Cembranoids from a Chinese Collection of the Soft Coral Lobophytum crassum.

Ten new cembrane-based diterpenes, locrassumins A-G (1-7), (-)-laevigatol B (8), (-)-isosarcophine (9), and (-)-7R,8S-dihydroxydeepoxysarcophytoxide (10), were isolated from a South China Sea collection of the soft coral Lobophytum crassum, together with eight known analogues (11-18). The structures of the new compounds were determined by extensive spectroscopic analysis and by comparison with previously reported data. Locrassumin C (3) possesses an unprecedented tetradecahydrobenzo[3,4]cyclobuta[1,2][8]annulene ring system. Compounds 1, 7, 12, 13, and 17 exhibited moderate inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC50 values of 8-24 μM.

1. Introduction

Soft corals of the genus Lobophytum (family Alcyoniidae) have proven to be a rich source of structurally diverse diterpenes, especially macrocyclic cembranoids characterized by their 14-membered carbocyclic skeleton. To date, numerous marine cembranoids and novel derivatives (mainly formed by dimerization, cyclic addition, or ring rearrangement) have been isolated from Lobophytum species and other genera including Sinularia and Sarcophyton [1]. Some of these metabolites merit further study because of their significant ecological and pharmacological bioactivities, such as antifouling, antifeeding, cytotoxic, antibacterial, antiviral, and anti-inflammatory properties [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. Species L. crassum is widely distributed in the tropical waters of the world and is well known to produce a variety of oxygenated cembranoids, the structural variety of which is often correlated with geographic variation and environmental conditions [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. However, the soft coral L. crassum in the South China Sea has been rarely examined chemically [22,23,24]. In the course of our investigation of bioactive substances produced by marine invertebrates from the South China Sea [25,26,27], a specimen of L. crassum was collected. Chemical examination of this specimen led to the isolation of 18 cembrane-based diterpenes, including nine new cembranoids (1, 2, and 4–10), an unprecedented diterpene possessing a tetradecahydrobenzo[3,4]cyclobuta[1,2][8]annulene ring system (3), and eight known analogues (11–18) (Figure 1). All compounds were tested for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMФ). This paper reports details of the isolation, structure elucidation, and biological evaluation of these compounds.

Figure 1

Structures of compounds 1–18.

2. Results and Discussion

Locrassumin A (1) was assigned a molecular formula of C22H32O5 according to its HRESIMS (m/z 399.2134 [M + Na]+, calcd for C22H32O5Na, 399.2147) and NMR data (Figures S1–S7). The 1H NMR spectrum showed signals for three olefinic protons (δH 7.70 d, J = 12.0 Hz, H-3; 6.80 t, J = 7.2 Hz, H-11; 6.24 d, J = 12.0 Hz, H-2), two methoxy groups (δH 3.77 s, H3-21; 3.75 s, H3-22), and three additional methyls (δH 1.18 s, H3-19; 1.00 d, J = 6.6 Hz, H3-16; 0.95 d, J = 6.6 Hz, H3-17), while the 13C NMR spectrum exhibited 22 carbon signals including two ester carbonyls, six olefinic carbons, and two carbons indicative of an epoxide (Table 1 and Table 2). These NMR data were very similar to those of the known cembranoids sarcrassin A [28] and sarcophytonolides B [29] and O (13) [30]. Detailed analysis of COSY and HMBC correlations (Figure 2) confirmed that 1 shared the same planar structure with those three analogues. The NOE correlations of H-3/H-15 (δH 3.20 m), H-2/H-5b (δH 2.55 m), H-2/H-14a (δH 2.42 m), and H3-19/H-6b (δH 1.57 m) (Figure 3) revealed that the geometries of the C-1/C-2 and C-3/C-4 double bonds and configurations of the 7,8-epoxy ring in 1 were identical to those in sarcrassin A [28]. In addition, the NOE correlation of H-10b (δH 2.15 m)/H-13a (δH 2.63 m) and the lack of an NOE correlation between H-11 and H2-13 revealed E geometry for the C-11/C-12 double bond. Thus, 1 was established as the 11E isomer of sarcrassin A [28].

Table 1

1H NMR data for 1–7 (CDCl3, 600 MHz) a.

