marine drugs Article
Biscembranoids and Cembranoids from the Soft Coral Sarcophyton elegans Wei Li, Yi-Hong Zou, Man-Xi Ge, Lan-Lan Lou, Yun-Shao Xu, Abrar Ahmed, Yun-Yun Chen, Jun-Sheng Zhang, Gui-Hua Tang and Sheng Yin * School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China;
[email protected] (W.L.);
[email protected] (Y.-H.Z.);
[email protected] (M.-X.G.);
[email protected] (L.-L.L.);
[email protected] (Y.-S.X.);
[email protected] (A.A.);
[email protected] (Y.-Y.C.);
[email protected] (J.-S.Z.);
[email protected] (G.-H.T.) * Correspondence:
[email protected]; Tel.: +86-20-3994-3090 Academic Editors: Kirsten Benkendorff and Vassilios Roussis Received: 13 February 2017; Accepted: 20 March 2017; Published: 23 March 2017
Abstract: 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. Keywords: Sarcophyton elegans; biscembranoids; cembranoids; NO inhibition
1. Introduction Biscembranoids are a group of unique tetraterpenoids exclusively occurring in the soft corals of the genera of Sarcophyton, Sinularia, and Lobophytum (family Alcyoniidae) [1–5]. Biosynthetically they are proposed to be derived from the Diels-Alder cycloaddition between two cembranoid monomers, forming a 14/6/14 (A/B/C) carbon ring system [6–10]. Since the first biscembranoid named methyl isosartortuoate was reported from the Chinese soft coral Sarcophyton tortuosum [1], more than 80 biscembranoids have been isolated so far [11]. The structural diversity of these biscembranoids mainly arise from the oxygenation and cyclization of the cembranoid-diene part (ring C), with maintaining of the basic structural features of cembranoid-dienophile moiety (ring A). Due to their significant biological activities (e.g., cytotoxic [2] and antibacterial [8] properties) and appealing architectures, biscembranoids have attracted broad interests from both natural product [12,13] and synthetic chemists [14,15] over the last decades. 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 [8,16–18]. 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 (3–7), Mar. Drugs 2017, 15, 85; doi:10.3390/md15040085
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and two known compounds (8 and 9). Compounds 1 and 2 represent the first example of A/B ring Subsequent chemical investigation led to the isolation of two new biscembranoids (1 and 2), five new trans-fused biscembranoids. Bioassay verified that(8 and 9). Compounds 1 and 2 represent the compounds 2 and 3 were responsible forfirst the NO cembranoids (3–7), and two known compounds example of A/B ring trans‐fused biscembranoids. Bioassay verified compounds and respectively. 3 were inhibitory activities of the EtOAc fraction, with IC beingthat 18.2 and 32.5 2 µM, 50 values responsible for the NO inhibitory activities of the EtOAc fraction, with 50 values being 18.2 and Herein, details of the isolation, structural elucidation, and NO inhibitory IC activities of these compounds 32.5 μM, respectively. Herein, details of the isolation, structural elucidation, and NO inhibitory are described. activities of these compounds are described.
2. Results and Discussion
2. Results and Discussion
The frozen sample of S. elegans was chopped and exhaustively extracted with 95% EtOH at room The frozen sample of S. elegans was chopped and exhaustively extracted with 95% EtOH at temperature (rt). After removal of solvent in vacuo, the residue was suspended in H2 O and then room temperature (rt). After removal of solvent in vacuo, the residue was suspended in H 2 O and partitioned sequentially with petroleum (PE) and EtOAc. Various column chromatographic then partitioned sequentially with ether petroleum ether (PE) and EtOAc. Various column separations of the EtOAc extract afforded compounds 1–9 (Figure 1). chromatographic separations of the EtOAc extract afforded compounds 1–9 (Figure 1).
Figure 1. Structures of compounds 1–9.
