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.

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.

Figure 1

Structures of metabolites 1–9.

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), which were designated with the assistance of the DEPT spectrum as five methyls (δC 28.7, 25.9, 24.0, 20.1, and 18.9), three methylenes (including δC 116.7, 45.1, and 27.7), six methines (including four oxymethines, δC 70.9, 75.3, 68.9, and 65.9; one terminal vinyl group methine, δC 139.9; and δC 41.7), and five quaternary carbons (including one ketone carbon, δC 209.2; three oxygenated quaternary carbons, δC 82.1, 77.4, and 76.2; and δC 45.2). The NMR data of 1 (Table 1) showed the appearance of a vinyl group (δC 139.9, CH and 116.7, CH2; δH 6.78, 1H, dd, J = 17.5 and 11.5 Hz; and δH 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 (δH 1.71) to C-1, 5, 9, and 10 (δC 209.2, 82.1, 41.7, and 58.1); both H3-18 and 19 (δH 1.24 and 1.21) to C-3, 4 (δC 65.9 and 45.2), and 5; H3-17 (δH 1.66) to C-7, 8 (δC 75.3 and 76.2), and 9; H3-16 (δH 1.49) to C-12, 13, and 14 (δC 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 (δH 3.34 and 2.64, each 1H) to a ketone carbon (δC 209.2); H-3 (δH 4.50) to C-2 (δC 45.1), 4, 18 (δC 24.0), and 19 (δC 20.1); 5-OH (δH 5.72) to C-4, 5 and 6; H-7 (δH 3.67) to C-6 (δC 27.7), 8, 9, and 17 (δC 25.9); 7-OH (δH 3.59) to C-6 and 7; H-9 (δH 2.72) to C-7, 8, 10, 11, 12 and 20; H-11 (δH 5.32) to C-12 and 13; 11-OH (δH 2.25) to C-9 and 11; H-12 (δH 4.06) to C-13 and 16 (δC 28.7); both H-14 (δH 6.78) and H2-15 (δH 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 (δC 65.9 and 68.9, respectively). As described above, 1 elucidated a new polyoxygenated chlorinated labdane diterpenoid, spongianol.

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

Figure 2

The selected COSY (▬), HMBC (→), and key NOESY (↔) correlations of 1.

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].

Figure 3

Calculated ECD curves of 3R,5R,7S,8R,9R,10S,11S,12S,13S-1 (1a) and 3S,5S,7R,8S,9S,10R,11R,12R,13R-1 (1b) and the experimental CD curve of 1.

Figure 4

The structural analogs of 10–12 [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 νmax 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 (δC 20.5, 19.6, 19.0, 18.9, 12.3, and 12.0; δH 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 δC 67.2 and δH 4.07, m; and one olefinic methane δC 119.9 and δH 5.66, br s), and five quaternary carbons (including one ketone carbon δC 197.7; one olefinic non-protonated carbon δC 164.3, and two oxygenated quaternary carbons δC 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.

Figure 5

The selected COSY (▬), HMBC (→), and key NOESY (↔) correlations of 2.

Figure 6

Structures of compounds 13–18.

The relative configuration of 2 was deduced by the analysis of NOE correlations (Figure 5). The observation of the NOE correlations of 5-OH (δH 3.34) with H-3 and 9-OH (δH 4.07 and 4.12, respectively); 9-OH with H-12α (δH 1.72); H-14 (δH 2.72) with H-12α and H-17 (δH 1.39); H-17 with H3-21 (δH 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 (ΔδC26,27, 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 ΔδC26,27 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 ΔδC26,27 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 δC 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.

Figure 7

(a) The ΔδC26,27 values of compounds 2 and 15–18; (b) the calculated ECD curves of 3S,5R,9R,10R,13R,14R,17R,20R,24S-2 (2a) and 3R,5S,9S,10S,13S,14S,17S,20S,24R-2 (2b), and the experimental CD curve of 2.

For compound 3, the IR absorption bands at νmax 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. It was found that C-16 of 3 resonated at a higher chemical shift (δC 28.0) than that of 19 (δC 27.7). The 0.3 ppm difference for carbon shifts of C-16 between both 3 and 19 was similar to those of 20 (δC 28.86) and 21 (δC 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.

Figure 8

The selected COSY (▬), HMBC (→), and key NOESY (↔) correlations of 3.

Figure 9

Structures of compounds 19–21.

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

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) showed the presence of two methyls (δC 29.9 and 25.4; δH 2.14 and 1.91, each 3H, s); five methylenes; one alkene methane (δC 115.3 and δH 5.69, br s); and three quaternary carbons (included one ketone carbon δC 209.4, one carbonyl carbon δC 169.5, and one alkene quaternary carbon δC 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 (δH 5.69) with H3-11 (δH 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.

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.

Figure 10

The selected COSY (▬) and HMBC (→) correlations of 4.

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). 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.

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.

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; − 14.5 (c 0.29, CH3OH); IR (neat) vmax 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; − 12.7 (c 0.49, CH3OH); IR (neat) vmax 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; + 38.5 (c 0.28, CH3OH); IR (neat) νmax 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) νmax 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.