Briarenols Q–T: Briaranes from A Cultured Octocoral Briareum stechei (Kükenthal, 1908)

Our continuous chemical study of a cultured octocoral Briareum stechei led to the isolation of four new briarane diterpenoids, briarenols Q–T (1–4). The structures of new metabolites 1–4 were established by spectroscopic methods, and compounds 3 and 4 were found to inhibit the generation of inducible nitric oxide synthase (iNOS) from RAW 264.7 stimulated by lipopolysaccharides (LPS).


Introduction
Since 1950, the nucleoside spongothymidine, which is obtained from the Caribbean sponge Cryptotethia [1], and the derivative of this nucleoside 1-β-D-arabinofuranosylcytosine (Ara-C) have been approved as the first marine-origin clinical medications used in treatment of leukemia in 1969. Marine natural products (MNPs) from marine invertebrates, such as Porifera and Cnidaria, played important roles in drug discovery due to their complex structures and interesting bioactivities [2,3]. Most of the pharmaceutical coral reef organisms are claimed to be endangered species. In order to protect natural populations and habitats of these marine organisms from overexploitation and to support bioactive materials for further study and medicinal use [4], a cultured octocoral Briareum stechei (Kükenthal, 1908), which was previously identified as Briareum excavatum (Nutting, 1911) [5], was studied for its interesting chemical constituents related to briarane-type diterpenoids. Herein, we report on the isolation, structure determination, and anti-inflammatory activity of four unreported isolates, briarenols Q-T (1-4) (Figure 1).

Results and Discussion
The octocoral B. stechei was harvested in an 80 ton culturing tank in April 2016. This target organism was freeze-dried and ground, followed by exhaustive extraction in a mixture of dichloromethane (CH2Cl2) and methanol (MeOH). Normal-and reverse-phase HPLC yielded four new briarane diterpenoids, briarenols Q-T (1-4).

Results and Discussion
The octocoral B. stechei was harvested in an 80 ton culturing tank in April 2016. This target organism was freeze-dried and ground, followed by exhaustive extraction in a mixture of dichloromethane (CH 2 Cl 2 ) and methanol (MeOH). Normal-and reverse-phase HPLC yielded four new briarane diterpenoids, briarenols Q-T (1-4).
Briarenol Q (1), [α] 25 D +22 (c 0.1, CHCl 3 ), was obtained as an amorphous powder with the molecular formula C 27 H 34 O 12 , based on its positive-mode high-resolution electrospray ionization mass spectrum (HRESIMS) at m/z 573.19426 (calculated for C 27 H 34 O 12 Na + , 573.19425), with 11 indices of hydrogen deficiency (IHDs). The IR spectrum showed the presence of hydroxy (ν max 3408 cm -1 ), γ-lactone (ν max 1783 cm -1 ), and ester carbonyl (ν max 1733 cm -1 ) groups. The 1 H, 13 C, and heteronuclear single quantum coherence (HSQC) spectra indicated the presence of seven methyls, two sp 3 methylenes, six sp 3 methines, three sp 3 quaternary carbons, two sp 2 methines, two quaternary olefinic carbons, and five ester-equivalents (Tables 1 and 2), accounting for all carbons and 33 of the 34 protons. Two of the sp 3 quaternary carbons had 13 C chemical shifts at δ C 80.8 and 94.0, indicative of an oxygen-bearing quaternary carbon and a hemiketal carbon, respectively. A tetracyclic scaffold was deduced by IHDs, as seven of the 11 unsaturation degrees could be assigned to a pair of carbon-carbon double bonds (δ C 138.4, C-5; 128.0, CH-6; 131.1, C-11; 122.0, CH-12) and five esters (δ C 164.6, 169.2, 169.7, 173.2, 174.9).  