Cytotoxic Serrulatane-Type Diterpenoids from the Gorgonian Euplexaura sp. and Their Absolute Configurations by Vibrational Circular Dichroism

Vibrational circular dichroism (VCD) method has become robust and reliable alternative for the stereochemical characterization of natural products. In this paper, three new serrulatane-type diterpenoids, euplexaurenes A–C (1–3), and a known metabolite, anthogorgiene P (4), were obtained from the South China Sea gorgonian Euplexaura sp. GXWZ-05. The absolute configuration of C-11 in 1–4, which was difficult to be determined by common means due to the high conformational flexibility of the eight-carbon aliphatic chain attached at C-4, was determined by VCD method, suggesting a new horizon to define the absolute configurations of natural products possessing chains. Compounds 1–4 were found to show selective cytotoxic activities against human laryngeal carcinoma (Hep-2) cell line with the IC50 values of 1.95, 7.80, 13.6 and 5.85 μM, respectively.

In the pharmaceutical chemistry and related fields, the absolute configuration is of prime importance in the interaction of drugs and organisms, since all receptors in the human body are chiral and probably exhibit different pharmacologic effects and pharmacokinetics between two enantiomers 1 . However, the determination of the absolute configurations for chiral natural products is one of the most challenge for natural product chemists. In current natural products research, X-ray diffraction and chiroptical methods are the most important and popular tools for determining the absolute configurations of novel natural products 1,2 . While, natural products are commonly available in small amounts from natural sources and usually do not bear heavy atoms, which often prevent direct assignment of the absolute configurations by X-ray diffraction method 2 . Vibrational circular dichroism (VCD) is one of the chiroptical method which, if combined with accurate quantum mechanical calculations, offers a powerful approach to the determination of absolute configurations in chiral natural products [3][4][5][6] . Interesting fact is VCD method has become robust and reliable alternative for the stereochemical characterization of natural products, especially in conditions not accessible to other methods.
Recently, in our continuing efforts to discover new bioactive substances with complicated absolute configurations from the South China Sea corals 7-11 , the gorgonian Euplexaura sp. GXWZ-05 attracted our attention due to the cytotoxic activity of its EtOAc extract. As a result, four serrulatane-type diterpenoids (Fig. 1), including three new compounds, euplexaurenes A-C (1-3), and a known compound, anthogorgiene P (4) 12 , were isolated. In order to determine the absolute configurations of 1-4, VCD chiroptical method was applied. Herein, we report the isolation and absolute configurations of the new compounds, as well as the cytotoxic activities of 1-4.  (Table 1). In the 13 C NMR spectrum, 20 carbon signals assignable to two quaternary carbons, eight methines, five methylenes, and five methyls were revealed ( Table 1). The above spectroscopic data suggested that 1 should be a serrulatane-type diterpenoid [12][13][14][15] . However, the traditional serrulatane-type diterpenoids nuclei only included a bicyclic system, which was not in accordance with the derived structure of the tricyclic nucleus in 1. Thus, a new connectivity type must be existed in 1 to form a novel tricyclic system. In the HMBC spectrum of 1, the correlations from H-5 to C-1 and C-8 ( Fig. 2) suggested the direct connection between C-5 and C-9 forming a 5,3,6-tricyclic unit of serrulatane-type diterpenoids. In fact, only one compound with this moiety (4) 12 was obtained from nature. It has been postulated that the 5,3,6-tricyclic moiety of the serrulatane-type diterpenoids was derived from bicyclic system of the traditional serrulatane-type diterpenoid via aromatic rearrangement 12 . In fact, the NMR spectra of 1 and 4 were similar. The main difference between 1 and 4 in the 1 H NMR spectra was the presence of an oxygen-bearing methine doublet at δ H 4.36 [1 H, t (8.0), H-8] in 1 instead of an olefinic methine singlet at δ H 5.39 (1 H, s, H-7) in 4. Accordingly, in the 13 C NMR spectrum of 1 the signal of one oxygen-bearing methine (δ C 75.0), one methine (δ C 33.9) and one methylene (δ C 38.4) were observed in place of the signals of carbonyl carbon (δ C 209.0) and trisubstituted double bond (δ C 178.0 and 123.5) in 4, respectively. The above NMR data suggested that the α, β-unsaturated ketone group in 4 was hydrogenated in 1, confirmed by the HMBC cross-peaks from H-8 to C-6, C-7, C-9, and from H-20 to C-5 and C-7 (Fig. 2). Detailed analysis of the 2D NMR spectra of 1 allowed the assignment for all of the proton and carbon resonances.  The relative configuration of the 5,3,6-tricyclic unit in 1 was deduced by NOESY experiments (Fig. 3). In the NOESY spectrum of 1, H-8 was found to show NOESY correlations with H-5, H 3 -19 and H 3 -20, and the NOESY correlation could be observed between H-4 and H-5, indicated that these protons should be on the same face of 1.

