Soft coral of the genus Sinularia has been found to be a rich source of bioactive secondary metabolites [1,2], such as acylated spermidine [3,4], lipids and fatty acids [5], cyclic sesquiterpene peroxides [6], sterols [7,8], and norditerpenes [9,10]. A number of them showed an array of biological activities such as cytotoxic activities [3,4] and inhibitory effect on LPS-induced TNF-α production [9,10]. As part of our ongoing investigation of new natural bioactive compounds from marine invertebrates in the South China Sea [11,12,13,14,15,16], the soft coral Sinularia sp. attracted our attention because the crude extract of Sinularia sp. showed lethal activity toward brine shrimp Artemia salina. Bioassay-guided fractionation of the active extracts led to the isolation of three new polyoxygenated sterols (1–3) and three known ones (4–6) [17,18] (Figure 1).

Compound 1 was obtained as a white, amorphous powder with [α]_D²⁵ −22.4 (c 0.05, CH₃OH). IR spectrum showed its absorption at 3410–3584 cm⁻¹ for hydroxy groups. The molecular formula was established as C₂₈H₄₈O₃ based on HRESIMS ([M + Na]⁺ 455.3498 (calcd for C₂₈H₄₈O₃Na 455.3496)) and NMR spectroscopic data. The ¹H-NMR (Table 1) and ¹³C-NMR (Table 2) spectra implied that compound 1 was a polyhydroxylated sterol. The ¹H-NMR spectrum of 1 revealed four methyl singlet signals at δ_H 0.70 (3H, s, H₃-18), 1.00 (3H, s, H₃-19), 1.23 (3H, s, H₃-26) and 1.23 (3H, s, H₃-27), and two methyl doublet signals at δ_H 0.81 (3H, d, J = 6.6 Hz, H₃-28) and 1.08 (3H, d, J = 6.6 Hz, H₃-21), as well as two oxymethines (δ_H 3.71 and 3.56), and an olefinic proton signal at δ_H 5.34 (br d, J = 4.8 Hz). The ¹³C-NMR and DEPT spectra of 1 displayed 28 signals which were assigned to four quaternary carbons, nine methines, nine methylenes, and six methyls. The olefinic carbon signals appearing at δ_C 140.8 (C) and 121.6 (CH) corresponded to one trisubstituted double bond. The ¹³C-NMR chemical shifts at δ_C 75.8 (CH), 75.2 (C) and 71.8 (CH) confirmed the presence of three oxygenated carbons. Detailed analysis of the ¹H-¹H COSY spectrum in combination with HMQC and HMBC (Figure 2) experiments allowed the assignment of all of the chemical shifts in the ¹H and ¹³C-NMR spectra and led to structure 1. Comparison of ¹H-NMR and ¹³C-NMR of 1 with those of the known compound 7 (patusterol A) [19], a hydroxylated steroid from the Kenyan soft coral Lobophytum patulum, further confirmed the structure of 1. The obvious differences between the two compounds are the chemical shifts at δ_H3.71 (1H, ddd, J = 12.0, 8.4, 3.0 Hz, H-23) in 1 vs. 3.71 (1H, br t,J = 2.6 Hz, H-1) in 7, and δ_C 75.8 (CH) in 1 vs. 72.5 (CH) in 7, indicating that the hydroxyl group in 1 is at C-23 not as 7 at C-1. In addition, the ¹H–¹H COSY correlations of H-23 with H-24 and H-22, and the HMBC correlations from H₃-28 to C-24, C-23 and C-25 enable the hydroxyl group to be placed at C-23. The relative stereochemistry of 1 was assigned on the basis of 2D NOESY experiment. In the NOE spectrum of 1, NOE correlations observed from H₃-18 to H-20 indicated that H-20 was in β disposition. The α orientation of H-9 and H-14, and β orientation of H-8 were also determined by NOESY experiment. The absolute configuration at C-3 and C-23 of 1 was tried to determine using the modified Mosher’s method [20]. The (S)- and (R)-MTPA esters 1r and 1s were prepared using (R)- and (S)-MTPA chloride, respectively. The determination of Δδ values (δ_S − δ_R) (Figure 3) for protons neighboring C-3 and C-23 should lead to the assignment of the configuration at C-3 and C-23 in 1. The configuration at C-24 in this and congener sterols was suggested as 24S on biogenetic grounds, since almost all the 24-methylsterols isolated from corals have 24S stereochemistry according to the literature [21,22]. According to the relatively small difference of Δδ values (δ_S − δ_R), the absolute configuration of 1 was tentatively determined as (3S,23R,24S)-ergost-5-ene-3β,23α,25-triol.