Figure 1. Structures of compounds 1–9. Compound 1, a colorless crystal, had the molecular formula C41H60O9, as established by
Compound 1, a colorless crystal, had the molecular formula C41 H60 O9 , as established by HRESIMS + (calcd. 719.4130), corresponding to 12 degrees of unsaturation HRESIMS at m/z 719.4121 [M + Na] + −1 indicated the presence of the hydroxyl and at m/z 719.4121 [M + Na] (calcd. 719.4130), corresponding to 12 degrees of unsaturation (DOUs). (DOUs). The IR absorption bands at 3408 and 1710 cm − 1 revealed the hydroxyl presence of carbonyl groups, respectively. Detailed analysis of the 1H‐NMR The IR absorption bands at 3408 and 1710 cm indicated the presence of the andseven carbonyl methyl groups [δ 0.92 (3H × 2, d, J = 6.7 Hz), 1.07 (3H, s), 1.08 (3H, s), 1.54 (3H, s), 1.70 (3H, s), and groups, respectively. HDetailed analysis of the 1 H-NMR revealed the presence of seven methyl groups 1.86 (3H, d, J = 1.2 Hz)], one methoxy group [δ H 3.63 (3H, s)], four oxymethine protons [δH 3.47 (1H, [δH 0.92 (3H × 2, d, J = 6.7 Hz), 1.07 (3H, s), 1.08 (3H, s), 1.54 (3H, s), 1.70 (3H, s), and 1.86 (3H, d, m), 3.89 (1H, dd, J = 10.2, 4.2 Hz), 4.07 (1H, d, J = 9.7 Hz), and 4.39 (1H, d, J = 9.7 Hz)], a terminal J = 1.2 Hz)], one methoxy group [δH 3.63 (3H, s)], four oxymethine protons [δH 3.47 (1H, m), 3.89 (1H, double bond [δH 5.21 (1H, s) and 5.47 (1H, s)], two olefinic protons [δH 4.88 (1H, d, J = 10.6 Hz) and dd, J = 10.2, 4.2 Hz), 4.07 (1H, d, J = 9.7 Hz), and 4.39 (1H, d, J = 9.7 Hz)], a terminal 13double bond 5.96 (1H, d, J = 1.2 Hz)], and a series of aliphatic methylene or methine multiplets. The C‐NMR [δH 5.21 (1H, s) and 5.47 (1H, s)], two olefinic protons [δH 4.88 (1H, d, J = 10.6 Hz) and 5.96 (1H, spectrum, in combination with DEPT experiments, resolved 41 carbon resonances attributable to d, J =three ketone groups (δ 1.2 Hz)], and a series of aliphatic methylene or methine multiplets. The 13 C-NMR spectrum, C 215.5, 215.4 and 203.4), a methyl ester group (δC 176.3 and 52.3), a terminal in combination with DEPT experiments, resolved 41 carbon resonances attributable to three ketone double bond (δ C 151.6 and 112.8), two trisubstituted double bonds (δ C 154.1, 136.9, 128.2, and 127.4), groups (δC 215.5, 215.4 and 203.4), aC 132.7 and 130.2), two sp methyl ester group (δC3 quaternary carbons (one oxygenated), 176.3 and 52.3), a terminal double bond a tetrasubstituted double bond (δ nine sp (δC 151.6 and3 methines (four oxygenated), 10 sp 112.8), two trisubstituted double3 methylenes, and seven methyls. As eight of the 12 DOUs bonds (δC 154.1, 136.9, 128.2, and 127.4), a tetrasubstituted 3 were accounted for by three ketones, an ester carbonyl group, and four double bonds, the remaining double bond (δC 132.7 and 130.2), two sp quaternary carbons (one oxygenated), nine sp3 methines (four DOUs required that 1 was tetracyclic. The aforementioned data are characteristic of a biscembranoid, oxygenated), 10 sp3 methylenes, and seven methyls. As eight of the 12 DOUs were accounted for closely related to those reported in the literature [5,12,22]. by three ketones, an ester carbonyl group,1 and four double bonds, the remaining DOUs required that 1 Detailed 2D NMR studies (HSQC, H–1H COSY, and HMBC experiments) further confirmed the was tetracyclic. The aforementioned data are characteristic of a biscembranoid, closely related to those presence of two highly oxygenated cembranoid units (a and b) in 1 (Figure 2). Three fragments, reported in the literature [5,12,22]. C‐2C‐1C‐14, C‐6C‐7C‐8, and C‐11C‐12C‐15C‐16 (C‐17), were first established in unit a by the 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 1 H–1 H COSY correlations. The connectivities of these fragments, three ketones, one double bond,
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1H– 1H COSY correlations. The connectivities of these fragments, three ketones, one double bond, one 1H COSY correlations. The connectivities of these fragments, three ketones, one double bond, one H– 1H–1H COSY correlations. The connectivities of these fragments, three ketones, one double bond, one 1H–1H COSY correlations. The connectivities of these fragments, three ketones, one double bond, one quaternary carbon, and one methyl ester were achieved correlations 3‐18/C‐8, C‐9, quaternary carbon, and one methyl ester were achieved by by HMBC correlations of of Hof 3H ‐18/C‐8, C‐9, one quaternary carbon, and one methyl ester were achieved byHMBC HMBC correlations H C-9, 3 -18/C-8, quaternary carbon, and one methyl ester were achieved by HMBC HMBC correlations of H3‐18/C‐8, C‐9, quaternary carbon, and one methyl ester were achieved by correlations 3‐18/C‐8, C‐9, and C‐10, H 3‐19/C‐4, C‐5, and C‐6, H 2‐2(H‐4)/C‐3, H 2‐11/C‐10, and C‐12, and H 2H ‐14/C‐13, and C‐20, and C‐10, H 3‐19/C‐4, C‐5, and C‐6, H 2‐2(H‐4)/C‐3, H 2‐11/C‐10, and C‐12, and H 2of ‐14/C‐13, and C‐20, andand C‐10, H C-10, H3 -19/C-4, C-5, and C-6, H -2(H-4)/C-3, H -11/C-10, and C-12, and H -14/C-13, and C-20, 2 2‐2(H‐4)/C‐3, H22‐11/C‐10, and C‐12, and H2‐14/C‐13, and C‐20, 2 3‐19/C‐4, C‐5, and C‐6, H and C‐10, H 3‐19/C‐4, C‐5, and C‐6, H 2‐2(H‐4)/C‐3, H2‐11/C‐10, and C‐12, and H2‐14/C‐13, and C‐20, which generated a methyl sarcoate moiety (ring A) commonly found in biscembranoids [23]. which generated a methyl sarcoate moiety (ring A) commonly found in biscembranoids [23]. which generated a methyl sarcoate moiety (ring A) commonly found in biscembranoids which generated a methyl sarcoate moiety (ring A) commonly found in biscembranoids [23]. which generated a methyl sarcoate moiety (ring A) commonly found in biscembranoids [23]. [23]. 1
