Briastecholide M, a New Polyoxygenated Briarane from Briareum stechei

Abstract

A formerly unpublicized briarane diterpenoid, briastecholide M (1), and its established analogue, brianodin B (2), were purified from Briareum stechei, an octocoral collected from Okinawan waters. Using spectroscopic methods, the structure of 1 was established. Functional study showed that 1 can reducing the release of inducible nitric oxide synthase (iNOS) but enhancing cyclooxygenase-2 (COX-2) protein expression.

Introduction

The Indo-Pacific genus Briareum is a group of octocorals of the family Briareidae that is known to include four distinguished species including B. stechei.¹⁾ Various diterpenoids have been obtained from theses intriguing marine invertebrates, including briaranes, a group of marine-origin diterpenoids featuring a bicyclo[8.4.0]tetradecane system. These briarane-skeleton compounds were reported to possess cytotoxicity²⁾ and anti-inflammatory activity.^3–5) We previously conducted substantial examinations of B. stechei, both wild- and cultured-type,^4–6) via which a new highly-oxygenated briarane, briastecholide M (1), and its known analogue, brianodin B (2),⁷⁾ were isolated (Fig. 1).

Fig. 1. Structures of Briaranes 1 (Briastecholide M) and 2 (Brianodin B)

Results and Discussion

Briastecholide M (1) was obtained as an amorphous powder and its high resolution electrospray ionization (HR-ESI)-MS spectrum established a molecular formula C₃₀H₄₂O₁₄ at m/z 649.24646 (Calcd for C₃₀H₄₂O₁₄Na, 649.24668). The IR absorptions at v_max 3447, 1773, 1735 cm⁻¹ consistent with the presences of hydroxy, γ-lactone, and ester functionalities, respectively. The ¹³C-NMR spectrum indicated the presence of 30 signals (Table 1), shown by the distortionless enhancement of polarization transfer (DEPT) spectrum to arise from eight methyls, two sp³-CH₂, seven sp³-CH, four sp³ quaternary carbons, three sp²-CH, one sp² quaternary carbon, and five sp² carbonyls. Based on the NMR data (Table 1), 1 was found to possess one disubstituted olefin (δ_H 5.69/δ_C 120.6, CH-13; δ_H 5.56/δ_C 141.6, CH-14), one trisubstituted olefin (δ_H 5.75/δ_C 126.2, CH-6; δ_C 139.2, C-5), three acetoxy groups (δ_H 2.21, 2.14, 2.10/δ_C 20.8, 22.0, 21.0/δ_C 169.5, 170.4, 173.0), and a γ-lactone moiety (δ_C 175.5, C-19). The additional ester group was clarified as an n-butyroxy group, and contiguous -CH₂-CH₂-CH₃ protons (δ_H 2.39/1.68/0.98) were revealed by ¹H–¹H correlation spectroscopy (COSY) (Fig. 2a). Based on correlations obtained from heteronuclear multiple bond connectivity (HMBC) study (Fig. 2a), the carbon signal at δ_C 173.5 was discovered to be related to the signals of the methylene protons at δ_H 2.39 and 1.68 and was accordingly allocated as the carbon atom of the n-butyrate carbonyl.

