Briarane-Related Diterpenoids from Octocoral Briareum stechei

A known polyoxygenated briarane, briaexcavatolide P (1), was isolated from a Formosan octocoral Briareum stechei. Moreover, the same species B. stechei, collected from Okinawan waters, yielded three chlorine-containing briaranes, including two new compounds, briastecholides B (2) and C (3) as well as a known analogue, briarenol R (4). The structures of 1–4 were established using spectroscopic methods. In addition, briarane 1 demonstrated anti-inflammatory activity in lipo-polysaccharide-induced RAW 264.7 mouse macrophage cells by suppressing the expression of inducible nitric oxide synthase (iNOS) protein.

Since 1977, when the first briarane-type diterpenoid was isolated from a Caribbean octocoral, Briareum asbestinum [11], hundreds of briarane-type diterpenoids have been obtained from various Briareum spp., and the compounds of this type are only found in marine invertebrates. The briarane-type diterpenoids have been reported to exhibit several biological effects, including anti-inflammatory activity [12], cytotoxicity [13,14], and antiviral activity [13,14]. In our continuing research on natural substances from the marine invertebrates originally distributed in the tropical Indo-Pacific Ocean, two samples of the octocoral Briareum stechei were collected from two positions surrounded by the Kuroshio current for their interesting chemical constituents. We report on a known polyoxygenated briarane, briaexcavatolide P (1) [15], from a Formosan B. stechei, and three chlorinated Molecules 2021, 26, 6861 2 of 10 briaranes, including two new metabolites, briastecholides B (2) and C (3), as well as a known analogue, briarenol R (4) [16] (Figure 1), from an Okinawan B. stechei. Isolates 1-4 were evaluated for their anti-inflammatory activity using the inhibition of inducible nitric oxide synthase (iNOS) in an in vitro pro-inflammatory model.

