Biscembranoids and Cembranoids from the Soft Coral Sarcophyton elegans

Two novel biscembranoids, sarelengans A and B (1 and 2), five new cembranoids, sarelengans C–G (3–7), along with two known cembranoids (8 and 9) were isolated from the South China Sea soft coral Sarcophyton elegans. Their structures were determined by spectroscopic and chemical methods, and those of 1, 4, 5, and 6 were confirmed by single crystal X-ray diffraction. Compounds 1 and 2 represent the first example of biscembranoids featuring a trans-fused A/B-ring conjunction between the two cembranoid units. Their unique structures may shed light on an unusual biosynthetic pathway involving a cembranoid-∆8 rather than the normal cembranoid-∆1 unit in the endo-Diels-Alder cycloaddition. Compounds 2 and 3 exhibited potential inhibitory effects on nitric oxide production in RAW 264.7 macrophages, with IC50 values being at 18.2 and 32.5 μM, respectively.

Sarcophyton elegans is a common soft coral species found on the sea shore of the South China Sea.Previous chemical investigations of S. elegans have led to the isolation of several cembranoids, tetracyclic diterpenoids, steroids, and biscembranoids, some of which showed antibacterial and cytotoxic activities [8,[16][17][18].In our early work aiming at the discovery of antitumor agents from this species, two cembranoids with anti-tumor cell migration properties were isolated [19].In our screening program aimed at the discovery of novel nitric oxide (NO) inhibitors from natural resources [20,21], the EtOAc fraction of the ethanolic extract of S. elegans showed a certain inhibitory activity against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages.Subsequent chemical investigation led to the isolation of two new biscembranoids (1 and 2), five new cembranoids (3)(4)(5)(6)(7), and two known compounds (8 and 9).Compounds 1 and 2 represent the first example of A/B ring trans-fused biscembranoids.Bioassay verified that compounds 2 and 3 were responsible for the NO inhibitory activities of the EtOAc fraction, with IC 50 values being 18.2 and 32.5 µM, respectively.Herein, details of the isolation, structural elucidation, and NO inhibitory activities of these compounds are described.

Results and Discussion
The frozen sample of S. elegans was chopped and exhaustively extracted with 95% EtOH at room temperature (rt).After removal of solvent in vacuo, the residue was suspended in H 2 O and then partitioned sequentially with petroleum ether (PE) and EtOAc.Various column chromatographic separations of the EtOAc extract afforded compounds 1-9 (Figure 1).Subsequent chemical investigation led to the isolation of two new biscembranoids (1 and 2), five new cembranoids (3)(4)(5)(6)(7), and two known compounds (8 and 9).Compounds 1 and 2 represent the first example of A/B ring trans-fused biscembranoids.Bioassay verified that compounds 2 and 3 were responsible for the NO inhibitory activities of the EtOAc fraction, with IC50 values being 18.2 and 32.5 μM, respectively.Herein, details of the isolation, structural elucidation, and NO inhibitory activities of these compounds are described.