Table 1. ¹H- and ¹³C-NMR Data for Briaranes 1 and 2, and Brianodin B

	1		2		Brianodin Bf)
C/H	δHa) (J in Hz)	δC,b) type	δHd) (J in Hz)	δC,e) type	δH (J in Hz)	δC
1		46.8, Cc)		46.8, Cc)		47.0
2	4.67 s	76.8, CH	4.66 s	76.9, CH	4.66 br s	78.4
3	5.08 dd (10.8, 8.4)	70.8, CH	5.07 dd (10.8, 8.0)	70.6, CH	5.07 d (11.2)	70.8
4	4.90 d (10.8)	76.6, CH	4.86 dd (10.8, 1.2)	76.8, CH	4.86 br dd (11.0, 0.8)	77.1
5		139.2, C		139.2, C		139.4
6	5.75 br d (9.6)	126.2, CH	5.75 ddq (10.0, 1.2, 1.2)	126.2, CH	5.75 br d (10.0)	126.5
7	5.95 d (9.6)	78.0, CH	5.93 dd (10.0, 1.2)	77.9, CH	5.93 d (10.0)	79.3
8		79.0, C		79.0, C		79.3
9	6.08 d (4.8)	65.2, CH	6.07 d (4.8)	65.2, CH	6.07 d (4.8)	65.6
10	2.97 d (4.8)	39.5, CH	2.96 d (4.8)	39.5, CH	2.96 d (4.8)	39.6
11		76.2, C		76.1, C		75.8
12	5.04 d (6.6)	72.1, CH	5.04 d (6.4)	72.0, CH	5.04 d (3.3)	72.2
13	5.69 dd (10.2, 6.6)	120.6, CH	5.68 dd (10.0, 6.4)	120.7, CH	5.68 m	121.1
14	5.56 d (10.2)	141.6, CH	5.55 d (10.0)	141.6, CH	5.55 d (4.5)	141.7
15	1.29 s	15.3, CH3	1.29 s	15.3, CH3	1.29 s	16.8
16	2.13 br d (1.2)	25.7, CH3	2.13 br d (1.2)	25.7, CH3	2.13 br d (1.6)	25.9
17		80.2, C		80.1, C		80.2
18	1.46 s	16.9, CH3	1.45 s	16.9, CH3	1.45 s	15.6
19		175.5, C		175.6, C		176.4
20	1.51 s	24.0, CH3	1.50 s	23.9, CH3	1.49 s	23.6
OH-3	2.26 d (8.4)		2.30 d (8.0)
OH-8	5.62 s		5.65 d (1.2)
OH-11	3.08 s		3.17 s
OH-17	2.12 s		2.18 s
OAc-2	2.21 s	169.5, C 20.8, CH3	2.21 s	169.5, C 20.8, CH3	2.21 s	169.9 21.0
OAc-4			2.15 s	171.0, C 21.2, CH3	2.15 s	171.3 21.4
OAc-9	2.14 s	170.4, C 22.0, CH3	2.15 s	170.4, C 22.1, CH3	2.15 s	170.9 22.3
OAc-12	2.10 s	173.0, C 21.0, CH3	2.10 s	172.9, C 21.0, CH3	2.10 s	172.7 21.2
n-OC(O)Pr-4	2.39 t (7.2) 1.68 m 0.98 t (7.2)	173.5, C 36.2, CH2 18.4, CH2 13.6, CH3

a) Spectrum recorded at 600 MHz in CDCl₃. b) Spectrum recorded at 150 MHz in CDCl₃. c) Multiplicity deduced from DEPT and HSQC spectra. d) Spectrum recorded at 400 MHz in CDCl₃. e) Spectrum recorded at 100 MHz in CDCl₃. f) Data were reported by Ishiyama et al., ¹H and ¹³C spectra recorded in CDCl₃, see Ref.⁷⁾

Fig. 2. Key COSY and HMBC Correlations of Compound 1 (a), and Its Stereoview and Calculated Distances (Å) between Particular Pairs of Protons That Have Crucial NOESY Correlations (b)

3D structure was created by ChemDraw software with MM2 force field.

The H-3/H-4, H-6/H-7, H-9/H-10, and H-12/H-13/H-14 spin systems, measured in the COSY spectrum, were appropriate to the regiochemistry of vicinal couplings in 1. The fused tricyclic network was established by HMBC experiments from the ²J- and ³J-¹H–¹³C correlations between protons and non-protonated carbons, such as H-2, H-3, H-10, H-13, H-14, H₃-15/C-1; H-4, H-7, H₃-16/C-5; H-9, H-10, H₃-18, OH-8, OH-17/C-8; H-9, H-10, H-12, H-13, H₃-20/C-11; H-9, H₃-18, OH-17/C-17; and H₃-18/C-19. The methyl groups Me-15, Me-16, Me-20, and Me-18 at C-1, C-5, C-11, and C-17 were established from HMBC correlations between H₃-15/C-1, C-2, C-10, C-14; H₃-16/C-4, C-5, C-6; H₃-20/C-10, C-11, C-12; and H₃-18/C-8, C-17, C-19. The hydroxy proton signal at δ_H 2.26 was revealed by its COSY correlation with H-3 (δ_H 5.08), which indicated its attachment to C-3. The oxymethine protons at δ_H 4.67 (H-2), 6.08 (H-9), 5.04 (H-12), and 4.90 (H-4) showed correlations with the ester carbonyls at δ_C 169.5, 170.4, 173.0, and 173.5, in the HMBC spectrum, which demonstrated the placement of acetoxy groups at C-2, C-9, C-12, and an n-butyroxy group at C-4. HMBC correlations from the hydroxy protons at δ_H 5.62 to δ_C 78.0 (CH-7)/79.0 (C-8)/65.2 (CH-9); and δ_H 2.12 to δ_C 79.0 (C-8)/80.2 (C-17), confirmed the presence of hydroxy groups at C-8 and C-17. Therefore, 13 of the 14 oxygen atoms in the molecular formula of 1 could be accounted for by the existence of one γ-lactone, four esters, and three hydroxy groups. The remaining oxygen atom had to be located at C-11, as a hydroxy group, indicated by its ¹³C-NMR chemical shift resonating at δ_C 76.2.