Figure 2), which were assembled with the assistance of a heteronuclear multiple-bond correlation (HMBC) experiment. The 2 Jor 3 J-1 H-13 C long-range correlations between H-2, H-3α, H-6/C-4, H-3α/C-2, H-9/C-1, and H-9/C-11 permitted elucidation of the main carbon skeleton of 1. A vinyl methyl at C-11 was confirmed by an allylic coupling between H-12 (δ H 5.56) and H 3 -20 (δ H 1.96) in the COSY experiment. The 2 Jor 3 J-1 H-13 C long-range correlations between H-2, H-3α, H-6/C-4, H-3α/C-2, H-9/C-1, and H-9/C-11 permitted elucidation of the main carbon skeleton of 1. A vinyl methyl at C-11 was confirmed by an allylic coupling between H-12 (δ H 5.56) and H 3 -20 (δ H 1.96) in the COSY experiment and by the HMBC between H 3 -20/C-11 and C-12. The ring junction Me-15 at C-1 was supported by the HMBC between H 3 -15/C-1, C-2, C-10, C-14, and H-2/C-15. Thus, the methyl-esterified carboxyl group at C-5 was supported by an HMBC between δ H 3.81 (3H, s, -OMe), with δ C 164.6 (C-16). The acetate ester at C-9 was established by a correlation between H-9 (δ H 6.05) and the acetate carbonyl at δ C 169.7 observed in the HMBC spectrum. The C-4 hydroxy group was concluded to be a part of a hemiketal constellation on the basis of a characteristic carbon signal at δ C 94.0 (a quaternary Mar. Drugs 2020, 18, 383 4 of 10 hemiketal carbon, C-4). An oxygenated quaternary carbon signal at δ C 80.8 showed a 3 J-coupling with the methyl protons at δ H 1.46 (H 3 -18). In total, 8 of the 12 oxygen atoms in the molecular formula could be accounted for the presence of a γ-lactone, an ester, a hemiketal, and an α,β-unsaturated methyl esterified carboxyl group. Thus, the remaining four oxygen atoms had to be positioned at C-2 and C-14 as acetoxy groups, respectively, as indicated by their 1 H and 13 C NMR chemical shifts (δ H 5.11, 1H, d, J = 7.2 Hz; δ C 72.1, CH-2; δ H 5.18, 1H, d, J = 4.2 Hz; δ C 71.8, CH-14), although no HMBC correlation was observed from H-2 and H-14 to any acetate carbonyl. These findings, together with the HMBC between H-17/C-19 and H 3 -18/C-17 and C-19, were used to establish the molecular framework of 1.   The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton   The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton , although no HMBC correlation was observed from H-2 and H-14 to any acetate carbonyl. These findings, together with the HMBC between H-17/C-19 and H3-18/C-17 and C-19, were used to establish the molecular framework of 1.  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton ), and nuclear Overhauser effect spectroscopy (NOESY;   The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton H-10 exhibited correlations with H-2, H-9, and H 3 -18, while H-14 correlated with H 3 -15, setting the Me-15 at C-1 as trans to H-10, as observed in all naturally occurring briarane-type diterpenoids [7]. Due to Me-18 at C-17 being α-oriented in the γ-lactone moiety, H-17 should be positioned on the β face. This proton showed a slight correlation with H-7, indicating that H-7 was β-oriented. H-7 showed a correlation with H-6, and a coupling constant (J = 4.8 Hz) was detected between H-7 and H-6, indicating that the dihedral angle between H-6 and H-7 is approximately 60 • , and that H-6 is β-oriented. The hydroxy proton at δ H 6.11 (OH-4) displayed a light correlation with H-2, setting the hydroxy group at C-4 in an S*-configuration. The NOESY spectrum also showed correlations of H-6/ H-7 and OH-4/H-2 and with H-12/H 3 -20, revealing the E-geometry and Z-geometry of C-5/C-6 and C-11/C-12 double bonds, respectively. The remaining stereogenic carbon, C-8, lacked a proton, but there were correlations between H-7/H-17 and H-7/H-9, indicating that C-8 was in an R*-configuration, as evidenced by modeling analysis. Based on the above findings, the relative configurations of stereogenic carbons of 1 were elucidated as 1R*,2S*,4S*,7S*,8R*,9S*,10S*,14S*, and 17R*. As briaranes 1-4 were isolated along with the known briaranes excavatolide A and briaexcavatolide F [8,9] from the same target organism, the absolute configurations of these two compounds were determined by single-crystal X-ray diffraction analysis [10]. Therefore, it is reasonable on biogenetic grounds to conclude that briaranes 1-4 have the same absolute configuration as those of excavatolide A and briaexcavatolide F. Based on the above findings, the configurations of the stereogenic carbon of 1 were elucidated as 1R,2S,4S,7S,8R,9S,10S,14S, and 17R (Supplementary Materials, Figures S1-S10).
