Briarane-type diterpenoids, a group of diterpenoids having a highly oxidized bicyclo[8.4.0] system with a γ-lactone group are found only in marine organisms and mainly from octocorals [1,2,3]. The compounds of this type are proven to possess various bioactivities such as anti-inflammatory, cytotoxicity, and antibacterial activity [1,2,3]. Octocorals belonging to the genus Briareum (family Briareidae) are recognized as rich sources of briarane-type diterpenoids (3,8-cyclized cembranoid) [1,2,3]. The samples for previous studies on the secondary metabolites of the Taiwanese octocoral B. excavatum were all collected along the southern coast of Taiwan [1,2,3].

HCMV is a highly ubiquitous pathogen in human population global prevalence 60~90%. For most healthy people, HCMV remains a long-term subclinical infection, however, in congenital neonates and in immunocompromised patients the virus can cause severe diseases. Of the FDA approved therapeutic agents, ganciclovir, foscarnet, and cidofovir are reported to have adverse effects on bone marrow and the kidneys. The first chemical investigation of B. excavatum (Figure 1) collected at Orchid Island off Taiwan during August 2008 afforded three new briarane-type diterpenoids, briacavatolides A–C (1–3) as well as two known briaranes, briaexcavatolide U (4) [4] and briaexcavatin L (5) [5] (Figure 2). The anti-HCMV (human cytomegalovirus) activity of 1–5 and their cytotoxicity against selected cell lines were evaluated.

Briacavatolide A (1) was obtained as a white powder. Its HRESIMS and NMR spectroscopic data established a molecular formula of C₂₄H₃₄O₁₁, implying the existence of eight double bond equivalents. The ¹³C NMR spectra showed signs of 24 carbons differentiated by DEPT into six methyls, two methylenes, eight methines and eight quaternary carbons. The ¹H and ¹³C NMR spectra (Table 1) of 1 indicated the presence of two acetoxyls (δ_H 1.99, 2.00; δ_C 21.5, 21.3, 170.4,170.4), a lactone (δ_H 6.18; δ_C 173.1), and a trisubstituted olefin (δ_H 5.27; δ_C 145.6, 121.8). A tetrasubstituted epoxide containing a methyl substituent was revealed from the signals of two quaternary oxygenated carbons (δ_C 65.2, 72.0) and a methyl (δ_C 9.2; δ_H 1.66, 3H). From the above data, metabolite 1 was found to be a tetracyclic compound. The structure and all of the ¹H and ¹³C chemical shifts of 1 were determined by the assistance of 2D NMR experiments, including ¹H-¹H COSY and HMBC experiments (Figure 3). By explanation of ¹H−¹H COSY correlations (Figure 3), it was possible to establish four partial structures of consecutive proton systems extending from H-2 to H-4; H₃-16 to H-6 through H-5 and H-6; H-9 to H-10; and H-12 to H-14. HMBC correlations (Figure 3) further led to the connectivities of the gross structure. By the above observations, the structure of metabolite 1 could be seen to be very similar to those of a known compound, briaexcavatin L [5] which was previously isolated from the soft coral B. excavatum. Also, it was found that the acetoxy groups attaching at the C-9 positions in briaexcavatin L were replaced by a hydroxy group by comparing the 1D and 2D NMR data of 1 with those of briaexcavatin L. On the basis of the above finding, and by the NOE correlations observed in the NOESY spectrum of 1 (Figure 3), it was found to be the 9-O-deacetyl derivative of briaexcavatin L.

Briacavatolide B (2), had a molecular formula of C₂₈H₃₈O₁₄ as deduced by HRESIMS. The IR spectrum of 2 indicated the presence of hydroxy (3448 cm⁻¹), γ-lactone (1777 cm⁻¹), and ester (1734 cm⁻¹) groups. From the ¹³C NMR data of 2 (Table 1), a trisubstituted olefin (δ_C 145.3, s, C-5; 125.4, d, CH-6) and five carbonyl resonances (δ_C 171.5, 170.6, 170.2, 168.3, 4×s, ester carbonyls; 170.2, s, C-19) were observed. The four esters were identified as acetates by the presence of four methyl resonances in the ¹H NMR spectrum of 2 at δ_H 2.26 (3H, s), 2.10 (3H, s), 2.00 (3H, s), and 1.98 (3H, s) (Table 1). The planar structure of 2 was determined by 2D NMR experiments (Figure 4).

The coupling information in the ¹H–¹H COSY experiment of 2 enabled the identification of the C-2/3/4, C-6/7, C-6/16 (by allylic coupling), C-9/10, and C-12/13/14 units. From these data, together with the results of an HMBC experiment of 2, the molecular framework of 2 could be further established. The HMBC correlations also revealed that the acetate groups are attached at C-2, C-9, C-14, and C-16; thus, the remaining hydroxy groups should be positioned at C-4, C-11, and C-12. The relative stereochemistry of 2 was elucidated from the NOE interactions observed in an NOESY experiment (Figure 4) and the configurations of all chiral centers of 2 were confirmed as being the same as those of 1 by comparison of the proton chemical shifts, coupling constants, and NOE correlations.

Briacavatolide C (3) was isolated as a white solid and had the molecular formula C₃₀H₄₂O₁₃, as determined by HRFABMS. The presence of hydroxyl, γ-lactone, and ester groups were evident from IR absorptions at 3500, 1779, and 1738 cm⁻¹, respectively. ¹H and ¹³C NMR spectral data (Table 1) revealed that 3 contains a trisubstituted double bond. The gross structure of 3 and all of the ¹H and ¹³C chemical shifts associated with the molecule were determined by a series of 2D NMR experiments (Figure 5). In the HMBC spectrum of 3, the n-butyrate positioned at C-12 was confirmed from the connectivity between H-12 (δ_H 4.80) with the carbonyl carbon (δ_C 172.5) of the n-butyryloxyl group. Furthermore, the HMBC correlations also revealed that three acetates were attached to C-2, C-9, and C-14. These data, together with the other ¹H-¹³C long-range correlations (Table 1), unambiguously established the molecular framework of 3. The relative configurations of 3 were identical to those of 1 except that of C-12. H-12 was found to exhibit NOE correlations (Figure 5) with H-13α, H-13β, and H₃-20, but not with H-10, revealing the β-orientation of H-12.

Briacavatolides A–C (1–3) and known compounds briaexcavatolide U (4) and briaexcavatin L (5) were evaluated for cytotoxicity against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells. No cytotoxic activity was detected for any of the isolated compounds against all cell lines at 100 μM. The compounds were also examined for antiviral activity against human cytomegalovirus (HCMV) using a human embryonic lung (HEL) cell line. Briacavatolide C (3) was found to have anti-HCMV activity with an IC₅₀ of 18 μM.

The first investigation of octocoral B. excavatum collected at Orchid Island has led to the isolation of three new briarane-type diterpenoids, briacavatolides A–C (1–3) as well as two known briaranes, briaexcavatolide U (4) and briaexcavatin L (5). Briacavatolide C (3)containing n-butyryloxyl groups was found to show anti-HCMV activity with an IC₅₀ of 18 μM.