2.1. Identification and Structure Elucidation
As part of the continuing investigation for biologically active constituents from Egyptian Red Sea costal soft corals [6,7,8,16], reported here is the chromatographic fractionation and purification of a methylene chloride:methanol (1:1) extract from S. ehrenbergi (Figure 1).
Compound 1 was obtained as white crystals with an optical rotation of −6.9 in CHCl3. HRESIFTMS analysis showed a molecular ion peak at m/z 387.2142 [M + Na]+ (calcd. 364.2250), corresponding to the molecular formula of C21H32O5. The IR spectrum showed characteristic bands at 3450 cm−1 (OH) and 1754 cm−1 (CO). The 1H NMR spectrum (Table 1) exhibited three oxygenated protons at δH 5.50 (d; J = 10.10 Hz); δH 3.24 (d; J = 6.90 Hz); and δH 3.37 (m). Only one olefinic proton at δH 4.94 d; J = 10.10 Hz was attributed to a tri-substituted double bond; four signals at δH 1.76 s, 1.86 s, 1.05 s, and 0.98 s were identified as methyls, in addition to one methyl of a methoxy group at δH 3.13 s. Twenty one carbon signals were observed in the 13C NMR spectrum and classified by DEPT analysis as five methyls (including one methyl of the methoxy group at δC 49.0), six methylenes, four methines, and six quaternary carbons (including the carbonyl group of the lactone ring δC 175.9 (Table 1)). The spectrum also revealed the presence of four olefinic carbon signals at δC 122.3, 164.3, 119.4, and 141.6; three oxymethine carbons at δC 81.0, 78.5, and 79.0; and one oxygenated quaternary carbon of an epoxy at δC 78.3. The most oxygenated down-field carbon signal indicated the presence of an ether linkage that was functionality confirmed by HRESIFTMS. These spectroscopic data were consistent with a cembrene diterpenoid based on spectroscopic data reported for other Sarcophyton species [4,13] (Figure 2). Six degrees of unsaturation were deduced, suggesting a tricyclic skeleton. The correlation of the oxygenated proton at δH 5.50 (d; J = 10.10 Hz) with the olefinic signal at δH 4.94 (d, J = 10.10 Hz) in DQF-COSY, as well as with quaternary olefinic carbons at δC 141.6 and δC 164.3, allowed the assignments of H-2, H-3, C-4, and C-1 of a cembrene diterpenoid, respectively [9,10].
The HMBC correlation of a methyl signal at δH 1.76 (s) with C-1 and a keto group at δC 175.9 allowed for the assignment of H-17 and C-16, respectively, and indicated the location of a lactone ring, including C-1/C-2. The observed HMBC correlation between H-3 and an olefinic methyl signal at δC 17.0 and a methylene signal at δC 40.9 allowed for the assignment of H-18 (δH 1.86, s) and H-5 [δH 2.07, t (J = 13.08)], respectively, which was confirmed by HMQC analysis. A doublet oxygenated methine signal at δH 3.24 (J = 6.90) correlated with a methyelene multiplet at δH 2.44/2.14 in DQF-COSY and C-5 in HMBC allowed for the assignment of H-7 and H2-6, respectively. Additionally, HMBC correlations of the methyl singlet at δH 1.05 with H-7 and an oxygenated quaternary carbon atom at δC 80.0, as well as the methyelene signal at δC 36.4, allowed for the assignment of H3-19 (δC 17.1), C-8, and C-9, respectively. The oxygenated signal at δH 3.37 (m) was assigned to H-11 (δC 79.0) based on an HMBC correlation with C-9 and a methyl signal at δC 17.6 (C-20). Correlations were observed between δH 1.45 (m, H-13)/δH 1.95 (m, H-14) and C-20 in DQF-COSY and HMBC analyses, respectively (Figure 3). The location of a characteristic methoxy group signal at δH 3.13 (δC 49.0) was confirmed to be at C-8 via an HMBC correlation. The complete assignment of 1, as well as the ether linkage between C-7/C-12 and the presence of the hydroxyl group at C-11, were established by NMR and HRESIFTMS data; structural confirmation including absolute configuration was established unambiguously using the anomalous scattering of Cu Kα radiation with the Flack parameter [17,18] being refined to 0.09 (3) (Figure 4).
The γ-lactone- (H-2) and olefinic-proton (H-3) vicinal coupling (10.10 Hz) established a cis configuration . The four methyl groups exhibited NOSEY correlations with alpha protons consistent with the X-ray assignment of all methyl groups below the ring (e.g., CH3-17 with H-14a, CH3-18 with H-2, CH3-19 with H-6a/H-10a, and CH3-20 with H-10a) and absolute stereochemistry of 8R and 12S (Figure 5). NOSEY correlations between H-7 and H-5b, as well as H-11 and H-14b, were also consistent with 7R and 11R configurations. From this consistent x-ray and NMR data, 1 was assigned as 2S,16:7R,12S-diepoxy-11α-hydroxy-8β-methoxy-16-keto-cembra-1E,3E-diene (sarcoehrenbergilid A).
