Targeted Isolation of Xenicane Diterpenoids From Taiwanese Soft Coral Asterospicularia laurae

Application of LC-MS/MS-based molecular networking indicated the ethanol extract of octocoral Asterospicularia laurae is a potential source for the discovery of new xenicane derivatives. A natural product investigation of this soft coral resulted in the isolation of four new xenicane diterpenoids, asterolaurins O–R (1–4), together with six known compounds, xeniolide-A (5), isoxeniolide-A (6), xeniolide-B (7), 7,8-epoxyxeniolide-B (8), 7,8-oxido-isoxeniolide-A (9), and 9-hydroxyxeniolide-F (10). The structures of isolated compounds were characterized by employing spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Asterolaurin O is the first case of brominated tricarbocyclic type floridicin in the family Xeniidae. Concerning bioactivity, the cytotoxic activity of those isolates was evaluated. As a result, compounds 1 and 2 demonstrated a selective cytotoxic effect against the MCF-7 cell line at IC50 of 14.7 and 25.1 μM, respectively.


Results and Discussion
The ethanol extract of A. laurae was partitioned between EtOAc and H 2 O to obtain an EtOAc-soluble layer. This layer was further partitioned between hexanes and 75% MeOH (aq) to remove the low polarity metabolites. The 75% MeOH layer was analyzed by the LC-MS/MS (negative ion mode). The MS/MS data were uploaded to the Global Natural Products Social Molecular Networking (GNPS, https://gnps.ucsd.edu/ (accessed on 3 February 2021)) website, and the output data were mapped to create correlated clusters.
A cluster ( Figure 1) with molecular weights of nodes between 276 and 384 was found to have the MS/MS fragment peaks of xenicane-type diterpenes ( Figure S45), suggesting this group of metabolites could be xenicane-type diterpenes. A de-replication work was subsequently executed by comparing those molecular weights of nodes to known xenicanes. This investigation indicated that A. laurae should be a rich sources of new xenicane-type diterpenes.

Results and Discussion
The ethanol extract of A. laurae was partitioned between EtOAc and H2O to obtain an EtOAc-soluble layer. This layer was further partitioned between hexanes and 75% MeOH(aq) to remove the low polarity metabolites. The 75% MeOH layer was analyzed by the LC-MS/MS (negative ion mode). The MS/MS data were uploaded to the Global Natural Products Social Molecular Networking (GNPS, https://gnps.ucsd.edu/ (accessed on 3 February 2021)) website, and the output data were mapped to create correlated clusters.
A cluster ( Figure 1) with molecular weights of nodes between 276 and 384 was found to have the MS/MS fragment peaks of xenicane-type diterpenes ( Figure S45), suggesting this group of metabolites could be xenicane-type diterpenes. A de-replication work was subsequently executed by comparing those molecular weights of nodes to known xenicanes. This investigation indicated that A. laurae should be a rich sources of new xenicanetype diterpenes.  Four new compounds named asterolaurins O-R (1-4) along with six known xenicane diterpenoids, xeniolide-A (5) [11], isoxeniolide-A (6) [12], xeniolide-B (7) [11], 7,8epoxyxeniolide-B (8) [13], 7,8-oxido-isoxeniolide-A (9) [11], and 9-hydroxyxeniolide-F (10) [14] (Figure 2) were isolated and purified by successive silica gel, Sephadex LH-20, and semi-preparative normal-phase and reversed-phase high performance liquid chromatography (HPLC) columns. Their structures were further elucidated by spectroscopic data and compared with the relative literature. Four new compounds named asterolaurins O-R (1-4) along with six known xenicane diterpenoids, xeniolide-A (5) [11], isoxeniolide-A (6) [12], xeniolide-B (7) [11], 7,8-epoxyxeniolide-B (8) [13], 7,8-oxido-isoxeniolide-A (9) [11], and 9-hydroxyxeniolide-F (10) [14] ( Figure 2) were isolated and purified by successive silica gel, Sephadex LH-20, and semipreparative normal-phase and reversed-phase high performance liquid chromatography (HPLC) columns. Their structures were further elucidated by spectroscopic data and compared with the relative literature. Asterolaurin O (1) was obtained as an amorphous, colorless gum. The infrared (IR) data indicated the presence of hydroxy (3424 cm -1 ) and ester carbonyl (1719 cm -1 ) functionalities. The presence of one bromine atom in 1 was apparent from the isotopic pattern in a 1:1 ratio observed for the quasi-molecular ion peaks at 451. 