The search for bioactive natural products from marine organisms has been remarkably successful and gorgonian corals have proven to be rich sources of interesting natural terpenoid derivatives [1,2]. In previous studies, two bisabolane-type sesquiterpenoids, (7S,10R)-(+)-10,11-epoxycurcuphenol and (+)-curcuphenol; a labdane-type diterpenoid, echinolabdane A; and a steroid analogue, 6-epi-yonarasterol B, had been isolated from a Formosan gorgonian coral identified as Echinomuricea sp. (Plexauridae) [3,4]. 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, we have further isolated a new clerodane-type diterpenoid, echinoclerodane A (1), from Echinomuricea sp. (Figure 1). In this paper, we describe the isolation, structure determination and biological activities of diterpenoid 1.

Echinoclerodane A (1) was isolated as an oil and its molecular formula was determined to be C₂₀H₃₀O₃ (m/z 341.2095 [M+Na]⁺) using HRESIMS. The IR spectrum of 1 showed bands at 3,318 and 1,741 cm^–1, consistent with the presence of hydroxy and ester carbonyl groups. The ¹³C-NMR for 1 confirmed the presence of 20 carbon signals (Table 1), which were characterized by the DEPT spectrum as three methyls, eight sp³ methylenes, three sp³ methines, three sp³ quaternary carbons, one sp² methine and two sp² quaternary carbons. A suite of resonances at δ_C 171.8 (s, C-15), 171.2 (s, C-13), 116.9 (d, C-14) and 99.2 (s, C-16), could be assigned to an α,β-unsaturated-γ-hydroxy-γ-lactone moiety. Thus, from the reported data, the proposed skeleton of 1 was suggested to be a diterpenoid with four rings.

From a ¹H–¹H COSY experiment (Table 1 and Figure 2), it was possible to establish the spin systems that map out the proton sequences from H-10/H₂-1/H₂-2/H₂-3, H₂-6/H₂-7/H-8/H₃-19, H₂-11/H₂-12 and H₂-12/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 H₂-2, H₂-3, H₃-17, H₂-18/C-4; H₂-6, H₃-17, H₂-18/C-5; H₂-1, H-8, H-10, H₂-11, H₃-19, H₃-20/C-9; H₂-12, H-14, H-16/C-13; and H-14, H-16/C-15, permitted elucidation of the carbon skeleton. The tertiary methyls at C-4 and C-9 were confirmed by the HMBC correlations between H₃-17/C-3, -4, -5 and H₃-20/C-8, -9. The methine unit at δ_C 99.2 (d, C-16) was more shielded than expected for an oxygenated C-atom and correlated with a methine proton at δ_H 6.01 (H-16) in the HMQC spectrum, and this proton showed a ²J-correlation and a ³J-correlation with C-13 and C-15, respectively, 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 offered by computer modeling [5], in which the close contacts of atoms in space calculated were consistent with the NOESY correlations (Figure 3). In the NOESY spectrum of 1, the correlations of H-10 with H₂-11 and H₃-19, indicated that these protons (H-10, H₂-11 and H₃-19) were situated on the same face and these were assigned as β protons, since the C-20 methyl is an α-substituent at C-9. An endo H-C18 proton exhibited a correlation with Me-20, suggesting that the cyclopropane moiety between C-4/5 was α-oriented. Based on the above findings, the main structure of 1 was elucidated unambiguously, and the chiral carbons for 1 were assigned as 4S*, 5S*, 8S*, 9S*, 10R* although the relative configuration for 16-hydroxy group could not be determined at this stage by this method. By comparison of the spectral data, echinoclerodane A (1) was found to be the 8-epimer of a known marine-derived clerodane-type diterpenoid, dytesinin A (2) (Figure 1), isolated from an Okinawa tunicate Cystodytes sp. [6].

The cytotoxicity of diterpenoid 1 against the 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 echinoclerodane A exhibited moderate cytotoxicity against MOLT-4, HL-60, DLD-1 and LoVo cells. The in vitro anti-inflammatory effects of diterpenoid 1 were also tested. Echinoclerodane A (1) displayed a significant inhibition effect on the generation of superoxide anion (inhibition rate 68.6%) and this compound showed a moderately inhibition effect (inhibition rate 35.4%) on the release of elastase by human neutrophils at a concentration of 10 μg/mL, respectively [7].

Clerodane-type diterpenoids are extensively present in terrestrial plants [14], and compounds of this type were also obtained from tunicates [6]. Octocorals have been proven to be rich sources of natural terpenoid derivatives and terpenoid analogues are often found in large amounts in marine invertebrates [15]. It is worth noting that the new clerodane metabolite 1 (echinoclerodane A) is the first clerodane-type derivative isolated from the marine organisms belonging to the phylum Cnidaria and this compound exhibited cytotoxicity and anti-inflammatory activity. The study material Echinomuricea sp. has begun to be transplanted to 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.