Briarenolides F and G, New Briarane Diterpenoids from a Briareum sp. Octocoral

Two new briarane diterpenoids, briarenolides, F (1) and G (2), were isolated from an octocoral identified as Briareum sp. The structures of briaranes 1 and 2 were established by spectroscopic methods and by comparison of the spectroscopic data with those of known briarane analogues. Briarenolide F was proven to be the first 6-hydroperoxybriarane derivative and this compound displayed a significant inhibitory effect on the generation of superoxide anion by human neutrophils.


Introduction
Among the diterpenoids isolated from octocorals, the briarane-type metabolites (3,8-cyclized cembranes) are a major group of compounds [1][2][3]. The compounds of this type were suggested to be of marine origin and the octocorals belonging to the genus Briareum have been proven to be the most important source of briarane-type compounds [4][5][6][7]. In previous studies, a series of interesting terpenoid derivatives, including briarane , cembrane [36] and carotenoid [37], had been isolated from the octocorals belonging to the genus Briareum that were distributed in the waters off Taiwan, at the intersection point of the Kuroshio current and the South China Sea surface current. In a continuation of our search for new substances from the Formosan marine invertebrates, the chemical constituents of a specimen octocoral identified as Briareum sp. (Briareidae) were studied. A fraction of its organic extract (fraction H, see Experimental Section) displayed inhibitory effects on the generation of superoxide anion (inhibition rate 36.8%) and the release of elastase (inhibition rate 90.3%) at a concentration of 10 μg/mL. We further isolated two new briarane-type diterpenoids, briarenolides, F (1) and G (2) (Figure 1), from the octocoral Briareum sp. In this paper, we report the isolation, structure determination and bioactivity of briaranes 1 and 2. (1) (2)

