Cembrene Diterpenoids with Ether Linkages from Sarcophyton ehrenbergi: An Anti-Proliferation and Molecular-Docking Assessment

Three new cembrene diterpenoids, sarcoehrenbergilid A–C (1–3), along with four known diterpenoids, sarcophine (4), (+)-7α,8β-dihydroxydeepoxysarcophine (5), sinulolide A (6), and sinulolide B (7), and one steroid, sardisterol (8), were isolated and characterized from a solvent extract of the Red Sea soft coral Sarcophyton ehrenbergi. Chemical structures were elucidated by NMR and MS analyses with absolute stereochemistry determined by X-ray analysis. Since these isolated cembrene diterpenes contained 10 or more carbons in a large flexible ring, conformer stabilities were examined based on density functional theory calculations. Anti-proliferative activities for 1–8 were evaluated against three human tumor cell lines of different origins including the: lung (A549), colon (Caco-2), and liver (HepG2). Sardisterol (8) was the most potent of the metabolites isolated with an IC50 of 27.3 µM against the A549 cell line. Since an elevated human-cancer occurrence is associated with an aberrant receptor function for the epidermal growth factor receptor (EGFR), molecular docking studies were used to examine preferential metabolite interactions/binding and probe the mode-of-action for metabolite-anti tumor activity.


Introduction
Cembrane diterpenoids are a large and structurally diverse group of natural products isolated from both terrestrial and marine organisms [1]. The 14-membered ring structure is biosynthetically formed from the cyclization of the geranylgeraniol precursor between carbons 1 and 14. The cembranoid diterpene, sarcophytol A, first isolated from the Okinawan soft coral Sarcophyton glaucum and found to exhibit strong inhibitory activity against tumor promoters [2], led to the subsequent isolation of hundreds of cytotoxic cembranoids from plant and marine sources [3]. Sarcophyton soft coral species are characterized by the production of cembrene-type diterpenoids [4][5][6][7][8][9][10] and these cyclic diterpenes usually exhibit cyclic ether, lactone, or furane moieties around the cembrane framework [11,12]. From a biomedical perspective, cembranoid diterpenes exhibit a diverse range of biological protection against tumors, inflammation, and fish toxins (ichthyotoxic), as well as microbial and/or viral infections [4,5,13]. With cancer occurrence and mortality associated with cancer increasing in the U.S. and around the world, the exploration of cytotoxic agents for improved cancer treatment via chemotherapy is a global priority. In fact, the World Health Organization (WHO) estimates that malignant neoplasms are ranked as the second leading cause of death globally. In 2012, 14.1 million newly-diagnosed cancer cases were reported, with 8.2 million deaths directly associated with cancer; these incidence and mortality numbers are estimated to increase by ca. 150% by 2030 [14,15]. In a continuing effort to characterize soft coral metabolites from the Red Sea with biological activity [6][7][8], herein is reported three new cembrene diterpenoids, as well as known diterpenoids and a polyoxygenated steroid isolated from Sarcophyton ehrenbergi. In this study, the anti-proliferative potential of the isolated compounds against three human tumor cell lines were evaluated. To probe the mode-of-action, molecular docking studies were performed with the epidermal growth factor receptor (EGFR), a large family of transmembrane receptors that normally regulate key events associated with cell growth, differentiation, and migration. An aberrant receptor function has been linked to elevated cancer occurrence.

Identification and Structure Elucidation
As part of the continuing investigation for biologically active constituents from Egyptian Red Sea costal soft corals [6][7][8]16], reported here is the chromatographic fractionation and purification of a methylene chloride:methanol (1:1) extract from S. ehrenbergi (Figure 1). framework [11,12]. From a biomedical perspective, cembranoid diterpenes exhibit a diverse range of biological protection against tumors, inflammation, and fish toxins (ichthyotoxic), as well as microbial and/or viral infections [4,5,13]. With cancer occurrence and mortality associated with cancer increasing in the U.S. and around the world, the exploration of cytotoxic agents for improved cancer treatment via chemotherapy is a global priority. In fact, the World Health Organization (WHO) estimates that malignant neoplasms are ranked as the second leading cause of death globally. In 2012, 14.1 million newly-diagnosed cancer cases were reported, with 8.2 million deaths directly associated with cancer; these incidence and mortality numbers are estimated to increase by ca. 150% by 2030 [14,15].
In a continuing effort to characterize soft coral metabolites from the Red Sea with biological activity [6][7][8], herein is reported three new cembrene diterpenoids, as well as known diterpenoids and a polyoxygenated steroid isolated from Sarcophyton ehrenbergi. In this study, the anti-proliferative potential of the isolated compounds against three human tumor cell lines were evaluated. To probe the mode-of-action, molecular docking studies were performed with the epidermal growth factor receptor (EGFR), a large family of transmembrane receptors that normally regulate key events associated with cell growth, differentiation, and migration. An aberrant receptor function has been linked to elevated cancer occurrence.

