Bioorganic & Medicinal Chemistry Letters 24 (2014) 228–232

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New anti-inflammatory cembranoid diterpenoids from the Vietnamese soft coral Lobophytum crassum Nguyen Phuong Thao a,b, Bui Thi Thuy Luyen a,b, Nguyen Thi Thanh Ngan b, Seok Bean Song b, Nguyen Xuan Cuong a, Nguyen Hoai Nam a, Phan Van Kiem a, Young Ho Kim b,⇑, Chau Van Minh a,⇑ a b

Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Nghiado, Caugiay, Hanoi, Viet Nam College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea

a r t i c l e

i n f o

Article history: Received 13 October 2013 Revised 12 November 2013 Accepted 14 November 2013 Available online 22 November 2013 Keywords: Lobophytum crassum Soft coral Cembranoid iNOS COX-2 anti-Inflammatory

a b s t r a c t Four new cembranoid diterpenes lobocrasols AD (1–4), were isolated from the methanol extract of the soft coral Lobophytum crassum. Their structures were elucidated by spectroscopic analysis and by comparison of the spectroscopic data with those of similar compounds previously reported in literature. The anti-inflammatory effects of isolated compounds were evaluated using NF-jB luciferase and reverse transcription polymerase chain reaction (RT-PCR). Compounds 1 and 2 significantly inhibited TNFainduced NF-jB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values of 6.30 ± 0.42 and 6.63 ± 0.11 lM, respectively. Furthermore, the transcriptional inhibition of these compounds was confirmed by a decrease in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression levels in HepG2 cells. Ó 2013 Elsevier Ltd. All rights reserved.

Nuclear transcription factor-jB (NF-jB) represents a family of Rel domain-containing proteins including RelA (p65), NF-jB1 (p50, p105), NF-jB2 (p52, p100), RelB, and c-Rel. The activation of NF-jB has been linked to multiple pathophysiological conditions such as cancer, arthritis, asthma, inflammatory bowel disease, and other inflammatory conditions.1,2 The induction of several pro-inflammatory mediators occurs as resulted by increase of iNOS and COX-2 activities.3,4 Therefore, suppression of iNOS and COX-2 activities is important for preventing inflammation in organs.5 NF-jB and the signaling pathways that regulate many physiological processes (including the innate and adaptive immune responses, cell death, and inflammation) have become the focus of intense drug discovery and development efforts.6,7 An increasing number of marine products have been found to display anti-inflammatory effects, such as microcolin A, scytonemin, malyngamides F acetate, phycocyanin, have been isolated from sponges, tunicates, algae, and other organisms.8 Soft corals belonging to the genus Lobophytum (class Coelenterata, subclass Octocorallia, and family Alcyonaceae) are a rich source of steroids and terpenoids. Most of isolated diterpenes are cembranoid compounds,9 which are often found in high concentrations (up to 5% dry weight) in soft corals and have possible chemical defense

⇑ Corresponding authors. Tel.: +82 42 821 5933; fax: +82 42 823 6566 (Y.H.K.); tel.: +84 4 37917053; fax: +84 4 37917054 (C.V.M.). E-mail addresses: [email protected] (Y.H. Kim), [email protected] (C.V. Minh). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.033

roles against predators such as fish as well as microorganisms and other corals.10,11 Many cembranoids exhibit various biological activities, such as acetylcholinesterase- and HIV-inhibition,11,12 as well as antitumor,13 antimicrobial,14 cytotoxicity,13,15 and antiinflammatory properties.16,17 Our previous investigations of Lobophytum crassum yielded steroidal constituents with anti-inflammatory activity.18 To discover additional novel bioactive substances in the Vietnamese marine invertebrates,18–21 we carried out further studies on the chemical constituents and biological activities of the soft coral L. crassum. In this Letter, we report the isolation, structure determination, and anti-inflammatory activity of four new cembranoids, lobocrasols AD (14), from this soft coral. The sample of L. crassum collected in Con Co, Quangtri, Vietnam in May 2013 and identified by Professor Do Cong Thung (the Institute of Marine Environment and Resources, VAST). A voucher specimen (LC0513) was deposited at the Institute of Marine Biochemistry (IMBC), VAST. A MeOH extract (75.0 g) of the soft coral L. crassum was suspended in H2O and successively extracted with n-hexane and CH2Cl2. The CH2Cl2 fraction was subjected to multiple separation steps over silica gel and YMC RP-18 column chromatography (CC) to afford new compounds 14.22 Compound 123 was obtained as a colorless oil with the molecular formula, C20H32O5, determined by Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) at m/z 353.23282 [M+H]+. The IR spectrum of 1 suggested the presence of hydroxy

