Chemistry and Bioactivity of Briaranes from the South China Sea Gorgonian Dichotella gemmacea

Seven new briarane diterpenoids, gemmacolides AZ–BF (1–7), were isolated together with eight known analogues (8–15) from the South China gorgonian Dichotella gemmacea. Their structures were elucidated based on detailed spectroscopic analysis and a comparison with reported data. In an in vitro bioassay, these compounds exhibited different levels of growth inhibition activity against A549 and MG63 cells, giving continuous evidences about the biological contribution of functional groups at C-2, C-12, C-13, and C-16. These compounds were also evaluated for their antibacterial and antifungal activities. Compound 8 exhibited a potential antibacterial activity against both Gram-positive bacterium Bacillus megaterium and Gram-negative bacterium Escherichia coli.

Gemmacolide AZ (1) was isolated as a white amorphous powder. The molecular formula C 31 H 42 O 13 was established by the HRESIMS. The IR spectrum showed strong absorption bands of hydroxyl (3468 cm −1 ), γ-lactone (1775 cm −1 ), and ester (1738 cm −1 ) functionalities. This observation was in agreement with the signals in the 13 C NMR and DEPT spectra (Table 1) for 9 sp 2 carbon atoms (5 × OC = O, CH = CH, CH = C) at lower field and 22 sp 3 carbon atoms at higher field (1 × C, 3 × CH, 2 × CH 2 , 7 × CH 3 , 2 × OC, 5 × OCH, 2 × OCH 2 ), accounting for seven double bond equivalents. The remaining double bond equivalents were due to the presence of four rings in the molecule. The 1 H and 13 C NMR spectra data (Tables 1 and 2) of 1 showed great similarity to those of gemmacolide AQ (16) [9], except for the absence of an acetoxy group. The missing group was readily assigned at C-13 due to the proton sequence of H-12/H 2 -13/H-14, established by 1 H-1 H COSY experiment ( Figure 1). The assignment was fully supported by the 1 H-1 H COSY and HMBC spectra as shown in Figure 1. The relative configuration of 1 at chiral centers was proven the same as that of 16 by a NOESY experiment (Figure 2), showing a β configuration of , and an α configuration of H-2, H-9, H-10, and Me-18. The geometry of the ∆ 3 double bond was assigned as (Z) based on the proton coupling constant between H-3 and H-4 (J = 10.6 Hz), while ∆ 5,6 was determined as (E) due to the NOESY correlation between H-6 and H 2 -16. The NMR assignments of 1 were further supported by a comparison with the reported data of gemmacolide N (17), an analogue obtained from the same species of animals with its absolute stereochemistry being determined by a detailed TDDFT/ECD analysis [12]. Compound 17 differs from 1 in the functional groups at C-14 and C-16, having acetoxy and primary hydroxyl groups instead of isovaleryl and methoxy groups, respectively. As compound 1 contained the same lactone and diene chromophores as gemmacolide N [12], and they differed only in the nature of the ester groups and R 5 , the ECD spectrum of gemmacolide N could be used as ECD reference for the configurational assignment. The absolute configuration of 1 is therefore determined as (1R,2S,7S,8S,9S,10S,11R,12R,14S,17R). 5.60, dd (10.6, 9.5) 5.55, dd (9.5, 9.4) 5.63, dd (9.9, 9.3) 5.56, dd (9.8, 9.5   Gemmacolide BA (2), a white amorphous powder, had the molecular formula of C36H50O15 on the basis of its HRESIMS. Its 1 H and 13 C NMR spectra data (Tables 1 and 2) were similar to those of gemmacolide AQ (16) [9]. However, an acetyl group in 16 was replaced by an isovaleryl group in 2. The three acetyl groups were assigned at C-2, C-9, and C-14 due to the obvious HMBC correlations from the secondary alcohol protons to the respective ester carbonyl groups. The location of two isovaleryl groups at C-12 and C-13 was supported by the obvious HMBC correlations from H-12 and H-13 to the respective carbonyl carbon of the isovaleryl groups. The relative configuration and the absolute configuration of 2 were proven to be the same as that of 16 on the basis of NOESY experiment and their similar ECD spectra.
