Sarcoehrenbergilides D–F: cytotoxic cembrene diterpenoids from the soft coral Sarcophyton ehrenbergi

A solvent extract of the soft coral Sarcophyton ehrenbergi afforded cembrene diterpenoids, sarcoehrenbergilid D–F (1–3). Chemical structures were established by modern spectroscopic techniques with absolute stereochemistries determined by circular dichroism (CD) and time-dependent density functional theory electronic CD calculations (TDDFT-ECD). Cytotoxicity activities for 1–3 were evaluated against three human cancer cell lines: lung (A549), colon (Caco-2) and liver (HepG2).


Introduction
So coral of the genus Sarcophyton (subclass Octocorallia; order Alcyonaceae; family Alcyoniidae) contain a diversity of cyclic diterpenes that usually contain ethers, lactones or furanes around a cembrane framework. 1,2 These cembrane diterpenoids exhibit a wide range of structural diversity and biological activity. [3][4][5][6][7][8][9][10] Cembranoids, the main metabolites identied in the genus Sarcophyton have been shown to serve as an effective chemical defense against natural predators of coral. 11 The leather coral Sarcophyton ehrenbergi (von Marenzeller, 1886) produces diverse metabolites with distinct chemical structures as well as promising biological activities. 8,[12][13][14][15][16][17] Additionally, prostaglandins (PGs) that regulate a broad range of physiological activities, have been isolated from S. ehrenbergi. 18,19 The Red Sea contains a high endemic biota including approximately 50 genera of hermatypic so coral. 20 While Red Sea marine invertebrates have been historically under-reported within the scientic literature, intensive investigation of Red Sea marine life has occurred over the past ten years. 8,[21][22][23] To continue efforts to identify new marine metabolites from Red Sea so coral, [6][7][8][22][23][24] herein we report three cembrene diterpenoids isolated from S. ehrenbergi (Fig. 1). Absolute stereochemistry of the newly reported compounds was determined by time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. All isolated metabolites were probed against three human cancer cell lines.

Results and discussion
Freshly collected S. ehrenbergi were rapidly frozen by placing in a À20 C chamber and kept frozen till time of extraction. The chromatographic separation of the methylene chloride : methanol (1 : 1) extract yielded three cembrene diterpenoids derivatives (Fig. 1 The IR spectrum showed absorption bands at n max 3450 cm À1 and 1754 cm À1 for hydroxyl and keto groups, respectively. The 13 C NMR and distortion less enhancement by polarization transfer (DEPT) spectrum showed 21 carbon signals, classied as ve methyls, six methylenes, four methines and six quaternary carbons (Table 1) (Fig. 2).
The signal at d H 5.45 (d; J ¼ 10.0 Hz) correlated with a proton signal at d H 5.14 (d, J ¼ 10.0 Hz) and quaternary olenic carbons at d C 147.0 and d C 163.0 in DQF-COSY and HMBC (Fig. 2), respectively, allowed for the assignments of H-2, H-3, C-4 and C-1, respectively. [8][9][10][23][24][25] Correlations in the HMBC spectrum showed several informative connections: H-3 to carbon signals at d C 13.6 (q, olenic) d C 34.6 (t), allowed for the assignment of H-18 (d H 2.02, s) and H-5 (d H 2.37, brd, J ¼ 14.0), respectively; methyl signal d H 1.83 (s) to C-1 and carbon signal at d C 174.0 (C]O) attributed to H-17 and C-16, respectively as well as supporting the location of C-1/C-2 lactone ring; methyl singlet at d H 1.11 to d C 73.5 (C-7), d C 37.0 and 78.5 allowed for the location of H 3 -19 (d C 13.6), C-9 and C-8, respectively; the oxygenated broad doublet at d H 3.57 (d C 79.0) to C-9 and C-20, was assigned to H-11. The assignment of H-7, H 2 -6 and C-5   2) as well as to C-20 in HMBC analyses (Fig. 2). An HMBC correlation established the site of a methoxy group (d H 3.20 s, d C 48.3 q) at C-8. The planar structure assignment of 1 and the C-7/C-12 ether linkage were proposed by 1D, 2D NMR and HREIMS data. The data comparison with those of sarcoehrenbergilid A, as previously reported, 23 suggested that 1 and sarcoehrenbergilid A, 23 differ only in stereochemistry.
The NOESY spectrum revealed that a g-lactone at H-2 (d H 5.45, d, J ¼ 10.0 Hz) correlated with CH 3 -18 (d H 2.02, s); a vicinal coupling with H-3 established a trans conguration and a borientation for H-2. 8 NOSEY correlations were observed between three methyl groups with alpha protons (e.g., CH 3 -20 with H-10a, CH 3 -19 with H-6a/H-10a, and CH 3 -17 with H-14a) (Fig. 3). H-7 and H-11 was assigned to a b-conguration based on NOSEY correlations with H-5b and H-14b, respectively. Absolute conguration was established by experimental and TDDFT-simulated ECD spectra. All possible conformations of 1 within energy window of 10 kcal mol À1 were generated and optimized at B3LYP/6-31G* level of theory. The rst 50 excitation states were then computed based on time-dependent density-functional theory (TDDFT) at B3LYP/6-31G* level in methanol by the PCM model. The generated TDDFT-ECD spectra were Boltzmann-weighted and compared to the experimental spectrum (Fig. 4). The TDDFT-simulated ECD spectrum was in a good agreement with the corresponding experimental ECD spectra (Fig. 4). This comparison revealed the absolute conguration and therefore 1 was assigned as 2S,16:7S,12S-diepoxy-11R-hydroxy-8R-methoxy-16-keto-cembra-1Z,3E-diene (sarcoehrenbergilid D).  The IR spectrum showed characteristic bands at n max 3445 cm À1 and 1747 cm À1 for hydroxyl and keto groups, respectively. The 13 C NMR spectrum revealed twenty carbon signals (Table 1) classied by DEPT as six quaternary, four methines, six methylenes and four methyls carbons. 1D and 2D NMR spectroscopic data were quite close to sarcoehrenbergilid A, 23 a formerly isolated diterpenoid from S. ehrenbergi except for an absence of methoxyl groups. For 2 there is an upeld carbon signal at d C 74.5 and a downeld methyl signal at d C 20.7 for C-8 and CH 3 -19, respectively.
Isolated metabolites 1-3 were tested for cytotoxic activity toward lung (A549), colon (Caco-2) and liver (HepG2) human cancer cell lines based on an MTT reduction assay (Fig. 5). 100 mM). Since primary necrosis is not easily differentiated from secondary necrosis that occurs with apoptosis, 27 the mode of action will not be considered. To differentiate these distinct biological events requires apoptotic assays accompanying necrosis measurements. A combined necrosis/apoptotic timecourse will be presented in a subsequent study to elaborate on mode of action.

