New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides

Abstract

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 Nephthea chabroli 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.

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 [δ_H 5.59 t (J = 8.0 Hz), 5.13 t (J = 7.2 Hz); δ_C 144.5 C, 121.6 CH, 130.9 C, 125.3 CH] (

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

^a Spectra were measured in CDCl₃ (400 MHz). ^b Spectra were measured in CDCl₃ (100 MHz).

). The NMR data [δ_H 0.25 dd (J = 15.2, 7.2 Hz), 0.36 m, 0.49 m, 0.78 dd (J = 14.8, 7.2 Hz); δ_C 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, (δ_H 0.52, 0.64, 1.62, 1.69). The presence of the oxygen bearing sp³ methine (δ_C 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).

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 (ν_max 1743 cm⁻¹). The ¹³C NMR spectrum of 2 (

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

^a Spectra were measured in CDCl₃ (100 MHz). ^b Spectra were measured in C₆D₆ (100 MHz).

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 [δ_H 2.03 (s), δ_C 20.8 (CH₃), and 170.1 (CO); δ_H 2.06 (s), δ_C 21.2 (CH₃), and 170.3 (CO)], two trisubstituted double bonds [δ_H 5.29 (brs), δ_C 137.9 (CH), and 128.0 (C); δ_H 5.57 (dd, J = 3.6, 3.2 Hz), δ_C 125.9 (CH), and 141.0 (C)], and two oxygen-bearing methines [δ_H 5.18 (ddd, J = 9.2, 4.8, 3.2 Hz), δ_C 74.7 (CH); δ_H 6.63 (d, J = 3.2 Hz), δ_C 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].

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. 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)	-

^a Spectra were measured in CDCl₃ (400 MHz). ^b Spectra were measured in C₆D₆ (400 MHz).

) (Figures S13 and S14) spectra showed the presence of (a) a secondary methyl (δ_C 15.5 CH₃; δ_H 0.35 d, J = 7.2 Hz), (b) a tertiary methyl (δ_C 16.4 CH₃; δ_H 0.54 s), (c) a secondary hydroxyl on C-1 (δ_C 71.7 CH; δ_H 3.32 brs), (d) a tertiary hydroxyl on C-10 (δ_C 72.6 C, (e) a COCH₃ group at C-6 (δ_C 214.3 CO, 34.3 CH₃; δ_H 1.82 s), and (f) an acetate at C-7 (δ_C 169.1 CO, 20.6 CH₃; δ_H 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.

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 δ_H 7.27 for CHCl₃ or δ_H 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 δ_C 77.0 for CDCl₃ or δ_C 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 F_254s 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; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = +102 (c 0.1, CHCl₃); IR (neat) ν_max 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; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = −25 (c 0.3, CHCl₃); IR (neat) ν_max 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; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = −54 (c 0.3, CHCl₃); IR (neat) ν_max 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; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = −87 (c 0.1, CHCl₃); IR (neat) ν_max 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.