Sinuhirtone A, An Uncommon 17,19-Dinorxeniaphyllanoid, and Nine Related New Terpenoids from the Hainan Soft Coral Sinularia hirta

Chemical investigation of the Hainan soft coral Sinularia hirta resulted in the isolation and identification of a library of sixteen structurally diverse terpenoids, including a dinorditerpenoid with an uncommon 17,19-dinorxeniaphyllane skeleton, namely sinuhirtone A (7), six new xeniaphyllane-type diterpenoids (1–6), one new norxeniaphyllanoid (8), two new norcaryophyllene-type sesquiterpenoids (9 and 10), together with six known related compounds (11–16). Compounds 1–3 are three new furanone-containing xeniaphyllane-type diterpenoids. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analysis and a series of quantum chemical calculations, including quantum mechanical-nuclear magnetic resonance (QM–NMR), time-dependent density functional theory–electronic circular dichroism (TDDFT–ECD), and optical rotatory dispersion (ORD) methods. A plausible biosynthetic connection between new compounds 1–9 was also proposed. New compounds 2–4, 7, and 8 were evaluated for in vitro cytotoxicity against four cancer cell lines.

Sinuhirfuranone A (1) was obtained as a colorless oil. Its molecular formula, C 20 H 28 O 3 , was deduced by HRESIMS ion peak at m/z 317.2115 ([M + H] + , calcd 317.2111), which suggested seven degrees of unsaturation. The IR spectrum of 1 showed typical absorption indicative of carbonyl group (ν max 1701 cm −1 ). Its 1 H NMR spectrum (Table 1) 6 and 196.2), one terminal double bond (δ C 114.5 and 150.5), and one ketone group (δ C 207.6) ( Table 2). One ketone and two double bonds accounted for three degrees of unsaturation, suggesting the presence of a tetracyclic ring system for 1. The 1 H-1 H COSY spectrum analysis of 1 defined two spin systems a and b (Figure 2)  , respectively. Based on HMBC experiment, fragments a and b could be connected by inserting the 8,19-exomethylene double bond and the oxygenated quaternary carbon C-4 (δ C 59.5), as indicated by the clear correlations from H 2 -19 to C-7/C-8/C-9 and H 3 -20 to C-3/C-4/C-5 ( Figure 2). Further HMBC correlations from H 3 -16 and H 3 -17 to C-14/C-15, from H 3 -18 to C-1/C-10/C-11/C-12, and from H-13 to C-12/C-14/C-15 constructed a xeniaphyllane skeleton. The presence of an epoxide group at C-4 and C-5 was deduced by the typical up-fielded 13 C NMR signals appearing at δ C 59.5 (C-4) and δ C 63.8 (C-5). The remaining cycle was deduced to be a furanone ring by the presence of an ether bridge between C-12 and C-15, as evidenced by the typical chemical shifts of an oxygenated carbon at δ C 88.7 and a ketone carbonyl at δ C 196.2 [12].      16) and the lack of correlation of H-5/H 3 -20. Thus, the RC of 1 was determined to be 1S*,4S*,5S*,9R*,11S*. Given the conformational flexibility of the core cyclononane ring in 1 that makes the assignment of RC using NOE interactions between remote protons potentially unreliable, a further QM-NMR calculation was performed to further confirm the RC for 1, which could be a showcase for the other cyclononane-containing compounds (2-10). Two possible configurations, 1a (1S*,4S*,5S*,9R*,11S*) and 1b (1S*,4R*,5R*,9R*,11S*), were calculated, and 1a showed a better result than 1b, verifying the RC of 1 to be 1S*,4S*,5S*,9R*,11S* ( Figure S81). The absolute configuration (AC) of compound 1 was further established by TDDFT-ECD calculation due to the presence of an α,β-unsaturated ketone group near the chiral centers. As shown in Figure 4, the Boltzmann-averaged ECD spectrum of (1S,4S,5S,9R,11S)-1 showed a highly matched ECD curve to the experimental curve of 1, allowing the assignment of the AC of 1.
The RC of compound 4 was assigned to be the same as 1 of 1S*,4S*,5S*,9R*,11S* through analysis of their NMR data and interpretation of their NOESY spectra, whereas the RCs of compounds 5 and 6 were determined to be the same as 2 of 1S*,4S*,9R*,11S* on the basis of the careful analysis of their NOESY spectra. To determine their ACs, TDDFT-ECD calculation was applied on compound 6 since the α,β-unsaturated ketone group was near its chiral center C-11 ( Figure 6). On the basis of the structural similarities of compounds 4-6, their ECD curves were compared with each other, resulting in the determination of the same absolute stereochemistry at C-1, C-9, and C-11 as 1S,9R,11S. The RC of compound 4 was assigned to be the same as 1 of 1S*,4S*,5S*,9R*,11S* through analysis of