Briarenolide E: The first 2-ketobriarane diterpenoid from an octocoral Briareum sp. (Briareidae)

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Briarenolide E: the ﬁrst 2-ketobriarane diterpenoid from an octocoral

Briareum sp. (Briareidae)

Pei-Han Hong

a,b

, Yin-Di Su

b,c

, Nai-Cheng Lin

b

, Yung-Husan Chen

b

, Yueh-Hsiung Kuo

d,e

,

Tsong-Long Hwang

f

, Wei-Hsien Wang

b,c,g

, Jih-Jung Chen

h

, Jyh-Horng Sheu

c,g,⇑

, Ping-Jyun Sung

a,b,c,g,i,⇑

a

Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan

b

National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan

c

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan

d

School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan

e_{Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung 404, Taiwan} f_{Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan}

g

Asia-Paciﬁc Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan

h

Department of Pharmacy, Tajen University, Pingtung 907, Taiwan

i

Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan

a r t i c l e

i n f o

Article history: Received 2 January 2012 Revised 16 January 2012 Accepted 19 January 2012 Available online 28 January 2012 Keywords: Briareum Briarenolide Briarane Superoxide anion Elastase

a b s t r a c t

A novel 2-ketobriarane diterpenoid, briarenolide E (1), was isolated from an octocoral Briareum sp. The structure of briarane 1 was elucidated by interpretations of spectral data. Compound 1 displayed mod-estly inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.

Previous chemical investigations of the chemical constituents of octocorals belonging to the genus Briareum (family Briareidae)1–3 collected off the waters of Taiwan have yielded a series of interest-ing new briarane-related diterpenoids (3,8-cyclized cembranoid), which possess a bicyclo[8.4.0] carbon skeleton. Over 500 naturally-occurring briarane-type metabolites have been isolated from various marine organisms, and all compounds of this type are recognized as being of marine origin, and in particular are produced by various octocorals.4–7 _{Owing to their interesting} chemical constituents and potential medicinal usage, octocorals belonging to the genus Briareum have been proven to be important target organisms.8,9 _{In our continuing studies of the chemical} constituents of an octocoral identiﬁed as Briareum sp., a novel 2-ketobriarane derivative, briarenolide E (1) was isolated. In this Letter, we describe the isolation, structural characterization, and bioactivity of briarane 1.

Sliced bodies of Briareum sp. (wet weight 6.32 kg, dry weight 2.78 kg) were extracted with a mixture of MeOH and DCM (1:1). The extract was partitioned between EtOAc and H2O. The EtOAc layer was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 100:1–pure EtOAc) to yield 18 fractions. Fraction 8 was puriﬁed by normal-phase HPLC, using a mixture of n-hexane and acetone (3:1) as the mobile phase to afford compound 1 (1.2 mg). Briarenolide E (1), ½

a

25D +28 (c 0.06, CHCl3); mp 117–118 °C, was isolated as a white powder that gave a pseudomolecular ion (M+Na)+at m/z 487.1946 in the HRESIMS, indicating the molecular formula C24H32O9(calcd for C24H32O9+Na, 487.1944) and implying nine degrees of unsaturation. IR absorptions were observed at 3456, 1776, 1745, and 1710 cm1_{, suggesting the presence of} hydroxy,

c

-lactone, ester, and ketone groups in 1. From the 1_H and13_{C NMR spectra (Table 1), 1 was found to possess a ketone} (dC 211.7, C-2), a

c

-lactone moiety (dC 176.0, C-19), two acetoxy groups (dH2.14, 2.13, each 3H s; dC21.0, 20.9, 2 acetate meth-yls; dC170.2, 170.2, 2 ester carbonyls), and a trisubstituted oleﬁn (dC145.5, C-5; 118.7, CH-6; dH5.35, 1H, dq, J = 9.2, 0.8 Hz, H-6). On the basis of the above unsaturation data, 1 was concluded to be a diterpenoid molecule possessing four rings. A disubstituted epox-ide was elucidated from the signals of two oxymethines at dC

⇑ Corresponding authors. Tel.: +886 7 5252000x5030; fax: +886 7 5255020 (J.-H.S.); tel.: +886 8 8825037; fax: +886 8 8825087 (P.-J.S.).

