Structurally Diverse Metabolites from the Soft Coral Sinularia verruca Collected in the South China Sea

Abstract

Nineteen metabolites with diverse structures, including the rare pyrroloindoline alkaloid verrupyrroloindoline (1), the unprecedented highly fused benzosesquiterpenoid verrubenzospirolactone (2), the new asteriscane-type sesquiterpenoid 10-deoxocapillosanane D (3), and the two new cyclopentenone derivatives (4S*,5S*)-4-hydroxy-5-(hydroxymethyl)-2,3-dimethyl-4-pentylcyclopent-2-en-1-one (4) and (S)-4-hydroxy-5-methylene-2,3-dimethyl-4-pentylcyclopent-2-en-1-one (5), were isolated from a South China Sea collection of the soft coral Sinularia verruca. Eleven previously described marine metabolites (7–15, 18, and 19) were also obtained as well as three new EtOH-adduct artifacts (6, 16, and 17). The structures of the new compounds were elucidated by extensive spectroscopic analysis and by comparison with previously reported data. Compounds 4, 5, and 16 showed protection against the cytopathic effects of HIV-1 infection with EC₅₀ values of 5.8–34 μM, and 4, 6, and 16 exhibited inhibition against LPS-induced NO production with IC₅₀ values of 24–28 μM.

Graphical Abstract

Soft corals of the genus Sinularia (family Alcyoniidae) are commonly distributed in the tropical coral reef systems of the world and are well known for being a rich source of biologically active secondary metabolites. In the past several decades, more than 50 Sinularia species have been chemically investigated, resulting in the discovery of hundreds of secondary metabolites including sesquiterpenoids, diterpenoids, polyhydroxylated steroids, cyclopentenones, and butenolides.¹ Many of these metabolites are structurally novel and display a wide range of significant ecological and pharmacological activities, such as antifouling, antifeedant, allelopathy, cytotoxic, antimicrobial, and anti-inflammatory properties.^2–8 In the course of our ongoing research on bioactive natural products from marine invertebrates of the South China Sea,^9–11 a previously unstudied soft coral, Sinularia verruca van Ofwegen, 2008, was collected. Detailed chemical examination of this specimen led to the isolation of 19 compounds with diverse structural scaffolds, including a rare pyrroloindoline alkaloid (1), a novel benzosesquiterpenoid (2), a new asteriscane-type sesquiterpenoid (3), and two new cyclopentenone derivatives (4 and 5). Eleven known compounds were also isolated from the chemically rich extract as well as three new EtOH-adduct artifacts (6, 16, and 17). All compounds were evaluated for in vitro antiviral activity against human immunodeficiency virus type 1 (HIV-1) and for inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ). This paper reports details of the isolation, structure elucidation, and bioactivity assess-ment of these compounds.

RESULTS AND DISCUSSION

Compound 1 had a molecular formula of C₁₇H₂₀N₂O₃ according to its HREASIMS data, implying nine degrees of unsaturation. The IR spectrum showed absorption bands attributed to hydroxy (3437 cm⁻¹), carbonyl (1677 cm⁻¹), and aromatic ring (1606 and 1466 cm⁻¹) functionalities. The presence of an ortho-disubstituted benzene ring was revealed by ¹H NMR signals at δ_H 7.31 (dd, J = 7.2, 1.2 Hz, H-3), 7.24 (td, J = 7.8, 1.2 Hz, H-5), 6.85 (t, J = 7.2 Hz, H-4), and 6.69 (d, J = 7.8 Hz, H-6) (Table 1) and ¹³C NMR signals at δ_C 150.9 (C, C-1), 131.1 (C, C-2), 131.0 (CH, C-5), 124.1 (CH, C-3), 120.3 (CH, C-4), and 111.1 (CH, C-6) (Table 2), as well as COSY correlations between H-3/H-4, H-4/H-5, and H-5/H-6 and HMBC correlations from H-3 to C-1 and from H-6 to C-2 (Figure 1). A disubstituted ethyl isopentenoate unit was established by HMBC correlations from methyl doublet H₃- 14 (δ_H 1.41 d, J = 6.6 Hz) to methine carbon C-11 (δ_C 46.3) and nonprotonated olefinic carbon C-12 (δ_c 101.7), from H-11 (δ_H 4.53 d, J = 6.6 Hz) to carbonyl carbon (3–15 (δ_C 167.4) and olefinic methine carbon C-13 (δ_C 142.6), from olefinic proton H-13 (δ_H 7.13 s) to C-15 and C-11, and from the oxymethylene protons at δ_H 4.13 (q, J = 7.2 Hz) to the carbonyl carbon C-15, in combination with COSY couplings between H₃-14 and H-11 and between a methyl triplet (δ_H 1.25 t, J = 7.2 Hz) and the oxymethylene protons mentioned above. On the basis of the remaining degrees of unsaturation, 1 had to be tetracyclic. HMBC correlations from H-3 to nonprotonated carbon C-7 (δ_C 84.1), from H-10 (δ_H 4.75 s) to C-1, C-2, and C-9 (δ_C 49.8, CH₂), from H₂-8 (δ_H 2.61 m) to C-2, and from H₂-9 to C-7, and a COSY correlation between H₂-8 and H₂-9, in combination with the chemical shifts of C-1, C-7, C-9, C-10, H-9, and H-10, allowed us to establish a 2,3,8,8a-tetrahydropyrrolo[2,3-b]indol-3a(1H)-ol moiety. Moreover, HMBC correlations from H-11 to C-1 and C-10 and from H-13 to C-9 and C-10 revealed that the ethyl isopentenoate unit was connected to the two N atoms of the fused indolin-3-ol and pyrrolidine moieties via C-11 and C-13, respectively. The relative configuration of 7S*, 10S*, and 11S* was assigned based on NOESY correlations between H-6/H-11, H₃-14/H-10, and H₂-8/H-3 (Figure 2). Compound 1 was given the name verrupyrroloindoline. The ethyl ester is possibly an artifact because EtOH was used during the extraction process; however no evidence of a nonesterified precursor of 1 was observed during the isolation procedure. In either case, the free acid or ethyl ester form of 1 represents a rare natural alkaloid class with a 2a, 5a-diazacyclopenta[jk]fluorene skeleton.¹²