Stereochemical analysis of 1 was completed using a nuclear Overhauser effect spectroscopy (NOESY) experiment and further supported by MM2 force field analysis,⁸⁾ demonstrating that a stable conformer was as shown in Fig. 2b. Briaranes were determined to have Me-15 in the β-orientation and proton H-10 in the α-orientation, evidenced by the lack of correlation between the two groups. H-10 was correlated with H-2/OH-8/H-9, which suggested that H-2, H-9, and the hydroxy group at C-8 were α-oriented; and correlations of H₃-15 with H-3 and H₃-20 indicated that Me-20 at C-11 and the hydroxy group at C-3 were β- and α-oriented, respectively. By modeling analysis, the correlations between H₃-15/H-3 and H-3/H-7 showed that H-7 was β-oriented. H-6 exhibited a cross peak with the vinyl methyl C-16, but not with H-7, and a large coupling constant was also noted between H-6 and H-7 (J = 9.6 Hz), which showed that the plane between H-6 and H-7 had a dihedral angle of about 180°,⁹⁾ representing the Z-geometry of ∆⁵. H-4 exhibited correlations with H-2 and H₃-16, suggesting the β-orientation of the 4-n-butyroxy group. H₃-18 was found to show associations with OH-8 and H-9, and reflected the α-orientation of Me-18 at C-17. The coupling constant of 10.2 Hz between H-13 and H-14 specified the cis geometry of ∆¹³. H-12 was observed to have associations with H-13/H₃-20/OH-11. From molecular model consideration, H-12 was found to be relatively close to H-13, H₃-20, and OH-11, and therefore H-12 should be placed on the β face.

Compound 1 seemed to be very similar to brianodin B, which was previously isolated from the octocoral Pachyclavularia sp.⁷⁾ (synonyms Briareum),¹⁾ and was also obtained in this study and presented as compound 2 (Fig. 1). It was clear that the acetoxy group at the C-4 position in 2 was replaced by an n-butyroxy group in 1; however, we found that the NMR data for brianodin B differed slightly from those of 2 reported in this article (Table 1). For example, the ¹³C-NMR data of C-2 (δ_C 78.4), C-7 (δ_C 79.3), C-15 (δ_C 16.8), and C-18 (δ_C 15.6), and the ¹H-coupling constant of H-14 (δ_H 5.55, 1H, d, J = 4.5 Hz) for brianodin B⁷⁾ were different by comparison with those of 2 (δ_C 76.9, C-2; 77.9, C-7; 15.3, C-15; 16.9, C-18; δ_H 5.55, 1H, d, J = 10.0 Hz, H-14). The structure of brianodin B is not in question; however, we suggest that the NMR data for brianodin B be re-examined.⁷⁾

The structures of briaranes 1 and 2 were resemblance to that of brianodin A,⁷⁾ which was also isolated from B. stechei, and whose absolute configuration was concluded by single-crystal X-ray diffraction study in a previous study.⁵⁾ According to the principles of biogenesis, it is reasonable to consider that briaranes 1 and 2 have the same absolute configuration as brianodin A. Hence, the configurations of the stereocarbons of both briaranes 1 and 2 should be assigned as 1S, 2R, 3S, 4S, 7S, 8S, 9S, 10S, 11S, 12R, 17S. It is worth noting that only 21 naturally-occurring briaranes were found to possess a hydroxy group at C-17, and all of the compounds of this type were isolated from octocorals belonging to the genus Briareum.^7,10–17) The compounds of this type (17-hydroxybriarane) could therefore be a chemical marker for the genus Briareum.

Using an in vitro pro-inflammatory assay with a RAW264.7 macrophage cell line, the effects of briaranes 1 and 2 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins from lipopolysaccharide (LPS)-stimulated macrophages were assessed. The results indicated that 1 at 10 µM reduced the expression of iNOS, but enhanced the release of COX-2 (Table 2). Briarane 2 was found to be inactive against the release of above two proteins, which indicated that the bulky butyrate at C-4 could significantly affect the biological activities of compounds of this category.

Table 2. Anti-inflammatory Effects of Briaranes 1 and 2 on iNOS and COX-2 Protein Expressions in Macrophage Cells

	iNOS	COX-2
Compound	Expression (% of LPS)		n
Control	2.40 ± 0.47	4.05 ± 0.51	3
Vehicle	100.00 ± 0.00	100.00 ± 0.00	3
1	82.50 ± 2.61	112.28 ± 8.41	3
2	93.73 ± 2.81	103.02 ± 9.72	3
Dexamethasone	41.36 ± 2.13	29.19 ± 0.64	3

Data were normalized to those of cells treated with LPS alone (vehicle), and dexamethasone-treated cells (at 10 µM) were used as a positive control for anti-inflammation response. Control: cells without LPS treatment. Data are presented as the mean ± standard error of the mean (S.E.M.).

