Abstract

1. Introduction

Soft corals of the genus Paralemnalia [1–7] and Lemnalia [8–12] have been found to be rich sources of sesquiterpenoids of nardosinane [1–3,9,12,13], neolemnane [3,4,10,11] and africanane-type [3,8] compounds and norsesquiterpenoids of nornardosinane-type [3,5,6,9,10] compounds. Our previous study on the secondary metabolites of a Taiwanese soft coral Paralemnalia thyrsoides, collected from the coast of Green Island, has resulted in the isolation of two norsesquiterpenoids [6], ten sesquiterpenoids [4,6,7] and three novel sesquiterpenoids derived from a nardosinane precursor [7]. In our continuing search for new and bioactive metabolites from Formosan soft corals, Paralemnalia thyrsoides collected from Orchid Island were chemically investigated for the first time, and the investigation has resulted in the isolation of five new nardosinane-type sesquiterpeneoids, paralemnolins Q–U (1–5), along with three known compounds 2-deoxylemnacarnol (6) [2], 2-deoxy-7-O-methyllemnacarnol (7) [14] and 2-oxolemnacarnol (8) [15] (Chart 1). The structures of sesquiterpenoids 1–5 were elucidated by spectroscopic analysis and the absolute configurations were established by application of modified Mosher’s method on 1 [16]. The inhibitory activity of compounds 1–8 against three human cancer cell lines was investigated, however, none of these metabolites was found to possess useful cytotoxicity. Furthermore, a study of the neuroprotective effect of these metabolites revealed that 1, 3, 6 and 7, in particular 6, could reduce 6-OHDA (6-hydroxydopamine)-induced neurotoxicity in neuroblastoma SH-SY5Y cells.

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Chart 1

Structures of metabolites 1–8.

2. Results and Discussion

Paralemnolin Q (1) was obtained as a white powder. The HRESIMS (m/z 273.1468 [M + Na]⁺) of 1 established the molecular formula C₁₅H₂₂O₃, appropriate for five degrees of unsaturation, and its IR spectrum revealed the presence of carbonyl (1735 cm⁻¹) and hydroxy (3429 cm⁻¹) groups. The ¹³C NMR and DEPT (Table 1) spectroscopic data showed signals of three methyls, three sp³ methylenes (including one oxymethylene appearing at δ_C 69.5), five sp³ methines, one sp² methine, one sp³ and two sp² quaternary carbons (including one carbonyl carbon appearing at δ_C 211.6). The above data accounted for two of the five degrees of unsaturation, indicating a tricyclic structure for 1. From the COSY spectrum measured in CDCl₃, it was possible to establish four proton sequences from H-1 to H₂-3, H-4 to H₃-14, H-8 to H-9 and H-11 to H₃-13 (Figure 1). Key HMBC correlations of H-6 to C-7; H-8 to C-7 and C-10; H-9 to C-1; H₃-13 to C-6, C-11 and C-12; H₃-14 to C-3, C-4 and C-5; and H₃-15 to C-4, C-5, C-6 and C-10, permitted the connection of the carbon skeleton. Furthermore, the HMBC cross-peak from H-8 to C-12 suggested that C-8 and C-12 were linked through an oxygen to form a tetrahydropyran ring. On the basis of the above analysis, the gross planar structure of 1 was established.

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Figure 1

Selected ¹H–¹H COSY (—) and HMBC (→) correlations of 1–3.

The relative configuration of 1 was elucidated by the analysis of NOE correlations, as shown in Figure 2. It was found that H₃-15 (δ_H 0.98, s) showed NOE interactions with H-1 (δ_H 4.22, dd, J = 12.0, 4.8 Hz), H-6 (δ_H 2.10, brs), and H₃-14 (δ_H 0.79, d, J = 6.4 Hz); therefore, assuming the β-orientation of H₃-15, all of H-1, H-6, and H₃-14 should also be positioned on the β face. Furthermore, H-4 (δ_H 2.02, ddq, J = 12.0, 4.0, 6.4 Hz) exhibited NOE correlations with H-11 (δ_H 2.39, ddq, J =3.6, 3.2, 7.2 Hz) and one proton of H₂-12 (δ_H 4.18, dd, J = 12.0, 3.2 Hz), revealing the α-orientation of H-11, and the β-orientations of H-8 and H₃-13. On the basis of the above findings and other detailed NOE correlations (Figure 2), the relative structure of 1 was determined. In order to resolve the absolute structure of 1, we determined the absolute configuration at C-1 using a modified Mosher’s method [15]. The (S)- and (R)-MTPA esters of 1 (1a and 1b, respectively) were prepared using the corresponding R-(−)- and S-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chlorides, respectively. The determination of the chemical shift differences (δ_S – δ_R) for the protons neighboring C-1 led to the assignment of the S configuration at C-1 of 1 (Figure 3). Thus, the absolute configuration of 1 has been determined.