Gemmacolide BB (3) was obtained as a white amorphous powder with the molecular formula of C38H52O17 being established by HRESIMS. The 1 H and 13 C NMR spectra of 3 showed similarity to those of compound 2 (Tables 1 and 2). The structure of 3 differed from that of 2 with the presence of an isovaleric acetyl group instead of an acetyl group at C-2 and an acetyl group replace of the isovaleryl group at C-12 (Tables 1 and 2). The conclusion was fully supported by the distinct HMBC correlations from H-2 to the carbonyl carbons of the isovaleric acetyl group at δ 167.8, and from H-12 to the acetyl ester carbon at δ 169.8, respectively. Its absolute configuration was proven to be the same as that of 2 on the basis of their similar ECD spectra.
Gemmacolide BC (4) was obtained as a white amorphous powder. Its HRESIMS demonstrated the same molecular formula as C34H46O15 by HRESIMS. Comparison of overall 1 H and 13 C NMR spectra data (Tables 1 and 2) of 4 with those of gemmacolide AQ (16) [9] revealed great similarity with the only difference of an appearance of an oxygenated methyl group at C-16 (δH 3.44, s; δC 58.5, CH3). The result was supported by the obvious HMBC correlations from the methoxyl protons to C-16. The absolute structure of 4 was proven to be the same as that of 16 by the analysis of NOESY and ECD spectra.
Gemmacolide BD (5) was obtained as a white amorphous powder and exhibited a molecular formula of C39H54O17 as deduced from its HRESIMS. 1 H and 13 C NMR spectra of 5 (Tables 1 and 3) were very similar to those of compound gemmacolide AB (18) [9]. The primary alcohol of the hydroxyacetyl group, however, was further isovalerianated. This conclusion was proved by the long-range correlation from secondary hydrogens of both the isovaleryl group and the hydroxyacetyl group to the carbonyl carbon of the isovaleryl group (δ 172.3), and from both H-2  Gemmacolide BA (2), a white amorphous powder, had the molecular formula of C36H50O15 on the basis of its HRESIMS. Its 1 H and 13 C NMR spectra data (Tables 1 and 2) were similar to those of gemmacolide AQ (16) [9]. However, an acetyl group in 16 was replaced by an isovaleryl group in 2. The three acetyl groups were assigned at C-2, C-9, and C-14 due to the obvious HMBC correlations from the secondary alcohol protons to the respective ester carbonyl groups. The location of two isovaleryl groups at C-12 and C-13 was supported by the obvious HMBC correlations from H-12 and H-13 to the respective carbonyl carbon of the isovaleryl groups. The relative configuration and the absolute configuration of 2 were proven to be the same as that of 16 on the basis of NOESY experiment and their similar ECD spectra.
Gemmacolide BB (3) was obtained as a white amorphous powder with the molecular formula of C38H52O17 being established by HRESIMS. The 1 H and 13 C NMR spectra of 3 showed similarity to those of compound 2 (Tables 1 and 2). The structure of 3 differed from that of 2 with the presence of an isovaleric acetyl group instead of an acetyl group at C-2 and an acetyl group replace of the isovaleryl group at C-12 (Tables 1 and 2). The conclusion was fully supported by the distinct HMBC correlations from H-2 to the carbonyl carbons of the isovaleric acetyl group at δ 167.8, and from H-12 to the acetyl ester carbon at δ 169.8, respectively. Its absolute configuration was proven to be the same as that of 2 on the basis of their similar ECD spectra.
Gemmacolide BC (4) was obtained as a white amorphous powder. Its HRESIMS demonstrated the same molecular formula as C34H46O15 by HRESIMS. Comparison of overall 1 H and 13 C NMR spectra data (Tables 1 and 2) of 4 with those of gemmacolide AQ (16) [9] revealed great similarity with the only difference of an appearance of an oxygenated methyl group at C-16 (δH 3.44, s; δC 58.5, CH3). The result was supported by the obvious HMBC correlations from the methoxyl protons to C-16. The absolute structure of 4 was proven to be the same as that of 16 by the analysis of NOESY and ECD spectra.
