Briarenolides M–T, new briarane diterpenoids from a Formosan octocoral Briareum sp.

Tetrahedron 72 (2016) 944e951

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Briarenolides MeT, new briarane diterpenoids from a Formosan octocoral Briareum sp. Yin-Di Su a, b, Zhi-Hong Wen a, c, Yang-Chang Wu d, e, f, g, Lee-Shing Fang h, Yu-Hsin Chen b, i, Yu-Chia Chang b, c, Jyh-Horng Sheu a, c, *, Ping-Jyun Sung a, b, e, g, j, * a

Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan, ROC National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan, ROC Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan, ROC d School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan, ROC e Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan, ROC f Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan, ROC g Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC h Department of Sport, Health, and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan, ROC i Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan, ROC j Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan, ROC b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2015 Received in revised form 12 December 2015 Accepted 23 December 2015 Available online 31 December 2015

Eight new briarane diterpenoids, designated briarenolides MeT (1e8), were isolated from an octocoral identified as Briareum sp. The structures of briaranes 1e8 were established by spectroscopic methods and by comparison of the spectroscopic data with those of known analogues. The relationship between 1 H and 13C NMR chemical shifts of 2-hydroxybriaranes possessing of (A) a D3,5(16)-conjugated diene moiety or (B) a D3,5-conjugated diene moiety are described. Briarenolides M (1), P (4), S (7), and T (8) were found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein and briarenolides N (2), P (4), and T (8) were found to inhibit the accumulation of the proinflammatory cyclooxygenase-2 (COX-2) protein in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Briarane Briarenolide Briareum Anti-inflammatory iNOS COX-2

1. Introduction Marine origin briarane-type natural products have been suggested to be produced by host octocorals,1,2 and compounds of this type have been shown to possess extensive bioactivities.3e9 In continuation of our research into new substances obtained from marine invertebrates collected off the waters of Taiwan, the

chemical constituents of an octocoral identified as Briareum sp. (Briareidae) were studied,10e16 and eight new briarane analogues, briarenolides MeT (1e8), were isolated. In this paper the isolation, structure determination, and anti-inflammatory activities of briaranes 1e8 and the relationship between 1H and 13C NMR chemical shifts of 2-hydroxybriaranes possessing (A) a D3,5(16)-conjugated diene or (B) a D3,5-conjugated diene moieties are described.

* Corresponding authors. Tel.: þ886 7 525 2000x5030; fax: þ886 7 525 5020(J.-H.S.); tel.: þ886 8 882 5037; fax: þ886 8 882 5087(P.-J.S.); e-mail addresses: sheu@mail. nsysu.edu.tw (J.-H. Sheu), [email protected] (P.-J. Sung). http://dx.doi.org/10.1016/j.tet.2015.12.058 0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951 OH 15

O

2

13 12

AcO

14

4

8

AcO

OH

6 7

9

H

17 19

18

O

Cl

5

OH

10

OH

O

O

16

1

11

20

OH

O 3

945

AcO O

OH

R

H

AcO

H

O

AcO

O

O

AcO

O

O

2: R=OAc 10: R=OCH3

1 R1

OCH3

3 OAc

R

AcO

HO

OAc

OCOPr

O Cl

R3 OH R2

H AcO

OH O O

H

Cl

OH O

O

HO

O

HO

H

O

HO

6: R=OH 16: R=OAc

2. Results and discussion Briarenolide M (1) was isolated as a white powder that gave a pseudomolecular ion (MþNa)þ at m/z 537.14965 in HRESIMS, indicating the molecular formula C24H31ClO10 (calcd for C24H31ClO10þNa, 537.14980) and implying nine degrees of unsaturation. Comparison of the 1H NMR and DEPT data with the molecular formula indicated that there must be two exchangeable protons, requiring the presence of two hydroxy groups, and this deduction was supported by a broad absorption in the IR spectrum at 3454 cm1. The IR spectrum of 1 also showed strong bands at 1778 and 1739 cm1, consistent with the presence of g-lactone and

R

O O

O

O

4: R1=R2=OH, R3=Cl 5: R1=R2=OAc, R3=OH 9: R1=OH, R2=OAc, R3=Cl 11: R1=R3=OH, R2=OAc

H

7

O

8: R=OH 17: R=OAc

ester groups. From the 1H and 13C NMR spectra (Table 1), 1 was found to possess a g-lactone moiety (dC 176.3, C-19), two acetoxy groups (dH 2.22, 2.11, each 3Hs; dC 21.8, 21.1, 2acetate methyls; dC 169.7, 170.9, 2acetate carbonyls), and a trisubstituted olefin (dH 6.03, 1H, d, J¼9.2 Hz, H-6; dC 137.8, C-5; 126.1, CH-6). On the basis of the above unsaturation data, 1 was concluded to be a diterpenoid molecule possessing five rings. Two disubstituted epoxides were elucidated from the signals of four oxymethines at dC 60.6 (CH-3), 58.0 (CH-4), 57.3 (CH-13), and 62.3 (CH-14), and further confirmed by proton signals at dH 3.38 (1H, dd, J¼9.6, 4.0 Hz, H-3), 4.16 (1H, br s, H-4), 3.17 (1H, d, J¼3.6 Hz, H-13), and 3.28 (1H, d, J¼3.6 Hz, H-14). From the 1He1H COSY spectrum of 1 (Table 1), it was possible to establish the separate system that maps out the proton sequences