Mar. Drugs
Similar to 1, the γ-lactone-(H-2) and olefinic-proton (H-3) vicinal coupling (10 Hz) established a cis configuration [8]. Also similar to 1, the NOSEY data for 2 showed a correlation between H-6b and H-7, indicating that the epoxy ring at C-7 is below the ring while H-6a correlates with CH 3 -19, establishing that the relative stereochemistry for the methyl is above the ring ( Figure 5). H-7, which is assumed to be in the beta position from the previous NOSEY correlation, also correlates with H-11, indicating that the other epoxide ring attachment is in an alpha configuration. Finally, a NOSEY correlation between H-10a and CH 3 -20 indicates that the methyl group is in an alpha orientation. Thus, 2 was confirmed to be 2S,16: 7R,11R-diepoxy-8β,12β-dihydroxy-16-keto-cembra-1E,3E-diene (sarcoehrenbergilid B).  (Table 1); four methyls, six methylenes, five methines, and five quaternary carbons. The spectroscopic data of 3 are similar to a previously isolated diterpenoid from S. trocheliophorum, trocheliophorol [21], except for the presence of a hydroxyl unit at C-12 (δ C 70.1) instead of an exomethylene. The location of the C-12 hydroxyl group was confirmed by HMBC correlations with a methyl singlet H 3 -20 (δ H 1.13 s); correlations were also observed between H 3 -20 and δ C 85.1 (C-11) and δ C 31.2 (C-13) (Figure 3). Similar to compounds 1 and 2, a γ-lactone-(H-2) and olefinic-proton (H-3) vicinal coupling (10 Hz) established a cis configuration [8]. A NOESY correlation between H-6a with H-7 indicated that the C-7 hydroxyl group is in a beta orientation and a H-6a correlation with CH 3 -19 indicated that the methyl at C-8 is in an alpha configuration ( Figure 5). A NOSEY correlation was also observed between H-6a and CH 3 -20, indicating that the hydroxyl at C-12 is in the beta orientation. Finally, a NOSEY correlation between CH 3 -20 and H-11 indicated that the epoxide connection at C-11 is the same as C-8, both in alpha configurations. From the above spectral data, 3 was established as 2S,16: 8S,11S-diepoxy-7β,12α-dihydroxy-16-keto-cembra-1E,3E-diene (sarcoehrenbergilid C).
With the large flexible ring systems for compounds 1-3, many conformers are possible. To predict the most stable form, molecular modeling calculations were performed to estimate the lowest energy state. Conformation ensembles were generated using a MMFF94 molecular mechanics force field within a 10 kJ/mol window, providing 101, 98, and 106 conforms for 1, 2, and 3, respectively. Each generated conformer was subjected to energy-minimization at a B3LYP/6-31G* level of theory and thereafter, the corresponding free energy was calculated on the optimized structure. The Boltzmann population was estimated based on the calculated relative free energies for each conformer with respect to the lowest free energy conformer at 298 K (Table S1). The lowest and next three higher free energy conformers for 1-3 are shown ( Figure 6). All conformations with populations higher than 1% are shown in Figures S22-S24. The optical rotation of all conformations was theoretically calculated and the results are summarized in Table S1. According to the calculated free energies and optical rotation, the lowest compound conform free energy is in strong agreement with the elucidated structures ( Figure 6). For 2, the structure is more stable than a previously reported conformer ( Figure S23, 2f) by −7.4 kJ/mol. In addition to the three new metabolites (1-3), four known compounds, sarcophine (4), (+)-7α,8β-dihydroxydeepoxysarcophine (5) [6], sinulolide A (6), and B (7) [22], and one steroid, sardisterol (8) [23], were identified from the coral extract ( Figure 2). 15,192 7 of 14 Figure 6. Optimized confirmers for 1-3, as well as the next three higher free-energy conformers.