Gemmacolide BD (5) was obtained as a white amorphous powder and exhibited a molecular formula of C39H54O17 as deduced from its HRESIMS. 1 H and 13 C NMR spectra of 5 (Tables 1 and 3) were very similar to those of compound gemmacolide AB (18) [9]. The primary alcohol of the hydroxyacetyl group, however, was further isovalerianated. This conclusion was proved by the long-range correlation from secondary hydrogens of both the isovaleryl group and the hydroxyacetyl group to the carbonyl carbon of the isovaleryl group (δ 172.3), and from both H-2 Gemmacolide BA (2), a white amorphous powder, had the molecular formula of C 36 H 50 O 15 on the basis of its HRESIMS. Its 1 H and 13 C NMR spectra data (Tables 1 and 2) were similar to those of gemmacolide AQ (16) [9]. However, an acetyl group in 16 was replaced by an isovaleryl group in 2. The three acetyl groups were assigned at C-2, C-9, and C-14 due to the obvious HMBC correlations from the secondary alcohol protons to the respective ester carbonyl groups. The location of two isovaleryl groups at C-12 and C-13 was supported by the obvious HMBC correlations from H-12 and H-13 to the respective carbonyl carbon of the isovaleryl groups. The relative configuration and the absolute configuration of 2 were proven to be the same as that of 16 on the basis of NOESY experiment and their similar ECD spectra.
Gemmacolide BB (3) was obtained as a white amorphous powder with the molecular formula of C 38 H 52 O 17 being established by HRESIMS. The 1 H and 13 C NMR spectra of 3 showed similarity to those of compound 2 (Tables 1 and 2). The structure of 3 differed from that of 2 with the presence of an isovaleric acetyl group instead of an acetyl group at C-2 and an acetyl group replace of the isovaleryl group at C-12 (Tables 1 and 2). The conclusion was fully supported by the distinct HMBC correlations from H-2 to the carbonyl carbons of the isovaleric acetyl group at δ 167.8, and from H-12 to the acetyl ester carbon at δ 169.8, respectively. Its absolute configuration was proven to be the same as that of 2 on the basis of their similar ECD spectra.
Gemmacolide BC (4) was obtained as a white amorphous powder. Its HRESIMS demonstrated the same molecular formula as C 34 H 46 O 15 by HRESIMS. Comparison of overall 1 H and 13 C NMR spectra data (Tables 1 and 2) of 4 with those of gemmacolide AQ (16) [9] revealed great similarity with the only difference of an appearance of an oxygenated methyl group at C-16 (δ H 3.44, s; δ C 58.5, CH 3 ). The result was supported by the obvious HMBC correlations from the methoxyl protons to C-16. The absolute structure of 4 was proven to be the same as that of 16 by the analysis of NOESY and ECD spectra.
