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 [1,2,3,4]. The compounds of this type are proven to possess various bioactivities such as anti-inflammatory, cytotoxicity, and antibacterial activity [1,2,3,4]. Octocorals belonging to the genus Briareum (family Briareidae) are recognized as rich sources of briarane-type diterpenoids (3,8-cyclized cembranoid) [1,2,3].

Human cytomegalovirus (HCMV) is a highly ubiquitous pathogen with a global prevalence of 60%~90% in the human population. For 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 drugs, ganciclovir, foscarnet, and cidofovir are reported to have adverse effects on bone marrow and the kidneys. The continued chemical investigation of octocoral B. excavatum (Figure 1) collected at Orchid Island off Taiwan during August 2008 [5] afforded three new briarane-type diterpenoids, briacavatolides D–F (1–3) (Figure 2) (not to be confused with the related briaexcavatolides). The anti-HCMV (human cytomegalovirus) activity of 1–3 and their cytotoxicity against selected cell lines were evaluated.

The sliced bodies of the Taiwanese octocoral B. excavatum were exhaustively extracted with acetone. The combined acetone extracts were concentrated to a brown gum, which was further partitioned between H₂O and EtOAc. The EtOAc-soluble portion was concentrated under reduced pressure and the residue was fractionated over silica gel 60 by column chromatography. The resulting fractions were further purified by RP-18 HPLC to yield the new compounds 1–3 (Figure 2).

Briacavatolide D (1) was obtained as a white powder. Its HRMS and NMR spectral data established a molecular formula of C₂₄H₃₂O₁₁, implying the existence of eight double bond equivalents. The ¹H and ¹³C NMR spectra (Table 1) of 1 indicated the presence of two acetoxyls (δ_H 1.97, 2.03; δ_C 21.2, 21.1, 171.8, 170.6), a γ-lactone (δ_H 6.22; δ_C 172.2), and a trisubstituted olefin (δ_H 5.71; δ_C 149.3, 124.0). A tetrasubstituted epoxide containing a methyl substituent was indicateded by the signals of two quaternary oxygenated carbons (δ_C 62.6, 71.1) and a methyl (δ_C 9.3; δ_H 1.67, 3H, s). A trisubstituted epoxide containing a methyl substituent was revealed from the signals of an oxymethine (δ_C 61.4; δ_H 3.05) and a quaternary oxygenated carbons (δ_C 63.5) and a methyl (δ_C 24.6; δ_H 1.33, 3H, s). From the above data, metabolite 1 was found to be a pentacyclic compound. The structure and the ¹H and ¹³C chemical shifts of 1 were assigned by the assistance of 2D NMR studies, including ¹H–¹H COSY and HMBC experiments (Figure 3). By analysis 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-7 through H-6; H-6 to H-7; H-9 to H-10; and H-12 to H-14 through H-13. HMBC correlations (Figure 3) further led to the connectivities of the gross structure. According to the above observations, metabolite 1 seemed to be very similar to 16-hydroxystecholide C acetate [6], which was previously isolated from the soft coral Solenopodium excavatum. By means of 1D and 2D NMR data it was found that the acetoxy groups at the C-4 and C-9 positions in 16-hydroxystecholide C acetate were replaced by hydroxy groups 1. On the basis of the above finding, and by the NOE correlations of 1 (Figure 3), bricavatolide D (1) was found to be the 4-O-deacetyl-9-O-deacetyl derivative of 16-hydroxystecholide C acetate.

Briacavatolide E (2) was isolated as a white solid and had the molecular formula C₂₈H₄₀O₁₂, as determined by HRESIMS. The presence of hydroxyl, γ-lactone, and ester groups were evident from IR absorptions at 3447, 1773, and 1734 cm⁻¹, respectively. ¹³C NMR spectral data (Table 1) revealed that 2 contains a trisubstituted double bond (δ_C 145.2, s, C-5; 122.0, d, C-6) and four carbonyl resonances (δ_C 171.0, 170.4, 170.2, and 172.5). Three esters were identified as two acetates and one n-butyrate by the presence of resonances in the ¹H NMR spectrum of 2 at δ_H 1.97 (3H, s), 1.96 (3H, s), 0.96 (3H, t), 1.60 (1H, m) and 2.27 (H, m) (Table 1).

The gross structure of 2 and all of the ¹H and ¹³C chemical shifts associated with the molecule were determined by a series of 2D NMR experiments (Figure 4). In the HMBC spectrum of 2, the n-butyrate positioned at C-12 was confirmed from the long-range coupling between H-12 (δ_H 4.82) with the carbonyl carbon (δ_C 171.0) of the n-butyryloxyl group. Furthermore, the HMBC correlations also revealed that two acetates were attached to C-2 and C-14. These data, together with the other ¹H-¹³C long-range correlations (Table 1), unambiguously established the molecular framework of 2. The relative configurations of 2 were identical to those of briaexcavatin L except that of C-12. H₃-20 were found to exhibit NOE correlations (Figure 4) with H-12 and H-9, revealing the β-orientation of H-12 [7]. Splitting patterns of H-12 and H-14 as triplets (3.0 Hz) indicated that both are β equatorial.

Briacavatolide F (3) had a molecular formula of C₂₈H₃₈O₁₃ as deduced by HRESIMS. The IR spectrum of 3 indicated the presence of hydroxy (3501 cm⁻¹), γ-lactone (1778 cm⁻¹), and ester (1736 cm⁻¹) groups. From the ¹³C NMR data of 3 (Table 1), a trisubstituted olefin (δ 147.4, C_q, C-5; 121.5, CH, C-6) and five carbonyl resonances (δ 170.5, 170.4, 169.6, 168.1, 170.9) were derived. Four esters were identified as acetates by the presence of four methyl resonances in the ¹H NMR spectrum of 3 at δ 2.22 (3H, s), 2.07 (3H, s), 2.04 (3H, s), and 2.00 (3H, s) (Table 1). The planar structure of 3 was determined by 2D NMR experiments (Figure 5). The coupling information in the ¹H–¹H COSY experiment of 3 enabled identification of the C-2/3/4, C-6/16 (by allylic coupling), C-6/7, C-9/10, and C-12/13/14 units. From these data, together with the results of an HMBC experiment of 3, the molecular framework of 3 could be further established. The HMBC data also revealed that the acetate groups are attached at C-2, C-9, C-12, and C-14; thus, the remaining hydroxy groups should be positioned at C-4 and C-11. The relative configurations of 3 elucidated from the NOE correlations (Figure 5) were the same as those of briaexcavatin L except C-11. NOE correlations from H₃-20 to H-10/H-12 and from H-10 to H-12 revealed a β-axial orientation of hydroxy at C-11 for 3 [7]. The dd splitting pattern of H-12 (12.0, 5.2 Hz) indicated that H-12 is α axial. The triplet splitting pattern of H-14 is (3.0 Hz) indicated that H-14 is an β equatorial position; this is confirmed by a cross signal with Me-15.

The cytotoxicity toward P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), A-549 (human lung epithelial carcinoma) tumor cells, and human embryonic lung (HEL) cells as well as anti-HCMV activity of briacavatolides D–F (1–3) are shown in Table 2. Compound 3 showed moderate anti-HCMV activity.

The continued investigation of Taiwanese octocoral B. excavatum collected at Orchid Island has led to the isolation of three new briarane-type diterpenoids, briacavatolides D–F (1–3). Briacavatolide F (3) was found to show anti-HCMV activity with an IC₅₀ of 22 μM.