Results and Discussion
The frozen sample of S. elegans was chopped and exhaustively extracted with 95% EtOH at room temperature (rt).After removal of solvent in vacuo, the residue was suspended in H2O and then partitioned sequentially with petroleum ether (PE) and EtOAc.Various column chromatographic separations of the EtOAc extract afforded compounds 1-9 (Figure 1).2), two sp 3 quaternary carbons (one oxygenated), nine sp 3 methines (four oxygenated), 10 sp 3 methylenes, and seven methyls.As eight of the 12 DOUs were accounted for by three ketones, an ester carbonyl group, and four double bonds, the remaining DOUs required that 1 was tetracyclic.The aforementioned data are characteristic of a biscembranoid, closely related to those reported in the literature [5,12,22].
Detailed 2D NMR studies (HSQC, 1 H-1 H COSY, and HMBC experiments) further confirmed the presence of two highly oxygenated cembranoid units (a and b) in 1 (Figure 2).Three fragments, C-2C-1C-14, C-6C-7C-8, and C-11C-12C-15C-16 (C-17), were first established in unit a by the  The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure 3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz) [25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4).Thus, compound 1 was given a trivial name sarelengan A.   The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure 3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz) [25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4).Thus, compound 1 was given a trivial name sarelengan A.   The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure 3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz) [25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4).Thus, compound 1 was given a trivial name sarelengan A. The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H 3 -18/H 2 -36b indicated that H-8 and CH 3 -18 occupied the axial bonds of ring B in a chair conformation (Figure 3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H 3 -18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz) [25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4).Thus, compound 1 was given a trivial name sarelengan A. The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure 3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz) [25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4).Thus, compound 1 was given a trivial name sarelengan A.    [13].The E geometry of Δ 30 was assigned by NOE correlation of H-30/H-32.Thus, compound 2 was determined as depicted and was given the trivial name sarelengan B.
It is worth noting that compounds 1 and 2 possessed a trans-fused A/B ring system, differing from those of the previously reported biscembranoids [8,10], suggesting that the coupling between two monemeric cembranoids in 1 and 2 may involve an unusual biosynthetic pathway.As shown in Scheme 1, the well-known soft coral metabolite, methyl sarcoate, was considered as the cembranoid-dienophile precursor, which underwent biohydrogenation to produce a dienophile intermediate 1,2-dihydro-methyl sarcoate.Lobophytone T isolated from Lobophytum pauciflorum [22], was served as the cembranoid-diene precursor, which underwent hydrolysis to generate intermediate i. Dehydration of i followed by a series redox reactions generated the epoxy intermediates (ii and iii) and the tetrahydrofuran-containing intermidiates (iv and v), respectively.The endo-Diels-Alder cycloaddition between methyl sarcoate-Δ 1 and ii-or iv-Δ 21(34) , Δ 35 generates the "normal biscembranoids" (A/B ring cis-fused), sarcophytolide H [13] or bisglaucumlide G [24], respectively, while the endo-Diels-Alder cycloaddition between methyl 1,2-dihydro-methyl sarcoate -Δ 8 and v-or iii-Δ 21(34) , Δ 35 produces the A/B ring trans-fused scaffold of 1 or 2, respectively.[13].The E geometry of ∆ 30 was assigned by NOE correlation of H-30/H-32.Thus, compound 2 was determined as depicted and was given the trivial name sarelengan B.
Compound 4, a colorless crystal, had the molecular formula C 20 H 30 O 5 , as established by HRESIMS.The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences arising from the carbon signals of C-5 (δ C 34.3 in 4; δ C 32.8 in 8) and C-18 (δ C 31.4 in 4; δ C 29.9 in 8), indicating that 4 was a C-4 epimer of 8. Detailed 2D analysis further supported that they shared the same planar structure.The structure of 4 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.08 (10) (Figure 5).Thus, compound 4 was given the trivial name sarelengan D.
