Numerous secondary metabolites including sesquiterpenoids, diterpenoids, meroditerpenoids, and steroids have been isolated from soft corals of the genus Nephthea [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. Previous bioassay results on these materials showed them to exhibit diverse biological properties including cytotoxic [4,5,6,7,18,20], anti-inflammatory [13,14,23,26] and antimicrobial activities [19]. The acetone extract of the soft coral Nephthea chabrolii (Figure 1) collected off the San-Hsian-Tai coast, Taiwan, in July 2008 was found to be cytotoxic towards P-388 mouse lymphocytic leukemia cell lines. Chromatographic fractionation led to the isolation of three new compounds, nebrosteroids N–P (1–3) (Figure 2).

Nebrosteroid N (1) had a molecular formula of C₂₉H₄₈O₄ as established by interpretation of its HRESIMS and NMR data. The IR spectrum of 1 indicated the presence of hydroxyl(s) (ν_max3393 cm⁻¹) and an ester group (ν_max 1738 cm⁻¹). Further, the ¹H NMR spectrum revealed the presence of a tertiary methyl (δ_H 0.71), three secondary methyls (δ_H 0.92, 0.87, and 0.86), and two oxymethines [δ_H 3.59 (1H, m), 3.82 (1H, d, J = 7.6 Hz)], and an oxymethylene [δ_H 3.99, 4.50 (J_AB = 11.8 Hz)]. The presence of a trisubstituted double bond was revealed by NMR signals [δ_H 5.54 (1H, br s), δ_C 129.6 (CH), 138.1 (C_q)] (Table 1). NMR data of 1 exhibited the presence of an acetoxyl group [δ_H 2.07 (3H, s), δ_C 21.1 (CH₃), 170.7 (C_q)]. The ¹³C NMR and DEPT spectra of 1 contained resonances for eleven sp³ methylenes, eight sp³ methines, two quaternary sp³ carbons, one sp² methine, one quaternary sp² carbon, and one carbonyl. Comparison of NMR chemical shift values of 1 with those of cholest-5-en-3β,7β,19-triol [27] reported from the black coral Antipathes subpinnata as well as its HMBC cross-peaks of H₂-19/C-1,C-5, C-9, C-10, carbonyl carbon at C-19 suggested that 1 may be a 19-acetyl analogue of cholesta-5-en-3β,7β,19-triol. Interpretation of the ¹H–¹H COSY spectrum led to partial structures I and II (Figure 3). Rings A and B were elucidated on the basis of HMBC cross-peaks (Figure 3) between H₂-19/C-1, C-5, C-9, C-10 and H₂-4, H-6/C-10, whereas rings C and D were completed based on HMBC correlations between H₃-18/C-12, C-13, C-14, C-17. The NOESY correlations (Figure 4) observed between H-11β and H₃-18, H-11β and H-19, H-19 and H-4β, H₃-18 and H-8, H₃-18 and H-20, H-3 and H-4α, H-6 and H-7, H-9 and H-14, and H-7 and H-14 in 1 confirmed that nebrosteroid N (1) was cholesta-5-en-3β,7β,19-triol 19-acetate.

Nebrosteroid O (2) was assigned the molecular formula of C₃₀H₄₈O₅ based on its HRESIMS and ¹³C NMR data. ¹³C NMR spectra of 2 showed the presence of five methyls, ten sp³ methylenes, nine sp³ methines, one sp² methylene, three quaternary sp³ carbons, and one quaternary sp² carbons. Analysis of the 1D and 2D NMR data (Table 1 and Figure 3) showed that 2 contains one primary acetoxy group [δ_H 2.11 (3H, s); δ_C 21.2 (q), 170.7 (s)], two secondary hydroxy groups at δ_H 3.72 (1H, m), 4.08 (1H, m) and δ_C 69.1 (d), 68.9 (d), one trisubstituted epoxy ring [δ_H 2.96 (1H, d, J = 2.5 Hz); δ_C 59.8 (d), 61.3 (s)] and one terminal methylene group [δ_H 4.65 (1H, s), 4.72 (1H, s); δ_C 156.7 (s), 106.0 (t)]. These spectral data resembled those for the armatinol A [16] except that 2 contained an additional hydroxyl function at C-11. The placement of this moiety was made on the basis of COSY (Figure 3) correlations between H-9, H-11 and H₂-12. The secondary hydroxyl was deduced to be α oriented based on H₂-19 and H₃-18 showing correlations to H-11 in the NOE spectrum (Figure 5).

Nebrosteroid P (3) had the formula of C₂₉H₅₀O₄ as determined by HRESIMS and NMR data thus required five double bond equivalents. The IR spectrum of 3 showed the absorptions for hydroxyl (ν_max 3432 cm⁻¹) and terminal methylene (ν_max 1639, 886 cm⁻¹). Its NMR data (Table 2) contain four methyls [δ_H 0.72 (3H, s), 0.94 (3H, d, J = 6.8 Hz), 1.02 (3H, d, J = 6.8 Hz), 1.03 (6H, d, J = 6.8 Hz); δ_C 12.5, 18.7, 21.9, 22.0)], two oxymethines [δ_H 3.88 (1H, m), 3.85 (1H, m); δ_C 67.5, 69.4], one oxymethylene [δ_H 4.30 (1H, d, J= 12.0 Hz), 4.93 (1H, d, J= 12.0 Hz); δ_C 65.9], a terminal methylene signal [δ_H 4.66 (s), 4.71 (s); δ_C 105.9 (CH₂), 156.9 (qC)], and a methoxy group [δ_H 3.17 (3H, s); δ_C 48.3]. The ¹³C NMR and DEPT spectra of 3 contained ten sp³ methylenes, eight sp³ methines, three quaternary sp³ carbons, one sp² methine, and one quaternary sp² carbon. These spectra data resembled those of armatinol B [16] except that 3 contained a tertiary methoxy instead of a tertiary hydroxyl at C-5. Based on the HMBC correlation from methoxyl protons to C-5 (Figure 3) and NOESY correlations from methoxyl protons to H-3 (Figure 6), nebrosteroid P was elucidated as 5α-methoxy-24-methylenecholestan-3β,6β,19-triol.

Nebrosteroids N–P (1–3) exhibited cytotoxicity against P-388 cell line with ED₅₀ of 0.9, 1.2, and 1.7 μg/mL, respectively (see Table 2). Nebrosteroids N–P (1–3) were also examined for their antiviral activity towards human cytomegalovirus (HCMV) using a human embryonic lung (HEL) cell line; all compounds were found to be inactive.

The first investigation of soft coral N. chabrolii collected at San-Hsian-Tai (Taitong County, Taiwan) has led to the isolation of three new 19-oxygenated steroids, nebrosteroids N–P (1–3). Nebrosteroids N–P (1–3) exhibited cytotoxicity against P-388 cell line with ED₅₀ of 0.9, 1.2, and 1.7 μg/mL, respectively. However, previously isolated cholestene derivatives, nebrosteroids I–K [13] did not show cytotoxicity. In order to rule out the possibility of 3 as an isolation artifact, a solution of 2 was kept at room temperature for three days in the presence of Si-60 or RP-18 gel in MeOH. However, the formation of 3 was not observed.