Structure Determination of Briaexcavatolide P from a Formosan Briareum stechei
Briarane 1 was obtained as an amorphous powder. The positive mode electrospray ionization mass spectrum (+)-ESIMS showed a peak at m/z 633 [M + Na] + and was found to have the molecular formula C 30 H 42 O 13 by the analysis of 13 C and 1 H NMR data. The result revealed that this compound had 10 degrees of unsaturation. Strong bands at 3488, 1783, and 1731 cm −1 in the IR spectrum indicated the presence of hydroxy, δ-lactone, and ester groups [17]. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra revealed that 1 had 30 carbons, including 8 methyls, 3 sp 3 methylenes, 9 sp 3 methines, 1 sp 2 methine, 3 sp 3 non-protonated carbons, and 6 sp 2 non-protonated carbons. Therefore, 1 was identified as having four rings. It was found that the spectroscopic data of 1 were identical to those of a known briarane, briaexcavatolide P, and 1 possessed the positive optical rotation value, [α] 25 D + 223 (c 0.04, CHCl 3 ), as that of briaexcavatolide P ([α] 27 D + 167 (c 1.0, CHCl 3 )) [15], suggesting that 1 is briaexcavatolide P.
The stereochemical evaluation of 2 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment. In the NOESY experiment (Figure 2b), H-10 correlated with H-2, H-9, H-12, and H3-18, respectively, indicating that these protons were situated on the same face and were assigned as α-protons; oppositely, C-15 methyl was determined as β-oriented at C-1 since H3-15 did not show correlation with H-10. The oxymethine proton H-14 exhibited an effect with H3-15 and no correlation with H-10, revealing that H-14 was β-oriented at C-14. One of the methylene protons at C-3 (δH 3.25) exhibited a correlation with H3-15, leading to its assignment as H-3β, while the other one was denoted as H-3α (δH 1.46). The correlation observed between H-3β and H-6 reflected the β-orientation of proton at C-6. The hydroxy protons at δH 3.76 (OH-8) and 5.52 (OH-9) displayed light correlations with H-2 and H-7, individually, setting the hydroxy groups at C-8, and the proton at C-7 were assigned as α-and β-oriented, respectively. Based on the above findings, the structure of 2 was established and the stereogenic carbons of 2 ), and (b) stereoview of 2 and calculated distances (Å) between selected protons with key NOESY ( d, J = 4.0 Hz, H-4), respectively.
According to the above and comparing the NMR data of 3 with those of the literature, the structure of 3 was highly similar to a known briarane, briarenol R (4) (Figure 1), which was originally isolated from a cultured B. stechei [16] and was also obtained in this study, except for a hydroxy group in 3 instead of an acetoxy group at C-2 in 4. The HMBC and COSY correlations, as shown in Figure 3a   The 13 C NMR signal of a methine unit at δ C 56.3 (CH-6) was more shielded than would be expected for an oxygenated C-atom. Furthermore, this carbon signal showed an HSQC correlation with a methine proton signal at δ H 5.21, which also exhibited a COSY crosspeak with H-7 in a 3 J-correlation, demonstrating the attachment of a chlorine atom at C-6. Together with the HMBC correlations between H-17/C-8, C-9, C-18, C-19 and H 3 -18/C-8, C-17, C-19, these data unambiguously established the molecular framework of 2.
Briastecholide C (3) was isolated as an amorphous powder that showed sodium adduct ions at m/z 477.1284 and 479.1253 (3:1) in (+)-HRESIMS, indicating the presence of a chlorine atom, and the molecular formula was established as C 22  . The IR spectrum of 3 showed the functionality signals of α,β-unsaturated ketonic group, ester carbonyl, γ-lactone, and OH stretching at 1672, 1735, 1757, and 3474 cm −1 , respectively. Based on the 13 C NMR data and unsaturated degree numbers, 3 was established as a tetracyclic briarane. In the 13 C and 1 H NMR (Table 1), HSQC, and HMBC spectra, an α,β-unsaturated ketonic group was deduced from the signals of three carbons at δ C 202.5 (C-12), 123.1 (CH-13), and 155.8 (CH-14). The presence of an exocyclic olefin was confirmed by the typical signals of one sp 2 methylene carbon at δ C 118.9 (CH 2 -16) and exomethylene proton signals at δ H 5.95 (1H, d, J = 2.8 Hz, H-16a) and 5.47 (1H, d, J = 2.8 Hz, H-16b). In addition, one γ-lactone, one