11  .0 (c), and 146.2 (d)) and a lactone carbonyl δc 176.9 (s), suggesting that 1 was tricyclic. Detailed inspection of 1 H and 13 C NMR spectra of 1 (Table 1) disclosed signals characteristic for the A ring and the side chain were similar to those in florlide A [15], such as two singlet methyl protons (δH 1.30 x2) on a quaternary carbon (δC 71.3, C-15) substituted by a hydroxyl group, were assigned to H-16 and H-17. Moreover, the diene resonance due to H-13 (δH 6.34, dd, J = 11.1, 15.3 Hz) was correspondingly coupled to H-12 (δH 6.15, d, J = 11.1 Hz) and H-14 (δH 5.94, d, J = 15.3 Hz). Additionally, the chemical shifts of diene protons combined with the appearance of lactone carbonyl signal, as well as an AB spin system oxymethylene at  Asterolaurin O (1) was obtained as an amorphous, colorless gum. The infrared (IR) data indicated the presence of hydroxy (3424 cm −1 ) and ester carbonyl (1719 cm −1 ) functionalities. The presence of one bromine atom in 1 was apparent from the isotopic pattern in a 1:1 ratio observed for the quasi-molecular ion peaks at 451. 11  and a lactone carbonyl δc 176.9 (s), suggesting that 1 was tricyclic. Detailed inspection of 1 H and 13 C NMR spectra of 1 (Table 1) disclosed signals characteristic for the A ring and the side chain were similar to those in florlide A [15], such as two singlet methyl protons (δ H 1.30 x2) on a quaternary carbon (δ C 71.3, C-15) substituted by a hydroxyl group, were assigned to H-16 and H-17. Moreover, the diene resonance due to H-13 (δ H 6.34, dd, J = 11.1, 15.3 Hz) was correspondingly coupled to H-12 (δ H 6.15, d, J = 11.1 Hz) and H-14 (δ H 5.94, d, J = 15.3 Hz). Additionally, the chemical shifts of diene protons combined with the appearance of lactone carbonyl signal, as well as an AB spin system oxymethylene at δ H 4.44 (1H, d, J= 12.0 Hz) and 5.06 (1H, br d, J = 12.0 Hz) implied that 1 should belong to a xeniolide B type pyran-cyclononane diterpenoid. Analysis of the 1 H-1 H COSY and HMBC spectra ( Figure 3) corroborated the plane structure of 1. COSY correlations between two de-shielded protons H-8 (δ H 4.09, d, 5.7; δ C 72.9) and H-9 (δ H 4.37, td, 5.7, 8.7; δ C 75.1), the latter one also correlating to H-10 (δ H 2.22, d, 5.7 and 2.24, d, 8.7; δ C 39.8), were observed. The other spin system for H-11a/H-4a/H-5/H-6 from the COSY spectrum as well as HMBC correlations from Me-18 to C-6, C-7, C-8, and C-19, from isolated AB quartet protons H 2 -19 to C-7, C-11, and C-11a, from H-11a to C-1, C-11, and C-19 had established the bicyclic [4.3.1] ring system in 1. Two hydroxyl groups were positioned at C-8 and C-11 due to some similar bicyclic [4.3.1] analogues being yielded from Xenia species, and possessed the same substitutes [15][16][17][18][19]. Thus, the residue bromine should be attached at the C-9 position to meet the data from NMR and mass, and the gross structure of 1 was identified. The relative stereochemistry of compound 1 was established from NOESY correlations ( Figure 3) and by comparison of its spectroscopic data to those of xeniolide analogues. The E geometry of the ∆ 13 double bond was established by the large coupling constant observed between H-13 and H-14 (J = 15.3 Hz). Furthermore, the geometry of the olefinic bond between C-4 and C-12 was concluded to be E, based on a strong NOESY correlation between H-4a (δ H 3.18) and H-13 was observed. The large coupling constant (J = 12.0 Hz) between H-4a and H-11a allowed us to assume H-4a was α-orientation, whereas H-11a was β-orientation. The NOESY correlations