An Anti-Inflammatory 2,4-Cyclized-3,4-Secospongian Diterpenoid and Furanoterpene-Related Metabolites of a Marine Sponge Spongia sp. from the Red Sea

Chemical investigation of a Red Sea Spongia sp. led to the isolation of four new compounds, i.e., 17-dehydroxysponalactone (1), a carboxylic acid, spongiafuranic acid A (2), one hydroxamic acid, spongiafuranohydroxamic acid A (3), and a furanyl trinorsesterpenoid 16-epi-irciformonin G (4), along with three known metabolites (−)-sponalisolide B (5), 18-nor- 3,17-dihydroxy-spongia-3,13(16),14-trien-2-one (6), and cholesta-7-ene-3β,5α-diol-6-one (7). The biosynthetic pathway for the molecular skeleton of 1 and related compounds was postulated for the first time. Anti-inflammatory activity of these metabolites to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophil cells and cytotoxicity of these compounds toward three cancer cell lines and one human dermal fibroblast cell line were assayed. Compound 1 was found to significantly reduce the superoxide anion generation and elastase release at a concentration of 10 μM, and compound 5 was also found to display strong inhibitory activity against superoxide anion generation at the same concentration. Due to the noncytotoxic activity and the potent inhibitory effect toward the superoxide anion generation and elastase release, 1 and 5 can be considered to be promising anti-inflammatory agents.


Results and Discussion
Compound 1 was obtained as a white powder. Its molecular formula C 20 H 26 O 5 was established by the molecular ion peak at m/z 369.1672 [M + Na] + in the HRESIMS, consistent with eight degrees of unsaturation. The IR spectrum showed absorptions of hydroxyl (3455 and 3401 cm −1 ) and lactone carbonyl (1752 cm −1 ) functionalities. The 13 C

