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

Abstract

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.

1. Introduction

Sponges of the genus Spongia are known to be abundant sources of chemical constituents with diverse structures and bioactivity [1,2]. So far, the 3,4-seco-3,19-dinorditerpenes [3], 5,5,6,6,5-pentacyclic diterpenes [4], furanoterpenes [5,6], spongian diterpenes [7,8], scalarane sesterterpenoids [9,10], sesquiterpene quinones [11,12], diverse terpenes [13,14], sterols [15,16], and macrolides [14,17], along with fatty acids [18,19] and halides [20,21], have been isolated from the genus Spongia sp. Our preliminary studies on the Red Sea sponge Spongia sp. resulted in the isolation of a series of new compounds, including one 5,5,6,6,5-pentacyclic diterpene, two new furanotrinorsesquiterpenoid acids, and a furanyl trinorsesterpenoid [22]. Our continuous investigation of the chemical constituents of this sponge has again afforded four new compounds, including spongianol (1), 3β,5α,9α-trihydroxy-24S-ethylcholest-7-en-6-one (2), (22E,24S)-ergosta-7,22-dien-3β,5α-diol-6,5-olide (3), and (Z)-3-methyl-9-oxodec-2-enoic acid (4) (Figure 1), along with five known metabolites: 10-hydroxykahukuene B (5) [23], pacifenol (6) [24], dysidamide (7) [25], 7,7,7-trichloro-3-hydroxy-2,2,6-trimethyl-4-(4,4,4-trichloro-3-methyl-1-oxobutylamino)-heptanoic acid methyl ester (8) [25], and the primary metabolite 2’-deoxynucleoside thymidine (9) [26]. The molecular structures of 1–9 were established by MS, IR, and detailed NMR spectroscopic analysis (Supplementary Figures S1–S45) and by comparison with the reported spectral data of related known compounds. The cytotoxicity of hepatocellular carcinoma (HCC) Huh7 cells and the anti-inflammatory and antibacterial activity of 1–9 were also evaluated.

2. Results and Discussion

Compound 1 was obtained as a colorless oil. The HRESIMS (Supplementary Figure S1) of 1 established the molecular formula C₂₀H₃₀³⁵Cl₂O₅, implying five degrees of unsaturation. The IR spectrum of 1 revealed the presence of the hydroxyl, carbonyl, and olefin from absorptions at 3420, 1698, and 1646 cm^–1, respectively. The ¹³C NMR spectroscopic data of 1 exhibited 20 carbon signals (

Table 1. ¹³C and ¹H NMR data for compounds 1–3.

