Antimicrobial Terpenoids from South China Sea Soft Coral Lemnalia sp.

Abstract

Chemical investigation of the South China Sea soft coral Lemnalia sp. afforded 13 structurally diverse terpenoids, including three new neolemnane sesquiterpene lineolemnenes E–G (1–3); a new aristolane-type sesquiterpenoid, 2-acetoxy-aristolane (4); four new decalin-type bicyclic diterpenes, named biofloranates A−D (5−8); a new serrulatane, named euplexaurene D (9); and a new aromadendrane-type diterpenoid cneorubin K (10), together with three known related compounds (11−13). The structures of the new compounds were elucidated by NMR spectroscopy, the Mosher’s method, and ECD analysis. Compounds 1–13 were tested in a wide panel of biological assays. Lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line. The isolated new diterpenes, except euplexaurene D (9), demonstrated moderate antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with a MIC of 4−64 μg/mL. Compound 2 exhibited a mild inhibitory effect against influenza A H1N1 virus (IC₅₀ = 5.9 µM).

1. Introduction

In the competition for food and living space for survival, marine organisms accumulate and secrete secondary metabolites that are deterrent, offensive, or even toxic to other organisms—that is, chemical defense substances [1]. Marine invertebrates, such as soft corals, grow slowly and lack physical defense capabilities, but they can survive in competitive marine environments. What they often depend on are chemical defense strategies, which represent a rich source of unexplored chemical structures and biological diversity with great potential for drug discovery [2].

The South China Sea is located in the tropics and subtropics, with a vast sea area and rich marine biological resources. It is a sea area with numerous coral distributions. Since the 1980s, researchers have studied the chemical composition of many kinds of corals collected from the South China Sea [3,4,5,6,7,8]. Many compounds with novel structures have been isolated, including steroids, terpenoids, alkaloids, etc. Among them, the marine soft coral genus Lemnalia has afforded structurally diverse novel terpenoids, including sesquiterpenes and diterpene glycosides, with promising biological activity [7].

In the course of our efforts to search for novel and bioactive natural marine products from soft corals and find more diverse and complex terpenoids, we obtained Lemnalia sp. (No. XSSC201907), a soft coral collected from the coast of Xisha Islands in the South China Sea. The acetone extracts were chemically investigated, resulting in the discovery of 13 structurally diverse terpenoids, including four new sesquiterpenes (1–4), six new diterpenoids (5–10), and three known related compounds (11–13) (Figure 1). In vitro cytotoxic, antimicrobial, and antiviral activity of the isolated compounds were investigated. Herein, their isolation, structure, and biological activity are described.

Figure 1.

Structures of compounds 1–13.

2. Results and Discussion

Compound 1 was isolated as a colorless oil; its HR-ESI-MS exhibited an ion peak at m/z 299.1616 [M + Na]⁺, corresponding to the molecular formula of C₁₇H₂₄O₃Na (six degrees of unsaturation). The ¹³C NMR spectrum of 1 showed 17 signals (Table 1), in combined ¹H NMR and HSQC experiments, indicating the presence of a ketone group (δ_C 201.2), an acetyl group (δ_C 170.1 and δ_C/δ_H 20.8/2.18), two double bonds (δ_C 140.4 and δ_C/δ_H 123.4/5.43; δ_C 127.8 and δ_C/δ_H 138.6/5.82), an oxygenated methine (δ_C/δ_H 85.5/5.66), a nonprotonated carbon (δ_C 46.2), two methylenes, four methines, and four methyls. The above signals revealed that 1 was a bicyclic structure. The neolemnane skeleton was established by extensive 2D NMR analysis [9]. The ¹H-¹H COSY spectrum revealed the connectivity of H-1/H₂-2/H₂-3/H-4/H₃-15 and H₂-10/H₂-11 (Figure 2). The vinyl methyl (δ_C/δ_H 13.0/1.51) attached to C-7 (δ_C 127.8) was confirmed by HMBC correlations from H₃-13 to C-6, C-7, and C-8. Together with further HMBC correlations from H-8 to C-6/C-7/C-13 and the carbonyl of the ester group, the planar structure of 1 was established. Moreover, established by HR-ESI-MS, compound 2 was found to possess the same molecular formula as 1. A comparison of the 1D and 2D NMR spectral data of 2 with those of 1 disclosed that they had the same planar structure.

Table 1.

NMR spectroscopic data (¹H 600 MHz, ¹³C 150 MHz, CDCl₃) for compounds 1–4.