Table 1 1 H and 13C NMR data, 1He1H COSY, and HMBC correlations for briarane 1 C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16a/b 17 18 19 20 9-OAc

d Ha

dCb

3.08 d (9.6)c 3.38 dd (9.6, 4.0) 4.16 br s 6.03 d (9.2) 5.32 d (9.2) 5.28 1.77 2.17 4.70 3.17 3.28 1.18 4.20 2.43 1.17

d (8.0) dd (8.0, 2.4) m d (4.8) d (3.6) d (3.6) s d (12.4); 4.09 d (12.4) q (7.2) d (7.2)

1.03 d (7.2) 2.22 s

12-OAc 2.11 s a b c d e



39.2 (C)d 76.1 (CH) 60.6 (CH) 58.0 (CH) 137.8 (C) 126.1 (CH) 76.7 (CH) 81.7 (C) 69.1 (CH) 36.4 (CH) 36.5 (CH) 71.8 (CH) 57.3 (CH) 62.3 (CH) 15.2 (CH3) 44.3 (CH2) 43.6 (CH) 6.2 (CH3) 176.3 (C) 9.7 (CH3) 169.7 (C) 21.8 (CH3) 170.9 (C) 21.1 (CH3)

Spectra recorded at 400 MHz in CDCl3 at 25 C. Spectra recorded at 100 MHz in CDCl3 at 25  C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o.¼not observed.

He1H COSY

1

HMBC(H/C)

H-3 H-2, H-4 H-3, H-6

C-1, -3, -14, -15 n.o.e C-5, -6

H-4, H-7, H2-16 H-6

C-4, -16 C-5, -6, -8

H-10 H-9, H-11 H-10, H-12, H3-20 H-11 H-14 H-13 H-6 H3-18 H-17

C-7, -8, -10, -11, -17, acetate carbonyl C-1, -2, -8, -9, -11, -15, -20 C-1, -12 C-10, -11, -13, -20, acetate carbonyl n.o. C-1, -10 C-1, -2 -10, -14 C-4, -5, -6 C-8, -18, -19 C-8, -17, -19

H-11

C-10, -11, -12 Acetate carbonyl Acetate carbonyl

946

Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951

from H-2/H-3/H-4, H-4/H-6 (by allylic coupling), H-6/H-7, and H-9/ H-10. These data, together with the HMBC correlations between H2/C-1, -3; H-4/C-5, -6; H-6/C-4; H-7/C-5, -6, -8; H-9/C-7, -8, -10; and H-10/C-1, -2, -8, -9, established the connectivity from C-1 to C-10 in the ten-membered ring (Table 1). The methylcyclohexane ring, which is fused to the ten-membered ring at C-1 and C-10, was elucidated from the 1He1H COSY correlations between H-10/H-11/ H-12, H-13/H-14, and H-11/H3-20 and from the HMBC correlations between H-2/C-14; H-9, H-10/C-11; H-10/C-20; H-11/C-1; H-12/C10; H-14/C-1, -10; and H3-20/C-10, -11, -12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H3-15/C-1, -2, -10, -14; and H-2, H-10/C-15. Furthermore, the acetate esters at C-9 and C-12 were established from the correlations between H-9 (dH 5.28), H-12 (dH 4.70) and the acetate carbonyls (dC 169.7 and 170.9, two ester carbonyls) observed in the HMBC spectrum of 1. The intensity of the (Mþ2þNa)þ isotope peak observed in ESIMS ((MþNa)þ:(Mþ2þNa)þ¼3:1) was strong evidence of the presence of a chlorine atom in 1. The methylene unit at dC 44.3 was more shielded than expected for an oxygenated C-atom and was correlated with the methylene protons at dH 4.20 and 4.09 in the HMQC spectrum. These methylene signals were 2J-correlated with C-5 (dC 137.8) and 3J-correlated with both C-4 (dC 58.0) and C-6 (dC 126.1), proving the attachment of a chloromethyl group at C-5 (Table 1). These data, together with the 1He1H COSY correlation between H-17 and H3-18 and the HMBC correlations between H-9/ C-17; H-17/C-8, -18, -19; and H3-18/C-8, -17, -19, were used to establish the molecular framework of 1. In all naturally-occurring briaranes, H-10 is trans to the C-15 methyl group, and these two groups are assigned as a- and b-oriented in most briarane derivatives.3e9 The relative configuration of 1 was elucidated from the interactions observed in the NOESY experiment and was found to be compatible with that of 1 offered by computer modeling (Fig. 1) and that obtained from vicinal proton coupling constant analysis. In the NOESY experiment of 1, the correlations of H-10 with H-2, H-11, and H-12, but not with H3-15 and H3-20, indicated that these protons (H-2, H-10, H-11, and H-12) were situated on the same face, and these were assigned as a protons, as the C-15 and C-20 methyls are b-substituents at C-1 and C11, respectively. H-14 showed correlations with H-13 and H3-15, and a lack of coupling was detected between H-12 and H-13, indicating that the dihedral angle between H-12 and H-13 is approximately 90 , and H-13 has a b-orientation at C-13. The E configuration of the C-5/-6 double bond was elucidated from the responses between the C-6 olefin proton (dH 6.03) and the C-16 methylene protons (dH 4.20 and 4.09). H-9 was found to show responses to H-11, H3-18, and H3-20. From modeling analysis, H-9