Results and Discussion
Briarenolide F (1) was isolated as a white powder. The molecular formula of 1 was established as C 28 H 40 O 12 (nine degrees of unsaturation) from a sodium adduct at m/z 591 in the ESIMS spectrum and further supported by HRESIMS (C 28 H 40 O 12 Na, m/z 591.2420, alculated 591.2417). The IR spectrum of 1 showed bands at 3498, 1789 and 1743 cm −1 , consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. The 13 C NMR and DEPT spectra of 1 showed that this compound had 28 carbons (Table 1), including seven methyls, four sp 3 methylenes, eight sp 3 methines, three sp 3 quaternary carbons, an sp 2 methine and five sp 2 quaternary carbons. From the 1 H and 13 C NMR spectra (Table 1) 8, qC-5). The presence of a tetrasubstituted epoxide containing a methyl substituent was established from the signals of two quaternary oxygenated carbons at δ C 68.8 (qC-8) and 58.4 (qC-17) and further confirmed by the proton signal of a methyl singlet at δ H 1.49 (3H, s, H 3 -18). Thus, from the above NMR data, five degrees of unsaturation were accounted for and 1 was identified as a tetracyclic compound.   The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H 3 -15/C-1, -2, -10, -14; H-2/C-15; and H-10/C-15. In addition, the carbon signal at δ C 173.1 (qC) was correlated with the signal of the methylene protons at δ H 2.27 in the HMBC spectrum and was consequently assigned as the carbon atom of the n-butyrate carbonyl. Additionally, the n-butyrate positioned at C-12 was confirmed by the connectivity between H-12 (δ H 4.98) and the carbonyl carbon (δ C 173.1, qC) of the n-butyrate. Furthermore, an acetate ester at C-2 was established by a correlation between H-2 (δ H 5.22) and the acetate carbonyl (δ C 170.6, qC) observed in the HMBC spectrum of 1.
The presence of a hydroxy group at C-9 was deduced from the 1 H-1 H COSY correlation between a hydroxy proton (δ H 2.95) and H-9 (δ H 4.59). The presence of a hydroperoxy group in 1 was supported by a hydroperoxy proton signal at δ H 8.71 as a broad signlet [22,32,38]. Due to absence of HMBC correlations for H-14 (δ H 4.88) and the hydroperoxy proton (δ H 8.71), the positions for the remaining acetoxy and hydroperoxy groups could not be determined by this method. By comparison the 1 H and 13 C NMR data of C-14 oxymethine for 1 (δ H 4.88; δ C 74.9) with those of a known briarane analogue, Figure 2) [10], which possesses a similar cyclohexane moiety as that of 1, the remaining acetoxy group in 1 was placed at C-14. Thus, the hydroperoxy group is positioned at C-6, an oxymethine at δ C 84.9 (CH), by analysis of the 1 H-1 H COSY correlations and characteristic NMR signal analysis. (1) In all naturally-occurring briaranes, H-10 is trans to the C-15 methyl group, and these two groups are assigned as α-and β-oriented in most briarane derivatives [4][5][6][7]. The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment and was found to be compatible with that of 1 offered by computer modeling (Figure 3) [39] and that obtained from vicinal proton coupling constant analysis. In the NOESY experiment of 1, the correlations of H-10 with H-2, H-9, H-11 and H-12, but not with H 3 -15 and H 3 -20, indicated that these protons (H-2, H-9, H-10, H-11 and H-12) were situated on the same face, and these were assigned as α protons, since the C-15 and C-20 methyls are β-substituents at C-1 and C-11, respectively. H-14 was found to exhibit an interaction with H 3 -15, but not with H-10, revealing the β-orientation of this proton. The configuration at C-9 is worthy of comment. H-9 was found to exhibit correlations with H-10, H-11, H 3 -18 and H 3 -20. From a consideration of molecular models, H-9 was found to be reasonably close to H-10, H-11, H 3 -18 and H 3 -20, while it was placed on the α face in 1. The C-16 vinyl methyl showed correlations with H-4 and H-6, demonstrating the Z configuration of Δ 4,5 and the hydroperoxy group at C-6 was α-oriented. The cis relationship between H-6 and H-7 was established by a correlation between H-6 and H-7 and a small coupling constant (J = 2.4 Hz) between these two protons. Moreover, an acetyl methyl (δ H 2.01) exhibited correlations with H-12 and H-2, further supporting an acetoxy group was positioned on the α-position at C-14 in 1. Based on the above findings, the configurations of all chiral carbons of 1 were assigned as 1S*, 2S*, 6S*, 7S*, 8R*, 9S*, 10S*, 11R*, 12S*, 14S*, 17R*, and the structure of 1 was established unambiguously. To the best of our knowledge, briarane derivatives possessing a hydroperoxy group are rarely found [22,32,38] and briarenolide F (1) is the first briarane derivative possessing a 6-hydroperoxy group. A double bond positioned at C-4(5) in briarane-type metabolites is also rarely found [31,[40][41][42].  , an acetate carbonyl (δ C 170.9, qC), an α,β-unsaturated-γ-lactone carbonyl (δ C 171.1, qC-19), a tetrasubstituted carbon atom bearing a carbon substituent (δ C 39.1, qC-1) and an oxymethine (δ C 77.2, CH-14). From the 1 H-1 H COSY experiment of 2 (Table 2), it was possible to establish the separate spin systems that map out the proton sequences from H 2 -2/H 2 -3/H 2 -4 and H 2 -9/H-10. These data, together with the HMBC correlations between H 2 -2/C-1, -3, -10; H 2 -3/C-2; H-4β/C-3; H-6/C-4, -7; and H 2 -9/C-7, -8, -10, established the connectivity from C-1 to C-10 in the ten-membered ring ( Table 2). The vinyl methyl at C-5 was confirmed by the HMBC correlations between H 3 -16/C-4, -5, -6; H-4β/C-16; and H-6/C-16, and further supported by the allylic coupling between H-6 and H 3 -16. The methylcyclohexene ring, which is fused to the ten-membered ring at C-1 and C-10, was elucidated by the 1 H-1 H COSY correlations between H-12/H 2 -13/H-14 and H-12/H 3 -20 (by allylic coupling) and by the HMBC correlations between H 2 -2/C-14, H 2 -9/C-11 and H 3 -20/C-10, -11, -12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H 3 -15/C-1, -2, -10, -14. In addition, the acetate ester at C-14 was established by a correlation between H-14 (δ H 4.78) and the acetate carbonyl observed in the HMBC spectrum of 2. The presence of a hydroxy group at C-7 was deduced from the HMBC correlations between the hydroxy proton (δ H 3.35, 1H, s, OH-7) and C-6, C-7, and C-8. The C-7 hydroxy group was concluded to be a part of hemiketal constellation on the basis of a characteristic carbon signal at δ C 106.7 (a quaternary hemiketal carbon, qC-7). These data, together with the HMBC correlations between H 3 -18/C-8, -17, -19, were used to establish the molecular framework of 2.
NOESY measurements were carried out in order to deduce the relative stereochemical features of 2 ( Figure 4). Thus, H 3 -15 gave a correlation with H-14, but not with H-10, indicating that H 3 -15 and H-14 are located on the same face (assigned as the β-face) and that H-10 lies on the opposite side, α-face. The NOESY spectrum showed correlations between H-6/H 3 -16 and H-12/H 3 -20, revealing the Z geometry of the C-5/6 and C-11/12 double bonds in 2. Due to the absence of NOESY correlations for the C-7 hydroxy group, the configuration at that chiral center could not be determined by this method. By comparison of the 13 C NMR chemical shifts of C-6 (δ C 124.6), C-7 (δ C 106.7) and C-8 (δ C 160.8) for 2 with those of an unnamed known 7β-hydroxybriarane analogue 4 (δ C 124.8, C-6; 106.2, C-7, 160.1, C-8), which was obtained from a Caribbean octocoral Briareum polyanthes [43] ( Figure 5), we deduced that the C-7 hydroxy group was β-oriented and the configuration of all the chiral carbons in 2 were assigned as 1R*, 7S*, 10S*, 14S*.  (2) The in vitro anti-inflammatory effects of briaranes 1 and 2 were tested. Briarenolide F (1) was found to display a significant inhibitory effect on the generation of superoxide anion by human neutrophils (Table 3). Table 3. Inhibitory effects of briaranes 1 and 2 on the generation of superoxide anion and the release of elastase by human neutrophils in response to FMLP/CB.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm −1 . The NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer. Coupling constants (J) are given in Hz. 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HMQC, HMBC and NOESY experiments. ESIMS and HRESIMS were recorded on a Bruker APEX II mass spectrometer. Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck), and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. HPLC was performed using a system comprised of a Hitachi L-7100 pump and a Rheodyne injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 μm) was used for HPLC.