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Figure 2

Key NOESY correlations for 1–3.

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Figure 3

¹H NMR chemical shift differences of MTPA esters of 1.

The HRESIMS spectrum of paralemnolin R (2) showed a molecular formula of C₁₅H₂₂O₃, the same as that of 1. The NMR data revealed the presence of an α,β-unsaturated ketone (δ_C 201.8 C), and one trisubstituted double bond (δ_C 143.6 CH, 133.9 CH). The above functionalities account for two of the five degrees of unsaturation, suggesting a tricyclic structure in 2. ¹H–¹H COSY and HMBC spectra (Figure 1) further revealed that 2 possesses an α,β-unsaturated ketone at C-7(C=O), C-8 and C-9. Furthermore, the HMBC cross-peak from H-12 to C-10 suggested that C-10 and C-12 are linked through an oxygen. On the basis of the above observations, and by the assistance of additional 2D NMR (¹H–¹H COSY and HMBC) correlations, it was possible to establish the planar structure of 2 as illustrated in Figure 1. The relative configurations of the six chiral centers at C-1, C-4, C-5, C-6, C-10 and C-11 in 2 were further determined on the basis of NOE correlations (Figure 2). It was found that H₃-15 showed NOE interactions with both H₃-14 and H-6, while H-4 was NOE correlated with H-11. Therefore, H-6, H₃-14 and H₃-15 were positioned on the same β-face, and in contrast, H-4 and H-11 should be placed on the α-face. Moreover, one of the methylene protons at C-2 (δ_H 2.18, dddd, J = 15.0, 15.0, 5.0, 3.5 Hz) exhibited NOE correlations with H-4 and was assigned as H-2α. The NOE correlation observed between H-2α and H-1 reflected the β-orientation of hydroxy group. Further NOE analysis revealed that 2 possesses the same configurations at C-4, C-5, C-10, and C-11, as in known compounds flavalins B-D [12]. Thus, the structure of 2 was fully established.

The HRESIMS of paralemnolin S (3) showed that it possesses the molecular formula C₁₅H₂₂O₃ (m/z 251.1634 [M + H]⁺). The IR spectrum of 3 showed the absorption of a carbonyl group (1719 cm⁻¹). The NMR data showed the presence of one trisubstituted epoxide (δ_H 3.31, 1H, brs; δ_C 62.8, C and 59.0, CH), and one ketone (δ_C 210.6, C). The above functionalities and ¹H and ¹³C NMR spectroscopic data (Tables 1 and ​and2)2) showed a polycyclic structure in 3. On the basis of the above results and by the assistance of ¹H–¹H COSY and HMBC spectroscopic analyses (Figure 1), the molecular framework of 3 could be established. This metabolite was found to be a rare nardosinane containing an oxacycloheptane. The 4S*, 5S*, 11R* configurations of 3 were revealed from the similar NOE interactions (Figure 2) as in 1 and 2. Moreover, by NOESY spectrum (Figure 2), it was found that the α-oriented H-1 showed NOE interactions with H₂-2, but not with H₃-15, indicating the α-orientation of H-1. Furthermore, the NOE correlation observed between one proton (δ_H 2.80, dd, J =19.2, 2.8 Hz) of H₂-8 with H₃-15 and H-9 reflected the β-orientation of H-9. From the above evidences and the other NOE correlations (Figure 3), the structure of 3 was determined.