of H-19 A /H-11a/H-19β/Me-18 revealed H 2 -19 and Me-18 were both on the β-side of 1. Based on the above results, we could deduce that the stereochemistry of ring junctions (C-7 and C-11) in the bicyclic [4.3.1] scaffold of 1 were the same with those of floridicins [17]. The NOESY correlations of H-10α/H-4a/H-6α/H-8 revealed those protons were on the α-side of 1. On the contrary, the NOESY correlations of H-6β/Me-18/H-19β/H-9/H-10β revealed that those protons were on the β-side of 1. Therefore, the structure of 1 (asterolaurin O) was assigned as 9α-bromo-florlide A on the basis of the above results. This structure represents the first case of brominated tricarbocyclic floridicin among the plethora of diterpenoid compounds already reported from corals. istry of ring junctions (C-7 and C-11) in the bicyclic [4.3.1] scaffold of 1 were the same with those of floridicins [17]. The NOESY correlations of H-10α/H-4a/H-6α/H-8 revealed those protons were on the α-side of 1. On the contrary, the NOESY correlations of H-6β/Me-18/H-19β/H-9/H-10β revealed that those protons were on the β-side of 1. Therefore, the structure of 1 (asterolaurin O) was assigned as 9α-bromo-florlide A on the basis of the above results. This structure represents the first case of brominated tricarbocyclic floridicin among the plethora of diterpenoid compounds already reported from corals. Asterolaurin P (2) was obtained as a pale yellowish amorphous gum with a molecular formula of C21H30O4 with 7 indices of hydrogen deficiency, as established based on its 13 C NMR data and an HRESIMS pseudo-molecular ion peak at m/z 369.20379 [M + Na] + (calcd for 369.20363). The IR spectrum indicated absorption bands due to hydroxyl (3454 cm -1 ) and ester carbonyl (1727 cm -1 ) functionalities, whereas the UV (λmax 237 and 215 nm) also supported a conjugated diene system. The structure of 1 was completely identified by a combination of 1D and 2D nuclear magnetic resonance experiments. The carbon resonances at δC 126.6 (CH), 131.8 (CH), 133.2 (qC),134.6 (qC), 137.3 (CH), and 146.4 (CH) in the 13 C NMR and DEPT spectra (Table 1) suggested the presence of three double bonds, and the quaternary carbon signal at δC 149.5 along with the methylene olefinic carbon signal at δC 115.3 indicated the presence of an exo-methylene double bond. Moreover, an ester δC 171.4 (qC) was also observed that implied that 2 was a bicyclic compound. The 1 H NMR spectrum (Table 1) confirmed the presence of an exo-methylene double bond by two singlet signals at δH 4.95 and 5.06. Three spin systems (I-III, Figure 1) were deduced from combined 1 H-1 H COSY ( Figure 4) and HSQC spectra of 1. Fragment I consisted of a sequence of three double bond methines, and fragment II started from an oxymethylene (δH 3.63, 4.11) and ended with the relative deshielding methylene (δH 2.19) as well as fragment Asterolaurin P (2) was obtained as a pale yellowish amorphous gum with a molecular formula of C 21 H 30 O 4 with 7 indices of hydrogen deficiency, as established based on its 13 C NMR data and an HRESIMS pseudo-molecular ion peak at m/z 369.20379 [M + Na] + (calcd for 369.20363). The IR spectrum indicated absorption bands due to hydroxyl (3454 cm −1 ) and ester carbonyl (1727 cm −1 ) functionalities, whereas the UV (λ max 237 and 215 nm) also supported a conjugated diene system. The structure of 1 was completely identified by a combination of 1D and 2D nuclear magnetic resonance experiments. The carbon resonances at δ C 126.6 (CH), 131.8 (CH), 133.2 (qC),134.6 (qC), 137.3 (CH), and 146.4 (CH) in the 13 C NMR and DEPT spectra (Table 1) suggested the presence of three double bonds, and the quaternary carbon signal at δ C 149.5 along with the methylene olefinic carbon signal at δ C 115.3 indicated the presence of an exo-methylene double bond. Moreover, an ester δ C 171.4 (qC) was also observed that implied that 2 was a bicyclic compound. The 1 H NMR spectrum (Table 1) confirmed the presence of an exo-methylene