Sinulariaone A: a novel diterpenoid with a 13-membered carbocyclic skeleton from an octocoral Sinularia species†

Hsuan-Jung Tseng‡ ^ab, Liang-Mou Kuo‡^c, Yu-Chi Tsai^d, Hao-Chun Hu^e, Po-Jen Chen^f, Su-Ying Chien^g, Jyh-Horng Sheu*^h and Ping-Jyun Sung*^behij
aGraduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944401, Taiwan
^bNational Museum of Marine Biology & Aquarium, Pingtung 944401, Taiwan. E-mail: pjsung@nmmba.gov.tw
^cDepartment of General Surgery, Chang Gung Memorial Hospital at Chiayi, Chiayi 613016, Taiwan
^dSchool of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404333, Taiwan
^eGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807378, Taiwan
^fClinical Immunology Laboratory, Department of Medical Research, E-Da Hospital, I-Shou University, Kaohsiung 824005, Taiwan
^gInstrumentation Center, National Taiwan University, Taipei 106319, Taiwan
^hDepartment of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804201, Taiwan. E-mail: sheu@mail.nsysu.edu.tw
ⁱChinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404394, Taiwan
^jPh.D. Program in Pharmaceutical Biotechnology, Fu Jen Catholic University, New Taipei City 242062, Taiwan

First published on 3rd April 2023

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1 Introduction

Octocorals of the genus Sinularia (phylum Cnidaria, sub-phylum Anthozoa, class Octocorallia, order Malacalcyonacea, family Sinulariidae)¹ are one of the most common marine invertebrates natively distributed throughout tropical and subtropical regions of the Indo-Pacific Ocean. Despite their ecological importance, the secondary metabolites, in particular terpenoid derivatives from these organisms were proven to have potential for biomedical uses.^2–4 In this research, we completed the preparation, structural identification, and cytotoxicity assessment of sinulariaone A (1), a diterpenoid featuring with a rare 13-membered carbocyclic skeleton and chlorofurancembranoid B (2)⁵ (Fig. 1), from an octocoral identified as Sinularia sp., collected from the waters of Taiwan, an area with high biodiversity at the intersection of the Kuroshio current, South China Sea surface current, and Mainland Coastal current.

2 Results and discussion

Sinulariaone A (1) was obtained as colorless prisms with the molecular formula determined to be C₂₀H₃₂O₂ by (+)-HRESIMS at m/z 327.22928 (calcd for C₂₀H₃₂O₂ + Na, 327.22945), corresponding to five double-bond equivalents (DBEs). The IR spectrum of 1 showed a strong absorption at ν_max 1716 cm⁻¹, consistent with a ketone moiety in the structure. The ¹³C spectrum (Table 1), in combination with the DEPT and HSQC spectrum, showed signals of 20 carbons, including a ketonic carbonyl (δ_C 210.0, C-17), four olefinic carbons (δ_C 149.8, C-1; 121.6, CH-2; 127.4, CH-6; 134.8, C-7), and two oxygenated carbons (δ_C 62.6, CH-10; 61.3, C-11), as well as five methyls, six aliphatic sp³ methylenes, and two aliphatic sp³ methines.