Mar. Drugs 2017, 15, 85 6 of 14 Compound 4, a colorless crystal, had the molecular formula C20H30O5, as established by HRESIMS.The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences arising from the carbon signals of C-5 (C 34.3 in 4; C 32.8 in 8) and C-18 (C 31.4 in 4; C 29.9 in 8), indicating that 4 was a C-4 epimer of 8. Detailed 2D analysis further supported that they shared the same planar structure.The structure of 4 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.08 (10) (Figure 5).Thus, compound 4 was given the trivial name sarelengan D. analysis further supported that they shared the same planar structure.The structure of 5 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = −0.01(10) (Figure 6).Thus, compound 5 was given the trivial name sarelengan E. Compound 6, a colorless crystal, exhibited a molecular formula of C20H30O5 as determined by HRESIMS.The 1 H and 13 C-NMR data of 6 showed high similarity to those of sarcophelegan B (9) Detailed 2D analysis further supported that they shared the same planar structure.The structure of 5 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = −0.01(10) (Figure 6).Thus, compound 5 was given the trivial name sarelengan E.
Mar. Drugs 2017, 15, 85 6 of 14 Compound 4, a colorless crystal, had the molecular formula C20H30O5, as established by HRESIMS.The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences arising from the carbon signals of C-5 (C 34.3 in 4; C 32.8 in 8) and C-18 (C 31.4 in 4; C 29.9 in 8), indicating that 4 was a C-4 epimer of 8. Detailed 2D analysis further supported that they shared the same planar structure.The structure of 4 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.08 (10) (Figure 5).Thus, compound 4 was given the trivial name sarelengan D. analysis further supported that they shared the same planar structure.The structure of 5 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = −0.01(10) (Figure 6).Thus, compound 5 was given the trivial name sarelengan E. Compound 6, a colorless crystal, exhibited a molecular formula of C20H30O5 as determined by HRESIMS.The 1 H and 13 C-NMR data of 6 showed high similarity to those of sarcophelegan B (9) Compound 6, a colorless crystal, exhibited a molecular formula of C 20 H 30 O 5 as determined by HRESIMS.The 1 H and 13 C-NMR data of 6 showed high similarity to those of sarcophelegan B (9) [19], except for the replacement of the ∆ 11 in 9 by an oxymethine (δ H 4.87; δ C 80.8) and a methine (δ H 2.69; δ C 44.1) in 6.Since 6 possessed the same DOUs as that of 9, the formation of an oxygen bridge between C-8 and the oxymethine (C-11) was suggested.This was supported by the downfield-shifted carbonyl at C-20 (δ C 175.3 in 6; δ C 168.8 in 9) and C-8 (δ C 90.0 in 6; δ C 79.7 in 9), although no direct HMBC correlation from H-11 to C-8 was available.The structure of 6 was further confirmed by converting 9 to 6 in a basic condition and the absolute configuration (1S, 4R, 8R, 11S, and 12R) was assigned by a single crystal X-ray diffraction analysis with Flack parameter = −0.05(13) (Figure 7).Thus, compound 6 was given a trivial name sarelengan F.
Mar. Drugs 2017, 15, 85 7 of 14 [19], except for the replacement of the  11 in 9 by an oxymethine (H 4.87; C 80.8) and a methine (H 2.69; C 44.1) in 6.Since 6 possessed the same DOUs as that of 9, the formation of an oxygen bridge between C-8 and the oxymethine (C-11) was suggested.This was supported by the downfield-shifted carbonyl at C-20 (C 175.3 in 6; C 168.8 in 9) and C-8 (C 90.0 in 6; C 79.7 in 9), although no direct HMBC correlation from H-11 to C-8 was available.The structure of 6 was further confirmed by converting 9 to 6 in a basic condition and the absolute configuration (1S, 4R, 8R, 11S, and 12R) was assigned by a single crystal X-ray diffraction analysis with Flack parameter = −0.05(13) (Figure 7).Thus, compound 6 was given a trivial name sarelengan F. Compound 7 had a molecular formula of C20H32O5 as established by HRESIMS data.The 1D NMR data of 7 were very similar to those of sartrolide D [28], a known cembranoid isolated from Sarcophyton trocheliophorum, with the only difference being due to the chemical shift of C-7 (C 66.1 in 7; C 72.7 in sartrolide D), indicating that 7 was a C-7 epimer of sartrolide D. Detailed 2D analysis further supported that they shared the same planar structure.The β-orientation of 7-OH was supported by the NOE correlations of H-2/H-7 and H-15.Thus, compound 7 was given the trivial name sarelengan G.
The known compounds, sartrolide E (8) [28] and sarcophelegan B (9) [19], were identified by comparison of their NMR data and optical rotation values with those reported in the literature.
All compounds were evaluated for their inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophages using the Griess assay, and quercetin was used as the positive control (IC50 = 20.0 μM).Compounds 2 and 3 showed moderate inhibitory activities with IC50 values being at 18.2 and 32.5 μM, respectively, while other compounds were inactive (inhibition <50% at 50 μM).To investigate whether the NO inhibition was caused by the cytotoxicity, the effects of compounds 2 and 3 on LPS-induced RAW 264.7 macrophages viability were measured using the MTT method.The results showed that 2 and 3 (up to 80 μM) had no significant cytotoxicity towards the cells in 24 h incubation.