double bond by two singlet signals at δ H 4.95 and 5.06. Three spin systems (I-III, Figure 1) were deduced from combined 1 H-1 H COSY ( Figure 4) and HSQC spectra of 1. Fragment I consisted of a sequence of three double bond methines, and fragment II started from an oxymethylene (δ H 3.63, 4.11) and ended with the relative deshielding methylene (δ H 2.19) as well as fragment III, which included a carbinolic proton (δ H 4.72; δ C 67.9) and was correlated with the fourth double bond methine (δ H 5.26; δ C 131.8) and with allylic methylene (δ H 2.34 and 2.50). These subunits were connected through key HMBC correlations (Figure 4) of H-1 (δ H 3.63, 4.11) with C-3 (δ C 171.4) and C-4a (δ C 52.0), of H-12 (δ H 6.53) with C-4a, of protons H-13 (δ H 6.76), H-14 (δ H 5.98), Me-16 (δ H 1.30), and methoxy (δ H 3.18) with C-15 (δ C 76.5), of Me-18 (δ H 1.70) with C-6 (δ C 40.9), C-7(δ C 133.2), and C-8 (δ C 131.8), as well as of exomethylene protons (δ H 4.95, 5.06) with C-10 (δ C 46.2), C-11(δ C 49.5), and C-11a (δ H 51.1). Based on the above results, the gross structure of asterolaurin P could be constructed. The coupling constant (J = 11.0 Hz) between H-4a and H-11a suggested a trans ring junction, which implied that H-4a was α-oriented. The Z geometry of the ∆ 4,12 double bond was deduced on the basis of the NOESY (Figure 4) cross-peaks H-12/H-4a, and the chemical shift of C-4a in 1 was shifted -9.8 ppm, compared with its ∆ 4,12 E isomer, due to an γ effect of C-13 [7]. Additionally, the chemical shift of H-13 at δ H 6.76, which is downfield-shifted to the corresponding E isomer (δ H 6.40) due to an anisotropic effect, occurred with the carbonyl group. Moreover, the E geometry of the ∆ 13 double bond was established by the large coupling constant observed between H-13 and H-14 (J = 15.7 Hz). On the other hand, the ∆ 7 double bond could be determined as an E configuration according to the 13 C chemical shift of Me-18, which was 17.3 rather than at 22-25 for Z configuration [20]. The large coupling constant (J = 11.0 Hz) between H-4a and H-11a suggested a trans-juncture of the two rings, which implied that H-4a was α-oriented. The NOESY correlations of H-4a with H-8, which presented quasi-axial on the α-face, and on the other hand, of H-11a with Me-18, exhibited a quasi-axial on the β-face as well as Me-18 and also showed correlation with H-9 revealed an α-orientation of the hydroxyl group at the C-9 position. Therefore, the structure of asterolaurin P was assigned as 2 based on the above results.
shift of Me-18, which was 17.3 rather than at 22-25 for Z configuration [20]. The large coupling constant (J = 11.0 Hz) between H-4a and H-11a suggested a trans-juncture of the two rings, which implied that H-4a was α-oriented. The NOESY correlations of H-4a with H-8, which presented quasi-axial on the α-face, and on the other hand, of H-11a with Me-18, exhibited a quasi-axial on the β-face as well as Me-18 and also showed correlation with H-9 revealed an α-orientation of the hydroxyl group at the C-9 position. Therefore, the structure of asterolaurin P was assigned as 2 based on the above results. The molecular formula of C20H28O5 was deduced for asterolaurin Q (3) from its HRESIMS data, being consistent with 7 indices of hydrogen deficiency. The IR absorptions at 3420 and 1704 cm −1 indicated the presence of hydroxy and ester carbonyl groups, respectively. The NMR spectral data of 3 revealed a ring A similar to those of 1 because of an AB system of H2-1 at δH 4.09 (dd, 4.1, 11.0) and 3.91 (d, 11.0 Hz) was observed. The diene system, H-13 at δH 6.57 (dd, J= 11.8, 15.1 Hz), was coupled to H-12 (δH 7.04, d, J = 11.8 Hz) and H-14 (δH 6.35, d, J = 15.1 Hz), whereas the downfield shift of H-12 attributed to an anisotropy effect occurred with carbonyl group at C-3 that implied the E form configuration of Δ 4,12 double bond in 3. In the aided DEPT spectra, 13   The molecular formula of C 20 H 28 O 5 was deduced for asterolaurin Q (3) from its