Briarane 2 (briarenol R) was isolated as an amorphous powder that showed two sodiated adduct ion peaks in (+)-HRESIMS at m/z 519.13912 and 521.13596 (3:1), which accounted for a chlorine atom in the molecular formula, C 24 H 29 35 ClO 9 (calculated for C 24 H 29 35 ClO 9 + Na, 519.13923) (10 degrees of unsaturation). The IR spectrum of 2 showed α,β-unsaturated ketone, ester carbonyl, γ-lactone, and broad OH stretching at 1682, 1742, 1783, and 3455 cm -1 , respectively. From the 13 C and 1 H NMR (Tables 1 and 2 [11] indicated the 3,4-epoxide group towards the α-side of the briarane system as that of 5. Furthermore, the 1D and 2D NMR spectra revealed that the signals corresponding to the 11α-hydroxy group in 5 were replaced by those of a proton in 2. Therefore, briarenol R (2) was assigned as having a structure with the same stereochemistry as 5 because of the stereogenic carbons that 2 has in common with 5, as confirmed by correlations observed in the NOESY spectrum ( Figure 3). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as 1S,2R,3R,4R,6S, 7R,8R,9S,10S,11R, and 17R (Supplementary Materials, Figures S11-S20). The molecular formula of briarenol S (3), containing a chlorine atom, was found to be C24H31ClO9 by (+)-HRESIMS at m/z 521.15499 (calculated for C24H31 35 ClO9, 521.15488). The IR spectrum showed absorptions at 3459 cm -1 due to hydroxy groups at 1777, 1737, and 1678 cm -1 , due to γ-lactone, ester carbonyl, and α,β-unsaturated ketonic carbonyl groups, respectively. The 13 C and 1 H NMR spectra (Tables 1 and 2) disclosed the signals due to five methyls, an sp 3 methylene, eight sp 3 methines, two sp 3 quaternary carbons, an sp 2 methylene, two sp 2 methines, an sp 2 quaternary carbon, and four carbonyl carbons. The low-field ester carbonyl carbon (δC 175.6), coupled with the IR absorption at 1777 cm -1 , suggested the presence of a γ-lactone moiety in 3. The high-field ketonic carbonyl carbon at δC 202.4 suggested that the carbonyl group was conjugated with a carbon-carbon double bond. The 1 H NMR spectrum (Table 2) showed the signals due to two secondary methyls, a tertiary metyl, two acetate methyls, an aliphatic methylene, three aliphatic methines, a methine bearing a chlorine atom, four oxymethines, an exomethylene, and two olefinic methines. These spectroscopic data, coupled with the degrees of unsaturation (IHDs = 9), suggested that compound 3 is a tricyclic diterpenoid with a γ-lactone, an α,β-unsaturated ketone, an exocyclic olefin, and two acetoxy groups. It was found that the spectroscopic data of 3 were similar to those of a known briarane, solenolide E (6), isolated from a soft coral identified as Solenopodium sp. (= Briareum sp.) [5], collected in the Western Caroline Islands of Palau [12], except that the signals corresponding to one of the C-4 methylene protons in 6 were replaced by signals for an acetoxy group in 3. In the NOESY experiment, H-4 showed a correlation with H-2, revealing that the acetoxy group at C-4 is on the β face in 3. The HMBC and COSY correlations, as shown in Figure 4, provided the gross structure for 3. Hence, briarenol S (3) was found to be the 4β-acetoxy derivative of 6 and the stereochemistry of the stereogenic carbons in 3 was deduced by NOESY analysis (Figure 4)    The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton  The stereochemical evaluation of 1 was approached using a nuclear Overh spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that by computer modeling [6] and that obtained from vicinal proton coupling constant ana  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton The molecular formula of briarenol S (3), containing a chlorine atom, was found to be C 24 H 31 ClO 9 by (+)-HRESIMS at m/z 521.15499 (calculated for C 24 H 31 35 ClO 9 , 521.15488). The IR spectrum showed absorptions at 3459 cm -1 due to hydroxy groups at 1777, 1737, and 1678 cm -1 , due to γ-lactone, ester carbonyl, and α,β-unsaturated ketonic carbonyl groups, respectively. The 13 C and 1 H NMR spectra (Tables 1 and 2) disclosed the signals due to five methyls, an sp 3 methylene, eight sp 3 methines, two sp 3 quaternary carbons, an sp 2 methylene, two sp 2 methines, an sp 2 quaternary carbon, and four carbonyl carbons. The low-field ester carbonyl carbon (δ C 175.6), coupled with the IR absorption at 1777 cm -1 , suggested the presence of a γ-lactone moiety in 3. The