The Chemically Highly Diversified Metabolites from the Red Sea Marine Sponge Spongia sp.

A polyoxygenated and halogenated labdane, spongianol (1); a polyoxygenated steroid, 3β,5α,9α-trihydroxy-24S-ethylcholest-7-en-6-one (2); a rare seven-membered lactone B ring, (22E,24S)-ergosta-7,22-dien-3β,5α-diol-6,5-olide (3); and an α,β-unsaturated fatty acid, (Z)-3-methyl-9-oxodec-2-enoic acid (4) as well as five known compounds, 10-hydroxykahukuene B (5), pacifenol (6), dysidamide (7), 7,7,7-trichloro-3-hydroxy-2,2,6-trimethyl-4-(4,4,4-trichloro-3-methyl-1-oxobu-tylamino)-heptanoic acid methyl ester (8), and the primary metabolite 2’-deoxynucleoside thymidine (9), have been isolated from the Red Sea sponge Spongia sp. The stereoisomer of 3 was discovered in Ganoderma resinaceum, and metabolites 5 and 6, isolated previously from red algae, were characterized unprecedentedly in the sponge. Compounds 7 and 8 have not been found before in the genus Spongia. Compounds 1–9 were also assayed for cytotoxicity as well as antibacterial and anti-inflammatory activities.

Metabolite 4 was isolated as a colorless oil. Its molecular formula was determined to be C 11 H 18 O 3 from the HRESIMS (m/z 221.1149 [M + Na] + ), indicating three degrees of unsaturation. The IR spectrum displayed the absorptions of the hydroxyl, ketone, and carboxylic acid groups (3445, 1700, and 1683 cm -1 , respectively). The NMR data (Table 3) showed the presence of two methyls (δ C 29.9 and 25.4; δ H 2.14 and 1.91, each 3H, s); five methylenes; one alkene methane (δ C 115.3 and δ H 5.69, br s); and three quaternary carbons (included one ketone carbon δ C 209.4, one carbonyl carbon δ C 169.5, and one alkene quaternary carbon δ C 163.5). The detailed analysis of 1 H-1 H COSY and HMBC correlations  Figure 10) assigned the positions of a carboxylic acid group, an olefinic double bond, and ketone functionalities to be at C-1, C-2, and C-9, respectively. Moreover, the NOE correlation observed for H-2 (δ H 5.69) with H 3 -11 (δ H 1.91) in 4 suggested the Z geometry of this double bond and consequently established the structure of 4 to be (Z)-3-methyl-9-oxodec-2-enoic acid.   10-Hydroxykahukuene B (5) [23] is a brominated diterpene with a rare prenylated chamigrane skeleton. To the best of our knowledge, two examples of this skeleton have been reported in the marine red alga Laurencia sp. [23,39,40], and 5 represent the first example of a metabolite with a prenylated chamigrane skeleton that has been isolated from the sponge. Pacifenol (6), the first trihalogenated compound with a chamigrane skeleton, was isolated by Sims and associates from the Californian red alga Laurencia pacifica [41]. After that, pacifenol was also isolated from other marine red algae, including L. caduciramulosa [42] and L. marianensis [24], among others. Mollusks of the genus Aplysia are known to be animals that do not biosynthesize the halogenated sesquiterpenes by themselves but obtain and accumulate these metabolites by ingesting alga and, in some cases, transform the alga metabolites into other compounds in the digestive gland [43,44]. Our present study is the first report to discover pacifenol in the sponge. Metabolites 7 and 8 have also been isolated from the Red Sea sponges Dysidea herbacea [45] and Lamellodysidea herbacea [25], but they were discovered for the first time in sponges of the genus Spongia in the present study.
It was previously known that sponges could take in and accumulate organohalides from environmental seawater and that these compounds might be transformed into chemical defense substances [46]. Macroalgae are important primary producers in coral reefs, and many species inhabit areas near sponges [47,48]. Algae synthesize secondary metabolites for competition and survival [49][50][51], and the red alga Laurencia sp. is known for producing diverse halides, many of which have been shown to have antibacterial activity [52,53]. The sponges could inhale these halides or even transform them chemically into compounds such as 1, 5, and 6 for their own use [46].
10-Hydroxykahukuene B (5) [23] is a brominated diterpene with a rare prenylated chamigrane skeleton. To the best of our knowledge, two examples of this skeleton have been reported in the marine red alga Laurencia sp. [23,39,40], and 5 represent the first example of a metabolite with a prenylated chamigrane skeleton that has been isolated from the sponge. Pacifenol (6), the first trihalogenated compound with a chamigrane skeleton, was isolated by Sims and associates from the Californian red alga Laurencia pacifica [41]. After that, pacifenol was also isolated from other marine red algae, including L. caduciramulosa [42] and L. marianensis [24], among others. Mollusks of the genus Aplysia are known to be animals that do not biosynthesize the halogenated sesquiterpenes by themselves but obtain and accumulate these metabolites by ingesting alga and, in some cases, transform the alga metabolites into other compounds in the digestive gland [43,44]. Our present study is the first report to discover pacifenol in the sponge. Metabolites 7 and 8 have also been isolated from the Red Sea sponges Dysidea herbacea [45] and Lamellodysidea herbacea [25], but they were discovered for the first time in sponges of the genus Spongia in the present study.
It was previously known that sponges could take in and accumulate organohalides from environmental seawater and that these compounds might be transformed into chemical defense substances [46]. Macroalgae are important primary producers in coral reefs, and many species inhabit areas near sponges [47,48]. Algae synthesize secondary metabolites for competition and survival [49][50][51], and the red alga Laurencia sp. is known for producing diverse halides, many of which have been shown to have antibacterial activity [52,53]. The sponges could inhale these halides or even transform them chemically into compounds such as 1, 5, and 6 for their own use [46].
Compounds 1-9 were tested for cytotoxicity using a resazurin assay in the HCC Huh7 cell line. Among them, compounds 5 and 8 showed weak cytotoxicity against the Huh7 cell line, with 17% and 32% inhibition toward the proliferation of Huh7 cells at 50 µM, respectively. Furthermore, 5 and 8 could inhibit the 43% and 53% proliferation of Huh7 cells at 200 µM, respectively. The growth inhibition assay of Staphylococcus aureus (S. aureus) was subsequently applied for compounds 1-9. The results showed that 9 displayed 31%, 37%, and 89% inhibition on the growth of S. aureus at 50, 100, and 200 µM, respectively.