No.	1	2	3	4	5	6	7
2	6.24, d (12.0)	6.23, d (12.0)	5.31, d (3.0)	6.02, dd (11.4, 1.8)	6.06, dd (11.4, 1.8)	5.29, d (10.8)	5.44, br d (9.6)
3	7.70, d (12.0)	7.62, d (12.0)	3.17, d (3.0)	5.87, d (11.4)	5.96, d (11.4)	5.19, d (10.8)	5.08, d (9.6)
5	2.63, m	2.66, m	1.92, m	2.28, m	2.26, m	2.40, m	2.32, m
	2.55, m	2.63, m	1.75, m	1.98, m	2.01, m	2.37, m	2.15, m
6	2.07, m	1.91, m	1.74, m	2.27, m	2.28, m	1.95, m	1.86, td (13.8, 3.0)
	1.57, m	1.71, m	1.57, m	2.04, m	2.03, m	1.92, m
							1.35, m
7	2.87, dd (9.6, 3.0)	2.64, m	3.58, d (10.8)	5.11, dd (10.2, 5.4)	5.09, dd (10.2, 4.8)	2.64, br t (3.6)	3.34, d (10.8)
9	1.82, m	2.05, m	1.97, m	2.25, m	2.20, m	2.15, m	2.31, m
	1.49, m	1.19, m	1.66, m	2.15, m	2.10, m	0.95, t (13.2)
10	2.18, m	2.12, m	1.87, m	1.82, m	1.82, m	2.28, m	5.53, m
	2.15, m	2.00, m	1.69, m	1.35, m	1.20, m	1.90, m
11	6.80, t (7.2)	6.60, dd (7.2, 4.2)	3.24, dd (7.2, 5.4)	3.79, br d (10.8)	3.44, br d (10.8)	5.12, dd (9.0, 6.6)	5.52, d (18.6)
13	2.63, m	2.69, m	2.08, m	2.26, m	2.08, m	2.29, m	1.67, m
	2.42, m	2.43, m	1.78, m	1.94, m	1.95, ddd (15.0, 6.0, 2.4)	1.99, m	1.53, m
14	2.42, m	2.83, m	1.94, m	2.98, m	2.84, ddd (15.0, 11.4, 6.0)	1.91, m	2.14, m
	2.28, m	2.37, m	1.72, m	2.17, m		1.71, m	1.69, m
					2.26, m
15	3.20, m	2.17, m	2.22, m	3.43, q (7.2)
16	1.00, d (6.6)	1.00, d (6.6)	1.00, d (6.6)	1.47,d (7.2)	1.49, s		4.51, d (11.4)
							4.46, d (11.4)
17	0.95, d (6.6)	1.08, d (6.6)	1.01, d (6.6)		3.82, d (12.6)	1.55, s	1.65, s
					3.44, d (12.6)
18				1.79, s	1.79, s	1.88, s	1.74, s
19	1.18, s	1.12, s	1.14, s	1.30, s	1.34, s	1.28, s	1.15, s
20				1.28, s	0.99, s	1.58, s	1.33, s
21	3.77, s	3.77, s	3.76, s				3.23, s
22	3.75, s	3.76, s	3.68, s

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

Table 2

13C NMR data for 1–7 (CDCl3, 150 MHz) a.

No.	1	2	3	4	5	6	7
1	158.8, C	158.7, C	148.3, C	133.7, C	140.9, C	71.7, C	132.4, C
2	120.4, CH	118.8, CH	113.8, CH	125.7, CH	120.4, CH	78.0, CH	84.0, CH
3	134.3, CH	136.6, CH	42.0, CH	121.7, CH	122.8, CH	119.8, CH	126.8, CH
4	127.9, C	127.7, C	50.0, C	138.2, C	137.3, C	144.4, C	139.6, C
5	23.2, CH2	23.6, CH2	24.4, CH2	40.4, CH2	40.5, CH2	37.7, CH2	35.6, CH2
6	27.2, CH2	26.8, CH2	29.9, CH2	26.4, CH2	26.3, CH2	25.3, CH2	26.2, CH2
7	60.7, CH	62.7, CH	73.7, CH	125.0, CH	124.9, CH	61.7, CH	71.2, CH
8	60.9, C	61.1, C	74.9, C	136.3, C	136.5, C	59.7, C	78.4, C
9	36.2, CH2	36.3, CH2	39.3, CH2	35.5, CH2	36.3, CH2	40.0, CH2	36.7, CH2
10	24.3, CH2	24.0, CH2	20.4, CH2	26.3, CH2	27.0, CH2	23.7, CH2	124.4, CH
11	142.5, CH	144.4, CH	43.5, CH	67.9, CH	70.8, CH	124.5, CH	138.9, CH
12	132.4, C	130.1, C	45.2, C	87.0, C	80.2, C	135.2, C	73.0, C
13	27.7, CH2	27.0, CH2	25.3, CH2	32.9, CH2	30.9, CH2	34.8, CH2	41.3, CH2
14	30.0, CH2	28.9, CH2	23.2, CH2	22.1, CH2	22.6, CH2	27.0, CH2	21.8, CH2
15	29.8, CH	36.8, CH	35.6, CH	51.0, CH	77.6, C	60.7, C	127.9, C
16	21.2, CH3	22.3, CH3	21.4, CH3	16.3, CH3	28.0, CH3	172.8, C	78.5, CH2
17	20.4, CH3	20.8, CH3	20.7, CH3	177.8, C	73.6, CH2	9.9, CH3	10.1, CH3
18	168.8, C	168.5, C	177.4, C	16.5, CH3	16.3, CH3	16.2, CH3	15.5, CH3
19	17.9, CH3	16.9, CH3	21.1, CH3	14.8, CH3	14.9, CH3	16.7, CH3	17.8, CH3
20	168.0, C	167.8, C	176.7, C	23.1, CH3	19.0, CH3	14.8, CH3	26.9, CH3
21	51.8, CH3	51.7, CH3	52.1, CH3				49.2, CH3
22	51.8, CH3	51.8, CH3	52.0, CH3

a The assignments were based on HMQC, HMBC, and COSY spectra.

Figure 2

Key COSY and HMBC correlations for 1 and 3–7.

Figure 3

Key NOE correlations and computer-generated models using MM2 force field calculations for 1–7.