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Analysis of ¹H (Table 1), ¹³C, and HSQC spectra illustrated that 1 contained two trisubstituted carbon–carbon double bonds (δ_H 5.13, 1H, dd, J = 6.8, 6.8 Hz/δ_C 127.4, CH-6; δ_C 134.8, C-7; δ_H 5.06, 1H, d, J = 10.0 Hz/δ_C 121.6, CH-2; δ_C 149.8, C-1) and an acetyl group (δ_H 2.06, 3H, s/δ_C 29.4, CH₃-18; δ_C 210.0, C-17). The ³J-proton-proton coupling information in the COSY spectrum led to the assignment of four continuous spin systems from H-2/H-3/H₂-4/H₂-5/H-6, H₂-8/H₂-9/H-10, H₂-12/H₂-13, and H-14/H₃-15 (H₃-16) (Fig. 2). The HMBC spectrum showed ²J- and ³J-heteronuclear correlations from neighbor protons to the non-protonated carbons such as H-3, H₂-12, H₂-13, H-14, H₃-15, H₃-16/C-1; H₂-5, H₂-8, H₂-9, H₃-19/C-7; H₂-12, H₂-13, H₃-20/C-11; and H-2, H-3, H₂-4, H₃-18/C-17 (Fig. 2), confirming the presence of central 13-membered carbon macrocyclic ring system.^6–10 The HMBC correlations from H₃-20/C-10, C-11, and C-12 indicated that Me-20 was placed at C-11. The presence of a vinyl methyl (Me-19) at C-7 was substantiated by the HMBC correlations from H-6, H-8′ (δ_H 2.18)/C-19 and H₃-19/C-6, C-7, C-8, and further confirmed by a long-range allylic coupling between H-6/H₃-19 (Fig. 2). The presence of an isopropyl group at C-1 was substantiated by the HMBC correlations from H-14, H₃-15, H₃-16 to C-1. The above analysis enabled the establishment of the carbon skeleton of 1. A trisubstituted epoxide containing a methyl substituent in 1 was established from the signals of an oxygenated quaternary carbon at δ_C 61.3 (C-11) and an oxymethine (δ_H 2.74, 1H, dd, J = 10.0, 4.0 Hz/δ_C 62.6, CH-10), and from the proton signal of a methyl at δ_H 1.30 (3H, s, H₃-20). An acetyl group at C-3 was confirmed by the HMBC correlations from the methine proton at δ_H 3.16 (H-3) to the ketonic carbonyl at δ_C 210.0 (C-17); the other HMBC correlations from the methyl protons resonating at δ_H 2.06 (H₃-18) to C-17 ketonic carbonyl (δ_C 210.0) and C-3 methine (δ_C 49.9), further supporting that this group was positioned at C-3.

The relative stereochemistry of 1 was determined based on correlations obtained from NOESY experiments. In the NOESY spectrum (Fig. 3), H-10 exhibited cross-peaks with H-3 and one of the diastereotopic methylene protons at C-12 (δ_H 1.07, H-12β); and H-12β was correlated with H-3 but not with H₃-20, which illustrated the β-orientations of H-3 and H-10, and the α-orientation of Me-20. The Z-form of Δ¹ and Δ⁶ was confirmed by NOESY correlations between H-2 (olefin proton)/H₃-15 (H₃-16); and H-6 (olefin proton)/H-3, respectively, and there were no NOESY correlations were found between H-6/H₃-19 (vinyl methyl) and H-2/H₂-13.

After the program of the above analysis, the gross structure of 1 displayed four possible relative configurations, including 1-3S*, 10R*, 11R*; 1-3S*, 10S*, 11R*; 1-3S*, 10R*, 11S*; and 1-3S*, 10S*,11S*. The four possible relative configurations were inputted into Spartan’16 and optimized at the MMFF94 level.^11–13 The predicted distance of key NOESY of possible configurations is shown in Table 2. The 1-3S*, 10R*, 11R* displayed the best result matching the experimental key NOESY correlations, and the calculated single optical rotation (SOR) value of 1-3S, 10R, 11R and 1-3R, 10S, 11S were +226 and −226, respectively. Comparing the calculated with the experimental SOR value of 1 (+236), the absolute configuration of 1 could be assigned as 3S, 10R, 11R.

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Due to the conformational mobility of the macrocycle, the stereochemistry of the stereogenic centers C-3, C-10, and C-11 of 1 would be further determined from an X-ray diffraction analysis. Regarding validation of the structure of 1, a single-crystal X-ray diffraction analysis was employed. The structure of 1 was fully established by X-ray crystallography, as observed by Cu Kα radiation (λ = 1.54178 Å) and the Flack parameter x = 0.0(3).^14,15 The X-ray structure (Fig. 4) demonstrates the location of an acetyl group at C-3 and an epoxy group between C-10/11 in the 13-membered macrocycle ring. Based on the X-ray diffraction analysis, the stereogenic centers in 1 were assigned as 3S, 10R, 11R. From the above findings, the structure, including the absolute configuration, of 1 was therefore elucidated unambiguously.

Chlorofurancembranoid B (2), a cytotoxic cembranoid toward human promyelocytic leukemia HL-60 cells, was reported in our previous publication, and its stereochemistry was established by combination of a NOESY experiment.⁵ Thus, in order to determine the absolute configuration. This compound has been crystallized, and the diffraction experiment was carried out with a diffractometer equipped with molybdenum radiation (Mo Kα, λ = 0.71073 Å) source. The ORTEP diagram (Fig. 5) showed the absolute configuration for all stereogenic centers were assigned as 3R, 4R, 7S, 8R, 11S, 12R.