was found to be close to H-11, H3-18, and H3-20, and H-9 was aoriented. H-3 was correlated with H-4 and H3-15, but not with H10, and a large coupling constant (J¼9.6 Hz) was detected between H-2 and H-3, indicating that the 3,4-epoxy group was a-oriented. In addition, H-7 showed a correlation with H-4, and a large coupling constant (J¼9.2 Hz) was detected between H-6 and H-7, indicating that the dihedral angle between H-6 and H-7 is approximately 180 , and H-7 was b-oriented. Furthermore, H-7 exhibited a correlation with H-17, indicating that H-17 and the 8-hydroxy group were b- and a-oriented in the g-lactone moiety, respectively, by modeling analysis. The spectroscopic data of 1 were found to be similar to those of a known briarane, briaexcavatolide M (9).17 By comparison of the NMR data of 1 with those of 9, it was found that the C-3/4 carbon-carbon double bond in 9 was replaced by an epoxide group in 1. Briarenolide N (2) was found to have the molecular formula C26H34O12 by HRESIMS at m/z 561.19410 (calcd for C26H34O12þNa, 561.19425), and its IR absorptions at 3420, 1772, and 1734 cm1, were typical of hydroxy, g-lactone, and ester carbonyl functionalities. It was found that the NMR data of 2 were similar to those of a known briarane, briarenolide H (10),14 except that the signals corresponding to the 6-methoxy group in 10 were replaced by those of an acetoxy group in 2 (Table 2). Therefore, briarenolide N (2) was assigned as having a structure with the same relative stereochemistry as briarenolide H (10) because of the chiral carbons that 2 has in common with 10. The molecular formula C25H34O10 of 3 (briarenolide O) was proposed by examination of the ESIMS pseudomolecular (MþNa)þ ion at m/z 517 and verified by HRESIMS at m/z 517.20458 (calcd for C25H34O10þNa, 517.20442). It was found that the NMR data of 3 were similar to those of briarenolide H (10).14 However, the 1H and 13 C NMR spectra (Table 2) revealed that the signals corresponding to the C-3/4 epoxide group in 10 were not present, and had been replaced by those of a carbon-carbon double bond in 3. On the basis of the above observations, 3 was found to be the 3,4-deoxy derivative of 10. Briarenolide P (4) was obtained as a white powder and was found to have the molecular formula C22H29ClO8 on the basis of HRESIMS at m/z 479.14446 (calcd for C22H29ClO8þNa, 479.14432). Its IR spectrum exhibited a broad OH stretch at 3420 cm1, a glactone carbonyl at 1770 cm1, and ester carbonyls at 1733 cm1. Furthermore, by comparison of the 1H and 13C NMR data of 4 (Table 2) with those of briaexcavatolide M (9),17 it was revealed that the signals corresponding to the 12-acetoxy group in 9 were replaced by a hydroxy group in 4, and briarenolide P (4) was found to be the 12-O-deacetyl derivative of 9.

Fig. 1. The stereoview of 1 (generated from computer modeling) and the calculated distances ( A) between selected protons with key NOESY correlations.

Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951

947

Table 2 1 H and13C NMR data for briaranes 2e5 C/H

2

3

d Ha 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc

dCb

3.55 dd (8.4, 4.0)c 3.34 dd (8.4, 4.4) 3.82 d (4.4) 5.90 d (10.4) 4.76 d (10.4) 5.35 1.81 2.04 4.58 3.23 3.25 1.16 5.72 5.46 2.66 1.17

d (8.8) dd (8.8, 2.4) m d (4.4) d (3.2) d (3.2) s s; s q (7.2) d (7.2)

1.02 d (7.2)

4

dH

38.4 (C)d 74.0 (CH) 62.3 (CH) 56.6 (CH) 135.2 (C) 74.0 (CH) 80.6 (CH) 80.4 (C) 69.5 (CH) 36.1 (CH) 35.8 (CH) 72.4 (CH) 57.3 (CH) 62.1 (CH) 15.5 (CH3) 123.7 (CH2) 45.6 (CH) 6.1 (CH3) 173.1 (C) 9.5 (CH3)

dC

5.19 dd (9.2, 3.6) 5.75 dd (12.0, 9.2) 6.04 d (12.0) 3.73 d (9.2) 4.53 d (9.2) 5.28 2.17 1.90 4.60 3.26 3.17 1.07 5.58 5.41 2.52 1.14

d (8.0) dd (8.0, 2.8) m d (4.4) d (3.6) d (3.6) s s; s q (7.2) d (7.2)

1.05 d (6.8)

5

dCe

dH

41.8 (C) 72.0 (CH) 134.1 (CH) 125.8 (CH) 138.4 (C) 85.1 (CH) 83.6 (CH) 80.5 (C) 69.8 (CH) 36.6 (CH) 37.2 (CH) 72.8 (CH) 57.7 (CH) 62.6 (CH) 14.7 (CH3) 120.0 (CH2)