1 a			2 b			3 b
#	δH	δC	#	δH	δC	#	δH	δC
1	–	209.2, C c	1	1.52, m	25.1, CH2	1	1.95, m	35.9, CH2
2α	2.64, dd (14.0, 6.0) d	45.1, CH2		2.34, dt (11.0, 3.0)			1.40, m
						2α	1.86, m	30.4, CH2
2β	3.34, t (13.0)		2α	1.93, m	30.1, CH2	2β	1.40, m
3	4.50, dd (13.0, 6.0)	65.9, CH	2β	1.51, m		3	3.79, m	67.6, CH
			3	4.07, m	67.2, CH	4α	2.25, m	46.2. CH2
4	–	45.2, C	4α	2.09, dd (11.5, 1.5)	37.2, CH2	4β	2.10, m
						5	–	104.3, C
5	–	82.1, C	4β	1.77, m		6	–	166.4, C
6α	2.18, tt (15.5, 3.5)	27.7, CH2	5	–	79.7, C	7	5.70, br s	115.3, CH
			6	–	197.7, C	8	–	159.7, C
6β	2.05, tt (15.5, 3.5)		7	5.66, br s	119.9, CH	9	2.26, m	51.7, CH
			8	–	164.3, C	10	–	43.1, C
7	3.67, t (3.5)	75.3, CH	9	–	74.6, C	11	1.82, m	25.3, CH2
8	–	76.2, C	10	–	41.8, CH		1.64, m
9	2.72, br s e	41.7, CH	11	1.76, m	28.8, CH2	12α	2.05, m	39.9, CH2
10	–	58.1, C		1.94, m		12β	1.42, m
11	5.32, t (2.5)	70.9, CH	12α	1.72, m	35.0, CH	13	–	46.6, C
12	4.06, d (2.5)	68.9, CH	12β	1.92, m		14	2.14, m	58.1, CH
13	–	77.4, C	13	–	45.4, C	15α	1.58, m	23.1, CH2
14	6.78, dd (17.5, 11.5)	139.9, CH	14	2.72, dd (9.5, 4.5)	51.7, C	15β	1.50, m
						16	1.77, m	28.0, CH2
15	5.21, d (11.5)	116.7, CH2	15α	1.64, m	22.5, CH2		1.33, m
	5.35, d (17.5)		15β	1.51, m		17	1.37, m	56.3, CH
16	1.49, s	28.7, CH3	16	1.40, m	27.7, CH2	18	0.62, s	12.4, CH3
17	1.66, s	25.9, CH3		2.01, m		19	1.06, s	17.8, CH3
18	1.24, s	24.0, CH3	17	1.39, m	56.0, CH	20	2.05, m	40.4, CH
19	1.21, s	20.1, CH3	18	0.61, s	12.0, CH3	21	1.01, d (6.0)	21.0, CH3
20	1.71, s	18.9, CH3	19	1.02, s	20.5, CH3	22	5.13, dd (15.0, 7.8)	135.2, CH
5-OH	5.72, br s		20	1.40, m	36.4, CH	23	5.21, dd (15.0, 8.4)	132.7, CH
7-OH	3.59, d (2.0)		21	0.94, d (6.0)	18.9, CH3	24	1.85, m	43.1, CH
11-OH	2.25, d (1.5)		22	1.05, m	33.7, CH2	25	1.47, m	33.2, CH
				1.39, m		26	0.82, d (6.6)	19.6, CH3
			23	1.38, m	26.3, CH2	27	0.84, d (7.2)	20.1, CH3
			24	0.93, m	46.0, CH	28	0.92, d (7.2)	18.0, CH3
			25	1.63, m	28.9, CH
			26	0.81, d (6.6)	19.0, CH3
			27	0.84, d (6.6)	19.6, CH3
			28	1.14, m	23.0, CH2
				1.32, m
			29	0.85, t (7.8)	12.3, CH3
			5-OH	3.34 br s	–
			9-OH	4.12 br s	–

^a13C and ¹H spectra recorded at 125 and 500 MHz in CDCl₃; ^{b 13}C and ¹H spectra recorded at 150 and 600 MHz in CDCl₃; ^c deduced from DEPT; ^d J values (Hz) in parentheses; ^e broad signal.