	1		2		3		4
Position	δH, Mult.	δC, Type	δH, Mult.	δC, Type	δH, Mult.	δC, Type	δH, Mult.	δC, Type
1	5.43, m	123.4, CH	5.49, m	125.1, CH	5.46, m	123.9, CH	5.17, br.s	118.1, CH
2a	2.07, m	26.1, CH2	2.09, m	26.0, CH2	2.06, m	26.0, CH2	5.35, m	68.9, CH
2b	2.01, m	—	2.09, m	—	2.06, m	—	—	—
3a	1.49, m	26.6, CH2	1.51, m	26.1, CH2	1.48, m	26.7, CH2	1.93, m	42.6, CH2
3b	1.37, m	—	1.51, m	—	1.41, m	—	1.57, m	—
4	2.26, ov	36.0, CH	2.41, m	33.2, CH	2.28, m	35.3, CH	1.95, m	37.9, CH
5	—	46.2, C	—	45.3, C	—	43.6, C	—	51.7, C
6	5.82, s	138.6, CH	5.22, s	147.5, CH	5.33, s	132.4, CH	0.85, d (6.5)	38.8, CH
7	—	127.8, C	—	128.0, C	—	137.4, C	1.22, br.t (6.3)	32.4, CH
8a	5.66, s	85.5, CH	5.16, s	84.8, CH	5.67, d (6.9)	73.7, CH	2.01, m	26.8, CH2
8b	—	—	—	—	—	—	1.74, m	—
9a	—	201.2, C	—	203.2, C	4.86, dd (9.9, 7.3)	79.5, CH	1.90, m	33.0, CH2
9b	—	—	—	—	—	—	1.59, m	—
10a	2.75, td (12.6, 3.8)	40.8, CH2	2.38, m	42.9, CH2	1.74, m	32.2, CH2	—	145.9, C
10b	2.16, m	—	2.08, m	—	1.57, dd (15.1, 9.3)	—	—	—
11a	2.59, m	32.6, CH2	2.33, m	34.3, CH2	2.22, dd (15.2, 9.4)	34.6, CH2	—	20.3, C
11b	2.30, ov	—	2.32, m	—	2.05, m	—	—	—
12	—	140.4, C	—	142.4, C	—	143.8, C	1.00, s	28.8, CH3
13	1.51, s	13.0, CH3	2.01, s	21.2, CH3	1.72, s	15.5, CH3	1.14, s	17.1, CH3
14	1.13, s	21.4, CH3	1.03, s	21.0, CH3	1.05, s	22.1, CH3	1.76, s	20.4, CH3
15	0.99, d (6.9)	17.8, CH3	0.95, d (6.9)	17.5, CH3	0.91, d (6.9)	17.6, CH3	1.07, d (7.1)	17.0, CH3
7-OAc	—	—	—	—	—	—	—	171.1, C
	—	—	—	—	—	—	2.04, s	21.5, CH3
8-OAc	—	170.1, C	—	170.6, C	—	170.1, C	—	—
	2.18, s	20.8, CH3	2.23, s	20.9, CH3	2.01, s	20.9, CH3	—	—
9-OAc	—	—	—	—	—	169.8, C	—	—
	—	—	—	—	2.12, s	20.9, CH3	—	—

^ov Overlapped signals, — absence of value.

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of 1–10.

The geometry of the 6,7-double bonds of compounds 1 and 2 was elucidated from NOE correlations (Figure 3). The absence of an NOE correlation for H₃-13/H-6 suggests an E geometry of the 6,7-double bond, which was different from the known lineolemnene A and B [10]. For compound 1, the NOESY correlations from H₃-14 to H₃-15, H-10a (δ_H 2.74) and from H-8 to H₃-14 and H-10a (δ_H 2.75) placed H-8, H₃-14, and H₃-15 on the same face. Thus, the relative configuration of 1 was elucidated as 4R*,5R*,8S*. For compound 2, H₃-15 was found to show NOE correlations with H₃-14 and H-10a (δ_H 2.38), and H-8 showed an NOE correlation with H-10b (δ_H 2.08) but not with H₃-14, suggesting that the relative configuration of 2 was 4S*,5S*,8S*. The absolute configurations (AC) of compounds 1 and 2 could be deduced from TDDFT/ECD calculations. A comparison of the experimental ECD spectrum of 1, 2， calculated (4S,5S,8R)-1 and calculated (4S,5S,8S)-2 (Figure 4) revealed that the AC of 1 and 2 was 4S,5S,8R and 4S,5S,8S, respectively. The NMR calculations were also conducted at the GIAO/mPW1PW91/6-31G (d, p) level. Finally, computed and experimental data were correlated, and the corresponding DP4+ probabilities were estimated. As shown in Figures S1 and S2, the experimental NMR data of compound 1 gave the best match for (4S,5S,8R)-1, and compound 2 gave the best match for (4S,5S,8S)-2.