E-mail addresses:[email protected](J.-H. Sheu),[email protected] (P.-J. Sung).

Tetrahedron Letters 53 (2012) 1710–1712

Contents lists available atSciVerse ScienceDirect

Tetrahedron Letters

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58.6 (CH-14) and 52.4 (CH-13) and further conﬁrmed by proton signals at dH 2.76 (1H, d, J = 3.6 Hz, H-14) and 3.07 (1H, d, J = 3.6 Hz, H-13). 1 2 3 4 6 5 7 8 9 10 11 12 1314 15 O O OH O H O AcO AcO 16 17 18 19 20 Briarenolide E (1)

From the1_H–1_{H COSY spectrum of 1 (Fig. 1}_and_{Table 1), it was} possible to establish the separate spin systems that map out the proton sequences from H2-3/H2-4, H-6/H-7, and H-9/H-10. These data, together with the HMBC correlations between H-4

a

/2, C-3, C-5, C-6; H-4b/C-2, C-3; H-6/C-4, C-7; H-7/C-5, C-6, C-8; H-9/ C-7, C-8, C-10; and H-10/C-1, C-2, C-8, C-9, established the connec-tivity from C-1 to C-10 in the 10-membered ring (Table 1and Fig. 1). The methyl group at C-5 was conﬁrmed by the HMBC cor-relations between H3-16/C-4, C-5, C-6; H-4

a

/C-16; and H-6/C-16; and further conﬁrmed by an allylic coupling between H-6 and H3-16 (J = 0.8 Hz). The methylcyclohexane ring, which is fused to the 10-membered ring at C-1 and C-10, was elucidated by the 1_H–1_{H COSY correlations between H-10/H-11/H-12, H-13/H-14,} and H-11/H3-20 and by the HMBC correlations between H-9, H-10/11; H-10/20; H-11/1, 10, 12, 13; H-12/10, C-11, C-13, C-14, C-20; H-14/C-1, C-10; and H3-20/C-10, C-11, C-12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H3-15/C-1, C-2, C-14; and H-10/ C-15. The 2-ketone group was elucidated by the HMBC correlations between H2-4, H-10, H3-15 and the ketone carbonyl (dC211.7, C-2).

Furthermore, the acetate esters at C-9 and C-12 were established by correlations between H-9 (dH5.15), H-12 (dH4.90) and the ace-tate carbonyls (dC 170.2, two ester carbonyls) observed in the HMBC spectrum of 1. Thus, the remaining hydroxy group is posi-tioned at C-8, an oxygen-bearing quaternary carbon at dC 81.1. These data, together with the 1_H–1_{H COSY correlation between} H-17 and H3-18 and the HMBC correlations between H-7, H-9/C-17; H-17/C-8, C-18, C-19, and H3-18/C-8, C-17, C-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-ori-ented in most briarane derivatives.4–7The relative conﬁguration of 1 was elucidated from the interactions observed in a NOESY exper-iment and was found to be compatible with that of 1 offered by computer modeling (Table 2)10_{and that obtained from vicinal} pro-ton coupling constant analysis. In the NOESY experiment of 1, the correlations of H-10 with H-3

a

, H-11, and H-12, but not with H3 -15 and H3-20, indicated that these protons (H-3

a

, H-10, H-11, and H-12) were situated on the same face, and these were assigned as

a

protons, since the C-15 and C-20 methyls are b-substituents at

:1_H-1_{H COSY} : HMBC O O OH O O 1 2 5 8 10 13 14 15 16 17 18 19 20 CH3CO O CH3CO O Figure 1. The1 H–1

H COSY and selective key HMBC correlations (protons?quater-nary carbons) of 1. Table 1 1 H and13 C NMR data,1 H–1

