Pregna-1,4,20-trien-3-one, a cytotoxic marine steroid from the marine soft coral Nephthea sp.

The title compound, C21H28O, was isolated from the cytotoxic lipid extract of the Fidjian soft coral Nephthea sp. The steroid showed inhibitory activity to human colon adenocarcinoma SW480 cells (IC50 = 2.5 µg ml−1). The molecular structure indicates that the A ring is almost planar (r.m.s. deviation = 0.032 Å), the B and C rings adopt chair conformations and the five-membered D ring is a half-chair. The B/C and C/D ring junctions are trans-fused.

The title compound, C 21 H 28 O, was isolated from the cytotoxic lipid extract of the Fidjian soft coral Nephthea sp. The steroid showed inhibitory activity to human colon adenocarcinoma SW480 cells (IC 50 = 2.5 mg ml À1 ). The molecular structure indicates that the A ring is almost planar (r.m.s. deviation = 0.032 Å ), the B and C rings adopt chair conformations and the five-membered D ring is a half-chair. The B/C and C/D ring junctions are trans-fused.
Bioassay-guided fractionation resulted in the isolation of pregna-1,4,20-triene-3-one as the major secondary metabolite. Xray analysis confirmed the structure proposed on the basis of spectral evidence, primarily NMR. Pregna-1,4,20-triene-3-one was first isolated independently in parallel by the groups of Fallis (Kingston et al., 1977) and Faulkner (Higgs & Faulkner, 1977) from the sea raspberry Gersemia rubiformis and an unidentified soft coral, respectively. Later on, the steroid was also obtained from soft corals of the genus Carijoa (Maia et al., 1998;Ciavatta et al., 2004), Cladiella (Zhang et al., 2003;Huang et al., 2006;Huang et al., 2009), Sinularia (Zhang et al., 2005 and Spongodes (Yan et al., 2004;Yan et al., 2007). However, this is the first time that pregna-1,4,20-triene-3-one has been isolated from a soft coral of the genus Nephthea. The molecule contains a fused four-ring system A/B/C/D ( Figure 2). Ring A with two double bonds is highly planar (Thompson et al., 1999), whereas the cyclohexane rings B and C adopt chair conformations. The five membered ring D exhibits a half-chair form. The B/C and C/D ring junctions are trans-fused. The methyl substituents at atoms C-10 and C-13 are oriented to the same side of the steroid nucleus. The vinyl group at C17 is attached equatorially to ring D. The absolute configuration could not be determined by x-ray, but could be established by direct comparison of optical rotation and 1 H NMR and 13 C NMR data with literature data (Ciavatta et al., 2004;Higgs & Faulkner, 1977;Kingston et al., 1977 and1979).
Despite its frequent occurrence and isolation of this metabolite, a biological activity was never attributed to its molecular structure. The cytotoxic effects of purified pregna-1,4,20-triene-3-one against mouse NIH/3 T3 and human SW480 (human colon adenocarcinoma) cell lines was investigated, and the compound showed no activity towards NIH/3 T3 tumor cells, but significant growth inhibitory activity towards SW480 tumor cells (IC 50 = 2.5 µg/ml). Sterols, particularly highly oxygenated steroids have been reported to be cytotoxic towards several cancer cell lines. However, in contrast to the examples known from the literature, it is noteworthy that pregna-1,4,20-triene-3-one exhibits cytotoxicity with its solely mono-oxygenated structure featuring a rare vinyl group at C-17.

Animal material
The soft coral Nephthea sp. (Figure 1) was collected in 1999 from Fiji Islands and stored in EtOH at -20°C until workup.
A voucher specimen has been deposited at the Institute for Pharmaceutical Biology, University of Bonn, voucher number CT199 NNNN.

Extraction and isolation
supplementary materials sup-2 The freeze-dried soft coral Nephthea sp. (250 g dry wt) was extracted repeatedly with CH 2 Cl 2 /MeOH (2:1, v/v) to produce 10.5 g of crude organic extract. A portion of the extract (5 g) was subjected to normal phase vacuum liquid chromatography (VLC), using stepwise gradient elution from hexanes containing increasing proportions of EtOAc followed by MeOH, to produce nine subfractions. The fraction eluting with 60% EtOAc in hexanes was found to be most active in cancer cell line assays. This fraction was further chromatographed on RP18 solid phase extraction (SPE) cartridges using a stepwise gradient solvent system of decreasing polarity starting from 70% aqueous MeCN to 100% MeCN. The most active fraction after SPE was then purified by normal phase HPLC [Knauer Eurospher-100Si-5 µm (250 x 8 mm), petroleum ether / acetone (85:15), 1.5 ml/min, refraction index detection] giving pure pregna-1,4,20-triene-3-one (9.3 mg). Colorless needles of (I) were prepared by slow evaporation from a methanol solution.

Refinement
All hydrogen atoms were placed in calculated positions with C-H distances ranging from 0.95 to 0.99Å and included in the refinement in riding motion approximation, with U iso = 1.2 U eq of the carrier atom.

Special details
Experimental. During data collection the crystal was in cold N 2 gas of a Kryoflex cooler (Bruker AXS).
Geometry. All e.s. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.