Figure 3.

Key NOESY correlations of 1–4 and 9–10.

Figure 4.

Experimental and calculated ECD spectra of compounds 1 and 2.

The molecular formula of 3, C₁₉H₂₈O₄, was established by HR-ESI-MS, requiring six degrees of unsaturation. The NMR data indicated the presence of two acetyl groups (δ_C/δ_H 170.1 and 20.9/2.01; δ_C/δ_H 169.8 and 20.9/2.12), and it was analogous of neolemnane [9]. Two oxygenated methines (δ_C/δ_H 79.5/4.86; δ_C/δ_H 73.7/5.67) were found according to the 1D NMR spectra. The placement of two acetyl groups at C-8 and C-9 was confirmed by HMBC correlations from H-8 (δ_H 5.67) to an acetyl carbon (δ_C 170.1), and from H-9 (δ_H 4.86) to another acetyl carbon (δ_C 169.8). The structure was further confirmed with the assistance of ¹H-¹H COSY and HMBC spectra, as shown in Figure 2. The E geometry of 6,7-double bond was deduced in the same manner as compounds 1 and 2. The relative stereochemistry of 3 was also deduced by the key NOE interactions. The observed NOE correlation (Figure 3) between H₃-14/H₃-15, H₃-14/H-9, H-9/8-OAc, and H-8/9-OAc and no NOE correlation with H₃-14/H-8 demonstrated that 8-OAc, H-9, H₃-14, and H₃-15 were close in space while H-8 and 9-OAc were distant, with an antiperiplanar orientation. As a result, the AC of 3 was proposed to be 4S,5S,8S,9S according to the NOE correlations and considering biosynthetic properties [9]. Compound 3 was the C-8 and C-9 epimer of lineolemnene C [10].

Compound 4 was also obtained as a colorless oil. The molecular formula was established as C₁₇H₂₆O₂ by HR-ESI-MS (m/z 285.1824 [M + Na]⁺). The NMR signals (δ_C/δ_H 118.1/5.17 and 145.9) (Table 1) showed the presence of a trisubstituted double bond. The ¹H–¹H COSY spectrum revealed the connectivity of H-1/H-2/H₂-3/H-4/H₃-15 and H-6/H-7/H₂-8/H₂-9, indicating that it was an aristolane-type sesquiterpenoid [11]. The location of an acetyl group (δ_C/δ_H 171.1 and 21.5/2.04) at position C-2 (δ_C/δ_H 68.9/5.35) was revealed by the relevant HMBC correlations. The third ring was a cyclopropane due to the key H₃-12/C-6/C-7/C-11 and H₃-13/C-6/C-7/C-11 HMBC correlations. The NOE correlations between H-2/H₃-15, H₃-15/H₃-14, and H-6/H₃-14 revealed the relative configuration of 4. ECD calculation was performed (Figure S3) to determine the absolute configuration of compound 4 as 2S,4S,5S,6R,7S. Compound 4 was named 2-acetoxy-aristolane. The acetoxy group was of natural origin but not artifact, which was confirmed by LC-HRMS analysis of the methanol extract of Lemnalia sp. (Figure S37). The protonated- molecular ion peak[M + H]⁺ of 4 was extracted successfully (Figure S37).

Biofloranate A (5) was also a colorless oil, and the molecular formula was established as C₂₁H₃₄O₃ (HR-ESI-MS: m/z 357.2400 [M + Na]⁺). The 1D NMR and HSQC spectra of 5 (Table 2 and Table 3) disclosed two trisubstituted olefinic bonds (δ_C/δ_H 134.7 and 123.6/5.47; δ_C/δ_H 136.7 and 121.2/5.39), an acetate group (δ_C/δ_H 176.7 and 51.7/3.71), and an oxygenated methine (δ_C/δ_H 72.1/3.85), which also indicated a bicyclic structure. A comparison of the ¹H and ¹³C NMR data of 5 with the aglycone of lemnabourside [12] showed that they had identical ring systems, including stereochemistry, and similar side chains. The planar structure was further established by the analysis of COSY and HMBC correlations. The orientation of 14-OH of 5 was assigned via Mosher’s method. Esterification of 5 with (R)- and (S)-MTPA chloride occurred at the C-14 hydroxyl group to give (S)- and (R)-MTPA ester derivatives. The observed Δδ_H (S-R) value distribution pattern (Figure 5, Figures S82 and S83) established the 14R absolute configuration for 5. However, the configuration of C-15 was difficult to determine.