Table 1.

¹H NMR Data for Compounds 1−6 (CDCl₃, 600 MHz)^a

no.	1	2	3	4	5	6
1		3.24, d (4.8)	2.28, d (15.6)
2		1.80, dd (12.0, 4.8)	1.99, d (15.6)
3	7.31, dd (7.2, 1.2)		2.22, m
4	6.85, t (7.2)	2.10, m 1.93, m	1.78, m 1.43, m
5	7.24, td (7.8, 1.2)	1.99, m 1.37, m	1.78, m 1.59, m	2.71, t (7.2)		2.34, t (6.6)
6	6.69, d (7.8)	2.56, m	4.83, br s	4.05, dd (10.8, 7.8)	6.05, s 5.53, s	1.81, m 1.39, m
				3.88, br t (8.4)
7				1.72, m 1.70, m	1.87, m 1.82, m	1.62, m 1.51, m
8	2.61, m	4.88, s	5.65, s	0.87, m	0.96, m 0.87, m	1.39, m
9	3.73, m 2.88, dd (18.6, 9.6)			1.24, m	1.22, m	0.94, t (7.2)
10	4.75, s	7.11, s	2.21, d (15.0) 2.01, d (15.0)	1.24, m	1.23, m	2.05, m 1.93, m
11	4.53, q (6.6)			0.85, t (6.6)	0.83, t (6.6)	2.04, m
12			1.04, s	1.73, s	1.81, s
13	7.13, s	1.96, s	1.03, s	1.99, s	2.00, s	1.70, s
14	1.41, d (6.6)	1.68, s	1.73, s			1.98, s
15		1.33, s	0.99, d (7.2)
3′		7.29, s
6′		6.54, s
7′		2.15, s
OEt	4.13, q (7.2)					4.11, q (6.6)
	1.25, t (7.2)					1.24, t (6.6)

^aThe coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.

Table 2.

¹³C NMR Data for Compounds 1−6 (CDCl₃, 150 MHz)^a

no.	1	2	3	4	5	6
1	150.9, C	36.6, CH	36.5, CH2	206.1, C	194.1, C	204.9, C
2	131.1, C	50.1, CH	142.4, C	136.3, C	139.4, C	136.6, C
3	124.1, CH	82.0, C	36.9, CH	169.3, C	166.3, C	166.7, C
4	120.3, CH	39.0, CH2	31.0, CH2	81.0, C	78.7, C	81.5, C
5	131.0, CH	25.1, CH2	33.3, CH2	59.7, CH	148.8, C	58.9, CH
6	111.1, CH	40.7, CH	71.5, CH	59.4, CH2	114.7, CH2	24.3, CH2
7	84.1, C	143.1, C	136.7, C	36.2, CH2	37.0, CH2	30.9, CH2
8	33.9, CH2	119.9, CH	122.8, CH	25.1, CH2	24.1, CH2	23.0, CH2
9	49.8, CH2	89.0, C	129.3, C	32.0, CH2	31.9, CH2	13.9, CH3
10	78.4, CH	153.5, CH	53.0, CH2	22.4, CH2	22.4, CH2	30.1, CH2
11	46.3, CH	128.0, C	29.2, C	14.0, CH3	13.9, CH3	31.0, CH2
12	101.7, C	174.0, C	29.6, CH3	7.7, CH3	8.2, CH3	173.1, C
13	142.6, CH	10.4, CH3	29.6, CH3	11.3, CH3	10.7, CH3	8.0, CH3
14	21.7, CH3	20.2, CH3	17.2, CH3			11.3, CH3
15	167.4, C	23.8, CH3	22.7, CH3
1′		146.4, C
2′		116.8, C
3′		115.4, CH
4′		147.4, C
5′		124.5, C
6′		119.7, CH
7′		15.6, CH3
OEt	14.5, CH3					14.2, CH3
	59.2, CH2					60.9, CH2

^aThe assignments were based on HMQC, HMBC, and COSY spectra.

Figure 1.

Key COSY and HMBC correlations for 1−6.

Figure 2.