ester, and one acetate methyl were confirmed by the NMR resonances at δ C 173.8 (C-19), 169.4 (ester carbonyl), and δ H 2.24 (3H, s)/δ C 21.9 (acetate methyl), respectively. A disubstituted epoxy group was identified by the chemical shifts of two oxymethine carbons at δ C 62. According to the above and comparing the NMR data of 3 with those of the literature, the structure of 3 was highly similar to a known briarane, briarenol R (4) (Figure 1), which was originally isolated from a cultured B. stechei [16] and was also obtained in this study, except for a hydroxy group in 3 instead of an acetoxy group at C-2 in 4. The HMBC and COSY correlations, as shown in Figure 3a  The 13 C NMR signal of a methine unit at δC 56.3 (CH-6) was more shielded than would be expected for an oxygenated C-atom. Furthermore, this carbon signal showed an HSQC correlation with a methine proton signal at δH 5.21, which also exhibited a COSY crosspeak with H-7 in a 3 J-correlation, demonstrating the attachment of a chlorine atom at C-6. Together with the HMBC correlations between H-17/C-8, C-9, C-18, C-19 and H3-18/C-8, C-17, C-19, these data unambiguously established the molecular framework of 2.
The stereochemical evaluation of 2 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment. In the NOESY experiment (Figure 2b), H-10 correlated with H-2, H-9, H-12, and H3-18, respectively, indicating that these protons were situated on the same face and were assigned as α-protons; oppositely, C-15 methyl was determined as β-oriented at C-1 since H3-15 did not show correlation with H-10. The oxymethine proton H-14 exhibited an effect with H3-15 and no correlation with H-10, revealing that H-14 was β-oriented at C-14. One of the methylene protons at C-3 (δH 3.25) exhibited a correlation with H3-15, leading to its assignment as H-3β, while the other one was denoted as H-3α (δH 1.46). The correlation observed between H-3β and H-6 reflected the β-orientation of proton at C-6. The hydroxy protons at δH 3.76 (OH-8) and 5.52 (OH-9) displayed light correlations with H-2 and H-7, individually, setting the hydroxy groups at C-8, and the proton at C-7 were assigned as α-and β-oriented, respectively. Based on the above findings, the structure of 2 was established and the stereogenic carbons of 2 ), HMBC ( The 13 C NMR signal of a methine unit at δC 56.3 (CH-6) was more shielded than would be expected for an oxygenated C-atom. Furthermore, this carbon signal showed an HSQC correlation with a methine proton signal at δH 5.21, which also exhibited a COSY crosspeak with H-7 in a 3 J-correlation, demonstrating the attachment of a chlorine atom at C-6. Together with the HMBC correlations between H-17/C-8, C-9, C-18, C-19 and H3-18/C-8, C-17, C-19, these data unambiguously established the molecular framework of 2.
According to the above and comparing the NMR data of 3 with those of the literature, the structure of 3 was highly similar to a known briarane, briarenol R (4) (Figure 1), which was originally isolated from a cultured B. stechei [16] and was also obtained in this study, except for a hydroxy group in 3 instead of an acetoxy group at C-2 in 4. The HMBC and COSY correlations, as shown in Figure 3a   The (+)-ESIMS mass spectra of 4 showed a pair of peaks at m/z 519/521 ([M + Na] + /[M + 2 + Na] + ) (3:1) with a relative intensity suggestive of a chlorine atom, indicating that the molecular formula of 4 was C 24 H 29 ClO 9 . The result revealed that this compound had 10 degrees of unsaturation. Strong bands at 3452, 1783, 1742, and 1680 cm −1 observed in the IR spectrum confirmed the presence of hydroxy, γ-lactone, ester, and α,β-unsaturated ketonic groups. The 13 C NMR and DEPT spectra revealed that 4 had 24 carbons, including 5 methyls, 1 sp 2 methylene, 9 sp 3 methines, 2 sp 2 methines, 2 sp 3 non-protonated carbons, and 5 sp 2 non-protonated carbons. Therefore, 4 was identified as having four rings. It was found that the spectroscopic data of 4 were identical to those of a known briarane, briarenol R [16], and these two compounds possessed negative optical rotation values ([α] 23 D -61 (c 0.01, CHCl 3 ) for 4 and [α] 22 D -55 (c 0.2, CHCl 3 ) for briarenol R [16]); thus, compound 4 was identified as briarenol R.

