The search for bioactive natural products from marine organisms has been remarkably successful [1] and octocorals have proven to be rich sources of interesting natural products [2,3]. In continuation of our search for new natural products from the marine invertebrates collected off the waters of Taiwan at the intersection of the Kuroshio current and the South China Sea surface current, a new bioactive substance, echinohalimane A (1) (Figure 1), was isolated from the gorgonian Echinomuricea sp. In this paper, we describe the isolation, structure determination and biological activities of echinohalimane A (1).

In previous studies, two bisabolane-type sesquiterpenoids, (7S,10R)-(+)-10,11-epoxycurcuphenol and (+)-curcuphenol [4]; a labdane-type diterpenoid, echinolabdane A [5]; a clerodane-type diterpenoid, echinoclerodane A [6]; and a steroid analogue, 6-epi-yonarasterol B [5], had been isolated from a Formosan gorgonian coral identified as Echinomuricea sp. (Plexauridae). We have further isolated a new halimane-type diterpenoid, echinohalimane A (1), from Echinomuricea sp.

Echinohalimane A (1) was isolated as a yellowish oil that gave a pseudomolecular ion (M + Na)⁺ at m/z 341.2089 in the HRESIMS, indicating the molecular formula C₂₀H₃₀O₃ (calcd for C₂₀H₃₀O₃ + Na, 341.2093) and implying six degrees of unsaturation. IR absorptions were observed at 3375 and 1755 cm⁻¹, suggesting the presence of hydroxy and ester groups in 1. The ¹³C NMR for 1 confirmed the presence of 20 carbon signals (Table 1), which were characterized by DEPT as four methyls, six sp³ methylenes, three sp³ methines, two sp² methines, two sp³ quaternary carbons and three sp² quaternary carbons. A suite of resonances at δ_C 172.2 (C-15), 171.6 (C-13), 116.7 (CH-14) and 99.4 (CH-16), could be assigned to an α,β-unsaturated-γ-lactone moiety [6]. A trisubstituted olefin was deduced from the ¹³C NMR data at δ_C 145.0 (C-10) and 118.1 (CH-1). Thus, from the reported data, the proposed skeleton of 1 was suggested to be a diterpenoid with three rings.

From the ¹H–¹H COSY experiment of 1 (Table 1 and Figure 2), it was possible to establish the spin systems that map out the proton sequences from H-1/H₂-2/H₂-3, H-5/H₂-6/H₂-7/H-8/H₃-17 and H₂-11/H₂-12, which was accomplished with the assistance of an HMBC experiment (Table 1 and Figure 2). The key HMBC correlations between protons and quaternary carbons of 1, including H₂-2, H₂-3, H-5, H₃-18, H₃-19/C-4; H-1, H₂-7, H-8, H₃-17, H₃-20/C-9; H₂-2, H-5, H-8, H₂-11, H₃-20/C-10; H₂-11, 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-9 were confirmed by the HMBC correlations between H₃-18/C-3, -4, -5, -19; H₃-19/C-3, -4, -5, -18; H₃-17/C-7, -8, -9; and H₃-20/C-8, -9, -10, -11. The methine unit at δ_C 99.4 (CH-16) was more shielded than that expected for an oxygenated C-atom and correlated with the methine proton at δ_H 5.98 (H-16) in the HMQC spectrum, and this proton showed ³J-correlations with C-14 and C-15 in the HMBC spectrum and concluded to be a part of a hemiketal constellation.

The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 ascertained using molecular mechanics calculations (MM2), which suggested the most stable conformations, as shown in Figure 3 [7], in which the close contacts of atoms in space calculated were consistent the NOESY correlations. In the NOESY experiment of 1, H-5 exhibited correlations with H₃-17 and a proton of C-11 methylene (δ_H 1.90), but not with H-8 and H₃-20, indicated that H-5 and Me-17 were situated on the same face in 1, and these were assigned as β protons, since the C-20 methyl is an α-substituent at C-9. The E-configuration of C-1/10 double bond was elucidated from a correlation between H-1 and H₃-20. Based on the above findings, the main structure of 1 was elucidated unambiguously, and the chiarl carbons for 1 were assigned as 5R*, 8S*, and 9S* although the configuration for 16-hydroxy group was not determined at this stage by this method.

The cytotoxicity of diterpenoid 1 toward K562 (human erythromyeloblastoid leukemia), MOLT-4 (human acute lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia), DLD-1 (human colorectal adenocarcinoma), LoVo (human colorectal adenocarcinoma) and DU-145 (human prostate carcinoma) cells was studied, and the results were shown in Table 2. These data showed that echinohalimane A (1) exhibited cytotoxicity toward MOLT-4, HL-60, DLD-1 and LoVo cells. Furthermore, the in vitro anti-inflammatory effects of diterpenoid 1 were tested and echinohalimane A (1) displayed a significant inhibitory effect on the release of elastase by human neutrophils (Table 3).

In general, halimane-type diterpenoids exist in terrestrial plants, and are rarely found in marine organisms [14]. The compounds of this type were found to possess a carbon skeleton intermediate between that of labdanes and clerodanes [15,16]. It is worth noting that echinohalimane A (1) is the first halimane-type derivative isolated from marine organisms belonging to phylum Cnidaria. The study material Echinomuricea sp. has begun to be transplanted in culturing tanks with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium, Taiwan, for the extraction of additional natural products in order to establish a stable supply of bioactive material.