Results and Discussion
On the opposite face of 1, the key NOESY cross-peaks between H-6 and H-10 suggested that these two protones should be cofacial. To determine the absolute configuration of 1, we applied modified Mosher's method using  1 s and 1r), respectively. The absolute configuration at C-8 in 1 was assigned as S deduced from the Δδ H values between the two MTPA esters (Fig. 4) following the MTPA rules 16 . Thus, the configuration of tricyclic nucleus for compound 1 was determined as 1 S, Euplexaurene B (2) was deduced to have the same molecular formula C 20 H 34 O as 1 by HRESIMS analysis. The 1 H NMR spectra of 2 ( Table 2) and 1 were almost identical, which suggested that they may be a pair of epimers. When comparing their NMR spectra, the signals attributable to H-8 were found to be slightly different (δ H 4.08 (1 H, d, J = 5.5 Hz), δ C 81.5 in 2 vs δ H 4.36 (1 H, t, J = 8.0 Hz), δ C 75.0 in 1), indicating that the noticeable difference between the epimers was the isomerization of C-8. This was supported by the NOESY crosspeaks of H-10/H-8. Hence, 2 is the 8-epi-isomer of 1.
Euplexaurene C (3) was found to have a molecular formula of C 20 H 28 O with seven degrees of unsaturation based on HRESIMS, revealing the loss of two hydrogen protons compared with that of 4. The 1 H and 13 C NMR data (Table 3)     Although serrulatane-type diterpenoids have been isolated from marine organisms [12][13][14][15] , they were mainly appeared in the form of bicyclic system [13][14][15] . Anthogorgiene P (4) was firstly isolated as a novel skeleton compound from a Chinese gorgonian Anthogorgia sp. 12 . Euplexaurenes A-C (1-3) represent the serrulatane-type diterpenoids characterized with a 5,3,6-tricyclic skeleton isolated from nature for the second time. A hypothesized biosynthetic pathway of 1-4 starting from geranylgeranyl pyrophosphat (GGPP) was proposed (Fig. 5). A key intermediate (4a) in the biosynthesis derives from GGPP by a ring closure and oxidation. Anthogorgiene P (4) is formed from 4a via aromatic rearrangement to form an unusual 5,3,6-tricyclic nucleus. Then, 4 is oxidized to form euplexaurene C (3), and hydrated to form euplexaurene B (2), respectively. Finally, the epimerization of 2 gives euplexaurene A (1). The new structural patterns found from this gorgonian specimen implied the presence of new biogenetic pathways within marine organisms to adopt different ecological environments.
The absolute configuration of C-11 in serrulatane-type diterpenoid was difficult to be determined by common means of NMR and ECD methods due to the high conformational flexibility of the eight-carbon aliphatic chain attached at C-4. In previous study, the stereochemistry of 4 was not assigned completely 12 . Meanwhile, the eight-carbon aliphatic chain as in 1-4 has been frequently found in terpenoids, ranging from bisabolene sesquiterpenes such as perezone 17 , to sterols such as desmosterol 18 , which was the last biogenetic intermediate in the biosynthesis of cholesterol 19 .
