Further casbane-type diterpenes from the soft coral Sinularia depressa
[ABSTRACT]
AIM: To study the minor diterpenes from the soft coral Sinularia depressa.
METHOD: The chemical constituents were isolated and purified by various chromatographic techniques, and the chemical struc- tures, including absolute configuration, were established on the basis of detailed analysis of spectroscopic data and by literature comparison with the data of related known compounds.
RESULTS: A new casbane-type diterpene, 2-epi-10-hydroxydepressin (1), was isolated and identified.
CONCLUSION: Compound 1 is a new casbane-type diterpene.
[KEY WORDS] Soft coral; Sinularia depressa; Casbane; Diterpene
Introduction
Soft corals of the genus Sinularia (Alcyoniidae) have been proven to be a prolific source of diterpene metabolites with intriguing structural features and various promising bioactivities [1-6]. In the course of an ongoing project seeking bioactive substances from Chinese marine organisms [7-10], a sample of the soft coral Sinularia depressa was collected off Lingshui Bay, Hainan Province, China. A chemical investiga- tion of the EtOAc extract of the collected sample resulted in the isolation and structure elucidation of nine rare cas- bane-type diterpenes [11], of which some showed promising antiproliferative and antibacterial activities. Casbane-type diterpenes are extremely rare in nature. A literature survey showed that until now about 39 casbane-type diterpenes had been isolated [11-13], of which fifteen were from marine sources (only soft coral belonging to the genus Sinularia). Continued investigation of the minor diterpenes of the soft coral S. de- pressa led to the isolation of an additional new casbane-type diterpene, named 2-epi-10- hydroxydepressin (1), structurally related to previously reported 10-hydroxydepressin (2) (Fig. 1) [11]. In this paper, the isolation and structure elucidation of this new compound are reported.
Results and Discussion
Freshly collected specimens of S. depressa were kept at −20 °C before extraction. The workup for the extraction and isolation of the casbane-type diterpenes was performed as previously reported [11]. This common procedure yielded the new compound 1.Compound 1 was obtained as an optically active, color- less oil. Its molecular formula, C20H30O2, was established by HR-ESI-MS from the quasimolecular ion peak at m/z 325.214 5 ([M + Na]+; Calcd. 325.214 4), suggesting the presence of six degrees of unsaturation. The IR spectrum showed absorp- tions indicative of a hydroxyl (3 411 cm−1) and , -unsaturated ketone (1 631 cm−1) groups. These observations were in agreement with the presence of signals in the 13C NMR spectrum for one secondary oxygenated carbon atom (C 65.1), a trisubstituted double bond (C 149.4 and 133.5), and a ketocarbonyl group (C 200.7) (Table 1). The 1H NMR spectrum of 1 demonstrated resonances of five methyls at δH
1.16 (3H, s, H-16), 1.09 (3H, s, H-17), 1.80 (3H, s, H-18), 1.58 (3H, s, H-19), and 1.77 (3H, s, H-20), and one oxygen- ated methine at δH 4.57 (1H, m, H-10). The presence of three trisubstituted double bonds was easily recognized by the 1H- and 13C NMR resonances {[δH 6.03 (1H, d, J = 10.4 Hz, H-3), 5.20 (1H, J = 10.5 Hz, br d, H-7), 5.15 (1H, d, J = 7.8 Hz,H-11)]; [C C-3], 140.9 (C-12), 133.5 (C-4), 131.5 (C-8), 128.5 (C-11), and 123.7 (C-7)}. These data account for four degrees of unsaturation, and the remaining two degrees of unsaturation were due to the presence of two rings in the molecule.
Three fragments a (C-13/C-14/C-1/C-2/C-3), b (C-6/C-7), and c (C-9/C-10/C-11)] were identified from the analysis of the 2D NMR spectra (1H-1H COSY and HMQC) (Fig. 2). On the basis of HMBC correlations, the three fragments a–c could be fully connected by inserting quater- nary carbons C-4, C-5, C-8, and C-12 to form a 14-membered ring. Finally, the HMBC correlations of H3-16/C-15, C-1, and C-2, and H3-17/C-15, C-1, and C-2, led to the connection from C-16 and C-17 to both C-1 and C-2 of fragment a through the quaternary carbon C-15 com- pleting the planar structure of 1 (Fig. 2).