high-field ketonic carbonyl carbon at δ C 202.4 suggested that the carbonyl group was conjugated with a carbon-carbon double bond. The 1 H NMR spectrum (Table 2) showed the signals due to two secondary methyls, a tertiary metyl, two acetate methyls, an aliphatic methylene, three aliphatic methines, a methine bearing a chlorine atom, four oxymethines, an exomethylene, and two olefinic methines. These spectroscopic data, coupled with the degrees of unsaturation (IHDs = 9), suggested that compound 3 is a tricyclic diterpenoid with a γ-lactone, an α,β-unsaturated ketone, an exocyclic olefin, and two acetoxy groups. It was found that the spectroscopic data of 3 were similar to those of a known briarane, solenolide E (6), isolated from a soft coral identified as Solenopodium sp. (= Briareum sp.) [5], collected in the Western Caroline Islands of Palau [12], except that the signals corresponding to one of the C-4 methylene protons in 6 were replaced by signals for an acetoxy group in 3. In the NOESY experiment, H-4 showed a correlation with H-2, revealing that the acetoxy group at C-4 is on the β face in 3. The HMBC and COSY correlations, as shown in Figure 4, provided the gross structure for 3. Hence, briarenol S (3) was found to be the 4β-acetoxy derivative of 6 and the stereochemistry of the stereogenic carbons in 3 was deduced by NOESY analysis (Figure 4)  The molecular formula of briarenol S (3), containing a chlorine atom, was found to be C24H31ClO9 by (+)-HRESIMS at m/z 521.15499 (calculated for C24H31 35 ClO9, 521.15488). The IR spectrum showed absorptions at 3459 cm -1 due to hydroxy groups at 1777, 1737, and 1678 cm -1 , due to γ-lactone, ester carbonyl, and α,β-unsaturated ketonic carbonyl groups, respectively. The 13 C and 1 H NMR spectra (Tables 1 and 2) disclosed the signals due to five methyls, an sp 3 methylene, eight sp 3 methines, two sp 3 quaternary carbons, an sp 2 methylene, two sp 2 methines, an sp 2 quaternary carbon, and four carbonyl carbons. The low-field ester carbonyl carbon (δC 175.6), coupled with the IR absorption at 1777 cm -1 , suggested the presence of a γ-lactone moiety in 3. The high-field ketonic carbonyl carbon at δC 202.4 suggested that the carbonyl group was conjugated with a carbon-carbon double bond. The 1 H NMR spectrum (Table 2) showed the signals due to two secondary methyls, a tertiary metyl, two acetate methyls, an aliphatic methylene, three aliphatic methines, a methine bearing a chlorine atom, four oxymethines, an exomethylene, and two olefinic methines. These spectroscopic data, coupled with the degrees of unsaturation (IHDs = 9), suggested that compound 3 is a tricyclic diterpenoid with a γ-lactone, an α,β-unsaturated ketone, an exocyclic olefin, and two acetoxy groups. It was found that the spectroscopic data of 3 were similar to those of a known briarane, solenolide E (6), isolated from a soft coral identified as Solenopodium sp. (= Briareum sp.) [5], collected in the Western Caroline Islands of Palau [12], except that the signals corresponding to one of the C-4 methylene protons in 6 were replaced by signals for an acetoxy group in 3. In the NOESY experiment, H-4 showed a correlation with H-2, revealing that the acetoxy group at C-4 is on the β face in 3. The HMBC and COSY correlations, as shown in Figure 4, provided the gross structure for 3. Hence, briarenol S (3) was found to be the 4β-acetoxy derivative of 6 and the stereochemistry of the stereogenic carbons in 3 was deduced by NOESY analysis (Figure 4)   and C-19, were used to establish the molecular framework of 1.  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton ), HMBC ( Mar. Drugs 2020, 18, x unsaturated methyl esterified carboxyl group. Thus, the remaining four oxygen atom positioned at C-2 and C-14 as acetoxy groups, respectively, as indicated by their 1 H an chemical shifts (δH 5.11, 1H, d, J = 7.2 Hz; δC 72.1, CH-2; δH 5.18, 1H, d, J = 4.2 Hz; δC 7 although no HMBC correlation was observed from H-2 and H-14 to any acetate carb findings, together with the HMBC between H-17/C-19 and H3-18/C-17 and C-19, w establish the molecular framework of 1.  The stereochemical evaluation of 1 was approached using a nuclear Overh spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that by computer modeling [6] and that obtained from vicinal proton coupling constant ana ), and NOESY ( , although no HMBC correlation was observed from H-2 and H-14 to any acetate carbonyl. These findings, together with the HMBC between H-17/C-19 and H3-18/C-17 and C-19, were used to establish the molecular framework of 1.  