General Experimental Procedures
X-ray data were collected using an Agilent Xcalibur Nova X-ray diffractometer (Agilent, Santa Clara, CA, USA).Melting points were measured on an X-4 melting instrument (SRI International, Silicon Valley, CA, USA) and are uncorrected.Optical rotations were measured on a Rudolph Autopol I automatic polarimeter (Perkin-Elmer, Waltham, MA, USA), UV spectra on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan), and IR spectra were determined on a Bruker The known compounds, sartrolide E (8) [28] and sarcophelegan B (9) [19], were identified by comparison of their NMR data and optical rotation values with those reported in the literature.
All compounds were evaluated for their inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophages using the Griess assay, and quercetin was used as the positive control (IC 50 = 20.0 µM).Compounds 2 and 3 showed moderate inhibitory activities with IC 50 values being at 18.2 and 32.5 µM, respectively, while other compounds were inactive (inhibition <50% at 50 µM).To investigate whether the NO inhibition was caused by the cytotoxicity, the effects of compounds 2 and 3 on LPS-induced RAW 264.7 macrophages viability were measured using the MTT method.The results showed that 2 and 3 (up to 80 µM) had no significant cytotoxicity towards the cells in 24 h incubation.

Animal Material
The soft coral S. elegans were collected from the Xisha Islands in the South China Sea, in October 2014, at a depth of 8-10 m of water.The biological material was frozen immediately until used and was identified by Cheng-Qi Fan from East China Sea Fisheries Research Institute.A voucher specimen (accession number: HLRZ201410) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.
Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1520904).Na, 373.1985).

Chemical Transformation of 9 to 6
Compound 9 (6 mg) was treated with NaOH (1% in MeOH, 1 mL) at room temperature for 2 h.The mixture was then diluted with 5 mL of H 2 O, followed by the extraction of EtOAc (3 × 5 mL).The organic layer was dried and evaporated to give a residue, which was purified on a flash silica gel column eluting with CH 2 Cl 2 to afford 6 (3.4 mg).The structure was confirmed by comparison of its NMR and optical rotation data with those of the natural product.

Cell Culture and Viability Assay
The RAW 264.7 mouse macrophage cell line was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China), and was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA).The cells were supplemented with 3.0 mM glutamine, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 10% heat-inactivated fetal bovine serum at 37 • C under a humidified atmosphere of

Figure 2 .
Figure 2. 1 H 1 H COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple-bond correlation spectroscopy) ( ) correlations of compounds 1 and 3.As for unit b, four spin systems of C-21-C-22, C-24-C-25-C-26, C-28-C-29-C-30, and C-32-C-33 were readily recognized from the 1 H-1 H COSY spectrum.The connections of these fragments, three double bonds, and one sp 3 quaternary carbon were achieved by HMBC correlations, generating the framework of a 14-membered carbon ring C.Although no HMBC correlation was observed between H-30 and C-27, the presence of the oxygen bridge between C-30 and C-27 were deduced by comparison of its 13 C-NMR data with those of bisglaucumlide H [24] bearing the similar tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule.The connection of units a and b was finally achieved by HMBC correlations of H3-18/C-8, C-9, and C-21, H-21/C-8, C-9, C-18, and C-35, and H3-37/C-34, C-35, and C-36, which formed a six-membered carbon ring B bridging these two parts.The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz)[25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure4).Thus, compound 1 was given a trivial name sarelengan A.

Figure 2 .
Figure 2. 1 H 1 H COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple-bond correlation spectroscopy) ( ) correlations of compounds 1 and 3.As for unit b, four spin systems of C-21-C-22, C-24-C-25-C-26, C-28-C-29-C-30, and C-32-C-33 were readily recognized from the 1 H-1 H COSY spectrum.The connections of these fragments, three double bonds, and one sp 3 quaternary carbon were achieved by HMBC correlations, generating the framework of a 14-membered carbon ring C.Although no HMBC correlation was observed between H-30 and C-27, the presence of the oxygen bridge between C-30 and C-27 were deduced by comparison of its 13 C-NMR data with those of bisglaucumlide H [24] bearing the similar tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule.The connection of units a and b was finally achieved by HMBC correlations of H3-18/C-8, C-9, and C-21, H-21/C-8, C-9, C-18, and C-35, and H3-37/C-34, C-35, and C-36, which formed a six-membered carbon ring B bridging these two parts.The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz)[25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure4).Thus, compound 1 was given a trivial name sarelengan A.