), which were designated with the assistance of the DEPT spectrum as five methyls (δ_C 28.7, 25.9, 24.0, 20.1, and 18.9), three methylenes (including δ_C 116.7, 45.1, and 27.7), six methines (including four oxymethines, δ_C 70.9, 75.3, 68.9, and 65.9; one terminal vinyl group methine, δ_C 139.9; and δ_C 41.7), and five quaternary carbons (including one ketone carbon, δ_C 209.2; three oxygenated quaternary carbons, δ_C 82.1, 77.4, and 76.2; and δ_C 45.2). The NMR data of 1 (Table 1) showed the appearance of a vinyl group (δ_C 139.9, CH and 116.7, CH₂; δ_H 6.78, 1H, dd, J = 17.5 and 11.5 Hz; and δ_H 5.35, 1H, d, J = 17.5 Hz, and 5.21, 1H, d, J = 11.5 Hz, respectively). The ¹H–¹H COSY experiment revealed the presence of four partial structures (Figure 2). The HMBC correlations of 1 (Figure 2) displayed from H₃-20 (δ_H 1.71) to C-1, 5, 9, and 10 (δ_C 209.2, 82.1, 41.7, and 58.1); both H₃-18 and 19 (δ_H 1.24 and 1.21) to C-3, 4 (δ_C 65.9 and 45.2), and 5; H₃-17 (δ_H 1.66) to C-7, 8 (δ_C 75.3 and 76.2), and 9; H₃-16 (δ_H 1.49) to C-12, 13, and 14 (δ_C 68.9, 77.4, and 139.9) suggested that the five methyls were positioned at C-4, 8, 10, and 13. Furthermore, the HMBC correlations were observed from both H₃-20 and H₂-2 (δ_H 3.34 and 2.64, each 1H) to a ketone carbon (δ_C 209.2); H-3 (δ_H 4.50) to C-2 (δ_C 45.1), 4, 18 (δ_C 24.0), and 19 (δ_C 20.1); 5-OH (δ_H 5.72) to C-4, 5 and 6; H-7 (δ_H 3.67) to C-6 (δ_C 27.7), 8, 9, and 17 (δ_C 25.9); 7-OH (δ_H 3.59) to C-6 and 7; H-9 (δ_H 2.72) to C-7, 8, 10, 11, 12 and 20; H-11 (δ_H 5.32) to C-12 and 13; 11-OH (δ_H 2.25) to C-9 and 11; H-12 (δ_H 4.06) to C-13 and 16 (δ_C 28.7); both H-14 (δ_H 6.78) and H₂-15 (δ_H 5.35 and 5.21, each 1H) to C-13, suggesting that a ketone, three hydroxyls, and one terminal vinyl functionalities were located on C-1, 5, 7, 11, and 14, respectively. The remaining two chlorines were positioned at C-3 and 12 (δ_C 65.9 and 68.9, respectively). As described above, 1 elucidated a new polyoxygenated chlorinated labdane diterpenoid, spongianol.

In the NOESY spectrum of 1, the NOE correlations (Figure 2) of H₃-20 with 11-OH, H₃-17, and H₃-19; 11-OH and H₃-19 with H₃-17; and H-14 with 11-OH and H₃-17 suggested that these protons were positioned at the same orientation. By contrast, the correlations of H-3 with H₃-18; 5-OH with H-9, H₃-18, and 7-OH; and H-12 with H-9 and H₃-16 revealed that these protons were on the same side. Furthermore, the stereochemistry of 1 was evidenced by the experimental CD (circular dichroism) and calculated ECD (electronic circular dichroism) spectra (Figure 3). The theoretical ECD curves of 3R,5R,7S,8R,9R,10S,11S,12S,13S-1 (1a) and its enantiomer 3S,5S,7R,8S,9S,10R,11R,12R,13R-1 (1b) were calculated at the B3LYP/6-311+G(d,p) (including a IEFPCM solvent model for MeOH) level of theory by the Gaussian 9.0 program [27,28]. The CD spectrum of 1 (Figure 3) showed the negative Cotton effect at 293 nm, which was found to be consistent with the calculated ECD of 1a (296 nm, Figure 3), and the absolute configuration of 1 was thus identified as 3R,5R,7S,8R,9R,10S,11S,12S and 13S. Furthermore, the absolute configurations of 1 were consistent with that of the structural analogs (3R,5S,6S,8S,9S,10R,13R)-3-bromo-6-hydroxy-8,13-epoxylabd-14-en-1-one (10), (1S,3R,5S, 6S,8S,9S,10R,13R)-1-acetoxy-3-bromo-6-hydroxy-8,13-epoxy-labd-14-ene (11) and paniculatol (12) (Figure 4) isolated from the red alga Laurencia sp. [29,30].