Locrassumin B (2) was also isomeric with sarcrassin A [28] and sarcophytonolides B [29] and O (13) [30] based on the compatible HRESIMS (m/z 399.2135 [M + Na]+, calcd for C22H32O5Na, 399.2147) and 1D and 2D NMR data (Figures S8–S14). The NOE correlations of H-2 (δH 6.23 d, J = 12.0 Hz)/H-15 (δH 2.17 m), H-3 (δH 7.62 d, J = 12.0 Hz)/H-5a (δH 2.66 m), H-3/H-14a (δH 2.83 m), and H3-19 (δH 1.12 s)/H-6b (δH 1.71 m) were indicative of 1E, 3Z, 7R*, and 8R* configurations (Figure 3), consistent with those of sarcophytonolide O (13) [30], while the NOE correlation of H-10a (δH 2.12 m)/H-13a (δH 2.69 m) and the lack of NOE correlation between H-11 and H2-13 allowed for the assignment of 11E, instead of 11Z as in 13. Thus, 2 was elucidated as the 11E isomer of sarcophytonolide O (13) [30].

Locrassumin C (3) had a molecular formula of C22H34O6 as determined by HRESIMS (m/z 417.2251 [M + Na]+, calcd for C22H34O6Na, 417.2253) and NMR data, requiring six degrees of unsaturation (Figures S15–S21). The IR absorptions at 3450 and 1726 cm−1 indicated the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum showed 22 carbon signals including two ester carbonyls (δC 177.4 and 176.7) and two olefinic carbons (δC 148.3, C; 113.8, CH) (Table 2), which accounted for three of the six degrees of unsaturation. Thus, 3 had to be tricyclic. COSY correlations established the subunits from C-2 to C-3, C-5 to C-7, C-9 to C-11, and C-13 to C-14, while their connectivities were completed by detailed analysis of HMBC correlations (Figure 2). The HMBC correlations from the olefinic proton H-2 (δH 5.31 d, J = 3.0 Hz), the aliphatic methine proton H-3 (δH 3.17 d, J = 3.0 Hz), and H2-13 (δH 2.08 m; 1.78 m) to the non-protonated carbon C-12 (δC 45.2), from H-3 and H2-13 to the non-protonated olefinic carbon C-1 (δC 148.3), and from H-2 to C-14 (δC 23.2, CH2) led to the establishment of a cyclohexene ring. The HMBC correlations from H3-19 (δH 1.14 s) to C-9 (δC 39.3, CH2) and two oxygenated carbons C-7 (δC 73.7, CH) and C-8 (δC 74.9, C) and from the aliphatic methine proton H-11 (δH 3.24 dd, J = 7.2, 5.4 Hz) and H2-6 (δH 1.74 m; 1.57 m) to another non-protonated carbon C-4 (δC 50.0) constructed a cyclooctane ring and revealed that C-7 and C-8 were hydroxylated and C-8 was also substituted by a methyl group. In addition, the HMBC correlations from H-3 to C-4 and from H-11 to C-12 finally connected the cyclohexene and cyclooctane rings to form an 8,4,6-tricarbocyclic nucleus. Further HMBC correlations from H-3 and H-11 to the two ester carbonyl carbons C-18 (δC 177.4) and C-20 (δC 176.7), from the methoxy protons H3-21 (δH 3.76 s) and H3-22 (δH 3.68 s) to C-18 and C-20, respectively, and from H2-13 to C-20 disclosed that C-4 and C-12 were substituted by methyl esters. Direct linkage of an isopropyl group to C-1 was inferred by the HMBC correlations from H3-16 (δH 1.00 d, J = 6.6 Hz) and H3-17 (δH 1.01 d, J = 6.6 Hz) to the non-protonated olefinic carbon C-1. Thus, the planar structure of 3 was established as depicted in Figure 1.

The relative configuration of 3 was determined on the basis of coupling constant and NOESY analysis (Figure 3). The NOE correlations of H-2/H-5a (δH 1.92 m), H-3/H-13b (δH 1.78 m), H-3/H-10a (δH 1.87 m), and H-11/H-5b (δH 1.75 m) suggested the trans fusions of the ring system and the opposite orientation of H-3 and H-11. In addition, the coupling pattern of H-7 (δH 3.58 d, J = 10.8 Hz) indicated its axial orientation (one large coupling due to dihedral angles of approximately 180° and 90° with the H2-6 protons), while the NOE correlations of H-7/H3-19, H-7/H-10a, and H3-19/H-10a suggested the same orientation of H-7, H3-19, and H-10a. Furthermore, the absolute configuration of the 7,8-diol was determined by an in situ dimolybdenum CD method [31,32], based on which the sign of the induced CD (ICD) bands at 310, 350, and 400 nm reflected the O-C-C-O torsion angle. After addition of dimolybdenum tetraacteate [Mo2(OAc)4] into a DMSO solution of 3, a metal complex was generated immediately and the ICD spectrum was acquired. The positive CD effects observed at 318 and 357 nm (Figure 4) allowed for the assignment of the 7R and 8S configurations. Accordingly, the configurations of the remaining chiral centers of 3 were assigned as 3R, 4R, 11R, and 12R.

Figure 4

ICD curve of 3 induced by Mo2(OAc)4 in DMSO.

It is interesting to note that 3 represents an unprecedented diterpenoid with a tetradecahydrobenzo[3,4]cyclobuta[1,2][8]annulene ring system, which could be derived from the coisolated cembranoids locrassumins A (1), B (2), or sarcophytonolide O (13) [30] via a series of isomerization, intramolecuar [2 + 2] cycloaddition between C-3/C-4 and C-11/C-12 double bonds [2], and hydrolysis reactions.