Key NOE correlations and computer-generated models for 1, 2, 4, and 6. Structures 1, 2, 4a, and 6 were generated using MM2 force field calculations, while 4b was produced by MD simulation and represents a higher energy conformer of the alternative 4S*,5R*-diastereomer that is not supported by the NOE data.

Compound 2 had a molecular formula of C₂₂H₂₄O₄ as determined by HRESIMS data, which implied 11 degrees of unsaturation. IR absorptions indicated the presence of hydroxy (3444 cm⁻¹), carbonyl (1743 cm⁻¹), and aromatic ring (1610, 1512, and 1450 cm⁻¹) functionalities. The ¹³C NMR spectrum exhibited 22 carbon signals including an ester carbonyl (δ_C and 10 additional sp² carbons, which indicated that 2 was pentacyclic. Signals for two oxygenated aliphatic carbons, four methyls, two methylenes, and three aliphatic methines were also present in the ¹³C NMR spectrum. A 1,2,4,5-tetrasubstituted benzene ring was established by HMBC correlations from H₃-7′ (δ_H 2.15 s) to C-4′ (δ_C 147.4, C), C-5′ (δ_C 124.5, C), and C-6′ (δ_C 119.7, CH), from H-3′ (δ_h 7.29 s) to C-1′ (δ_C 146.4, C) and C-5′, and from H-6′ (δ_H 6.54 s) to C-2′ (δ_C 116.8, C) and C-4′, while an α,β-unsaturated-γ-lactone moiety was recognized by HMBC correlations from H₃-13 (δ_H 1.96 s) to the carbonyl carbon C-12 (δ_C 174.0) and two olefinic carbons C-10 (δ_C 153.5, CH) and C-11 (δ_C 128.0, C) and from H-10 (δ_H 7.11, s) to the oxygenated nonprotonated carbon C-9 (δ_C 89.0) (Figure 1). COSY correlations between H-1 (δ_H 3.24 d, J = 4.8 Hz)/H-2 (δ_H 1.80 dd, J = 12.0, 4.8 Hz), H-2/H-6 (δ_H 2.56 m), H-6/H-5 (δ_H 1.99 m; 1.37 m), and H-5/H-4 (δ_H 2.10 m; 1.93 m) defined an extended proton spin system. HMBC correlations from H₃-14 (δ_H 1.68 s) to the aliphatic methine carbon C-6 (δ_C 40.7) and two olefinic carbons C-7 (δ_C 143.1, C) and C-8 (δ_C 119.9, CH), from H₃-15 (δ_H 1.33 s) to the other oxygenated nonprotonated carbon C-3 (δ_C 82.0), an aliphatic methine carbon C-2 (δ_C 50.1), and a methylene carbon C-4 (δ_C 39.0), and from H-1 to C-8 and two carbons (C-9 and C-10) of the α,β-unsaturated-γ-lactone moiety allowed us to establish the 5,6-membered ring system C/D, in which C-3 and C-7 were substituted by methyls, C-7/C-8 composed a double bond, and C-9 was a spiro carbon that was also a constituent of the α,β-unsaturated-γ-lactone. Direct linkage of the substituted benzene ring to C-1 was indicated by the chemical shift of H-1 and confirmed by HMBC correlations from H-1 to C-1′, C-2′, and C-3′. An ether linkage between C-3 and C-1′ was supported by their chemical shifts (δ_C 82.0 and 146.4, respectively) and the remaining unsaturation equivalent, which thus required C-4′ to be hydroxylated. The relative configurations of the stereogenic centers in 2 were assigned on the basis of coupling constants and NOESY analysis. The coupling constant values J_H-1/_H-2 (4.8 Hz) and J_H-2/_H-6 (12.0 Hz) uggested the cis and trans fusions of rings B/D and C/D, respectively, while NOESY correlations between H₃-15/H-1, H-1/H-2, and H-1/H-10 indicated that H₃-15, H-1, H-2, and H-10 had the same facial orientation (Figure 2). Thus, compound 2 was established to be a highly fused benzosesquiterpenoid lactone, and it was named verrubenzo-spirolactone. It is interesting to note that 2 represents a novel skeleton that could be derived from capillobenzopyranol 12¹³ via a series of oxidation and cyclization reactions.

Compound 3 was assigned a molecular formula of C₁₅H₂₄O based on its HRESIMS data, requiring four degrees of unsaturation. The ¹³C NMR spectrum showed 15 carbon signals including four olefinic carbons, indicating that 3 must be bicyclic. A 4,4-dimethylcyclopent-1-ene moiety was established by HMBC correlations from H₃-12 (δ_H 1.04 s) and H₃-13 (δ_H 1.03 s) to C-1 (δ_C 36.5, CH₂), C-10 (δ_C 53.0, CH₂), and C-11 (δ_C 29.2, C) and from H₂-1 (δ_H 2.28 d, J = 15.6 Hz; 1.99 d, J = Hz) and H₂-10 (δ_H 2.21 d, J = 15.0 Hz; 2.01 d, J = 15.0 Hz) to two nonprotonated olefinic carbons C-2 (δ_C 142.4) and C-9 (δ_C 129.3). COSY correlations established a spin system from H-3 to H-6 and extended to H₃-15. Additional HMBC correlations from H₃-14 (δ_H 1.73 s) to oxygenated carbon C-6 (δ_C 71.5, CH) and two olefinic carbons C-7 (δ_C 136.7, C) and C-8 (δ_C 122.8, CH), from H-8 (δ_H 5.65 s) to C-10 and C-2, and from H₃-15 (δ_H 0.99 d, J = 7.2 Hz) to C-2 allowed connection of the structural subunits to define an asteriscanetype sesquiterpenoid. Accordingly, the planar structure of 3 was established as 6-hydroxyasterisca-2(9),7-diene. The relative configurations at C-3 and C-6 and the Z configuration of the C-7/C-8 double bond in 3 were suggested to be the same as those of the known analogues capillosananes B—D¹⁴ from Sinularia capillosa based on their very similar NMR data. As such, compound 3 was assigned as 10-deoxocapillosanane D.

