New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides.

A novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E-G (2-4), were isolated by cytotoxicity-guided fractionation from soft corals Nephtheachabroli and Paralemnalia thyrsoides. The structures of the new compounds were determined by extensive analysis of spectroscopic data.

1. Introduction

Soft corals belonging to the genera Nephthea and Paralemnalia have proven to be a rich source of bioactive terpenoids [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. In searching for bioactive compounds from marine organisms, the crude extracts of soft corals Nephthea chabroli and Paralemnalia thyrsoides have been found to exhibit cytotoxicity against murine lymphcytic leukemis cell line, with IC50 values of 12.0 and 15.2 μg/mL, respectively. Cytotocixity-guided fractionation of soft corals N. chabroli and P. thyrsoides led to the isolation and characterization of a novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E–G (2–4) (Figure 1). The structures of these metabolites were established by extensive spectroscopic analysis.

Figure 1

Structures of Metabolites 1–4.

2. Results and Discussion

The molecular formula of chabrolin A (1) was determined as C20H32O from its HR-FAB-MS, 13C NMR, and DEPT spectroscopic data. The 13C NMR (Figure S2) and DEPT spectrum of 1 exhibited the presence of four methyl, seven sp3 methylene, three sp3 methine, two sp2 methine, two sp3 quaternary, and two sp2 quaternary carbons. The presence of two trisubstituted olefins in 1 was shown by the NMR data [δH 5.59 t (J = 8.0 Hz), 5.13 t (J = 7.2 Hz); δC 144.5 C, 121.6 CH, 130.9 C, 125.3 CH] (Table 1). The NMR data [δH 0.25 dd (J = 15.2, 7.2 Hz), 0.36 m, 0.49 m, 0.78 dd (J = 14.8, 7.2 Hz); δC 8.6 CH2, 15.6 CH, 23.7 CH] (Table 1) pointed to a 1,2-disubstituted cyclopropane ring in 1. The 1H NMR spectrum (Figure S1) displayed signals for four tertiary methyl groups, (δH 0.52, 0.64, 1.62, 1.69). The presence of the oxygen bearing sp3 methine (δC 74.1 CH) was shown in the 13C NMR spectrum. By interpretation of 1H-1H COSY correlations (Figure 2 and Figure S3), it was possible to establish four partial structures of contiguous proton systems extending from H-1 to H-5, from H-7 to H-8, from H-10 to H-12, and from H-14 to H-16. HMBC correlations (Figure S5) of (a) CH3-18 to C-5, C-6, C-7, and C-16, (b) H2-19 to C-8, C-9, C-10, C-13, and C-14, (c) H-8 to C-10, (d) CH3-20 to C-12, C-13, C-14, and C-19 connected four partial structures concluding the planar structure of 1, as shown in Figure 2. The above functionalities revealed that chabrolin A (1) possesses a novel diterpene tricyclic skeleton. The relative configuration of all chiral centers in 1 was deduced from a NOESY experiment (Figure S6). Assuming the β-orientation of H3-18, NOESY correlations of H3-18/H-15b, H3-18/H-8, H-8/H-10, H-10/H-11b, H-11b/H-14, and H-14/H-15b suggested all to be on the β face of the molecule. NOESY correlations of H-15a/H-16, H-16/H-19a, H-15a/H3-20, H-16/H3-20, and H2-19/H3-20 suggested H-15a, H-16, H2-19, and H3-20 were on the α face of the molecule (Figure 3).

Table 1

NMR spectral data of 1.