The molecular formula of locrassumin D (4) was determined to be C20H30O3 on the basis of HRESIMS (m/z 341.2094 [M + Na]+, calcd for C20H30O3Na, 341.2093) and NMR data, implying six degrees of unsaturation (Figures S22–S28). The IR absorption at 1707 cm−1 indicated the presence of a carbonyl functionality. A carbonyl carbon (δC 177.8) and six olefinic carbons were evident by 13C NMR data (Table 2), requiring a bicyclic structure for the remaining two degrees of unsaturation. A conjugated diene was easily recognized by a COSY correlation between the two olefinic protons H-2 (δH 6.02 dd, J = 11.4, 1.8 Hz) and H-3 (δH 5.87 d, J = 11.4 Hz) as well as the HMBC correlations from H3-18 (δH 1.79 s) to two olefinic carbons C-3 (δC 121.7, CH) and C-4 (δC 138.2, C) and from H-3 to C-1 (δC 133.7, C). Further COSY correlations established the other four subunits from C-5 to C-7, C-9 to C-11, C-13 to C-14, and C-15 to C-16, while HMBC correlations from H3-18 to a methylene carbon C-5 (δC 40.4, CH2), from H3-19 (δH 1.30 s) to two olefinic carbons C-7 (δC 125.0, CH) and C-8 (δC 136.3, C) and a methylene carbon C-9 (δC 35.5, CH2), from H3-20 (δH 1.28 s) to two oxygenated carbons C-11 (δC 67.9, CH) and C-12 (δC 87.0, C) and a methylene carbon C-13 (δC 32.9, CH2), from H3-16 (δH 1.47 d, J = 7.2 Hz) to the ester carbonyl C-17 (δC 177.8) and C-1, and from H-2 to a methylene carbon C-14 (δC 22.1, CH2) (Figure 2) finally connected the subunits to form a cembrane skeleton, in which C-1/C-2, C-3/C-4, and C-7/C-8 formed double bonds and C-11 was hydroxylated. In addition, an ester linkage between C-12 and C-17 could be inferred by the chemical shifts of C-12 and C-17 and the remaining one degree of unsaturation in the molecule. The geometries of 3E and 7E were indicated by the diagnostic chemical shifts of C-18 and C-19 (<20 ppm) [33,34] and confirmed by the NOE correlations of H-3/H-5b (δH 1.98 m) and H-7 (δH 5.11 dd, J = 10.2, 5.4 Hz)/H-9a (δH 2.25 m), respectively. The coupling constant value of JH-2/H-3 (11.4 Hz) and NOE correlations of H-2/H3-18 and H-3/H-14a (δH 2.98 m) suggested the trans-axial orientation of H-2 and H-3, and 1E geometry. An NOE correlation of H-2/H-15 (δH 3.43 q, J = 7.2 Hz) established that these two protons were on the same face of the molecule and thus H-15 had an α-orientation (Figure 3). Furthermore, the significant NOE correlations of H-11 (δH 3.79 br d, J = 10.8 Hz)/H-3 and H-11/H-14a, and the lack of an NOE correlation between H-11 and H3-20 were in agreement with the 11R* and 12S* configurations. Thus, 4 was elucidated as (11R*,12S*,15R*,1E,3E,7E)-11-hydroxycembra-1,3,7-trien-17,12-olide.

Locrassumin E (5) had a molecular formula of C20H32O3 as determined by HRESIMS (m/z 343.2247 [M + Na]+, calcd for C20H32O3Na, 343.2249) and NMR data, indicating five degrees of unsaturation (Figures S29–S35). The preliminary analysis of 1D NMR data revealed that compound 5 had a structure closely related to that of 4. The only difference was loss of signals for a methyl doublet, an ester carbonyl, and an aliphatic methine and the appearance of signals for an additional methyl singlet (δH 1.49 s, H3-16) and two additional oxygenated sp3 carbons (δC 77.6, C, C-15; 73.6, CH2, C-17). Thus, 5 should be a 17-deoxo-15-hydroxy derivative of 4. This assumption was supported by the HMBC correlations from the methyl singlet H3-16 to an olefinic carbon C-1 (δC 140.9, C) and the two oxygenated carbons C-15 and C-17 (Figure 2). The ether linkage between C-12 and C-17 was confirmed by HMBC correlations from the oxymethylene protons H2-17 (δH 3.82 d, J = 12.6 Hz; 3.44 d, J = 12.6 Hz) to the oxygenated carbon C-12 (δC 80.2, C). The geometries of C-3/C-4 and C-7/C-8 double bonds and relative configurations at C-11 and C-12 were consistent with those in 4 as indicated by the similar chemical shifts of C-18 and C-19 and compatible NOE relationships [H-3 (δH 5.96 d, J = 11.4 Hz)/H-5b (δH 2.01 m), H-7 (δH 5.09dd, J = 10.2, 4.8 Hz)/H-9a (δH 2.20 m), and H-11 (δH 3.44 br d, J = 10.8 Hz)/H-3], as well as the lack of NOE correlation between H3-20 and H-11 (Figure 3). In addition, the coupling constant value of JH-2/H-3 (11.4 Hz) and the NOE correlations of H-3/H-14a (δH 2.84 ddd, J = 15.0, 11.4, 6.0 Hz), H-2 (δH 6.06 dd, J = 11.4, 1.8 Hz)/H3-18 (δH 1.79 s), and H-2/H3-16 allowed the assignment of 1E and α-orientation of H3-16. Thus, 5 was determined as (11R*,12S*,15R*,1E,3E,7E)-12,17-epoxycembra-1,3,7-trien-11,15-diol.

