The search for new natural products from marine organisms has been remarkably successful, and gorgonian corals have been proven to be rich sources of interesting natural terpenoid derivatives [1,2]. In a previous study, two sesquiterpenoid phenols, (7S,10R)-(+)-10,11-epoxycurcuphenol and (+)-curcuphenol [3], were isolated from the Formosan gorgonian coral Echinomuricea sp. (family Plexauridae). In continuation of our search for new natural substances from marine invertebrates collected off the waters of Taiwan at the intersection point of the Kuroshio current and the South China Sea surface current, we have further isolated a new labdane-type diterpenoid, echinolabdane A (1), and a new steroid derivative, 6-epi-yonarasterol B (2), from Echinomuricea sp. In this paper, we describe the isolation, structural characterization and bioactivity of new compounds 1 and 2 (Figure 1).

Echinolabdane A (1) was isolated as an oil, and the molecular formula for this compound was determined using HRESIMS to be C₂₀H₃₀O₃ (6° of unsaturation) (m/z 341.2095 [M + Na]⁺, calculated as 341.2093). An IR absorption at 1765 cm⁻¹ suggested the presence of a γ-lactone group in 1. The ¹³C NMR data for 1 confirmed the presence of 20 carbon signals (Table 1), which were characterized by DEPT as four methyls, seven sp³ methylenes, three sp³ methines, an sp² methine, three sp³ quaternary carbons and two sp² quaternary carbons. A suite of resonances at δ_C 170.6 (C-15), 169.3 (C-13), 117.0 (CH-14) and 100.9 (CH-16) could be assigned to the α,β-unsaturated-γ-lactone moiety. From the reported data, the carbon skeleton of 1 was suggested to be a diterpenoid derivative with four rings.

From the ¹H–¹H COSY analysis of 1 (Table 1 and Figure 2), it was possible to establish the spin systems that map out the proton sequences from H₂-1/H₂-2/H₂-3, H-5/H₂-6/H₂-7, H-9/H₂-11/H₂-12 and H-12a/H-14 (by allylic coupling), which was accomplished with the assistance of an HMBC experiment (Table 1 and Figure 2). The key HMBC correlations between the protons and quaternary carbons of 1, including H-3a, H-5, H₃-17, H₃-18/C-4; H₂-6, H-7b, H-9, H-11b, H-16, H₃-19/C-8; H-1, H-5, H₂-6, H-9, H₃-20/C-10; H-12a, H-14, H-16/C-13; and H-14, H-16/C-15, permitted the elucidation of the carbon skeleton of 1. The tertiary methyls at C-4, C-8 and C-10 were confirmed by the HMBC correlations between H₃-17/C-3, C-4, C-5, C-18; H₃-18/C-3, C-4, C-5, C-17; H₃-19/C-7, C-8, C-9; and H₃-20/C-1, C-5, C-9, C-10. Furthermore, an HMBC correlation between H-16 (δ_H 6.07) and an oxygenated quaternary carbon at δ_C 82.5 (C-8) suggested the presence of a C-8/16 ether linkage in 1. The methine unit at δ_C 100.9 (CH-16) was more shielded than expected for an oxygenated C-atom and was correlated with the methine proton at δ_H 6.07 (H-16) in the HMQC spectrum, and this proton showed a ²J-correlation with C-13 and showed ³J-correlations with C-8, C-14 and C-15 in the HMBC spectrum, and was concluded to be a part of a hemiketal moiety.

The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 offered by computer modeling (Figure 3) [4], in which the close contacts of atoms in space calculated were consistent with the NOESY correlations. In the NOESY analysis of 1, the correlations of H-9 with H-5 and H-16, but not with H₃-19 and H₃-20, indicated that these protons (H-5, H-9 and H-16) were situated on the same face, and these were assigned as α protons, since the C-19 and C-20 methyls are β-substituents at C-8 and C-10, respectively. The Z-configuration of the C-13/14 double bond was elucidated from a correlation between H-14 (δ_H 5.82) and H-12b (δ_H 2.91). From the above evidence, the relative configurations of the chiral carbons of 1 were assumed to be 5S*, 8R*, 9R*, 10S* and 16R*. On the basis of the above findings, the structure of 1 was elucidated.

The in vitro cytotoxicity of labdane 1 was studied, and this compound exhibited weak cytotoxicity toward HL-60 (human acute promyelocytic leukemia) cells (IC₅₀ = 19.1 μg/mL).

