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Mar 23, 2017 - the genera of Sarcophyton, Sinularia, and Lobophytum (family Alcyoniidae) [1–5]. Biosynthetically they are proposed to be derived from the ...
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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‐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  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.  H1H H COSY (correlation spectroscopy) ( 1H1H 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