40.3 (C) 76.1 (CH) 137.0 (CH) 124.9 (CH) 140.9 (C) 126.1 (CH) 79.3 (CH) 82.0 (C) 70.1 (CH) 37.1 (CH) 41.0 (CH) 70.0 (CH) 59.1 (CH) 62.8 (CH) 15.1 (CH3) 46.3 (CH2)

4.16 d (10.0) 5.89 dd (11.2, 10.0) 6.20 br d (11.2) 5.79 d (8.8) 5.09 d (8.8) 5.19 1.81 1.86 3.87 3.24 3.21 1.15 4.20 4.19 2.37 1.17

45.2 (CH) 6.0 (CH3) 175.0 (C) 9.2 (CH3)

dH

d (6.4) dd (6.4, 2.8) m d (4.8) d (3.6) d (3.6) s s; s q (7.2) d (7.2)

43.2 (CH) 6.4 (CH3) 175.7 (C) 9.1 (CH3)

1.06 d (7.2)

dC 40.3 (C) 78.8 (CH) 131.0 (CH) 129.0 (CH) 145.3 (C) 122.0 (CH) 79.3 (CH) 81.4 (C) 69.7 (CH) 36.9 (CH) 37.5 (CH) 71.6 (CH) 57.1 (CH) 62.2 (CH) 16.1 (CH3) 63.4 (CH2)

5.23 d (10.0) 5.61 dd (11.6,10.0) 6.32 br d (11.6) 5.78 d (8.8) 5.13 d (8.8) 5.19 2.03 2.12 4.81 3.15 3.00 1.21 4.58 4.33 2.29 1.16

d (6.8) m m d (4.8) d (3.2) d (3.2) s d (16.0) d (16.0) q (7.2) d (7.2)

43.3 (CH) 6.4 (CH3) 176.4 (C) 9.4 (CH3) 170.2 (C) 21.2 (CH3)

1.06 d (7.2) 2.06 s

6-OAc 2.10 s 9-OAc 2.23 s 12-OAc 2-OH 6-OCH3 a b c d e

2.12 s 2.31 d (4.0)

169.9 (C) 21.0 (CH3) 170.3 (C) 21.9 (CH3) 170.7 (C) 21.2 (CH3)

2.14 s 2.13 s 2.01 d (3.6) 3.31 s

170.4 (C) 21.9 (CH3) 170.4 (C) 21.2 (CH3)

169.8 (C) 21.1 (CH3)

2.18 s

169.9 (C) 21.8 (CH3) 170.4 (C) 21.0 (CH3)

2.18 s 2.11 s

56.1 (CH3)



Spectra recorded at 400 MHz in CDCl3 at 25 C. Spectra recorded at 100 MHz in CDCl3 at 25  C. J values (in Hz) in parentheses. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. The 13C NMR data for this compound were assigned with the assistance of HMQC and HMBC spectra.

The new briarane briarenolide Q (5) had the molecular formula C26H34O11 as determined by HRESIMS at m/z 545.19926 (calcd for C26H34O11þNa, 545.19933). It was found that the spectroscopic data (IR, 1H, and 13C NMR) of 5 were similar to those of briaexcavtolide N (11).17 However, the 1H and 13C NMR spectra (Table 2) revealed that the signals corresponding to the 2-hydroxy group in 11 were replaced by those of an acetoxy group in 5, and briarenolide Q (5) was found to be the 2-O-acetyl derivative of 11. From the characteristics of the chemical shifts, it was apparent that the 2-hydroxybriaranes possessed of (A) a D3,5(16)-conjugated diene moiety, such as briarenolide O (3), brianthein X (12),18 briaexcavatolide H (13),19 and briaexcavatin T (14),20 or (B) a D3,5conjugated diene moiety, such as briarenolide P (4), briaexcavatolide M (9),17 briaexcavatolide N (11),17 and briarenolide I (15)14 in their structures. We summed up the chemical shifts for olefinic protons H-2, -3, and H-4, in addition to the chemical shifts for the C-2 oxymethine and the olefinic carbons C-3, -4, and C-5 (Table 3) of the above compounds. By comparison of the NMR data, it was found that the 1H NMR data for H-2 and H-4 were shifted upfield and downfield, respectively; the 13C NMR data for C-2, -3, and C-5 were shifted downfield, while the 2-hydroxybriarane analogues possessing a D3,5(16)-conjugated diene moiety comparing with the 2-hydroxybriaranes possessing a D3,5-conjugated diene moiety. The new briarane briarenolide R (6) had the molecular formula C20H27ClO6 as determined by HRESIMS at m/z 421.13886 (calcd for C20H27ClO6þNa, 421.13884). It was found that the spectroscopic data (IR, 1H, and 13C NMR) of 6 were similar to those of solenolide E (16).21 However, the 1H and 13C NMR spectra (Table 4) revealed that the signals corresponding to an acetoxy group in 16 were replaced

by those of a hydroxy group in 6. On the basis of the above observations, the structure of 6 was found to be the 2-O-deacetyl derivative of 16. Briarenolide S (7) had the same molecular formula as that of solenolide E (16),21 C22H29ClO7, as determined by HRESIMS at m/z 463.14949 (calcd for C22H29O7ClþNa 463.14940), which implied eight degrees of unsaturation. Based on the 1H and 13C NMR spectra (Table 4), 7 was found to possess an acetoxy group (dH 2.29, 3Hs; dC 167.6, acetate carbonyl; 20.8, acetate methyl), a trisubstituted

Table 3 NMR chemical shifts for H-2, H-3, H-4 and C-2, C-3, C-4, and C-5 of 2hydroxybriaranes possessing (A) a D3,5(16)-conjugated diene moiety or (B) a D3,5conjugated diene moiety.

