New cytotoxic isoprenoid derivatives from the Red Sea soft coral Sarcophyton glaucum

Chemical investigation of the soft coral Sarcophyton glaucum collected from the Red Sea led to isolation of 11 isoprenoidal metabolites (1–11). A new sesquiterpenoid, 6-oxo-germacra-4(15),8,11-triene (1), a new natural cembranoid, sarcophinediol, along with two known sesquiterpenoids (2 and 3) and seven known cembranoids (5–11) was obtained. The structures of the compounds were established based on their NMR, MS, IR and UV spectral data. All compounds were evaluated for their cytotoxicity employing three cancer cell lines (HepG2, MCF-7 and HCT116). Compounds 4 and 6 showed significant cytotoxicity towards HepG2 with IC50 values of 18.8 ± 0.07 and 19.9 ± 0.02 μM; respectively. Compounds 5–7 exhibited potent cytotoxicity against MCF-7 cells with IC50 values of 9.9 ± 0.03, 2.4 ± 0.04 and 3.2 ± 0.02 μM, respectively. Compounds 1, 4 and 5 showed significant activities towards HCT116 cells with IC50 values of 29.4 ± 0.03, 19.4 ± 0.02 and 25.8 ± 0.03 μM, respectively.


Introduction
The genus Sarcophyton is known for its richness in cembrane-type diterpenoids, biscembranoids, sesquiterpenoids and steroids. Up to date, more than 300 natural cembranoid derivatives were isolated (Wahlberg & Eklund 1992;Cheng et al. 2008;Cheng, Chuang, et al. 2010. Cembranoid diterpenes are a common class of natural metabolites in marine milieu (Hegazy et al. 2012). The basic chemical structural patterns typically featured a common 14-membered carbocyclic nucleus, festooned with carbon -carbon double bond(s) and methyl groups; this framework is easily exposed to oxidation, photochemical, rearrangement and enzymatic processes leading to the formation of cyclic ether, lactone, pyran or furan moieties around the cembrane skeleton (Fahmy et al. 2004(Fahmy et al. , 2006Szymanski et al. 2012). Unconventional cembranoids containing 1,2-or 1,3-or 1,4-membered variants were also identified. The cembranoid derivatives play an important role in the biomedical perspective (Fahmy et al. 2006;Huang et al. 2006;Cheng, Chuang, et al. 2010;Szymanski et al. 2013).
Compound 1 was isolated as colourless oil with ½a 22 showed resonances for 15 carbons, differentiated by DEPT NMR experiment into two methyl, six methylene, four methine and three quaternary carbons. Four of the five elements of unsaturation, as indicated by the molecular formula of 1, are attributed to three CvC double bonds and a carbonyl carbon; thus, the molecule is monocyclic. 1 H NMR data of 1 showed one methyl singlet, one methyl doublet and four exomethylene proton signals. All the spectral data are in agreement with the germacrane-type sesquiterpene skeleton with carbonyl group and disubstituted carbon -carbon double bond. Regarding the germacrane-carbon skeleton, the isopropyl group linked to C-7 should have been converted into isopropenyl group owing to the absence of correlation of the two doublet methyl signals in 1 H NMR and COSY spectra, and the appearance instead of one singlet methyl signal. The HMBC correlations observed between the singlet methyl protons signal resonating at d H 1.60 ppm and the exomethylene protons signals resonating at d H 4.74 and 4.72 ppm on one side and the methine carbon signal appearing at d C 52.7 on the other side establish unambiguously the position of the isopropenyl moiety. Extensive interpretation of the COSY spectrum indicated the presence of the following correlations sequence: the doublet methyl protons resonating at d H 0.84, the aliphatic methine proton signal at d H 1.78, the olefinic proton at d H 4.95, the olefinic proton signal at d H 4.95 and the methine proton at d H 2.46, this sequence in turn establishes the position of the double bond to be in position six or eight depending on the germacrane numbering system. The HMBC correlations between H-7 signal with the carbonyl -carbon resonating at d C 204.3, the isolated methylene carbon at d C 37.3 and the olefinic carbon signal at d C 132.6 indicated the position of the keto group at C-6 and the exocyclic methylene protons at d H 5.23 and 5.08 ppm.
The relative stereochemistry of the asymmetric centres C-7 and C-10 was established on the basis of the NOESY experiments. The absence of NOE between Me-14 and H-7 suggested the cofacial orientation of H-7 and H-10. The geometry of the double bond between C-8 and C-9 was found to be cis owing to the low value of coupling constant (J). Accordingly, the relative configuration of 1 was deduced as (8Z,7R*,10S*). From the previous discussion, compound 1 can be identified as 6-oxo-germacra-4(15),8,11-triene.
Compound 4 was isolated as yellowish oil (  and H-20 are positioned on C-15, C-4, C-8 and C-12, respectively. An extensive computer survey on soft corals indicated that Sarcophyton is abundantly produced cembranoids. Careful investigation of these literature data and comparing it with the obtained data indicated that 4 has C-14 cembrane skeleton (Fahmy et al. 2006;Huang et al. 2006;Cheng, Chuang, et al. 2010Lu et al. 2010;Szymanski et al. 2013). Compound 4 was obtained as a mixture of two epimers (4 and 4a), which proved to be inseparable. 13 C and 1 H NMR spectra of the mixture showed duplicated signals and the difference between them ranged from^0.01 to 0.05 ppm. The optical rotation value ½a 22 D ¼ þ1.0 (c ¼ 0.01, CHCl 3 ) confirms that they are diasteroisomers. Extensive study of the NMR spectral data indicated that the orientation of two positions can be assigned; the oxiran ring and the hydroxyl group at C-15. The former is proved by 13 C, 1 H and 2D NOESY NMR assignments. Thus, 4 has 15S* and 15R*. Extensive literature survey revealed that compound 4 is a new natural product which was previously published as semi-synthetic (Sarcophinediol), which was published without NMR data (Fahmy et al. 2004;Szymanski et al. 2012).
The antiproliferative activity of compounds 1-11 was evaluated against three cancer cell lines (HepG2, MCF-7 and HCT116) with reference to the standard anticancer drug (doxorubicin), using Sulforhodamine B assay. The data in Table S1 highlight considerable antiproliferative activity. The tested compounds showed observable activity towards HepG2 cells in the range of 18.8 -734.3 mM. Compounds 4 and 6 showed significant cytotoxicity towards HepG2 with IC 50 values of 18.8^0.07 and 19.9^0.02 mM, respectively. The compounds showed activity against MCF-7 cells in the range 1.88-57.0 mM. It is noteworthy to report that compounds 5, 6 and 7 exhibited potent activity against MCF-7 cells with IC 50 values of 9.9^0.03, 2.4^0.04 and 3.2^0.02 mM, respectively. Furthermore, the compounds showed cytotoxic activity towards HCT116 cells in a concentration range of 21.8 -86.7 mM, whilst compounds 1, 4 and 5 showed significant activity towards HCT116 with IC 50 values of 29.4^0.03, 19.4^0.02 and 25.8^0.03 mM, respectively.

