NPC
Natural Product Communications
Antiproliferative Sesquiterpenes from the Red Sea Soft Coral Sarcophyton glaucum
2007 Vol. 2 No. 0 1-3
Swapnali S. Sawanta, Diaa T.A. Youssef b, Paul W. Sylvestera, Vikram Wali and Khalid A. El Sayed a,* a
Department of Basic Pharmaceutical Sciences, College of Pharmacy, 700 University Avenue, University of Louisiana at Monroe, Monroe, Louisiana 71209 b
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
[email protected] Received: July 7th, 2006; Accepted: July 29th, 2006
The Red Sea soft coral Sarcophyton glaucum was investigated for its sesquiterpene content. A novel 1,2-dioxolane sesquiterpene alcohol, dioxosarcoguaiacol (1) along with the known (+)-alloaromadendrene are reported. The structure of the new sesquiterpene was determined on the basis of extensive spectral analyses. (+)-Alloaromadendrene showed potent inhibition of the proliferation of the highly malignant + SA mammary epithelial cells at a dose of 20µM. Keywords: Sarcophyton glaucum, soft coral, sesquiterpenes, antiproliferative.
Soft corals are marine invertebrates that lack physical means of defense and yet, are one of the most abundant fauna encountered in this extremely competitive environment [1]. This is mainly due to the chemical defense mechanisms mediated through the production of bioactive secondary metabolites [2]. Sarcophyton is one of the common genera among the soft corals that can produce a single diterpene in up to 10% yield of their dry weight for defense, reproduction, and possibly pheromonal roles. Among the secondary metabolites of Sarcophyton, the cembranoid diterpenes and polyhydroxysteroids have been thoroughly investigated [3]. However, relatively few sesquiterpenes and insufficient biological activity data have been reported for the genus Sarcophyton sp. [4]. Hence, this study was aimed at exploring the chemistry and anticancer potential of sesquiterpenes isolated from the Red Sea soft coral S. glaucum. Reinvestigation of the chemical content of S. glaucum led to the isolation of a novel 1,2-dioxolane3-ol sesquiterpene, dioxosarcoguaiacol (1) and the known (+)-alloaromadendrene (2). The HRMS [M+Na]+ spectrum of 1 suggested a molecular formula of C15H22O3, with five degrees of unsaturation. The 1H and 13C NMR data (Table 1)
suggested the presence of a three ring system with two olefinic moieties. The 2D NMR data also suggested a guaiane backbone for the molecule, similar to previously reported sesquiterpenes from this soft coral [4]. The presence of one olefinic methine and three olefinic quaternary carbons indicated the presence of one trisubstituted and one tetrasubstituted double bond systems. The assignment of quaternary olefinic carbons resonating at δ 145.0 (C-1) and δ 132.5 (C-5) was based on their 3J-HMBC correlations with H3-15 (δ 1.14) and H3-14 (δ 1.06) methyl doublets, respectively. The olefinic proton singlet H-6 showed 3J-HMBC correlation with the quaternary olefinic carbons C-1 and 2J-HMBC correlations with the quaternary olefinic carbons C-5, and C-7. This confirmed the conjugated system Δ1,5 and Δ6,7. The two methyl singlets H3-12 (δ 1.39) and H3-13 (δ 1.45) showed 3J-HMBC correlation with the quaternary carbon C-7 (δ 152.8). Both H3-12 and H313 also showed 2J-HMBC correlations with the oxygenated quaternary carbon at δ 85.4 (C-11). The proton H-6 also showed a 3J-HMBC correlation with the dioxygenated quaternary carbon C-8 (δ 103.6), confirming the connection of a 1,2-dioxolane system with the guaiane skeleton. The guaiane moiety accounted for two out of three rings, and the third
2 Natural Product Communications Vol. 2 (0) 2007
15 HO 10 1 O 8 O 5 11 7 6
H 3 H 14
1
2
Figure 1: Structures of dioxosarcoguaiacol (1) and (+)-alloaromadendrene (2).