their NMR data and interpretation of their NOESY spectra, whereas the RCs of compounds 5 and 6 were determined to be the same as 2 of 1S*,4S*,9R*,11S* on the basis of the careful analysis of their NOESY spectra. To determine their ACs, TDDFT-ECD calculation was applied on compound 6 since the α,β-unsaturated ketone group was near its chiral center C-11 ( Figure 6). On the basis of the structural similarities of compounds 4-6, their ECD curves were compared with each other, resulting in the determination of the same absolute stereochemistry at C-1, C-9, and C-11 as 1S,9R,11S.  3 for 7, δC 47.4 for 14), indicating that the terminal double bond at C-8 in 14 was oxidized to be a ketone carbonyl group in 7. It is worth noting that sinuhirtone A, featuring an uncommon 17,19-dinorxeniaphyllane-type skeleton, is the first example of dinorditerpene from marine soft coral.
Sinuhirtone A (7) was isolated as colorless oil with the chemical formula of C 18  . The NMR data of 7 were strongly reminiscent of those of co-occurring gibberosin A (14). The overall comparison of the 13 C NMR data of 7 and 14 revealed that the differences between them mainly happened at C-8 (δ C 213.3 for 7, δ C 150.8 for 14) and its neighboring carbons C-7 (δ C 37.6 for 7, δ C 29.4 for 14), C-6 (δ 25.0 for 7, δ C 30.2 for 14), and C-9 (δ C 51.3 for 7, δ C 47.4 for 14), indicating that the terminal double bond at C-8 in 14 was oxidized to be a ketone carbonyl group in 7. It is worth noting that sinuhirtone A, featuring an uncommon 17,19-dinorxeniaphyllane-type skeleton, is the first example of dinorditerpene from marine soft coral.
Sinuhirtone B (8) was also isolated as colorless oil. Its molecular formula, C 19 H 26 O 3 , was established by HREIMS at m/z 302.1883 ([M] + , calcd 302.1876), which suggested seven degrees of unsaturation. The 1 H and 13 C NMR data of 8 were almost identical to those of 14 except that C-13 and C-14 were both shifted downfield from δ C 30.6 and 36.8 in 14 to δ C 132.7 and 137.8 in 8, respectively, indicating that C-13 and C-14 were oxidized to be a double bond, which was in agreement with the 2 mass-units loss of molecular weight in 8 (Tables 2 and 3).  (Figure 7). Based on the assignment of RCs of compounds 7 and 8, we further decided to perform the TDDFT-ECD calculation to assign the AC of 8 as well as compare the ECD curves of 7 and 8. As shown in Figure 8, both the ECD spectra of 7 and 8 displayed the positive CEs at around 260 nm and negative CEs at 310 nm, which highly matched the calculated ECD of (1S,4S,5S,9R,11S)-8.    Sinuhirtin F (9) was isolated as colorless oil. Its molecular formula was deduced to be C14H22O2 based on the HRESIMS pseudo-molecular ion peak at m/z 223.1689 ([M + H] + , calcd 223.1693), indicating four degrees of unsaturation. Its 1 H NMR spectrum (Table 3) (Table 3) displayed signals for one singlet methyl at δ H 1.24 and four singlet olefinic protons at δ H 4.79, 4.83, 4.96, and 5.05. The 13 C NMR, DEPT, and HSQC spectra indicated 14 carbon signals in 9, including one methyl (δ C 21.6), five sp 3 methylenes (δ C 29.9, 32.3, 32.5, 33.2, and 40.3), three sp 3 methines (δ C 40.0, 58.1, and 75.7), one sp 3 quaternary carbon (δ C 70.7), and two terminal double bonds (δ C 110.0, 114.6, 150.9, and 151.1) ( Table 2). Two double bonds accounts for two out of the four degrees of unsaturation, suggesting a bicyclic ring system for 9. The planar structure of 9 was further elucidated by detailed 2D NMR analysis. (δ C 75.7), and from H 3 -12 to C-1 (δ C 58.1)/C-10 (δ C 40.3)/C-11 (δ C 70.7), constructing a norcaryophyllene skeleton. Thus, the planar structure of 9 was established as shown in Figure 1.
Sinuhirtone A (7) features the first 17,19-dinorxeniaphyllane skeleton, and sinuhirfuranones A-C (1-3) possess the rare 2,2-dimethylfuran-3-one group, which are structurally different from other xeniaphyllanoids. However, they do share the same 4/9 bicyclic ring system, indicating that they should be biosynthetic related. Therefore, a plausible biosynthetic connection between 1 and 9 was proposed. As shown in Scheme 1, geranylgeranyl pyrophosphate (GGPP) could be envisaged as an early linear diterpene precursor to form the xeniaphyllane-type skeleton (i) under double-bond migration and ring closure steps, while new compounds were generated after a series of different reactions, including epoxidation, allylic oxidation, elimination, double-bond oxidation, and/or reduction, and so on.

