Gorgonian corals of the genus Junceella (Ellisellidae) are common in subtropical and tropical waters in a number of places around the world, such as the South China Sea and Indo-Pacific Ocean, and are well known as a source of highly oxidized diterpenes of the briarane class (3,8-cyclized cembranoids) [1]. Many in vitro and in vivo studies on diterpenes isolated from gorgonians showed a variety of biological activities including anti-tumor, anti-inflammatory, antiplasmodial, antibacterial, antiviral, antimalarial and antioxidant, as well as ecologically relevant activities such as fish-feeding deterrence. Diterpenes isolated from gorgonian corals have a large structural diversity with 40 different diterpene classes being represented [2]. Recently, three new 8-hydroxybriarane diterpenoids (junceols A–C) and four new briarane diterpenoids (juncenolides H–K) were reported from a chemical investigation of Junceella juncea Pallas collected off the southern Taiwan coast, and some of these metabolites were found to exhibit inhibitory effects on superoxide anion generation and elastase release by human neutrophils [3,4]. Fourteen new briarane diterpenes, juncins O–ZII, were isolated from the EtOH/CH₂Cl₂ extract of a South China Sea sample of J. juncea and some of them have been shown to exhibit potent antifouling and antifeedant activities [5,6,7]. A bioassay-guided fractionation of the acetone extract of a Taiwanese collection of J. juncea led to the identification of seven new diterpenoids, juncenolides A–G [8,9,10,11]. Moreover, a chemical investigation of the Indian Ocean gorgonian J. juncea resulted in the isolation of eight new briarane-type diterpenoids, juncins G–N [12,13,14]. A new briarane diterpenoid with antifungal activity was also isolated [15]. In continuation of our research programs oriented towards discovering new metabolites from the gorgonians collected off Taiwanese waters, we reinvestigated J. juncea. Examination of different chromatographic fractions of an AcOEt-soluble extract of the Taiwanese J. juncea Pallas resulted in the isolation of three new briaranes, designated juncenolides M–O (1–3) (Figure 1). Their structures were elucidated through detailed spectroscopic analyses, mainly 2D NMR experiments (¹H, ¹H COSY, HQMC, HMBC). The relative stereochemistry of the chiral centers and the geometry of the double bonds were deduced from NOESY spectra.

Compound 1 was isolated as a colorless amorphous solid. The molecular formula was determined to be C₂₈H₃₅ClO₁₂ (11 degrees of unsaturation) from the HR-ESI-MS data (m/z 621.1711 ([M + Na]⁺)), which also showed a M + 2 peak at m/z 623.1685 (3:1), indicating the presence of one chlorine atom. Its IR bands revealed the presence of a hydroxyl group (3402 cm⁻¹), a five-membered lactone (1779 cm⁻¹) and ester groups (1741 cm⁻¹). ¹H- and ^{13C-NMR data} (Table 1 and Table 2) indicated the presence of four acetates (δ_H 2.18, s; 2.07, s; 1.97, s; 1.95, s) and δ_C 170.2 × 2; 170.0 × 2; 21.6; 21.3; 21.0; 20.8, one carbonyl carbon (δ_C 175.3), and two double bonds (δ_C 126.1; 128.1; 131.7; 140.0), suggesting four rings in the structure. The protons of CH₂-20 (δ_H 3.54, br. s; 2.74, br. s), its corresponding carbon (δ_C 50.2) and the quaternary carbon at δ_C 58.1, were assigned to an exocyclic epoxide [16]. The presence of a γ-lactone ring was ascertained by the carbonyl carbon at δ_C 175.3 (C-18), and the O-bearing carbons at δ_C 78.5 (C-7) and 81.2 (C-8), and confirmed the HMBC correlations (Figure 2) of Me-19/C-8, C-17, C-18 and C-7/C-8, C-18 [16,17]. Four OAc groups were attached to C-2, C-9, C-13 and C-14 by the observation of HMBC correlations (Figure 2). A tertiary methyl signal (δ_H 1.09, Me-15) correlated with a quaternary carbon (C-1), two oxymethines at δ_C 74.2 and 73.8, and the CH at δ_C 41.4, (C-10), implying oxygenation at C-2 and C-14. HMBC correlations of Me-15/C-1, C-2, C-10, C-14, CH₂-16/C-4, C-5, C-6, CH-7/C-5, C-8, C-18, CH-9/C-8, C-11, and CH-10/C-1, C-8, C-11, C-20, as well as ¹H–¹H COSY connectivities between CH-2/CH-3/CH-4, CH-6/CH-7, CH-9/CH-10, CH₂-12/CH-13/CH-14 (Figure 1), suggested that compound 1 possesses 8-hydroxybriarane-type diterpenoid skeleton together with an exocyclic epoxy group which was corroborated by HMBC correlations of CH₂-12/C-11, C-20.