H COSY, and HMBC correlations for 1

Position dHa dHb 1H–1H COSY HMBC (1H?13C) 1 48.6 (qC) 2 211.7 (qC) 3a 3.15 m 36.1 (CH2) H-3b, H2-4 n.o.c b 2.57 m H-3a, H2-4 n.o. 4a 2.41 dd (14.0, 2.0)d 28.2 (CH2) H2-3, H-4b C-2, C-3, C-5, C-6, C-16 b 2.54 m H2-3, H-4a C-2, C-3 5 145.5 (qC) 6 5.35 dq (9.2, 0.8) 118.7 (CH) H-7, H3-16 C-4, C-7, C-16 7 5.15 d (9.2) 78.1 (CH) H-6 C-5, C-6, C-8, C-17 8 81.1 (qC) 9 5.15 d (9.2) 67.8 (CH) H-10 C-7, C-8, C-10, C-11 C-17, acetate carbonyl 10 2.68 dd (9.2, 2.8) 33.6 (CH) H-9, H-11 C-1, C-2, C-8, C-9, C-11, C-15, C-20 11 2.72 m 32.0 (CH) H-10, H-12, H3-20 C-1, C-10, C-12, C-13 12 4.90 d (5.6) 72.0 (CH) H-11 C-10, C-11, C-13, C-14, C-20, acetate carbonyl 13 3.07 d (3.6) 52.4 (CH) H-14 n.o. 14 2.76 d (3.6) 58.6 (CH) H-13 C-1, C-10 15 1.17 s 15.8 (CH3) C-1, C-2, C-14 16 1.92 d (0.8) 23.8 (CH3) H-6 C-4, C-5, C-6 17 2.35 q (7.2) 43.2 (CH) H3-18 C-8, C-18, C-19 18 1.18 d (7.2) 6.3 (CH3) H-17 C-8, C-17, C-19 19 176.0 (qC) 20 1.02 d (7.2) 10.5 (CH3) H-11 C-10, C-11, C-12 9-OAc 170.2 (qC) 2.14 s 21.0 (CH3) Acetate carbonyl 12-OAc 170.2 (qC) 2.13 s 20.9 (CH3) Acetate carbonyl a Spectra measured at 400 MHz in CDCl3at 25 °C. b Spectra measured at 100 MHz in CDCl3at 25 °C. c

n.o. = not observed.

d

J values (in hertz) in parentheses.

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C-1 and C-11, respectively. H-13 showed correlations with H-12 and H-14, as well as the lack of coupling was detected between H-12 and H-13, indicating the dihedral angle between H-12 and H-13 is approximately 90° and H-13 has an

a

-orientation at C-13. 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 when H-9 was

a

-oriented. The C-16 vinyl methyl showed correlations with H-3

a

and H-6, and a large coupling con-stant was detected between H-6 and H-7 (J = 9.2 Hz), indicating the Z-conﬁguration of the C-5/6 double bond; in addition, the dihedral angle between H-6 and H-7 is approximately 180°, and H-7 has a b-orientation at C-7.11 _{Furthermore, H-7 exhibited a correlation} with H-17, suggesting that H-17 and the 8-hydroxy group are b-and

a

-oriented in the

c

-lactone moiety, respectively, by modeling analysis. Based on the above ﬁndings, the structure of 1 was estab-lished unambiguously.

It is worth noting that a briarane analogue possessing a 2-keto group, 1 (briarenolide E), was discovered for the ﬁrst time in this study. The in vitro anti-inﬂammatory effects of 1 were tested. Briarenolide E (1) displayed modestly inhibitory effects on the gen-eration of superoxide anion (inhibition rate 23.7%) and the release of elastase (inhibition rate 28.3%) by human neutrophils at a con-centration of 10

l

g/mL.12–14_{Owing to structural complexity, it is}

difﬁcult to obtain sufﬁcient amounts of the bioactive metabolite 1 for further study of its potential medicinal usage from natural sources. The octocoral Briareum sp. has begun to be transplanted into tanks using our highly-developed aquaculture technology for the extraction of natural products to establish a stable supply of bioactive materials.

