Norsesquiterpenoids from the octocoral Paralemnalia thyrsoides (Ehrenberg 1834)

Three norsesquiterpenoids, pathyspirolactones A (1) and B (2), and napalilactone (3), featuring a γ-spirolactone moiety, were isolated from the cultured octocoral Paralemnalia thyrsoides. The structures of 1–3 were determined by analyzing spectroscopic data, DP4+ computation, specific optical rotation, and X-ray diffraction. In addition, we explored the absolute configurations of pathyspirolactone A (1) and its conformation of the cyclohexane ring to resolve the stereochemical confusion of those of norsesquiterpenoid compounds. Furthermore, we proved that pathyspirolactone B (2) was the first bromine-containing norsesquiterpenoid reported from octocorals.


Introduction
Octocorals of the genus Paralemnalia (family Nephtheidae) represent a rich source of natural substances with intriguing and unique structural features. Among these metabolites, sesquiterpenoids 1-10 and norsesquiterpenoids [11][12][13][14] are representative compounds for the natural products from Paralemnalia spp. Paralemnalia thyrsoides (Ehrenberg 1834) is one of the most common marine invertebrates natively distributed throughout tropical and subtropical regions of the Indo-Pacic Ocean.
Corals were described by Shi-Zhen Li in his ancient herbal Compendium of Chinese Materia Medica, published in 1596, as "sweet, neutral and non-toxic; used to remove eye vision obstruction; clear abiding static blood; blow the powder to nose to stop nose bleeding; brighten the eye and calm the spirit; stop epileptic seizure; apply to the eye to improve oater." 15 In connection with our continuing studies of marine organisms with the aim of informing new natural products, in this research, we completed the preparation and structural identi-cation of two nor-sesquiterpenoids, pathyspirolactones A (1) and B (2), and a known analogue, napalilactone (3) (Fig. 1), obtained from the cultured octocoral identied as P. thyrsoides.