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(3441 cm1) and epoxy (1250 and 909 cm1) groups. The NMR spectroscopic features indicated that 1 is a cembranoid diterpene, which are typical constituents of soft coral species. The 13C NMR spectrum of 1 displayed twenty carbon signals, and DEPT experiments indicated the presence of four methyl, six methylene, five methine, and five quaternary carbons (see Table 1); including two trisubstituted olefins [dC 124.54 (CH, C-3), 142.92 (C, C-4), 123.84 (CH, C-11), 135.28 (C, C-12)], four oxygen-bearing carbons [dC 77.68 (CH, C-2), 74.81 (CH, C-7), 75.65 (C, C-8), 98.42 (CH, C16)], and one epoxy functionality [dC 71.24 (C, C-1) and 68.26 (C, C-15)]. The NMR spectroscopic data of 1 were similar to those of laevigatol A,24 except that the signals of C-7/C-8 epoxide ring in laevigatol A occurred as two hydroxy groups in 1 [dC 74.81 (C-7)/ dH 3.39 (1H, m, H-7) and dC 75.65 (C-8)]. The NMR data of 1 were assigned by comparison with those of laevigatol A, and by HMBC analysis. The methyl protons H-19 (dH 1.27) exhibited HMBC correlations with C-7 (dC 74.81)/C-8 (dC 75.65)/C-9 (dC 38.16), confirming the locations of two additional hydroxy groups at C-7 and C-8. Detailed analysis of other correlations in the HMBC spectrum further characterized the planar structure of compound 1 (see Fig. 2). The relative stereochemistries at C-1, C-2, C-7, C-8, C-15, and C16 of 1 were assigned by comparison of 1H and 13C NMR data with those of similar reported compounds and further supported by NOESY data. The b-orientation of the C-1/C-15 epoxy group, H-2, and hydroxy group at C-16 were assigned by agreement of 13C NMR data for C-1 (dC 71.24), C-2 (dC 77.68), C-15 (dC 68.26), C-16 (dC 98.42), and C-17 (dC 11.34) of compound 1 with the corresponding data of laevigatol A24 at dC 71.3 (C-1), 78.1 (C-2), 69.0 (C-15), 99.2 (C-16), and 11.8 (C-17), respectively. This was further confirmed by a correlation of H-2 (dH 4.84) with H-18 (dH 1.86) and those of H-17 (dH 1.45) with H-3 (dH 5.33) and H-16 (dC 5.26) in the NOESY (see Fig. 2). The 13C NMR chemical shifts at C-7 (dC 74.81), C-8 (dC 75.65), and C-19 (dC 25.77) of 1 were similar to those of (+)7b,8b-dihydroxydeepoxysarcophytoxide,25 sinumaximol A, and

Table 1 NMR spectroscopic data of compounds 1 and 2 C

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

1

2

dCa,b

dCa,c mult. (J in Hz)

dCa,b

dCa,c mult. (J in Hz)

71.24 77.68 124.54 142.92 34.71 31.32 74.81 75.65 38.16 23.23 123.84 135.28 33.75 24.70 68.26 98.42 11.34 18.23 25.77 16.35

— 4.84 5.33 — 2.23 1.61 3.39 — 1.68 2.10 5.11 — 1.95 1.56 — 5.26 1.45 1.86 1.27 1.60

71.19 77.50 125.28 139.22 34.95 26.52 72.87 75.83 37.00 23.84 124.62 134.84 35.40 26.27 68.21 98.30 11.20 15.82 24.33 15.00 — — —

— 4.83 d (11.0) 5.38 d (11.0) — 2.00 m/2.40 m 1.50 m/1.82 m 3.43 t (9.0) — 1.55 m/1.83 m 2.02 m/2.21 m 4.81d — 1.92 m/2.20 m 1.65 m/1.73 m — 5.20 d (4.0) 1.42 s 1.80 s 1.15 s 1.55 s 3.06 d (9.0) 2.62 s 5.26 br s

d (10.5) d (10.5) m m/1.68 m m m m/2.22 m t (7.5) m/2.22 m m/2.00 m br s s d (1.0) s s

Assignments were confirmed by HSQC, HMBC, and NOESY experiments. a Measured in CDCl3. b 125 MHz. c 500 MHz. d Overlapped signal.

18

HO

7

6

9 10

H

4

5

8

HO

19 11 12

3

O

16 15

2 1

13 14

O

OH

H HO

O

OH

HO O

17

20

2

1 H HO

O

H HO

HO

O

HO OH

OH

OH

OH

3

4

Figure 1. Structures of compounds 14 from the soft coral L. crassum.

