Unusual pyranosyl cembranoid diterpene from Sarcophyton trocheliophorum

2.1 9-Hydroxy-10,11-dehydro-sarcotrocheliol

Compound 1 was obtained as optically active colorless oil using various chromatographic techniques. On thin-layer chromatography (TLC), compound 1 displayed a pink color reaction, which later turned to brown with the anisaldehyde/sulfuric acid developmental stain. The molecular weight of 1 was determined as 322 Dalton by EI-MS. The molecular formula of 1 was deduced as C₂₀H₃₄O₃ by HRMS ((+)-ESI) (Table 1), ¹H and ¹³C NMR, indicating the presence of four double-bond equivalents. The ¹H and ¹³C NMR spectroscopic data (Table 2) of compound 1 showed resonances due to 20 carbons categorized by a heteronuclear single-quantum coherence experiment into five methyl, five methylene, seven methine (among them three sp² and two sp³ oxy methines), and three quaternary carbons. Signals attributed to four olefinic carbons (δ_C=135.5 [δ_H=5.41], δ_C=127.8 [δ_H=5.86], δ_C=124.0 [δ_H=5.28], δ_C=140.9 ppm), accounting for two unsaturated double bonds, and implying that compound 1 contains bicyclic skeleton.

In the ¹H, ¹³C NMR, and heteronuclear multiple-quantum coherence spectra, compound 1 featured resonances corresponding to five methyl groups: three tertiary methyl groups [C-18 (δ_H=1.03, δ_C=23.7 ppm), C-19 (δ_H=1.31, δ_C=29.3 ppm), C-20 (δ_H=1.83, δ_C=17.6 ppm)], and isopropyl doublet methyl signals [C-16 (δ_H=0.76, δ_C=20.4 ppm), C-17 (δ_H=0.88, δ_C=20.6 ppm)]; the in-between isopropyl methine carbon signal (C-15) was deduced at δ_C=29.1 (δ_H=1.20) ppm. The NMR spectra revealed further two doublet of doublet oxygenated methine functions resonating at δ_C=71.8 (δ_H=4.62, C-1) ppm and δ_C=75.4 (δ_H=3.46, C-6) ppm, and two quaternary oxygenated carbons at δ_C=75.0 (C-5) and δ_C=73.4 (C-9), along with a non-oxygenated methine signal appeared at δ_C=45.4 (δ_H=1.34, C-2) ppm (Table 2).

The planar constitution of 1 was finally confirmed, as depicted in Fig. 2, based on ¹H–¹H COSY and HMBC experiments. An extensive study of the ¹H–¹H COSY correlations established two proton sequences: the coupling between the olefinic proton shown at δ_H=5.28 (H-14) and the oxymethine proton at δ_H=4.62 ppm (H-1), as well as this proton (H-1) and the methine proton at δ_H=1.34 ppm (H-2), which in turn is correlated with both the isopropyl methine proton at δ_H=1.20 ppm (H-15) and the methylene protons found at δ_H=1.62, 1.36 ppm (H₂-3); signals of the last methylene protons displayed a COSY correlation to CH₂-4 (δ_H=2.26 ppm), confirming the connectivity of H-14/H-1/H-2/H-15 and H-2/H₂-3/H₂-4 (Fig. 2).

The HMBC couplings shown from H-1 to C-3, C-5, C-13 established the presence of a pyrane ring. The ²J HMBC correlation from CH₃-18 to C-5 confirmed their direct attachment. The last methyl group (CH₃-18) showed two further HMBC correlations to C-4 (δ_C=34.0 ppm), and C-6 (δ_C=75.4 ppm), proving the direct connectivity of C-4/C-5/C-6. The isopropyl group connectivity to the C-2 position was confirmed by the ³J HMBC correlations from both isopropyl methyl signals (H₃-16, H₃-17) toward C-2 (δ_C=45.4 ppm). The second sequence of proton patterns were identified through the shown HMBC correlations between the olefinic methyl H₃-20 (δ_H=1.83 ppm) and C-13 (δ_C=140.9 ppm, ²J), CH-14 (δ_C=124.0 ppm, ³J), and CH₂-12 (δ_C=42.3 ppm, ³J), proving the direct attachment of the corresponding 2-methyl-butylene partial structure to the pyrane ring (C₁₄–C₁). Based on the ¹H–¹H COSY data, the two olefinic protons at δ_H=5.86 ppm (CH-11) and δ_H=5.41 ppm (CH-10, d, J=15.7 Hz) are vicinal, forming one transoid olefinic double bond [–CH=CH–]. The last double bond (H-10/H-11) showed a direct attachment to CH₂-12 (δ_H=2.76, 2.54 ppm) indicated by COSY correlation with H-11 along with HMBC correlations from H-10 (³J) and H-11 (²J) toward C-12. Proton signals of the last double bond (H-10/H-11) exhibited further two HMBC correlations versus the sp³ quaternary-oxygenated carbon C-9 (δ_C=73.4 ppm), to which the singlet tertiary methyl CH₃-19 (δ_H=1.31, δ_C=29.3 ppm) is attached. The proton signals of CH₃-19 displayed further HMBC correlations to H-10 and CH₂-8, in which CH₂-8 (δ_H=1.84, 1.53 ppm) proton signals displayed a COSY correlation to CH₂-7 (δ_H=1.72, 1.30 ppm). CH₂-7 showed a COSY correlation with the oxymethine CH-6 (δ_H=3.46 ppm). The last methylene protons (CH₂-7) exhibited two further HMBC correlations at C-8 and C-6, confirming their location in between C-8 and C-6. Accordingly, connections between different parts of 1 were proven. The geometries of the double bonds at C-10, C-11, and C-14 are trans owing to the values of the coupling constant of H-10 (J=15.7 Hz), and chemical shifts of allylic methylene groups δ_C>30 ppm (C-12 and C-8 have δ_C=42.3 and 39.9 ppm, respectively) [19], [24]. The remaining 2D NMR correlations are in full agreement with structure 1 (Figs. 1 and 2). The relative configuration of 1 was proposed on the basis of the NOE cross peaks (Figs. 2 and 3) and by comparison with NOE spectra of our isolated 2, which has been recently reported from the Red Sea soft coral S. trocheliophorum as well [19]. The NOE observed between H-1/CH₃-18, H-1/CH-14, H-1/CH₃-20, and H-1/CH-2 suggested the cofacial orientation of H-1, CH₃-18, CH₃-20 (β), and H-2. In addition, the NOE correlation between CH₃-18/H-6 and H-6/CH₃-19 suggested their cofacial orientations as depicted in Fig. 2. Thus, the cumulative analysis of ESI-HRMS, ¹H, ¹³C, COSY, heteronuclear single-quantum coherence, and HMBC spectroscopic data (Table 2; Fig. 2) established compound 1 as new pyrane-based cembranoid diterpene, which was thereby designated as 9-hydroxy-10,11-dehydro-sarcotrocheliol.