Results and discussion
Pathyspirolactone A (1) was isolated as an amorphous powder. NMR data coupled with the [M + Na] + peak in the HRESIMS at m/z 277.14129 suggested a molecular formula C 14 H 22 O 4 (calcd for C 14 H 22 O 4 + Na, 277.14103) that indicated four degrees of unsaturation. IR spectrum analysis showed that 1 had absorption peaks at n max 3422, 1762, and 1700 cm −1 , suggesting that the structure of 1 included hydroxy, g-lactone, and ketonic groups. The 13 C NMR and DEPT spectra revealed that compound 1 had 14 carbons (Table 1), including three methyls, ve sp 3 methylenes, two sp 3 methines (including one oxymethine), two sp 3 quaternary carbons (including one oxygenated quaternary carbon), one ester carbonyl and one ketonic carbonyl carbon. Therefore, according to the aforementioned data, two degrees of unsaturation were accounted for, and compound 1 was identied as having two rings.
The relative stereochemistry of 1 was deduced mainly from the NOE interactions in the NOESY experiment, Chem3D  13 C NMR spectroscopic data of pathyspirolactones A (1) and B (2), napalilactone (3), and epi-pathylactone  16 These data were recorded at 500 MHz for 1 H and 125 MHz for 13 C in CDCl 3 . i The coupling pattern and coupling constants were assigned as "two similar coupling constants (J ¼ 4.2, 2.9 Hz)" in the content text of the ref. 16. Model, and vicinal 1 H-1 H coupling constant analysis. In accordance with convention, when analyzing the stereochemistry of 1, the tertiary C-14 methyl group at C-5 was assigned to the b-face, anchoring stereochemical analysis. In the NOESY experiment ( Fig. 3), the correlations of H 3 -14 with H-1 and H 2 -9 showed that these protons were positioned on the same face of the molecule, and therefore they were assigned as b-protons.
One of the methylene protons at C-3 (d H 1.41) exhibited a correlation with H 3 -14, leading to its assignment as H-3b, while the other was denoted as H-3a (d H 1.61). The correlation between H-3b and H 3 -13 reected the b-orientation of the CH 3 -13 group at C-4. The conguration at the cyclohexane ring in 1 is worthy of comment. H-1 was found to exhibit correlations with H 3 -14, as well as coupling between H-1 and H 2 -2 (J ¼ 11.6, 4.8 Hz), indicating that both H-1 and C-14 methyl at C-5 should be oriented at b-axial positions. Therefore, based on the above ndings, the congurations of the stereogenic carbons of 1 were determined as (1S*, 4S*, 5R*, 6R*). It is very interesting to note that the structure of 1 as we presented herein had been reported and named epi-pathylactone A, a synthetic product by Coelho and Diaz in 2002. 16 Although these two compounds possessed the same relative congurations (1S*, 4S*, 5R*, 6S*) ( Fig. 3 and 4 16 demonstrated that H-1 had signicantly different dihedral angles with H-2a and H-2b in both compounds, respectively. Furthermore, the 1 H-NMR spectra of compound 1 at temperatures of 0, 25, and 50 C were also measured to discover the 1 H chemical shi changes in those spectra. However, the critical proton NMR signal on C-1 (d H 3.70) in the different temperature experiments was quite similar (Fig. S27-S29 †). Therefore, compound 1 was proposed as a new conformational isomer and did not undergo conformational interconversion aer heat.
Based on the Newman projection analysis, H-1 in 1 expressed anti with H-2a and gauch with H-2b; H-1 in epi-pathylactone A showed gauch with the methylene protons on C-2 ( Fig. 5), indicating H-1 should be in a b-axial position in 1 and b-equatorial in epi-pathylactone A. In addition, the conformational variation in the cyclohexane ring led to the structural difference in those compounds, resulting in the absolute conguration of 1 being hard to discuss by the reference comparison.
Due to electronegativity effects, the methine unit at d C 58.2 was more shielded than expected for oxygenated carbon and was correlated to the methine proton at d H 4.59 in the HSQC spectrum, proving the attachment of a bromide atom at C-1. These data, together with the 1 H-1 H COSY and HMBC correlations, established the molecular framework of 2 (Fig. 6). The NMR data of 2 were found to be close to those of a known halogenated norsesquiterpenoid, napalilactone (3), 17 which was also isolated in this study (Fig. 1), except for replacing a chlorine atom at C-1 in 3 (d H 4.36, 1H, dd, J ¼ 10.4, 4.4 Hz/d C 63.8, CH-1) (Fig. S25 and S26 †) with a bromine atom in 2 (d H 4.59, 1H, dd, J ¼ 12.0, 4.4 Hz 4.0 Hz/d C 58.2, CH-1). To the best of our knowledge, compound 2 is the rst bromine-containing norsesquiterpenoid reported from octocorals.
Moreover, the absolute conguration of napalilactone (3) was fully established by a single-crystal X-ray diffraction analysis with the Flack parameter x ¼ −0.04 (4). 18,19 The computergenerated ORTEP diagram (Fig. 8) showed the absolute conguration of stereogenic centers of 3 were 1R, 4S, 5R, 6R. Based on the principles of biogenetics and the above DP4+ analysis, pathyspirolactone B (2) can be veried as the same absolute conguration as 3.
Based on the past reports, Paralemnalia spp. showed a promising anti-inammatory effect and cytotoxic activity. 7,8,10,14 Therefore, in in vitro anti-inammatory activity tests, upregulation of pro-inammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated RAW 264.7 macrophage cells were evaluated using immunoblot analysis. At a concentration of 10 mM, norsesquiterpenoids 1 and 2 were found to be inactive to reduce the level of iNOS and COX-2 in relation to control cells stimulated with LPS only. Using trypan blue staining to measure the cytotoxic effects of the compounds, it was observed that 1 and 2 did not induce cytotoxicity in RAW 264.7 macrophage cells.

Conclusions
In summary, we obtained three norsesquiterpenoids, including pathyspirolactones A (1) and B (2), and napalilactone (3) from octocoral P. thyrsoides. We further explored the conformation of  the cyclohexane ring moiety to resolve the stereochemical confusion of those of norsesquiterpenoid compounds. Moreover, we demonstrated the absolute congurations of pathyspirolactones based on analyzing spectroscopic data, specic optical rotation, DP4+ computation, and X-ray diffraction. However, because the screening platforms were limited and lots of material were consumed in physical and spectral experiments, the other possible bioactivities for the new interesting natural substances will not be assayed at this stage.