Figure 2. H COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple‐bond 11H− 1H COSY (correlation spectroscopy) ( 11 Figure 2. HH COSY (correlation spectroscopy) ( ) and key HMBC (heteronuclear multiple‐bond ) and key HMBC (heteronuclear multiple-bond Figure 2. ) and key HMBC (heteronuclear multiple‐bond Figure 2. H1H H COSY (correlation spectroscopy) ( 1H1H COSY (correlation spectroscopy) ( correlation spectroscopy) ( ) correlations of compounds 1 and 3. Figure 2. ) and key HMBC (heteronuclear multiple‐bond correlation spectroscopy) ( ) correlations of compounds 1 and 3. correlation spectroscopy) ( ) correlations of compounds 1 and 3. correlation spectroscopy) ( ) correlations of compounds 1 and 3. 1
1
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
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 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 1H COSY spectrum. The connections of these fragments, three were readily recognized from the H–C-21–C-22, As for unit b, four spin systems 1of C-24–C-25–C-26, C-28–C-29–C-30, and C-32–C-33 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 1H– 1H COSY spectrum. The connections of these fragments, three 1H– 1H COSY spectrum. The connections of these fragments, three 3 were readily recognized from the were readily recognized from the double bonds, and one sp quaternary carbon were achieved by HMBC correlations, generating the 1 H–1 H COSY spectrum. The connections of these fragments, three were readily recognized from the 1 1 were readily recognized from the H– H COSY spectrum. The connections of these fragments, three 3 quaternary carbon were achieved by HMBC correlations, generating the 3 quaternary carbon were achieved by HMBC correlations, generating the double bonds, and one sp double bonds, and one sp framework of a 14‐membered carbon ring C. Although no HMBC correlation was observed between double bonds, and one sp3 quaternary carbon were achieved by HMBC correlations, generating the 3 quaternary carbon were achieved by HMBC correlations, generating the double bonds, and one sp H‐30 and C‐27, the presence of the oxygen bridge between C‐30 and C‐27 were deduced by framework of a 14‐membered carbon ring C. Although no HMBC correlation was observed between framework of a 14‐membered carbon ring C. Although no HMBC correlation was observed between framework of C‐27, a 14-membered carbon ring C.those Although no HMBCC‐30 correlation waswere observed between 13C‐NMR framework of a 14‐membered carbon ring C. Although no HMBC correlation was observed between comparison of the its data with of bisglaucumlide [24] bearing similar H‐30 and presence the oxygen bridge between and C‐27 deduced H‐30 and C‐27, the presence of of the oxygen bridge between C‐30 H and C‐27 were the deduced by by H-30 and C-27, the presence of the oxygen bridge between C-30 and C-27 were deduced by comparison H‐30 and C‐27, the presence of the oxygen bridge between C‐30 and C‐27 were deduced by tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule. The 13 13C‐NMR comparison C‐NMR data with those bisglaucumlide [24] bearing the similar comparison of of its its data with those of of bisglaucumlide H H [24] bearing the similar 13 C-NMR 13C‐NMR connection of units a and b was finally achieved by HMBC correlations of H 3[24] ‐18/C‐8, C‐9, and C‐21, oftetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule. The its data with those of bisglaucumlide H [24] bearing the similar tetrahydrofuran rings, comparison of its data with those of bisglaucumlide H bearing the similar tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule. The H‐21/C‐8, C‐9, C‐18, and and H3‐37/C‐34, C‐35, and C‐36, which formed a six‐membered tetrahydrofuran rings, as well as on consideration of the unsaturation degrees of the molecule. The asconnection of units a and b was finally achieved by HMBC correlations of H well as on consideration of C‐35, the unsaturation degrees of the molecule. The connection of units a connection of units a and b was finally achieved by HMBC correlations of H 3‐18/C‐8, C‐9, and C‐21, 3‐18/C‐8, C‐9, and C‐21, carbon ring B bridging these two parts. connection of units a and b was finally achieved by HMBC correlations of H 3‐18/C‐8, C‐9, and C‐21, and b was finally achieved by HMBC correlations of H -18/C-8, C-9, and C-21, H-21/C-8, C-9, C-18, H‐21/C‐8, C‐9, C‐18, and C‐35, and 3‐37/C‐34, C‐35, and C‐36, which formed a six‐membered 3and H‐21/C‐8, C‐9, C‐18, and C‐35, and H3H ‐37/C‐34, C‐35, C‐36, which formed a six‐membered The relative configuration of 1 was established based on a NOESY experiment