Anti-Proliferative Activity against Cancer Lines
Isolated compounds 1-8 were evaluated for their anti-proliferative activity against three human tumor cell lines originating from the lung (A549), colon (Caco-2), and liver (HepG2) tissue based on an MTT reduction assay. The treatment of a human lung tumor cell line revealed differential antiproliferative effects ( Table 2). Among all tested compounds, sardisterol (8) had the most potent effect on A549 cells with a concentration-dependent loss of cell proliferation compared to a DMSO solvent control. Sardisterol was isolated for the first time from the marine soft coral S. digitatum [23]. To the best of our knowledge, this is the first report of anti-proliferative activity for 8 against tumor cells (Table 2 and Figure S25). The treatment of HepG2 cells with increasing concentrations of 1-8 revealed differential anti-proliferative potential (Table 2 and Figure S27). Compounds 3 and 8 showed a moderate inhibition (IC50 = 53.8 and 56.8 μM, respectively). Compounds 1, 3, 5, 6, and 7 exhibited IC50 values between 63.1 and 98.6 μM.

Anti-Proliferative Activity against Cancer Lines
Isolated compounds 1-8 were evaluated for their anti-proliferative activity against three human tumor cell lines originating from the lung (A549), colon (Caco-2), and liver (HepG2) tissue based on an MTT reduction assay. The treatment of a human lung tumor cell line revealed differential anti-proliferative effects ( Table 2). Among all tested compounds, sardisterol (8) had the most potent effect on A549 cells with a concentration-dependent loss of cell proliferation compared to a DMSO solvent control. Sardisterol was isolated for the first time from the marine soft coral S. digitatum [23]. To the best of our knowledge, this is the first report of anti-proliferative activity for 8 against tumor  (Table 2 and Figure S25). The treatment of HepG2 cells with increasing concentrations of 1-8 revealed differential anti-proliferative potential ( Table 2 and Figure S27). Compounds 3 and 8 showed a moderate inhibition (IC 50 = 53.8 and 56.8 µM, respectively). Compounds 1, 3, 5, 6, and 7 exhibited IC 50 values between 63.1 and 98.6 µM.

Molecular Docking Studies
The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that is overexpressed in many tumor cell types and reported as a cause for some non-small-cell lung carcinomas [24]. Based on the hypothesis that anti-proliferation activity with the A549 cell line is associated with EGFR inhibition [25], receptor binding to an ATP binding site domain of EGFR kinase by isolated metabolites was examined. Doxorubicin, a known effector molecule that binds to the ATP binding domain of EGFR, was initially used as a positive control. The ability of 8 (the most active metabolite) and 4 (the least active) were examined using molecular docking simulations. Simulations were conducted with AutoDock 4.2 software with an initial assessment of docking done by comparing the docked pose of the co-crystallized inhibitor ligand erlotinib with the experimental pose (PDB code: 1M17). To further provide a predictive picture as to where the compounds are positioned in terms of direct interactions with EGFR, docking scores for the newly isolated natural products are provided and two other EGFR inhibitors, afatinib and gefitinib, as well as the positive control doxorubicin (Table S2). Per the predicted binding poses, AutoDock accurately reproduced the crystal structure observed binding modes of erlotinib, afatinib, and gefitinib (Figure 7a-c). To reveal the binding features and possible interactions, compounds were subjected to molecular docking simulation followed by AMBER-based molecular mechanical minimization and MM/GBSA binding energy calculations. The calculated binding energies are in good agreement with experimental data, with a correlation coefficient R 2 of 0.96 (Table S2, Figure S28). Per the minimized doxorubicin complex (Figure 7d (Figure 7h). Several van der Waals and hydrophobic interactions were observed between the added marine ligand and amino acids in the active site including Leu 865 , Leu 694 , and Val 702 . To assess the stability of the predicted ligand-receptor complex, a short molecular dynamics simulation of 2.5 ns was performed for 8 in the complex with the EGFR receptor. The corresponding hydrogen bond distance between 8 and a carboxylate oxygen atom of Asp 776 was then measured and averaged over the simulation time ( Figure S29). According to the calculation, 8 can be considered stable, with an average hydrogen bond length of 2.16 Å with Asp 776 .

