Briarenones A‒C, New Briarellin Diterpenoids from the Gorgonian Briareum violaceum

Three new eunicellin-derived diterpenoids of briarellin type, briarenones A‒C (1‒3), were isolated from a Formosan gorgonian Briareum violaceum. The chemical structures of the compounds were elucidated on the basis of extensive spectroscopic analyses, including two-dimensional (2D) NMR. The absolute configuration of 1 was further confirmed by a single crystal X-ray diffraction analysis. The in vitro cytotoxic and anti-inflammatory potentialities of the isolated metabolites were tested against the growth of a limited panel of cancer cell lines and against the production of superoxide anions and elastase release in N-formyl-methionyl-leucyl-phenyl-alanine and cytochalasin B (fMLF/CB)-stimulated human neutrophils, respectively.

Taking into account the unsaturation degrees mentioned above, the 22 carbon signals in the 13 C NMR spectrum of 1 (Table 1) [13,16,24,25] or an asbestinin-related [15,17,18] structure for 1. However, since the protons of two methyl groups (δH 1.99 ppm, s, H3-20 and 1.09 ppm, d, J = 7.2 Hz, H3-17), but not one methyl group, showed 3 JCH correlations in the heteronuclear multiple bond correlation (HMBC) spectrum with two of the angular methine carbons (δC 51.1 and 48.3 ppm for C-10 and C-14, respectively), the briarellin-type structure was designated for 1 ( Figure 2). Three partial structures were assigned by the analysis of proton correlation spectroscopy ( 1 H-1 H COSY), including that assigned by the allylic correlation of the H3-20 (δH 1.99 ppm) with the olefinic proton H-12 (δH 5.95 ppm, s) ( Figure 2). The connectivity of these partial structures, the positions of the hydroxyl, acetoxyl, and ketone carbonyl, and the ring-fusion carbons of THF and oxepane rings were deduced from the complete correlation analyses of the HMBC spectrum as illustrated in Figure 2. Furthermore, a comparison of the 13 C NMR data of 1 with those of pachyclavulariaenone F (4), isolated from Pachyclavularia violacea [25], verified the replacement of the C-4 hydroxymethine group (δC 69.7 ppm, CH) in 4 by a methylene group (δC 33.6 ppm, CH2) in 1. This was associated with a significant upfield chemical shift at C-5 (∆δC -8.5 ppm) in 1 relative to that in 4. The planar structure of 1 was accordingly Briarenone A (1) was obtained as a needle crystal, [α] 25 D +224.4 (CHCl 3 ). Its molecular formula C 22 H 32 O 6 was determined by the sodium adduct peak [M + Na] + at m/z 415.2089 in the high-resolution electrospray ionization mass spectrometry (HRESIMS), inferring seven degrees of unsaturation. The infrared (IR) absorption bands at ν max 3477, 1728, and 1670 cm −1 indicated the presence of hydroxyl, ester carbonyl, and α,β-unsaturated carbonyl functionalities, respectively. As the NMR spectra of 1 showed numerous broad and very weak signals due to the likely presence of a mixture of slowly interconverting conformers on the NMR time scale, remeasuring the spectra at −10 • C allowed us to have better resolution for the signals. The 1 H NMR spectrum displayed the signals of four methyls, including one olefinic (δ H 1.99 ppm, s), two methyls attached to oxygen-bearing carbons (δ H 1.27 ppm, s and 1.28 ppm, s), and one secondary methyl (δ H 1.09 ppm, d, J = 7.2 Hz), signifying the terpenoid nature of 1. Furthermore, the NMR data (Table 1) indicated the existence of an acetyl (δ C 171.3 ppm, δ C /δ H 21.5/2.08 ppm) and an α,β-unsaturated ketone (δ C 198.0, 157.0, and δ C /δ H 128.0/5.95 ppm). Taking into account the unsaturation degrees mentioned above, the 22 carbon signals in the 13 C NMR spectrum of 1 (Table 1) are, thus, ascribable to the presence of a tetracyclic diterpenoid acetate. Three ring-juncture methines (δ C /δ H 51.1/2.71, 48.3/2.5, and 36.8/3.13 ppm), two tetrahydrofuran (THF)-oxymethines (δ C /δ H 83.4/3.72 and 77.9/4.79 ppm), and one oxymethylene (δ C /δ H 63.7/3.62 and 3.34 ppm) suggested a briarellin-related [13,16,24,25] or an asbestinin-related [15,17,18] structure for 1. However, since the protons of two methyl groups (δ H 1.99 ppm, s, H 3 -20 and 1.09 ppm, d, J = 7.2 Hz, H 3 -17), but not one methyl group, showed 3 J CH correlations in the heteronuclear multiple bond correlation (HMBC) spectrum with two of the angular methine