Cytotoxic Cembranoids from the Red Sea Soft Coral ...

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Leopold Jirovetz. Vienna ..... Gross H, Wright AD, Beil W, Konig G. (2004) Two new bicyclic cembranoides from a new Sarcophyton species and determination.
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Natural Product Communications

Cytotoxic Cembranoids from the Red Sea Soft Coral Sarcophyton glaucum

2011 Vol. 6 No. 12 1809 - 1812

Mohamed-Elamir F. Hegazya,b, Ahmed A. El-Beihb,c,*, Alaa Y. Moustafad, Abdelhamed A. Hamdyc, Montaser A. Alhammadye, Rehab M. Selimc, Mohamed Abdel-Rehimb,f,g and Paul W. Paré h a

Chemistry of Medicinal Plants Department, National Research Centre, Dokki, Giza 12622, Egypt Pharmaceutical Research Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki 12622, Cairo, Egypt c Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Giza 12622, Egypt d Zoology Department, Faculty of Science, Sohag University, Egypt e National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada, Egypt f Department of Chemistry & Biomedical Sciences, Faculty of Technology and Science, Karlstad University, SE-651 88 Karlstad, Sweden g Department of Clinical Pharmacology & DMPK, Astra Zeneca, R&D, Södertälje, 15185 Södertälje, Sweden h Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA b

[email protected] Received: June 21st, 2011; Accepted: August 3rd, 2011

One new cembrane diterpene, 2R,7R,8R-dihydroxydeepoxysarcophine (1), together with three known compounds, 7α,8β-dihydroxydeepoxysarcophine (2), 7β-acetoxy-8α-hydroxydeepoxysarcophine (3), and sarcophine (4), have been isolated from the Red Sea soft coral Sarcophyton glaucum. Their structures were determined using 1D and 2D NMR spectroscopy. 7β-Acetoxy-8α-hydroxydeepoxysarcophine (3) exhibits cytotoxic activity against HepG2, HCT-116, and HeLa cells with IC50 values of 3.6, 2.3, and 6.7 µg/mL, respectively. Keywords: Sarcophyton glaucum, cembrane diterpenes, dihydroxydeepoxysarcophine, sarcophine.

Marine organisms, comprising more than half of the total global diversity, offer an enormous source of novel and biologically active compounds. The soft corals (phylum Cnidaria, class Anthoza, subclass Octocorallia, order Alycyonacea) have proven to be a biochemical warehouse for terepenoids. Nearly 30 species of soft corals of the genus Sarcophyton, from different geographical areas, have been chemically examined. They contain antiproliferative sesquiterpenes [1] and an impressive series of cembranoid diterpenes [2]. Many cembrane-type compounds have been shown to exhibit cytotoxicity and anti-inflammatory activity [3-5]. Herein, we report the isolation of the new compound 2R,7R,8R-dihydroxydeepoxysarcophine (1), together with three known cembranolides, 7α,8β dihydroxydeepoxysarcophine (2), 7β-acetoxy-8α-hydroxydeepoxysarcophine (3), and sarcophine (4), from the Red Sea soft coral S. glaucum and cytotoxic activity against HepG2, HCT-116, HeLa, and MCF-7 cells. Structures of these isolated metabolites were elucidated through extensive 1D and 2D spectroscopic analyses.

Compound 1 was obtained as colorless crystals, [α]D25 –21.2 (c 1.86, MeOH). The HR-CI-MS exhibited a [M+Na]+ ion at m/z 357.20371 (calc. 357.20363), suggesting a molecular formula C20H29O4Na and six degrees of unsaturation that was supported by NMR data. The IR spectrum indicated the presence of an α,β unsaturated-γ-lactone group (1750 and 1686 cm–1), a ketone group (1707 cm–1), an olefinic group (1669 cm–1), and broad absorption for OH stretching (3000–3353 cm–1) functionalities. A literature survey and careful comparison of the 1H and 13C NMR spectroscopic data of 1 (Table 1) revealed that it was in part close to the known compounds, 7β,8α-dihydroxydeepoxysarcophine (1a) and 7α,8β dihydroxydeepoxysarcophine (2) [6a]. The 13C NMR and

1810 Natural Product Communications Vol. 6 (12) 2011

Table 2: Comparison of 1H and compounds 1, 2, and 1a.