Compound 2 was obtained as a white powder with an optical rotation of −3.7 (c 0.0027, CHCl3). HRESIFTMS analysis showed a molecular ion peak at m/z 373.1986 [(M + Na)+] (calcd. 350.2093), implying six degrees of unsaturation. The IR spectrum exhibited characteristic bands at 3447 cm−1 (OH) and 1747 cm−1 (CO). 13C NMR and DEPT spectral data (Table 1) showed 20 carbon resonances that distributed in the configuration of four methyls, six methylenes, four methines, and six quaternary carbons. Chemical shift data indicated the same cembrenoid backbone, containing diagnostic carbon signals associated with the lactone ring including a carbonyl signal C-16 (δC 175.6), three olefinic carbons at C-15, C-1, C-3, and C-4 (δC 122.5, 164.4, 117.8, and 146.0, respectively), and C-2 (δC 79.6). The spectra data closely matched a cemberene compound reported by Sawant et al. in 2004 , except for a large down field carbon signal difference at C-12 compared with the previously published structure tertiary carbon, which had a methyl substitution (δC 38.0). Compound 2 was proposed to contain a hydroxylated quaternary carbon at C-12 which would explain the downfield shift to δC72.8 and a 16 AMU addition compared to the previously published compound . The addition of a hydroxyl group at C-12 was consistent with a H3-20 downfield shift from δH 0.88 to δH 1.16 and a C-20 downfield shift from δC 17.2 to δC 23.8 without versus with a C-12 hydroxyl group. HMBC correlations of H3-20 (δH 1.16 s) with C-12 (δC 72.8), C-11 (δC 78.5), and C-13 (δC 35.0) were also consistent with the hydroxylation of C-12 (Figure 3).
Similar to 1, the γ-lactone- (H-2) and olefinic-proton (H-3) vicinal coupling (10 Hz) established a cis configuration . Also similar to 1, the NOSEY data for 2 showed a correlation between H-6b and H-7, indicating that the epoxy ring at C-7 is below the ring while H-6a correlates with CH3-19, establishing that the relative stereochemistry for the methyl is above the ring (Figure 5). H-7, which is assumed to be in the beta position from the previous NOSEY correlation, also correlates with H-11, indicating that the other epoxide ring attachment is in an alpha configuration. Finally, a NOSEY correlation between H-10a and CH3-20 indicates that the methyl group is in an alpha orientation. Thus, 2 was confirmed to be 2S,16: 7R,11R-diepoxy-8β,12β-dihydroxy-16-keto-cembra-1E,3E-diene (sarcoehrenbergilid B).
Compound 3 was obtained as a white powder with a negative optical rotation of −6.6. HRESIFTMS analysis exhibited a molecular ion peak at m/z 373.1985 [(M + Na)+] (calcd. 350.2093), corresponding to the molecular formula C20H30O5 with six degrees of unsaturation. The IR spectrum showed characteristic bands at 3445 cm−1 (OH) and 1747 cm−1 (CO). Twenty carbon resonances were exhibited in the 13C NMR and DEPT spectrum (Table 1); four methyls, six methylenes, five methines, and five quaternary carbons. The spectroscopic data of 3 are similar to a previously isolated diterpenoid from S. trocheliophorum, trocheliophorol , except for the presence of a hydroxyl unit at C-12 (δC 70.1) instead of an exomethylene. The location of the C-12 hydroxyl group was confirmed by HMBC correlations with a methyl singlet H3-20 (δH 1.13 s); correlations were also observed between H3-20 and δC 85.1 (C-11) and δC 31.2 (C-13) (Figure 3).
Similar to compounds 1 and 2, a γ-lactone- (H-2) and olefinic-proton (H-3) vicinal coupling (10 Hz) established a cis configuration . A NOESY correlation between H-6a with H-7 indicated that the C-7 hydroxyl group is in a beta orientation and a H-6a correlation with CH3-19 indicated that the methyl at C-8 is in an alpha configuration (Figure 5). A NOSEY correlation was also observed between H-6a and CH3-20, indicating that the hydroxyl at C-12 is in the beta orientation. Finally, a NOSEY correlation between CH3-20 and H-11 indicated that the epoxide connection at C-11 is the same as C-8, both in alpha configurations. From the above spectral data, 3 was established as 2S,16: 8S,11S-diepoxy-7β,12α-dihydroxy-16-keto-cembra-1E,3E-diene (sarcoehrenbergilid C).
With the large flexible ring systems for compounds 1–3,