HRESIMS data, being consistent with 7 indices of hydrogen deficiency. The IR absorptions at 3420 and 1704 cm −1 indicated the presence of hydroxy and ester carbonyl groups, respectively. The NMR spectral data of 3 revealed a ring A similar to those of 1 because of an AB system of H 2 -1 at δ H 4.09 (dd, 4.1, 11.0) and 3.91 (d, 11.0 Hz) was observed. The diene system, H-13 at δ H 6.57 (dd, J= 11.8, 15.1 Hz), was coupled to H-12 (δ H 7.04, d, J = 11.8 Hz) and H-14 (δ H 6.35, d, J = 15.1 Hz), whereas the downfield shift of H-12 attributed to an anisotropy effect occurred with carbonyl group at C-3 that implied the E form configuration of ∆ 4,12 double bond in 3. In the aided DEPT spectra, 13 C NMR resonances at δ C 116.5 (CH 2 ) and 120.1 (CH 2 ) indicated the presence of two exo methylene double bonds, which were confirmed by the observation of four doublet signals at δ with C-10 and C-11a, allowed the construction of a cyclononane ring with two exomethylene functionalities at C-7 and C-11. Considering the molecular formula of 3 as well as the chemical shifts of C-8 (δ C 83.1), C-9 (δ C 70.5), and C-15 (δ C 71.5), three hydroxy groups were attached at the abovementioned positions. Herein, the gross structure of 3 was assigned. The trans junction of the two rings was suggested by coupling constant (J = 9.2 Hz) between H-4a and H-11a, and the configuration of H-4a could be assumed as α-oriented. Additionally, the NOESY correlations (Figure 4) of H-19/H-4a/H-13 and also the correlations of H-5α/H-13/H-6α/H-18/H-8 revealed those protons were on the same α face of the structure. On the other hand, the NOESY cross-peaks of H-11a/H-5β/H-9 implied that H-9 was β-orientation. Therefore, the structure of asterolaurin P was assigned as 3 on the basis of the above results. junction of the two rings was suggested by coupling constant (J = 9.2 Hz) between H-4a and H-11a, and the configuration of H-4a could be assumed as α-oriented. Additionally, the NOESY correlations (Figure 4) of H-19/H-4a/H-13 and also the correlations of H-5α/H-13/H-6α/H-18/H-8 revealed those protons were on the same α face of the structure. On the other hand, the NOESY cross-peaks of H-11a/H-5β/H-9 implied that H-9 was β-orientation. Therefore, the structure of asterolaurin P was assigned as 3 on the basis of the above results. Compound 4 was isolated as an amorphous gum, and its molecular formula was established as C21H32O5 by HREIMS and NMR spectral data. The 1 H and 13 C NMR spectra of 4 showed some characteristic signals in the cyclononane skeleton as B ring moiety, similar to those of compounds 8 and 9. Two singlets at δH 5.  (Figure 6) supported the structure in which three spin fragments were connected in the aid of key HMBC corrections of Me-18 with C-6, C-7, and C-8, of H-19 with C-10, C-11, and C-11a, of an acetal proton H-3 (δH 5.17, brs; δC 99.2) with C-1, C-4, C-12, and methoxyl carbon (δC 55.1). Therefore, the structure of 4 could be established unambiguously. The 4(12) E-configuration and the E-geometry for Δ 13 double bonds were determined by NOESY correlation (Figure 6) between H-13 and H-4a, and the coupling constant between H-13 and H-14 (14.9), respectively. Ring junction proton H-4a was assumed as an α-orientation, and H-11a was β-orientation due to the coupling constant between these two protons. On the β-face, H-11a showed the NOESY cross peak with Me-18, in turn coupled with H-9 revealed an α-orientation of hydroxyl group at the C-9 position. Besides, NOESY correlations of H-19A/H-4a/H-8 unveiled αorientation of H-8. Based on the above results, we could infer that Me-18 was a β-quasiaxial orientation, whereas H-7 and H2-19 were α-quasi-axial orientations. Thus, the relative stereochemistry of the cyclononane ring system was similar to that of asterolaurin A   (Figure 6) supported the structure in which three spin fragments were connected in the aid of key HMBC corrections of Me-18 with C-6, C-7, and C-8, of H-19 with C-10, C-11, and C-11a, of