Compound 4 had a molecular formula of C₁₃H₂₂O₃ as determined by HRESIMS data, requiring three degrees of unsaturation. IR absorptions at 3393, 1693, and 1647 cm⁻¹ indicated the presence of hydroxy, carbonyl, and olefinic functionalities. The ¹³C NMR spectrum showed 13 carbon signals including a conjugated ketone carbonyl (δ_C 206.1) and two nonprotonated olefinic carbons at δ_C 169.3 and 136.3 (Table 2), which accounted for two of the three degrees of unsaturation; therefore 4 was monocyclic. The ¹H NMR spectrum exhibited three methyl signals including two olefinic methyl singlets (δ_H 1.99 s, H₃-13; 1.73 s, H₃-12) and a methyl triplet (δ_H 0.85 t, J = 6.6 Hz, H₃-11) (Table 1). In addition, a tertiary oxygen-bearing carbon (δ_C 81.0, C-4), a secondary alcohol (δ_C 59.4, C-6; δ_H 4.05 dd, J = 10.8, 7.8 Hz, H-6a; 3.88 br t, J = 8.4 Hz, H-6b), an aliphatic methine (δ_C 59.7, C-5; δ_H 2.71 t, J = 7.2 Hz, H-5), anad four aliphatic methylenes were recognized from the ¹H and ¹³C NMR data. An n-pentyl unit was established by COSY correlations between H₃-11/H₂-10 (δ_h 1.24 m), H₂-7 (δ_h 1.72 m; 1.70 m)/H₂-8 (δ_H 0.87 m), and H₂-8/H₂-9 (δ_H 1.24 m) and HMBC correlations (Figure 1) from H₃-11 and H₂-7 to the aliphatic methylene carbon C-9 (δ_C). Finally, the gross structure of 4 was established as 4-hydroxy-5-(hydroxymethyl)-2,3-dimethyl-4-pentylcyclopent-2-en-1-one based on HMBC correlations from H₃-12 to the ketone carbonyl C-1 and the two olefinic carbons C-2 and C-3, from H₃-13 to C-2, C-3, and the oxygenated nonprotonated carbon C-4, from H₂-7 to C-3, C-4, and the methine carbon C-5, and from the oxymethylene H₂-6 to C-1, C-5, and C-4. COSY correlations between H-5 and H₂-6 were observed, and an NOE correlation between H₂-6 and H₂-7 could establish their cis relationship (4a in Figure 2) in the cyclopent-2-enone nucleus, which is much more rigid than a typical five-membered ring system, such as a cyclopentane ring. In order to support this assignment, a set of 500 conformers of the trans isomer 4b (Figure 2) were modeled, and the distance between H₂-6 and H₂-7 was determined to be 3.6–4.8 Å, which is greater than the typical limit for seeing NOE correlations. Compound 4 was found to be closely related to the previously reported metabolite (+)-sinularone J, which has a methoxy in place of the hydroxy at C-6.¹⁵

The closely related analogue 16 was also isolated and assigned a molecular formula of C₁₅H₂₆O₃ based on HRESIMS data. It provided NMR data (Tables 1 and 2) that were very similar to those of 4, except there were additional signals for an ethoxy group (δ_C 66.8 (CH₂), 15.2 (CH₃); δ_H 3.54 2H, q, J = 6.6 Hz; 1.22 3H, t, J = 6.6 Hz). Linkage of the ethoxy group to C-6 in 16 was indicated by the deshielded chemical shift of C-6 (δ_C 66.8) in comparison with that of 4 (C-6, δ_C 59.4) and further confirmed by HMBC correlations from H₂-6 (δ_H 3.96 dd, J = 9.6, 4.0 Hz; 3.52 dd, J = 10.2, 9.6 Hz) to the ketone carbon C-1 (δ_C 202.7) and to the oxymethylene carbon of the ethoxy group (Figure 1). The configurations at C-4 and C-5 in 16 were tentatively determined to be the same as those in 4 since it showed similar NOE correlations between H₂-6 and H₂- 7 (δ_H 1.86 ddd, J = 13.2, 12.0, 4.2 Hz; 1.78 ddd, J = 13.2, 12.6, 5.4 Hz) and it had the same sign of specific rotation (${[\alpha ]}_{\text{D}}^{25}+7$, MeOH). The co-occurrence of 16 and 4 in the extract and the fact that EtOH was used during the extraction process suggest that compound 16 is likely a 6-O-ethyl artifact of 4.