General Experimental Procedures
Measurements of circular dichroisms, optical rotations, and IR spectra were carried out on a Jasco J-715 CD spectrometer, JASCO P-1020 polarimeter, and FT/IR-4100 infrared spectrophotometer (JASCO Corporation, Tokyo, Japan), respectively. ESIMS was performed on a Bruker APEX II (Bruker, Bremen, Germany) mass spectrometer, and HRESIMS was performed on a Bruker APEX II and Impact HD Q-TOF mass spectrometers (Bruker, Bremen, Germany). The NMR spectra were recorded on a Varian 400MR FT-NMR at 400 and 100 MHz for 1 H and 13 C, respectively; a Varian Unity INOVA500 FT-NMR (both Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz for 1 H and 13 C, respectively; or a JEOL ECZ600R FT-NMR (Japan) at 600 and 150 MHz for 1 H and 13 C, respectively. Silica gel and reversed-phase (RP-18, 230-400 mesh) silica gel were used for column chromatography and analytical thin-layer chromatography (TLC) analysis (Kieselgel 60 F-254, 0.2 mm, Merck, Darmstadt, Germany), respectively. The isolation and purification of compounds by highperformance liquid chromatography (HPLC) were achieved using a 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 in March 2016 off the Red Sea coast of Jeddah, Saudi Arabia (21 • 22 11.08 N, 39 • 06 56.62 E). A voucher sample (RSS-1) was deposited at the Department of Pharmacognosy, College of Pharmacy, King Saud University, Saudi Arabia.
CAM-B3LYP/6-311+G(d,p) level of theory. The range-separated functional CAM-B3LYP is recommended for ECD calculations [27]. The bulk solvent effect of methanol was taken into account with the integral equation formalism polarizable continuum model (IEFPCM solvent model for MeOH). All calculations were performed by the Gaussian 09 program [28]. The calculated ECD curves were converted using GaussSum 2.2.5 and illustrated using Microsoft Excel.

Cytotoxicity Assay
The cytotoxicity assay was performed using the methods described in a previous paper [57,58]. Huh7 cells were cultured in a 96-well plate containing 100 µL of culture medium in triplicate and treated with indicated concentrations of compounds for 72 h. At the assay time point, resazurin (Cayman Chemical) was added and incubated for 4 h at 37 • C. The DMSO wells was defined as the control and assigned a relative cell viability of 100%. Sorafenib, the positive control, inhibited the 52% proliferation of Huh7 cells at 12.5 µM.

Antibacterial Assay
The antibacterial assay was performed using the methods described in a previous paper [59]. S. aureus was cultured in Lysogeny broth (LB) in a shaker-incubator at 37 • C for 24 h. The cultures were then diluted to an absorbance at 600 nm of 0.04 using sterile LB. The diluted bacteria aliquots were placed (100 µL per well) into 96-well flat-bottom plates. Tested compounds (cpd) were then added to the final concentration at 50 µM, 100 µM, and 200 µM, respectively. Background controls (1% DMSO in LB solution), positive controls (1% DMSO in the diluted bacteria solution), and known drug controls (tetracyclin; inhibited the 99% growth of bacteria at 50 µM) 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
Human neutrophils were isolated from the blood of healthy adult volunteers and enriched by using dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic lysis, as described previously [56]. Then, neutrophils were incubated in Ca 2+ -free HBSS buffer (pH 7.4, ice-cold).

Conclusions
New metabolites (1-4) along with five known compounds (5-9) were isolated from a Red Sea sponge, Spongia sp. Compounds 5 and 8 showed weak cytotoxicity to HCC Huh7 cells, while 9 displayed significant inhibition against S. aureus. Furthermore, compounds 7 and 8 exhibited notable activity to inhibit elastase release and weaker inhibitory activity toward superoxide anion generation. Both compounds 6 and 9 also showed inhibition against elastase release. Although compound 5 was found to be inactive in the present study, its antibacterial activity against S. aureus and E. coli has been reported [23]. It is noteworthy that some of the isolates from this sponge were also found in red algae, which suggests that the specific metabolites of sponges could have originated from alga and would be accumulated and/or transformed into metabolites in sponges.