6-epi-Yonarasterol B (2) had a molecular formula of C₃₀H₄₈O₅ as indicated by HRESIMS at m/z 511.3396 (calculated for C₃₀H₄₈O₅Na, 511.3399). The ¹³C NMR and DEPT spectra of 2 exhibited the presence of seven methyls, seven sp³ methylenes, nine sp³ methines, two sp² methines, three sp³ quaternary carbons and two sp² quaternary carbons (Table 2). The IR spectrum of 2 showed absorptions due to α,β-unsaturated ketone (1671 cm⁻¹) and ester (1732 cm⁻¹) groups. The presence of a conjugated enone system in 2 was also indicated by ¹H (δ_H 6.68, 1H, ddd, J = 10.5, 5.5, 2.5 Hz, H-3; 6.15, 1H, dd, J = 10.5, 2.0 Hz, H-2) and ¹³C (δ_C 205.8, C-1; 140.7, CH-3; 128.8, CH-2) NMR spectra (Table 2). The presence of a secondary acetoxy group was evident from the IR (1732 cm⁻¹), ¹H (δ_H 2.11, 3H, s, acetate methyl; 5.06, 1H, dd, J = 12.0, 5.5 Hz, H-6) and ¹³C (δ_C 21.2, acetate methyl; 171.5, acetate carbonyl; 75.1, CH-6) NMR spectra. IR absorption at 3392 cm⁻¹ and ¹H NMR signals at δ_H 3.91 (1H, br s, H-11) and ¹³C NMR at δ_C 66.9 (CH-11) indicated the presence of a secondary hydroxy group. It was found that the structure of 2 is similar to the structures of known sterols yonarasterol B (3) [5] and stoloniferone K (4) [6] (Figure 1). All C-H correlations of 2 were detected in the HMQC experiment. The ¹H–¹H COSY spectrum exhibited partial structures A, B and C (Table 2 and Figure 4). In the HMBC spectrum, the partial structure A could be connected to B through three quaternary carbons C-1, C-5, C-10 and Me-19 (Table 2 and Figure 4). Partial structure B could be connected to C through the remaining quaternary carbon C-13 and Me-18. Based on the above findings, the planar structure of 2 was concluded, as shown in Figure 4.

The configuration of two chiral centers (C-20 and C-24) in the side chain of 2 was elucidated by comparison of ¹³C NMR spectral data (δ_C 36.0, 39.0) with those of 3 (δ_C 36.3, 39.1) [5], 4 (δ_C 36.3, 39.1) [6]and (24S)-24-methylcholest-5-en-3β-ol (δ_C 36.26, 39.17) [7]. The configuration of the ring junctures in 2 was confirmed by NOESY analysis, as shown in Figure 5. The NOESY correlations between H-4β/Me-19; H-6/H₃-19; H-8/H-11; H-8/Me-18; H-9/H-14; H-11/Me-18; H-11/Me-19; H-12β/Me-18; H-12α/H-17; and H-14/H-17 suggested that the 6-acetoxy and 11-hydroxy groups were α-oriented and all ring fusions in 2 were of a trans configuration. The coupling constants of H-6 and H-7a/b (J = 12.0, 5.5 Hz) suggested that H-6 was an axial hydrogen. This result further supported that the 6-acetoxy was α-oriented in 2. Due to the fact that coupling pattern of H-11 in 2 appeared as a broad singlet in the ¹H NMR spectrum of 2, it is difficult to elucidate the relative stereochemistry of the 11-hydroxy group in 2 by vicinal coupling constant analysis; however, H-11 showed significant correlations with H-8, Me-18 and Me-19 in the NOESY analysis of 2, which suggested that the 11-hydroxy group in 2 was α-oriented.

The in vitro anti-inflammatory effects of compounds 1 and 2 were tested (Table 3). 6-epi-yonarasterol B (2) was found to show significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.

Labdane derivatives exist extensively in terrestrial plants [14], and compounds of this type have also been obtained from various marine organisms, including red algae belonging to the genus Laurencia [15,16,17,18,19]; sponges Chelonaplysilla erecta [20], Raspaciona aculeata [21], Agelas sp. [22]; and nudibranch Austrodoris kerguelensis [23]. It is worth noting that echinolabdane A (1) is the first labdane derivative to be isolated from marine organisms belonging to the phylum Cnidaria. The labdane analogue possessing a tetracyclic skeleton with an oxepane ring jointed to a γ-lactone ring by a hemiketal moiety, 1 (echinolabdane A), was discovered for the first time in this study. Furthermore, 6-epi-yonarasterol B (2) is the first steroid derivative to be isolated from a gorgonian coral belonging to the genus Echinomuricea, and this compound was found to exhibit significant anti-inflammatory activities. The gorgonian coral Echinomuricea sp. has begun to be transplanted in tanks for the extraction of natural products in order to establish a stable supply of bioactive material.