Cembranoids from Octocoral Lobophytum crassum (von Marenzeller, 1886)

Two cembranoids, including a new compound, lobocrassin I (1), as well as a known analogue, lobohedleolide (2), were obtained by solvent extraction from octocoral Lobophytum crassum. This study employed a spectroscopic approach to establish the structures of these two cembranoids, and utilized single-crystal X-ray diffraction analysis to determine their absolute configurations. The results of biological activity assays demonstrated that cembranoid 2 exhibited bioactivity against the protein expressions of inducible nitric oxide synthase (iNOS) lipopolysaccharide (LPS)-treated RAW 264.7 mouse macrophage cells.


Introduction
Cembrane-type diterpenoids are a group of 14-membered macrolides obtained from terrestrial and marine organisms, with novel structures and extensive bioactivities [1]. Octocorals belonging to the genera Lobophytum, Sarcophyton, and Sinularia are currently known to be critical sources for the supply of cembranoids [2]. In connection with our known to be critical sources for the supply of cembranoids [2]. In connection with our 51 continuing studies of marine invertebrates with biomedical potential, we have focused 52 considerable attention on invertebrates found in the marine habitat of the waters around 53 Taiwan, with the aim of informing new drug development. In this research, we completed 54 the preparation, structural identification, and anti-inflammatory activity assessment of a 55 new cembranoid, lobocrassin I (1), as well as a known cembranoid, lobohedleolide (2) [3-56 6] (Figure 1), obtained from L. crassum (von Marenzeller, 1886) [7,8]. Lobophytum crassum 57 is a rich cembranoid-containing octocoral distributed extensively in tropical Indo-Pacific 58 Ocean, including Taiwanese waters where the Kuroshio current and South China Sea sur-59 face current converge to provide high biodiversity and the cembrane-type diterpenoids 60 prepared from soft coral L. crassum were proven to have the potential to be used as thera-61 peutic agents to treat inflammation [9][10][11][12][13][14][15]. This paper reported details of the isolation, 62 structure determination, and biological evaluation of cembranoids 1 (lobocrassin I) and 2 63 (lobohedleolide) (Figure 1). 64 Figure 1. Structures of lobocrassin I (1) and lobohedleolide (2). 65

Results and Discussion
Freshly-collected L. crassum was frozen and subsequently freeze-dried, powdered, and extracted with a solvent mixture of methanol/dichloromethane (MeOH/CH 2 Cl 2 ) at a 1:1 ratio to give an extract that was subsequently separated by organic solvent ethyl acetate (EtOAc)-water partitioning. The EtOAc layer was collected and loaded onto a column chromatograph with silica gel, and subsequently separated using high performance liquid chromatography (HPLC), yielding cembranoids 1 and 2. The known compound was elucidated as lobohedleolide (2) by analysis of its spectroscopic data and comparison with previously reported values [3].