Table 2.

¹H NMR (600 MHz) spectroscopic data for compounds 5–10.

Position	5	6	7	8	9	10
	δH, Mult.	δH, Mult.	δH, Mult.	δH, Mult.	δH, Mult.	δH, Mult.
1a	1.85, m	1.84, m	1.78, m	1.84, ov	1.84, m	1.88, ov
1b	1.36, ov	1.36, m	1.36, ov	1.37, ov	1.52, ov	1.47, qd (11.5, 8.3)
2a	1.98, ov	1.97, m	1.99, ov	1.98, ov	1.53, ov	2.40, m
2b	1.92, ov	1.91, ov	1.95, ov	1.91, ov	1.35, ov	2.31, m
4	5.47, br.s	5.47, br.s	5.45, br.s	5.48, br.s	0.87, ov	1.71, ov
5	2.04, dd (9.3, 4.7)	2.02, m	2.04, ov	2.04, m	0.80, ov	0.54, br.t (9.5)
6	1.52, ov	1.48, m	1.51, dt (7.4, 3.1)	1.51, m	1.14, m	0.66, td (10.9, 10.1, 6.5)
7a	1.78, ov	1.75, ov	1.83, ov	1.84, ov	1.33, ov	1.85, ov
7b	1.78, ov	1.75, ov	1.78, ov	1.77, ov	0.83, ov	0.89, m
8a	5.39, br.s	5.39, br.s	5.39, br.s	5.40, br.s	1.57, ov	1.75, dd (12.9, 7.1)
8b	—	—	—	—	0.51, m	1.60, ov
9	—	—	—	—	1.64, dd (11.9, 6.0)	—
10	1.95, ov	1.93, ov	1.94, ov	1.94, ov	—	1.92, td (11.1, 6.5)
11	1.78, ov	1.78, ov	1.83, ov	1.79, ov	1.51, ov	—
12a	1.38, ov	1.18, ov	1.36, ov	1.30, ov	1.46, m	1.26, m
12b	1.22, ov	1.18, ov	1.36, ov	1.21, ov	1.21, m	1.16, m
13a	1.51, m	1.69, ov	2.27, m	1.19, m	2.04, dd (13.9, 6.3)	2.06, m
13b	1.36, ov	1.62, ov	2.27, m	1.19, m	1.92, dq (14.5, 7.5)	2.06, m
14a	3.85, dd (8.0, 3.8)	1.39, ov	6.88, t (7.8)	1.37, ov	5.12, br.t (6.2)	5.09, t (7.1)
14b	—	1.15, m	—	1.08, ov	—	—
15	2.54, qd (7.2, 3.5)	—	—	1.61, m	—	—
16a	—	—	—	3.40, dd (10.4, 6.6)	1.61, s	1.60, s
16b	—	—	—	3.49, dd (10.5, 5.9)	—	—
17	1.18, d (7.2)	1.39, s	4.33, br.s	0.90, d (6.7)	1.69, s	1.67, s
18	0.83, d (6.8)	0.79, d (6.7)	0.84, d (6.9)	0.81, d (6.8)	0.90, d (6.7)	1.04, s
19a	1.68, s	1.69, s	1.70, s	1.69, s	1.28, s	4.90, s
19b	—	—	—	—	—	4,79, s
20	1.68, s	1.68, s	1.69, s	1.69, s	0.93, d (6.4)	1.12, s
OMe	3.71, s	3.78, s	3.78, s	—	—	—

^ov Overlapped signals, — absence of value.

Table 3.

¹³C NMR (150 MHz) spectroscopic data for compounds 5–10.