H HO

O

OH

HO O

HMBC

NOESY

Figure 2. Key HMBC and NOESY correlations of 1.

sinumaximol H21 suggesting for b-orientation of both hydroxy groups at C-7 and C-8, which was further supported by a NOESY correlation of H-3 (dH 5.33) with H-5 (dH 2.23) and those of H-7 (dH 3.39) with H-5 (dH 2.23) and H-19 (dH 1.27). Thus, the structure of diterpenoid 1 was established and termed lobocrasol A. The FTICRMS of lobocrasol B (2)23 exhibited a pseudo-molecular ion peak at m/z 353.23272 [M+H]+, confirming its molecular formula as C20H32O5. The 1H and 13C NMR data of 2 were similar to those of 1 (see Table 1), apart from significantly different chemical shifts for the oxymethine group at C-7 and the methylene carbon at C-6, thereby suggesting a different configuration at C-7 and/or C-8 in 2 relative to that in 1. The 13C NMR chemical shifts at C-6 (dC 26.52), C-7 (dC 72.87), C-8 (dC 75.83), and C-19 (dC 24.33) of 2 were similar to those of 7a,8b-dihydroxydeepoxysarcophine at dC 26.7 (C-6), 72.5 (C-7), 75.4 (C-8), and 24.2 (C-19)26 and different from those of 1 (see Table 1) and 7b,8a-dihydroxydeepoxysarcophine at dC 27.8 (C-6), 72.3 (C-7), 78.0 (C-8), and 26.4 (C-19),26 thereby indicating a 7a,8b-dihydroxyl configuration in 2. The relative configurations at C-1, C-2, C-15, and C-16 of 2 were identical to those of 1 based on agreement of 1H and 13C NMR data between these two compounds and NOESY data. Thus, the gross structure of lobocrasol B (2) was elucidated as a C-7 epimer of lobocrasol A (1). Compound 323 was obtained as a colorless oil. The FTICRMS spectrum exhibited a pseudo-molecular ion peak at m/z 353.23246 [M+H]+, which is consistent with the molecular formula C20H32O5. The 13C NMR spectrum of 3 showed twenty carbon atom signals, which were identified using DEPT and HSQC spectra as three methyl, seven methylene, five methine, and five quaternary carbons. The NMR spectroscopic data of 3 (see Table 2) were similar to those of sinumaximol G, except for the presence of an oxygenated quaternary carbon, a 1,1-disubstituted double bond, and

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Table 2 NMR spectroscopic data of compounds 3 and 4 C

3 dC

4 a,b

dC

a,c

mult. (J in Hz)

dC

b,d

dC

c,d

mult. (J in Hz)

dCb,d

dCc,d mult. (J in Hz) — 4.27 5.09 — 1.95 2.25 1.15 3.33 — 1.90 5.57 5.32 — 1.44 1.29 — 4.29 4.98 4.99 1.55 0.99 1.12 5.67 3.74 3.93 5.23

1 2 3 4 5

81.59 84.69 121.06 142.39 37.20

— 4.42 d (9.0) 5.21 dd (1.0, 9.0) — 2.21 m/2.28 m

79.96 82.99 120.14 139.13 35.20

— 4.25 d (9.0) 5.07 d (9.0) — 2.02 m/2.17 m

80.23 82.64 123.32 137.84 35.61

6 7 8 9 10 11 12 13 14 15 16 17

28.29 72.53 75.54 44.54 125.38 140.71 74.07 36.08 30.85 153.08 70.42 105.72

1.55 3.57 — 2.21 5.64 5.51 — 1.55 1.55 — 4.45 5.06 5.13 1.80 1.21 1.27

26.73 70.36 73.43 43.21 123.10 139.87 71.71 35.38 29.93 152.97 68.74 104.36

1.35 m/1.85 m 3.34e — 2.02 m/2.23 m 5.44 m 5.35 d (16.0) — 1.35 m/1.42 m 1.32 m/1.42 m — 4.29 m 4.93 br s 4.97 br s 1.67 s 1.05 s 1.10 s 4.76 s 3.74 d (7.5) 4.07 s 4.38 s

26.17 68.94 72.89 42.82 123.83 138.87 71.96 35.65 28.11 153.04 68.61 105.12

18 19 20 1-OH 7-OH 8-OH 12-OH

18.27 23.32 26.54

m/2.02 m dd (2.0, 10.0) m/2.41 m m d (16.0) m/1.65 m m dd (2.0, 4.5) t (2.0) t (2.0) d (1.0) s s