The molecular formula of compound 5 was determined to be C₁₃H₂₀O₂ by HRESIMS, indicating four degrees of unsaturation. NMR data analyses revealed that 5 had a structure closely related to that of 4. The only difference was loss of signals for an aliphatic methine and a secondary alcohol and the appearance of signals for an exocyclic double bond (δ_C 148.8 (C), 114.7 (CH₂); δ_H 6.05 s, 5.53 s). HMBC correlations from the exomethylene protons to a ketone (δ_C 194.1, C-1) and a hydroxylated nonprotonated carbon (δ_C 78.7, C-4) revealed the exocyclic double bond was located at C-5/C-6. Thus, compound 5 was elucidated as 4-hydroxy-5-methylene-2,3-dimethyl-4-pentylcyclopent-2-en-1-one. The measured ECD spectrum of 5 was similar to the calculated ECD spectrum for the S-enantiomer, allowing the tentative assignment of the 4S configuration for 5 (Figure 3).

Figure 3.

Experimental ECD and calculated ECD spectra for 5.

A molecular formula of C₁₆H₂₆O₄ was established for compound 6 by HRESIMS data, which required four unsaturation equivalents. The 2,3-dimethylcyclopent-2-enone nucleus was recognized from diagnostic NMR signals for conjugated ketone carbonyl C-1 (δ_C 204.9), two non-protonated olefinic carbons C-2 (δ_C 136.6) and C-3 (δ_C 166.7), and two olefinic methyls (δ_C 11.3, 8.0; δ_H 1.98, 1.70). An n-butyl group was established by COSY correlations between H₃-9 (δ_H 0.94 t, J = 7.2 Hz)/H₂-8 (δ_H 1.39 m), H₂-8/H₂-7 (δ_H 1.62 m; 1.51, m), and H₂-7/H₂-6 (δ_H 1.81 m; 1.39 m). In addition, an ethylpropanoate unit was constructed by a COSY correlation between two vicinal methylenes H₂-10 (δ_H 2.05 m; 1.93 m) and H₂-11 (δ_H 2.04 m) and the coupling between an oxymethylene (δ_H 4.11 q, J = 6.6 Hz) and the second methyl triplet (δ_H 1.24 t, J = 6.6 Hz), in combination with HMBC correlations from H₂-10, H₂-11, and the oxymethylene protons to ester carbonyl C-12 (δ_C 173.1). HMBC correlations from H₂-10 to olefinic carbon C-3, oxygenated nonprotonated carbon C-4 (δ_C 81.5), and methine C-5 (δ_C 58.9), along with a COSY correlation between H₂-6 and the methine proton H-5 (δ_H 2.34 t, J = 6.6 Hz), allowed connection of the ethylpropanoate and n-butyl units to C-4 and C-5, respectively. This indicated that C-4 was hydroxylated. Thus, compound 6 was established as 4-hydroxy-4-(ethyl-propanoate)-2,3-dimethyl-5-butylcyclopent-2-en-1-one, probably an ethanolysis artifact formed from the co-occurring analogue sinularone D (8).¹⁶ The absolute configurations at C-4 and C-5 were tentatively assigned to be identical to those in 8 based on biogenetic considerations and similar NOE correlations between H₂-6 and H₂-10 (Figure 2).

Eleven known compounds were also isolated from the S. verruca extract and identified as sinularones A (7),¹⁶ D (8),¹⁶ G (9),¹⁶ and I (10),¹⁶ (S)-5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one (11),¹⁷ capillobenzopyranol (12),¹³ flexibilisqui-none (13),¹⁸ 5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2-methylpentanoic acid methyl ester (14),¹⁹ capillosanane I (15),¹⁴ (Z)-N-(4-hydroxyphenethyl)-3-methyldodec-2-enamide,²⁰ and (Z)-N-(3-hydroxy-4-methoxyphenethyl)-3-methyl-dodec-2-enamide,²⁰ by comparison of their ¹H and ¹³C NMR and MS spectroscopic data, as well as specific rotations, with those reported in the literature.

A final compound, 17, was isolated, and its molecular formula determined to be C₂₁H₃₆O₅ on the basis of HRESIMS data. Therefore, compound 17 was isomeric with compound 10, and its NMR data also corresponded very closely with the NMR data for 10. HMBC correlations from H₃-18 to the carbonyl carbon C-1 and two olefinic carbons C-2 and C-3 and from H₃-19 to the two olefinic carbons and the acetal carbon C-4 disclosed the same α,β-unsaturated-2,3-dimethyl-γ-lactone unit as found in butenolide 10. An ethoxy group [δ_C 58.3 (CH₂), 15.2 (CH₃); δ_H 3.40 and 3.14 (both 1H, dq, J = 12.0, 6.6 Hz), 1.17 (3H, t, J = 6.6 Hz)] and 12 aliphatic methylenes were also similar to those seen in the artifact sinularone I (10).¹⁶ An HMBC correlation from the oxymethylene protons to the acetal carbon C-4 revealed the location of the ethoxy group at C-4 in 17, instead of forming an ethyltridecanoate unit as in 10. Thus, compound 17 was defined as 13-(2-ethoxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl)tridecanoic acid, which has not previously been reported in the chemical literature, and it was named isosinularone I. The absolute configuration at C-4 was tentatively assigned to be the same as that of 10 based on its similar specific rotation ${[\alpha ]}_{\text{D}}^{25}+6$, MeOH).