The HRESIMS of metabolite 2 showed a molecular ion peak [M + Na]⁺ at 483.3446 m/z, which established the molecular formula C₂₉H₄₈O₄, implying six degrees of unsaturation. The IR absorption bands at ν_max 3458–3291, 1682, and 1654 cm⁻¹ revealed the presence of the hydroxyl, ketone, and olefin, respectively. The NMR spectroscopic data of 2 (Table 1) displayed six methyls (δ_C 20.5, 19.6, 19.0, 18.9, 12.3, and 12.0; δ_H 1.02, 3H, s; 0.84, 3H, d, J = 6.6 Hz; 0.81, 3H, d, J = 6.6 Hz; 0.94, 3H, d, J = 6.0 Hz; 0.85, 3H, t, J = 7.8 Hz and 0.61, 3H, s); six methylenes; five methines (including one oxygenated methane δ_C 67.2 and δ_H 4.07, m; and one olefinic methane δ_C 119.9 and δ_H 5.66, br s), and five quaternary carbons (including one ketone carbon δ_C 197.7; one olefinic non-protonated carbon δ_C 164.3, and two oxygenated quaternary carbons δ_C 79.7 and 74.6, respectively). The detailed analyses of ¹H–¹H COSY and HMBC correlations (Figure 5) established the molecular skeleton of 2. Furthermore, a comparison of the NMR data of 2 with the similar structures 3β,5α,9α-trihydroxy-(22E,24R)-23-methylergosta-7,22-dien-6-one (13) and 3β,5α,9α-trihydroxy-(24S)-ergost-7-en-6-one (14) [31] (Figure 6, Supplementary Table S1) confirmed that 2 is a new polyoxygenated sterol.

The relative configuration of 2 was deduced by the analysis of NOE correlations (Figure 5). The observation of the NOE correlations of 5-OH (δ_H 3.34) with H-3 and 9-OH (δ_H 4.07 and 4.12, respectively); 9-OH with H-12α (δ_H 1.72); H-14 (δ_H 2.72) with H-12α and H-17 (δ_H 1.39); H-17 with H₃-21 (δ_H 0.94); and H₃-21 with H₂-12 elucidated that these protons were cofacial. Furthermore, a comparison of the NMR data of 2 with those of similar analogs, 13 and 14 (Figure 6), confirmed the configuration of the steroidal nucleus ([31], Supplementary Table S1). The chemical shift differences of C-26 and C-27 (Δδ_C^26,27, useful for assignment of the absolute configuration of C-24 of this side chain [32,33,34]) for compound 2 and the related known 24S and 24R analogs are summarized in Supplementary Table S2. In 24S analogues 15 (CDCl₃) and 17 (C₅D₅N), the Δδ_C^26,27 values were found to be 0.55 and 0.62, respectively, while those for 24R analogues 16 (CDCl₃) and 18 (C₅D₅N) were 0.77 and 0.73, respectively (Figure 6) [32,35]. The Δδ_C^26,27 values for 2 were found to be 0.59 and 0.62 in CDCl₃ and C₅D₅N, respectively, indicating that C-24 of 2 had an S-configuration (Figure 7a). In addition, the δ_C values for C-20, C-24, to C-27 were shown to be much more similar to those of compound 15 than those of 16. Thus, the 24S configuration of 2 was established. Furthermore, the stereochemistry of 2 was evidenced by the experimental CD and calculated ECD spectra (Figure 7b). The theoretical ECD curves of 3S,5R,9R,10R,13R,14R,17R,20R,24S-2 (2a) and its enantiomer 3R,5S,9S,10S,13S,14S,17S,20S,24R-2 (2b) were calculated at the CAM-B3LYP/6-311+G(d,p) (including a IEFPCM solvent model for MeOH) level of theory by the Gaussian 9.0 program [27,28]. The tendency of the experimental CD spectrum of 2 (Figure 7b) was similar to that of the calculated ECD of 2a (Figure 7b); furthermore, the absolute configuration of 2 was identified. On the basis of the above observations, the molecular structure of 2 was determined as 3β,5α,9α-trihydroxy-24S-ethylcholest-7-en-6-one.

For compound 3, the IR absorption bands at ν_max 3393 and 1670 cm⁻¹ revealed the presence of the hydroxy and α,β-unsaturated ester groups, respectively, and the HRESIMS m/z 445.3316 [M + H]⁺ established the molecular formula of 3 to be C₂₈H₄₄O₄. The structure of 3 was established by analyses of ¹H–¹H COSY and HMBC experiments (Figure 8), and the planar structure of 3 was found to be as same as that of (22E,24R)-ergosta-7, 22-dien-3β,5α-diol-6,5-olide (19) (Figure 9) [36]. Compound 3 exhibited almost the same NMR data as those of 19 except for the chemical shifts of C-16 and C-26 to C-28 (Supplementary Table S3). The chemical shifts of C-16 and C-24 to C-28 for 3 and the related known 24S and 24R analogs 19–21 [32,36] are summarized in

Table 2. The ¹³C NMR data at C-16 and C-24–C-28 of 3 and related compounds 19−21.