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton Our present study has led to the isolation of a new briarane, briarenol T (4). Its molecular formula C 24 H 32 O 8 was deduced from (+)-HRESIMS at m/z 471.19879 (calculated for C 24 H 32 O 8 + Na, 471.19894). The IR spectrum showed absorptions that indicated three different carbonyl types: γ-lactones (ν max 1772 cm −1 ), esters (ν max 1740 cm -1 ), and α,β-unsaturated ketones (ν max 1683 cm -1 ). The latter structural feature was confirmed by the presence of signals at δ C 202.6 (C-12), 154.6 (CH-14), and 124.1 (CH-13) in the 13 C NMR spectrum (Table 1), and the presence of a mutually coupled pair of doublet signals in the 1 H NMR spectrum at δ H 5.85 (H-13) and 6.39 (H-14) (J = 10.2 Hz) corresponding to the αand β-olefinic protons, respectively ( Table 2). The spectroscopic data of 4 were similar to those of a known diterpene, cavernulin B (7) (Figure 1), isolated from a sea pen, Cavernularia sp., collected from the Eastern Coast of Bay of Bengal near Digna, India [13], except that the signals corresponding to the 12-hydroxy group in 7 disappeared and were replaced by a ketone group in 4, as assessed by comparing the related spectroscopic data of 4 with those of 7. The locations of functional groups were confirmed by 2D NMR correlations ( Figure 5), and hence the structure of briarenol T was assigned as 4, and the configurations of the stereogenic carbons were elucidated as 1S,2S,7S,8R,9S,10S,11R, and 17R ( Figure 4) (Supplementary Materials, Figures S32-S41).
Mar. Drugs 2020, 18, x 7 of 10 Our present study has led to the isolation of a new briarane, briarenol T (4). Its molecular formula C24H32O8 was deduced from (+)-HRESIMS at m/z 471.19879 (calculated for C24H32O8 + Na, 471.19894). The IR spectrum showed absorptions that indicated three different carbonyl types: γ-lactones (νmax 1772 cm −1 ), esters (νmax 1740 cm -1 ), and α,β-unsaturated ketones (νmax 1683 cm -1 ). The latter structural feature was confirmed by the presence of signals at δC 202.6 (C-12), 154.6 (CH-14), and 124.1 (CH-13) in the 13 C NMR spectrum (Table 1), and the presence of a mutually coupled pair of doublet signals in the 1 H NMR spectrum at δH 5.85 (H-13) and 6.39 (H-14) (J = 10.2 Hz) corresponding to the α-and βolefinic protons, respectively ( Table 2). The spectroscopic data of 4 were similar to those of a known diterpene, cavernulin B (7) (Figure 1), isolated from a sea pen, Cavernularia sp., collected from the Eastern Coast of Bay of Bengal near Digna, India [13], except that the signals corresponding to the 12hydroxy group in 7 disappeared and were replaced by a ketone group in 4, as assessed by comparing the related spectroscopic data of 4 with those of 7. The locations of functional groups were confirmed by 2D NMR correlations (  The effects of briaranes 1-4 on the release of iNOS and COX-2 from LPS-stimulated RAW 264.7 macrophage cells were assessed (Table 3). Briaranes 3 and 4 were found to inhibit the release of iNOS to 78.50 and 79.95%, respectively. It is interesting to note that 2 enhanced the expression of COX-2 to 112.96%, as compared to results of the cells stimulated with LPS only (Supplementary Materials, Figures S37).  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton ), HMBC (  The stereochemical evaluation of 1 was approached using a nuclear Overh spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that by computer modeling [6] and that obtained from vicinal proton coupling constant ana ), and NOESY (  The stereochemical evaluation of 1 was approached using a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and was found to be compatible with that of 1 offered by computer modeling [6] and that obtained from vicinal proton coupling constant analysis. Proton The effects of briaranes 1-4 on the release of iNOS and COX-2 from LPS-stimulated RAW 264.7 macrophage cells were assessed (Table 3). Briaranes 3 and 4 were found to inhibit the release of iNOS to 78.50 and 79.95%, respectively. It is interesting to note that 2 enhanced the expression of COX-2 to 112.96%, as compared to results of the cells stimulated with LPS only (Supplementary Materials, Figure S37).