The interactions obtained using nuclear Overhauser effect spectroscopy (NOESY) and the data of vicinal 1 H-1 H coupling constants revealed the relative stereochemistry of 1 (Figure 3). Biogenetically, in most cases the proton at C-1 is β-oriented in naturallyoccurring cembranoids from Lobophytum spp. [2]. The NOESY cross-peak of H-1 and H-2 suggested that these two protons were β-oriented. The vinyl methyl H 3 -18 exhibited a NOESY response with H-2, but not with H-3, and a NOESY correlation was observed between H-3 and H-15, demonstrating the trans configuration of ∆ 3 , and H-15 was therefore α-oriented in the γ-lactone moiety. The trans relationship between H-1 and H-15 was established from a large coupling constant (J = 12.0 Hz) for these two vicinal protons. H-11 showed a NOESY correlation with one of the C-13 methylene protons (δ H 2.01), but not with H 3 -20, indicating the trans configuration of ∆ 11 . Furthermore, olefin proton H-7 exhibited a NOESY correlation with one of the C-14 methylene protons (δ H 1.82), also demonstrating the cis geometry of ∆ 7 . Based on the aforementioned results, the configurations of the stereogenic centers of 1 were assigned as (1S*,2R*,15S*) (Supplementary Materials, Figures S1-S10). Single-crystal X-ray diffraction was used to confirm the structure of 1. The data sug-127 gested E-geometries of the C-3/4, C-11/12, and Z-geometry of the C-7/8 carbon-carbon 128 double bonds in 1; in addition, the absolute configurations of the stereogenic carbons of 1 129 were confirmed as (1S,2R,15S) based on an Oak Ridge thermal-ellipsoid plot (ORTEP) of 130 1 (Figure 4). According to the X-ray determined structure of 1, the carboxylic acids formed 131 dimers, in which the monomer units were held together by hydrogen bonds. Single-crystal X-ray diffraction was used to confirm the structure of 1. The data sug-127 gested E-geometries of the C-3/4, C-11/12, and Z-geometry of the C-7/8 carbon-carbon 128 double bonds in 1; in addition, the absolute configurations of the stereogenic carbons of 1 129 were confirmed as (1S,2R,15S) based on an Oak Ridge thermal-ellipsoid plot (ORTEP) of 130 1 (Figure 4). According to the X-ray determined structure of 1, the carboxylic acids formed 131 dimers, in which the monomer units were held together by hydrogen bonds. Single-crystal X-ray diffraction was used to confirm the structure of 1. The data suggested E-geometries of the C-3/4, C-11/12, and Z-geometry of the C-7/8 carbon-carbon double bonds in 1; in addition, the absolute configurations of the stereogenic carbons of 1 were confirmed as (1S,2R,15S) based on an Oak Ridge thermal-ellipsoid plot (ORTEP) of 1 (Figure 4). According to the X-ray determined structure of 1, the carboxylic acids formed dimers, in which the monomer units were held together by hydrogen bonds. showed a NOESY correlation with one of the C-13 methylene protons (δH 2.01), but not 119 with H3-20, indicating the trans configuration of ∆ 11 . Furthermore, olefin proton H-7 ex-120 hibited a NOESY correlation with one of the C-14 methylene protons (δH 1.82), also 121 demonstrating the cis geometry of ∆ 7 . Based on the aforementioned results, the configura-122 tions of the stereogenic centers of 1 were assigned as (1S*,2R*,15S*) (Supplementary Ma-123 terials, Figures S1-S10). Single-crystal X-ray diffraction was used to confirm the structure of 1. The data sug-127 gested E-geometries of the C-3/4, C-11/12, and Z-geometry of the C-7/8 carbon-carbon 128 double bonds in 1; in addition, the absolute configurations of the stereogenic carbons of 1 129 were confirmed as (1S,2R,15S) based on an Oak Ridge thermal-ellipsoid plot (ORTEP) of 130 1 (Figure 4). According to the X-ray determined structure of 1, the carboxylic acids formed 131 dimers, in which the monomer units were held together by hydrogen bonds. Cembranoid 2 was obtained as colorless prisms, showing a sodiated ESIMS quasimolecular ion peak at m/z 353, and was found to have the molecular formula C 20 H 26 O 4 by analysis of 13 C and 1 H NMR data (see Materials and Methods). The result revealed that this compound had 8 degrees of unsaturation. Strong bands at 3665~2398 (broad), 1760, and 1682 cm −1 in the IR spectrum indicated the presence of α,β-unsaturated carboxyl and γ-lactone groups. The 13 C NMR and DEPT spectra revealed that 2 had 20 carbons, including two methyls, six sp 3 methylenes, one sp 2 methylene, two sp 3 methines (one bearing a heteroatom), three sp 2 methines, and six sp 2 non-protonated carbons (one ester carbonyl and one carboxyl group). Therefore, 2 was identified as having two rings. It was found that the NMR data of 2 were almost identical to those of a known compound, lobohedleolide [3], and these two compounds were found to possess positive optical rotation value, suggesting that compound 2 is lobohedleolide, although the 13 C chemical shift for the carboxyl carbon in 2 (δ C 171.7, C-19) was different from that reported (δ C 173.2) [3] (Supplementary Materials, Figures S11-S19).