Position	δH a (J in Hz)	δC b, Type	COSY	HMBC	NOESY
1	1.62 s	17.6, CH3	3	2, 3, 17	-
2	-	130.9, C	-	-	-
3	5.13 t (7.2)	125.3, CH	1, 4, 17	1, 17	17
4	1.86 m; 2.04 m	22.8, CH2	3, 5	-	-
5	1.35 m	45.3, CH2	4	3, 6, 7, 16, 18	16
6	-	39.7, C	-	-	-
7a	2.24 m	40.9, CH2	8	6, 8, 9, 16	16
7b	1.79 m		8	6, 8, 9, 10	8, 18
8	5.59 t (8.0)	121.6, CH	7	6, 7, 10	10, 18
9	-	144.5, C	-	-	-
10	4.29 s	74.1, CH	11, 19	8	8, 11b
11a	2.05m	32.4, CH2	12	10,	-
11b	1.86 m		12	10,	14
12	1.41 m; 1.77 m	33.6, CH2	11	-	-
13	-	37.4, C	-	-	-
14	0.78 dd (14.8, 7.2)	15.6, CH	15, 16	12, 13, 16, 20	11b, 15b, 18
15a	0.49 m	8.6, CH2	14, 16	6, 13, 16	20
15b	0.36 m		14, 16	6, 13, 16	14, 18,
16	0.25 dd (15.2, 7.2)	23.7, CH	14, 15	-	5, 20
17	1.69 s	25.7, CH3	3	1, 2, 3	3
18	0.52 s	15.9, CH3	-	5, 6, 7, 16	8, 10, 14, 15a
19a	2.08 m	38.9, CH2	10	8, 9, 10, 13, 14	16, 20
19b	2.16 m		10	8, 9, 10, 13, 14	20
20	0.64 s	23.2, CH3	-	12, 13, 14, 19	15a, 16, 19

Spectra were measured in CDCl3 (400 MHz). Spectra were measured in CDCl3 (100 MHz).

Figure 2

Selected 1H-1H COSY (blue bold lines) and HMBC (red arrows) correlations of 1–4.

Figure 3

Key NOESY correlations for 1–4.

HRESIMS of parathyrsoidin E (2) exhibited a pseudomolecular ion peak at m/z 343.1887 [M + Na]+, consistent with the molecular formula C19H28O4, and six degrees of unsaturation. The IR spectrum of 2 revealed the presence of ester carbonyl group (νmax 1743 cm−1). The 13C NMR spectrum of 2 (Table 2 and Figure S8) displayed 19 carbon signals, and a DEPT experiments confirmed the presence of five methyl, four sp3 methylene, three sp3 methine, two sp2 methine, one sp3 quaternary, two sp2 quaternary, and two carbonyl carbons. The 13C and 1H NMR spectra (Figure S7) revealed the presence of two acetoxy groups [δH 2.03 (s), δC 20.8 (CH3), and 170.1 (CO); δH 2.06 (s), δC 21.2 (CH3), and 170.3 (CO)], two trisubstituted double bonds [δH 5.29 (brs), δC 137.9 (CH), and 128.0 (C); δH 5.57 (dd, J = 3.6, 3.2 Hz), δC 125.9 (CH), and 141.0 (C)], and two oxygen-bearing methines [δH 5.18 (ddd, J = 9.2, 4.8, 3.2 Hz), δC 74.7 (CH); δH 6.63 (d, J = 3.2 Hz), δC 71.0 (CH)]. Thus, the bicyclic structure of 2 was revealed. From the 1H-1H COSY spectrum (Figure S9) of 2, it was also possible to identify two different structural units (Figure 2), which were assembled with the assistance of HMBC experiments (Figure S11). Key HMBC correlations (Figure 2) from H-2 to C-1, C-4, C-8, C-13, and C-14, H2-6 to C-4, C-5, and C-8, H2-11 to C-1, C-9, C-10, and C-12, H3-13 to C-1, C-2, C-8, and C-12, H3-14 to C-2, C-3, and C-4, and H3-15 to C-1, C-11, and C-12 permitted the connection of the carbon skeleton. Thus, 2 was identified as a neolemnane-type new compound, on the basis of the above analysis and NMR data comparison with its stereoisomers, paralemnolins E and [9].

Table 2

13C NMR spectral data of 2–4.