VCD spectroscopy is one such chiroptical technique that sheds new light on many important phenomena studies intensively. The interplay of VCD spectra of chiral molecules in the liquid state and computational studies has led to a remarkably detailed picture of the systems. In recent years, this technique has provided a powerful physicochemical method for the assignment of absolute configurations in natural products. Especially, compared to the other chiroptical methods, VCD present many advantages, since it could be applied to virtually any molecules without the requirement of either UV or Vis chromophores. In present research, VCD has opened a new horizon to define the absolute configurations at C-11 in 1-4. As the low yields of 1-3, compound 4 was chosen to test its experimental VCD spectrum. Thus, the two C-11 diastereomers of 4 were investigated by quantum chemical TDDFT calculations of their VCD spectra. Conformational searches were performed using MMFF94S force field for (1 S,4 R,5 R,9 R, 10 Table 2. NMR spectroscopic data for compound 2 a . a Spectra measured at 500 MHz in CDCl 3 . 4.6 kcal/mol were then re-optimized at the B3LYP/6-311 + G(d) level. The IR and VCD frequencies were calculated for all these structures at the B3LYP/6-311 + G(d) level, and the conformational populations were obtained by means of the ΔG = −RT lnK equation to generate the Boltzmann-averaged IR and VCD spectra. The experimental IR and VCD spectra were measured in CDCl 3 at room temperature. The comparison of the two VCD spectra was superimposed in Fig. 6. All of the calculated IR signals of (1 S,4 R,5 R,9 R,10 S,11 S)-4 had agreements with the experimental IR signals, while the signals of 3, 4, 9, and 10 in the calculated IR spectrum of (1 S,4 R,5 R,9 R,10 S,11 R)-4 had disagreements (the signals labeled in red) (Fig. 6) with the corresponding signals in the experimental spectrum. This suggested that the structure of (1 S,4 R,5 R,9 R,10 S,11 S)-4 was closer to the real case. Furthermore, the calculated VCD spectra were compared with the experimental VCD spectrum, respectively. Most of the calculated VCD signals of (1 S,4 R,5 R,9 R,10 S,11 R)-4 had the identity with the experimental results, however, the signals of 3, 4, 9, and 10 (the signals labeled in red) (Fig. 6) did not match the experimental signals. For (1 S,4 R,5 R,9 R,10 S,11 S)-4, the calculated VCD spectrum compared well with the experimental VCD spectrum. Therefore, based on the IR  Table 3. NMR spectroscopic data for compound 3 a . a Spectra measured at 500 MHz in CDCl 3 .  100 μM). Preliminary structure-activity analysis suggested that the hydroxy group at C-8 may increase the cytotoxic activity, and the presence of 8α-OH contributed more to the activity than 8β-OH. In addition, the antibacterial activities of 1-4 were also tested toward several pathogenic bacteria. But none of the tested compounds showed any activity (MIC > 25.0 μM).
In conclusion, four serrulatane-type diterpenoids (1-4) with potent cytotoxicity against Hep-2 were isolated from the gorgonian Euplexaura sp. VCD experiment combined with accurate quantum mechanical calculation method was carried out to assign their absolute configurations. It could be concluded that VCD is one of important chiroptical methods for the structural elucidation of natural products.

Methods
General Experimental Procedures. Optical rotations were measured on an Optical Activity Limited AA-55 polarimeter. ECD spectra were obtained on a Bio-logic MOS-450 circular dichroism spectrometer. IR and VCD spectra were acquired using a BioTools ChiralIR-2X spectrophotometer. NMR spectra (500 MHz for 1 H NMR and 125 MHz for 13 C NMR) were measured on a Bruker AV-500 spectrometer. ESIMS spectra were obtained using a Micromass Q-TOF spectrometer. Preparative HPLC was performed on a Shimadzu LC-20AT HPLC system with a SPD-M20A detector using a Waters C 18 semi-preparative column (250 × 19 mm, 5 μm). Silica gel (200-300 mesh, Qing Dao Marine Chemical Inc.), Sephadex LH-20 (Pharmacia, Co.) and ODS (40-63