Position	3 a (22E,24S)	19 b (22E,24R)	20 c (22E,24S)	21 c (22E,24R)
C-16	28.0	27.7	28.86	28.58
C-24	43.1	42.8	43.12	42.90
C-25	33.2	33.0	33.28	33.16
C-26	19.6	19.9	19.69	20.02
C-27	20.1	19.6	20.19	19.69
C-28	18.0	17.6	18.08	17.68

^{a 13}C spectra recorded at 150 MHz in CDCl₃; ^{b 13}C spectra recorded at 150 MHz in CDCl₃ [36]; ^{c 13}C spectra recorded at 25.16 MHz in CDCl₃ [32].

. It was found that C-16 of 3 resonated at a higher chemical shift (δ_C 28.0) than that of 19 (δ_C 27.7). The 0.3 ppm difference for carbon shifts of C-16 between both 3 and 19 was similar to those of 20 (δ_C 28.86) and 21 (δ_C 28.58). In addition, the chemical shifts of C-24 and C-26−28 of 3 showed closer chemical shift values to compound 20 than to 21. On the basis of the above analysis, compound 3 was determined as (22E,24S)-ergosta-7,22- dien-3β,5α-diol-6,5-olide. Compound 3 is the fourth member of the group of steroids with a rare seven-membered lactone B ring, and the other three members, astersterol A, fortisterol, and (22E,24R)-ergosta-7,22-dien-3β,5α-diol-6,5-olide, were isolated previously from the starfish [37], the marine sponge [38], and the fungus Ganoderma resinaceum [36], respectively.

Metabolite 4 was isolated as a colorless oil. Its molecular formula was determined to be C₁₁H₁₈O₃ 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. ¹³C and ¹H NMR data for compound 4.

4
Position	δH	δC
1	-	169.5, C a
2	5.69, br s b	115.3, CH
3	-	163.5, C
4	2.62, t (7.5) c	33.1, CH2
5	1.48, quin (7.5)	27.8, CH2
6	1.33, quin (7.5)	29.0, CH2
7	1.60, quin (7.5)	23.5, CH2
8	2.43, t (7.5)	43.6, CH2
9	-	209.4, C
10	2.14, s	29.9, CH3
11	1.91, s	25.4, CH3

¹³C and ¹H spectra recorded at 125 and 500 MHz in CDCl₃. ^a Deduced from DEPT; ^b broad signal; ^c J values (Hz) in parentheses.

) 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 ¹H–¹H COSY and HMBC correlations of 4 (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 H₃-11 (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].

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.

The anti-inflammatory activities of compounds 1–9 inhibiting superoxide anion (O₂⁻) generation and elastase release in fMLF/CB-stimulated human neutrophils [54,55,56] were also evaluated (

Table 4. Effects of compounds on superoxide anion generation and elastase release in fMLF/CB-induced human neutrophils.

Compound	Superoxide Anion		Elastase Release
	IC50 (μM) a	Inh% (20 μM)	IC50 (μM)	Inh% (20 μM)
1	>20	15.65 ± 7.56	>20	16.31 ± 4.66 *
2	>20	−0.04 ± 3.90	>20	6.28 ± 3.04
3	>20	18.10 ± 2.29 **	>20	13.08 ± 2.01 **
4	>20	7.81 ± 3.87	>20	18.53 ± 3.57 **
5	>20	3.25 ± 4.06	>20	13.27 ± 3.81 *
6	>20	15.51 ± 7.55	>20	20.00 ± 4.87 *
7	>20	25.24 ± 4.68 **	17.23 ± 2.45	55.96 ± 3.88 ***
8	>20	22.38 ± 3.95 **	14.60 ± 2.24	60.80 ± 6.49 ***
9	>20	15.58 ± 0.58 ***	>20	21.22 ± 4.71 *
LY294002	1.91 ± 0.79	88.71 ± 1.50 ***	2.94 ± 0.13	79.50 ± 1.95 ***

Results are presented as mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control value (DMSO). ^a Concentration necessary for 50% inhibition.