The relative configuration of 1 was determined on the basis of NOESY experiment, MM2 minimized energy calculated molecular modeling (Figure 3), and comparison with other naturally occurring briarane diterpenoids. Briarane-type diterpenoids were previously reported to contain the Me-15 in the β-orientation and H-10 in the α-orientation. As expected, there is no NOE correlation between CH-10 and Me-15. The orientations of CH-10 and the methyl group Me-15 should be opposite. According to MM2 study, we thus concluded that compound 1 had the Me-15 in the β-orientation and CH-10 in the α-orientation as reported [18]. NOESY correlations of CH-10/H-2, CH-9, CH_α-12, Me-15/CH₂-20, CH-14, CH₂-20/CH-13, CH_β-12 and CH-14/CH-13 suggested the β-orientation of CH-13 and CH-14 and α-orientation of CH-2 and CH-9, a β-oriented exocyclic epoxy group attached to cyclohexane moiety as previously assigned from the ¹H- and ¹³C NMR data of C-11 and C-20 [19] and the α-orientation of CH-2. The cis configuration of the C-3/C-4 double bond was suggested by the NOESY correlations (Figure 3) between CH-3/CH-4 and the J value (9.6 Hz). Compound 1 appears to be the chlorinated derivative of juncenolide B isolated from the same species previously [9]. They have the same configuration of chiral centers. Based on the above interpretation, compound 1 is a new chlorinated briarane-type diterpene designated juncenolide M.

Compound 2, isolated as a colorless solid, and had a molecular formula of C₂₄H₃₄O₈, deduced from HR-ESI-MS at m/z 473.2155 ([M + Na]⁺), showing eight degrees of unsaturation. The presence of hydroxyl, a γ-lactone ring, and ester groups were consistent with IR absorptions at 3481, 1772 and 1732 cm⁻¹. The ¹H and ^{13C NMR} (Table 1 and Table 2), revealed the presence of an exomethylene (δ_C 150.8, 113.3), one trisubstituted double bond (δ_C 144.8, 120.5), two OAc carbonyl (C=O) (δ_C 170.4, 169.3) and one γ-lactone carbonyl (C=O) (δ_C 175.9), which accounted for five degrees of unsaturation and were suggestive of a tricyclic briarane bearing a γ-lactone ring. The carbonyl signal at δ_C 175.9 (C-18) was ascribed to a γ-lactone ring with the oxymethine at δ_C 78.3 (C-7) and the O-bearing quaternary carbon at δ_C 82.2 (C-7). The proton singlets at δ_H 5.05 and 4.92 (δ_C 113.3) were assigned to the exocyclic methylene group and correlated to C-10, C-12, and C-11 in the HMBC spectrum (Figure 2), suggesting the presence of a C-11/C-20 double bond. HMBC correlations of Me-15/C-1, C-2, C-14, C-10, Me-16/C-4, C-5, C-6, CH-7/C-5, C-8, C-18, CH-9/C-8, CH-10/C-11, C-1, C8 and the ¹H, ¹H COSY correlations of CH-2/CH₂-3/CH₂-4, CH-6/CH-7, CH-9/CH-10, CH₂-12/CH₂-13/CH-14 revealed the tricyclic skeleton of 2. Furthermore, two OAc groups positioned at C-9 and C-14 were established by the key correlations observed in the HMBC spectrum of 2. NOESY experiment revealed that the absence of correlation between and suggested orientation of and disposition of compound 2. The NOESY correlations (Figure 3) between Me-15/CH-14; CH-10/CH-2, CH-9, CH-9/Me-19, and CH-7/CH-17 were in agreement with the β-orientation of CH-7, CH-14 and CH-17, and α-orientation of CH-2, CH-9 and Me-19. Therefore, compound 2 is a new tricyclic briarane bearing a γ-lactone ring, and was given the name of juncenolide N.