Materials and methods 3.1. General
Silica gel GF 254 (Merck, Darmstadt, Germany) was used for analytical thin layer chromatography (TLC). Preparative thin layer chromatography was performed on aluminium oxide plates (20 cm £ 20 cm) of 250 mm thickness. 1D and 2D NMR spectra were recorded in CDCl 3 on Bruker (Karlsruhe, Germany) AVANCE III WM 600 MHz spectrometers and 13 C NMR spectra at 150 MHz. Tetramethylsilane was used as internal standard. Plates were sprayed with 50% sulphuric acid in methanol and heated at 1008C for 1 -2 min.

Animal material
Soft coral S. glaucum (Order Alcyonacea, Family Alcyoniidae) was collected from the north of Jeddah Saudi Arabia Red Sea coast (21829 0 31 00 N 39811 0 24 00 E) in Jeddah, at a depth of 5 -10 m, in January 2014. This collection was done by SCUBA divers. After collection, this material was immediately subjected to extraction. A voucher specimen (SC-2014-10) has been deposited in the faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. S. glaucum is called elephant ear coral or green toadstool coral. It has a thick smooth, single stalk with a flared smooth mushroom-shaped top that can be folded or shaped into a funnel. The flesh is firm and soft, yet can be easily torn. The polyps can retract all the way, giving them a smooth look.

Extraction and isolation
The fresh soft coral S. glaucum (5.0 kg) was minced and exhaustively extracted with a mixture of CH 2 Cl 2 :MeOH (2:1 v/v, 24 h for each batch, 228C, 10 L £ 3), and then the combined extracts were concentrated under vacuum to yield viscous blackish residue. The residue was partitioned between diethyl ether and water; the organic layer was dried (30 g) and then fractionated on normal phase silica gel (NP-Silica), eluted stepwise with n-hexane containing increasing amounts of diethyl ether and then increasing polarity with EtOAc. A total of 100 fractions (F 1-100) were collected. The fractions were investigated by TLC pattern using UV lamp and/or 50%-sulfuric acid in methanol as spraying reagent. Fraction F 3 eluted with n-hexane:diethtyl ether (9.5:0.5, 300.00 mg) was purified by preparative TLC using the solvent system n-hexane: diethyl ether (9.5:0.5). The band with R f ¼ 0.96 (violet-red colour with sulphuric acidmethanol) was taken to give compound 2 as colourless oil (22.00 mg); the band with R f ¼ 0.88 (pink colour with sulphuric acid-methanol) was taken to give compound 1 as colourless oil (1.50 mg) and the band with R f ¼ 0.80 (UV active and exhibited dark violet colour with sulphuric acid-methanol) was taken to give compound 11 as colourless oil (9.00 mg). Fraction F 13 eluted with n-hexane:diethyl ether (8:2, 125.00 mg) was purified by preparative TLC using the solvent system n-hexane:diethyl ether (8:2) to give two bands. The first band with R f ¼ 0.71 (violet colour with sulphuric acid -methanol) was taken to give colourless oil (16 mg), compound 6; the second band with R f ¼ 0.50 (violet colour with sulphuric acid-methanol) was taken to give colourless oil (4.50 mg), compound 3. Fraction F 41 eluted with n-hexane:EtOAc (9:1, 123.00 mg) was purified by preparative TLC using the solvent system n-hexan:ethylacetate (8:2). The band with R f ¼ 0.25 (pink colour with sulphuric acid -methanol) was taken to give colourless oil (30.60 mg), compound 7; the band with R f ¼ 0.50 (reddish colour with sulphuric acid -methanol) was taken to give colourless oil (12.00 mg), compound 5 and the band with R f ¼ 0.69 (violet colour with sulphuric acid -methanol) was taken to give colourless oil (11.50 mg), compound 9. Fraction F 50 eluted with n-hexane:EtOAC (8:2, 70.00 mg) was purified by RP-18 HPLC (MeOH -H 2 O, 65:35) which yielded 8 (3.00 mg) and 11 (2.00 mg). Fraction F 52 eluted with n-hexane:EtOAc (8:2, 70.00 mg) was purified by RP-18 HPLC (MeOH -H 2 O, 65:35) which yielded 4 (8.00 mg).