Animal Material
Specimens of the octocorals Briareum sp. were collected by hand using scuba equipment off the coast of southern Taiwan in July 2011 and stored in a freezer until extraction. A voucher specimen (NMMBA-TW-SC-2011-77) was deposited in the National Museum of Marine Biology and Aquarium. This organism was identified by comparison with previous descriptions [44][45][46][47].

Extraction and Isolation
Sliced bodies of Briareum sp. (wet weight 6.32 kg, dry weight 2.78 kg) were extracted with a mixture of methanol (MeOH) and dichloromethane (DCM) (1:1). The extract was partitioned between ethyl acetate (EtOAc) and H 2 O. The EtOAc layer was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 100:1-pure EtOAc) to yield 18 fractions A-R. Fraction H was chromatographed on silica gel and eluted using n-hexane/acetone (stepwise, 40:1-pure acetone) to afford 45 fractions H1-H45. Fraction H11 was separated by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and EtOAc (5:2) as the mobile phase to afford compound 2 (0.4 mg). Fraction H16 was further purified by normal-phase HPLC using a mixture of n-hexane and acetone as the mobile phase (7:2) to afford compound 1 (2.3 mg).

Molecular Mechanics Calculations
Implementation of the MM2 force field [39] in CHEM3D PRO software from CambridgeSoft Corporation (version 9.0, Cambridge, MA, USA; 2005) was used to calculate the molecular models.

Superoxide Anion Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [48,49]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Conclusions
Briarane-type natural products (3,8-cyclized cembranoid) were found in various marine organisms, particularly with the octocorals belonging to the genus Briareum (family Briareidae) [4][5][6][7]. It is interesting to note that the briarane-type natural products are major constituents of the extracts of octocorals Briareum spp. distributed in the tropical and subtropical Indo-Pacific Ocean. In the past 35 years, over 500 briarane analogues have been obtained and the number is still increasing based on their structural complexity and interesting bioactivities. It is worth noting that only three hydroperoxybriarane analogues have been isolated to date [22,32,38] and that briarenolide F (1) is the first 6-hydroperoxybriarane. 7-Hydroxybriarane derivatives are also rarely found [43,[50][51][52]; the new briarane, briarenolide G (2) was the first 7-hydroxybriarane derivative isolated from the octocorals collected off the waters of Taiwan. The study material Briareum sp. has begun to be transplanted in tanks for the extraction of natural products in order to establish a stable supply of bioactive material.