Table 1: NMR spectroscopic data of compound 1. Position

δH (J in Hz)

δC

HMBC

1 2 3 4 5 6

-163.1 C 5.43 (dd, 10.0, 1.5) 80.1 CH C-1, C-3, C-4 4.95 (dd, 10.0, 1.0) 118.7 CH C-5, C-18 -146.2 C 2.24 (m) 36.5 CH2 C-3, C-4, C-6, C-7, C-18 1.60 (m) 29.1 CH2 C-4, C-5, C-7 1.79 (m) C-4, C-5, C-7, C-8 7 3.33 (dd, 10.0, 1.5) 73.8 CH C-5, C-6, C-8, C-19 8 -75.3 C C-7, C-8, C-10, C-11, C-19 9 1.68 (m) 38.4 CH2 C-12 10 2.10 (m) 22.4 CH2 11 5.24 (td, 7.5, 1.0) 126.6 CH C-9, C-10, C-13, C-20 12 -135.3 C 13 2.37 (m) 36.7 CH2 C-1, C-11, C-12, C-14, C-20 14 2.10 (m) 26.1 CH2 C-1, C-2, C-12, C-13, C-15 2.62 (m) C-1, C-2, C-12, C-13, C-15 15 -123.1 C 16 -175.1 C 17 1.88 (brt, 1.5) 9.3 CH3 C-1, C-15, C-16 18 1.90 (d, 1.0) 17.7 CH3 C-3, C-4, C-5 19 1.20 (s) 24.3 CH3 C-7, C-8, C-9 20 1.64 (s) 16.1 CH3 C-11, C-12, C-13 Recorded in CDCl3 and obtained at 500 and 125 MHz for 1H and 13C NMR, respectively.

18

O

O 6

HO

16

4

7 5

3

2 15

8

19

HO

1 13

11

9 10

14

17

12

20

A

Hegazy et al.

1 H-2

5.43, dd (10.0, 1.5) H-7 3.33, dd (10.0, 1.5) H-11 5.24, td (7.5, 1.0) H3-19 1.20 (s)

2

C NMR spectroscopic data of

1a

5.57, dq (13.6, 2.4) 3.47, d (13.5) 4.98 (m) 1.18 (s)

13

5.55, dq (10.1, 1.3) 3.66, dd (9.6, 9.5) 5.10, dd (6.7, 6.6) 1.54 (s)

1

2

1a

C-2

80.1

79.3

79.1

C-7

73.8

72.5

72.3

C-11

126.6

125.2

124.0

C-19

24.3

24.2

26.4

in the 1H NMR spectrum of 1 are the downfield chemical shift of H-11, which resonates at δ 5.24 (td, J = 7.5, 1.0), and the upfield chemical shift of H-7 [δ 3.33 (dd, J = 10.0, 1.5)] rather than the values reported for compounds 2 and 1a [6b] (Table 2). These data indicate differences in the stereochemistry of the three chiral carbons C-2, C-7, and C-8 compared with those of 1a and 2. The relative configuration of 1 was determined on the basis of the study of the coupling constants and NOESY experiments. The H-2 and H-3 vicinal coupling constant (10.0 Hz), as well as a NOESY correlation of H-2 with H318, establish a trans configuration between the γ-lactone (H-2) and the olefinic protons (H-3); the proton signal H-5 (2.24, m) showed clear correlations with the H3-18 methyl protons (1.90, d, J= 1.0); the oxygenated proton at C-7 (δH 3.33, dd, J =10.0, 1.5) showed a clear correlation with the H-6 (1.60, m) and H3-19 (1.20, s) protons. From this information, we noted that H-2, H-7, and H3-19 all faced in the same direction (Figure 1b).

B

Figure 1: (A) 1H-1H COSY (bold lines) and key HMBC (arrows) correlations of 1; (B) Selected NOESY correlations in compound 1.