Figure 2 .
Figure 2. 1 H 1 H COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple-bond correlation spectroscopy) ( ) correlations of compounds 1 and 3.As for unit b, four spin systems of C-21-C-22, C-24-C-25-C-26, C-28-C-29-C-30, and C-32-C-33 were readily recognized from the 1 H-1 H COSY spectrum.The connections of these fragments, three double bonds, and one sp 3 quaternary carbon were achieved by HMBC correlations, generating the framework of a 14-membered carbon ring C.Although no HMBC correlation was observed between H-30 and C-27, the presence of the oxygen bridge between C-30 and C-27 were deduced by comparison of its 13 C-NMR data with those of bisglaucumlide H [24] bearing the similar tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule.The connection of units a and b was finally achieved by HMBC correlations of H3-18/C-8, C-9, and C-21, H-21/C-8, C-9, C-18, and C-35, and H3-37/C-34, C-35, and C-36, which formed a six-membered carbon ring B bridging these two parts.The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz)[25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure4).Thus, compound 1 was given a trivial name sarelengan A.

Figure 2 .
Figure 2. 1 H 1 H COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple-bond correlation spectroscopy) ( ) correlations of compounds 1 and 3.As for unit b, four spin systems of C-21-C-22, C-24-C-25-C-26, C-28-C-29-C-30, and C-32-C-33 were readily recognized from the 1 H-1 H COSY spectrum.The connections of these fragments, three double bonds, and one sp 3 quaternary carbon were achieved by HMBC correlations, generating the framework of a 14-membered carbon ring C.Although no HMBC correlation was observed between H-30 and C-27, the presence of the oxygen bridge between C-30 and C-27 were deduced by comparison of its 13 C-NMR data with those of bisglaucumlide H [24] bearing the similar tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule.The connection of units a and b was finally achieved by HMBC correlations of H3-18/C-8, C-9, and C-21, H-21/C-8, C-9, C-18, and C-35, and H3-37/C-34, C-35, and C-36, which formed a six-membered carbon ring B bridging these two parts.The relative configuration of 1 was established based on a NOESY experiment and analysis of coupling constants.The strong NOE interactions of H-8/H-22 and H3-18/H2-36b indicated that H-8 and CH3-18 occupied the axial bonds of ring B in a chair conformation (Figure3).Thus, ring A and B were trans-fused.The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were cofacial on ring A and were arbitrarily assigned as β-orientation.The NOE correlations of H-22/H-8 and H-26 and H-26/H-30 suggested that these protons were cofacial on ring C and were assigned as β-orientation, while correlations of H-21/H3-18 and H-32 suggested that these protons were α-oriented.The trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two protons (J = 9.7 Hz)[25].Finally, the structure of 1 including the absolute configuration was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure4).Thus, compound 1 was given a trivial name sarelengan A.