Compound 2 was obtained as a white, amorphous powder with [α]D²⁵ −45.6 (c 0.90, CH₃OH). Its positive ion HRESIMS revealed a pseudo molecular ion peak at m/z 515.3711 [M + Na]⁺ (calcd for C₃₀H₅₂O₅Na 515.3712), corresponding to the molecular formula C₃₀H₅₂O₅, possessing five degrees of unsaturation. IR spectrum showed its absorption at 3234–3587 cm⁻¹ for hydroxyl groups, and 1652 cm⁻¹ for carbonyl (acetate) group. Comparison of the ¹H-NMR (Table 1) and ¹³C-NMR (Table 2) data of 2 with those of the known compound 4 [17], revealed that 2 shares the same structure nucleus as 4, differing from 4 only at the side chain where the C-25 was oxygenated, in agreement with the mass data. The oxygenation of the C-25 caused the ¹³C-NMR resonance of C-26 and C-27 to be shifted significantly downfield (from δ_C 21.5/21.6 to 26.0/27.2) and two singlet methyl signals appeared in 2 [δ_H 1.09 (3H, s), 1.10 (3H, s)] instead of two doublet methyls in 4 [δ_H 0.77 (3H, d, J = 6.6 Hz), 0.78 (3H, d, J = 6.6 Hz)] in the ¹H-NMR spectrum. According to these data, compound 2 was assigned as the 25-OH derivative of 4. In addition, the HMBC (Figure 2) correlation from H-6 to ester carbonyl carbon at δ_C 172.1 (CH₃CO), suggesting that the acetoxy group was positioned at C-6. The assigned relative configuration at C-6 was confirmed by that H-6 (δ_H 4.68 (1H, t, J = 2.4 Hz)) was coupled with H-7α (equatorial) (δ_H 1.47) with a small coupling constant of 2.4 Hz. Consequently, H-6 was in equatorial orientation, indicating the location of the acetoxy group was at axial position. Furthermore, the NOE cross-peaks observed between H₃-19 (δ_H1.14) and both H-4β (δ_H 1.77) and H-2β (δ_H 2.01), and the absence of NOE correlations between H₃-19 and both H-3 and H-6 implied that H-3 and H-6 are both α oriented. At the same time the NOE correlations observed between H-4α and both H-3 and H-6 further confirmed that H-3 and H-6 were α oriented. The configuration of the side chain of 2 was also confirmed by NOE correlations from H₃-18 to H-20. So the structure of compound 2 was determined as (24S)-ergostane-6-acetate-3β,5α,6β,25-tetraol.

Compound 3 was obtained as a white, amorphous powder with [α]_D²⁵ −26.6 (c 0.50, CH₃OH). It was found to have the same molecular formula (C₃₀H₅₂O₅) as 2, as determined from high-resolution mass measurements which revealed a pseudo molecular ion peak at m/z 515.3710 [M + Na]⁺, (calcd for C₃₀H₅₂O₅Na 515.3712). Both compounds (2 and 3) showed similarity in the ¹H NMR and ¹³C NMR spectra (Table 1 and Table 2), with the most significant difference being the chemical shifts of C-5 (δ_C 75.5, C in 2 vs. δ_C 30.7, CH in 3) and C-12 (δ_C 41.0, CH₂ in 2 vs. δ_C 73.7, CH in 3). This indicated that the location of a hydroxyl group in 3 was different from that of in 2. The established planar structure of 3 was further supported by the 2D NMR spectra. The diagnostic HMBC correlation from H₃-18 to C-12 and ¹H–¹H COSY correlations between H-12 and H-11 (Figure 2) led the location of the hydroxyl group at C-12. The relative configuration of C-12 was established by comparison with the known compound 8 (3α-acetoxy-12α-hydroxy-5β-cholan-24-oic acid). The small coupling constant of H-12 (δ_H 3.99, 1H, t, J = 2.6 Hz) in 8 means that H-12 is at equatorial position according to the literature [23]. Whereas the large coupling constant of H-12 (δ_H 4.31, 1H, td, J = 7.2, 0.6 Hz) in 3 supported the axial position of H-12. Moreover, NOE correlations observed from both H-14 and H-9 to H-12 and the absence of NOE correlations between H₃-18 and H-12 also implied H-12 was at α orientation. The chemical shift of H-3 (δ_H 4.09) suggested that 3-OH was β oriented and H-5 was α oriented by comparison with the ¹H-NMR data of 3β-hydroxy-5α-oxygenated A/B trans sterols [24,25]. Based on the above analysis, the relative configuration of 3 was assigned, and the structure was elucidated as (24S)-ergostane-6-acetate-3β,6β,12β,25-tetraol.

The structures of compounds 4, 5 and 6 were identified as 24(S)-methylcholestane-3β,5α,6β-triol-6-monoacetate [17], 24-methylenecholestane-3β,5α,6β-triol-6-monoacetate [17], and ergost-24(28)-en-3β,5α,6β-triol [18], respectively, by comparison of their spectroscopic data with those in the literature.

All the isolated compounds (1–6) were evaluated for their cytotoxic activity against a panel of five human tumor cell lines (Hela, HL-60, K562, A-549 and SMMC-7721) and lethality toward brine shrimp A. salina. Only compound 5 exhibited moderate cytotoxicity against K562 cell line with an IC₅₀ value of 3.18 μM. Moreover, compound 5 also displayed strong lethality toward brine shrimp A. salina with a LC₅₀ value of 0.96 μM. For the other compounds, no cytotoxic activity at the concentration of 10 μM and no lethality toward brine shrimp at 25 μg/mL were found.

In our continuing discovery for biological secondary metabolites from marine invertebrates in the South China Sea, this study provided a series of polyoxygenated sterols. The discovery of new compounds 1–3 has added to an extremely diverse and complex array of soft coral sterols. Further studies should be conducted to unambiguously establish their absolute configurations by total synthesis as well as to understand their biological/ecological roles in the life cycle of the animal.