). Compounds 7 and 8 exhibited medium inhibitory activity against elastase release (55.96 ± 3.88 and 60.80 ± 6.49%, respectively) at 20 μM, with IC₅₀ values of 17.23 ± 2.45 and 14.60 ± 2.24 μM, respectively. However, compounds 7 and 8 showed weak inhibition of superoxide anion generation (25.24 ± 4.68% and 22.38 ± 3.95%, respectively) at 20 μM. Furthermore, compounds 6 and 9 were found to display inhibitory activity to some extent (20.00 ± 4.87% and 21.22 ± 4.71%, respectively) against elastase release at 20 μM.

3. Materials and Methods

3.1. 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 ¹H and ¹³C, respectively; a Varian Unity INOVA500 FT-NMR (both Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz for ¹H and ¹³C, respectively; or a JEOL ECZ600R FT-NMR (Japan) at 600 and 150 MHz for ¹H and ¹³C, 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 high-performance 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).

3.2. 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.

3.3. Extraction and Separation

The freeze-dried material Spongia sp. (550 g dry wt) was minced and extracted exhaustively with EtOAc/MeOH/CH₂Cl₂ (1:1:0.5). The solvent-free extract was suspended in water and partitioned with CH₂Cl₂, EtOAc, and then n-BuOH saturated with water to obtain CH₂Cl₂ (18.47 g), EtOAc (0.78 g), and n-BuOH (1.0 g) fractions. The CH₂Cl₂ fraction was chromatographed over a silica gel column using EtOAc in n-hexane (0% to 100%, stepwise) and then MeOH in EtOAc (0% to 100%, stepwise) to yield 12 fractions (F1–F12).

Fraction F3 (0.986 g) eluted with n-hexane/EtOAc (9:1) was re-chromatographed over a RP-18 column using H₂O in MeOH (100% to 0%, stepwise) to give six subfractions (F3-1 to F3-6). F3-5 (54.6 mg, eluted with MeOH/H₂O 8:2) was isolated using RP-18 HPLC (MeOH/H₂O 9:1) to give six subfractions (F3-5-1 to F3-5-6); F3-5-2 (32.5 mg) was further purified on RP-18 HPLC (MeOH/H₂O 7.5:2.5) to afford 6 (1.6 mg).

F5 (1.706 g) eluted with n-hexane/EtOAc (6.5:3.5) was re-chromatographed over a RP-18 column using H₂O in MeOH (100% to 0%, stepwise) to give eight subfractions (F5-1 to F5-8). F5-4 (32.4 mg, eluted with MeOH/H₂O 6:4) was isolated using RP-18 HPLC (MeOH/H₂O 7:3) to give eight subfractions (F5-4-1 to F5-4-8). F5-4-1 (29.5 mg) was further purified on RP-18 HPLC (CH₃CN /H₂O 4:6) to afford 7 (5.8 mg) and 8 (1.7 mg). F5-6 (48.2 mg, eluted with MeOH/H₂O 1:0) was separated on RP-18 HPLC (IPA/MeOH 1:19) to give six subfractions (F5-6-1 to F5-6-6), F5-6-1 (27.6 mg) was purified on RP-18 HPLC (MeOH/H₂O 8:2) to afford 1 (2.9 mg) and 5 (4.6 mg).

