Marine natural products are diverse in terms of chemical structures as well as biological activities. The Red Sea serves as an epicenter for marine bio-diversity with a high endemic biota. Indeed of the 180 soft corals species identified world-wide, approximately 40% are native to the Red Sea [1]. Soft corals are marine invertebrates possessing a vast range of terpenoid metabolites. These terpenes, mostly cembranoids, represent the main chemical defense for coral against natural predators [2]. Soft corals of the genus Sarcophyton (family Alcyoniidae) are particularly rich in cembrane terpenes [3]. Cembranoids contain a 14-membered macro cyclic skeleton and exhibit a wide range of biological activities including anti-tumor, neuro-protective, antimicrobial, calcium-antagonistic, and anti-inflammatory activity [4,5,6,7]. The cembranoid diterpene sarcophine has been investigated since 1998 for its potential as a chemo-preventive agent [8], cytotoxic agent, anti-microbial agent [9], competitive cholinesterase inhibitor [10], noncompetitive phosphofructokinase inhibitor [11], and a Na⁺, K⁺-ATPase inhibitor [12]. Recent studies focusing on the treatment of human diseases have shown that sarcophine and sarcophine derivatives (e.g., hydroxylated sarcophine) are potent cancer chemo-preventive agents [8,9,13,14,15].

Cancer chemoprevention is based on chemical constituents that block, inhibit, or reverse the development of cancer in normal or pre-neoplastic tissue [16]. During the past 20 years, thousands of novel marine metabolites have been identified and assayed for anticancer activity [17]. Most of these drug leads are identified by high-throughput in vitro screening via a cost-effective testing of cancer cell lines derived from human and rodent sources. Indeed several marine-derived drug leads have reached phase II human clinical trials based on promising anticancer results, although toxicity testing has mostly screened out such candidate drugs. Sarcophine anti-tumor potency appears to at least in part involve inhibition of cell transformation that can be induced in vitro by 12-O-tetradecanoyl phorbol-13-acetate (TPA) with irreversible acquisition of tumorigenicity [7,13]. In many cases, carcinogenesis is initiated by pro-carcinogens in combination with phase I enzymes such as cytochrome P₄₅₀ 1A and oxidative stress leading to DNA damage. This process can be mitigated at least in part by phase II detoxification enzymes such as glutathione S-transferases (GSTs), quinone reductase (QR), and epoxide hydrolase (mEH).

Herein, we report the isolation of three new and two known cembranolides (Chart 1) from an ethyl acetate extraction of the Red Sea soft coral Sarcophyton glaucum. Structures of these isolated metabolites were elucidated by 1D and 2D spectroscopic techniques, while the absolute configuration of 1 and 2 were confirmed by X-ray diffraction and circular dichroism (CD) analyses. Compounds 2 and 3 were found to be promising inhibitors of cytochrome P₄₅₀ 1A activity as well as inducers of GST and QR activity in in vitro assays.

Freshly collected specimens of S. glaucum were immediately frozen in dry ice and kept at −20 °C until ready for organic-solvent extraction. The EtOAc-soluble fraction was subjected to normal and reverse phase chromatography to afford new hydroperoxyl cembranolides (1 and 2), a cembrene derivative 8-epi-sarcophinone (3) along with two known cembranolides, ent-sarcophine (4) [18,19], and sarcophine (5) [18,19,20,21].

Preliminary ¹H NMR analysis established that all fractions shared a common carbon skeleton, differing either in the degree of oxidation or the configuration of one or more chiral centers. Precedent from soft coral literature led to the assumption of a cembranoid-skeleton backbone [19]. Compound 1 was obtained as colorless crystals, +12.6 (c 0.09, CHCl₃). The HR-FAB-MS exhibited a [M + Na]⁺ ion at m/z 371.18281, indicating a molecular formula of C₂₀H₂₈O₅Na and seven degrees of unsaturation that was supported by NMR data. An IR spectrum indicated the presence of an α,β-unsaturated-γ-lactone (1750 and 1686 cm⁻¹), a carbonyl (1707 cm⁻¹), an olefin (1669 cm⁻¹), an epoxide (1256 cm⁻¹) and a broad absorption band for OH stretching (3000–3353 cm⁻¹). The ¹³C NMR and DEPT spectrum (Table 1) exhibited 20 carbon signals establishing: three methyls, seven methylenes, four methines, and six quaternary carbons. The spectrum also revealed the presence of an exomethylene functionality at δ_C 113.4/144.5, two oxymethine carbons at δ_C 60.8 and 86.5, one oxygenated quaternary carbon at δ_C 61.1, and two olefinic carbons at δ_C 119.6 and 146.0.