an acetal proton H-3 (δ H 5.17, brs; δ C 99.2) with C-1, C-4, C-12, and methoxyl carbon (δ C 55.1). Therefore, the structure of 4 could be established unambiguously. The 4(12) E-configuration and the E-geometry for ∆ 13 double bonds were determined by NOESY correlation (Figure 6) between H-13 and H-4a, and the coupling constant between H-13 and H-14 (14.9), respectively. Ring junction proton H-4a was assumed as an α-orientation, and H-11a was β-orientation due to the coupling constant between these two protons. On the β-face, H-11a showed the NOESY cross peak with Me-18, in turn coupled with H-9 revealed an α-orientation of hydroxyl group at the C-9 position. Besides, NOESY correlations of H-19 A /H-4a/H-8 unveiled α-orientation of H-8. Based on the above results, we could infer that Me-18 was a β-quasi-axial orientation, whereas H-7 and H 2 -19 were α-quasi-axial orientations. Thus, the relative stereochemistry of the cyclononane ring system was similar to that of asterolaurin A [4]. Additionally, the NOESY correlations of H-3 with H 2 -5 showed β-quasi-axial orientation revealed αorientation of methoxyl group at C-3 position. Thus the structure of asterolaurin R was unambiguously established as shown in Figure 6.

Global Natural Product Social Molecular Networking
Equal divisions (10 µL) of the MeOH-layer of Taiwanese soft coral A. laurae were dispensed into 96-well plates, dried under nitrogen, and resuspended in DMSO (10 µL), and 10 µL of a DMSO aliquot was injected into an Agilent 6545XT AdvanceBio LC/Q-TOF (quadrupole time-of-flight) equipped with an Agilent 1290 Infinity II LC system, eluting with an ACQUITY UPLC BEH C 18 column (1.7 µm, 2.1 × 100 mm, flow rate: 0.4 mL/min, Waters). The elution program, using water (A) and acetonitrile (B), both with 0.1% formic acid as mobile phases, started with a 5% isocratic elution for 1 min and was then followed by a linear gradient from 5% to 99.5% B until 16 min, and then maintained 99.5% B as a solvent system for 10 min followed by re-equilibration period for 2 min before the next injection. The UPLC-Q-TOF-(-)MS/MS data acquired for all samples at a fixed collision energy of 40 eV were converted from RAW data files to mzXML file format using the ProteoWizard MSconvert software [23] and uploaded to the Global Natural Products Social Molecular Networking Web server to create a molecular network [24]. The resulting spectral networks were imported into Cytoscape version 3.8.2 [25]. Careful review of these GNPS data associated with the Comprehensive Marine Natural Products Database and Reaxys ® database highlighted a promising cluster (Figure 1 and Figure S45), as a possible source of new xenicane diterpenoids.

Cytotoxic Assays
Breast (MCF-7), oral (Ca9-22), and ovarian (SK-OV-3) cancer cell lines were available from the American Type Culture Collection (ATCC, Manassas, VA, USA) or the Japanese Collection of Research Bioresources (JCRB) Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan). The cell viability was detected by MTS assay at 72 h treatment as previously described [24].

Conclusions
With the assistance of molecular networking-based de-replication strategy, xenicane diterpenes were targeted and obtained from the marine soft coral A. laurae. Among ten isolated compounds, asterolaurins O-Q (1-3) were identified as new xeniolides (possessing a δ-lactone-cyclononane skeleton), and asterolaurin R (4) was a new xenicin (containing an 11-oxabicyclo[7.4.0]tridecane ring system with an acetal functionality). It is noteworthy that asterolaurin O (1) was the first case of natural brominated tricarbocyclic floridicins yielded from the family Xeniidae. Moreover, compared with other asterolaurins obtained from the genus Asterospicularia, asterolaurin O (1) showed potent inhibition toward MCF-7 cells. This finding suggests that brominated xenicane-type diterpenes were worthy for further cytotoxic evaluations.

Conflicts of Interest:
The authors declare no conflict of interest.