All of the compounds obtained from the S. verruca extract were tested for anti-HIV-1 activity in a cell-based HIV screening assay.^21–24 Compounds 4, 5, and 16 were found to be moderately protective against the cytopathic effects of in vitro HIV-1 infection with EC₅₀ values of 34, 5.8, and 30 μM, respectively, and maximum protection rates of 86%, 52%, and 75%, respectively. However, their IC₅₀ values for cytotoxicity against the CEM-SS host cells were determined to be 79, 6.3, and 66 μM, indicating low therapeutic indices. The other compounds tested showed no detectable antiviral activity in this assay.

In addition, all compounds were evaluated for their in vitro anti-inflammatory activities.²⁵ In the primary assay, 4, 6, and 16 showed moderate inhibition against lipopolysaccharide-induced NO production in mouse peritoneal macrophages with IC₅₀ values of 28, 24, and 26 μM, respectively, whereas no inhibitory effect (inhibition <50%) was observed for the other compounds at 30 μM.

EXPERIMENTAL SECTION

General Experimental Procedures.

Optical rotations, UV, and IR spectra were measured using a PoLAAR 3005 digital polarimeter, a TU 1901 spectrometer, and a Bruker Equinox 55 spectrometer, respectively. ¹H and ¹³C NMR spectra were acquired with a Bruker Avance-600FT NMR spectrometer using TMS as an internal standard. HRESIMS data were obtained using a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Column chromatography was performed using silica gel (200–300 mesh, Qingdao Marine Chemistry Co. Ltd.), Sephadex LH-20 (GE Healthcare Biosciences AB), and ODS (50 μm, YMC). Silica gel GF₂₅₄ (Qingdao Marine Chemistry Co. Ltd.) was used for TLC analysis. HPLC separation was performed using an Agilent 1100 series instrument equipped with a VWD G1314A detector and a YMC-Pack C₁₈ column (10 μm, 250 × 10 mm).

Animal Material.

Specimens of the soft coral Sinularia verruca van Ofwegen, 2008, were collected from the inner coral reef of Ximao Island, Hainan Province, China, in November 2011, at a depth of 8 m and frozen immediately after collection. The specimen was identified by one of the authors (X.L.). A voucher specimen (HS201102) was deposited at the Laboratory of Marine Natural Products Chemistry, Wenzhou Medical University, China.

Extraction and Isolation.

The frozen soft coral S. verruca (wet weight: 1.20 kg) was homogenized and extracted with 95% EtOH at room temperature (rt). The concentrated extract was partitioned between H₂O and EtOAc. Evaporation of EtOAc in vacuo yielded a dark residue of 22.5 g. The EtOAc fraction (22.0 g) was subjected to silica gel vacuum column chromatography, eluting with a gradient of EtOAc/petroleum ether (1:20, 1:10, 1:5, 1:3, and 1:2), to obtain eight fractions (A1–A8). Fraction A3 (700.2 mg) was chromatographed on a Sephadex LH-20 column, eluting with CH₂Cl₂/MeOH (1:1), to afford four fractions (A3a–A3d). Fraction A3b (100.5 mg) was further separated on an ODS column, eluting with MeOH/H₂O (75:25, 80:20), to afford 8 (6.4 mg) and 11 (26.1 mg). Fraction A3d (60.0 mg) was purified by semipreparative HPLC, using MeOH/H₂O (75:25) as eluent, to yield 5 (6.0 mg) and 15 (22.5 mg). Fraction A4 (1.1 g) was subjected to a Sephadex LH-20 column, eluting with CH₂Cl₂/MeOH (1:1), to obtain three fractions (A4a–A4c). Fraction A4c (798.1 mg) was further separated on an ODS column, eluting with MeOH/H₂O (70:30, 75:25, 80:20, 85:15, and 90:10), to afford seven fractions (A4c1–A4c7). Fraction A4c2 (40.0 mg) was purified by HPLC (MeOH/H₂O, 60:40) to yield 9 (3.5 mg) and 1 (1.5 mg). Fraction A4c3 (19.8 mg) was purified by HPLC (MeOH/H₂O, 62:38) to afford 6 (3.3 mg). Fraction A4c4 (30.2 mg) was purified by HPLC (MeOH/H₂O, 70:30) to yield 13 (14.0 mg) and 14 (2.2 mg). Fraction A4c5 (70.3 mg) was purified by HPLC (MeOH/H₂O, 70:30) to obtain (Z)-N-(3-hydroxy-4-methoxyphenethyl)-3-methyldodec-2-enamide (5.0 mg), 3 (6.3 mg), and 7 (2.6 mg). Fraction A4c6 (90.2 mg) was purified by HPLC (MeOH/H₂O, 80:20) to obtain 17 (5.2 mg), 12 (11.3 mg), and (Z)-N-(4-hydroxyphenethyl)-3-methyldodec-2-enamide (23.5 mg). Fraction A6 (1.5 g) was subjected to a Sephadex LH-20 column, using CH₂Cl₂/MeOH (1:1) as eluent, to obtain three fractions (A6a–A6c). Fraction A6c (580.1 mg) was fractionated on an ODS column, eluting with MeOH/H₂O (65:35, 70:30, 75:25, and 80:20), to afford five fractions (A6c1–A6c5). Fraction A6c2 (19.3 mg) was purified by HPLC (MeOH/H₂O, 70:30) to yield 16 (2.6 mg) and 2 (2.0 mg). Following the same protocol as for fraction A6c2, 4 (1.2 mg) and 10 (1.3 mg) were separated from fraction A6c4 (10.5 mg) using MeOH/H₂O (80:20) as a mobile phase.