The low field oxymethine carbon at δ_C 86.5 (C-11) suggested the presence of a peroxide functionality that is consistent with the presence of a broad singlet at δ_H 8.25 in the ¹H NMR spectrum [18]. ¹³C NMR analysis indicated that two oxygens contribute to an α,β-unsaturated-γ-lactone with appropriate signals at δ_C 174.5 and 78.9 for the carbonyl and oxymethine carbons, respectively. The olefinic methyl group at δ_H 1.85 (H₃-17) exhibited an HMBC correlation with a low-filed ¹³C NMR resonance for a keto group in association with the α,β-unsaturated-γ-lactone ring at δ_C 174.5 (C-16). Carbon signals at δ_C 124.2 (C-15) and 162.2 (C-1) were consistent with α and β olefinic carbons of the α,β-unsaturated-γ-lactone system. The carbon signal at δ_C 78.9 (C-2) is consistent with an oxymethine carbon while the oxymethine proton at δ_H 5.50 (d, J = 15.0 Hz; H-2) exhibited a strong correlation with a one-proton doublet at δ_H 5.09 (J = 15.0 Hz; H-3) in the ¹H-¹H COSY spectrum (Figure 1). The olefinic methyl group at δ_H 1.94 (H-18) also shows an HMBC correlation with an olefinic methine at δ_C 119.6 (C-3). The methyl signal at δ_H 1.27 (H-19) indicates a proximal oxygen functionality identified from ¹³C NMR to be an epoxide. The location of the epoxide ring at C7/C-8 was detected from HMBC correlations (Figure 1), as there are clear correlations between C-7 (δ_C 60.8) and H-6 (2.59, td, J = 5.0, 13.5 Hz; 2.39, m), H-5 (2.20, m; 2.39, m), H₃-19, and H-9 (1.30, m; 1.79, m); and between C-8 (δ_C 61.1) and H-7 (2.50, dd, J = 4.5, 8.5 Hz), H-9, H-10, and H-6. A triplet-like signal at δ_H 4.35 (J = 5.0 Hz; H-11) revealed the presence of a peroxide at δ_C 86.5 that showed a strong correlation with methylene signals at δ_H 1.50 (m) and 1.70 (m) (H-10) in the ¹H-¹H COSY spectrum (Figure 1). The position of the peroxide was established through HMBC correlation between H-11 and C-9 (32.1, t), C-10 (26.7, t), C-12 (144.5, s), and C-13 (30.1, t). Exomethylene protons at δ_H 5.12 (s) and 5.16 (s) (H₂-20) showed strong correlations with carbon signals at δ_C 113.4 (C-20) and δ_C 144.5 (C-12) in HMQC and HMBC analyses, respectively.

Comparison of the above data with those structural relatives isolated from the same species [22,23], strongly indicated a cembranoid molecular framework containing the rare 11-peroxid-12(20)-exomethylene as confirmed by X-ray analysis (Figure 2). The relative configuration of 1 was determined on the basis of coupling constants and NOESY experiments. The vicinal coupling constant of 15.0 Hz between H-2 and H-3 as well as a NOESY correlation of H-2 with H₃-18 established a trans configuration between the γ-lactone (H-2) and the olefinic proton (H-3). In order to confirm the position of the peroxyl group, as well as the relative stereochemistry, X-ray structure analysis was performed. The absolute stereochemistry of 1 at C-2 was determined via circular dichroism (CD) analysis (Figure 3). The observed positive Cotton effect {[θ]₂₄₈ +0.7} followed by a negative value {[θ]₂₂₅ −3.23} observed in the CD spectrum for the electronic transitions of the 2(5H)-furanone moiety, indicated a left hand (M) helix configuration for the five-membered α,β-unsaturated-γ-lactone ring [24]. Supporting CD data for 1, CD spectral comparison between 1 and ent-sarcophine (4) indicated the same R absolute configuration for the two compounds at C-2 [18,19,21,22]. Therefore, 1 was assigned as 11(S)-hydroperoxylsarcoph-12(20)-ene.