The cytotoxicity of 1 against cancer cells HL-60 and HepG2 (human hepatoma cell line) were investigated. The assay used in this study was performed as described in previous publications.^16,17 The results are shown in Table 3. According to the outcomes of cytotoxic assays, diterpenoid 1 showed cytotoxicity towards human promyelocytic leukemia HL-60 cells, with an IC₅₀ value of 38.01 μM.

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3 Conclusions

In this study, the chemical composition of an octocoral identified as Sinularia sp. was screened, resulted in the isolation of a novel diterpenoid, sinulariaone A (1). It is to note that diterpenoid 1, involving an uncommon 13-membered carbocyclic carbon system, which was suggested biosynthesized from the common 14-membered carbocyclic cembrane analogues by ring contraction,^6,7 however, to be one of a kind, this is the first time to obtain a 13-membered carbocyclic cembranolide analogue featuring with an acetyl group at C-3. The structure of 1, including the absolute configuration, was determined by spectroscopic methods and further confirmed by a single-crystal X-ray diffraction analysis and this compound showed cytotoxicity toward the HL-60 tumor cells. In addition, the absolute configuration of a known cytotoxic cembranoid, chlorofurancembranoid B (2), was determined using a single-crystal X-ray diffraction analysis with the molybdenum radiation source, with the material obtained in previous study.⁵

4 Experimental

4.1 General experimental procedures

Optical rotation values were measured using a JASCO P-1010 digital polarimeter. IR spectra were obtained with a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer. NMR spectra were recorded on a 400 MHz Jeol ECZ NMR spectrometer using the residual CHCl₃ (δ_H 7.26 ppm) and CDCl₃ signals (δ_C 77.0 ppm) as internal standards for ¹H and ¹³C NMR, respectively; coupling constants (J) are presented in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system. Column chromatography was carried out with silica gel (230–400 mesh, Merck). TLC was performed on plates precoated with silica gel 60 F₂₅₄ (Merck) and RP-18W/UV₂₅₄ (0.15 mm-thick, Macherey-Nagel), then sprayed with 10% H₂SO₄ solution followed by heating to visualize the spots.

4.2 Animal material

Specimens of Sinularia sp. were collected on Turtle Island, Yilan County, Taiwan. The samples were stored in a freezer at −20 °C until extraction. A voucher specimen was deposited in the National Museum of Marine Biology & Aquarium, Taiwan (NMMBA-TW–SC–2018-0619). Identification of this organism was performed by comparison with previous descriptions.^1,18

4.3 Extraction and isolation

Freeze-dried and sliced bodies (wet/dry weight = 510/172 g) of the coral specimens were extracted with a mixture of MeOH/CH₂Cl₂ (1:1) to give 17.8 g of crude extract, which was partitioned between EtOAc and H₂O. The EtOAc extract (6.8 g) was subjected to silica gel column chromatography (Si C. C.) and eluted with gradients of n-hexane/EtOAc (100% n-hexane–100% EtOAc, stepwise) to furnish 14 sub-fractions A–N. Fraction D was chromatographed by Si C. C. and eluted with a mixture of CH₂Cl₂/EtOAc (20:1) to obtain 18 sub-fractions D1–D18. Fraction D13 was further separated by Si C. C. and eluted with CH₂Cl₂ to afford 1 (3.5 mg).

4.4 Structural characterization of undescribed compound

4.5 Single-crystal X-ray crystallography of sinulariaone A (1)

Suitable colorless prisms of 1 were obtained from a solution of MeOH. The crystal (0.600 × 0.484 × 0.138 mm³) was identified as being of the orthorhombic system, space group P2₁2₁2₁ (#19), with a = 9.0173(3) Å, b = 10.8659(3) Å, c = 18.6542(6) Å, V = 1827.76(10) ų, Z = 4, D_calcd = 1.106 Mg m⁻³ and λ (Cu Kα) = 1.54178 Å. Intensity data were obtained on a crystal diffractometer (Bruker, model: D8 Venture) up to a θ_max of 69.999°. All measurement data of 18147 reflections were collected, of which 3472 were independent. The structure was solved by direct methods and refined by a full-matrix least-squares on F² procedure.^19,20 The refined structural model converged to a final R₁ = 0.0601; wR₂ = 0.1637 for 3095 observed reflections [I > 2σ(I)] and 199 variable parameters; and the absolute configuration was established from the Flack parameter x = 0.0(3).^14,15 Crystallographic data for the structure of sinulariaone A (1) were submitted to the Cambridge Crystallographic Data Center (CCDC) with supplementary publication number CCDC 2226689 (data can be obtained from the CCDC website at https://www.ccdc.cam.ac.uk/conts/retrieving.html).