carbons (δ C 51.1 and 48.3 ppm for C-10 and C-14, respectively), the briarellin-type structure was designated for 1 ( Figure 2). Three partial structures were assigned by the analysis of proton correlation spectroscopy ( 1 H-1 H COSY), including that assigned by the allylic correlation of the H 3 -20 (δ H 1.99 ppm) with the olefinic proton H-12 (δ H 5.95 ppm, s) ( Figure 2). The connectivity of these partial structures, the positions of the hydroxyl, acetoxyl, and ketone carbonyl, and the ring-fusion carbons of THF and oxepane rings were deduced from the complete correlation analyses of the HMBC spectrum as illustrated in Figure 2. Furthermore, a comparison of the 13 C NMR data of 1 with those of pachyclavulariaenone F (4), isolated from Pachyclavularia violacea [25], verified the replacement of the C-4 hydroxymethine group (δ C 69.7 ppm, CH) in 4 by a methylene group (δ C 33.6 ppm, CH 2 ) in 1. This was associated with a significant upfield chemical shift at C-5 (∆δ C -8.5 ppm) in 1 relative to that in 4. The planar structure of 1 was accordingly established (Figure 2). The relative configuration of 1 at C-1, C-2, C-3, C-6, C-7, C-9, C-10, C-14, and C-15, was assigned by the analysis of nuclear rotating-frame Overhauser effect spectroscopy (ROESY) correlations (Figure 2), which were found to be consistent with that of 4 as illustrated in Figure 2 , and H-6 should be positioned in its α-face. In order to confirm the molecular structure of 1, including the absolute configuration, a single-crystal X-ray structure analysis was further performed (Figure 3). The compound was slowly crystallized from MeOH as a monohydrate C 22 H 32 O 6 ·H 2 O, with H-bond holding the H 2 O molecule in a hydrophilic pocket. From a dataset collected using copper radiation, the X-ray crystallographic analysis (Tables S1-S3, Supplementary Materials) of 1 determined the absolute configurations of briarenone A (1) as 1S,2R,3R,6S,7S,9R,10R,14S,15S, which forms a hydrogen bond from 7-OH with a molecule of water.  Briarenone B (2) was obtained as a white powder. It possessed a molecular formula of C 20 H 28 O 4 , as established from the sodiated ion peak in the HREIMS (m/z 355.1880 [M + Na] + ) of 2, accounting for seven degrees of unsaturations. The IR absorptions at ν max 3420 and 1670 cm −1 indicated the presence of hydroxyl and conjugated carbonyl functionalities, respectively. The NMR data of 2, measured in CDCl 3 at −10 • C, were comparable to those of the framework of 1 except in the replacement of an sp 3 oxycarbon (δ C 74.6 ppm, C, C-7) and a methyl group (δ C /δ H 23.7/1.28 ppm, CH 3 , CH 3 -19) in 1 by an exomethylene group (δ C /δ H 148.3 ppm, C and 117.7/5.35 and 5.18 ppm, CH 2 , respectively) in 2. Analysis of NMR data of 2 (Table 1) and correlations found in the 1 H-1 H COSY and HMBC spectra ( Figure 4) enabled the establishment of the gross structure of 2. Furthermore, the observed NOE correlations for 2 ( Figure 5) assigned similar β-positioning for H-1, H-10, H-14, and H 3 -17, and α-orientation for H-2, H-9, and H 3 -18 as found in 1. Moreover, one of the exomethylene protons (δ H 5.18 ppm, s) exhibited NOE interaction with H-10 (δ H 2.97 ppm, br d, J = 7.0 Hz), while the other one (δ H 5.35, s) displayed NOE with the hydroxymethine proton at C-6 (δ H 4.07 ppm, dd, J = 11.0, 4.5 Hz). A molecular modeling investigation revealed that H-6 did not exhibit NOE interaction in the nuclear Overhauser effect spectroscopy (NOESY) with the methylene protons at C-8 (distances >3.7 Å). This designated the α-orientation of 6-OH and, hence, the 6R configuration as shown in Figure 6. Moreover, it was found that eunicellins with similar substitutions in the ten-membered ring, but with an 6β-OH ( Figure 6), displayed distinctive chemical shifts at C-6 (δ C < 74.0 ppm), C-7 (δ C ≥ 150.0 ppm), and H-6 (δ H > 4.25 ppm) [27][28][29][30] than those assigned for 2 (δ 78.3, 148.3, and 4.07 ppm, respectively). This observation also suggests the α-orientation of 6-OH in 2. On the basis of the above findings, the absolute configuration of 1, and the biogenetic consideration, the configuration 1S, 2R,3R,6R,9R,10R,14S,15S was, thus, established for 2.   (Table 1) and correlations found in the 1 H-1 H COSY and HMBC spectra (Figure 4) enabled the establishment of the gross structure of 2. Furthermore, the observed NOE correlations for 2 ( Figure 5) assigned similar β-positioning for H-1, H-10, H-14, and H3-17, and α-orientation for H-2, H-9, and H3-18 as found in 1. Moreover, one of the exomethylene   The HREIMS (m/z 355.1880 [M + Na] + ) and NMR data (Table 1) indicated a molecular formula C 20 H 28 O 4 for briarenone C (3), inferring that 3 is an isomer of 2. Therefore, it was found that 3 possessed an α,β-unsaturated carbonyl (IR: ν max 1668 cm −1 δ C 198.1 ppm, C, 156.4 ppm, C, and δ C /δ H 128.6/5.91 ppm, CH) and a hydroxy methine group (IR: ν max 3299 cm −1 ; δ C /δ H 65.0/4.84 ppm, CH). However, compound 3 differs in the presence of a trisubstituted double bond (δ C /δ H 135.4/5.52 ppm, CH and δ C 128.8 ppm, C), instead of a 1,1-disubstituted double bond in 2, with the appearance of an olefinic methyl (δ C /δ H 29.2/1.93 ppm). The hydroxyl group was found to be linked to the allylic carbon (C-5) due to the HMBC correlation observed from the olefinic methyl (δ H 1.93 ppm, H 3 -19) Mar. Drugs 2019, 17, 120 5 of 10 to the sp 2 methine carbon (δ C 135.4 ppm, C-6), which in turn exhibited 1 H-1 H COSY correlation with H-5 (δ H 4.84 ppm, dd, J = 8.5, 8.5 Hz). Moreover, comparison of NMR data of 3 with those of pachyclavulariaenone D pointed out that 3 is the 5-hydroxy isomer of pachyclavulariaenone D [25]. The gross structure of 3 was further elucidated from the full 2D NMR spectroscopic correlation analyses of 3 (Figure 4). The NOE interaction of H 3 -19 with the olefinic methine protons H-6 (δ H 5.52 ppm, d, J = 8.5 Hz) ( Figure 5), in addition to the downfield shift of C-19 (δ C 29.2 ppm), indicated the Z geometry of the 6,7-double bond [31]. Moreover, the NOE correlations for H-2/H 3 -18, H 3 -18/H-5, H-5/H-8α (δ H 2.74 ppm, d, J = 14.5 Hz), and H-8α/H-9 disclosed the 5S configuration. These NOEs were further validated by a molecular modeling study ( Figure 6). Finally, full NOE correlation analysis ( Figure 5) coupled with the previous identification of the absolute configuration of 1, which was co-isolated along with 3, designated the 1S,2R,3R,5S,9R,10R,14S,15S configuration for 3.
Although briarellin-type diterpenoids (e.g., briarellins A-S) were discovered initially from Caribbean gorgonians of genus Briareum [12][13][14][15][16], since 1995, numerous related analogs were successfully isolated and identified from Pachyclavularia violacea inhabiting Taiwanese [24,25] and Indonesian [26] waters. Unlike the previously identified briarellins, the structures of briarenones A- Figure 6. A partial structure of eunicellin-derived diterpenoids: cladiellisin [27], 3-acetyl cladiellisin [28], pachycladin B [29], and klysimplexins C and E [30]. Although briarellin-type diterpenoids (e.g., briarellins A-S) were discovered initially from Caribbean gorgonians of genus Briareum [12][13][14][15][16], since 1995, numerous related analogs were successfully isolated and identified from Pachyclavularia violacea inhabiting Taiwanese [24,25] and Indonesian [26] waters. Unlike the previously identified briarellins, the structures of briarenones A-C (1-3), isolated in this study from B. violaceum, were found to be similar to those of pachyclavulariaenones [24,25] in possessing the three ring-juncture protons (H-1, H-10, and H-14) cis to each other and forming the cis fusion of the cyclohexane, cyclodecane, and oxepane rings. Also, the determination of the absolute configurations of 1-3 by NOE correlation analysis coupled with X-ray crystallographic analysis of 1 using copper radiation, could imply the absolute configurations for similar briarellins isolated from the genera of Briareum and Pachyclavularia, such as pachyclavulariaenones A-G [24,25], to be similar to that of 1. Compounds of this kind possessing a six-membered carbocyclic ring cis-fused to both of the ten-membered carbocyclic and seven-membered ether rings, making three ring-junction protons (H-1, H-10, and H-14) cis to each other, including those reported from Pachyclavularia violacea [24][25][26], can be considered as a useful chemotaxonomic marker in classification of gorgonians and in approaching a resolution of the argument for considering the gorgonian species of Pachyclavularia to be the same as those of genus Briareum [26,32].