The molecular formula of locrassumin F (6) was determined to be C20H28O4 by its HRESIMS (m/z 355.1883 [M + Na]+, calcd for C20H28O4Na, 355.1885) and NMR data, requiring seven degrees of unsaturation (Figures S36–S42). The NMR data of 6 (Table 1 and Table 2) were found to be very similar to those of the co-occurring analogue ent-sarcophine (12) [35]. The only difference was attributed to the absence of a tetrasubstituted double bond, while presenting an additional tetrasubstituted epoxy (δC 71.7, C, C-1; 60.7, C, C-15), indicating that 6 was probably a C-1/C-15 epoxylation derivative of 12. The presence of an α,β-epoxy-γ-lactone was further supported by the HMBC correlations from H3-17 (δH 1.55 s) to the carbonyl carbon C-16 (δC 172.8) and two oxygenated carbons C-1 and C-15 and from the oxymethine proton H-2 (δH 5.29 d, J = 10.8 Hz) to C-16, while the substructure from C-3 to C-13 was established as identical to that in 12 based on the HMBC and COSY correlations as depicted in Figure 2. The relative configurations at C-1, C-7, and C-8, as well as the geometries of the two double bonds were assigned similarly to those in 12 by the compatible NMR data including the NOE relationships of H-2/H3-18 (δH 1.88 s), H-3 (δH 5.19 d, J = 10.8 Hz)/H-5b (δH 2.37 m), H-3/H-7 (δH 2.64 br t, J = 3.6 Hz), H-7/H-9b (δH 0.95 t, J = 13.2 Hz), H-11 (δH 5.12 dd, J = 9.0, 6.6 Hz)/H-13b (δH 1.99 m), and the lack of NOE correlations of H-7/H3-19 and H-11/H3-20. In addition, the NOE correlations of H-3/H-14a (δH 1.91 m) and the lack of an NOE correlation between H-2 and H2-14 suggested the α-orientation of C-1/C-15 epoxy ring. Thus, 6 was elucidated as (1R*,2R*,7R*,8R*,15R*,3E,11E)-7,8:1,15-diepoxycembra-3,11-dien-16,2-olide.

Locrassumin G (7) was assigned a molecular formula of C21H34O4 on the basis of HRESIMS (m/z 373.2351 [M + Na]+, calcd for C21H34O4Na, 373.2355) and NMR data, implying five degrees of unsaturation (Figures S43–S49). The 13C NMR spectrum showed six olefinic carbon signals (Table 2), requiring 7 to be a bicyclic molecule according to the remaining two degrees of unsaturation. A methyl-bearing dihydrofuranring was indicated by the 13C NMR signals at δC 132.4 (C, C-1), 127.9 (C, C-15), 84.0 (CH, C-2), 78.5 (CH2, C-16), 10.1 (CH3, C-17) and 1H NMR signals at δH 5.44 (br d, J = 9.6 Hz, H-2), 4.51 (d, J = 11.4 Hz, H-16a), 4.46 (d, J = 11.4 Hz, H-16b), 1.65 (s, H3-17), and further confirmed by the HMBC correlations from H3-17 to C-1, C-15, and C-16 and from the oxymethylene protons H2-16 to C-2 (Figure 2). The COSY correlation between H-2 and an olefinic proton H-3 (δH 5.08 d, J = 9.6 Hz) revealed that C-3/C-4 was located by a double bond. The other two olefinic carbons at δC 138.9 (CH, C-11) and 124.4 (CH, C-10) and two olefinic proton signals at δH 5.53 (m, H-10) and 5.52 (d, J = 18.6 Hz, H-11) were attributed to a 1,2-disubstituted double bond. These NMR data were similar to those of the known cembranoid (2S*,7S*,8S*,12R*,1Z,3E,10E)-7,8:2,16-diepoxycembra-1(15),3,10-trien-12-ol [27]. The difference arose from the absence of a trisubstituted epoxy in 7. The HMBC correlations from H3-19 (δH 1.15 s) to two oxygenated carbons C-7 (δC 71.2, CH) and C-8 (δC 78.4, C) and a methylene carbon C-9 (δC 36.7, CH2) and from the methoxy protons (δH 3.23 s, H3-21) to C-8 disclosed that C-7 and C-8 were substituted by a hydroxy and a methoxy group, respectively. Further HMBC correlations from H3-20 (δH 1.33 s) to an olefinic carbon C-11, an oxygenated carbon C-12 (δC 73.0, C), and a methylene carbon C-13 (δC 41.3, CH2) confirmed the location of a double bond at C-10/C-11 and C-12 was hydroxylated. The coupling constant value of JH-2/H-3 (9.6 Hz) and the NOE correlations of H-2/H3-18 (δH 1.74 s) and H-3/H-5a (δH 2.32 m) suggested the trans-axial orientation of H-2 and H-3 and 3E, which was also implied by the chemical shifts of C-18 (<20 ppm) [23,24], while the coupling constant value of JH-10/H-11 (18.6 Hz) indicated the 10E geometry. In addition, NOE correlations of H-3/H-14b (δH 1.69 m), H-7 (δH 3.34 d, J = 10.8 Hz)/H-14b, H-7/H-10, and H3-19/H-10, H3-20/H-10, and H3-20/H-14a (δH 2.14 m) were in agreement with the relative configurations of 7R*, 8S*, and 12S* (Figure 3). Thus, 7 was defined as (2R*,7R*,8S*,12S*,1Z,3E,10E)-8-methoxy-2,16-epoxycembra-1(15),3,10-trien-7,12-diol.