Experimental

General

The optical rotation was measured on a JASCO P-1010 polarimeter. The IR spectra were recorded on a THERMO Scientific Nicolet iS5 FT-IR spectrophotometer. One-dimensional (1D) and two-dimensional NMR spectra were recorded on spectrometers (JEOL; model: ECZ 400S and ECZ 600R, respectively) in solution in CDCl₃ [residual CHCl₃ (δ_H = 7.26 ppm) and CDCl₃ (δ_C = 77.0 ppm) were used as internal references]. For positive-mode (ESI)-MS and (HR-ESI)-MS, the results were acquired from the BRUKER SolariX FT mass spectrometer. Silica gel 60 (mesh#: 230–400, MERCK, Germany) were used for column chromatography. Pre-coated silica gel plates (MERCK, Kieselgel 60 F₂₅₄, 0.25 mm), were used for TLC. For normal-phase HPLC separation, a system equipped with a normal-phase column (YMC-Pack SIL, 25.0 × 2.0 cm, 5 µm; Sigma-Aldrich, U.S.A.) was used. The system included a pump (HITACHI, L 7110, Japan) coupled with an injection port (RHEODYNE, model: 7725). For reverse-phase HPLC separation, a system that consisted of a pump (HITACHI, L 2130) with a diode array detector (HITACHI, L 2455) equipped with a reverse-phase column (Luna, 5 µm, C18(2) 100 Å, 25.0 × 2.1 cm) was used.

Animal Material

Specimens of the octocoral Briareum stechei were hand-harvested by a SCUBA diver off the coast of Ie Island, Okinawa, Japan (26°44′21.8″N, 127°48′33.8″E) in 2019. Specimen identification was carried out by comparing the morphology and coral sclerites with samples from earlier studies.^1,18) A voucher specimen was deposited at the NMMBA, Taiwan (NMMBA-TW-OK-2019-001).

Extraction and Isolation

A total of 167.0 g (wet weight) of Briareum stechei was dried (dry weight 84.8 g) and minced, followed by solvent extraction with MeOH and dichloromethane (1 : 1, v/v) to give extract (10.9 g) after evaporation under reduced pressure. This crude was then partitioned using ethyl acetate (EtOAc) and water to afford the EtOAc-soluble fraction (3.6 g). The EtOAc-soluble layer was loaded for silica column chromatography using n-hexane and n-hexane/EtOAc of increasing polarity, to give fractions A–K. Fraction G (98/196.0 mg) was separated by NP-HPLC using an isocratic system (71% n-hexane in acetone; 5 mL/min) to give subfractions G1–G9. Fraction G6 was then purified by RP-HPLC on a Luna C18(2) column using an isocratic system (65% of MeOH in H₂O; 5 mL/min) to give 1. Fraction H (95.0 mg) was separated by NP-HPLC on a YMC-Pack SIL column using an isocratic system (n-hexane/acetone) to yield subfractions H1 − 10. Fraction H9 was purified by RP-HPLC on a Luna C18(2) column using an isocratic system (65% MeOH in H₂O; 5 mL/min) to yield 2.

Briastecholide M (1): Colorless oil (0.4 mg); [α]_D²² −80° (c = 0.02, CHCl₃); IR (KBr) ν_max 3447, 1773, 1735 cm⁻¹; data of ¹H-NMR (CDCl₃, 600 MHz) and ¹³C-NMR (CDCl₃, 150 MHz) are listed in Table 1; ESI-MS m/z: 649 (M + Na)⁺; HR-ESI-MS m/z 649.24646 (Calcd for C₃₀H₄₂O₁₄Na, 649.24668).

Brianodin B (2): Colorless oil (0.5 mg); [α]_D²⁵ −58° (c = 0.03, CHCl₃) [ref.⁷⁾ [α]_D²⁵ −6 (c = 1.0, CHCl₃)]; IR (KBr) ν_max 3408, 1762, 1735 cm⁻¹; data of ¹H-NMR (CDCl₃, 400 MHz) and ¹³C-NMR (CDCl₃, 100 MHz) are listed in Table 1; ESI-MS m/z: 621 (M + Na)⁺.

Molecular Mechanics Calculations

The implementation of the MM2 hydrocarbon force field⁸⁾ in 3D Ultra software from CambridgeSoft Corporation (version 15.00) was used to create the 3D model.

In Vitro Inflammatory Assay

A RAW264.7 cell line was used to establish an in vitro assay to assess the bioactivities of briaranes 1 and 2 on LPS-induced inflammation in macrophages, in terms of stimulation of the release of iNOS and COX-2, as shown in earlier studies.^19,20)

Acknowledgments

The authors thank Hsiao-Ching Yu and Chao-Lien Ho from National Sun Yat-sen University for their assistance in collecting MS data (MS 0006 00) and the performance of NMR (NMR 001100) (MOST 111-2731-M-110-001). This work was supported by fundings from the NMMBA and the NSTC (MOST 111-2320-B-291-001), Taiwan, awarded to P.-J.S.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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