Results and Discussion
Compound 1 was obtained as a white powder. Its molecular formula C 20 H 26 O 5 was established by the molecular ion peak at m/z 369.1672 [M + Na] + in the HRESIMS, consistent with eight degrees of unsaturation. The IR spectrum showed absorptions of hydroxyl (3455 and 3401 cm −1 ) and lactone carbonyl (1752 cm −1 ) functionalities. The 13 C NMR spectroscopic data of 1 exhibited 20 carbon signals (Table 1), which were assigned by the assistance of DEPT spectrum showing thirteen carbon signals of a diterpene, including three ring-juncture methyls (δ C 26.9, 22.6, and 14.0; δ H 1.24, 1.14, and 0.84) and a 3,4disubstituted furan ring (δ C 137.1, CH; 134.8, CH; 136.8, C; and 119.6, C and δ H 7.06, 1H, br s and 7.09, 1H, br s) [30,31,35,44]. On the basis of the number of unsaturations, 1 was, thus, suggested to be a pentacyclic 3,4-disubstituted furan diterpenoid. The NMR spectroscopic data of 1 and 2D NMR corraltions ( Figure 2) were similar to those of the previously described sponalactone (8) [30], except that a hydroxymethyl in 8 was replaced by a methyl at C-8 in 1. Compound 1 also possesses the same B, C, and D rings as 9 [32] (Scheme 1). The relative and absolute configurations of 1 were established on the basis of nuclear Overhauser effect (NOE) correlation analysis ( Figure 3) and by comparison of the observed NOE correlations with those of the related compounds [30,31], the observed pyridineinduced solvent shifts [45], and biogenetic consideration. The NOESY spectrum of 1 showed NOE correlations of H 3 -17/H 3 -20 and H-5/H-9, depicting the 5R*,8R*,9S*,10R*configuration. H-1 displayed NOE interactions with the β-oriented H 3 -20 and H-11α (δ H 1.68, m), indicating the α-orientation of the H-1. Furthermore, the NOE correlations of H-5/H 3 -18, H 3 -18/H-19α (δ H 3.92) and H-19β (δ H 4.37)/H 3 -20 disclosed the α-and β-orientations of H 3 -18 and the γ-lactone ring, respectively, and the α-orientation of the hydroxyl at C-2, accordingly. The analysis of the pyridine-induced deshielding effect of the axial hydroxy groups was also employed to support the configuration of 1. Therefore, the significant pyridine-induced downfield shifts (∆δ = δ CDCl3 − δ C6D5N ) exerted on H-5 (∆δ H = −0.24 ppm) could only be approached when 1-OH was axially oriented on the same α-face of the molecule. Also, H 3 -18 exhibited pyridine-induced downfield shift (∆δ H = −0.14 ppm) due to the vicinal effect of 2-OH, which should be syn to H 3 -18 [45]. On the basis of the above findings, we propose that 1 can be derived from an intermediate spongian 9, which was biosynthesized from the mevalonic acid pathway, after oxygenation of the six-membered ring A and a subsequent ring contraction and formation of a fivemembered carbocycle, as illustrated in Scheme 1. The relative and absolute configurations of 1 were established on the basis of nuclear Overhauser effect (NOE) correlation analysis ( Figure 3) and by comparison of the observed NOE correlations with those of the related compounds [30,31], the observed   (Figure 2) correlations. The methylene protons H2-6 (δH 2.25, dt, J = 7.6, 7.2 Hz, 2H) was found to be further correlated with the olefinic proton (δH 5.22, dd, J = 7.2, 7.2 Hz, H-7) in 2. The detailed analysis of HMBC correlations ( Figure 2) resolved the carbon positions of the furan ring, olefinic double bond, and the carboxyl group to be at C-1-C-4, C-7/C-8, and C-11, respectively. Furthermore, the methyl group was positioned at C-8. The furanyl H-2 (δH 6.27, s), H-4 (δH 7.20, s), and the olefinic proton H-7 (δH 5.22, dd, J = 7.2, 7.2 Hz, 2H) displayed HMBC correlations with the sp 3 carbon C-5 (δC 24.8, CH2), and H3-12 (δH 1.61, s) showed HMBC correlations with C-7 (δC 124.7, CH) and C-9 (δC 34.2, CH2), while the signal of H2-9 (δH 2.32, dd, J = 7.6, 7.6 Hz, 2H) was found to be correlated with the carboxyl carbon (C-11, δC 180.0). Moreover, the NOE correlations observed for H 3 -12 with H 2 -6 but not with H-5 and the chemical shift of C-12 (δC < 20 ppm) assigned the E-configuration of the 7,8-double bond [47]. Therefore, 2 was determined to be a furanotrinorsesquiterpenoid carboxylic acid with the structure of (E)-7-(furan-3-yl)-4methylhept-4-enoic acid. The literature search showed that this compound had been prepared as a synthetic intermediate during the total syntheses of the furanosesquiterpenoids and dendrolasins [42,48], however, its NMR data had not been reported. Therefore, this is the first report of 2 as a natural product, with the NMR data assigned and reported for the first time. the α-and β-orientations of H 3 -18 and the γ-lactone ring, respectively, and the α-orientation of the hydroxyl at C-2, accordingly. The analysis of the pyridine-induced deshielding effect of the axial hydroxy groups was also employed to support the configuration of 1. Therefore, the significant pyridine-induced downfield shifts (∆δ = δCDCl 3 − δC 6 D 5 N) exerted on H-5 (∆δH = −0.24 ppm) could only be approached when 1-OH was axially oriented on the same α-face of the molecule. Also, H 3 -18 exhibited pyridine-induced downfield shift (∆δH = −0.14 ppm) due to the vicinal effect of 2-OH, which should be syn to H 3 -18 [45]. On the basis of the above findings, we propose that 1 can be derived from an intermediate spongian 9, which was biosynthesized from the mevalonic acid pathway, after oxygenation of the six-membered ring A and a subsequent ring contraction and formation of a five-membered carbocycle, as illustrated in Scheme 1.  