New Antiproliferative Cembrane Diterpenes from the Red Sea Sarcophyton Species

The combination of liquid chromatography coupled to high resolution mass spectrometry (LC-HRESMS)-based dereplication and antiproliferative activity-guided fractionation was applied on the Red Sea-derived soft coral Sarcophyton sp. This approach facilitated the isolation of five new cembrane-type diterpenoids (1–5), along with two known analogs (6 and 7), as well as the identification of 19 further, known compounds. The chemical structures of the new compounds were elucidated while using comprehensive spectroscopic analyses, including one-dimensional (1D) and two-dimensional (2D) NMR and HRMS. All of the isolated cembranoids (1–7) showed moderate in vitro antiproliferative activity against a human breast cancer cell line (MCF-7), with IC50 ranging from 22.39–27.12 µg/mL. This class of compounds could thus serve as scaffold for the future design of anticancer leads.


Introduction
Marine natural products are characterized by their diversity in chemical structures, along with biological activities [1]. The Red Sea is considered to be one of the most important marine spots comprising high biodiversity. About 40% of the soft corals that are identified worldwide are native to the Red Sea, however only a few species that have been chemically examined in the last decades [1][2][3]. The genus Sarcophyton (Phylum Cnidaria; Order Alcyonacea; Family Alcyoniidae) contains 46 species with typically mushroom-or toadstool-shaped appearance and it is considered one of the most most abundant Red Sea soft corals [1]. Sarcophyton sp. are well recognized as a rich source for a wide range of terpenoid metabolites. Macrocyclic cembrane-type diterpenoids and their derivatives represent the main chemical defense for Sarcophyton species against natural predators [3][4][5]. Previous studies concerning the biological activities of cembranoid analogues revealed that they exhibited a wide range of biological activities, including ichthyotoxic [6], anti-inflammatory [7], anti-bacterial [8], neuroprotective [9], and antitumor [10] properties. In addition, a number of Sarcophyton-derived cembranoid analogues (e.g., sarcophine and its hydroxylated derivatives) were investigated as potential competitive cholinesterase inhibitors [11], selective noncompetitive phosphofructokinase inhibitors [12], and a Na + , K + -ATPase inhibitors [13].
Cancer chemoprevention is based on chemical compounds that inhibit or reverse the development of cancer in normal or pre-neoplastic tissue [14]. A lot of novel marine metabolites were identified as anticancer leads during the past 20 years [15]. Our previous work on sarcophine (7) and its analogs has illustrated the ability of this class of compounds to inhibit growth, proliferation, and migration of the prostate and breast metastatic cancer cell lines PC-3 and MDA-MB-231 [6].
Looking for new natural products with cytotoxic activity that is applicable in cancer therapy is of utmost importance. The discovery process has improved with the evolution of new spectroscopic techniques. Liquid chromatography coupled to high resolution mass spectrometry (LC-HRESMS) can generate information-rich data sets that can assist in the dereplication of previously reported natural products that are present in the crude extracts prior to a tedious isolation attempt. New biological material was collected and extracted in order to extend our previous investigation of the Red Sea Sarcophyton species [6]. Subsequently, bioactivity-guided isolation assisted by LC-HRESMS metabolic profiling led to the isolation of five new cembrane-type diterpenoids (1-5, Figure 1) that are structurally related to sarcophine (7) which was also isolated along with another known cembranoid, sinumaximol G (6). Testing of all isolated compounds against the MCF-7 breast cancer cell line showed a moderate in vitro growth inhibitory activity.

Results and Discussion.
Extraction and fractionation on normal phase (NP) silica gel while using medium pressure liquid chromatography (MPLC) afforded seven major fractions. LC-HRESMS dereplication of these fractions using commercial and public databases led to the putative identification of 19 compounds,

Results and Discussion
Extraction and fractionation on normal phase (NP) silica gel while using medium pressure liquid chromatography (MPLC) afforded seven major fractions. LC-HRESMS dereplication of these fractions using commercial and public databases led to the putative identification of 19 compounds, which were previously reported from Sarcophyton sp., in addition to 17 molecules with mass data showing no hits in MS databases, most of them in fractions 6 and 7 (Table S1). These LC-HRESMS findings, together with the in vitro cytotoxicity results against MCF-7 cell line using the acquired MPLC fractions, led to prioritization of fractions 6 and 7 for further chromatographic purification on silica gel and Sephadex LH-20 to isolate the active components. Preliminary 1 H NMR spectroscopic analysis revealed that all the major compounds in fractions 6 and 7 had a cembranoid backbone [16,17], differing either in the degree of oxidation or the configuration of one or more chiral centers.

