Sarcophyolides B–E, New Cembranoids from the Soft Coral Sarcophyton elegans

Four new cembrane-type diterpenoids, sarcophyolides B–E (1–4), along with 11 known analogues were isolated from the soft coral Sarcophyton elegans. The structures of new compounds 1–4 were established on the basis of spectroscopic analysis and chemical conversion. The new cembranoids sarcophyolides B (1) and lobocrasol were found to exhibit potent inhibition against A2780 human ovarian tumor cells.


Introduction
Since the first cembranoid (+)-cembrene was reported five decades ago, numerous cembranoids have been isolated from marine organisms, plants, and insects [1][2][3]. Their basic structural patterns typically featured a common 14-membered carbocyclic nucleus and unconventional cembranoids OPEN ACCESS containing a 12-membered carbon skeleton or 13-membered variants. Some of the typical terpenoids are known as chemical defense tools to protect soft corals against natural predators, as feeding deterrents or act by virtue of their toxicity [4][5][6]. From a pharmaceutical point of view, cembranoids have been reported to exhibit various biological activities, such as having antitumor, ichthyotoxic, antiinflammatory, neuroprotective, antibacterial, antiangiogenic, antimetastatic, and antiosteoporotic properties [7][8][9][10][11][12][13]. Soft corals, belonging to the genus Sarcophyton (Alcyoniidae), are well recognized as a rich source of macrocyclic cembrane-type diterpenoids and biscembranoids. The structural patterns of cembranoids from the genus Sarcophyton vary notably due to geographic location and species differentiation [14]. It is a challenging work to uncover new natural products from known species of marine organisms distributed in new locations. In our continuing search for the chemical diversity from the soft corals inhabited in various locations of South China Sea, the specimen Sarcophyton elegans was collected. Primary HPLC-ESIMS and 1 H NMR examinations on the EtOAc extracts revealed the spectroscopic signals representing a diverse array of terpenoids. Further chromatographic separation and purification resulted in the isolation of four new cembranoids ( Figure 1) in addition to 11 known analogues.

