Echinohalimane A, a Bioactive Halimane-Type Diterpenoid from a Formosan Gorgonian Echinomuricea sp. (Plexauridae)

A new halimane-type diterpenoid, echinohalimane A (1), was isolated from a gorgonian, identified as Echinomuricea sp. The structure of 1 was determined by spectroscopic methods and this compound was found to exhibit cytotoxicity toward various tumor cells and display an inhibitory effect on the release of elastase by human neutrophils. Echinohalimane A (1) is the first halimane analogue from the marine organisms belonging to phylum Cnidaria.


Introduction
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).
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 H 3 -17 and a proton of C-11 methylene (δ H 1.90), but not with H-8 and H 3 -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 H 3 -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).

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm −1 . NMR spectra were recorded on a Varian Inova 500 or a Varian Mercury Plus 400 NMR spectrometers using the residual CHCl 3 signal (δ H 7.26 ppm) as internal standard for 1 H NMR and CDCl 3 (δ C 77.1 ppm) for 13 C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded by a Bruker APEX II mass spectrometer. Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck); spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. HPLC was performed using a system comprised of a Hitachi L-7100 pump, a Hitachi L-7455 photodiode array detector and a Rheodyne injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 μm) was used for HPLC.

Animal Material
Specimens of the gorgonian coral Echinomuricea sp. were collected by hand using scuba equipment off the coast of the southern Taiwan and stored in a freezer until extraction. This organism was identified by comparison with previous descriptions [8,9]. A voucher specimen (NMMBA-TW-GC-125) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
The freeze-dried and minced material of Echinomuricea sp. (wet weight 1.68 kg, dry weight 428 g) was extracted with a mixture of methanol (MeOH) and dichloromethane (CH 2 Cl 2 ) (1:1). The residue was partitioned with ethyl acetate (EtOAc) and H 2 O. The EtOAc layer was partitioned between MeOH and n-hexane. The n-hexane layer was separated by silica gel and eluted using n-hexane/EtOAc/MeOH to yield 21 fractions A-U. Fraction M was separated on silica gel and eluted using a mixture of n-hexane/EtOAc to yield 10 fractions M1-M10. Fraction M3 was further purified by normal phase HPLC (n-hexane/acetone, 10:1) to yield compound 1 (33.7 mg).

Molecular Mechanics Calculations
Implementation of the MM2 force field [7] in CHEM3D PRO software from CambridgeSoft Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to calculate molecular models.

Superoxide Anion Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [12,13]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Conclusions
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.