1. Introduction

Soft corals belonging to the genus Clavularia (class Octocorallia, order Alcyonacea, family Clavulariidea), inhabiting abundantly in the South China Sea, have been proved to be a rich source of structurally diverse and biologically active terpenoids, prostanoids and steroids [1–4]. Remarkably, the genus Clavularia is really a productive factory of dolabellane type diterpenoids and many of them showed significant cytotoxic activities [5, 6]. The complex and unique structures of dolabellane diterpenoid have also attracted the attention of synthetic chemists for their total synthesis [7, 8].

In the course of our ongoing research aiming at searching for the biologically active substances from South China Sea soft corals [9–12], we made a collection of the title sample C. viridis off the Xisha Islands, Hainan Province, China. Chemical investigation of the Et₂O-soluble fraction of the acetone extract of this animal resulted in the isolation of a new dolabellane-type diterpenoid, clavirolide G (1), and a related one, clauvdiol A (3) [13] (Fig. 1). This paper describes the isolation and structure elucidation of these diterpenoids.

2. Results and discussion

The structural determination of the isolate 3 is straightforward since it is a crystal suitable for X-ray diffraction analysis allowing to unambiguously recognize its structure as depicted in Fig. 2, the same as clauvdiol A [13], a dolabellane-type diterpenoid previously isolated from the same species by Su et al. However, it may be worth to point out that in the literature [13], the absolute configuration (AC) of 3 was tentatively assigned based mainly on the interpretation of CD profile of 3 and biogenetic consideration. In our case, the AC of 3 was, for the first time, unambiguously elucidated by using X-ray crystallography with Cu Kα radiation on a single crystal of 3.

Clavirolide G (1) was obtained as an optically active, colorless oil. The molecular formula, C₂₀H₂₈O₂, consistent with seven degrees of unsaturation, was determined by HREIMS (m/z 300.2091 [M]⁺, calcd. 300.2089) suggesting the diterpenoid nature of the metabolite. Further, ¹H NMR and ¹³C NMR data (Table 1) showed similarities with those of the co-occurring 3 indicating that 1 is also a dolabellane-type diterpenoid, rare among octacorals. The UV absorption of 1 at 230 nm (log ε = 4.95), together with IR absorptions at 1712, and 1094 cm^-1 and ¹³C NMR signals at δ 166.7 (C-20), 163.1 (C-12), and 119.8 (C-18), suggested the presence of a conjugated lactone system. In addition, the ¹³C NMR signals at δ 150.6 (C-4) and 111.6 (C-16) indicated an exocyclic methylene function. Signals in the ¹³C NMR spectrum at δ 130.3 (C-8) and 132.6 (C-7), together with a signal accounting for one vinylic hydrogen in the ¹H NMR spectrum at δ 5.37 (dd, lH, J = 5.2, 9.3 Hz, H-7), established the presence of one trisubstituted double bond. The E configuration of this double bond was indicated by the ¹³C NMR chemical shift of the vinyl methyl group (δ 16.8, CH₃) [14]. These data, coupled with the degrees of unsaturation (seven), suggested that compound 1 to be tricyclic. Detailed analysis of the 1D and 2D NMR spectra (¹H-¹H COSY, HSQC, HMBC, NOESY) of 1 allowed us to propose the structure of clavirolide G as 1 (Fig. 1).

Literature checking revealed that the NMR data of 1 were strongly reminiscent of the structurally related dolabellane-type diterpenoids, e.g. clavulactone (2) [2, 15] and 3 [13]. A comparison the overall ¹³C NMR data of 1 and 2, 3 (Table 1) readily recognized that 1 and 2 shared the common partial structures for the rings a and b (Fig. 1), while 1, like 3, possessed the same olefins at △⁷ and △⁴⁽¹⁶⁾ for the ring c. These spectroscopic evidences give further support that compound 1 is an analogue of the model compound 2 [15]. In fact, 1 differs from 2 only by the isomerization of the olefin at △³, dehydrogenation between C-7 to C-8 and reduction of the C-6 ketone. Finally, the AC at C-1, C-10, and C-11 of 1 were assigned as R, R and S, respectively, the same as those of already determined for clavulactone (2) [2, 15] and other related analogues [2, 13, 16], by both direct CD spectra comparison [15, 17, 18] and biogenetic consideration.