and analysis of H‐21/C‐8, C‐9, C‐18, and C‐35, and H 3 ‐37/C‐34, C‐35, and C‐36, which formed a six‐membered and C-35, and H3 -37/C-34, C-35, and C-36, which formed a six-membered carbon ring B bridging carbon ring B bridging these two parts. carbon ring B bridging these two parts. coupling constants. The strong NOE interactions of H‐8/H‐22 and H 3‐18/H2‐36b indicated that H‐8 carbon ring B bridging these two parts. The relative configuration of 1 was established based on a NOESY experiment and analysis of these two parts. The relative configuration of 1 was established based on a NOESY experiment and analysis of and CH 3‐18 occupied the axial bonds of ring B in a chair conformation (Figure 3). Thus, ring A and B 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 coupling constants. The strong NOE interactions of H‐8/H‐22 and H 3‐18/H 2‐36b indicated that H‐8 trans‐fused. The NOE of correlations of H‐14a/H‐1 and H‐12 that these protons Thewere relative configuration 1 was established based on aindicated NOESY experiment andwere analysis of coupling constants. The strong NOE interactions of H‐8/H‐22 and H 3‐18/H2‐36b indicated that H‐8 cofacial on ring A and were arbitrarily assigned as β‐orientation. The NOE correlations of H‐22/H‐8 and CH 3 ‐18 occupied the axial bonds of ring B in a chair conformation (Figure 3). Thus, ring A and B and CH 3 ‐18 occupied the axial bonds of ring B in a chair conformation (Figure 3). Thus, ring A and B coupling constants. The strong NOE interactions of H-8/H-22 and H3 -18/H2 -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 and H‐26 and H‐26/H‐30 suggested that these protons were cofacial on ring C and were assigned as were trans‐fused. The NOE correlations H‐14a/H‐1 and H‐12 indicated that these protons were were trans‐fused. The NOE correlations of of H‐14a/H‐1 and H‐12 indicated that these protons were and CH 3 -18 occupied the axial bonds of ring B in a chair conformation (Figure 3). Thus, ring A and β‐orientation, while correlations of H‐21/H 3‐18 and and H‐32 suggested that these protons were were trans‐fused. The NOE correlations of H‐14a/H‐1 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 Bcofacial on ring A and were arbitrarily assigned as β‐orientation. The NOE correlations of H‐22/H‐8 were trans-fused. The NOE correlations of H-14a/H-1 and H-12 indicated that these protons were α‐oriented. The trans‐relationship of H‐33 and H‐32 was implied by the large coupling constant 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 and H‐26 and H‐26/H‐30 suggested that these protons were cofacial on ring C and were assigned as cofacialbetween on ring these A and were arbitrarily β-orientation. Theof NOE correlations of H-22/H-8 two protons (J = 9.7 assigned Hz) [25]. as Finally, the structure 1 including the absolute 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 β‐orientation, while correlations of that H‐21/H 3‐18 and H‐32 suggested that these protons were and H-26 and H-26/H-30 suggested these protons were cofacial on ring C and were assigned as configuration was confirmed by a single crystal X‐ray crystallographic analysis using anomalous β‐orientation, while correlations of 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 α‐oriented. The trans‐relationship H‐33 and H‐32 was implied by the large coupling constant scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4). Thus, compound 1 was given a β-orientation, while correlations of H-21/H and H-32 suggested that these protons were constant α-oriented. 3 -18 α‐oriented. The trans‐relationship of H‐33 and H‐32 was implied by coupling between these two protons = 9.7 Hz) [25]. Finally, the structure of 1 large including the absolute between these two protons (J (J = 9.7 Hz) [25]. Finally, the structure of the 1 including the absolute trivial name sarelengan A. Thebetween trans-relationship of H-33 and H-32 was implied by the large coupling constant between these two 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 configuration was confirmed by a single crystal X‐ray crystallographic analysis using anomalous protons (J = 9.7 Hz) [25]. Finally, the structure of 1 including the absolute configuration was confirmed 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 scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4). Thus, compound 1 was given a bytrivial name sarelengan A. atrivial name sarelengan A. single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation, scattering of Cu Kα radiation, Flack parameter = 0.04 (13) (Figure 4). Thus, compound 1 was given a Flack parameter = 0.04 (13) (Figure 4). Thus, compound 1 was given a trivial name sarelengan A. trivial name sarelengan A.
Figure 3. Key NOE (nuclear overhauser effect) correlations of 1.
Figure 3. Key NOE (nuclear overhauser effect) correlations of 1. Figure 3. Key NOE (nuclear overhauser effect) correlations of 1. Figure 3. Key NOE (nuclear overhauser effect) correlations of 1.
Figure 3. Key NOE (nuclear overhauser effect) correlations of 1.
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Figure 4. ORTEP diagram of 1. Figure 4. ORTEP diagram of 1.