General Experimental Procedures
Optical rotations were measured on a PerkinElmer 241MC polarimeter. IR spectrum was recorded on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Madison, WI, USA). 1 H and 13 C NMR spectra were acquired on a Bruker AVANCE III 500 and 600 spectrometer. Chemical shifts are reported with the residual CHCl3 (δH 7.26 ppm) as the internal standard for 1 H NMR spectrometry and CDCl3 (δC 77.2 ppm) for 13 C NMR spectrometry. The LREIMS and HREIMS data were recorded on a Finnigan-MAT-95 mass spectrometer (Finnigan-MAT, San Jose, CA, USA). HRESIMS spectra were recorded on an Agilent G6250 Q-TOF (Agilent, Santa Clara, CA, USA). All solvents used for column chromatography and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (Dikma Technologies Inc., Beijing, ), respectively. Sephadex LH-20 (Pharmacia, Peapack, NJ, USA) was also used for column chromatography. Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 100-200 and 300-400 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical TLC. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatograph equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, CA, USA). An Agilent semipreparative XDB-C18 column (5 μm, 250 × 9.4 mm) was employed for the purification.

General Experimental Procedures
Optical rotations were measured on a PerkinElmer 241MC polarimeter. IR spectrum was recorded on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Madison, WI, USA). 1 H and 13 C NMR spectra were acquired on a Bruker AVANCE III 500 and 600 spectrometer. Chemical shifts are reported with the residual CHCl 3 (δ H 7.26 ppm) as the internal standard for 1 H NMR spectrometry and CDCl 3 (δ C 77.2 ppm) for 13 C NMR spectrometry. The LREIMS and HREIMS data were recorded on a Finnigan-MAT-95 mass spectrometer (Finnigan-MAT, San Jose, CA, USA). HRESIMS spectra were recorded on an Agilent G6250 Q-TOF (Agilent, Santa Clara, CA, USA). All solvents used for column chromatography and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (Dikma Technologies Inc., Beijing, China), respectively. Sephadex LH-20 (Pharmacia, Peapack, NJ, USA) was also used for column chromatography. Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 100-200 and 300-400 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical TLC. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatograph equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, CA, USA). An Agilent semipreparative XDB-C18 column (5 µm, 250 × 9.4 mm) was employed for the purification.