4.6 Single-crystal X-ray crystallography of chlorofurancembranoid B (2)

Suitable colorless prisms of 2 were obtained from a solution of MeOH. The crystal (0.388 × 0.145 × 0.028 mm³) was identified as being of the triclinic system, space group P1 (#1), with a = 10.1046(4) Å, b = 10.4180(3) Å, c = 10.9661(4) Å, V = 1066.93(7) ų, Z = 2, D_calcd = 1.167 Mg m⁻³ and λ (Mo Kα) = 0.71073 Å. Intensity data were obtained on a crystal diffractometer (Bruker, model: D8 Venture) up to a θ_max of 29.998°. All measurement data of 38323 reflections were collected, of which 12414 were independent. The structure was solved by direct methods and refined by a full-matrix least-squares on F² procedure.^19,20 The refined structural model converged to a final R₁ = 0.0561; wR₂ = 0.1188 for 8865 observed reflections [I > 2σ(I)] and 468 variable parameters; and the absolute configuration was established from the Flack parameter x = −0.01(3).^14,15 Crystallographic data for the structure of chlorofurancembranoid B (2) were deposited with the Cambridge Crystallographic Data Center (CCDC) as supplementary publication number CCDC 2208811 (data can be obtained from the CCDC website at https://www.ccdc.cam.ac.uk/conts/retrieving.html).

4.7 In silico calculations

The conformational search and calculated SOR results were carried out with the same method published as ref. 11–13. The brief procedure was described as follows. First, we optimized the minimized energy of the structure in the MM2 level and outputted an xyz file. Then, we submitted the file into spartan'16 software (Wavefunction Inc.; Irvine, CA, USA) at MMFF94 to generate conformational search results. The output data were imported into the Gaussian 09 software (Gaussian Inc.; Wallingford, CT, USA) and optimized using the time-dependent density functional theory (TDDFT) methodology at the B3LYP/6-31G* level in the gas phase and the B3LYP/6-31(d) levels in the solvent phase for SOR calculation, and the GIAO-DFT at the PCM/mpw1pw91/6-311 + g(d,p) level in the solvent phase for GIAO-NMR DP4+ analysis. The results were averaged by the proportion of each conformer.

4.8 In vitro cytotoxic assay

The cytotoxicity assay used in this study was performed as described in previous publications.^16,17

Author contributions

Hsuan-Jung Tseng: investigation, analysis of results. Liang-Mou Kuo: investigation, analysis of results. Yu-Chi Tsai: investigation, software, modelling and simulation. Hao-Chun Hu: software, modelling and simulation. Po-Jen Chen: data curation, methodology. Su-Ying Chien: formal analysis, X-ray analysis. Jyh-Horng Sheu: conceptualization, supervision, visualization. Ping-Jyun Sung: analysis of results, conceptualization, visualization, supervision, writing-original draft, writing-reviewing and editing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors are grateful to Hsiao-Ching Yu and Chao-Lien Ho, of the High Valued Instrument Center, National Sun Yat-sen University, for obtaining the mass (MS 006500) and NMR (NMR 001100) spectra (NSTC 112-2740-M-110-002), and to the Instrumentation Center, National Taiwan University, for providing X-ray facilities (NSTC 112-2740-M-002-006, XRD 000200). This work was mainly funded by grants from the National Museum of Marine Biology & Aquarium, the National Science and Technology Council (MOST 109-2320-B-291-001-MY3, 111-2320-B-291-001, and 110-2314-B-242-003) and Chang Gung Memorial Hospital (CMRPG6L0311-3), Taiwan, awarded to L.-M. K. and P.-J. S. All funding is gratefully acknowledged.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: HRESI-MS, 1D and 2D NMR spectra of 1; experimental and calculated SOR values of 1; X-ray crystallo-graphic data of 1 and 2. CCDC 2226689 and 2208811. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3ra01589k

‡ These authors have contributed equally to this work.

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