Position	5	6	7	8	9	10
	δC, Type	δC, Type	δC, Type	δC, Type	δC, Type	δC, Type
1	24.6, CH2	24.6, CH2	24.5, CH2	24.7, CH2	29.5, CH2	26.3, CH2
2	30.8, CH2	30.8, CH2	30.7, CH2	30.8, CH2	36.3, CH2	32.1, CH2
3	134.7, C	134.6, C	134.8, C	134.5, C	80.4, C	157.8, C
4	123.6, CH	123.8, CH	123.4, CH	123.8, CH	39.0, CH	41.6, CH
5	36.3, CH	36.4, CH	36.2, CH	36.3, CH	23.1, CH	32.7, CH
6	39.1, CH	38.9, CH	38.7, CH	39.0, CH	42.5, CH	26.6, CH
7	24.6, CH2	24.6, CH2	24.5, CH2	24.6, CH2	25.3, CH2	20.4, CH2
8	121.2, CH	121.4, CH	121.0, CH	121.4, CH	31.7, CH2	44.6, CH2
9	136.7, C	136.7, C	136.6, C	136.6, C	30.9, CH	75.1, C
10	39.5, CH	39.6, CH	39.4, CH	39.5, CH	33.6, C	57.9, CH
11	31.8, CH	35.6, CH	31.6, CH	31.7, CH	38.3, CH	23.2, C
12	31.4, CH2	31.6, CH2	34.5, CH2	35.9, CH2	34.6, CH2	43.2, CH2
13	31.9, CH2	21.7, CH2	26.6, CH2	25.0, CH2	26.1, CH2	25.4, CH2
14	72.1, CH	40.6, CH2	146.1, CH	33.5, CH2	125.0, CH	124.8, CH
15	44.0, CH	74.8, C	130.4, C	35.8, CH	131.3, C	131.1, C
16	176.7, C	177.9, C	168.0, C	68.4, CH2	17.7, CH3	17.6, CH3
17	10.6, CH3	26.0, CH3	57.2, CH2	16.6, CH3	25.7, CH3	25.7, CH3
18	13.2, CH3	13.3, CH3	13.1, CH3	13.3, CH3	16.1, CH3	13.7, CH3
19	23.9, CH3	24.0, CH3	23.8, CH3	24.0, CH3	27.9, CH3	105.5, CH2
20	21.7, CH3	21.8, CH3	21.6, CH3	21.7, CH3	18.8, CH3	20.5, CH3
OMe	51.7, CH3	52.7, CH3	51.7, CH3

Figure 5.

Δδ_H values (Δδ (in ppm) = δ_S-δ_R) obtained for (S)- and (R)-MTPA esters of compound 5 in pyridine-d₅.

Compound 6 shared the same molecular formula as 5. The ¹H and ¹³C NMR spectra of 6 (Table 2 and Table 3) were also similar to those of 5, except that the oxygenated carbon was quaternary (δ_C 74.8) instead of a methine. As a result, the hydroxy was connected to C-15 instead of C-14. The orientation of 15-OH present in 3 was hard to determine, leading to the unknown relative configuration of C-15. Compound 6 was given the name biofloranate B.

Biofloranate C (7) had a molecular formula of C₂₁H₃₂O₃ as determined by HR-ESI-MS data, requiring six degrees of unsaturation. Comparing the molecular formula of 7 with 5 and 6, two hydrogens were missing, corresponding to the presence of one more trisubstituted double bond (δ_C/δ_H 130.4 and 146.1/6.88). An oxygenated methylene H-17 (δ_C/δ_H 57.2/4.33) showed HMBC correlations with C-14, C-15, and C-16. Key H₂-13/H-14 ¹H-¹H COSY correlation and H-14/C-12/C-13/C-15/C-16/C-17 HMBC correlations (Figure 2) suggested that the extra double bond was C-14−C-15. The H-14/16-OCH₃ NOESY correlation placed H-14 and the methoxy group on the same side (Figure S59). Thus, the structure of 7 was established unambiguously. Biofloranate D (8) was obtained as a colorless oil and the formula was assigned as C₂₀H₃₄O by HR-ESI-MS (m/z 313.2336, [M + Na]⁺). The 1D NMR data of 8 (Table 2 and Table 3) also corresponded closely to those of aglycone of lemnabourside [12]. Compared with aglycone, the 1D NMR data revealed the absence of an aldehyde group but the presence of an oxygenated methylene (δ_C/δ_Ha/δ_Hb 68.4/3.49/3.40). The structure of 8 was also confirmed by ¹H-¹H COSY and HMBC correlations, as shown in Figure 2. The AC of C-15 was also difficult to determine.