Extraction and Isolation
The frozen animals (210 g, dry weight) were cut into pieces and extracted exhaustively with acetone at room temperature (3 × 3.0 L). The organic extract was evaporated to give a brown residue, which was partitioned between Et 2 O and H 2 O. The Et 2 O solution was concentrated under reduced pressure to give a dark-brown residue (9.8 g), which was fractionated by silica gel column chromatography and eluting with a step gradient (0-100% diethyl ether (EE) in petroleum ether (PE)), yielding twelve fractions (Fr. A-L). Fr.

QM-NMR Calculational Section
Conformational search was performed by using the torsional sampling (MCMM) approach and OPLS_2005 force field within an energy window of 21 kJ/mol. Conformers above 1% Boltzmann populations were re-optimized at the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for chloroform. Frequency analysis was also carried out to confirm that the re-optimized geometries were at the energy minima. Subsequently, NMR calculations were performed at the PCM/mPW1PW91/6-31G(d) level, as recommended for DP4+. NMR shielding constants were calculated by using the GIAO method. Finally, shielding constants were averaged over the Boltzmann distribution obtained for each stereoisomer and correlated with the experimental data [12].

TDDFT-ECD Calculational Section
Conformational search was carried out by using the torsional sampling (MCMM) method and OPLS_2005 force field within an energy window of 21 kJ/mol. Conformers above 1% Boltzmann populations were re-optimized at the MPW1PW91/6-31G(d) level with the IEFPCM solvent model for MeCN. Frequency analysis was also carried out to confirm that the re-optimized geometries were at the energy minima. ECD spectra were obtained by TDDFT calculations showed with the identical functional basis set and solvent model as the energy optimization. At last, the Boltzmann-averaged ECD spectra of the compounds were obtained with SpecDis 1.62 [23].

Optical Rotation Calculational Section
The specific optical rotation calculations for compounds were carried out by using Information Gaussian 09. The conformers were further optimized at the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for chloroform, all of which were subjected to specific optical rotation calculations at the B3LYP/6-311+G(d) level in chloroform with SMD model. The calculated specific optical rotation values of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The predicted ORD curves were generated from specific rotations calculated at four different wavelengths [24].

Bioassay
Cytotoxicity assays were carried out by using Capan-1 (human pancreatic cancer), A549 (human lung cancer), HT-29 (human colon carcinoma), and SNU-398 (human hepatocellular carcinoma), following a previously described procedure for a modification of the MTT colorimetric method [25,26]. To measure the cytotoxicity of tested compounds, five concentrations with three replications were performed on each cell line. Vincristine was used as a positive control for the bioassays using Capan-1, A549, HT-29, and SNU-398 cell lines, respectively.

Conclusions
In summary, the systematic chemical investigation of S. hirta of South China Sea yielded a series of terpenoids 1-10 with different skeletons, which increased the chemical diversity and complexity of marine terpenoids. The stereochemistry of these compounds was assigned by QM-NMR, TDDFT-ECD, and optical rotation calculations, which provided different approaches to deal with the challenges of stereochemistry determination of the complex molecules without single crystals. Structurally, sinuhirtone A (7) represents the first example of 17,19-dinorxeniaphyllane-type skeleton, whereas sinuhirfuranones A-C (1-3) possess the 2,2-dimethylfuran-3-one group, which is rare among marine diterpenoids. Although some new compounds did not show obvious anti-cancer activities, our proposed biogenetic pathway of these interesting compounds should impel further study on their accumulation for more extensive biological evaluation to understand the role they play on the life cycle of soft coral and to find their possible medicinal application.