The spectroscopic data analysis and comparison of NMR and HRESIMS data revealed that the structures of compounds 8–10 were identical to the known cembranoids laevigatol B [36], (+)-isosarcophine [37], and (+)-7S,8R-dihydroxydeepoxysarcophytoxide [38], respectively. However, the antipodal specific rotations of 8–10 ( −17 (c 0.10, CHCl3); −263 (c 0.10, CHCl3); −99 (c 0.10, CHCl3), respectively) in comparison with those of the three known analogues ( +7.7 (c 1.00, CH2Cl2); +235.3 (c 0.14, CHCl3); +140.0 (c 0.48, CHCl3), respectively) suggested 8–10 to be enantiomers of the previously reported analogues, and named (–)-laevigatol B, (–)-isosarcophine, and (–)-7R,8S-dihydroxydeepoxysarcophytoxide, respectively.

Eight known compounds were also isolated from the L. crassum extract and identified as (–)-sarcophytoxide (11) [39], ent-sarcophine (12) [35], sarcophytonolide O (13) [30], sartrolide G (14) [40], emblide (15) [41], sarcrassin D (16) [28], ketoemblide (17) [42], and methyl sarcotroate B (18) [2], by comparison of their 1H and 13C NMR, MS spectroscopic data (Figures S50–S68), and specific rotations, with those reported in the literature.

All compounds were tested for their in vitro anti-inflammatory activities [43]. Nitric oxide (NO) is an important signaling molecule that is involved in the regulation of diverse physiological and pathological processes. Overproduction of NO is associated with various human diseases, particularly acute and chronic inflammation, while the level of NO can reflect the degree of inflammation. In the primary assay, compounds 1, 7, 12, 13, and 17 showed moderate inhibition against lipopolysaccharide-induced NO production in mouse peritoneal macrophages with IC50 values of 8–24 μM, whereas no inhibitory effect was observed for the other compounds (IC50 > 30 μM) (Table 3).

Table 3

Inhibitory Activity against LPS-Induced NO Production a.

Compound	IC50 (μM)	CC50 b (μM)
1	17 ± 3	>60.0
7	13 ± 2	>60.0
12	24 ± 2	>60.0
13	8 ± 1	>60.0
17	12 ± 2	>60.0

a The other compounds were inactive at 30 μM; b CC50: cytotoxicity against mouse peritoneal macrophages.

3. Materials and Methods

3.1. General Experimental Procedures

1H and 13C NMR spectra were acquired with a Bruker Avance-600FT NMR spectrometer (Bruker, Munich, Germany) using TMS as an internal standard. HRESIMS data were recorded using a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). UV spectra were measured with a TU 1901 spectrometer (Puxi Ltd., Beijing, China). IR spectra were obtained using a Bruker Equinox 55 spectrometer (Bruker, Munich, Germany). Optical rotations were measured with a PoLAAR 3005 digital polarimeter (Optical Activity Ltd., Cambridgeshire, UK). Silica gel (200–300 mesh, Qingdao Marine Chemistry Co. Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-sciences AB, Uppsala, Sweden), and ODS (50 μm, YMC, Tokyo, Japan) were used for column chromatography. TLC analysis was carried out using silica gel GF254 (Qingdao Marine Chemistry Co. Ltd., Qingdao, China). Semipreparative HPLC was performed using an Agilent 1100 series instrument equipped with a VWD G1314A detector (Agilent, Palo Auto, Santa Clara, CA, USA) and a YMC-Pack C18 column (10 μm, 250 × 10 mm, YMC, Tokyo, Japan).

3.2. Animal Material

Specimens of the soft coral Lobophytum crassum von Marenzeller, 1886 were collected from the inner coral reef of Meishan, Hainan Province, China, in April 2014, at a depth of 6 m and frozen immediately after collection. The identification was carried out by one of the authors (X.L.). A voucher specimen (HS201404) was deposited at the Laboratory of Marine Natural Products Chemistry, Wenzhou Medical University, China.