Euplexaurene D (9) was isolated as a white powder, and HR-EI-MS analysis (found 290.2604, M⁺) showed the molecular formula C₂₀H₃₄O, suggesting four degrees of unsaturation. As the only sp² group, the trisubstituted olefinic bond (δ_C/δ_H 131.3 and 125.0/5.12) was deduced from the 1D NMR data, indicating the tricyclic structure of 9. In the ¹³C NMR spectrum (Table 3), 20 carbon signals were assignable to three quaternary carbons, six methines, six methylenes, and five methyls. Comparing the ¹³C NMR spectrum of 9 with that of reported euplexaurene A [13] showed that they have identical ring systems, and the main difference was the position of the hydroxy group. Measuring the 2D NMR spectrum (Figure 2) enabled the assignment of all resonances in the ¹H NMR and ¹³C NMR spectra (Table 2 and Table 3). The relative configurations of 9 were assigned by NOESY correlations between H₃-19/H-4, H₃-19/H₃-20, H-6/H₃-19, and H-6/H₃-18 (Figure 4).

Cneorubin K (10) was obtained as a colorless oil, with the molecular formula C₂₀H₃₂O determined from HR-EI-MS (m/z 270.2334 [M − H₂O]⁺), indicating five degrees of unsaturation. The NMR spectra revealed the presence of a trisubstituted olefinic bond (δ_C/δ_H 131.1 and 124.8/5.09) and an exocyclic double bond (δ_C/δ_H 157.8 and 105.5/4.90/4.79), which accounted for two degrees of unsaturation, and the remaining three degrees of unsaturation required a tricyclic system in 10. The aromadendrane diterpenoid skeleton [14] was established by extensive ¹H-¹H COSY and HMBC analysis, as shown in Figure 2. In particular, the exocyclic double bond was C-3/C-19 and oxygenated nonprotonated carbon was C-9 (δ_C 75.1), which were confirmed by HMBC correlations from H₂-19 to C-2/C-3 and H₃-20 to C-10/C-8. The relative stereochemistry of 10 was deduced by the key NOE interactions. The NOE correlations between H-5/H-10, H₃-20/H-4, H₃-20/H₂-7b, H₃-20/H₃-18, H₃-18/H₂-7b and H₃-18/H-4 revealed the relative configuration of 10 (Figure 3). ECD calculation was performed (Figure S4) to determine the absolute configuration of compound 10 as 4S,5R,6R,9R,10R,11S.

According to previously reports, all diterpenoids from Lemnalia have a biflorane-based skeleton [7]. In the present study, diterpenoids separated from the Lemnalia organism possessed different ring structures, characterized as biflorane, 5,3,6-tricyclic, 5,7,3-tricyclic, and 10-membered carbocyclic skeletons, and were biogenetically related to each other. A geranylgeranyl-PP (GGPP, C20) has been regarded as the same biosynthetic precursor of these diterpenoids, and we further propose biosynthesis for these diterpenoids, as illustrated in Scheme 1.

Scheme 1.

Plausible biosynthetic pathway of diterpenoids 5–13.

All of the isolated compounds (1–13) were evaluated by a wide panel of biological assays, including cytotoxic activity (A-549, HepG2, HeLa, and CCRF-CEM cell lines) and antibacterial (Bacillus subtilis and Staphylococcus aureus) and antiviral (H1N1 and HSV-1) activity (as shown in Table S1). As a result, lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line (IC₅₀ 15.9 µM). Most of the diterpenoids exhibited corresponding antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Moreover, sesquiterpenes 1–4 exhibited anti-H1N1 virus activity with 77.6–100% inhibition at a concentration of 30 µM, and compound 2 exhibited activity with IC₅₀ = 5.9 µM.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations and UV spectra were measured on a Jasco P-1010 Polarimeter polarimeter (JASCO, Tokyo, Japan) and ThermoFisher Evolution 201/220 spectrophotometer (Thermo Scientific, Waltham, MA, USA). in MeOH at 20 °C., respectively. NMR spectra were recorded on a Bruker AVANCE NEO 600 spectrometer (BrukerBiospin AG, Fällanden, Germany) with a 5 mm inverse detection triple resonance (H-C/N/D) cryoprobe. ¹H chemical shifts were referenced to the residual CDCl₃ (7.24 ppm) and ¹³C chemical shifts were referenced to the CDCl₃ (77.0 ppm) solvent peaks. High-resolution electrospray ionization mass spectra (HRESIMS) were performed on an Agilent 6230 TOF LC/MS system (Agilent Technologies Inc., Palo Alto, CA, USA) and Thermo Scientific TM Q Exactive PlusTM (Thermo Scientific, Waltham, MA, USA). High-resolution electron impact mass spectra (HREIMS) were recorded on JEOL AccuTOF LC-plus 4G (JEOL, Tokyo, Japan). Reversed-phase HPLC purifications were performed on a Shimadzu LC-20AT HPLC system equipped with a quaternary gradient solvent delivery unit and a photodiode array detector (Shimadzu, Kyoto, Japan), using a semipreparative Cosmosil ODS column (250 mm × 10.0 mm i.d., 5 µm, Cosmosil, Nakalai Tesque Co. Ltd., Kyoto, Japan). Column chromatography was performed on a silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Cosmosil 75 C₁₈ (75 μm, Nakalai Tesque Co. Ltd., Kyoto, Japan).