Position	2, δH a (Type)	3, δH b (Type)	4, δH b (Type)
1	43.2, C	71.7, CH	68.9, CH
2	137.9, CH	30.86, CH2	35.8, CH2
3	128.0, C	29.0, CH2	28.7, CH2
4	71.0, CH	30.94, CH	35.4, CH
5	74.7, CH	46.1, C	43.9, C
6	30.2, CH2	59.5, CH	58.5, CH
7	31.2, CH2	71.6, CH	69.4, CH
8	141.0, C	23.1, CH2	27.4, CH2
9	125.9, CH	29.6, CH2	114.0, CH
10	23.1, CH2	72.6, C	143.6, C
11	26.2, CH2	214.3, C	206.9, C
12	38.3, CH	34.3, CH3	33.8, CH3
13	23.6, CH3	15.5, CH3	15.3, CH3
14	21.4, CH3	16.4, CH3	21.1, CH3
15	15.0, CH3	-	-
4-OAc	170.3, C 21.2, CH3	-	-
5-OAc	170.1, C 20.8, CH3	-	-
7-OAc	-	169.1, C 20.6, CH3	169.0, C 20.4, CH3

Spectra were measured in CDCl3 (100 MHz). Spectra were measured in C6D6 (100 MHz).

The Z geometry of the 2,3-double bond was established by a NOESY correlation (Figure S12) between H-2 and H3-14 and the relative configurations of all the chiral centers in 2 was established by analysis of NOESY correlations as shown in Figure 3. Assuming that the β-orientation of H3-13 (δ 1.05, s) and H3-13 showed NOESY correlations with H3-15 (δ 0.87, d, J = 6.8 Hz), H-7β (δ 2.48, m), and H-2 (δ 5.29 brs), H3-15 and H-2 should also be positioned on the β-face. NOESY correlations observed from H-12 (δ 1.71 m) to H-4, H-6α (δ 1.83, m) to H-5 (δ 5.18, ddd, J = 9.2, 4.8, 3.2 Hz), and H-5 to H-4 (δ 6.63, d, J = 3.2 Hz) reflected the α-orientation of H-12, H-4, and H-5. Therefore, 2 was found to be the C-5 epimer of paralemnolin E [9].

Parathyrsoidin F (3) was obtained as a colorless oil. The HRESIMS of 3 established the molecular formula C16H26O5, implying four degrees of unsaturation. The DEPT spectrum (Table 2) of 3 evidenced four methyl, four sp3 methylene, three sp3 methine, two sp3 quaternary, and two carbonyl carbons. In turn, the 1H and 13C NMR (Table 2 and Table 3) (Figures S13 and S14) spectra showed the presence of (a) a secondary methyl (δC 15.5 CH3; δH 0.35 d, J = 7.2 Hz), (b) a tertiary methyl (δC 16.4 CH3; δH 0.54 s), (c) a secondary hydroxyl on C-1 (δC 71.7 CH; δH 3.32 brs), (d) a tertiary hydroxyl on C-10 (δC 72.6 C, (e) a COCH3 group at C-6 (δC 214.3 CO, 34.3 CH3; δH 1.82 s), and (f) an acetate at C-7 (δC 169.1 CO, 20.6 CH3; δH 1.52 s). IR absorption at 3421 cm−1 and two absorptions at 1736 and 1714 cm−1 and the NMR signals indicated the presence of a hydroxyl, an acetate, and a methyl ketone (Table 2 and Table 3). The structure of 3 was determined by COSY (Figure 2 and Figure S15) and HMBC correlations (Figure 2 and Figure S17), and the latter correlations determined the acetate to be at C-7 (71.6 CH). The relative configuration of 3 was established from NOESY cross-peaks (Figure 3 and Figure S18). Assuming the β-orientation of H3-14 (δ 0.54 s), which exhibited NOESY correlations with H-1 (δ 3.32, d, J = 5.2 Hz), H-6 (δ 2.99, d, J = 6.8 Hz), H-7 (δ 5.16, ddd, J = 12.4, 6.8, 4.8 Hz), and H3-13 (δ 0.35, d, J = 7.2 Hz), the β-orientation of H-1, H-6, H-7, and H3-14 were suggested (Figure 3). One of the methylene protons at C-2 (δ 1.75, m) exhibited a NOESY correlation with H-1 and was identified as H-2β, while the other (δ 1.92, m) was assigned to H-2α. The NOESY correlation observed between H-2α and 10-OH (δ 6.68, d, J = 1.2 Hz) indicated the α-orientation of the 10-OH.