It is interesting to note that although the structures of 1 and the co-occurring 3 are formally quite different, they are biogenetically related. As outlined in Scheme 1, the △¹¹ olefin migration accompanying the hydroxyl group shifting from C-18 to C-20 will generate the intermediate 4, of which further oxidation at C-20 gives 5 and successive esterification between the C-20 carboxyl group and C-10 hydroxyl group will yield the compound 1.

In light of the observation that many dolabellane diterpenoids displayed promising anti-tumoral activities, the cytotoxicity of compounds 1 and 3 were tested. In the bioassay, only the new compound 1 exhibited moderate cytotoxic activity against KB and HL-60 cell lines with IC₅₀ values of 15.4 μmol/L and 17.8 μmol/L, respectively, with adriamycin as the positive control (IC₅₀ = 0.04 μmol/L and 0.02 μmol/L).

3. Conclusion

The Xisha soft coral C. viridis has been proved to be able to produce various dolabellane diterpenoids. To our knowledge, till now, nine such diterpenoids, namely clavulactone (2) [2], clavirolides A–F [2, 13, 16], clavudiol A (3) and B [13, 16], were isolated from the title species. The present work is the second chemical study on the same species from the same location. The discovery of clavirolide G has added a new member of this class of diterpenes, of which the numbers are expanding. The real producer responsible for these unique metabolites is a matter worth to discuss. It is reported that they were initially found either from sea hare [19, 20] or algae [21], latter found in seawhips [22] and Aplysia [23]. Further studies should be conducted to verify the true origin of these metabolites and to understand their real ecological role played in the life cycle of the title animal.

4. Experimental 4.1. General experimental procedures

ECD spectrum was recorded on a J-810 spectropolarimeter. UV spectrum was recorded on a Shimadzu UV-2550 spectrophotometer. IR spectrum was recorded on a Nicolet Magna FT-IR 750 spectrometer. Optical rotations were measured on a PerkinElmer 241MC polarimeter. NMR spectra were measured on a Bruker DRX-400 (400 MHz for ¹H and 100 MHz for ¹³C NMR) spectrometer (Bruker Biospin AG, Fällanden, Germany) in CDCl₃, chemical shifts are referenced to the residual solvent signal (δ_H 7.26 ppm; δ_C 77.2 ppm). EIMS and HREIMS spectra were recorded on a FinniganMAT-95 mass spectrometer (Finnigan MAT, San Jose, CA, USA). Commercial silica gel (Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, China, 200–300 and 400–500 mesh) was used for column chromatography, and precoated silica gel GF₂₅₄ plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for TLC. Sephadex LH-20 (Pharmacia, USA) was also used for column chromatography. All solvents were of analytical grade. KB, HL-60 and A549 cells were bought from AmericanType Culture Collection (ATCC, Rockville, MD).

4.2. Collection of biological materials

Specimens of the Clavularia sp. (class Octocorallia, order Alcyonacea, family Clavulariidea) were collected from Xisha Island, Hainan Province, P.R. China, in March 2013. The soft coral was identified as C. viridis by Prof. H. Hang of South China Sea Institute of Oceanology, Chinese Academy of Sciences. Freshly collected coral tissue was frozen on-site and stored at -20 ℃ until processed. The voucher specimen (No. 13XS-49) is available for inspection at the Shanghai Institute of Materia Medica, CAS.