Compound 2 was obtained as color-less crystals, −20.1 (c 0.1, CHCl₃) with much of the spectral data identical to 1 (Table 1). The HR-FAB-MS showed an [M + Na]⁺ ion at m/z 371.18293 indicating a molecular formula C₂₀H₈O₅Na and seven degrees of unsaturation that was supported by NMR data. The analysis of ¹H, ¹³C NMR and DEPT spectra revealed the presence of four methyls, five methylenes, five methines (two of them oxygenated, δ_C 59.0, and 79.2) , and six quaternary carbons (two of them oxygenated, δ_C 59.2, and 84.0). NMR spectra also revealed the presence of four olefinic functionalities at δ_C 118.9, 124.6, 136.1 and 146.7. The presence of an α,β-unsaturated-γ-lactone functionality was assigned based on NMR parallels with 1. From HMBC (Figure 1), a methyl unit (1.43, s; H₃-20) was observed proximal to C-12 determined from correlations between C-12 (δ_C 84.0) and H₃-20 (1.43, s), H-11 (5.56, d, J = 16.0 Hz), H-10 (5.42, ddd, J = 16.0, 10.5, 7.5 Hz), H-13 (2.07, td, J = 13.0, 4.5 Hz; 1.41, dd, J = 4 Hz, overlapped with H₃-18). HMBC correlations (Figure 1) were also observed between C-7 (δ_C 59.0) and H-6 (1.77, m, 2H), H-5 (2.02, m; 2.38, dt, J = 4.5, 13.5 Hz), H₃-19 (1.30, s), and H₂-9 (2.25, m; 2.46, m), and C-8 (δ_C 59.2) and H-7 (2.53, dd, J = 5.0, 6.0 Hz), H₂-9 (2.25, m; 2.46, m), H-10 (5.42), and H₂-6 (1.77, m, 2H) indicating the same epoxide location as in 1 bridging C-7 and C-8. The olefinic proton signal at δ_H 5.56 (H-11, d, J = 16.0 Hz) showed an HMBC correlation with an oxygenated carbon at δ_C 84.0 (C-12), a methyl signal at δ_C 22.8 (C-20), and an olefinic carbon at δ_C 124.6 (C-10) establishing that the peroxyl and double bond functionalities are located at C-12 and C-10/C-11, respectively.

The combined spectral data indicated a cembranoid molecular framework containing a rare 12-peroxid-10-ene. This chemical configuration was confirmed by X-ray analysis (Figure 2) and HMBC correlations (Figure 1). The relative configuration of 2 was determined on the basis of coupling constants and NOESY experiments. The germinal coupling between H-2 and H-3 (16.0 Hz) and a NOESY correlation between H-2 and H₃-18 indicated a trans configuration between the γ-lactone (H-2) and olefinic protons (H-3). The absolute stereochemistry of 2 was determined via CD analysis with the CD spectra (Figure 3) of 2 nearly equivalent with 1 and 4 establishing the same (R) configuration at C-2 [18,19,21,22]. Therefore, compound 2 was assigned to be 12-hydroperoxylsarcoph-10-ene (2).

Compound 3 was obtained as a color-less oil, +19.2 (c 0.1, CHCl₃). The HR-FAB-MS showed an [M + Na]⁺ ion at m/z 339.19313 suggesting a molecular formula of C₂₀H₂₈O₃Na that was supported by NMR data. Spectral data suggested that 3 was similar to the sarcophinone previously isolated from the soft coral Sarchophyton molle Tix [23], except for an up-field shift for H₃-19 (δ_H 1.06) and an increase in its coupling constant (7.5 Hz) in comparison with sarcophinone H₃-19 (δ_H 1.13, J = 6.4 Hz). This up-field shift for such a methyl attached to a methine carbon can be explained by an alternative stereochemistry since the β-configuration methyl group is down-field relative to the α-stereochemistry [23,24].

