Nardosinane-Type Sesquiterpenoids from the Formosan Soft Coral Paralemnalia thyrsoides

Five new nardosinane-type sesquiterpenoids, paralemnolins Q–U (1–5), along with three known compounds (6–8), were isolated from the Formosan soft coral Paralemnalia thyrsoides. The structures of new metabolites were elucidated on the basis of extensive spectroscopic methods, and the absolute configuration of 1 was determined by the application of Mosher’s method on 1. Among these metabolites, 1 and 3 are rarely found nardosinane-type sesquiterpenoids, possessing novel polycyclic structures. Compounds 1, 3, 6 and 7 were found to possess neuroprotective activity.


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 C 15 H 22 O 3 , 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 13 C NMR and DEPT (Table 1) spectroscopic data showed signals of three methyls, three sp 3 methylenes (including one oxymethylene appearing at δ C 69.5), five sp 3 methines, one sp 2 methine, one sp 3 and two sp 2 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 CDCl 3 , it was possible to establish four proton sequences from H-1 to H 2 -3, H-4 to H 3 -14, H-8 to H-9 and H-11 to H 3 -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; H 3 -13 to C-6, C-11 and C-12; H 3 -14 to C-3, C-4 and C-5; and H 3 -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.  The relative configuration of 1 was elucidated by the analysis of NOE correlations, as shown in Figure 2. It was found that H 3 -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 H 3 -14 (δ H 0.79, d, J = 6.4 Hz); therefore, assuming the β-orientation of H 3 -15, all of H-1, H-6, and H 3 -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 H 2 -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 H 3 -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.
The HRESIMS spectrum of paralemnolin R (2) showed a molecular formula of C 15 H 22 O 3 , 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. 1 H-1 H 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 ( 1 H-1 H 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 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 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) showed a polycyclic structure in 3. On the basis of the above results and by the assistance of 1 H-1 H 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 H 2 -2, but not with H 3 -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 H 2 -8 with H 3 -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 C 15 H 24 O 4 (m/z 269.1742 [M + H] + ). The IR spectrum of 4 showed the absorption of a hydroxy group (3361 cm −1 ). Comparison of the 1 H and 13 C NMR spectroscopic data (Tables 1 and 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    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 C 15 H 24 O 4 . 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 H 3 -15 showed NOE correlations with one proton of H 2 -3 (δ H 1.56, m) and the other proton of H 2 -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.

General Experimental Procedures
Melting points were determined using a Fisher-Johns melting point apparatus. Optical rotations were measured on a JASCO P-1020 polarimeter. Ultraviolet spectrum was recorded on a JASCO V-650 spectrophotometer. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. The NMR spectra were recorded on a Varian 400MR FT-NMR (or Varian Unity INOVA500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1 H and 100 MHz (or 125 MHz) for 13 C in CDCl 3 . ESIMS data were obtained with a Finnigan LCQ ion-trap mass spectrometer. HRESIMS data were recorded on a LTQ Orbitrap XL mass spectrometer. Silica gel (Merck, 230-400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 µm).

Animal Material
Soft coral P. thyrsoide was collected by hand using SCUBA off the coast of Orchid Island, located off Taiwan's southeastern coast, in August 2008, at a depth of 10-15 m, and stored in a freezer until extraction. A voucher sample was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.