Gemmacolide BD (5) was obtained as a white amorphous powder and exhibited a molecular formula of C 39 H 54 O 17 as deduced from its HRESIMS. 1 H and 13 C NMR spectra of 5 (Tables 1 and 3) were very similar to those of compound gemmacolide AB (18) [9]. The primary alcohol of the hydroxyacetyl group, however, was further isovalerianated. This conclusion was proved by the long-range correlation from secondary hydrogens of both the isovaleryl group and the hydroxyacetyl group to the carbonyl carbon of the isovaleryl group (δ 172.3), and from both H-2 and the secondary hydrogens of the hydroxyacetyl group to the carbonyl carbon of the hydroxyacetyl group (δ 166.6). The same absolute geometry was obtained on the basis of the ECD spectrum. Gemmacolide BE (6) was found to be a white amorphous powder, having the molecular formula of C 36 H 50 O 15 based on the HRESIMS. 1 H and 13 C NMR spectra (Tables 1 and 3) of 6 resembled to those of gemmacolide AS (19) [8] except for a hydrogen at C-13 in 19 was replaced by a hydroxyl group in 6. The assignment was in agreement with its NMR shift values (δ H-13 4.07, br s; δ C-13 67.4, CH), compared to those of the ester group substitution in gemmacolide P, gemmacolide AC, gemmacolide AD, gemmacolide AF, gemmacolide AG, gemmacolide AR, and gemmacolide AT (δ H-13 5.08-5.10, d, J = 2.7-3.5; δ C-13 66. CH) [8,9,12], and further supported by the proton sequences from H-12 to H-14 established by the 1 H-1 H COSY experiment. The hydroxyl group was assigned as an α-orientation due to the NOESY correlation of H-13 with H-15. The two isovaleryl groups were deduced to be attached to C-14 and C-16 based on the 2D NMR ( 1 H-1 H COSY, HMBC) analysis and a comparison to those reported data of analogues [7][8][9][10][11][12]. The relative and absolute configuration of 6 was also proven to be the same as those of 19 by the NOESY and ECD experiments.
Gemmacolide BF (7) was a white amorphous powder and had the same molecular formula of C 36 H 50 O 15 as that of 6 as deduced from its HRESIMS. The structure of 7 differed from that of 6 only in the sequence of substituent groups. The hydroxyl, isovaleryl, and acetoxy groups at C-13, C-14, and C-12 in 6 were instead assigned at C-12, C-13, and C-14 in 7, respectively. The location of hydroxyl at C-12 was supported by 1 H and 13 C NMR spectra data (δ H-12 3.48, br d, J = 4.7; δ C-12 75.3, CH), compared to those of ester group substitution (δ H-12 4.88-4.93, br d, J = 2.8-3.5; δ C-12 72. CH) [8,9,12]. A β-orientation of H-12 was deduced from its NOESY correlation with H-20b. Two isovaleryl groups were attached at C-13 and C-16 due to the HMBC correlations of H-13 and H-16 with the respective carbonyl carbon of the isovaleryl groups. The assignment was supported by the proton sequence of H-12/H-13/H-14, as deduced from the 1 H-1 H COSY experiment. The established structure of 7 was further supported by a detailed analysis of its 1D NMR and 2D NMR data. Its absolute configuration was determined as (−)-(1S,2S,3Z,5E,7S,8S,9S,10S,11R,12R,13S,14R,17R) based on NOESY and ECD experiments.
The compounds were evaluated for their antimicrobial and tumor cell growth inhibition activities. In the in vitro bioassays, compounds 1-9 exhibited a different level of growth inhibition against cell lines A549 and MG63 ( Table 4). The activity of 5 with 2-isovaleric acetyl substitution marked decreased compared with its 2-glycolyl analogue (IC 50 for A549 = 19.4 µM, for MG63 = 22.8 µM) [9]. The previous conclusion that 13-isovalerate may decrease the activity [9] was further supported by comparing the activity of 1 and 9 with those of 16 (IC 50 = 28.7 and > 100 µM for A549 and MG63, respectively) [9] and 8, respectively. Comparing the activity of 2 and 8 with respect to those of gemmacolides AT (IC 50 > 30.0 µM for both A549 and MG63) [8] and N (IC 50 > 50.5 µM for both A549 and MG63) [12] supported the conclusion that 12-O-isovalerate gave a positive contribution to the activity as mentioned previously [9,11,12]. The replacement of an isovaleryl group by an acetyl group at C-14 would increase the activity as observed in 4 and juncenolide D [12]. The 16-substituents displayed an activity order as 16-OH > 16-OMe when the activity of 2 and 3 was compared with those of 9 (IC 50 > 30.0 µM for both A549 and MG63) and gemmacolide AL (IC 50 > 37.8 µM for both A549 and MG63) [9], respectively. In antimicrobial test in vitro, compounds 4 and 8 showed potential antibacterial activity against the Gram-negative bacterium Escherichia coli, while 8 exhibited significant antibacterial activity against the Gram-positive bacterium Bacillus megaterium. Compounds 1, 2, 4, 5, and 8 exhibited weak antifungal activity against both Septoria tritici and Microbotryum violaceum, while compound 9 only displayed weak activity against S. tritici (Table 5).