Table 1: 13C and 1H NMR data of dioxosarcoguaiacol (1). Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Compound 1 δC 145.0, C 35.0, CH2 29.8, CH2 44.5, CH 132.5, C 115.1, CH 152.8, C 103.6, C 37.9, CH2 31.7, CH2 85.4, C 27.0, CH3 24.8, CH3 20.2, CH3 19.9, CH3
δΗ 2.62, m, 2.25, m 1.94, m, 1.40, m 2.64, m 5.58, s 2.10, dd (13.5, 5.2) 1.64, t (13.2) 2.74, m 1.39, s 1.45, s 1.06, d (6.6) 1.14, d (6.6)
Compound 2 was found to be the known (+)-alloaromadenrene, an enantiomer of the terrestrial (-)-alloaromadendrene. (+)-Alloaromadenrene has been previously reported from S. glaucum, but without any report of its biological activity [7]. The identity and configuration was confirmed by comparing the 1H and 13C NMR and the optical rotation data with previous reports [8-9]. Cells per Well (1x10-5)
ring was then assigned as a 1,2-dioxolane ring between C-7 and C-8. Recently, plant-derived guaiane dimers with similar 1,2-dioxolane-3-ol moieties were reported from Xylopia species [5]. The relative stereochemistry of 4 was based on NOESY data and by comparing the 1H and 13C proton chemical shifts and coupling constants (Js) with those of Xylopia guaiane dimers. This comparison led to the assignment of H-9a (δ 2.10, dd, J = 13.5, 5.2 Hz) as α-oriented [5]. H-9a also showed a strong NOESY correlation with H3-15, indicating the α orientation of this methyl group. Furthermore, the α-oriented H-9a showed NOESY correlation with H-2a (δ 2.25), which in turn correlated with H-4, suggesting its αorientation. Thus, the C-14 methyl can then be assigned as β-oriented. The available spectral data were not enough to assign the stereochemistry of the quaternary chiral center C-8 (Figure 1). The 1,2dioxolane-3-ol sesquiterpene is unprecedented in marine-derived secondary metabolites. Cembranoids, the major terpenes of Sarcophyton species are known to be unstable and yield peroxides and smaller molecular weight terpenes on auto-oxidation [6]. Dioxosarcoguaiacol (1) was detected by TLC in the fresh animal crude extract, confirming its natural identity.
Author A et al.
(+)-Alloaromadendrene 3.5 3.0 2.5 2.0 1.5
*
1.0
*
0.5
N.D. 0
10
20
25
30
Treatment Dose (μM) Figure 2: Effect of various doses of (+)-alloaromadendrene on the growth of neoplastic +SA mouse mammary epithelial cells in vitro. Cells were initially plated at a density of 5x104/well in 24-well culture plates and maintained for 4 days in control or treatment serum-free defined media. Vertical bars represent the mean viable cell number/well ± SEM for 6 replicates in each group. *p < 0.05, as compared with untreated controls.
The effects of the sesquiterpenes, dioxosarcoguaiacol (1) and (+)-alloaromadendrene (2) on highly malignant +SA mammary epithelial cell growth after a 4-day culture period were investigated. Treatment with 0-100 µM of dioxosarcoguaiacol had no significant inhibitory effect on +SA cell growth, as compared to untreated control (data not shown). In contrast, treatment with 20-30 µM of (+)alloaromadendrene significantly inhibited +SA cell growth, as compared to untreated control (Figure 1). In addition, treatment with 30 µM (+)alloaromadendrene was found to be cytotoxic and reduced viable cell numbers to undetectable levels (Figure 1). Interestingly, a previous study indicated that the plant enantiomer of alloaromadendrene was completely inactive when examined for its effects on more than ten human and murine cancer cell lines [10]. Experimental Isolation: The soft coral Sarcophyton glaucum was collected from the Red Sea, Hurghada, Egypt in June 2003. A voucher specimen (03RS24) is deposited in the Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at
Antiproliferative Sesquiterpenes from Sarcophyton glaucum
Monroe. The frozen soft coral (1.9 kg) was extracted four times with isopropanol in a percolator at room temperature. The extract (260 g) was then concentrated under vacuum and chromatographed on silica gel using n-hexane: EtOAc gradient elution to yield the less polar (+)-alloaromadendrene (2, 100 mg), the new dioxosarcoguaiacol (1, colorless oil, 50 mg), and the known cembranoids, namely sarcophine, 16-deoxysarcophine, 2-epi-16deoxysarcophine, sarcophytonin A, and (+)sarcophytoxin [11-12]. Antiproliferative assay: The highly malignant +SA mouse mammary epithelial cell line was used to investigate the antiproliferative effects of the sesquiterpenes, dioxosarcoguaiacol and (+)alloaromadendrene. +SA cells were maintained in serum-free defined media containing 10ng/mL epidermal growth factor (EGF), as described previously [13]. Cells were initially plated at a density of 5x104/well (6 wells/group) in 24-well culture plates and maintained on either control or treatment media throughout the 4-day culture period.
Natural Product Communications Vol. 2 (0) 2007 3
On day 4, viable cell number was determined by 3(4,5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay [13]. Dioxosarcoguaiacol (1) [α]D: +35.4º (c 1.00, CHCl3). Rf : 0.3 (toluene-EtOAc 4:1). IR (neat): 3628-3100, 2944, 2836, 1631, 1461, 1234, 1021,756 cm-1. UV/Vis λmax (MeOH) nm (log ε): 280 (3.4), 267 (3.6), 259 (3.6) 1 H NMR: Table 1. 13 C NMR: Table 1. HRESITOF: m/z [M + Na+] calcd for C15H22O3Na: 273.1463; found: 273.1467. Acknowledgments - This publication was made possible by the NIH Grant Number P20RR16456 from the BRIN Program of the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. Philip Morris USA Inc. is also acknowledged for financial support.
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