The HRESIMS of paralemnolin T (4) showed that it possesses the molecular formula C₁₅H₂₄O₄ (m/z 269.1742 [M + H]⁺). The IR spectrum of 4 showed the absorption of a hydroxy group (3361 cm⁻¹). Comparison of the ¹H and ¹³C NMR spectroscopic data (Tables 1 and ​and2)2) of compounds 4 and 8 [15] suggested that the structure of 4 should be very similar to that of 8, with the exception of signals assigned to C-2, where a ketone (δ_C 198.0, C) in 8 was replaced by one hydroperoxy-bearing methine (δ_H 4.37, 1H, t, J = 4.0 Hz, δ_C 77.8, CH) in 4. Furthermore, H₃-15 was found to show NOE correlations with H-6, H₃-14 which further correlates with H₃-13, and one proton of H₂-3 (δ_H 1.57, m), and the other proton of H₂-3 (δ_H 1.93, m) showed an NOE correlation with hydroperoxy proton (δ_H 7.83, brs), suggesting that H-2, H-6, H₃-13, H₃-14 and H₃-15 should be placed on the same β-face and in contrast, hydroperoxy group should be positioned on the α-face. Further analysis of other NOE correlations revealed that 4 possesses the same relative configurations at C-4, C-5, C-6, C-7 and C-11 as those of 8 (Figure 4). Based on the above results, the structure of 4 was established.

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Figure 4

Key NOE correlations of 4 and 5.

Paralemnolin U (5) was isolated as a white solid. Its HRESIMS exhibited a [M + H]⁺ ion peak at 269.1746 m/z, establishing a molecular formula of C₁₅H₂₄O₄. By 2D NMR spectroscope data, including COSY, HMQC, and HMBC, compound 5 was shown to possess the same molecular framework as that of 4. Furthermore, the NMR data of 5 were very similar to those of 4, suggesting that 5 is an isomer of 4. By NOESY spectrum (Figure 2), it was found that the β-oriented H₃-15 showed NOE correlations with one proton of H₂-3 (δ_H 1.56, m) and the other proton of H₂-3 (δ_H 1.84, m) showed NOE correlation with H-2, indicating the β-orientation of the hydroperoxy group. On the basis of the above findings and other NOE correlations (Figure 4), 5 was revealed to be the C-2 epimer of 4.

In order to explore the biological activities of the isolated compounds, cytotoxicity of these compounds against the proliferation of a limited panel of cancer cell lines, including mouse melanoma (B16), human epithelial carcinoma (HeLa), human hepatoma carcinoma (HepG2) cell lines, was evaluated. The results showed that all of the compounds were not cytotoxic toward the above cancer cells (IC₅₀’s > 20 μg/mL). Furthermore, the neuroprotective assay of 1–8 using 6-OHDA-induced neurotoxicity in neuroblastoma SHSY5Y, a human dopaminergic neuron often used for study of Parkinson’s disease [17], was performed by a method reported previously [18]. It was observed that the cytotoxicity of 6-OHDA on SH-SY5Y cells could be reduced by pretreatment with 1, 3, 6 and 7 at various concentrations. The relative neuroprotective activities of 1 at 10⁻², 10⁻¹, 1 and 10 μM were 8.7 ± 2.5, 20.2 ± 14.0, 16.2 ± 3.5 and 11.2 ± 1.3%, 3 at 10⁻⁴, 10⁻³, 10⁻² and 10⁻¹ μM were 6.1 ± 2.6, 16.2 ± 5.1, 25.2 ± 3.4 and 10.2 ± 5.3%, 6 at 10⁻², 10⁻¹, 1 and 10 μM were 10.1 ± 1.9, 44.8 ± 4.5, 30.2 ± 1.2 and 38.9 ± 2.7%, and 7 at 10⁻⁴, 10⁻³ and 10⁻² μM were 13.5 ± 3.5, 24.5 ± 7.9 and 16.7 ± 2.2%, respectively (Figure 5). From the neurological activity results, we suggest that further investigation of 1, 3, 6 and 7 for their therapeutic potential against neurodegenerative diseases is worthwhile.

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Figure 5

The neuroprotective effects of 1, 3, 6 and 7 on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pre-incubated for 1 h with the indicated concentration of test compound and then stimulated with 6-OHDA (20 μM) or vehicle. Relative neuroprotection of control (without the treatment of 6-OHDA and compound) and 6-OHDA-treated alone group were taken to be 100% and 0%, respectively. The experiment was repeated three times. * Significantly different from the 6-OHDA-treated alone group (P < 0.05).

3. Experimental Section

4. Conclusions

Our present investigation demonstrated that the Formosan soft coral Paralemnalia thyrsoides is a good source of bioactive substances. In our investigation of new and bioactive metabolites from the Formosan soft corals, this is the first study of P. thyrsoides collected from Orchid Island. From the neurological activity results, compounds 1, 3, 6 and 7, in particular 6, deserve further study for therapeutic potential against neurodegenerative diseases.

Supplementary Material

Acknowledgements

Footnotes

References