Compound 2, a colorless oil, had the molecular formula C41H60O8, as determined by HRESIMS Compound 2, a colorless had the molecular formula C41spectra H60 O8 ,of as2 determined by HRESIMS + (calcd. ion at m/z 703.4193 [M + Na]oil, 703.4180). The 1D NMR exhibited most of the ionstructural features found in 1, with major difference being the absence of a tetrahydrofuran ring and at m/z 703.4193 [M + Na]+ (calcd. 703.4180). The 1D NMR spectra of 2 exhibited most of the structural featuresof found in 1, with major difference being the double absencebond of ain tetrahydrofuran ring the replacement a terminal double bond by a trisubstituted 2. The relatively and the replacement of asignals terminal double bond by trisubstituted bond in 2. The upfield‐shifted carbon of an oxymethine (δCa 64.0, C‐26) and double a quaternary carbon (δrelatively C 61.2, upfield-shifted carbon signals of an oxymethine (δ 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 C 1 1 C-27) suggested the presence3‐39/C‐26 and C‐27, as well as of an epoxy ring in 2 [26]. The ring was located 2at C-26 and C-27 by HMBC correlations of H H– epoxy H COSY correlation of H ‐25/H‐26. The 3‐40 to C‐30 bytrisubstituted double bond was located at C‐30 and C‐31 by HMBC correlations from H HMBC correlations of H3 -39/C-26 and C-27, as well as 1 H–1 H COSY correlation of H2 -25/H-26. 1H COSY correlation of H2‐29/H‐30. The planar structure of 2 was further and C‐31, and 1H– The trisubstituted double bond was located at C-30 and C-31 by HMBC correlations from H3 -40 to confirmed by detailed analyses of its 2D NMR data. The configuration of 2 was assigned to be the C-30 and C-31, and 1 H–1 H COSY correlation of H2 -29/H-30. The planar structure of 2 was further 13C‐NMR data. In particular, the NOESY same as by that of 1 by comparison and confirmed detailed analyses of its of 2Dtheir NMRNOESY data. The configuration of 2 was assigned to be the same 3‐18/H‐36b 13 confirmed the trans‐fused ring, while the correlations of H‐8/H‐22 and H as that of 1 by comparison of their NOESY and C-NMR data. In particular,A/B the NOESY correlations trans‐relationship of H‐32 and H‐33 was assigned by NOE correlations of H‐33/H 3‐37 and H‐32/H‐21, of H-8/H-22 and H3 -18/H-36b confirmed the trans-fused A/B ring, while the trans-relationship of as well as the large coupling constant (J = 10.0 Hz) between H‐32 and H‐33. The NOE correlations 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 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 coupling constant (J = 10.0 Hz) between H-32 and H-33. The NOE correlations of H-22/H-8 and CH3‐39 on the epoxy ring (S56, Supplementary Materials), which was further confirmed by H-26/H-22 and H-28b indicated that H-26 was β-oriented and in a trans-position of CH3 -39 on the comparison of the NMR data of C‐26 and C‐27 in 2 with those of known analogue sarcophytolide H epoxy ring (S56, Supplementary Materials), which was further confirmed by comparison of the NMR [13]. The E geometry of Δ30 was assigned by NOE correlation of H‐30/H‐32. Thus, compound 2 was data of C-26 and C-27 in 2 with those of known analogue sarcophytolide H [13]. The E geometry of determined as depicted and was given the trivial name sarelengan B. ∆30 wasIt is worth noting that compounds 1 and 2 possessed a trans‐fused A/B ring system, differing assigned by NOE correlation of H-30/H-32. Thus, compound 2 was determined as depicted and was given the trivial name sarelengan B. from those of the previously reported biscembranoids [8,10], suggesting that the coupling between It is worth noting that compounds 1 and 2 possessed a trans-fused A/B ring system, differing two monemeric cembranoids in 1 and 2 may involve an unusual biosynthetic pathway. As shown in from those 1, of the reported biscembranoids [8,10], suggesting that considered the couplingas between Scheme the previously well‐known soft coral metabolite, methyl sarcoate, was the cembranoid‐dienophile precursor, which underwent biohydrogenation to produce a dienophile two monemeric cembranoids in 1 and 2 may involve an unusual biosynthetic pathway. As shown in intermediate 1,2‐dihydro‐methyl sarcoate. Lobophytone T isolated from Lobophytum pauciflorum [22], Scheme 1, the well-known soft coral metabolite, methyl sarcoate, was considered as the was served as the cembranoid‐diene underwent hydrolysis cembranoid-dienophile precursor, which precursor, underwentwhich biohydrogenation to produceto a generate dienophile intermediate i. Dehydration of i followed by a series redox reactions generated the epoxy intermediate 1,2-dihydro-methyl sarcoate. Lobophytone T isolated from Lobophytum pauciflorum [22], intermediates (ii and iii) and the tetrahydrofuran‐containing intermidiates (iv and v), respectively. was served as the cembranoid-diene precursor, which underwent hydrolysis to generate intermediate i. 1 and ii‐ or iv‐Δ21(34), Δ35 generates the The endo‐Diels‐Alder cycloaddition between methyl sarcoate‐Δ Dehydration of i followed by a series redox reactions generated the epoxy intermediates (ii and “normal biscembranoids” (A/B ring cis‐fused), sarcophytolide H [13] or bisglaucumlide G [24], iii) and the tetrahydrofuran-containing intermidiates (iv and v), respectively. The endo-Diels-Alder respectively, while the endo‐Diels‐Alder cycloaddition between methyl 1,2‐dihydro‐methyl sarcoate cycloaddition between methyl sarcoate-∆1 and ii- or iv-∆21(34) , ∆35 generates the “normal ‐Δ8 and v‐ or iii‐Δ21(34), Δ35 produces the A/B ring trans‐fused scaffold of 1 or 2, respectively.