General experimental procedures
Circular dichroism was measured on JASCO 810 spectropolarimeter. HREIMS data were collected on a JEOL JMS-700 instrument (Tokyo, Japan). NMR spectra were recorded on a Bruker 500 NMR spectrometer (Japan). JASCO P-2200 polarimeter and JASCO FT/IR-6300 spectrometer was used for optical rotation and infrared measurements, respectively.

Animal material
Sarcophyton ehrenbergi coral was collected from the Red Sea on the Egyptian coast at Hurghada, in March 2016 and identied by Dr M. Al-Hammady. A voucher specimen (03RS27/1) was deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.
3.4.2 MTT cytotoxicity assay. The cytotoxicity of tested compounds was investigated by a MTT assay. Cell lines were seeded and incubated overnight allowing cell adhesion to the plate well (5 Â 10 3 cells per well; 96-well plate in a volume of 100 mL). To generate concentration-dependent curves, sample concentration was varied (100, 50, 25, 12.5, 6.25 mM) for a total well volume of 200 mL for 48 h. MTT solution (5 mg ml À1 ) was added (100 mL per well) for 90 min before measurements. 28,29 Aer medium removal, dark blue formazan crystals formed in viable cells were dissolved in 100 mL of DMSO, followed by shaking for 10 min (200 rpm). The absorbance was recorded at 492 nm using a microplate reader (Sunrise™ microplate reader, Tecan Austria Gmbh, Grödig, Austria) for cell viability measurement. IC 25 values were expressed as a concentration of tested compound that inhibits 50% cell growth in comparison with a vehicle control (quadrate to octuplet treatment) by nonlinear regression model analyses using GraphPad Prism® v 6.0 soware.

Computational functional theory calculations
Conformational analysis was performed using Omega2 soware 30 to obtain the possible conformers for 1-3 within energy window value of 10 kcal mol À1 . All resulting conformers were optimized at B3LYP/6-31G* level of theory using Gaussian09 soware. 30 Frequency calculations were then performed on the optimized structures to ensure the nature of the local minima as well as to estimate the Gibbs free energies. Time-dependent density functional theory (TDDFT) calculations with incorporating a polarizable continuum model (PCM) using methanol as a solvent were carried out at the B3LYP/6-31G* level of theory to calculate the rst y excitation states. Electronic circular dichroism (ECD) spectra were nally generated using SpecDis 1.71 (SpecDis 2017 (ref. 31 and 32)) by applying Gaussian band shapes with sigma ¼ 0.20-30 eV. The generated ECD spectra were Boltzmann-averaged.

Conclusions
Cembrene diterpenoids (1-3) were isolated and identied from the S. ehrenbergi so coral. The isolated compounds were tested against three human cancer cell lines, which resulted in 2 being the most potent compound against lung A549 cancer cell. The absolute stereochemistry of 1-3 were conrmed by comparing experimental and TDDFT-simulated ECD spectra.

Conflicts of interest
There are no conicts to declare.