15 Compound 1 Compound 2 10

DEPT spectra (Table 1) revealed the presence of four olefinic functionalities at C-3 (δ 118.7), C-11 (δ 126.6), C-12 (δ 135.3), and C-4 (δ 146.2), two oxymethine carbons at C-7 (δ 73.8) and C-2 (δ 80.1), and one oxygenated quaternary carbon at C-8 (δ 75.3). The carbon signal resonances at C-15 (δ 123.1), C-1 (δ 163.1), and C-16 (δ 175.1) were assigned to the α,β -unsaturated-γlactone functionality that was confirmed by comparison of NMR data for 1 with 4. From the 1H NMR and 1H-1H COSY spectra, the following proton correlations were assigned H-2/H-3, H2-5/H2-6/H-7, H2-9/H2-10/H-11, and H2-13/H2-14. The HMBC correlations H-2/C-1, H-2/C-4, H-3/C-5, H2-9/C-7, H2-10/ C-12, H-11/C-13, H2-13/C-1, and H2-14/C-2 confirmed the connectivities of the 14-membered ring from C1–C14. The position of the methyl groups H3-18 (δ 1.90), H3-19 (δ 1.20), and H3-20 (δ 1.64) was deduced from the HMBC correlations H-3/C-18, H3-18/C-4, H3-19/C-8, H3-19/C-7, H3-19/C-9, and H3-20/C-12. The last methyl group, H3-17 (δ 1.88), was connected to the α,β-unsaturated-γ-lactone ring from the HMBC correlations with the olefinic carbons C-1 (δ 163.1) and C-15 (δ 123.1) and the carbonyl carbon C-16 (δ 175.1). The most significant differences observed

Abs 5

0 210

230

250

270

290

310

330

350

-5

Wave lenght (nm)

Figure 2: Circular dichroism spectra of compounds1 and 2 in MeOH.

The absolute stereochemistry of 1 at C-2 was determined via circular dichroism (CD) analysis (Figure 2). The positive and negative Cotton effect ([θ]248 +1.12; [θ]225 –2.19) observed in the CD spectrum for the electronic transitions of the 2(5H)-furanone moiety indicate a lefthanded (M) helix for the five-membered α,β -unsaturatedγ-lactone ring [6c]. Consistent with this interpretation, the similar optical rotation for 1 {[α]D25 –21.2 (c 1.86, MeOH)} and ent-sarcophine {[α]D25 = –16.0 (c 1.19, MeOH)} [7], confirm the same R absolute configuration at C-2, C-7, and C-8 [6-8].These data establish the isolation and identification of 7β ,8β-dihydroxydeepoxysarcophine, a newly identified natural product. Compounds 2–4 were previously reported from the soft coral S. glaucum [6b,9] with no report of cytotoxicity. The

Cembrane diterpenes from Sarcophyton glaucum

Natural Product Communications Vol. 6 (12) 2011 1811

Table 3: Cytotoxic effect of different samples against different human cancer cell lines, using MTT assay (n=4); data expressed as the mean value of IC50 (g/mL) ± S.E. Sample results are compared with paclitaxel. Compound 1 2 3 Paclitaxel

Hep-G2 38.8 ± 1.0 23.5 ± 2.4 3.6 ± 1.0 0.49 ± 0.1

Cell line HCT-116 26.3 ± 2.7 15.6 ± 1.9 2.3 ± 1.5 0.4 ± 0.1

Experimental

HeLa > 50 44.1 ± 5.4 6.7 ± 0.8 0.6 ± 0.08

cyototoxic activity of compounds 1–3 was tested against HepG2, HCT-116, HeLa, and MCF-7 cells. 7-Acetoxy8-hydroxydeepoxy-sarcophine (3) showed strong cytotoxic activity against HepG2, HCT-116, and HeLa cells with IC50 values of 3.6, 2.3, and 6.7 g/mL, respectively, while no activity against MCF-7 cells was observed for compounds 1 and 2. Using MTT assay, the effect of isolated compounds on the growth of various cell lines was studied after 48 h of incubation. As shown in Table 3, compound 3 showed a potential cytotoxic activity (low IC50 values 10 g/mL). Paclitaxel, a known anti-cancer drug, was used as a positive control.  

*

HeLa

Treated

HCT-116

Control

*

Treated

HepG2

Control

*

Treated

Control 0

20

40

60

80

DNA fragm entation (%)

(A)

significantly different from the control (p< 0.01–0.001), in the following order: HCT-116 >HeLa> HepG2.

(B)

Figure 3: (A) Type of cell death was investigated in HepG2, HCT-116, and HeLa cells after treatment with compound 3, using acridine orange/ethidium bromide staining to compare between the percentage of necrotic cells (grey segment) and apoptotic cells (black segment). (B) Effect of compound 3 (grey bars) on the percentage of DNA fragmentation in different cancer cell types compared with untreated cells (black bars). The fragmented DNA was determined with the diphenylamine reaction. Values are means of three measurements (mean ± SD), where (*) represent the p