Bioactivty of Isolated Briaranes
It has been well documented that the microbial LPS can activate toll-like receptor-4 (TLR-4) located in mammal cell membrane surface, triggering inflammatory responses through the activation of intracellular signal transduction and the upregulation of pro-inflammatory protein iNOS [20]. Therefore, the determination of the inhibited rate of pro-inflammatory protein iNOS expression in LPS-stimulated macrophage cells can be used as an in vitro screening model for anti-inflammatory compounds [21][22][23]. The anti-inflammatory effect related to the release of iNOS from LPS-stimulated RAW 264.7 macrophage cells by briaranes 1-4 was assessed. In a concentration of 10 µM, briaexcavatolide P (1) reduced the release of iNOS (46.53%) as compared to results of the vehicle group, which did not, while briaranes 2-4 slightly reduced iNOS (Table 2 and Figure 4). These findings seem to be consistent with the results in the literature that demonstrated most briarane-type natural products can potentially be claimed to be anti-inflammatory agents [12]. Structure-activity relationships between 3 and 4 showed that the functional groups at C-2 might did not affect their activities. Data were normalized to those of cells treated with LPS alone, and cells treated with dexamethasone were used as a positive control. Data are expressed as the mean ± SEM (n = 3).

General Experimental Procedures
A digital polarimeter (model P-1010; Jasco Corp., Tokyo, Japan) was used to determine the optical rotations of the samples. IR spectra were collected using a spectrophotometer (model Nicolet iS5 FT-IR; Thermo Fisher Scientific, Waltham, MA, USA). 1 H and 13 C NMR spectra were recorded on ECZ-400 spectrometer (Jeol Ltd., Tokyo, Japan) for solutions in acetone-d 6 or CDCl 3 (with residual acetone (δ H 2.04 ppm) and acetone-d 6 (δ C 206.7, 29.8 ppm) or with residual CHCl 3 (δ H 7.26 ppm) and CDCl 3 (δ C 77.0 ppm), as internal standards). For coupling constants (J), the results are given in frequency units, Hz. For positive mode ESIMS and HRESIMS, the results were obtained using a SolariX FTMS mass spectrometer (7 Tesla; Bruker, Bremen, Germany). The extracted samples were separated by column chromatography with silica gel (range, 230−400 mesh; Merck, Darmstadt, Germany). Thin-layer chromatography plates with silica gel coated with fluorescent indicator F 254 were employed. For visualization, the plates were charred with 10% (v/v) aqueous sulfuric acid solution, then heated at 105 • C until spots were observed. For normal-phase HPLC (NP-HPLC) separation, a system containing a pump (Hitachi model L-7110; Tokyo, Japan) and an injection interface (No. 7725i; Rheodyne, Rohnert Park, CA, USA) was employed, which was equipped with a silica preparative column with a dimension of 250 × 20 mm and a 5 µm particle size (YMC-Pack SIL; Sigma-Aldrich, St. Louis, MO, USA). For reverse-phase HPLC (RP-HPLC) separation, a system composed of a pump (model L-2130, Hitachi, Tokyo, Japan) and a diode-array detector (model L-2455, Hitachi, Tokyo, Japan) was used, which was equipped with a C18 preparative column with a dimension of 250 × 21 mm and a 5 µm particle size (Luna, C18(2) 100Å, AXIA; Phenomenex, Torrance, CA, USA).

Animal Material
The specimens of Formosan Briareum stechei used for this study were collected from Orchid Island, Taitung These two samples were identified based on their morphology and micrographs of the coral sclerites using comparison as described in a previous study [8].

Okinawan Briareum stechei
Freeze-dried and sliced bodies (wet/dry weight = 618/305 g) of the coral specimen were extracted with a 1:1 mixture of MeOH and dichloromethane (CH 2 Cl 2 ) to give 42.7 g of crude extract, which was then subjected to liquid-liquid partition between EtOAc and H 2 O. The EtOAc phase (15.1 g) was applied on Si C.C. and eluted with a gradient solvent system of n-hexane/EtOAc mixtures (100% n-hexane−100% EtOAc, stepwise) to obtain 11 subfractions A-K. Fraction F was further subjected to the NP-HPLC with a solvent system of n-hexane/EtOAc mixture (3:2; flow rate = 5 mL/min) to yield 10 subfractions

Conclusions
In a continuation of our search for briaranes from B. stechei, briaexcavatolide P (1) found in this study was previously isolated from B. excavatum collected in the waters of Taiwan. In addition, two new chlorinated briaranes, briastecholides B (2) and C (3), together with a known analogue, briarenol R (4), were further identified from B. stechei. This octocoral is originally flourishing in the waters of Okinawa, where the Kuroshio current and South China Sea surface current converge to provide high biodiversity. Moreover, the structures, especially the absolute configurations of compounds 1-4, were determined based on spectroscopic data and biogenetic consideration. In bioassay, compound 1 displayed moderate activity against LPS-induced iNOS production. Accordingly, the diverse diterpenoids and their potential pharmacological effects of B. stechei demonstrated it worthy of further exploration.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.