3.2. Animal Material

Soft coral Lemnalia sp. (no. XSSC201907) was collected from the Xisha Islands of the South China Sea in April 2019 by scuba at a depth of 10 m. It was identified by Prof. Pingjyun Sung, National Museum of Marine Biology and Aquarium (NMMBA), Taiwan, China.

3.3. Extraction and Isolation

A sample of Lemnalia sp. (1.9 kg) was freeze-dried and extracted with acetone (1:1, v/v, 5 times). Then the extracting solution was concentrated to yield 33.8 g of residue. The residue was suspended in H₂O (500 mL) and partitioned with Et₂O (500 mL, 4 times) to yield an Et₂O solvent extract (25.5 g). The Et₂O solvent extract was separated on a Sephadex LH-20 column eluted with petroleum ether (PE)-CH₂Cl₂-MeOH (2:1:1, v/v/v), leading to 4 fractions (Fr.1–Fr.4). Fr.2 (10.4 g) was chromatographed over a silica gel column (PE/EtOAc, from 50:1 to 1:1) to give 9 fractions (Fr.2.1−Fr.2.9). Fr.2.3 was further separated on an ODS column (MeCN/H₂O, from 50 to 100%) and purified by semipreparative HPLC (MeOH/H₂O, 97:3, 3 mL/min) to give compound 8 (15.7 mg). Fr.2.5 was also separated on the ODS column (MeCN/H₂O, from 40 to 100%) to give 4 fractions (Fr.2.4.1–Fr.2.4.4). Fr.2.4.2 was purified by semipreparative HPLC (MeOH/H₂O, 87:13, 3 mL/min) to give compounds 9 (12.0 mg), 10 (4.1 mg), 11 (7.8 mg), and 13 (7.8 mg). Fr.2.4.3 was purified by semipreparative HPLC (MeOH/H₂O, 92:8, 3 mL/min) to give compounds 6 (10.0 mg) and 12 (3.5 mg). Fr.2.6 was further separated on the ODS column (MeCN/H₂O, from 40 to 100%) and purified by semipreparative HPLC (MeOH/H₂O, 88:12, 3 mL/min) to give compounds 4 (9.8 mg), 5 (10.0 mg), and 7 (5.0 mg). Fr.2.8 was separated on the ODS column (MeCN/H₂O, from 40 to 80%) and purified by semipreparative HPLC (MeOH/H₂O, 75:25, 3 mL/min) to give compounds 1 (11.0 mg), 2 (23.5 mg), and 3 (28.0 mg).

Lineolemnene E (1): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ −21 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 204 (2.53) nm; ¹H and ¹³C NMR spectroscopic data, Table 1; HRESIMS m/z 299.1616 [M + Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

Lineolemnene F (2): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +214 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.49) nm; ¹H and ¹³C NMR spectroscopic data, Table 1; HRESIMS m/z 299.1618 [M + Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

Lineolemnene G (3): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +39 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.42) nm; ¹H and ¹³C NMR spectroscopic data, Table 1; HRESIMS m/z 343.1880 [M + Na]⁺ (calcd for C₁₉H₂₈O₄, 343.1886).

2-Aristolanol acetate (4): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ −31 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 203 (2.24) nm; ¹H and ¹³C NMR spectroscopic data, Table 1; HRESIMS m/z 285.1824 [M + Na]⁺ (calcd for C₁₇H₂₆O₂, 285.1831).

Biofloranate A (5): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +34 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.03) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HRESIMS m/z 357.2400 [M + Na]⁺ (calcd for C₂₁H₃₄O₃, 357.2406).