Structural Elucidation of New Compounds
Sarcophyolide B (1) was isolated as a colorless oil with the molecular formula of C 20 H 32 O 2 based on the high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR) data, implying five degrees of unsaturation. The IR absorptions at 3406 and 1604 cm −1 suggested the presence of hydroxy and olefinic groups. The 1 H NMR displayed the signals for five methyls, two olefinic protons at δ H 5.26 (1H, d, J = 11.5 Hz, H-2) and 5.49 (1H, dd, J = 3.0, 5.0 Hz, H-11), a hydroxymethine δ H 5.04 (1H, brd, J = 9.0 Hz, H- 14), and a number of alkyl protons for methylene and methine groups. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra exhibited a total of 20 carbon resonances, involving four olefinic carbons and three oxygen-bearing sp 3 carbons. Diagnostic NMR data (Tables 1 and 2) through COSY and heteronuclear multiple quantum coherence (HMQC) analyses indicated compound 1 to be a cembrane-based diterpenoid. The COSY relationships connected the protons to form three subunits from C-2 to C-7, C9 to C-11, and C-13 to C-14, in addition to an isopropyl group. The connectivity of the subunits was accomplished by the HMBC correlations. The observed HMBC interactions from the methyl protons of isopropyl group (δ H 1.12 and 1.13, d) to an olefinic carbon at δ C 150.6 (qC, C-1) and, in turn, the olefinic proton H-2 correlating to the methine carbon C-15 (δ C 26.9, CH), indicated a double bond to be resided at C-1/C-2, while an isopropyl group is positioned at C-1. The HMBC relationships from H 3 -18 (δ H 1.19, s) to C-3 (δ C 53.2, CH), C-4 (δ C 87.6, qC), and C-5 (δ C 32.8, CH 2 ); from H-3 (δ H 3.11, brd, J = 11.5 Hz) to C-1, C-4, C-7, and C-8 (δ C 82.8, qC); and from H-7 (δ H 1.90) to C-4 and C-2 (δ C 120.9, CH) revealed a capnosane-based cembranoid bearing a 3,7-cyclopentane ring [15], in which an oxygen atom and a methyl group were co-positioned at C-4. Additional HMBC relationships were conducted to assign the linkage of a methyl group H 3 -19 (δ H 1.23, s) at oxygenated carbon C-8, while the second olefinic group was resided at C-11/C-12 ( Figure 2A). A hydroxy group was evident to be located at C-14 (δ C 72.9, CH) according to the COSY relationship between a D 2 O exchangeable proton at δ H 4.60 (br) and H-14, while H-14 coupled to C-14 in HMQC. The above functional groups are accounted for four degrees of unsaturation, the remaining site is, thus, assumed to be contributed by an ether bridge across C-4 and C-8.    The NMR data of sarcophyolide C (2) closely resembled those of compound 1, while 2D NMR data analysis established the structure of 2 to be a homolog of 1. The major difference was found concerning the upfield-shifted C-4 (δ C 81.5) and C-8 (δ C 74.7) and the molecular weight of 2 having 18 amu more than that of 1, while the degrees of molecular unsaturation in 2 are four instead of five, based on the HRESIMS data (m/z 345.2406 [M + Na] + ). These findings disclosed the structure of 2 to be a 4,8-dihydroxylated derivative of 1. The closely similar NOE interactions of 2 and 1 in association with the chemical conversion from 1 to 2 under acidic solution (Figure 3) indicated the configurations of 2 to be the same as those of 1.
The absolute configurations of the stereogenic centers in 1 and 2 were further proved by their single-crystal X-ray diffraction analysis using Flack's method ( Figure 4).  The HRESIMS data (m/z 343.2239 [M + Na] + ) of sarcophyolide D (3) was in accordance with a molecular formula of C 20 H 32 O 3 with five degrees of unsaturation. The NMR data of compound 3 were compatible to those of sarcophytol L [15], except for the presence of two olefinic bonds instead of three bonds in the known analog. Analysis of 13 C NMR in association with 2D NMR data revealed 3 presenting two epoxy carbons (δ C 58.1 and 59.5), residing at C-11 and C-12 according to the HMBC correlations of H 3 -20 (δ H 1.49, s) to C-11 (δ C 59.5, CH), C-12 (δ C 58.1, qC), and C-13 (δ C 45.6, CH 2 ).
Thus, the structure of 3 was determined as an 11,12-epoxidated sarcophytol L. The relative configurations of the stereogenic centers in 3 from C-1 to C-7 were in agreement with those of sarcophytol L due to the similar NOE and NMR data. Additional NOE relationships from H-14 to H 3 -20 and H-3, and from H-11 to H 3 -16 and H-13a ( Figure 5) assigned a trans geometry of the epoxy group, while H 3 -20 is oriented in the same face as H-14. The 2D NMR (COSY, HMQC, and HMBC) data analysis revealed the gross structure of sarcophyolide E (4) closely related to a known cembranoid derived from sarcophtolide through oxymercuration [17]. The only difference was due to C-12 in which a quaternary carbon (δ C 71.8) of 4 was replaced by a methine carbon of the known analog. The hydroxylated C-12 was supported by its HMBC correlations with H 3 -20 (δ H 0.98, s) and a hydroxyl proton OH-12 (δ H 4.46, s). The relative configurations of 4 were determined on the basis of NOE interactions. The NOE correlations between H-7 and H-11 and H 3 -18, and between H 3 -18 and H-2 informed a cis-geometry of the epoxy bond and 3E of the olefinic bond. In addition, the NOE interactions between H-7 and OH-8 (δ H 4.40, s) and between H-11 and OH-12 in association with the absence of the interactions of H-11/H 3 -20 and H-7/H 3 -19 revealed the opposite orientation of H-7 and H-11 toward their vicinal methyl groups ( Figure 6). Based on the CD rule for α,β-unsaturated-γ-lactone [18], the Cotton effects due to the n→π* and π*→π* transitions of the α,β-unsaturated lactone chromophore correlated directly to the absolute configuration of the stereogenic center at C(γ). Thus, the positive Cotton effects for n→π* (252 nm) and negative π*→π* (226 nm) of 4 ( Figure 7) indicated that it follows p-helicity rule, demonstrating 2R configuration.  Based on the spectroscopic analyses and comparison of the NMR data with those reported in literature, 11 known cembranoids were identical to: sarcophytol L [19], 13α-hydroxysarcophytol L [19], sarcophyolide A [20], sarcophine [21], sarcophinone [22,23], 7α-hydroxy-Δ 8(19) -deepoxysarcophine [24], 4β-hydroxy-Δ 2(3) -sarcophine [24], 7α,8β-dihydroxydeepoxysarcophine [25], 1,15β-epoxy-2-epi-16deoxysarcophine [3], sarcophytol Q [26], and lobocrasol [27]. Lobocrasol presented as a unique skeleton that was isolated from soft coral for the second time.