(A)

(B)

OH 2

3

4

5

16

OH 2

3

16 4

5

R

Compounds/dH and dC

H-2

H-3

H-4

C-2

C-3

C-4

C-5

(A)

5.19 5.23 5.15 5.20 4.16 4.18 4.13 4.07

5.75 5.21 5.83 5.84 5.89 5.87 5.82 5.84

6.04 5.83 5.84 5.88 6.20 6.22 6.27 6.13

72.0 72.2 71.7 72.6 76.1 75.8 75.6 75.3

134.1 135.6 135.1 134.1 137.0 136.8 136.0 137.8

125.8 126.1 126.1 126.5 124.9 126.2 125.1 123.2

138.4 138.0 137.0 136.9 140.9 140.8 145.4 139.1

(B)

briarenolide O (3) brianthein X (12) briaexcavatolide H (13) briaexcavatin T (14) briarenolide P (4) briaexcavatolide M (9) briaexcavatolide N (11) briarenolide I (15)

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Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951

Table 4 1 H and13C NMR data for briaranes 6 and 7 C/H

6

7

d Ha 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc

dCb 45.8 (C)d 79.5 (CH) 29.7 (CH2) 25.2 (CH2) 143.3 (C) 66.8 (CH) 76.9 (CH) 86.3 (C) 71.1 (CH) 39.5 (CH) 45.3 (CH) 203.7 (C) 125.2 (CH) 155.4 (CH) 21.1 (CH3) 118.2 (CH2)

3.87 d (9.2)c 2.18e2.01 m 2.39 m; 2.28 m 4.92 br s 5.40 d (2.8) 3.49 d (6.8) 3.07 dd (6.8, 4.8) 2.79 qd (7.6, 4.8) 5.94 6.26 1.53 5.72 5.26 3.27 1.17

dd (10.4, 0.8) d (10.4) s d (2.0); d (2.0) q (7.6) d (7.6)

43.0 (CH) 8.0 (CH3) 177.8 (C) 14.5 (CH3)

1.22 d (7.6)

dH

dC

4.93 d (9.2) 2.47e2.33 m 2.37 m; 1.72 m 5.86 d (9.6) 5.24 d (9.6) 3.75 dd (8.8, 8.0) 2.95 dd (8.0, 4.0) 3.05 qd (7.2, 4.0) 5.97 6.30 1.44 4.04 3.98 3.43 1.20

d (10.4) d (10.4) s d (11.2); d (11.2) q (7.2) d (7.2)

1.24 d (7.2) 2.29 s

8-OH 9-OH

45.1 (C) 81.3 (CH) 20.9 (CH2) 24.4 (CH2) 141.2 (C) 127.5 (CH) 76.6 (CH) 85.0 (C) 69.2 (CH) 38.8 (CH) 44.3 (CH) 203.1 (C) 126.3 (CH) 154.3 (CH) 20.0 (CH3) 48.5 (CH2) 41.9 (CH) 6.4 (CH3) 177.8 (C) 14.5 (CH3) 167.6 (C) 20.8 (CH3)

3.19 br s 4.54 d (8.8)

Spectra recorded at 400 MHz in CDCl3 at 25  C. b Spectra recorded at 100 MHz in CDCl3 at 25  C. c J values (in Hz) in parentheses. d Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. a

olefin (dH 5.86, 1H, d, J¼9.6 Hz, H-6; dC 141.2, C-5; 127.5, CH-6), and a g-lactone moiety (dC 177.8, C-19). 13C NMR resonances at dC 203.1 (C-12), 126.3 (CH-13), and 154.3 (CH-14), as well as lH NMR signals at dH 6.30 (1H, d, J¼10.4 Hz, H-14) and 5.97 (1H, d, J¼10.4 Hz, H-13), further confirmed the presence of an a,b-unsaturated ketone. Spectral comparison showed that 7 was closely related to solenolide E (16),21 a compound possessing the same molecular formula and an identical substitution pattern on the 6-membered ring. Spectral comparison of 7 with 16 suggested a similar briarane skeleton. It was found that the exocyclic carbon-carbon double bond between C-5/16 and the chloride atom at C-6 in 16 were replaced by a chloromethyl group and a carbon-carbon double bond between C-5/6 in 7. Briarenolide T (8) had the molecular formula C28H40O12 as deduced from HRESIMS at m/z 591.24103 (calcd for C28H40O12þNa, 591.24120). Its IR spectrum exhibited a broad OH stretch at 3446 cm1, a g-lactone carbonyl group at 1783 cm1, and ester carbonyl groups at 1734 cm1. Carbonyl resonances in the 13C NMR spectrum of 8 confirmed the presence of a g-lactone and three ester groups (Table 5). Two of the esters were identified as acetates by the presence of two methyl resonances in the 1H NMR spectrum at dH 2.15 (3H, s) and 2.08 (3H, s) (Table 5). The other ester was found to be an n-butyrate based on 1H NMR studies, which revealed seven contiguous protons (dH 2.30, 2H, t, J¼7.6 Hz; 1.63, 2H, sext, J¼7.6 Hz; 0.96, 3H, t, J¼7.6 Hz). However, due to their slight distortion, a set of smaller, extraneous 1H NMR peaks were found for the methylene protons attached at the n-butyrate carbonyl. From data obtained from an HMBC experiment for 8 (Table 5), the molecular framework of 8 could be further established. These data also revealed that the carbon signal at dC 173.9 (C) was correlated with the signals of the methylene protons of the n-butyrate at dH 2.30 and 1.63 in the