Figure 4 .
Figure 4. ORTEP diagram of 1. Compound 2, a colorless oil, had the molecular formula C41H60O8, as determined by HRESIMS ion at m/z 703.4193 [M + Na] + (calcd.703.4180).The 1D NMR spectra of 2 exhibited most of the structural features found in 1, with major difference being the absence of a tetrahydrofuran ring and the replacement of a terminal double bond by a trisubstituted double bond in 2. The relatively upfield-shifted carbon signals of an oxymethine (δC 64.0, C-26) and a quaternary carbon (δC 61.2, C-27) suggested the presence of an epoxy ring in 2 [26].The epoxy ring was located at C-26 and C-27 by HMBC correlations of H3-39/C-26 and C-27, as well as 1 H-1 H COSY correlation of H2-25/H-26.The trisubstituted double bond was located at C-30 and C-31 by HMBC correlations from H3-40 to C-30 and C-31, and 1 H-1 H COSY correlation of H2-29/H-30.The planar structure of 2 was further confirmed by detailed analyses of its 2D NMR data.The configuration of 2 was assigned to be the same as that of 1 by comparison of their NOESY and 13 C-NMR data.In particular, the NOESY correlations of H-8/H-22 and H3-18/H-36b confirmed the trans-fused A/B ring, while the trans-relationship of H-32 and H-33 was assigned by NOE correlations of H-33/H3-37 and H-32/H-21, as well as the large coupling constant (J = 10.0 Hz) between H-32 and H-33.The NOE correlations of H-22/H-8 and H-26/H-22 and H-28b indicated that H-26 was β-oriented and in a trans-position of CH3-39 on the epoxy ring (S56, Supplementary Materials), which was further confirmed by comparison of the NMR data of C-26 and C-27 in 2 with those of known analogue sarcophytolide H[13].The E geometry of Δ 30 was assigned by NOE correlation of H-30/H-32.Thus, compound 2 was determined as depicted and was given the trivial name sarelengan B.It is worth noting that compounds 1 and 2 possessed a trans-fused A/B ring system, differing from those of the previously reported biscembranoids[8,10], suggesting that the coupling between two monemeric cembranoids in 1 and 2 may involve an unusual biosynthetic pathway.As shown in Scheme 1, the well-known soft coral metabolite, methyl sarcoate, was considered as the cembranoid-dienophile precursor, which underwent biohydrogenation to produce a dienophile intermediate 1,2-dihydro-methyl sarcoate.Lobophytone T isolated from Lobophytum pauciflorum[22], was served as the cembranoid-diene precursor, which underwent hydrolysis to generate intermediate i. Dehydration of i followed by a series redox reactions generated the epoxy intermediates (ii and iii) and the tetrahydrofuran-containing intermidiates (iv and v), respectively.The endo-Diels-Alder cycloaddition between methyl sarcoate-Δ 1 and ii-or iv-Δ21(34) , Δ 35 generates the "normal biscembranoids" (A/B ring cis-fused), sarcophytolide H[13] or bisglaucumlide G[24], respectively, while the endo-Diels-Alder cycloaddition between methyl 1,2-dihydro-methyl sarcoate -Δ 8 and v-or iii-Δ21(34) , Δ 35 produces the A/B ring trans-fused scaffold of 1 or 2, respectively.

Figure 5 .
Figure 5. ORTEP diagram of 4. Compound 5, a colorless crystal, gave a [M + Na] + ion at m/z 373.1984 in the HRESIMS corresponding to the molecular formula C20H30O5.The 1D NMR spectra of 5 showed high similarity to those of sarcophelegan B (9) [19], except for the slight difference of carbon signals of C-5 (C 38.1 in 5; C 36.5 in 9) and C-18 (C 26.9 in 5; C 29.3 in 9), indicating that 5 was a C-4 epimer of 9. Detailed 2Danalysis further supported that they shared the same planar structure.The structure of 5 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = −0.01(10) (Figure6).Thus, compound 5 was given the trivial name sarelengan E.

Figure 5 .
Figure 5. ORTEP diagram of 4.Compound 5, a colorless crystal, gave a [M + Na] + ion at m/z 373.1984 in the HRESIMS corresponding to the molecular formula C20H30O5.The 1D NMR spectra of 5 showed high similarity to those of sarcophelegan B (9)[19], except for the slight difference of carbon signals of C-5 (C 38.1 in 5; C 36.5 in 9) and C-18 (C 26.9 in 5; C 29.3 in 9), indicating that 5 was a C-4 epimer of 9. Detailed 2D analysis further supported that they shared the same planar structure.The structure of 5 including the absolute configuration (1R, 4S, and 8R) was secured by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, Flack parameter = −0.01(10) (Figure6).Thus, compound 5 was given the trivial name sarelengan E.
a in methanol-d 4 ; b in CDCl 3 .