The in vitro anti-inflammatory activities of compounds 1-3 were also measured against the release of elastase and the production of superoxide anions in N-formyl-methionyl-leucyl-phenyl-alanine and cytochalasin B (fMLF/CB)-activated neutrophils. However, the compounds did not show either cytotoxic or anti-inflammatory activities in the tested in vitro models.

Animal Material
The soft coral B. violaceum was collected from Jihui Fish Port (Taitung, Taiwan) by scuba divers at a depth of 10-15 m on March 2013 and stored in a freezer at −20 • C until extraction. Moreover, the soft coral was taxonomically identified by Prof. Chang-Feng Dai, National Taiwan University, Taipei. A voucher specimen was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung.

Extraction and Isolation
The freeze-dried soft coral (0.5 kg) was extracted with EtOAc (3 × 3 L) and filtered. The filtrate was evaporated in vacuo to yield the EtOAc extract (3.90 g). The extract was fractionated by Si gel column chromatography using EtOAc in n-hexane (6.25% to 100%) as a gradient elution system to afford 21 fractions (A01-A21). Fraction A04 was further fractionated on an RP-18 Si gel column, using MeOH-H 2 O (2.5:1) as a mobile phase, into eight subfractions (A0401-A0408). Compound 1 (7.4 mg) was obtained from A405 after two-step purification on RP-HPLC using MeOH-H 2 O (3:2) and acetyl nitrite (CH 3 CN)-H 2 O (1:2). Subfractions A402 and A403 were combined together on the basis of their similar TLC chromatogram and were further separated on RP-HPLC using CH 3   Intensity data of single-crystal X-ray diffraction were measured on a Bruker APEX DUO diffractometer. Of the 13326 reflections collected, only 3658 independent reflections [R(int) = 0.0331] with I > 2σ(I) were used for the analysis. The structure was solved by direct method and refined by a full-matrix least squares on F2 method. The refinement converged to a final R 1 = 0.0401, wR 2 = 0.1064, with goodness-of-fit = 1.091. For coordinates corresponding to the absolute stereochemistry represented, absolute structure parameter 0.04 (6) was obtained [33]. for 10 min with fMLF (100 nM)/CB (0.6 and 0.5 µg·mL −1 for superoxide anion generation and elastase release, respectively). The anti-inflammatory activities of compounds 1-3 were measured with examining the inhibition of fLMF/CB-induced human neutrophils producing superoxide anion and elastase, using UV spectrometer detection at wavelengths of 550 nm and 405 nm, respectively [36,37].

Conclusions
Three new briarellin type diterpenoids, briarenones A-C (1-3), were identified from Briareum violaceum inhabiting Taiwanese waters. The compounds have three cis ring-juncture protons (H-1, H-10, and H-14) due to the cis fusion of the cyclohexane, cyclodecane, and oxepane rings. The molecular structures of this type may be considered as a useful chemotaxonomic marker in the identification of some species of genus Briareum. The absolute configurations of the compounds were assigned on the basis NOE correlation analysis coupled with a single-crystal X-ray diffraction analysis for briarenone A. The isolated metabolites showed no in vitro cytotoxicity against DLD-1, HT-29, and HuCC-T1 cells and did not inhibit the superoxide anion generation or elastase release in fMLF/CB-stimulated neutrophils. Compounds 1-3 did not exhibit cytotoxic and anti-inflammatory activities in this study; however, more biological activity screening should be carried out to discover their pharmaceutical potential. Moreover, the absolute configuration of 1 analyzed by X-ray diffraction would be useful for elucidation of the structurally similar metabolites isolated from the genera Briareum and Pachyclavularia.
Author Contributions: J.-H.S. designed the experiment. Y.C. isolated the compounds and performed spectroscopic data measurement and structure interpretation. A.F.A. performed the spectroscopic data analysis, final structure determination, and preparation of the manuscript. R.S.O. contributed to data analysis and editing of the manuscript. C.-F.D. contributed to species identification of the soft coral.