General Experimental Procedures
Commercial silica gel (Yantai, China, 200-300; 400-500 mesh) and RP silica gel (Merck, Darmstadt, Germany, 43-60 µm) were used for column chromatography (CC). Precoated silica gel plates (Yantai, China, HSGF-254) and RP silica gel (Macherey-Nagel, Düren, Germany, RP-18 F254) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with an anisaldehyde-sulphuric acid reagent. The NMR spectra were recorded at 300 K on a Bruker DRX 400 spectrometer (Ettlingen, Germany). Chemical shifts are reported in parts per million (δ), with use of the residual CHCl 3 signal (δ H 7.26 ppm) as an internal standard for 1 H NMR and CDCl 3 (δ C 77.0 ppm) for 13 C NMR; Coupling constants (J) are reported in Hz. 1 H NMR and 13 C NMR assignments were complemented 1 H-1 H COSY, HSQC, HMBC, and NOESY experiments. The following abbreviations are used to describe spin multiplicity: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br s = broad singlet; dd = doublet of doublets; ov = overlapped signals. Optical rotations were measured in CHCl 3 with an Autopol IV polarimeter at the sodium D line (590 nm). Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet, Madison, WI, USA); peaks are reported in cm −1 . UV absorption spectra were recorded on a Varian Cary 100 UV-Vis spectrophotometer (Palo Alto, CA, USA); peaks wavelengths are reported in nm. Circular dichroism spectra were recorded with a JASCO J-715 circular dichroism spectropolarimeter (Mary's Court Easton, MD, USA). The MS and HRMS were performed on a Q-TOF Micro mass spectrometer (Bruker, Bremen, Germany), resolution 5000. An isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound. Semi-preparative RP-HPLC was performed on an Agilent 1100 system (Santa Clara, CA, USA) equipped with a refractive index detector using an YMC Pack ODS-A column (Kyoto, Japan, particle size 5 µm, 250 mm × 10 mm).

Animal Material
The South China Sea gorgonian coral Dichotella gemmacea (3.5 kg, wet weight) was collected from the South China Sea in August 2007 and identified by Xiu-Bao Li, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (ZS-3) was deposited in the Second Military Medical University.

Extraction and Isolation
The frozen specimen was extracted ultrasonically three times with acetone and MeOH, respectively. The combined residue was partitioned between H 2 O and EtOAc to afford 16.1 g of an EtOAc extract. The EtOAc extract was further partitioned between MeOH and hexane, affording IR (film) ν max 3469, 1777, 1742 cm −1 ; 1 H NMR spectroscopic data, see Table 3; 13 C NMR spectroscopic data, see Table 1

Agar Diffusion Test for Biological Activity
Compounds 1-9 were dissolved in acetone at a concentration of 2 mg/mL; 25 µL of the solution (0.05 mg) were pipetted onto a sterile filter disc (Schleicher & Schuell,Dassel,Germany,9 cm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organize. The test organisms were the Gram-negative bacterium Escherichia coli (Coli), the Gram-positive bacterium Bacillus megaterium (Meg) (both grown on NB medium), the fungus Microbotryum violaceum (Vio), and Septoria tritici (Tri) (both grown on MPY medium). Commencing at the outer edge of the filter disc, the diameter of zone of inhibition was measured in mm. Reference substances were ketoconazole (0.05 mg), penicillin (0.05 mg), and streptomycin (0.05 mg), with the diameter (φ) of zone of inhibition being 18.0, 18.0, 30.0, 25.0; 26.0, 18.0, 14.0, 12.0; and 18.0, 11.0, 16.0, 11.0 mm, respectively.