General Experimental Procedures
Specific rotation was measured with a JASCO P-2200 polarimeter (JASCO Corporation, Tokyo, Japan) and the IR spectra were collected on a JASCO FT/IR-6300 spectrometer (JASCO Corporation, Tokyo, Japan). HR-ESI-FT-MS was carried out using a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer (Waltham, MA, USA) at the Natural Science Center for Basic Research and Development (N-BARD), Hiroshima University. The 1 H (600 MHz) and 13 C (150 MHz) NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer (JEOL Ltd, Tokyo, Japan) with tetramethylsilane as an internal standard. Purification was run on a Shimadzu HPLC system equipped with a RID-10A refractive index detector and compound separation was performed on YMC-Pack ODS-A (YMC CO., LTD., Tokyo, Japan, 250 × 4.6 mm i.d., 5 μm) and (250 × 10 mm i.d., 5 μm) columns for analytical and preparative separation, respectively. Chromatography separation included normal-phase Silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany), which was used for column chromatography. Pre-coated silica gel plates (Kieselgel 60 F254, 0.25 mm, Merck, Darmstadt, Germany) were used for TLC analyses. Spots were visualized by heating after spraying with 10% H2SO4.

Animal Material
Soft coral Sarcophyton ehrenbergi was collected from the Egyptian Red Sea off the coast of Hurghada in March 2015. The soft coral was identified by M Al-Hammady with a voucher specimen (03RS27) deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

General Experimental Procedures
Specific rotation was measured with a JASCO P-2200 polarimeter (JASCO Corporation, Tokyo, Japan) and the IR spectra were collected on a JASCO FT/IR-6300 spectrometer (JASCO Corporation, Tokyo, Japan). HR-ESI-FT-MS was carried out using a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer (Waltham, MA, USA) at the Natural Science Center for Basic Research and Development (N-BARD), Hiroshima University. The 1 H (600 MHz) and 13 C (150 MHz) NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer (JEOL Ltd, Tokyo, Japan) with tetramethylsilane as an internal standard. Purification was run on a Shimadzu HPLC system equipped with a RID-10A refractive index detector and compound separation was performed on YMC-Pack ODS-A (YMC CO., LTD., Tokyo, Japan, 250 × 4.6 mm i.d., 5 µm) and (250 × 10 mm i.d., 5 µm) columns for analytical and preparative separation, respectively. Chromatography separation included normal-phase Silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany), which was used for column chromatography. Pre-coated silica gel plates (Kieselgel 60 F 254 , 0.25 mm, Merck, Darmstadt, Germany) were used for TLC analyses. Spots were visualized by heating after spraying with 10% H 2 SO 4 .

Animal Material
Soft coral Sarcophyton ehrenbergi was collected from the Egyptian Red Sea off the coast of Hurghada in March 2015. The soft coral was identified by M Al-Hammady with a voucher specimen (03RS27) deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

X-ray Crystallography Data
Data collection was performed with a Bruker SMART-APEX II ULTRA CCD area detector with graphite monochromated Cu Kα radiation (λ = 1.54178 Å) at the Center for Analytical Instrumentation, Chiba University, Japan. The structure was solved by direct methods using SHELXS-97 [26]. Refinements were performed with SHELXL-2013 [27] using full-matrix least squares on F 2 . All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in idealized positions and refined as riding atoms isotropically. Crystal data: C 21

Cell Proliferation Assay
Anti-proloiferative studies were performed using a modified MTT (3-[4,5]-2,5-diphenyltetrazolium bromide) assay based on a previously published method [28,29]. Appropriate cell densities of exponentially growing A549, Caco-2, or HepG2 cells (5000-10000 cells/well) were seeded onto 96-well plates. After a 24 h incubation period with 5% CO 2 at 37 • C, stock test compounds (1-8) dissolved in dimethyl sulfoxide (DMSO) were added at concentrations of 100, 50, 25, 12.5, and 6.25 µM in culture medium (final DMSO concentration in medium = 0.1%, by volume). After 48 h of incubation, MTT solution in PBS (5 mg/mL) was added to each well, after which the incubation was resumed for a further 90 min. The formation of intracellular formazan crystals (mitochondrial reduction product of MTT) was confirmed by a phase contrast microscopic examination. At the end of the incubation period, the medium was removed, and 100 µL of DMSO was added to each well to dissolve formed formazan crystals with shacking for 10 min (200 rpm). Dissolved crystals were quantified by reading the absorbance at 492 nm (OD) on a microplate reader (Sunrise™ microplate reader, Tecan Austria Gmbh, Grödig, Austria) and were used as a measure of cell proliferation.