The location of the ketone carbonyl group at C-8 was determined from HMBC data that established clear correlations with H-8 (δ_H 2.44, m)/H₃-19 (δ_H 1.06, d, J = 7.5), H-9 (δ_H 1.56 and 1.95, m, 2H), H-6 (δ_H 2.06 and 2.73, m, 2H), and H-5 (δ_H 2.66 and 2.76, m, 2H) (Figure 1). The absolute stereochemistry at C-2 was determined by CD analysis in which the spectrum was dominated by negative and positive Cotton effects {[θ]₂₄₅ −1.9, [θ]₂₂₀ +4.8} (Figure 3) due to the electronic transitions of the 2(5H)-furanone moiety [15]. These Cotton effects indicated a right-handed (P) helix for the five-membered α,β-unsaturated-γ-lactone ring. Similar CD spectra for 3 and sarcophine (5) show a common S configuration at C-2 [11,12,14,15]. Compound 3 was therefore identified as 8-epi-sarcophinone. There are two reports that have the same structure as 3 and are referred to as iso-sarcophinone [25,26]; however with an absence of spectral data, direct comparisons cannot be made. In a more comprehensive study of iso-sarcophinone by Su et al. [23] full ¹H and ¹³C NMR data is provided and the reported compound is an epimer of 3 with the opposite stereochemistry at C-8; this epimer of 3 has also been named as iso-sarcophinone. Since only spectral data comparisons are possible for the Su et al. study [23] and the NMR data for compound 3 reported here are not consistent with iso-sarcophinone, we propose that iso-sarcophinone has not been isolated in the present study but instead a new 8-epi-sarcophinone as shown in 3. Whether 8-epi-sarcophinone was isolated and not appropriately named or iso-sarcophinone was isolated but incorrectly identified by Czarkie et al. [25] is uncertain with an absence of key spectral data. With this study, spectral data is now available for both iso-sarcophinone [23] and 8-epi-sarcophinone.

To examine the anti-cancer activity of characterized S. glaucum metabolites, individual components were assayed for inhibition of the phase I enzyme cytochrome P₄₅₀ 1A since the enzyme in combination with pro-carcinogens and/or oxidative stress can lead to DNA damage. Compounds 2, 3, and 4 were identified as inhibitors of Cyp1A activity (p < 0.01) with IC₅₀ values of 2.7, 3.7 and 3.4 nM respectively (Figure 4), compared with the initial activity of β-naphthoflavone-stimulated cells. Assayed compounds 1 and 5 exhibited insignificant inhibition of Cyp1A activity (p > 0.05).

To examine induction of protective enzymes of oxidative stress by characterized S. glaucum metabolites, individual components were assayed for induction of glutathione-S-transferase activity, quinone reductase (QR) and epoxide hydrolase (mEH). GSTs are responsible for the detoxification of a wide range of substrates including xenobiotics as well as occupational and environmental carcinogens such as pesticides and polycyclic aromatic hydrocarbons [27]. Total GST activity was investigated in cultured Hepa1c1c7 cells. Forty eight hours after murine hepatoma cell culture incubation with 10 µg/mL of each metabolite, total GSTs activity was significantly induced by 2–3 (p < 0.01 and p < 0.05, respectively) (Figure 5A). While free thiols that serve as non-enzymatic antioxidants assisting in counteracting the deleterious effect of ROS were significantly elevated in cell cultures only when treated with 2 (10 µg/mL) (p < 0.05) (Figure 5A).

QR that is induced coordinately with other Phase II enzymes such as GSTs and contributes to quinone detoxification was investigated in murine hepatoma cell culture. After 48 h incubation, 2–3 resulted in a significant induction of QR activity (p < 0.01 and p < 0.05, respectively) (Figure 5B). In contrast, epoxide hydrolase mEH, an important metabolic enzyme that catalyzes the addition of water to alkene epoxides and arene oxides [28] was significantly elevated in cell cultures only when treated with 4 (10 µg/mL) (p < 0.05) (Figure 5C).

Three new (1–3) and two known cembranolides (4 and 5) were isolated and chemically characterized from the Red Sea soft coral Sarcophyton glaucum. The absolute configuration of 1 and 2 were confirmed by X-ray diffraction and circular dichroism (CD) analyses. Compounds 2 and 3 were found to be promising inhibitors of cytochrome P₄₅₀ 1A activity as well as inducers of GST and QR activity in in vitro assays.