MS, UV, IR, NMR, and CD spectra (Supplementary Figures S1-S51 and Figures 1−4). Furthermore, the cytotoxicity, antibacterial and anti-inflammatory activities of 1−5 were assayed. Herein, we report the isolation, structural elucidation, and bioactivity evaluation of these compounds.
The NMR data of compound 3 are also similar to those of 1 (Table 1); however, notable differences were observed for NMR chemical shifts of CH2-7, CH2-12, and CH3-17. A comparison of their HMBC correlations also showed distinct differences in the correlation of H2-12. These protons correlated with the ester carbonyl carbon in 1 (Figure 2), whereas the same protons correlated with the oxygenated methylene carbon in 3 ( Figure  2). Furthermore, the NOE correlations of H3-17 with H-7β and H3-20, and of H-5 with H-7α and H-9 suggested a 5R*,8R*,9R*,10R* relative configuration for 3 ( Figure 3).
A high similarity in the 13 C and 1 H NMR data between metabolites 3 and 4 (Table 1)
The 13 C and 1 H NMR data of compound 2 are very similar to those of 1 (  (Figures 2 and 3).
The NMR data of compound 3 are also similar to those of 1 (Table 1); however, notable differences were observed for NMR chemical shifts of CH 2 -7, CH 2 -12, and CH 3 -17. A comparison of their HMBC correlations also showed distinct differences in the correlation of H 2 -12. These protons correlated with the ester carbonyl carbon in 1 (Figure 2), whereas the same protons correlated with the oxygenated methylene carbon in 3 ( Figure 2). Furthermore, the NOE correlations of H 3 -17 with H-7β and H 3 -20, and of H-5 with H-7α and H-9 suggested a 5R*,8R*,9R*,10R* relative configuration for 3 ( Figure 3).
A high similarity in the 13 C and 1 H NMR data between metabolites 3 and 4 (Table 1) was observed, with the exception being that the 16-oxymethylene group (δ H 4.67 and 4.61, each 1H, d, J = 17.5 Hz; δ C 71.3, CH 2 ) in γ-lactone of 3 was converted to an acetal (δ H 5.88, 1H, d, J = 4.0 Hz; δ C 97.5, CH) in 4. The detailed analyses observed by 2D NMR experiments confirmed that 4 is a 16-hydroxylated derivative of 3 ( Figure 2). However, due to the lack of NOE interaction of H-16 with other protons in 4 (Figure 3), the configuration of C-16 was determined by comparison of the calculated ECD with experimental CD spectra (Figure 4c). The experimental CD curve of 4 was found to be quite similar to 4a (5R,8R,9R,10R and 16R) rather than 4b (5S,8S,9S,10S and 16S) or 4c (5R,8R,9R,10R and 16S) (Figure 4c). On the basis of the above results, the structure of 4 was determined, and named secodinorspongin D.
It is suggested that compounds 1-4 share the same biogenetic origin, as they are obtained from the same organism. Consequently, we propose that 1-4 might be biosynthesized from the spongian diterpene (i.e., 6, Scheme 1). The initial oxidation at C-3 and C-19 and the subsequent decarboxylation resulting in the loss of C-19 and dehydration, generate a methyl cyclohexene moiety. Further oxidative cleavage on the double bond of the cyclohexene ring and oxidation at C-2 give intermediate 7 (Scheme 1). With the loss of CO 2 from the β-keto acid moiety and the subsequent oxidation at the relevant position, diterpenes 1-4 may be generated, as illustrated in Scheme 1.

Animal Material
The sponges Spongia sp. were collected from the coast of the Red Sea

Cytotoxicity Assay
The human hepatocellular carcinoma (HCC) Huh7 cells were used in the resazurin assay (Cayman Chemical) to evaluate the cytotoxicity of compounds (Supplementary Table S13). The method was performed as described previously [18,19], and the detailed process of the cytotoxicity assay was the same as in our previous publications [12,25]. Sorafenib, the positive control, inhibited 52% of the growth of Huh7 cells at 12.5 µM, and the DMSO controls were assigned 100% of relative cell-viability.

Antibacterial Assay
The antibacterial assay was processed using the previously reported methods [26]. The culture and dilution of S. aureus were performed as previously described [12,25]. The bacteria aliquots were plated (100 µL/well of 96-well plate) with the tested compounds (cpd) at concentrations of 50 µM, 100 µM, and 200 µM. A total of 1% DMSO in LB solution (background control), 1% DMSO in the diluted bacteria solution (positive control), and 0.5 µg/mL tetracycline (known-drug control) were plated on the same plate. After incubation at 37 • C for 16 h, the plate was measured by the absorbance at 600 nm (A), and then the percentage of S. aureus growth was measured using the following equation: [(Acpd-Acpd_basal) − Abackground control]/[(Apositive control − Apositive control_basal) − Abackground control] × 100.

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

Inhibition of Superoxide Anion Generation
The method was performed as described previously [20,21], and the detailed process of incubating and treating neutrophils was the same as in our previous publications [12,25]. After cytochalasin B (CB, 1 µg/mL) for 3 min, the 100 nM fMLF for 10 min (fMLF/CB) was used to activate neutrophils, and the positive control LY294002 [2-(4-morpholinyl)-8phenyl-1(4H)-benzopyran-4-one] was used. The wavelength 550 nm (spectrophotometer U-3010, Hitachi, Tokyo, Japan) was used to measure the generation of the superoxide anion.

Inhibition of Elastase Release
The method was performed as described previously [20,21], and the detailed process of incubating and treating neutrophils was the same as in our previous publications [12,25]. The fMLF (100 nM)/CB (0.5 µg/mL) for 10 min, was used to activate neutrophils, and the wavelength 405 nm (spectrophotometer U-3010, Hitachi, Tokyo, Japan) was used to measure the generation of elastase release.

Conclusions
The new dinorditerpenoid lactones 1−4 have the rare 3,4-seco-3,19-dinorspongian structure. In the previous study, compounds of this skeleton [1,2] did not show cytotoxicity, and the same situation was also found for metabolites 1−4. However, the current study revealed that compound 1 possessed notable inhibition against the growth of S. aureus, while 4 and 5 exhibited in vitro anti-inflammatory potential through the inhibitory activity against the generation of the superoxide anion and elastase release in fMLF/CBinduced human neutrophils. In prior studies, most of the spongian diterpenoids were not found to exhibit conspicuous biological activities [1,2,4,6,12,13]; however, few analogs with 3,19-dihydroxyl-2-one fragment in the A-ring and/or furano D-ring [2][3][4][8][9][10][11]13,27] were reported to have potent cytotoxicity, anti-inflammatory, and anti-viral activities. In this study, compounds 1−5, which lack the aforementioned functionality, could be responsible for the deficiency of cytotoxic and anti-inflammatory activities.