General experimental procedures
Optical rotation values were measured using a JASCO P-1010 digital polarimeter. IR spectra were obtained with a Thermo Scientic Nicolet iS5 FT-IR spectrophotometer. NMR spectra were recorded on a 400 MHz Jeol ECZ NMR spectrometer using the residual CHCl 3 (d H 7.26 ppm) and CDCl 3 signals (d C 77.0 ppm) as internal standards for 1 H and 13 C 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 F 254 (Merck) and RP-18W/UV 254 (0.15 mm-thick, Macherey-Nagel), then sprayed with 20% H 2 SO 4 solution followed by heating to visualize the spots. NP-HPLC was performed using a system comprised of a Hitachi L-5110 pump and a Rheodyne 7725i injection port with a normal-phase column (Galaksil ® EF-SiO 2 , 5 mm 120Å, S/N E08210401; Galak Co., Wuxi, CN). RP-HPLC was performed using a system comprised of a Hitachi L-2130 pump, a Hitachi L-2455 photodiode array detector, and a Rheodyne 7725i injection port with a reversephase column (Supelco, Ascentis ® C18, 581343-U, 250 mm Â 10 mm, 5 mm).

Animal material
Specimens of Paralemnalia thyrsoides (Ehrenberg, 1834) used for this study were collected from the culturing tank in the NMMBA in June 2021. A voucher specimen was deposited in the NMMBA (voucher no.: NMMBA-TW-SC-2021-902). Identication of the species of this organism was performed by comparison as described in previous studies. [20][21][22]

Extraction and isolation
The freeze-dried and sliced bodies of the coral specimen (wet/ dry weight ¼ 2031/299 g) were extracted by 95% EtOH to yield a crude extract I (31.7 g) and continued to be extracted by the mixture of MeOH/CH 2 Cl 2 (1 : 1) to give an extract II (29.5 g). Then extracts I and II were partitioned with EtOAc and H 2 O to obtain the EtOAc-soluble layers A (8.9 g) and B (1.8 g), respectively. The EtOAc layers A and B were then combined, placed in a silica column, and eluted by hexanes/EtOAc (pure hexanes to pure EtOAc, stepwise) to yield ten fractions A-J. Fraction G (121.3 mg) was further separated by normal-phase HPLC on Galaksil ® EF-SiO 2 column with a mixture of n-hexane and acetone (65 : 35) at a rate of 5 mL min −1 to give four subfractions (G 1 -4). G 2 (63.2 mg) was subjected to the normalphase HPLC system with an isocratic solvent system of nhexane and ethyl acetate (80 : 20, 5 mL min −1 ) to yield 12 fractions (G 2 A-L). G 2 L (48.6 mg) was then separated by the normal-phase HPLC system using a mixture of n-hexane and acetone (80 : 20, 5 mL min −1 ) to obtain 15 subfractions (G2L1-15). G2L10 (10.9 mg) was further puried by reverse-phase HPLC on Ascentis ® C18 column with an isocratic solvent system of MeOH and H 2 O (55 : 45, 5 mL min −1 ) to obtain compound 1 (0.2 mg). Fraction D (710.1 mg) was subjected to the normal-phase HPLC system with a mixture of n-hexane and ethyl acetate (85 : 15, 5 mL min −1 ) to give nine subfractions (D1-9). D7 (18.2 mg) was further puried by the reverse-phase HPLC using a mixture solvent system of MeOH : H 2 O (50 : 50, 5 mL min −1 ) to obtain compounds 2 (0.3 mg) and 3 (11.6 mg).

In silico calculations
The structure was optimized the minimized energy in MM2 level and outputted as an xyz le. The le was submitted into spartan'16 soware (Wavefunction Inc.; Irvine, CA, USA) at MMFF94 to generate conformational search results. The output data were imported into the Gaussian 09 soware (Gaussian Inc.; Wallingford, CT, USA) and optimized using the timedependent density functional theory (TDDFT) methodology at the B3LYP/6-31G* level in the gas phase, and at 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. All the computed SOR and NMR results were averaged by the proportion of each conformer. 24,25

Conflicts of interest
There are no conicts to declare.