Verrupyrroloindoline (1): colorless oil; ${[\alpha ]}_{\text{D}}^{25}-22$ (c 0.06, CHCl₃); UV (MeOH) λ_max (log ε) 205 (3.96), 242 (3.55), 288 (3.76); IR (KBr) v_max 3437, 2932, 2873, 1677, 1607, 1466, 1383, 1237, 1148, 1105, 1048, 756 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 323.1369 [M + Na]⁺ (calcd for C₁₇H₂₀N₂O₃Na, 323.1372).

Verrubenzospirolactone (2): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+14$ (c 0.10, CHCl₃); UV (MeOH) λ_max (log ε) 203 (4.68), 296 (3.37); IR (KBr) ν_max 3444, 2929, 2862, 1743, 1647, 1610, 1512, 1450, 1413, 1381, 1186, 1049 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 375.1567 [M + Na]⁺ (calcd for C₂₂H₂₄O₄Na, 375.1572).

10-Deoxocapillosanane D (3): colorless oil; ${[\alpha ]}_{\text{D}}^{25}-230$ (c 0.04, CHCl₃); UV (MeOH) λ_max (log ε) 240 (3.73); IR (KBr) ν_max 3432, 2925, 1645, 1459, 1385, 1051, 997 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 203.1791 [M + H − H₂O]⁺ (calcd for C₁₅H₂₃, 203.1800).

(4S*,5S*)-4-Hydroxy-5-(hydroxymethyl)-2,3-dimethyl-4-pentylcy-clopent-2-en-1-one (4): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+19$ (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 232 (3.68); IR (KBr) ν_max 3393, 2923, 2855, 1693, 1647, 1382, 1048 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 249.1465 [M + Na]⁺ (calcd for C₁₃H₂₂O₃Na, 249.1467).

(S)-4-Hydroxy-5-methylene-2,3-dimethyl-4-pentylcyclopent-2-en-1-one (5): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+6$ (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 259 (3.72); ECD (MeOH) 257 (Δε +2.53) nm; IR (KBr) ν_max 3454, 2927, 2861, 1694, 1631, 1386, 1033 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 231.1359 [M + Na]⁺ (calcd for C₁₃H₂₀O₂Na, 231.1361).

(4S,5S)-4-Hydroxy-4-(ethylpropanoate)-2,3-dimethyl-5-butylcy-clopent-2-en-1-one (6): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+14$ (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 229 (3.77); IR (KBr) ν_max 3446, 2930, 2866, 1776, 1707, 1650, 1461, 1384, 1165, 1018 cm⁻¹; ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 305.1726 [M + Na]⁺ (calcd for C₁₆H₂₆O₄Na, 305.1729).

Sinularone D (8): colorless oil; ${[\alpha ]}_{\text{D}}^{25}-5$ (c 0.10, MeOH); lit. ${[\alpha ]}_{\text{D}}^{23}-3$ (c 0.09, MeOH).¹⁶

(4S*,5S*)-4-Hydroxy-5-(ethoxymethyl)-2,3-dimethyl-4-pentylcy-clopent-2-en-1-one (16): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+7$ (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 233 (3.63); IR (KBr) ν_max 3440, 2928, 2865, 1700, 1651, 1458, 1382, 1110, 1042 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 3.96 (1H, dd, J = 9.6, 4.0 Hz, H-6a), 3.54 (2H, q, J = 6.6 Hz), 3.52 (1H, dd, J = 10.2, 9.6 Hz, H-6b), 2.84 (1H, dd, J = 10.2, 4.0 Hz, H-5), 1.98 (3H, s, H₃-13), 1.86 (1H, ddd, J = 13.2, 12.0, 4.2 Hz, H-7a), 1.78 (1H, ddd, J = 13.2, 12.6, 5.4 Hz, H-7b), 1.71 (3H, s, H₃-12), 1.22 (3H, t, J = 6.6 Hz), 1.23 (2H, m, H-9), 1.23 (2H, m, H-10), 0.85 (1H, m, H-8a), 0.85 (3H, t, J = 6.6 Hz, H₃-11), 0.80 (1H, m, H-8b); ¹³C NMR (150 MHz, CDCl₃) δ 202.7 (C, C-1), 168.2 (C, C-3), 136.1 (C, C-2), 81.3 (C, C-4), 66.8 (CH₂, C-6), 66.8 (CH₂), 59.4 (CH, C-5), 36.6 (CH₂, C-7), 32.1 (CH₂, C-9), 25.2 (CH₂, C-8), 22.4 (CH₂, C-10), 15.2 (CH₃), 14.0 (CH₃, C-11), 11.3 (CH₃, C-13), 7.8 (CH₃, C-12); HRESIMS m/z 277.1776 [M + Na]⁺ (calcd for C₁₅H₂₆O₃Na, 277.1780).