The molecular formula of 3 was established as C₂₉H₃₈O₁₃ from the molecular peak at m/z 617.2212 [M + Na]⁺ in the HR-ESI-MS. The NMR data (Table 1 and Table 2) revealed the basic features of a 8-hyoxybriarane type diterpenoid with a γ-lactone, one exomethylene double bond, one trisubstituted double bond (Table 1 and Table 2), and four acetate esters. The C-20/C-11 exomethylene double bond was assigned with the aid of HMBC correlations of CH₂-20/C-10, C-11, C-12. The signal of CH-6 showed correlations to C-4, C-5 and C-16 in HMBC, revealing the C-5/C-6 trisubstituted double bond, and a COOMe group attached to C-5 (Figure 2). The four acetates were deduced to be located at C-2, C-4, C-9, and C-14 by HMBC correlations of the oxymethines at δ_H 4.86 (CH-2), 5.93 (CH-4), 5.56 (CH-13), and 4.69 (CH-14) to their respective acetate carbonyls. The ¹H,¹H COSY connectivities (Figure 2) of CH-2/CH₂-3/CH-4, CH-6/CH-7, CH-9/CH-10, CH₂-12/CH₂-13/CH-14, as well as HMBC correlations of Me-15/C-1, C-2, C-10, C-14, CH-4/C-16, CH-7/C-8, C-18, CH-9/C-8, C-17, CH-10/C-1, C-8, C-11, CH₂-12/C-11 and Me-19/C-8, C-17, C-18, confirmed the tricyclic skeleton of 3. The NOESY experiments (Figure 3) showed the relative configuration of compound 3. Due to the α-orientation of CH-10, the methyl group Me-15 at the ring junction should be β-oriented as no NOE correlation was observed between CH-10 and Me-15. NOESY spectrum clearly displayed the interactions between Me-15/CH-14, CH-10/CH-9, CH-2, CH-2/CH-4, and CH-9/CH-19, indicating that the OAc at C-2, C-4 and C-9 are β-oriented, whereas the OAc at C-14 is in the α-position. Thus, compound 3 is a new briarane ester with a γ-lactone skeleton, and designated juncenolide O.

The isolated briaranes 1–3 were tested on inhibitory effects of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB at a concentration of 10 μg/mL. As illustrated in Table 3, compounds 2 and 3 showed moderate inhibitory activities against elastase release with 29.0 ± 5.6%, and 35.9 ± 7.4%, respectively. Furthermore, compound 3 also exhibited moderate inhibitory activity against superoxide anion with 27.6 ± 7.0%.

Three new diterpenoids, named juncenolides M–O (1–3), were isolated from the Taiwanese gorgonian Junceella juncea Pallas. Compound 1 is a new chlorinated briarane, compound 2 is a new brierane with a free hydroxy at C-2, while compound 3 contains a rare methyl ester at C-5. The anti-inflammatory activities tested on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB were evaluated. As a result, compounds 2 and 3 showed moderate inhibitory activities against elastase release at 10 μg/mL.