3.3. Extraction and Isolation

The frozen soft coral Lobophytum crassum (wet weight: 1.06 kg) was homogenized and extracted with 95% EtOH at room temperature (r.t.). The concentrated extract was partitioned between EtOAc and H2O. Evaporation of EtOAc in vacuo afforded a dark residue of 30.0 g. The EtOAc fraction (15.0 g) was subjected to silica gel vacuum column chromatography, eluting with a gradient of EtOAc/petroleum ether (1:30, 1:10, 1:5, 1:3, and 1:2), to yield seven fractions (A1–A7). Fraction A3 (300.5 mg) was further fractionated on a silica gel column, eluting with a gradient of acetone/petroleum ether (1:15 and 1:10), to afford three fractions (A3a–A3c). Fraction A3b (30.5 mg) was purified by semipreparative HPLC, using MeOH/H2O (75:25) as eluent, to afford 9 (2.2 mg). Fraction A3c (62.1 mg) was purified by HPLC, eluting with MeOH/H2O (70:30), to yield 11 (15.8 mg). Fraction A4 (2.1 g) was chromatographed on a Sephadex LH-20 column, using CH2Cl2/MeOH (1:1) as a mobile phase, to obtain three fractions (A4a–A4c). Fraction A4b (1.1 g) was further subjected to an ODS column, eluting with a gradient of MeOH/H2O (70:30, 75:25, 80:20, 85:15, and 90:10), to afford six fractions (A4b1–A4b6). Fraction A4b1 (100.3 mg) was purified by HPLC (MeOH/H2O, 65:35) to obtain 6 (5.6 mg) and 12 (17.4 mg). In the same manner, fractionsA4b2 (120.0 mg) and A4b3 (98.2 mg) were eluted with MeOH/H2O (70:30) to yield 14 (6.0 mg), 17 (18.7 mg), 15 (9.1 mg), 4 (3.8 mg), and 16 (6.7 mg), while fraction A4b4 (103.5 mg) was purified with MeOH/H2O (75:25) to afford 1 (7.0 mg), 2 (8.0 mg), and 13 (28.7 mg). Fraction A5 (620.7 mg) was separated on a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) to yield three fractions (A5a–A5c). Fraction A5b (271.4 mg) was further fractionated on an ODS column, eluting with MeOH/H2O (70:30 and 75:25), to afford three fractions (A5b1–A5b3). Fraction A5b1 (58.9 mg) was purified by HPLC (MeOH/H2O, 65:35) to obtain 8 (3.3 mg) and 18 (9.6 mg). In the same manner, fraction A5b3 (16.8 mg) was eluted with MeOH/H2O (60:40) to afford 5 (3.2 mg). Fraction A6 (421.5 mg) was fractionated on a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) to yield four fractions (A6a–A6d). Fraction A6b (37.2 mg) was purified by HPLC (MeOH/H2O, 70:30) to afford 7 (3.5 mg) and 3 (3.8 mg). Following the same protocol as for fraction A6b, 10 (5.6 mg) was separated from fraction A6c (26.1 mg).

Locrassumin A (1): colorless oil; +9 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 212 (4.08), 284 (4.25); IR (KBr) νmax 2967, 2870, 1708, 1624, 1436, 1384, 1263, 1198, 1109, 1067, 866, 762 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 399.2134 [M + Na]+ (calcd. for C22H32O5Na, 399.2147).

Locrassumin B (2): colorless oil; +385 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 214 (4.05), 284 (4.22); IR (KBr) νmax 2955, 2872, 1708, 1631, 1435, 1384, 1264, 1191, 1121, 1070 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 399.2135 [M + Na]+ (calcd. for C22H32O5Na, 399.2147).

Locrassumin C (3): colorless oil; +44 (c 0.10, CHCl3); IR (KBr) νmax 3450, 2966, 2871, 1726, 1459, 1379, 1222 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 417.2251 [M + Na]+ (calcd. for C22H34O6Na, 417.2253).

Locrassumin D (4): colorless oil; −50 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 204 (3.55), 243 (3.21); IR (KBr) νmax 3442, 2930, 1707, 1646, 1460, 1383, 1081 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 341.2094 [M + Na]+ (calcd. for C20H30O3Na, 341.2093).

Locrassumin E (5): colorless oil; +127 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 203 (3.96), 251 (4.13); IR (KBr) νmax 3449, 2924, 2858, 1742, 1446, 1378, 1271, 1085, 917, 873 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 343.2247 [M + Na]+ (calcd. for C20H32O3Na, 343.2249).

Locrassumin F (6): colorless oil; −16 (c 0.10, CHCl3); IR (KBr) νmax 2930, 1777, 1451, 1383, 1320, 1243, 1104, 975 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 355.1883 [M + Na]+ (calcd. for C20H28O4Na, 355.1885).

Locrassumin G (7): colorless oil; −48 (c 0.07, CHCl3); IR (KBr) νmax 3446, 2931, 2860, 1452, 1377, 1272, 1090, 979 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 373.2351 [M + Na]+ (calcd. for C21H34O4Na, 373.2355).

(–)-Laevigatol B (8): colorless oil; −17 (c 0.10, CHCl3); IR (KBr) νmax 3435, 2928, 2858, 1662, 1450, 1384, 1031 cm−1; 1H NMR (600 MHz, CDCl3) δ 5.23 (1H, t, J = 1.8 Hz, H-17a), 5.16 (1H, d, J = 10.2 Hz, H-3), 5.10 (1H, t, J = 1.8 Hz, H-17b), 5.06 (1H, t, J = 7.8 Hz, H-11), 4.78 (1H, d, J = 10.2 Hz, H-2), 4.64 (1H, dt, J = 13.2, 1.8 Hz, H-16a), 4.45 (1H, dt, J = 13.2, 1.8 Hz, H-16b), 2.69 (1H, t, J = 4.2 Hz, H-7), 2.32 (1H, m, H-13a), 2.30 (2H, m, H2-5), 2.27 (1H, m, H-10a), 2.17 (1H, m, H-14a), 2.12 (1H, ddd, J = 12.6, 4.2, 3.0 Hz, H-9a), 1.94 (1H, m, H-10b), 1.83 (1H, m, H-6a), 1.83 (3H, s, H3-18), 1.81 (1H, m, H-13b), 1.65 (1H, m, H-6b), 1.60 (3H, s, H3-20), 1.26 (1H, m, H-14b), 1.26 (3H, s, H3-19), 0.96 (1H, td, J = 13.2, 3.0 Hz, H-9b); 13C NMR (150 MHz, CDCl3) δ 152.5 (C, C-15), 139.4 (C, C-4), 136.2 (C, C-12), 123.4 (CH, C-11), 121.6 (CH, C-3), 106.2 (CH2, C-17), 84.5 (CH, C-2), 81.2 (C, C-1), 69.0 (CH2, C-16), 62.1 (CH, C-7), 59.8 (C, C-8), 40.0 (CH2, C-9), 37.6 (CH2, C-5), 34.4 (CH2, C-13), 32.7 (CH2, C-14), 25.5 (CH2, C-6), 23.7 (CH2, C-10), 16.6 (CH3, C-19), 16.3 (CH3, C-18), 15.2 (CH3, C-20); HRESIMS m/z 341.2090 [M + Na]+ (calcd. for C20H30O3Na, 341.2093).