Metabolite 2 was isolated as a colorless oil. Its molecular formula was determined to be C 12 H 16 O 3 from the HREIMS (m/z 231.0992 [M + Na] + ), indicating the four degrees of unsaturation. The IR spectrum displayed the absorptions of carboxylic acid (3105-2857 and 1708 cm -1 ) and olefin (1654 cm −1 ). The NMR data ( methyl (δ C 15.9; δ H 1.61, 3H, s) and a carbonyl group (δ C 180.0, C). Other 1 H NMR signals in the shielded region (δ H 2.25-2.47, 8H) were attributable to four methylene groups, as depicted from the COSY (Figure 2) correlations. The methylene protons H 2 -6 (δ H 2.25, dt, J = 7.6, 7.2 Hz, 2H) was found to be further correlated with the olefinic proton (δ H 5.22, dd, J = 7.2, 7.2 Hz, H-7) in 2. The detailed analysis of HMBC correlations (Figure 2) resolved the carbon positions of the furan ring, olefinic double bond, and the carboxyl group to be at C-1-C-4, C-7/C-8, and C-11, respectively. Furthermore, the methyl group was positioned at C-8. The furanyl H-2 (δ H 6.27, s), H-4 (δ H 7.20, s), and the olefinic proton H-7 (δ H 5.22, dd, J = 7.2, 7.2 Hz, 2H) displayed HMBC correlations with the sp 3 carbon C-5 (δ C 24.8, CH 2 ), and H 3 -12 (δ H 1.61, s) showed HMBC correlations with C-7 (δ C 124.7, CH) and C-9 (δ C 34.2, CH 2 ), while the signal of H 2 -9 (δ H 2.32, dd, J = 7.6, 7.6 Hz, 2H) was found to be correlated with the carboxyl carbon (C-11, δ C 180.0). Moreover, the NOE correlations observed for H 3 -12 with H 2 -6 but not with H-5 and the chemical shift of C-12 (δ C < 20 ppm) assigned the E-configuration of the 7,8-double bond [46]. Therefore, 2 was determined to be a furanotrinorsesquiterpenoid carboxylic acid with the structure of (E)-7-(furan-3-yl)-4-methylhept-4-enoic acid. The literature search showed that this compound had been prepared as a synthetic intermediate during the total syntheses of the furanosesquiterpenoids and dendrolasins [42,47], however, its NMR data had not been reported. Therefore, this is the first report of 2 as a natural product, with the NMR data assigned and reported for the first time. Metabolite 3 exhibited almost the same NMR data as those of 2 ( Table 2) from C-1 to C-6, with the carbon chemical shifts of the trisubstituted double bond (δ C 139.7, C and 115.5, CH; δ H 5.34, dd, J = 6.0, 6.0 Hz, 1H) and the carbonyl group (δ C 176.1, C) in 3 showing significant differences of ∆δ C −6.0, +9.2, and −3.9 ppm as compared with those of the corresponding carbons in 2, respectively. As illustrated by 1 H-1 H COSY correlations (Figure 2), the double bond has been isomerized from the C-7/C-8 position in 2 to the C-8/C-9 position in 3. However, the IR spectrum displayed the absorptions of the hydroxyl and NH groups (3407-2858 cm -1 ), carbonyl group (1705 cm -1 ), and olefin (1634 cm -1 ) functionalities. Furthermore, the HREIMS m/z 246.1098 [M + Na] + established the molecular formula of 3 to be C 12 H 17 NO 3 and the chemical shift of the carbonyl group (176.1 ppm), showing that a hydroxamic acid moiety [48][49][50][51] replaced a carboxylic acid group at C-11 in 3. The IR absorptions 3432, 1769, and 1647 cm -1 revealed the presence of hydroxyl, carbonyl, and olefin functionalities, respectively. Moreover, it was found that the NMR data of 4 was the same as those of irciformonin G (10) [52] in all aspects except for those at positions 17 and 18−20 (Table 4), proposing 4 as an isomer of 10. By using Mosher's method [53,54], the 15R absolute configuration in 4 was established based on the calculated ∆δ H (δ S − δ R ) values of protons neighboring C-15 of (S)-and (R)-α-methoxy-α-(trifluoromethyl)-phenylacetyl (MTPA) esters 4a and 4b, respectively (Figure 4). After the assignment of the 15R configuration, the 13 C NMR data of C-15 to C-20 of 4 were further compared with the corresponding data of irciformonin G (10), (+)-sponalisolide A (11), and 8-epi-(+)sponalisolide A (12) [42] of known absolute configurations (Table 4 and Figure 5). The 15R,16R-configuration of 4 was, thus, confirmed as those of the 7R, 8R configured 12, while 10 and 11 possessed the same configurations (R,S) at the corresponding asymmetric carbons. From the above findings, compound 4 was, thus, identified as 16-epi-irciformonin G.          (Table 3), in particular two-dimensional (2D) NMR correlations, the structure of 5 was established to be identical to that of the known (-)-sponalisolide B [42]. However, the coupling constants and spin-spin splitting patterns of the proton H 2 -6 (δ H 2.25, dt, 2H, J = 7.5, 7.0 Hz at 500 MHz in CDCl 3 ) were wrongly assigned. We, herein, reanalyzed the spectrum and provided the correct NMR data for 5.
With the aim of discovering bioactive compounds from these isolates, the cytotoxic activities of the isolated compounds 1−7 against the proliferation of three cancer cell lines including murine leukemia (P388), human bile duct carcinoma (HuCCT), and human colon adenocarcinoma (DLD-1), and a human dermal fibroblast cell line (CCD-966SK) were evaluated, using the Alamar Blue assay [55,56]. The results indicated that none of the tested metabolites exhibited cytotoxic activity (IC 50 > 20 µg/mL).
The anti-inflammatory activities of compounds 1−7 on inhibition of superoxide anion (O 2 − ) generation and elastase release in the fMLF/CB-stimulated human neutrophils [57][58][59] were also evaluated. The results ( 10 µM, with the IC 50 values of 3.37 ± 0.21 and 4.07 ± 0.60 µM, respectively. Compound 5 was also found to display significant inhibitory activity against the superoxide anion generation (IC 50 = 5.31 ± 1.52 µM), and the percentage of inhibition was 67.12 ± 6.00% at 10 µM. Due to the noncytotoxic character and the potent activity toward the superoxide anion generation and elastase release, 1 and 5 can be considered to be the promising anti-inflammatory agents. Percentage of inhibition (Inh %) at 10 µM. Results are presented as mean ± SEM (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the control (DMSO). a Concentration necessary for 50% inhibition (IC 50 ). b The compound is not considered to be anti-inflammatory when IC 50 value is >10 µM. c A phosphatidylinositol-3-kinase inhibitor was used as a positive control.