Fig. 3:

Selected three-dimensional NOESY correlations of 9-hydroxy-10,11-dehydro-sarcotrocheliol (1).

2.2 Sarcotrocheliol (2), sarcophytol A (3), and sarcophytonin A (4)

Compounds 2–4 were obtained as colorless oils using a series of chromatographic techniques, and displayed physicochemical properties common to diterpenes (see the Experimental section). The molecular formulas of compounds 2–4 were established as C₂₂H₃₆O₃, C₂₀H₃₂O, and C₂₀H₃₀O, respectively, by ESI-HRMS, ¹H, and ¹³C NMR. Based on the full 1D and 2D NMR spectroscopic data (Figs. 1 and 2; Tables 3 and 4) along with HRMS analysis, structures of compounds 2–4 have been confirmed as sarcotrocheliol acetate (2), (+)-sarcophytol A (3), and (−)-sarcophytonin A (4, also known as deoxysarcophytoxide). Sarcotrocheliol acetate (2) has been recently reported from Red Sea soft coral S. trocheliophorum, and to have significant antibacterial activities against Staphylococcus aureus, Acinetobacter spp., and methicillin-resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentration (MIC) range 1.53–4.34 mm [19]. The low value of the reported optical activity of compound 2 (lit. [19]: [α]D20=+8.4 (c=0.1, CHCl₃)) compared with our isolated one ([α]D20=+74 (c=0.15, CHCl₃)) is an indication that the reported 2 is mostly containing some impurities.

2.3 Biological activity

The crude extract of S. trocheliophorum and isolated compounds 1–4 are biologically inactive against Bacillus subtilis, S. aureus, Streptomyces viridochromogenes (Tü 57), Escherichia coli, Candida albicans, Mucor miehei, Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus subspicatus, Rhizoctonia solani, and Pythium ultimum at 40 μg per disk. Compounds 1–4 and the crude extract were further examined for their cytotoxic activity against brine shrimp at a concentration 10 μg mL⁻¹ (24 h). However, compounds 1–4 demonstrated no cytotoxicity, whereas the crude extract of S. trocheliophorum exhibited low cytotoxicity with a mortality rate of 1.7% [17], [18].

3.1 Extraction, isolation, and purification

Details of extraction and chromatographic purification of the soft coral S. trocheliophorum were reported recently [17], [18], in which 10 fractions were obtained. Fraction FIII (7.2 g) was subjected to size exclusion fractionation on a Sephadex LH-20 column [dichloromethane (DCM)/40% MeOH] afforded two sub-fractions FIIIA (3.2 g) and FIIIB (2.8 g). Sub-fraction IIIA was deduced as a mixture of fatty acid and has been discarded. Sub-fraction FIIIB showed several UV-absorbing bands on TLC, which stained as violet on spraying with anisaldehyde/sulfuric acid and heating, indicating diterpene components. As a result, sub-fraction FIIIB was applied to silica gel column chromatography (eluted with cyclohexane-DCM gradient), followed by preparative thin-layer chromatography (cyclohexane-DCM, 2:1) and Sephadex LH-20 (DCM/40% MeOH), and afforded 9-hydroxy-10,11-dehydro-sarcotrocheliol (1, 169.5 mg), sarcotrocheliol acetate (2, 119.1 mg), sarcophytol A (3, 25.3 mg), and sarcophytonin A (4, 142.2 mg) as colorless oils. The remaining fractions and sub-fractions obtained from the soft coral S. trocheliophorum extract lacked the diterpenes analogs based on TLC and high-performance liquid chromatography analyses.

3.2 Antimicrobial activity and brine shrimp microwell cytotoxic assays

Details of our general antimicrobial activity testing were reported recently [17], [18]. The cytotoxicity assay was performed according to Takahashi et al. [25] and Sajid et al. [26] screening.