Identification of the Isolated Compounds
Compound 1 was obtained as colorless oil. Its molecular formula was established as C 22 H 32 O 6 that is based on HRESIMS data ( Figure S1) that showed an [M + H] + ion at m/z 393.2264 (calcd m/z 393.2272). The 1 H NMR spectrum (Tables 1 and 2), together with the multiplicity-edited HSQC (Figures S2-S5), were in harmony with a sarchophine (7) skeleton that was previously isolated from other Sarcophyton species [6,16,18]. 1 H NMR and 13 C NMR (Table 2), together with multiplicity-edited HMQC, showed the following characteristic resonances: (i) the presence of an α,β-unsaturated-γ-lactone functionality: ; and finally, H 2 -14 and δ C 161.4 (C-1)/79.2 (C-2) established the connectivity of the 14-membered ring. The positions of the methyl groups were recognized through HMBC correlations between δ H 1.85 (H 3 -18, s) and C-3 and C-4; δ H 1.20 (H 3 -19, s) and C-9; δ H 1.36 (H 3 -20, s) and C-11 and C-12; and, δ H 1.86 (H 3 -17, s) and C-1, C-15, and C-16. These NMR spectroscopic features were very similar to the reported spectroscopic data of the known compound sinumaximol G, which was previously isolated from the Soft Coral Sinularia maxima [16] and also isolated in the present work (Compound 6). The 1 H NMR and 13 C NMR spectroscopic data of compound 1 showed differences in the chemical shifts of C-7/δ C 74.4 and H-7/δ H 4.82, which indicated changes of the substitution at C-7. In addition, the presence of additional resonances in the NMR spectral data for acetate moiety [δ C 170.3 (C-1'), δ C/H 21.1/2.08 (C-2')], along with the HMBC correlations of H 3 -2'/C-1', H-7/C-1', and H-7/C-19, suggested that the OH group in sinumaximol G at C-7 has been acetylated in compound  The molecular formula of compound 2 was determined to be the same as that of sinumaximol G (6) (C 20 H 30 O 5 ); m/z 351.2160 [M + H] + ion (calcd m/z 351.2166) ( Figure S10). The one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic features (Tables 1 and 2; Figures S11-S15) also indicated that 2 had an almost identical structure as 1 and sinumaximol G (6) [16]. The difference between those two compounds was the deshielding of the H 3 -19 methyl protons (δ H 1.34), which indicated that the configuration of the methyl group at C-8 is altered to the β-configuration.  The molecular formula of compound 2 was determined to be the same as that of sinumaximol G (6) (C20H30O5); m/z 351.2160 [M + H] + ion (calcd m/z 351.2166) ( Figure S10). The one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic features (Tables 1 and 2; Figures S11-S15) also indicated that 2 had an almost identical structure as 1 and sinumaximol G (6) [16]. The difference between those two compounds was the deshielding of the H3-19 methyl protons (δH 1.34), which indicated that the configuration of the methyl group at C-8 is altered to the β-configuration. The 1 H-1 H NOESY correlation ( Figure 3) between H3-19 (δH 1.34) and H3-18 (δH 1.83) further confirmed this change, and hence compound 2 was identified as a new natural product, which we named 8-episinumaximol G (2).
Compounds 6 and 7 were previously isolated from the Soft Corals Sinularia maxima and Sorcophytum glaucum, and identified as sinumaximol G (6) [16] and sarcophine (7) [18], respectively. Their structures were confirmed based on a comparison of their HRMS and NMR data with literate.

Antiproliferative Activity
Our previous in vitro screening results showed that, among several cancer cell lines, sarchophine (7), along with other analogs, were only active against breast (MDA-MB-231) and prostate (PC-3) cancer cell lines [6]. We consequently chose a breast carcinoma cell line (MCF-7) to assess the in vitro anticancer effect of the isolated compounds (1-7). All of the tested compounds induced dose-dependent cell death with IC 50 values (Table 3) of (22.39-27.12 µg/mL). Although all of the tested compounds have significant cytotoxic potentials against MCF-7, they have slightly reduced (approx. two-fold) anticancer effects in comparison to the standard anticancer drug doxorubicin (IC 50 : 12.78 µg/mL). In the light of these results, along with the previous reports [6,16,[19][20][21] on other cembrane-type diterpenoids, we can conclude that the presence of an α, β-unsaturated-γ-lactone moiety is an essential feature for the antiproliferative properties of these class of compounds. Additionally, changing in the orientation or acetylation of hydroxyl groups at C-7, C-8, and C-12 almost have no effect on their cytotoxicity.