General Procedures
Measurements of optical rotations and IR spectra were carried out on a JASCO P-1020 polarimeter and FT/IR-4100 infrared spectrophotometer (JASCO Corporation, Tokyo, Japan), respectively. ESIMS and HRESIMS were performed on a Bruker APEX II (Bruker, Bremen, Germany) mass spectrometer. The NMR spectra were recorded on a Varian 400MR FT-NMR at 400 and 100 MHz for 1 H and 13 C, respectively or a Varian Unity INOVA500 FT-NMR at 500 and 125 MHz for 1 H and 13 C, respectively (Varian Inc., Palo Alto, CA, USA). Silica gel or reversed-phase (RP-18, 230-400 mesh) silica gel was used for column chromatography and analytical thin-layer chromatography (TLC) analysis (Kieselgel 60 F-254, 0.2 mm, Merck, Darmstadt, Germany), respectively. Isolation and purification of compounds by high-performance liquid chromatography (HPLC) were achieved using an Hitachi L-2455 HPLC apparatus (Hitachi, Tokyo, Japan) equipped with a Supelco C18 column (250 × 21.2 mm, 5 µm, Supelco, Bellefonte, PA, USA).

Animal Material
The sponge Spongia sp. was collected during March 2016, off the Red Sea Coast at Jeddah, Saudi Arabia (21 o 22 11.08 N, 39 o 06 56.62 E). A voucher sample (RSS-1) has been deposited at the Department of Pharmacognosy, College of Pharmacy, King Saud University, Saudi Arabia.

Statistical Analysis
Data are displayed as the mean ± SEM and comparisons were performed by one-way ANOVA with Dunnett analysis. All results were obtained from more than 3 biological replicates. A p value of 0.05 or less was considered to be significant. The software Prism (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis.

Conclusions
The chemical investigation of dichloromethane-soluble fraction of the organic extract of a Red Sea sponge Spongia sp. resulted in the isolation and identification of a rare A-ring contracted secospongian diterpenoid 17-dehydroxysponalactone (1) and three new furanonorterpenoids 2-4. Compound 1 was found to be noncytotoxic but was shown to exhibit potent inhibitory activity against the superoxide anion generation and elastase release in the fMLF/CB-induced neutrophils, and 5 was also found to display strong inhibitory activity against the superoxide anion generation. Therefore, 1 and 5 are the promising candidates for further development of anti-inflammatory agents.