Isosinularone I (17): colorless oil; ${[\alpha ]}_{\text{D}}^{25}+6$ (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 204 (4.24), 228 (sh); IR (KBr) ν_mal 3448, 2926, 2861, 1727, 1636, 1383, 1277, 1120, 1074 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 3.40 (1H, dq, J = 12.0, 6.6 Hz), 3.14 (1H, dq, J = 12.0, 6.6 Hz), 2.34 (2H, m, H₂-16), 1.98 (1H, ddd, J = 13.8, 12.0, 3.6 Hz, H-5a), 1.86 (3H, s, H₃-19), 1.84 (3H, s, H₃-18), 1.67 (1H, m, H-5b), 1.62 (2H, m, H₂-15), 1.30 (2H, m, H-6), 1.25 (16H, H₂-7-H₂-14), 1.17 (3H, t, J = 6.6 Hz); ¹³C NMR (150 MHz, CDCl₃) δ 178.4 (C, C-17), 172.0 (C, C-1), 156.6 (C, C-3), 126.9 (C, C-2), 110.1 (C, C-4), 35.8 (CH₂, C-5), 34.1 (CH₂, C-16), 29.5 (CH₂, C-7), 29.5 (CH₂, C-8), 29.5 (CH₂, C-9), 29.5 (CH₂, C-10), 29.4 (CH₂, C-11), 29.4 (CH₂, C-12), 29.2 (CH₂, C-13), 29.1 (CH₂, C-14), 24.9 (CH₂, C-15), 22.6 (CH₂, C-6), 10.9 (CH₃, C-19), 8.4 (CH₃, C-18); HRESIMS m/z 367.2495 [M − H]⁻ (calcd for C₂₁H₃₅O₅, 367.2484).

Conformational Modeling.

The electrostatic potential of molecule 4b was calculated at the Hartree–Fock level with the 6–31G* basis set using Gaussian09. The general AMBER force field (gaff) and the ff99SB force field were used for 4b and CHCl₃ molecules, respectively. Molecule 4b was solvated in a box of CHCl₃ molecules with a shell of 10 Å. Energy minimization and molecular dynamic (MD) simulation were performed with the AMBER 11 packages. First, in order to remove bad contacts, the whole system was subjected to 500 steps of minimization without any restriction. Then, the system was heated gradually from 0 to 300 K in the NVT ensemble for 100 ps and equilibrated at 300 K for 100 ps prior to MD simulation. The 500 ps of MD simulation was accomplished at 300 K under a constant pressure of 1 bar. The particle mesh Ewald method was employed to deal with the long-range electrostatic interactions under periodic boundary conditions. The SHAKE method was used to constrain hydrogen atoms, and the time step was set to 2 fs. Finally, Ptraj, a module of AMBER 11 packages, was used to monitor the distance between H₂-6 and H₂-7 in 4b.

ECD Calculation.

The MD simulations were accomplished for a pair of enantiomers of 5 following the same protocol as depicted for 4b except that methanol was used as solvent for 5. The lowest energy conformers were optimized by CAM-B3LYP/6–311++G** in methanol. The solvent effects were included using the polarizable continuum model. The theoretical ECD spectra were obtained according to Boltzmann weighting of each conformer.

Anti-HIV-1 Activity Assay.

The XTT-tetrazolium primary NCI screen for anti-HIV activity was performed as described previously.^21–24 The CEM-SS human lymphocytic target cell line was maintained in RPMI 1640 medium (Gibco) without phenol red and supplemented with 5% fetal bovine serum, 50 μg/mL gentamicin, and 2 mM l-glutamine (complete medium). Exponentially growing cells were pelleted and resuspended in complete medium at a concentration of 2 × 10⁵ cells/mL. Frozen virus stock solution (Haitian variant HIV-1_RF, 3.54 × 10⁶ SFU/mL) was thawed immediately prior to use and resuspended in complete medium to yield 1.2 × 10⁵ SFU/mL. Each test compound was dissolved in 100% DMSO and then diluted to the desired initial concentrations in complete medium.

Uninfected CEM-SS cells were plated in 50 μL of complete medium at a density of 1 × 10⁴ cells. Diluted HIV-1 virus was then added in a volume of 50 μL to yield a multiplicity of infection of 0.6. Appropriate cell, virus, and positive control (AZT) were incorporated in each experiment, and the final volume in each microtiter well was 200 μL. Quadruplicate and duplicate wells were used for virus-infected and uninfected cells, respectively. Plates were incubated for 6 days at 37 °C in an atmosphere containing 5% CO₂. Subsequently, aliquots of cell-free supernatant were removed from each well. Cellular growth then was estimated on the remaining contents of each well using the XTT assay as previously described.^23,24

Assay for Inhibition of Nitric Oxide Production.

A previously established protocol²⁵ was followed except that 30 mM dexamethasone in DMSO was used as the positive control and each test compound (30 mM in DMSO) was diluted to 1–30 μM before the experiment.

Chart 1.