(–)-Isosarcophine (9): colorless oil; −263 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 204 (4.24); IR (KBr) νmax 2924, 2854, 1754, 1676, 1446, 1384, 1287, 1089, 998 cm−1; 1H NMR (600 MHz, CDCl3) δ 5.45 (1H, d, J = 9.6 Hz, H-2), 4.98 (1H, d, J = 9.0 Hz, H-7), 4.85 (1H, d, J = 9.6 Hz, H-3), 2.54 (1H, dd, J = 10.2, 2.4 Hz, H-11), 2.54 (1H, m, H-14a), 2.46 (1H, m, H-6a), 2.37 (1H, m, H-5a), 2.32 (1H, m, H-9a), 2.22 (1H, m, H-5b), 2.13 (1H, m, H-6b), 2.10 (1H, m, H-10a), 2.10 (1H, m, H-14b), 2.00 (1H, m, H-13a), 1.99 (1H, m, H-9b), 1.85 (3H, s, H3-17), 1.68 (3H, s, H3-18), 1.68 (3H, s, H3-19), 1.32 (3H, s, H3-20), 1.26 (1H, m, H-10b), 1.05 (1H, m, H-13b); 13C NMR (150 MHz, CDCl3) δ 174.6 (C, C-16), 161.1 (C, C-1), 144.9 (C, C-4), 133.7 (C, C-8), 125.2 (CH, C-7), 123.4 (C, C-15), 120.6 (CH, C-3), 78.4 (CH, C-2), 62.0 (CH, C-11), 60.8 (C, C-12), 38.7 (CH2, C-5), 37.1 (CH2, C-13), 36.7 (CH2, C-9), 24.2 (CH2, C-10), 23.9 (CH2, C-6), 23.6 (CH2, C-14), 15.9 (CH3, C-20), 15.1 (CH3, C-18), 14.8 (CH3, C-19), 8.8 (CH3, C-17); HRESIMS m/z 339.1931 [M + Na]+ (calcd. for C20H28O3Na, 339.1936).

(–)-7R,8S-Dihydroxydeepoxysarcophytoxide (10): colorless oil; −99 (c 0.10, CHCl3); IR (KBr) νmax 3443, 2925, 2857, 1446, 1379, 1000, 945 cm−1; 1H NMR (600 MHz, CDCl3) δ 5.54 (1H, m, H-2), 5.14 (1H, d, J = 10.2 Hz, H-3), 4.92 (1H, dd, J = 9.6, 3.6 Hz, H-11), 4.50 (2H, s, H2-16), 3.57 (1H, d, J = 10.8 Hz, H-7), 2.53 (1H, ddd, J = 13.8, 10.8, 8.4 Hz, H-14a),2.39 (H, td, J = 12.6, 2.4 Hz, H-5a), 2.23 (1H, m, H-10a), 2.18 (1H, m, H-5b), 2.11 (1H, m, H-10b), 1.93 (2H, m, H2-13),1.87 (1H, m, H-6a), 1.85 (3H, s, H3-18), 1.81 (1H, m, H-9a), 1.70 (1H, m, H-9b), 1.68 (1H, m, H-14b), 1.64 (3H, s, H3-17), 1.63 (3H, s, H3-20), 1.51 (1H, m, H-6b), 1.19 (3H, s, H3-19); 13C NMR (150 MHz, CDCl3) δ 139.1 (C, C-4), 135.9 (C, C-12), 133.3 (C, C-1), 127.9 (C, C-15), 126.8 (CH, C-3), 124.2 (CH, C-11), 84.0 (CH, C-2), 78.5 (CH2, C-16), 75.5 (C, C-8), 72.9 (CH, C-7), 37.0 (CH2, C-9), 36.7 (CH2, C-13), 35.7 (CH2, C-5), 26.7 (CH2, C-6), 25.3 (CH2, C-14), 24.3 (CH3, C-19), 23.7 (CH2, C-10), 16.0 (CH3, C-18), 15.4 (CH3, C-20), 10.2 (CH3, C-17); HRESIMS m/z 343.2240 [M + Na]+ (calcd. for C20H32O3Na, 343.2249).

3.4. Assay for Inhibition of Nitric Oxide Production

A previously established protocol [43] 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 at r.t. before the experiment.

4. Conclusions

This is a further chemical examination on the soft coral Lobophytum crassum from the South China Sea, which presents ten new cembrane-based diterpenes (1–10) to enrich the chemical diversity of secondary metabolites from Lobophytum species. Compound 3 possesses an unprecedented tetradecahydrobenzo[3,4]cyclobuta[1,2][8]annulene skeleton which could be derived from 1, 2, or 13. Compounds 1, 7, 12, 13, and 17 exhibited moderate inhibition against lipopolysaccharide-induced NO production in mouse peritoneal macrophages with IC50 values of 8–24 μM. These results add to a growing class of diverse cembranoid structures that are known to inhibit NO production.