Table 5 1 H and13C NMR data, 1He1H COSY, and HMBC correlations for 8 C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-OAc

dHa 3.68 br s 5.70 d (6.0)c 6.32 d (6.0) 5.60 dd (8.0, 1.6) 5.90 d (8.0) 4.11 2.79 2.47 3.94 1.95 4.90 1.27 1.98

d (9.6) dd (9.6, 4.8) m ddd (12.0, 4.8, 3.6) m; 1.72 m dd (3.2, 2.8) s s

1.57 s 0.99 d (6.8) 2.08 s 2.15 s

4-OC(O)CH2CH2CH3 10 20 30 40 2.30 t (7.6) 1.63 sext (7.6) 0.96 t (7.6) b c d e

1

HMBC(H/C)

H-3 H-2, H-4 H-3

C-1, -3, -4, -14 C-5, acetate carbonyl C-2, -3, -5, -6, -16, n-butyrate carbonyl

H-7, H3-16 H-6

n.o.e C-5, -6

H-10 H-9, H-11 H-10, H-12, H3-20 H-11, H2-13 H-12, H-14 H2-13

C-7, -8, C-1, -8, C-10 C-14 n.o. n.o. C-1, -2, C-4, -5,

d

14-OAc

a

He1H COSY

dCb 43.6 (C) 83.4 (CH) 74.5 (CH) 65.8 (CH) 136.9 (C) 128.0 (CH) 73.1 (CH) 70.2 (C) 65.0 (CH) 42.4 (CH) 35.1 (CH) 67.4 (CH) 30.3 (CH2) 79.5 (CH) 20.6 (CH3) 18.1 (CH3) 59.0 (C) 9.3 (CH3) 172.2 (C) 8.7 (CH3) 170.3 (C) 20.8 (CH3) 170.4 (C) 21.7 (CH3) 173.9 (C) 35.8 (CH2) 18.0 (CH2) 13.6 (CH3)

Spectra recorded at 400 MHz in CDCl3 at 25  C. Spectra recorded at 100 MHz in CDCl3 at 25  C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o.¼not observed.

H-6

-11 -9, -11, -20

-10, -14 -6

C-8, -17, -19 H-11

C-10, -11, -12 Acetate carbonyl Acetate carbonyl

H2-30 H2-20 , H3-40 H2-30

C-10 , -30 , -40 , n-butyrate carbonyl C-10 , -20 , -40 , n-butyrate carbonyl C-20 , -30

Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951

HMBC spectrum of 8, and was consequently assigned to the carbon atom of the n-butyrate carbonyl group. The n-butyrate positioned at C-4 was confirmed by the connectivity between H-4 (dH 6.32) and the carbonyl carbon (dC 173.9) of the n-butyrate group. Furthermore, the HMBC correlation revealed that an acetoxy group was attached to C-3. Thus, the remaining acetoxy group attached at C-14 and the remaining hydroxy groups positioned at C-2, C-9, and C-12 were suggested by key 1He1H COSY correlations (Table 5) and characteristic NMR signal analysis. The relative stereochemistry of 8 was confirmed as being similar to that of a known metabolite, briaexcavatolide O (17),22 by comparison of the NMR data and coupling constant analysis for the chiral centers. In the NOESY experiment of 8 (Fig. 2), H-10 exhibited responses to H-3, H-11, and H-12, but not with H3-15 and H3-20. These results indicated that H-3/-10/-11 and H-12 are situated on the same face of the molecule and can be assigned as a protons, as the C-15 and C-20 methyls are b-substituents at C-1 and C-11. H-2 correlated with H-3, H-14, and H3-15, suggesting that H-2 is aoriented. H-14 exhibited correlations with H-2 and H3-15, confirming the b-orientation of this proton. The signal of H-9 showed correlations with H3-18 and H3-20, but not with H3-15; and H3-18 correlated with H3-20, but not with H-11, indicating that H-9 and H3-18 are a- and b-oriented, respectively, in 8. Moreover, H-4 was found to exhibit a correlation with H-7, but not with H-3, which indicated that H-4 and H-7 should be placed on the b face in 3 by molecular modeling analysis. Furthermore, the Z configuration of the C-5/-6 double bond was elucidated from the response between the C-6 olefin proton (dH 5.60) and the C-16 vinyl methyl (dH 1.98). In in vitro anti-inflammatory activity tests, the upregulation of the pro-inflammatory iNOS and COX-2 protein expression of LPSstimulated RAW264.7 macrophage cells was evaluated using immunoblot analysis. At a concentration of 10 mM, briarenolides M (1), P (4), S (7), and T (8) were found to significantly reduce the levels of iNOS to 49.6, 58.4, 57.4, and 53.5%, respectively, and briarenolides N (2), P (4), and T (8) were found to significantly reduce the levels of COX-2 to 53.9, 59.1, and 59.3%, respectively, relative to the control cells stimulated with LPS only (Fig. 3). 3. Experimental 3.1. General experimental procedures Melting points were determined on a Fargo apparatus and are uncorrected. Optical rotation values were measured with a Jasco P1010 digital polarimeter. IR spectra were obtained on a Jasco FTIR 4100 spectrophotometer; absorptions are reported in cm1. NMR spectra were obtained using a Varian Mercury Plus 400 NMR