4.3. Extraction and isolation

The frozen specimens of C. viridis (dry weight, 60 g) were cut into pieces and exhaustively ultrasonic extracted with acetone at room temperature (1.0 L × 4). The solvent-free acetone extract was partitioned between Et₂O and H₂O. The organic phase was evaporated under reduced pressure to give a dark-red residue (0.9 g), which was subjected to silica gel CC (200–300 mesh) and eluted with PE in Et₂O (0–100%, gradient) to yield six fractions (A–F). Fraction D was first purified by Sephadex LH-20 (PE/CH₂Cl₂/ MeOH, 2:1:1), and then fraction B2 (41.0 mg) repeatedly purified by silica gel CC (400–500 mesh) using a PE/Et₂O gradient as eluent (98:2–90:10) affording compound 1 (20.4 mg) and 3(25.9 mg). All the spectra are deposited in Supporting information.

Clavirolide G (1): Colorless oil; α_D²⁰ -96.8 (c 0.20, CHCl₃); UV (MeOH) λ_max (log ε): 206 (5.28), 230 (4.95) nm; CD (MeOH): 206 (∆ε -36.5), 227 (∆ε +14.5) nm, 255 (∆ε -46.0) nm; IR (KBr, cm^-1) ν_max 2932, 1712, 1441, 1309, 1094, 1001; ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR(CDCl₃, 100 MHz) spectral data see Table 1; HREIMS m/z 300.2091 [M]⁺ (calcd for C₂₀H₂₈O₂: 300.2091).

Clauvdiol A (3): Colorless crystals; α_D²⁰ -62.3 (c 0.13, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 1.09 (3H, s, Me-15), 1.39 (3H, s, Me-20), 1.44 (3H, s, Me-19), 1.69 (3H, s, Me-17), 3.25 (1H, t, J = 11.8 Hz, H-9), 4.12 (1H, dd, J = 11.4, 2.5 Hz, H-10), 4.63 (1H, s, H-16), 4.71 (1H, s, H-16), 5.33 (1H, dd, J = 9.4, 5.2 Hz, H-7); ¹³C NMR (CDCl₃, 100 MHz) see Table 1; EIMS m/z 304 [M]⁺.

4.4. Single-crystal X-ray diffraction analysis of clauvdiol A (3)

A colorless block crystal of 3 was obtained by recrystallization from PE-CH₂Cl₂. A single crystal with dimensions of 0.32 mm × 0.18 mm × 0.15 mm was used for X-ray diffraction studies on a Bruker APEX-II CCD diffractometer employing graphite-monochromated Cu Kα radiation (l.54178 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least squares difference Fourier techniques. All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters.

Crystal data: C₂₀H₃₂O₂ (Mr = 304.45), orthorhombic with space group P2₁2₁2₁, with a = 8.5414(3) Å, b = 10.4689(3) Å, c = 20.7809 (7) Å, V = 1858.21(10) ų, T = 170 K, Z = 4. D_x = 1.0088 mg/m³, F(000) = 672. Independent reflections 3281 [R_int = 0.039], Flack parameter = 0.03(7).

4.5. Cytotoxicity assays

The cytotoxicity of compounds 1 and 3 against KB cell lines has been tested by using the sulforhodamine B (SRB) method [24] and the cytotoxicity against HL-60 and A549 cell lines has been tested by using the Cell Counting Kit-8 (CCK-8) method [25], according to the protocols described in previous literatures. Cells were seeded into 96-well plates and grown for 24 h and then treated with increasing concentrations of compounds and cultured for further 72 h. Inhibition rates of cell proliferation after compounds treatments were determined by the SRB and CCK-8 methods. ADR (adriamycin) was used as the positive control with IC₅₀ values of 0.04 μmol/L, 0.02 μmol/L and 0.13 μmol/L on KB, HL-60 and A549 cancer cells, respectively.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (Nos. 81520108028, 21672230, 41506187, 816013016) and the SKLDR/SIMM Project (No. SIMM1501ZZ-03).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.01.004.