Density Functional Theory Calculations
The conformational structures for 1-3 were generated using Omega2 software (version 2.5.1.4, OpenEye Scientific Software, Santa Fe, NM, USA) [30]. In the conformational search, the energy window value was set to 10 kcal/mol and all stereogenic centers were considered; other parameters were set to default. The geometry of each generated conform was then energetically optimized at the B3LYP/6-31G* level of theory using Gaussian09 software (Revision E.01, Gaussian, Inc., Wallingford, CT, USA) [31]. All optimized conformers were subjected to a vibrational frequency calculation to confirm the minimum energy states and the corresponding free energies were obtained. The relative Boltzmann population of each conformer was then valued at 298 K. Optical rotations were predicted at the same level of theory.

Molecular Docking Studies
Molecular docking in the ATP binding site of EGFR kinase domain was performed using AutoDock 4.2 software (version 4.2, The Scripps Research Institute, La Jolla, CA, USA) [32]. The crystal structure of the EGFR kinase domain complexed with erlotinib (PDB code: 1M17 [33]) was taken as the template for all docking calculations. Water molecules were deleted and all missing hydrogen atoms were added based on the protonation state of the protein. The receptor pdbqt file was then prepared according to AutoDock protocol [34]. The grid center was centered on an erlotinib inhibitor, and the grid box size was set to 60 × 60 × 60 points with a grid spacing of 0.375 Å.
The number of Autodock GA runs was set to 50 and maximum number of energy evaluations was set to 2,500,000. Other AutoDock parameters were set to their default values. 3D structures were constructed and minimized using an MMFF94S force field with the help of SZYBKI software (version 1.9.0.3, OpenEye Scientific Software, Santa Fe, NM, USA) [35]; atomic charges were assigned using a gasteiger method. Prior to the binding energy calculation, all docked complexes were minimized using AMBER14 software (version 14, University of California, San Francisco, CA, USA) [36]. The studied compounds and receptor were described by the general AMBER force field (GAFF) [37] and AMBER force field 14SB [38], respectively. The atomic partial charges of the studied inhibitors were evaluated using the restrained electrostatic potential (RESP) approach at the HF/6-31G* level. For minimization, the truncated Newton linear conjugate gradient method with LBFGS preconditioning was used. The convergence criterion for the energy gradient was 10 −9 kcal/mol·Å. A cutoff value of 999 Å and a generalized Born solvent model were used. On the basis of the minimized complex structures, the binding energies were calculated using the molecular mechanical-generalized Born surface area (MM-GBSA) approach.
The estimated MM-GBSA binding energies were correlated to experimental binding energies, which were calculated based on A549 inhibitory constants using the following equation: ∆G exp = RT ln(IC 50 ), where IC 50 in µM and T = 298.15 K. To assess the compound 8 stability inside the EGFR active site, a short molecular mechanical simulation of an 8-EGFR complex was first performed using the AMBER software. For the MD simulation, the complex was neutralized and solvated with TIP3P water molecules. The complex was then energetically minimized, heated gradually over a period of 50 ps to 300 K, and equilibrated for 500 ps. The data were then collected over a 2.5 ns simulation with a time step of 2 fs and the SHAKE option to constrain all bonds involving hydrogen atoms was used.

Conclusions
Three new (1-3) and five previously reported (4-8) terpenoids were isolated and chemically characterized from the Red Sea soft coral S. ehrenbergi. The eight identified compounds exhibited differential antiproliferative potential against three human cancer cell lines, with lung A549 cell being the most sensitive to compound treatment. The present study establishes S. ehrenbergias as a new source of sardistrol and a possible antiproliferative candidate against lung cancer. Molecular docking studies are consistent with the binding of 8 to the EGFR kinase domain and the inhibition of cell growth. Molecular docking studies supported high inhibitory activity for 8 versus 6 or 7 with the EGFR kinase domain.