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spectrometer, using the residual CHCl3 signal (dH 7.26 ppm) as the internal standard for 1H NMR and CHCl3 (dC 77.1 ppm) for 13C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system. Column chromatography was performed on silica gel (230e400 mesh, Merck). TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck); spots were visualized by spraying with 10% H2SO4 solution followed by heating. Normal-phase HPLC (NPHPLC) was performed using a system comprised of a Hitachi L-7110 pump, a Hitachi L-7455 photodiode array detector, a Rheodyne 7725 injection port, and a semi-preparative normal-phase column (Hibar 25025 mm, LiChrospher Si 60, 5 mm, Merck). Reversephase HPLC (RP-HPLC) was performed using a system comprised of a Hitachi L-2130 pump, a Hitachi L-2455 photodiode array detector, a Rheodyne 7725 injection port, and a reverse-phase column (Luna 5u C18(2) 100A AXIA Packed, 25021.2 mm). 3.2. Animal material Specimens of the octocorals Briareum sp. were collected by hand using scuba equipment off the coast of southern Taiwan in July, 2011, and stored in a freezer until extraction. A voucher specimen (NMMBA-TW-SC-2011-77) was deposited in the National Museum of Marine Biology & Aquarium, Taiwan. 3.3. Extraction and isolation Sliced bodies of Briareum sp. (wet weight, 6.32 kg; dry weight, 2.78 kg) were extracted with a mixture of methanol (MeOH) and dichloromethane (DCM) (1:1). The extract was partitioned between ethyl acetate (EtOAc) and H2O. The EtOAc layer was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 100:1epure EtOAc) to yield 18 fractions, AeR. Fractions MeP were combined and further separated on silica gel and eluted using n-hexane/ EtOAc (stepwise, 100:1epure EtOAc) to afford 30 subfractions, M1eM30. Fraction M12 was chromatographed on silica gel and eluted using DCM and MeOH (stepwise, 100:1epure MeOH) to afford 34 subfractions, M12-1eM12-34. Fraction M12e13 was further separated by reverse-phase C-18 column chromatography and eluted with MeOH/H2O (stepwise, 20:80epure MeOH) to afford 1 (20:80, 14.5 mg). Fraction M12e27 was repurified by RP-HPLC using acetonitrile (CH3CN) and H2O (40:60) to afford 6 and 7, respectively. Fraction M12e30 was purified by RP-HPLC using CH3CN and H2O (40:60) to afford 8. Fraction M18 was separated by NP-HPLC using a mixture of DCM and acetone (15:1) as the mobile phase to obtain 28 subfractions, M18-1eM18-28. Fraction M18e27 was further separated by RP-HPLC using MeOH/H2O (30:70) to afford 5 and 4,

Fig. 2. The stereoview of 8 (generated from computer modeling) and the calculated distances ( A) between selected protons with key NOESY correlations.

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Fig. 3. Effects of compounds 1e8 on pro-inflammatory iNOS and COX-2 protein expression in the LPS-stimulated murine macrophage cell line RAW264.7. (A) The relative density of iNOS immunoblot; (B) the relative density of COX-2 immunoblot. The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percent change relative to that of the LPS-stimulated group. Briarenolides M (1), P (4), S (7), T (8), and dexamethasone (Dex) significantly inhibited LPS-induced iNOS protein expression (<60%) and briarenolides N (2), P (4), T (8), and Dex significantly inhibited LPS-induced COX-2 protein expression (<60%) in macrophages. The experiments were repeated three times (*p<0.05, significantly different from the LPS-stimulated group).

ESIMS: m/z 517 (MþNa)þ; HRESIMS: m/z 517.20458 (calcd for C25H34O10þNa, 517.20442).

respectively. Fraction Q was chromatographed on silica gel and eluted using n-hexane/EtOAc (stepwise, 4:1epure EtOAc) to yield 20 subfractions, Q1eQ20. Fraction Q1 was further separated by column chromatography on silica gel and eluted with DCM and acetone (stepwise, 30:1epure acetone) to yield 20 subfractions, Q1-1eQ1-20. Fraction Q1e12 was purified by RP-HPLC using MeOH and H2O (35:65) to afford 3. The residues of Q1e12 were repurified by RP-HPLC using CH3CN and H2O (35:65) to afford 2.