Acknowledgments

This work was supported by Grants from the National Dong Hwa University; The National Museum of Marine Biology and Aquarium (Grant No. 100100101 and 100200311); the Division of Marine Biotechnology, Asia-Paciﬁc Ocean Research Center, National Sun Yat-sen University, (Grant No. 00C-0302-05); The Tai-wan Department of Health Clinical Trial and Research Center of Excellence (Grant No. DOH 101-TD-B-111-004); and the National Science Council (Grant No. NSC 100-2325-B-291-001 and 98-2320-B-291-001-MY3), Taiwan, awarded to Y.-H.K. and P.-J.S. References and notes

1. Bayer, F. M. Proc. Biol. Soc. Wash. 1981, 94, 902–947.

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4. Sung, P.-J.; Sheu, J.-H.; Xu, J.-P. Heterocycles 2002, 57, 535–579.

5. Sung, P.-J.; Chang, P.-C.; Fang, L.-S.; Sheu, J.-H.; Chen, W.-C.; Chen, Y.-P.; Lin, M.-R. Heterocycles 2005, 83, 195–204.

6. Sung, P.-J.; Sheu, J.-H.; Wang, W.-H.; Fang, L.-S.; Chung, H.-M.; Pai, C.-H.; Su, Y.-D.; Tsai, W.-T.; Chen, B.-Y.; Lin, M.-R.; Li, G.-Y. Heterocycles 2008, 75, 2627–2648.

7. Sung, P.-J.; Su, J.-H.; Wang, W.-H.; Sheu, J.-H.; Fang, L.-S.; Wu, Y.-C.; Chen, Y.-H.; Chung, H.-M.; Su, Y.-D.; Chang, Y.-C. Heterocycles 2011, 83, 1241–1258. 8. Berrue, F.; Kerr, R. G. Nat. Prod. Rep. 2009, 26, 681–710.

9. Hanson, J. R. Nat. Prod. Rep. 2009, 26, 1156–1171. 10. Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127–8134.

11. Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identiﬁcation of Organic Compounds, 7th ed.; John-Wiley & Sons, Inc.: Hoboken, NJ, USA, 2005. pp 171–172.

12. In the in vitro anti-inflammatory bioassay, the inhibitory effect on the generation of superoxide anions and the release of elastase by activated neutrophils were used as indicators. To indicate significant activity of pure compounds, an inhibition rate >40% is required (inhibition rate <10%, not active, 20% >inhibition rate >10%, weakly anti-inflammatory; 40% >inhibition rate >20%, modestly anti-inflammatory). Diphenyl indonium (DPI) and elastatinal were used as reference compounds in anti-inflammatory activity testing. DPI displayed an inhibitory effect on superoxide anion generation (IC50= 0.9lg/mL), and elastatinal exhibited an inhibitory effect on elastase

release (IC50= 30lg/mL) by human neutrophils, respectively.

13. Hwang, T.-L.; Wang, C.-C.; Kuo, Y.-H.; Huang, H.-C.; Wu, Y.-C.; Kuo, L.-M.; Wu, Y.-H. Biochem. Pharmacol. 2010, 80, 1190–1200.

14. Yu, H.-P.; Hsieh, P.-W.; Chang, Y.-J.; Chung, P.-J.; Kuo, L.-M.; Hwang, T.-L. Free Radic. Biol. Med. 2011, 50, 1737–1748.

Table 2

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

Briarenolide E (1) H/H (Å) 1 7 10 9 8 11 12 13 14 17 3 4 5 6 2 15 16 18 19 20 H-3a/H-10 2.610 H-3a/H3-16 2.259 H-6/H3-16 2.285 H-7/H-17 2.844 H-9/H-11 2.555 H-9/H3-18 2.398 H-9/H3-20 2.147 H-10/H-11 2.432 H-10/H-12 2.371 H-12/H-13 2.551 H-13/H-14 2.544