The relative configuration of 1 was subsequently eluci- dated by detailed analysis of its 13C NMR and ROESY spectra, and by comparison with those of related model compounds [11, 13]. The E geometry of all three double bonds 3(4) 7(8) and 11(12) was deduced by the C values of CH3-18, CH3-19, and CH3-20 (< 20 ppm) [14]. There are three chiral centers (C-1, C-2, and C-10) in the molecule. The junc- tion of the two rings at carbons C-1/C-2 was suggested to be trans based on the 13C NMR chemical shifts of the geminal methyls C-16 and C-17 (C 21.9 and 23.9, respectively) that are significantly different in comparison with those of the co-occurring model compound 2 (C 15.9 and 29.0, respec- tively) [11]. The proposed configuration of C-1/C-2 was further supported by the nOe correlation between H-1 and H3-16, and H-2 and H3-17 in the ROESY experiment. Due to the flexible nature of the14-membered macrocylic ring, the configuration of C-10 could not be deduced by a nOe experiment. However, since the 13C NMR chemical shift of carbon at C-10 (C = 65.1) of 1 is almost identical with that of the model com- pound 3 (C-10, C = 66.2), a casbane-type diterpene recently isolated from South China Sea soft coral Sinularia sp. [13], and based on the biogenetic considerations, the relative configura- tion of C-10 in 1 was tentatively assumed to be same as that of 3. Consequently, the only difference between the structures of 1 and 2 happened at the configuration of the junction cen- ters (C-1 and C-2), and therefore compound 1 is the C-2 epimer of 2. The remaining question needing to be answered is the as- signment of absolute configuration for C-1, C-2, and C-10. To determine the absolute configuration of C-10, a modified Mosher’s method was applied. Unfortunately, the experiment was unsuccessful probably due to the limited amount of sam- ple. The absolute configuration at the junction asymmetric centers (C-1 and C-2) of 1 was determined by comparison of its CD spectrum with that of 2. The CD method has been developed for the configuration assignment of natural prod- ucts, and it was found especially useful for large flexible natural products [15-18]. In previous work, it was reported that the absolute configuration of C-2 adjacent to an enone chromophore (C-3/C-4/C-5) in the casbane-type diterpenes could significantly influence the CD curve, namely a pair of C-2 epimers displaying opposite CD spectra [11]. Since the CD profile of compound 1 [(c 2.5 × 10−3 mol·L–1, n-hexane), λ (Δ) 202 (−12.1) and 232 (+2.2) nm] is opposite to that of 2 [11] [(c 9.93 × 10−4 mol·L–1, n-hexane), λ (∆) 202 (27.6) and 252 (−17.7) nm]. This implies that the absolute stereochemis- try of C-2 in compound 1 is opposite to that in compound 2, thus the absolute configurations at the ring junction carbons in compound 1 had to be 1S, 2R. As mentioned above, the diterpenes with casbane fram- work are extremely rare in nature. The discovery of 2-epi-10-hydroxydepressin (1) is an important addition to the family of marine casbane-type diterpenes. This appears to be the fourth report of diterpenes with the casbane framework from the marine sources. Based on the observation that ma- rine casbane-type diterpenes appear to be exclusively present in the soft coral of the genus Sinularia, further studies should be conducted to establish the biosynthetic origin of these compounds and to understand their real ecological roles in the life cycle of the soft coral. Experimental Apparatus and reagents Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectrum was recorded on a Nicolet Magna FT-IR 750 spectrometer. NMR spectra were measured on a Varian Mercury-400 spectrometer with the residual CDCl3 (H 7.26; C 77.0) as an internal standard. The HR-ESI-MS data were recorded on a Q-TOF Micro LC-MS mass spec- trometer. All solvents were of analytical grade (Shanghai Chemical Plant, Shanghai, China). Commercial SiO2 gel (Qingdao Haiyang Chemical Group Co., 200−300 and 400−600 mesh) was used for column chromatography, and precoated SiO2 gel plates (Yantai Zifu Chemical Group Co., G60 F-254) were used for analytical TLC. Sephadex LH-20 gel (Amersham Biosciences) was also used for column chro- matography. Plant material Specimens of Sinularia depressa were collected at Ling- shui Bay, Hainan Province, China, in July 2004, at a depth of 20 m and were frozen immediately after collection. A voucher specimen of S. depressa (LS-338) is available for inspection at the Shanghai Institute of Materia Medica, CAS. The frozen material (510 g dry weight) was cut into small pieces and extracted exhaustively with acetone at room temp (3 × 1.5 L). The organic extract was evaporated to give a residue which was partitioned between Et2O and H2O. The Et2O solution was concentrated under reduced pressure to give a dark brown residue (9.6 g). Extraction and isolation The Et2O extract was fractionated by gradient Si gel column chromatography (CC) eluting with a step gradient (0−100% acetone in light petroleum ether) to yield three diter- pene-containing fractions [A (900 mg), B (1.2 g), C (500 mg)]. Fraction C was fractionated by Si gel CC (light petroleum ether−Et2O, 6 : 4 and 5 : 5) to give a mixture which was further purified on Sephadex LH-20 column (light petroleum ether- CHCl3-CH3OH, 2 : 1 : 1) and followed by reversed-phase HPLC (semi-preparative ODS-HG-5, MeCN−H2O, 55 : 45, 2.0 mL·min−1, tR 29 min) to yield Hexamethonium Dibromide compound 1 (1.9 mg).