General
Optical rotations were measured on a Perkin-Elmer 243B polarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FTIR spectrometer. 1 H and 13 C NMR and 2D NMR spectra were measured on an Avance-500 FT 500 MHz NMR spectrometer using TMS as an internal standard, while δ values are expressed in parts per million (ppm), and J values are reported in Hertz (Hz). HRESIMS data were obtained from Bruker APEX IV instrument. Low pressure column chromatography was carried using silica gel (160-200 and 200-300 mesh). The GF 254 silica gel for TLC was provided by Qingdao Marine Chemistry Co., Ltd. (Qingdao, China).

Animal Material
The soft coral Sarcophyton elegans was collected from Xidao Island, Hainan, China, in 2002, and kept frozen until extraction. The specimen was identified by Dr. Leen van Ofwegen (National Museum of National History, Naturalis). The soft coral (HSE-17) was deposited at State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China.

Extraction and Isolation
The frozen soft coral Sarcophyton elegans (3.5 kg, wet weight) was homogenized and extracted with EtOH. The concentrated extract was desalted through dissolving in MeOH to yield a residue (100 g) after evaporation. This residue was defatted by partitioning between H 2 O and petroleum ether, and then the H 2 O fraction was extracted with EtOAc. The EtOAc fraction (7.4 g) was subjected to Si gel column chromatography eluting with a gradient of petroleum ether (PE)-acetone to obtain eight subfractions (SF1-SF8). SF2 (1.2 g) was subsequently subjected to Si gel column chromatography eluting with PE-EtOAC (5:1) to yield 1 (9.0 mg), 3 (4.2 mg), and 2 (4.8 mg). SF3 (1.0 g) was treated by the same process as SF2 to yield 4 (2.3 mg). From SF7 (890 mg) and SF8 (320 mg) fractions, 5 (5.

Mosher Reaction
Compound 1 (0.01 mmol), together with DMAP (4-dimethylaminopyridine, 0.01 mmol) and DCC (dicyclohexylcarbodiimide, 0.01 mmol), were dissolved in methylene dichloride (2 mL) at 0 °C, and then (R)-or (S)-MPA (0.01 mmol) was added to the solution. After stirring at room temperature for 24 h, the mixture was evaporated under reduced pressure to obtain a residue, which was separated using a reversed phase semipreparative HPLC with 95% CH 3 CN-H 2 O as a mobile phase to yield (R)-MPA ester or (S)-MPA ester.

Chemical Conversion
To a solution of 1 (1 mg/mL) in acetone 5% HCl (0.2 mL) was added. After stirring for 2 h at room temp. the reaction mixture was extracted with EtOAc (0.5 mL). The organic layer was concentrated to yield a product 1a. Specific rotations, ESIMS, Rf-values of TLC, and 1 H NMR data indicated the structure of 1a to be identical to 2.

Conclusions
Present work provided a number of new cembranoids, which enriched the cembranoid family. Capnosane-type cembranoids with 3,7-fused carbobicyclic skeleton are a group of uncommon derivatives, derived from soft corals, while the unique ether bridge across C-4/C-8 in 1 is reported for the first time. These findings implied that the species of genus Sarcophyton are potential sources, waiting for the discovery of structurally unique chemical diversity.