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 -∆8 and v- or iii-∆21(34) , ∆35 produces the A/B ring trans-fused scaffold of 1 or 2, respectively.
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Scheme 1. Proposed biogenesis of compounds 1 and 2. Scheme 1. Proposed biogenesis of compounds 1 and 2.
Compound 3, a colorless oil, had the molecular formula C21H28O4 as determined by HRESIMS at Compound 3, a colorless oil, had the molecular formula C21 H281H‐NMR data of 3 showed two O4 as determined by HRESIMS + (calcd. 367.1880), implying eight DOUs. The m/z 367.1871 [M + Na] + (calcd. 367.1880), implying eight DOUs. The 1 H-NMR data of 3 showed at m/z 367.1871 [M + Na] methyl groups [δH 1.43 (3H, s) and 1.96 (3H, d, J = 1.1 Hz)], an isopropyl group [δH 1.12 (3H × 2, d, J = two6.8 Hz), 2.52 (1H, m)], four olefinic protons [δ methyl groups [δH 1.43 (3H, s) and 1.96 H(3H, d, J = 1.1 Hz)], an isopropyl group [δH 1.12 (3H 5.64 (1H, s), 5.99 (1H, t, J = 6.4 Hz), 6.29 (1H, d, J = 12.1 × 2, d, J = 6.8 Hz), 2.52 (1H, m)], four olefinic protons [δH 5.64 (1H, s), 5.99 (1H, t, J = 6.4 Hz), Hz), and 7.42 (1H, d, J = 12.1, 1.1 Hz)], and a series of aliphatic methylene or methine multiplets. The 13 6.29 C‐NMR (1H, d, Jspectrum, = 12.1 Hz), 7.42 (1H, with d, J =DEPT 12.1, 1.1 Hz)], and aresolved series of21 aliphatic in and combination experiments, carbon methylene resonances or 13 C-NMR methine multiplets. The in a combination with DEPT resolved attributable to one ketone group spectrum, (δC 209.6), methyl ester group (δCexperiments, 169.2 and 51.5), four 21 3 trisubstituted double bonds (δto C one 187.7, 164.1, group 152.1, (δ 136.6, 130.1, 121.7, 119.3, and 100.4), one spand carbon resonances attributable ketone 209.6), a methyl ester group (δ 169.2 C C 3 methine, four sp3 methylenes and four methyls. oxygenated quaternary carbon, one sp 51.5), four trisubstituted double bonds (δC 187.7, 164.1, 152.1, 136.6, 130.1, 121.7, 119.3, and 100.4), 3 methine, structural of a cembranoid. The oneAforementioned sp3 oxygenatedinformation quaternaryindicated carbon, that one 3 sppossessed four sp3features methylenes and four methyls. 1H–1H gross structure of 3 was further established by analysis of its 2D NMR data (Figure 2). In the Aforementioned information indicated that 3 possessed structural features of a cembranoid. The gross COSY spectrum four partial structures, C‐2–C‐3, C‐9–C‐10–C‐11, C‐13–C‐14, and C‐16–C‐15–C‐17 1 structure of 3 was further established by analysis of its 2D NMR data (Figure 2). In the H–1 H were established by a series of 1H–1H COSY correlation (Figure 2). The connectivities of the four COSY spectrum four partial structures, C-2–C-3, C-9–C-10–C-11, C-13–C-14, and C-16–C-15–C-17 structural fragments, the double bonds, the methyls, and the methyl ester were achieved by analysis were established by a series of 1 H–1 H COSY correlation (Figure 2). The connectivities of the four of the HMBC correlations. In particular, HMBC correlations from H‐15 to C‐1, C‐2, and C‐14, from structural fragments, the double bonds, the methyls, and the methyl ester were achieved by analysis H3‐18 to C‐3, C‐4, and C‐5, from H3‐19 to C‐7, C‐8, and C‐9, from H‐6 to C‐5, C‐7, and C‐8, and from of the HMBC correlations. In particular, HMBC correlations from H-15 to C-1, C-2, and C-14, from H2‐13 to C‐11 and C‐20 constructed a 14‐membered carbon ring. As the above‐mentioned structure H3 -18 to C-3, C-4, and accounted C-5, from for H3 -19 to C-7, C-8, andDOUs, C-9, from H-6 to C-5,one C-7, andrequired C-8, andthe from elucidation already seven of the eight the remaining DOU H2 -13 to C-11 and C-20 constructed a 14-membered carbon ring. As the above-mentioned structure presence of an additional ring. The severely down‐field shifted carbon signals of C‐5 (C 187.7) and elucidation already accounted for seven of the eight DOUs, the remaining one DOU required the C‐8 (C 91.3) implied the presence of an ether bond between C‐8 and C‐5 to form a furan‐3(2H)‐one presence of anwas additional The severely down-fieldof shifted carbon signals of C-5 (δC of 187.7) and C-8 ring. This further ring. supported by comparison its 13C‐NMR data with those synthetic (δC2,2,5‐trimethyl‐3(2H)‐furanone [27]. The configuration of C‐8 was tentatively assigned as S based on 91.3) implied the presence of an ether bond between C-8 and C-5 to form a furan-3(2H)-one ring. This was further supported by comparison of its 13 C-NMR data with those of synthetic the biogenetic consideration of this compound class, while the 1E, 3E, 5Z, 11Z configurations were determined by NOESY correlations of H‐2/H 3‐16, H‐2/H3‐18, H‐6/H 3‐18, and H‐11/H 2‐13, respectively. 2,2,5-trimethyl-3(2H)-furanone [27]. The configuration of C-8 was tentatively assigned as S based on Thus, compound 3 was determined as shown in Figure 1 and was given the trivial name sarelengan C. the biogenetic consideration of this compound class, while the 1E, 3E, 5Z, 11Z configurations