General Experimental Procedures
Silica gel 60 (Natland, 63-200 µm) and solvent systems consisting of n-hexane-EtOAc (9.5:0.5 to 70:30) were used for column chromatography. Pre-coated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F 254 , 0.25 mm) were used for TLC analyses. 1% vanillin in concentrated H 2 SO 4 was used as the visualizing reagent. LC/MS was conducted on a Thermo MS system (LTQ XL/LTQ Orbitrap Discovery) coupled to a Thermo Instruments HPLC system with Accela PDA detector: The data were processed using Xcalibur 2.0.7. 1 H and 13 C NMR spectra were recorded in CDCl 3 on a JEOL ECA-600 spectrometer (600 MHz for 1 H and 150 MHz for 13 C, respectively). All of the chemical shifts (δ) are given in ppm units with reference to TMS as an internal standard, and coupling constants (J) are reported in Hz.

Extraction and Fractionation
The soft coral Sarcophyton sp. was collected from the Egyptian Red Sea off the coast of Hurghada (GPS coordinates N 27 • 15048", E 33 • 4903") at depths of 5-7 m in March 2016 and then frozen for storage. A voucher specimen (NIOF404/2016) was reserved at the National Institute of Oceanography and Fisheries, Red Sea Branch, Invertebrates Department. The frozen soft coral (700 g) was extracted four times with isopropanol. The extract was concentrated under vacuum to give 70 g raw material and then chromatographed on silica gel while using n-hexane/EtOAc to afford seven fractions. Only fractions 6 and 7 exhibited in vitro cytotoxicity against MCF-7 cell lines. In addition, LC-HRESMS dereplication results were used to prioritize those fractions, as they showed several unknown molecular formulas. Further chromatographic separation of fractions 6 and 7 on silica gel while using a gradient of n-hexane/EtOAc yielded compounds 1-7, which were purified on Sephadex LH-20 using 10% aqueous MeCN.

In Vitro Antiproliferative Activity
The human breast cancer cell line (MCF-7) was obtained from the American Type Culture Collection (ATCC, Manassas, USA). They were seeded in 96 well microtiter plates at a concentration of 1000-2000 cells/well, 100 µL/well. After 24 h, the cells were incubated for 72 h with the compounds to be tested. Dulbecco's Modified Eagle Medium (DMEM) with 10% foetal calf serum, sodium pyruvate, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 • C and 5% CO 2 was used as culture medium. The medium was discarded at the end of the incubation. The cells were fixed with 150 µL cold trichloroacetic acid with 10% final concentration for 1 h at 4 • C. The plates were washed with distilled water (automatic washer Tecan, Germany) and then stained with 50 µL 0.4% Sulforhodamine B dissolved in 1% acetic acid for 30 min at room temperature in the dark. The plates were washed with 1% acetic acid to remove unbound dye and air-dried (24 h). The dye was solubilized with 150 µL/well of 10 mM tris base (pH 7.4) for 5 min. on a shaker at 1600 rpm. The optical density (OD) of each well was spectrophotometrically measured at 490 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted and the mean values of each tested compound and doxorubicin concentration was calculated. The experiment was repeated three times for each tested compound. The percentage of cell survival was calculated by using the following formula: surviving percent = [O.D. (treated cells)/O.D. (control cells)] x100. The IC 50 values (the concentrations of compound required to produce 50% inhibition of cell growth) were also calculated.

Conclusions
The present study provides an additional chemical characterization of the Red Sea soft coral Sarcophyton. LC-HRESMS-based dereplication, in combination with biological activity-guided fractionation, allowed for the accelerated characterization of further new bioactive cembrane-type diterpenoids. All of the isolated compounds demonstrated moderate in vitro antiproliferative activity against breast cancer cell line MCF-7. This finding adds to our existing knowledge and understanding regarding the cytotoxic activity of cembranoid diterpenes, indicating that this class of compounds can be utilized as a potential scaffold for the future design of potent anticancer agents.