Lobohedleolide (2) was first isolated from octocoral Lobophytum hedleyi, collected from the coral reefs of Yayeyama Islands of Okinawa, Japan [3]. The structure of lobohedleolide was revealed by spectroscopic analysis, and its absolute configuration was concluded from X-ray study of its p-bromophenacyl ester derivative [3]. In this study, the structure of 2 was determined by single-crystal X-ray diffraction directly for the first time, and the absolute configuration for this compound was elucidated as (1S,2S) ( Figure 5). Because the structure of 2 has been established by a single-crystal X-ray diffraction analysis, the authors suggested that the 13 C chemical shift of carboxyl carbon in structure for lobohedleolide should be re-examined [3].
Mar. Drugs 2021, 19, x FOR PEER REVIEW 5 of 10 Cembranoid 2 was obtained as colorless prisms, showing a sodiated ESIMS quasi-136 molecular ion peak at m/z 353, and was found to have the molecular formula C20H26O4 by 137 analysis of 13 C and 1 H NMR data (see Materials and Methods). The result revealed that 138 this compound had 8 degrees of unsaturation. Strong bands at 3665~2398 (broad), 1760, 139 and 1682 cm −1 in the IR spectrum indicated the presence of α,β-unsaturated carboxyl and 140 γ-lactone groups. The 13 C NMR and DEPT spectra revealed that 2 had 20 carbons, includ-141 ing two methyls, six sp 3 methylenes, one sp 2 methylene, two sp 3 methines (one bearing a 142 heteroatom), three sp 2 methines, and six sp 2 non-protonated carbons (one ester carbonyl 143 and one carboxyl group). Therefore, 2 was identified as having two rings. It was found 144 that the NMR data of 2 were almost identical to those of a known compound, lobo-145 hedleolide [3], and these two compounds were found to possess positive optical rotation 146 value, suggesting that compound 2 is lobohedleolide, although the 13 Figures S11-S19). 149 Lobohedleolide (2) was first isolated from octocoral Lobophytum hedleyi, collected 150 from the coral reefs of Yayeyama Islands of Okinawa, Japan [3]. The structure of lobo-151 hedleolide was revealed by spectroscopic analysis, and its absolute configuration was con-152 cluded from X-ray study of its p-bromophenacyl ester derivative [3]. In this study, the 153 structure of 2 was determined by single-crystal X-ray diffraction directly for the first time, 154 and the absolute configuration for this compound was elucidated as (1S,2S) ( Figure 5). 155 Because the structure of 2 has been established by a single-crystal X-ray diffraction anal-156 ysis, the authors suggested that the 13 C chemical shift of carboxyl carbon in structure for 157 lobohedleolide should be re-examined [3].  The anti-inflammatory effects of lobocrassin I (1) and lobohedleolide (2) were as-161 sessed by measuring their effects on proinflammatory proteins/enzymes inducible nitric 162 oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) production from lipopoly-saccha-163 rides (LPS)-stimulated RAW264.7 cells (a murine macrophage cell line). Western blotting 164 results showed that cembranoid 2 at 10 µM inhibited iNOS expression, which reduced the 165 protein level to 28.50%, as compared with cells in the control group treated with LPS only 166 ( Table 2). In previous studies, cembranoid 2 was reported to exhibit extensive bioactivities 167 such as cytotoxic [3,5], antiviral [4, 16,17], and anti-inflammatory [16,[18][19][20] activities, and 168 the anti-inflammatory activity of 2 has been evaluated and revealed that 2 is effective 169 against both carrageenin induced edema and cotton pellet implantation rat models [18]. 170 Lobocrassin I (1) did not show activity, implying that the exo-methylene substituent at C-171 15 enhanced the bioactivity in comparison with cembranoid 2. The anti-inflammatory effects of lobocrassin I (1) and lobohedleolide (2) were assessed by measuring their effects on proinflammatory proteins/enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) production from lipopoly-saccharides (LPS)-stimulated RAW264.7 cells (a murine macrophage cell line). Western blotting results showed that cembranoid 2 at 10 µm inhibited iNOS expression, which reduced the protein level to 28.50%, as compared with cells in the control group treated with LPS only (Table 2). In previous studies, cembranoid 2 was reported to exhibit extensive bioactivities such as cytotoxic [3,5], antiviral [4, 16,17], and anti-inflammatory [16,[18][19][20] activities, and the antiinflammatory activity of 2 has been evaluated and revealed that 2 is effective against both carrageenin induced edema and cotton pellet implantation rat models [18]. Lobocrassin I (1) did not show activity, implying that the exo-methylene substituent at C-15 enhanced the bioactivity in comparison with cembranoid 2.