F7 (1.505 g) eluted with n-hexane/EtOAc (2.5:7.5) was re-chromatographed over a RP-18 column using H₂O in MeOH (100% to 0%, stepwise) to give eight subfractions (F7-1 to F7-8). F7-3 (146.3 mg, eluted with MeOH/H₂O 4:6) was isolated using RP-18 HPLC (MeOH/H₂O 1:1) to give 10 subfractions (F7-3-1 to F7-3-10). F7-3-7 (13.2 mg) was purified on RP-18 HPLC (CH₃CN/H₂O 2.8:7.2) to afford 4 (4.5 mg). F7-6 (67.0 mg, eluted with MeOH/H₂O 1:0) was isolated using RP-18 HPLC (isopropanol/MeOH 1:19) to give nine subfractions (F7-6-1 to F7-6-9); F7-6-5 (16.1 mg) was further separated on RP-18 HPLC (MeOH/H₂O 93:7) to afford 3 (1.0 mg); F7-6-7 (25.7 mg) was purified on RP-18 HPLC (CH₃CN/H₂O 8:2) to afford 2 (4.9 mg).

3.3.1. Spongianol (1)

Colorless oil; ${\left[\alpha \right]}_{25}^{\mathrm{D}}$ − 14.5 (c 0.29, CH₃OH); IR (neat) v_max 3420, 2979, 2919, 1698, and 1646 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 443 and 445 [M + Na]⁺; HRESIMS m/z 443.1364 and [M + Na]⁺ (calcd for C₂₀H₃₀³⁵Cl₂O₅Na, 443.1363).

3.3.2. 3β,5α,9α-Trihydroxy-24S-ethylcholest-7-en-6-one (2)

White powder; ${\left[\alpha \right]}_{25}^{\mathrm{D}}$ − 12.7 (c 0.49, CH₃OH); IR (neat) v_max 3291, 2959, 2872, 1682 and 1654 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 483 [M + Na]⁺; HRESIMS m/z 483.3446 [M + Na]⁺ (calcd for C₂₉H₄₈O₄Na, 483.3445).

3.3.3. (22E,24S)-Ergosta-7,22-dien-3β,5α-diol-6,5-olide (3)

Colorless crystal; ${\left[\alpha \right]}_{25}^{\mathrm{D}}$ + 38.5 (c 0.28, CH₃OH); IR (neat) ν_max 3393, 2954, 2917, 2849, and 1670 cm^–1; ¹H NMR and ¹³C data, see Table 1; HRESIMS m/z 445.3316 [M + H]⁺ (calcd for C₂₈H₄₅O₄, 445.3312).

3.3.4. (Z)-3-Methyl-9-oxodec-2-enoic Acid (4)

Colorless oil; IR (neat) ν_max 3445, 2923, 2859, 1700, 1683, and 1647 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; HRESIMS m/z 221.1149 [M + Na]⁺ (calcd for C₁₁H₁₈O₃Na, 221.1148).

3.4. DFT and TD-DFT Calculations

The preliminary geometry optimization of conformers was simulated using the DFT approach at the B3LYP/6-31G(d) level of theory [27]. The ECD spectra were simulated by using the time-dependent DFT (TD-DFT) approach at the B3LYP/6-311+G(d,p) or 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.

3.5. 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.

3.6. 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.

3.7. 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 Ca²⁺-free HBSS buffer (pH 7.4, ice-cold).

3.7.1. Superoxide Anion Generation

Neutrophils (6 × 10⁵ cells/mL) incubated (with 0.6 mg/mL ferricytochrome c and 1 mM Ca²⁺) in HBSS at 37 °C were treated with DMSO (as a control) or the tested compound for 5 min. Neutrophils were primed with 1 μg/mL cytochalasin B (CB) for 3 min before being activated by 100 nM fMLF for 10 min. The change in superoxide anion generation was spectrophotometrically measured at 550 nm (U-3010, Hitachi, Tokyo, Japan) [54,55]. LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one] was used as a positive control.

3.7.2. Elastase Release

Neutrophils (6 × 10⁵ cells/mL) incubated (with 100 μM MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide and 1 mM Ca²⁺) in HBSS at 37 °C were treated with DMSO or the tested compound for 5 min. Neutrophils were, then, activated with fMLF (100 nM)/CB (0.5 μg/mL) for 10 min. The change in elastase release was spectrophotometrically measured at 405 nm (U-3010, Hitachi, Tokyo, Japan) [55].

4. 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.