Polyoxygenated Klysimplexane- and Eunicellin-Based Diterpenoids from the Gorgonian Briareum violaceum

Three new polyoxygenated diterpenoids with a rare 4-isopropyl-1,5,8a-trimethylperhydrophenanthrane structure of the klysimplexane skeleton, briarols A‒C (1‒3), and one eunicellin-based diterpenoid, briarol D (4), were isolated from Briareum violaceum, a gorgonian inhabiting Taiwanese waters. The chemical structures of these compounds were determined by employing extensive analyses of NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Metabolites 1‒3 were found to possess the rarely found skeleton of the diterpenoid klysimplexin T. All isolated compounds showed very weak cytotoxic activity against the growth of three cancer cell lines. A plausible biosynthetic pathway for briarols A‒C from the coexisting eunicellin diterpenoid briarol D (4) was postulated.


Results and Discussion
The lyophilized organism was extracted with ethyl acetate (EtOAc) followed by chromatographic fractionation of solvent-free extract on silica (Si) gel. Fractions showing 1 H NMR signals characteristic of polyoxygenated terpenoids were separated mainly by a reverse-phase (RP) column and high-performance liquid chromatography (RP-HPLC), yielding diterpenoids 1-4 ( Figure 1). The spectra of these compounds are given in the Supplementary Materials ( Figures S4-S40). The IR absorption bands at νmax 3413-3464 cm −1 and the four 13 C NMR signals, resonating at the region of δC 70.7 to 81.9 ppm, disclosed the multi-hydroxylated pattern of the isolated compounds.  (1)(2)(3)(4) and the klysimplexane skeleton (5).

Results and Discussion
The lyophilized organism was extracted with ethyl acetate (EtOAc) followed by chromatographic fractionation of solvent-free extract on silica (Si) gel. Fractions showing 1 H NMR signals characteristic of polyoxygenated terpenoids were separated mainly by a reverse-phase (RP) column and high-performance liquid chromatography (RP-HPLC), yielding diterpenoids 1-4 ( Figure 1). The spectra of these compounds are given in the Supplementary Materials ( Figures S4-S40). The IR absorption bands at ν max 3413-3464 cm −1 and the four 13 C NMR signals, resonating at the region of δ C 70.7 to 81.9 ppm, disclosed the multi-hydroxylated pattern of the isolated compounds.
An inspection of NOESY correlations (Figure 3) enabled us to assign the relative configurations of the seven chiral carbons C-1, C-6, C-8, C-9, C-10, C-11, and C-14 in 4. The NOE correlations observed for the β-oriented ring-juncture proton H-1 with the protons of the 9-oxymethine and one of the 14-isopropyl methyls reflected the α-orientation of H-14 and 9-OH. Furthermore, the NOE observed for H-1/H 3 -18 and H-2/H-4 combined with upfield chemical shift (δ C < 20 ppm) observed for C-18 (δ C 18.3 ppm) determined the E-geometry of the olefinic bond [27]  Deprotonation at C-18 in 8 can produce 2, while reduction at C-3 and dehydrogenation at C-14/C-15 in 8 gives 3. Subsequently, the epoxidation of the olefinic double bond in metabolite 3 affords 1 (Scheme 1). To the best of our knowledge, the biosynthesis of the klysimplexane-and eunicellin-type diterpenoids is limited to marine invertebrates, and there are no analogous structures in terrestrial natural products.
The in vitro cytotoxicity of the new diterpenoid metabolites (1-4) was assessed against the cancer cell lines of human colon cholangiocellular carcinoma (HuCC-T1), human colon carcinoma (HT-29), and human colon adenocarcinoma (DLD-1). The results showed that all compounds only exhibited very weak cytotoxicity against the tested cancer cells, with the IC 50 values ranging from 220.75 to 238.88 µM as compared to doxorubicin hydrochloride (IC 50 1.38 to 2.24 µM). Because of the low yield (< 2.5 mg) and the consumption of the isolated metabolites in measurements of spectroscopic data and cytotoxicity, we suggest that further investigation on other biological activities should be carried out once these tetradroxylated diterpenoid molecules, in particular those with the rare klysimplexane skeleton, can be obtained in sufficient quantities. of polyoxygenated terpenoids, were combined and subfractionated on Si gel CC using acetone-hexane (1:2.5), affording the subfractions F1 and F5. Subfraction F4 was separated on RP-18 Si gel CC using acetyl nitrite (CH 3 CN)-H 2 O (1.5:1 then 1.2:1) to give compounds 2 (1.5 mg), 3 (2.0 mg), and 4 (2.2 mg), respectively. Compound 1 (2.4 mg) was obtained from subfraction F5 with a 3-step purification process with RP-18 Si gel CC using MeOH-H 2 O (1.5:1 then 5:1), RP-HPLC using CH 3

Cytotoxicity Assay
Cancer cell lines (HT-29, HuCC-T1, and DLD-1) were obtained from the American Type Culture Collection (ATCC). Compounds 1-4 were evaluated for the cytotoxic activity using an Alamar blue assay as previously described [28,29]. The intensity of the produced color was measured at 570 nm using an ELISA plate reader.

Conclusions
Three new polyoxygenated diterpenoids of the rare klysimplexane-skeleton, along with a non-ether bridged eunicellin diterpenoid, were discovered from the gorgonian coral Briareum violaceum and named briarols A-D, respectively. A possible biosynthetic pathway for briarols A-C from the coexisting eunicellin diterpenoid was postulated for the first time. Although the compounds did not show potent cytotoxic activity against the tested cancer lines, other possible bioactivities for these metabolites might be worthwhile for further screening. It is noteworthy to mention that this is the first discovery of these rare klysimplexane-type metabolites from a gorgonian coral since the isolation of klysimplexin T from the cultured soft coral Klyxum simplex a decade ago.