were determined by NOESY correlations of H-2/H3 -16, H-2/H3 -18, H-6/H3 -18, and H-11/H2 -13,
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respectively. Thus, compound 3 was determined as shown in Figure 1 and was given the trivial name Mar. Drugs 2017, 15, 85 6 of 14 sarelengan C. Mar. Drugs 2017, 15, 85 6 of 14 by Compound 4, a colorless crystal, had the molecular formula C20 H30 O5 , as established Compound 4, a colorless crystal, had the molecular formula C20H30O5, as established by HRESIMS. HRESIMS. The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences arising Compound 4, a colorless crystal, had the molecular formula C20H30O5, as established by HRESIMS. arising from the carbon signals ofC 34.3 in 4; C-5 (δC 34.3 in 4; δC 32.8 in 8) and C-18 (δ C 29.9 in 8), indicating 31.4 in 4; δC 29.9 in 8), from the carbon signals of C‐5 ( C 32.8 in 8) and C‐18 ( C 31.4 in 4; C The NMR data of 4 were very similar to those of sartrolide E (8) [28], with slight differences arising indicating that 4 was a C-4of epimer of 8. 2D Detailed 2Dfurther analysis further supported that they shared that 4 was a C‐4 epimer 8. Detailed supported that they shared the same from the carbon signals of C‐5 ( C 34.3 in 4; Canalysis 32.8 in 8) and C‐18 ( C 31.4 in 4; C 29.9 in 8), indicating theplanar same planar structure. The structure of 4 including the absolute configuration (1R, 4S, and structure. structure of 4 including the absolute configuration and the 8R) same was 8R) that 4 was a C‐4 The epimer of 8. Detailed 2D analysis further supported that (1R, they 4S, shared was secured a single crystal X-ray crystallographic analysis using anomalous of Cu secured by by a single crystal X‐ray using anomalous scattering Cu was Kα planar structure. The structure of crystallographic 4 including the analysis absolute configuration (1R, 4S, scattering and of 8R) Kαradiation, radiation, Flack parameter = 0.08 (10) (Figure 5). Thus, compound 4 was given the trivial name Flack parameter = 0.08 (10) (Figure 5). Thus, compound 4 was given the trivial name secured by a single crystal X‐ray crystallographic analysis using anomalous scattering of Cu Kα sarelengan D. sarelengan D. radiation, Flack parameter = 0.08 (10) (Figure 5). Thus, compound 4 was given the trivial name sarelengan D.
Figure 5. ORTEP diagram of 4. Figure 5. ORTEP diagram of 4. Figure 5. ORTEP diagram of 4.
Compound 5, a colorless crystal, gave a [M + Na]+ ion at m/z 373.1984 in the HRESIMS Compound 5, a colorless crystal, gave aO[M + Na]++ ion at m/z 373.1984 in the HRESIMS corresponding to the molecular formula C 20H30a 5[M . The 1D NMR spectra of 5 showed high similarity Compound 5, a colorless crystal, gave + Na] ion at m/z 373.1984 in the HRESIMS corresponding to the molecular formula C H O5 . The 1D NMR spectra of 5 showed highC 38.1 in similarity to those of sarcophelegan B (9) [19], except for the slight difference of carbon signals of C‐5 ( corresponding to the molecular formula C2020H30 30O5. The 1D NMR spectra of 5 showed high similarity to those of sarcophelegan BC 26.9 in 5; (9) [19], except for the slight difference of carbon signals of C-5 (δC 38.1 5; C 36.5 in 9) and C‐18 ( C 29.3 in 9), indicating that 5 was a C‐4 epimer of 9. Detailed 2D to those of sarcophelegan B (9) [19], except for the slight difference of carbon signals of C‐5 ( C 38.1 in in 5; δ 36.5 in 9) and C-18 (δ 26.9 in 5; δ 29.3 in 9), indicating that 5 was a C-4 epimer of 9. Detailed C C C analysis further supported that they shared the same planar structure. The structure of 5 including 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 2Dthe absolute configuration analysis further supported(1R, that4S, they shared the same planar structure. TheX‐ray structure of 5 including and 8R) was secured by a single crystal crystallographic analysis further supported that they shared the same planar structure. The structure of 5 including theanalysis absolute configuration secured by by a single crystal X-ray crystallographic using anomalous (1R, scattering of 8R) Cu was Kα radiation, Flack parameter = X‐ray −0.01 (10) (Figure 6). the absolute configuration (1R, 4S, 4S, and and 8R) was secured a single crystal crystallographic Thus, compound 5 was given the trivial name sarelengan E. analysis using anomalous scattering of Cu Kα Kα radiation, Flack parameter = −= 0.01 (10)(10) (Figure 6). 6). Thus, analysis using anomalous scattering of Cu radiation, Flack parameter −0.01 (Figure
compound 5 was given the trivial name sarelengan E. Thus, compound 5 was given the trivial name sarelengan E.