Table 3

1H NMR spectral data of 2–4.

Position	2, δH a (J in Hz)	3, δH b (J in Hz)	4, δH b (J in Hz)
1	-	3.52 d (5.2)	3.76 m
2	5.29 brs	α: 1.92 m; β: 1.75 m	α: 1.13 m; β: 1.73 m
3	-	α: 1.16 m; β: 0.98 m	α: 1.13 m; β: 1.04 m
4	6.63 d (3.2)	1.86 m,	1.63 m
5	5.18 ddd (9.2, 4.8, 3.2)	-	-
6	α: 1.83 m; β: 1.94 m	2.99 d (6.8)	3.09 d (5.2)
7	α: 2.10 ddd (14.8, 12.4, 4.4); β: 2.48 m	5.16 ddd (12.4, 6.8, 4.8)	5.38 ddd (10.0, 6.4, 5.2)
8	-	α: 1.92 m; β: 1.64 ddt (12.4, 4.0, 4.0)	α: 2.42 m; β: 2.34 m
9	5.57 dd (3.6, 3.2)	α: 2.22 ddd (14.0, 4.0, 4.0); β: 1.42 td (14.0, 4.0)	5.67 dd (6.4, 2.8)
10	2.03 m	-	-
11	α: 1.78 m; β: 1.39 m	-	-
12	1.71 m	1.82 s	1.96 s
13	1.05 s	0.35 d (7.2)	0.54 d (6.8)
14	1.68 d (1.2)	0.54 s	0.82 s
15	0.87 d (6.8)	-	-
4-OAc	2.06 s	-	-
5-OAc	2.03 s	-	-
7-OAc	-	1.52 s	1.63 s
12-OH	-	6.68 d (1.2)	-

Spectra were measured in CDCl3 (400 MHz). Spectra were measured in C6D6 (400 MHz).

The spectral evidence of parathyrsoidin G (4) (C16H24O4) suggested that this compound was a dehydrated form of 3. 1H-1H COSY correlations (Figure S21) from H-6 to H-9 through H-7 and H2-8 and from H-1 to H3-14 through H2-2, H2-3, and H-4, together with HMBC correlations from H3-13 to C-4, C-5, C-6, C-10, H-6 to C-5, C-7, C-8, C-10, and H-9 to C-1 (Figure 2 and Figure S23) and NOESY correlations (Figure 3 and Figure S24) between H3-13/H-1, H3-13/H-6, H3-13/H-7, H-6/H-7, and H-6/H3-14, confirmed that 4 was a dehydrated analogue of 3.

Cytotoxicity of Compounds 1–4 against the proliferation of a limited panel of cancer cell lines was evaluated against mouse lymphocytic leukemia (P-388), human colon adenocarcinoma (HT-29), and human lung epithelial carcinoma (A-549). Compounds 1–4 displayed cytotoxicity against P-388, with ED50 values of 3.18, 2.59, 3.31, and 2.49 μg/mL, respectively. However, Compounds 1–4 were not cytotoxic to A549 and HT-29 cell lines. Compounds 1–4 were also examined for the antiviral activity against human cytomegalovirus (HCMV), but none of them showed such activity.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were determined with a JASCO P1020 digital polarimeter (Tokyo, Japan). UV and IR spectra were obtained on JASCO V-650 (Tokyo, Japan) and JASCO FT/IR-4100 spectrophotometers (Tokyo, Japan), respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer (Santa Clara, CA, USA) at 400 MHz for 1H and 100 MHz for 13C. 1H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δH 7.27 for CHCl3 or δH 7.15 for C6D6, and coupling constants are expressed in Hertz (Hz). 13C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δC 77.0 for CDCl3 or δC 128.0 for C6D6. MS were recorded by a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Silica gel 60 (Merck, Darmstadt, Germany, 230–400 mesh) and LiChroprep RP-18 (Merck, 40–63 µm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) (Tokyo, Japan) was carried out using a Hitachi L-7100 pump (Tokyo, Japan) equipped with a Hitachi L-7400 UV detector (Tokyo, Japan) at 220 nm together with a semi-preparative reversed-phased column (Merck, Hibar LiChrospher RP-18e, 5 µm, 250 mm × 25 mm).

