Excavatoids E and F: Discovery of Two New Briaranes from the Cultured Octocoral Briareum excavatum

Two new briarane-related diterpenoids, designated as excavatoids E (1) and F (2), were isolated from the cultured octocoral Briareum excavatum. The structures of compounds 1 and 2 were established on the basis of extensive spectral data analysis. Briaranes 1 and 2 were found to exhibit moderate inhibitory effects on elastase release by human neutrophils.

Based on previous studies, all naturally occurring briarane-type diterpenoids have the C-15 methyl group trans to H-10, and these two groups are assigned as βand α-oriented, respectively, as shown in most briarane derivatives [42][43][44]. The relative stereochemistry of 1 was established from a NOESY experiment (Figure 3   The IR spectrum showed bands at 3,487, 1,779, and 1,737 cm -1 , consistent with the presence in 2 of hydroxy, γ-lactone, and ester groups. From the 13 C-NMR data of 2 (Table 1), a trisubstituted olefin was deduced from the signals of two carbons at δ C 146.2 (s, C-5) and 117.6 (d, CH-6). A methyl-containing tetrasubstituted epoxy group was confirmed from the signals of two quaternary oxygenated carbons at δ C 70.6 (s, C-8) and 64.5 (s, C-17), and from the chemical shifts of a tertiary methyl group (δ H 1.73, 3H, s, H 3 -18; δ C 10.3, q, CH 3 -18) (Table 1) The planar structure of 2 was determined mainly by 2D NMR studies. The coupling information in the 1 H-1 H COSY spectrum of 2 enabled identification of the proton sequences H-2/3/4, H-4/6 (by allylic coupling), H-6/7, H-6/H 3 -16 (by allylic coupling), H-9/10, and H-12/13/14 (Table 2). These data, together with the correlations observed in an HMBC experiment of 2 (Table 2), the molecular framework of 2 could be further established. The HMBC correlations also indicated that the acetoxy groups should attach at C-2, -9, -12, and C-14. Thus, the remaining hydroxy group has to be positioned at C-11, as indicated by characteristic NMR signal analysis. The relative stereochemistry of 2 was elucidated from the NOE interactions observed in a NOESY experiment (Figure 4). In the NOESY spectrum of 2, correlations were observed between H-10 with H-2, -9, -12, and H 3 -20, indicating that these protons should be positioned on the α face in 2 and Me-20 was positioned on the equatorial direction in the methylcyclohexane ring of 2. One proton attaching at C-3 (δ H 2.65) was found to exhibit a correlation with H 3 -15 and was assigned as H-3β proton. H-7 showed a correlation with H-3β, confirming the β-orientation for this proton. Furthermore, H 3 -18 was found to show correlations with H-9, H 3 -20, and H-7, and from molecular models, was found to be reasonably closed to H-9, H 3 -20, and H-7; therefore, H 3 -18 should be placed on the β face in the γ-lactone ring of 2. The Z-configuration of the C-5/C-6 double bond was elucidated by an interaction between H-6 (δ H 5.25) and H 3 -16 (δ H 2.02). On the basis of the above results, the structure of 2, including the relative configuration, was elucidated.   In previous study, several diterpenoid derivatives of potential medical interest were isolated from a cultured gorgonian coral Erythropodium caribaeorum [45]. Because all corals are claimed to be threatened species, we wanted to maintain and culture these interesting specimens as sources of new and interesting natural products in our continuing search for novel substances from marine organisms originally collected in Taiwan waters, in the hope of identifying extracts that exhibit bioactivity. Briaranes 1 and 2 displayed moderate inhibitory effects on elastase release by neutrophils, and briarane 1 exhibited weak inhibitory effects on superoxide anion generation by human neutrophils at 10 μg/mL, respectively (Table 3). Furthermore, these two compounds were not cytotoxic toward the CCRF-CEM (human T-cell acute lymphoblastic leukemia) and DLD-1 (human colon adenocarcinoma) cells (ED 50 > 40 μg/mL). The possible bioactivity for these two compounds will be further studied if we can obtain enough material from the cultured B. excavatum in the future.

General Experimental Procedures
Melting points were determined on a Fargo apparatus and were uncorrected. Optical rotation values were measured with a JASCO P-1010 digital polarimeter at 25 °C. Infrared spectra were obtained on a Varian Digilab FTS 1000 FT-IR spectrometer. The NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C, in CDCl 3 , respectively. Proton chemical shifts were referenced to the residual CHCl 3 signal (δ 7.26 ppm). 13 C-NMR spectra were referenced to the center peak of CDCl 3 at δ 77.1 ppm. ESIMS and HRESIMS data 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, a Hitachi photodiode array detector L-7455, and a Rheodyne 7725 injection port. A semi-preparative reverse phase column (Hibar 250 × 10 mm, LiChrospher 100 RP-18e, 5 μm, Merck) was used for HPLC.

Animal Material
Specimens of the octocoral Briareum excavatum were collected and transplanted to a 0.6-ton cultivating tank located in the NMMBA, Taiwan, in December 2003, and the material for this research work was collected from the tank in December 2006. This organism was identified by comparison with previous descriptions [46][47][48]. A voucher specimen was deposited in the National Museum of Marine Biology & Aquarium, Taiwan (NMMBA-CSC-001).

Extraction and Isolation
The freeze-dried and minced material of B. excavatum (wet weight 672 g, dry weight 270 g) was extracted with a mixture of MeOH and CH 2 Cl 2 (1:1) at room temperature. The residue was partitioned between EtOAc and H 2 O. The EtOAc layer was separated on Sephadex LH-20 and eluted using MeOH/CH 2 Cl 2 (2:1) to yield three fractions A-C. Fraction C was separated on silica gel and eluted using hexane/EtOAc (stepwise, 20:1-pure EtOAc) to yield fractions 1-9. Fraction C7 was separated by reverse phase HPLC, using the mixtures of MeOH, CH 3

Human Neutrophil Superoxide Anion Generation and Elastase Release
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide generation was carried out according to the procedures described previously [49,50]. 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.