Figure 6. ORTEP diagram of 5.
Figure 6. ORTEP diagram of 5. Figure 6. ORTEP diagram of 5. Compound 6, a colorless crystal, exhibited a molecular formula of C20H30O5 as determined by HRESIMS. The 1H and 13C‐NMR data of 6 showed high similarity to those of sarcophelegan B (9) Compound 6, a colorless crystal, exhibited a molecular formula of C 20H 30O 5 as determined by
HRESIMS. The 1H and 13C‐NMR data of 6 showed high similarity to those of sarcophelegan B (9)
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Mar. Drugs 2017, 15, 85 Compound 6,
a colorless crystal, exhibited a molecular formula of C20 H30 O5 as determined7 of 14 by HRESIMS. The and 13 C-NMR data of 6 showed high similarity to those of sarcophelegan B (9) [19], 11 [19], except for the replacement of the C 80.8) and a methine (H except for the replacement of the ∆11 in 9 in 9 by an oxymethine ( by an oxymethine (δH 4.87;Hδ 4.87; C 80.8) and a methine (δH 2.69; 2.69; C 44.1) in 6. Since 6 possessed the same DOUs as that of 9, the formation of an oxygen bridge δC 44.1) in 6. Since 6 possessed the same DOUs as that of 9, the formation of an oxygen bridge between between C‐8 and the oxymethine (C‐11) was suggested. This was supported by the downfield‐shifted C-8 and the oxymethine (C-11) was suggested. This was supported by the downfield-shifted carbonyl 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 at C-20 (δC 175.3 in 6; δC 168.8 in 9) and C-8 (δC 90.0 in 6; δC 79.7 in 9), although no direct HMBC HMBC correlation from H‐11 to available. C‐8 was The available. The of confirmed 6 was further confirmed correlation from H-11 to C-8 was structure of 6structure was further by converting 9 toby converting 9 to 6 in a basic condition and the absolute configuration (1S, 4R, 8R, 11S, and 12R) was 6 in a basic condition and the absolute configuration (1S, 4R, 8R, 11S, and 12R) was assigned by a single assigned by a single crystal X‐ray diffraction analysis with Flack parameter = −0.05 (13) (Figure 7). crystal X-ray diffraction analysis with Flack parameter = −0.05 (13) (Figure 7). Thus, compound 6 was Thus, compound 6 was given a trivial name sarelengan F. given a trivial name sarelengan F. 1H
Figure 7. ORTEP diagram of 6. Figure 7. ORTEP diagram of 6.
Compound formula of of CC2020H H32 32O O55 as as established established by by HRESIMS HRESIMS data. The Compound7 7had had a a molecular molecular formula data. The 1D1D NMR data of 7 were very similar to those of sartrolide D [28], a known cembranoid isolated from NMR data of 7 were very similar to those of sartrolide D [28], a known cembranoid isolated from C 66.1 in Sarcophyton trocheliophorum, with the only difference being due to the chemical shift of C‐7 ( Sarcophyton trocheliophorum, with the only difference being due to the chemical shift of C-7 (δC 66.1 in 7; 7; 72.7 in sartrolide D), indicating that 7 was a C‐7 epimer of sartrolide D. Detailed 2D analysis δCC72.7 in sartrolide D), indicating that 7 was a C-7 epimer of sartrolide D. Detailed 2D analysis further supported that theythat shared theshared same planar structure. The structure. β-orientation of β‐orientation 7-OH was supported by the further supported they the same planar The of 7‐OH was NOE correlations of H-2/H-7 and H-15. Thus, compound 7 was given the trivial name sarelengan G. supported by the NOE correlations of H‐2/H‐7 and H‐15. Thus, compound 7 was given the trivial The known compounds, sartrolide E (8) [28] and sarcophelegan B (9) [19], were identified by name sarelengan G. comparison of their NMR data and optical rotation values with those reported in the literature. The known compounds, sartrolide E (8) [28] and sarcophelegan B (9) [19], were identified by All compounds were evaluated for their inhibitory effects on the NO production in LPS-induced comparison of their NMR data and optical rotation values with those reported in the literature. RAW macrophages the Griess quercetin was used as the positive controlin All 264.7 compounds were using evaluated for assay, their and inhibitory effects on the NO production (IC = 20.0 µM). Compounds 2 and 3 showed moderate inhibitory activities with IC values 50 50 used being LPS‐induced RAW 264.7 macrophages using the Griess assay, and quercetin was as the at 18.2 and 32.5 (IC µM, respectively, while other compounds were inactive (inhibition