General Experimental Procedures
A digital polarimeter (model P-1010; JASCO Corp., Tokyo, Japan) was used to determine optical rotations of the samples. IR spectra were collected using a spectro-photometer (model Nicolet iS5 FT-IR; Thermo Fisher Scientific, Waltham, MA, USA). 1 H and 13 C NMR spectra were recorded on an ECZ-400 spectrometer (Jeol Ltd., Tokyo, Japan) for solutions in CDCl 3 (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 were given in frequency units, Hz. For 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 to 400 mesh; Merck, Darmstadt, Germany). The purity of a compound can be determined by Thin-layer chromatography (TLC), a method for analyzing mixtures by separating the compounds in the mixture. The TLC 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 seen. For normal-phase HPLC separation, a system containing a pump (Hitachi model L-7110; Tokyo, Japan) and an injection interface (No. 7725; Rheodyne) was employed, which was equipped with a semi-prep column with a dimension of 2 × 25 cm, 5 µm particle size (Sigma, St. Louis, MO, USA). For reverse-phase HPLC separation, a system composed of a pump (Hitachi model L-2130, Tokyo, Japan) and a diode-array detector (LaChrom L-2455, Hitachi, Tokyo, Japan) were used, which was equipped with a column with a dimension of 2.1 × 25 cm, 5 µm particle size (Phenomenex, Torrance, CA, USA).

Soft Coral Specimens
The soft coral L. crassum was manually collected by an underwater diver with a breathing apparatus from the marine habitat around Southern Taiwan on 23 July 2020. The specimens were frozen directly after harvesting. Identification of the specimens was performed by one of the authors of this study (Y.-T.Y.) by assessment of the features and comparison with the characteristics reported in the literature [7,8]. A representative sample of the soft coral (voucher no.: NMMBA-TW-SC-2020-0723) was stored in the National Museum of Marine Biology and Aquarium, Taiwan.

Cembranoid Compound Preparation
Lobophytum crassum (wet/dry weight = 1174 g/591 g) was crushed and then extracted with a mixture of MeOH and CH 2 Cl 2 (1:1) to give an extract (22.4 g), that was next separated by solvent-partition with EtOAc and H 2 O. The EtOAc extract (7.34 g) was then placed in an SiO 2 column and washed with an eluent of hexanes/EtOAc (by stepwise-gradient increase from 100:1-pure EtOAc) to yield 12 fractions A−L. Fraction J (294 mg) was then purified by semi-prep normal-phase HPLC to give eight fractions J1-J8. Fraction J3 (161.0 mg) was then purified by semi-prep reverse-phase HPLC (MeOH:H 2 O = 65:35 (v/v); at a rate of 5.0 mL/min) to afford compounds 2 (47.1 mg) and 1 (16.6 mg).