Biofloranate B (6): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +33 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 201 (2.05) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HRESIMS m/z 357.2395 [M + Na]⁺ (calcd for C₂₁H₃₄O₃, 357.2406).

Biofloranate C (7): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ −105 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.03) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HRESIMS m/z 355.2251 [M + Na]⁺ (calcd for C₂₁H₃₂O₃, 355.2249).

Biofloranate D (8): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +30 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.05) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HRESIMS m/z 313.2511 (calcd for C₂₀H₃₄O, 313.2508).

Euplexaurene D (9): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ +21 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 201 (1.98) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HREIMS m/z 290.2604 [M]⁺ (calcd for C₂₀H₃₄O, 290.2610).

Cneorubin K (10): colorless oil; {${[\alpha ]}_{\mathsf{D}}^{25}$ −24 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 202 (2.04) nm; ¹H and ¹³C NMR spectroscopic data, Table 2 and Table 3; HREIMS m/z 270.2334 [M-H₂O]⁺ (calcd for C₂₀H₃₀, 270.2348).

3.4. Computational Methods

Geometry optimization was performed using Gaussian 09W (Gaussian Inc., Wallingford, CT, USA) and the B3LYP functional at the 6-311+G(d,p) level of theory as the method described in a reported article [15].

The theoretical calculation of ¹³C NMR chemical shifts of epimers 1 and 2 were carried out by geometry optimization [16]. The molecular structures were first minimized by MMFF. All torsional angles were systematically varied to search for the global minimum. The molecular mechanics minimized structures were then imported to Gaussian for all subsequent steps. Geometry optimization was conducted at the B3LYP/6-31G (d, p) level of theory. IEF-PCM solvation in chloroform was used for the stereoisomers. Chloroform was the solvent in which experimental NMR spectra were acquired. ¹³C NMR shielding constants were calculated at the GIAO/mPW1PW91/6-31G (d, p) level, with IEF-PCM solvation in the chloroform solvent.

3.5. Cytotoxic Activity Assay

Cell lines were purchased from the American Type Cultural Collection (ATCC, Manassas, VA, USA). The cytotoxicity of the compounds was evaluated against the A549, HepG2, Hela and CCRF-CEM with the Cell Counting Kit-8 (CCK-8) as the reported method [17]. Chidamide was used as a positive control.

3.6. Antibacterial Assays

The antibacterial activities were evaluated with the broth dilution assay [18]. Bacterial strains, Bacillus subtilis (CMCC (B) 63501), Staphylococcus aureus (CMCC (B) 26003) were used, and penicillin G as a positive control.

3.7. Antiviral Assays

The antiviral activities against the influenza A virus (H1N1) and (HSV-1) were evaluated as the literature reported [19]. Acyclovir was used as a positive control.

4. Conclusions

Marine invertebrates have been regarded as an important source of bioactive secondary metabolites. The acetone extracts of soft coral Lemnalia sp. (no. XSSC201907) were chemically investigated, resulting in the discovery of four new sesquiterpenes (1–4) and six new diterpenoids (5–10), along with three known related diterpenoids. Most of the isolated diterpenes were found to exhibit moderate antimicrobial activity against Bacillus subtilis and Staphylococcus aureus. Sesquiterpene lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line. Lineolemnene (2) exhibited medium inhibitory activity against influenza A H1N1 virus.

Supplementary Materials

The following are available online https://www.mdpi.com/article/10.3390/md19060294/s1. Table S1 Antibacterial and antiviral activity of the isolated compounds 1–13; Figures S1–S2 Calculated and experimental ECD spectra of compounds 4 and 10; Figures S3–S81 HRESIMS, 1D and 2D NMR spectra of all new compounds 1–10; Figures S82–S83 ¹H-NMR for (S)-MTPA and (R)-MTPA esters of compound 5; Figures S84–S89 1D NMR spectra of known compounds 11–13.

Author Contributions

X.Y. (Xia Yan) performed isolation, structure determination of the compounds and wrote the manuscript. J.L. performed the extraction of the sample. S.H. and T.L. collected the soft coral by scuba. S.H., B.W. and X.Y. (Xiaojun Yan) revised the manuscript. H.O. performed the cytotoxic, anti-inflammatory and antimicrobial bioassays. W.W. performed the antiviral bioassays. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2018YFC0310900), Ningbo Municipal Natural Science Foundation (2019A610201), and the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Materials file associate with this article.

Conflicts of Interest

The authors declare no conflict of interest.