3.2. Animal Material

The octocoral N. chabroli was collected by hand using scuba at Green Islang, Taitong County, Taiwan, in August 2015, at a depth of 6 m. A voucher specimen (GN-100) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

The octocoral P. thyrsoides was collected by hand using scuba at San-Hsian-Tai, Taitong County, Taiwan, in July 2008, at a depth of 6 m. A voucher specimen (SST-07) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

3.3. Extraction and Separation

The frozen soft coral was chopped into small pieces and extracted with acetone in a percolator at room temperature. The acetone extract of N. chabroli was concentrated to a brown gum, which was partitioned with EtOAc and H2O. The EtOAc-soluble residue (50 g) was subjected to Si 60 CC using n-hexane–EtOAc mixtures of increasing polarity for elution. Fraction 10, eluted with n-hexane–EtOAc (1:10), was purified by reverse-phase HPLC (MeOH–H2O, 85:15) to afford 1 (2.9 mg).

The frozen soft coral was chopped into small pieces and extracted with acetone in a percolator at room temperature. The acetone extract of P. thyrsoides was concentrated to a brown gum, which was partitioned with EtOAc and H2O. The EtOAc-soluble residue (10 g) was subjected to Si 60 CC using n-hexane–EtOAc mixtures of increasing polarity for elution. Fraction 8, eluted with n-hexane–EtOAc (30:1), was purified by reverse-phase HPLC (MeOH–H2O, 95:5) to afford 2 (3.0 mg). Fraction 15, eluted with n-hexane–EtOAc (1:1), was purified by reverse-phase HPLC (MeOH–H2O, 55:45) to afford 3 (3.0 mg) and 4 (1.0 mg).

Chabrolin A (1): Colorless amorphous solid; = +102 (c 0.1, CHCl3); IR (neat) νmax 3439, 2965, 1635, and 772 cm−1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 311 [M + Na]+; (+)-HRESIMS m/z 311.23465 (calcd. for C20H32ONa, 311.23454).

Parathyrsoidin E (2): Colorless amorphous solid; = −25 (c 0.3, CHCl3); IR (neat) νmax 2964, 2927, 1743, 1455, 1369, 1247, and 871 cm−1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 343 [M + Na]+; (+)-HRESIMS m/z 343.1887 (calcd. for C19H28O4Na, 343.1885).

Parathyrsoidin F (3): Colorless amorphous solid; = −54 (c 0.3, CHCl3); IR (neat) νmax 3421, 2926, 1736, 1714, 1376, 1245, 1111, and 859 cm−1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 321 [M + Na]+; (+)-HRESIMS m/z 321.1676 (calcd. for C16H26O5Na, 321.1678).

Parathyrsoidin G (4): Colorless amorphous solid; = −87 (c 0.1, CHCl3); IR (neat) νmax 3421, 2964, 1739, 1714, 1362, 1236, and 869 cm−1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 303 [M + Na]+; (+)-HRESIMS m/z 303.1573 (calcd for C16H24O4Na, 303.1572).

3.4. Cytotoxicity Assay

Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [20,21]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. To measure the cytotoxic activities of tested compounds, five concentrations with three replications were performed on each cell line. Mithramycin was used as a positive control.

3.5. Anti-HCMV Assay

To determine the effects of natural products upon the HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as IC50 (50% inhibitory concentration), or a compound concentration required to reduce virus-induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [22].

4. Conclusions

Cytotoxicity-guided fractionation of the ethyl acetate extract of the soft corals N. chablroli and P. thyrsoides led to the isolation of a novel diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton) as well as three new sesquiterpenoids. Compounds 1–4 displayed cytotoxicity against P-388, with ED50 values of 3.18, 2.59, 3.31, and 2.49 μg/mL, respectively. However, Compounds 1–4 were not cytotoxic to A549 and HT-29 cell lines and did not show anti-HCMV activity. A plausible biosynthetic pathway of 1 is proposed in Scheme 1.

Scheme 1

A plausible biosynthetic pathway of 1.