3.3.4. Briarenolide P (4). White powder (2.0 mg); mp 247e248  C; 1 1 [a]25 D 11 (c 0.1, CHCl3); IR (neat) nmax 3420, 1770, 1733 cm ; H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 2); ESIMS: m/z 479 (MþNa)þ, 481 (Mþ2þNa)þ; HRESIMS: m/z 479.14446 (calcd for C22H29ClO8þNa, 479.14432).

3.3.1. Briarenolide M (1). White powder (14.5 mg); mp 190e191  C; 1 1 [a]25 D 58 (c 0.7, CHCl3); IR (neat) nmax 3454, 1778, 1739 cm ; H 13 (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR data (see Table 1); ESIMS: m/z 537 (MþNa)þ, 539 (Mþ2þNa)þ; HRESIMS: m/z 537.14965 (calcd for C24H31ClO10þNa, 537.14980).

3.3.5. Briarenolide Q (5). White powder (2.9 mg); mp 149e150  C; 1 1 [a]25 D þ6 (c 0.1, CHCl3); IR (neat) nmax 3446, 1772, 1735 cm ; H 13 (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR data (see Table 2); ESIMS: m/z 545 (MþNa)þ; HRESIMS: m/z 545.19926 (calcd for C26H34O11þNa, 545.19933).

3.3.2. Briarenolide N (2). White powder (2.5 mg); mp 274e275  C; 1 1 [a]25 D 10 (c 0.1, CHCl3); IR (neat) nmax 3420, 1772, 1734 cm ; H 13 (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR data (see Table 2); ESIMS: m/z 561 (MþNa)þ; HRESIMS: m/z 561.19410 (calcd for C26H34O12þNa, 561.19425).

3.3.6. Briarenolide R (6). White powder (3.0 mg); mp 169e170  C; 1 [a]25 D 1 (c 0.2, CHCl3); IR (neat) nmax 3328, 1770, 1748, 1682 cm ; 1 13 H (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR data (see Table 4); ESIMS: m/z 421 (MþNa)þ, 423 (Mþ2þNa)þ; HRESIMS: m/z 421.13886 (calcd for C20H27ClO6þNa, 421.13884).

3.3.3. Briarenolide O (3). White powder (2.0 mg); mp 156e157  C; 1 1 [a]25 D 47 (c 0.1, CHCl3); IR (neat) nmax 3446, 1772, 1740 cm ; H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 2);

3.3.7. Briarenolide S (7). White powder (3.5 mg); mp 176e177  C; 1 [a]25 D 4 (c 0.2, CHCl3); IR (neat) nmax 3445, 1769, 1732, 1682 cm ; 1 H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table

Y.-D. Su et al. / Tetrahedron 72 (2016) 944e951

4); ESIMS: m/z 463 (MþNa)þ, 465 (Mþ2þNa)þ; HRESIMS: m/z 463.14949 (calcd for C22H29ClO7þNa, 463.14940). 3.3.8. Briarenolide T (8). White powder (0.7 mg); mp 223e224  C; 1 1 [a]25 D þ26 (c 0.1, CHCl3); IR (neat) nmax 3446, 1783, 1734 cm ; H 13 (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR data (see Table 5); ESIMS: m/z 591 (MþNa)þ; HRESIMS: m/z 591.24103 (calcd for C28H40O12þNa, 591.24120).

3.4. Molecular mechanics calculations Implementation of the MM2 force field23 in CHEM3D PRO software from Cambridge Soft Corporation, Cambridge, MA, USA (ver 9.0, 2005) was used to calculate molecular models.

3.5. In vitro anti-inflammatory assay According to our previous and other studies including in vitro anti-inflammatory activity assays, we used an LPS-induced RAW264.7 murine macrophage cell line that was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).24e27 The in vitro anti-inflammatory activities of compounds 1e8 were measured by examining the inhibition of lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory iNOS (inducible nitric oxide synthase) and cyclooxygenase-2 (COX-2) protein expression in macrophage cells using Western blotting analysis.27e29 Briefly, inflammation in macrophages was induced by incubating them for 16 h in a medium containing only LPS (10 ng/mL) without compounds. For the anti-inflammatory activity assays, Briarenolides MeT (1e8) and dexamethasone (10 mM) were added to the cells 10 min before LPS challenge. The cells were then subjected to Western blot analysis. The immunoreactivity data were calculated with respect to the average optical density of the corresponding LPS-stimulated group. The RAW264.7 macrophage cell viability was determined after treatment with alamar blue (Invitrogen, Carlsbad, CA, USA), a tetrazolium dye that is reduced by living cells to fluorescent products. This assay is similar in principle to the cell viability assay using 3-(4,5-dimethyldiazol-2-yl)-2,5diphenyltetrazolium bromide and has been validated as an accurate measure of the survival of RAW264.7 macrophage cells.30,31 For statistical analysis, the data were analyzed by one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls post hoc test for multiple comparisons. A significant difference was defined as a p-value of <0.05.

Acknowledgements This research was supported by grants from the Asia-Pacific Ocean Research Center, National Sun Yat-sen University; the National Dong Hwa University; the National Museum of Marine Biology & Aquarium; and the Ministry of Science and Technology (Grant No. MOST 103-2325-B-291-001, MOST 104-2325-B-291-001, and MOST 104-2320-B-291-001-MY3), Taiwan, awarded to J.-H.S. and P.-J.S.

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Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2015.12.058.

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