18.06 23.47 26.83 — — — —

16.06 22.23 29.17 — — — —

d (9.0) d (9.0) m dt (3.0, 13.0) m/1.76 m dd (6.0, 10.0) m/2.34 m m d (15.5) m/1.58 m m/1.64 m t (2.5) s s s s s s d (6.0) s s

Assignments were confirmed by HSQC, HMBC, and NOESY experiments. a Measured in CD3OD. b 125 MHz. c 500 MHz. d Measured in DMSO-d6. e Overlapped signal.

an oxymethylene group in 3 instead of a fully substituted double bond, an a,b-conjugated lactone carbonyl carbon, and a tertiary methyl group in sinumaximol G.21 Assignment of the NMR data of 3 was performed by comparison with the corresponding values of sinumaximol G and further confirmed based on HMBC analysis (see Fig. 3). The HMBC cross peaks of H-18 (dH 1.80) with C-3 (dC 121.06)/C-4 (dC 142.39) and those of H-20 (dH 1.27) with C-11 (dC 140.71)/C-12 (dC 74.07) indicated the two double bonds at C-3/C4, C-10/C-11 and oxygenated quaternary carbon at C-12. The presence of a 1,1-disubstituted double bond (dC 105.72 and 153.08) and the additional oxygenated quaternary carbon at C-1, was also confirmed using HMBC correlations between H-17 (dC 5.06/5.13, each t, J = 2.0 Hz) and C-1 (dC 81.59)/C-15 (dC 153.08)/C-16 (dC 70.42).

H HO

O

HO OH OH

HMBC

NOESY

Figure 3. Key HMBC and NOESY correlations of 3.

Methyl protons H-19 (dH 1.21) correlated with C-7 (dC 72.53)/C-8 (dC 75.54)/C-9 (dC 44.54), confirming placement of the two hydroxy groups at C-7 and C-8 (see Fig. 3). The relative configuration of 3 was elucidated by comparison of 1 H and 13C NMR data with those of similar reported compounds and based on NOESY interactions. The 13C NMR chemical shifts in CD3OD of 3 at C-7 (dC 72.53), C-8 (dC 75.54), and C-19 (dC 23.32) were similar to those of 2 suggesting for the same 7a,8b-dihydroxy configuration for both compounds. Moreover, an agreement of the 13 C NMR data in CD3OD of 3 at C-1 (dC 81.59) and C-2 (dC 84.69) with those of sinumaximol D at dC 81.1 (C-1) and 83.8 (C-2)21 and laevigatol B at dC 81.6 (C-1) and 84.8 (C-2)24 indicated the same configuration at C-1 and C-2 for these three compounds. To further confirm the configuration of the hydroxy group at C-1 of 3, 1D and 2D NMR spectra were obtained again in DMSO-d6 (see Table 2). Clear NOE interactions (in DMSO-d6) between H-2 (dH 4.25) and H3-18 (dH 1.67)/1-OH (dH 4.76) suggested that 1-OH, H-2, and H-18 were all in a b-orientation (see Fig. 3). Additionally, the 13C NMR chemical shifts for C-12 (dC 71.71) and C-20 (dC 26.83) in DMSO-d6 of 3 were closely similar to that of sinumaximol G at dC 72.3 (C-12) and 26.2 (C-20),21 suggesting b-orientation of the hydroxy group at C-12 in both compounds. Thus, the structure for compound 3 was established and named as lobocrasol C. Lobocrasol D (4)23 was obtained as a colorless oil. Its molecular formula was determined to be C20H32O5 by FTICRMS at m/z 353.23272. The 1H and 13C NMR data of 4 were closely related to those of 3. However, the 13C NMR spectrum of 4 revealed a substantial difference in the C-20 signal at dC 29.17 compared to that of 3 at dC 26.83, suggesting an opposite configuration of the hydroxy group at C-12 of these two compounds. The a-configuration of the hydoxy group at C-12 in 4 was confirmed by the strong NOESY correlation of H-20 (dH 1.12) with H-10 (dH 5.57) instead that of H11 as was observed for 3. Careful comparison of the 1H and 13C

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0.1 μM

1.0 μM

10.0 μM

14000 12000

Luciferase activity

10000 8000 6000 4000 2000

TNFα 0 (10 ng/mL)

DMSO

+

+

+

+

+

+

Pos.

1

2

3

4

Compounds Figure 4. Effects of compounds 14 on the TNFa-induced NF-jB luciferase reporter activity in HepG2 cells. The values are mean ± SD (n = 3). Suf: Sulfasalazine (1.0 lM) was used as a positive control (Pos.).

NMR data of 4 with those of 3 in combination with NOESY data confirmed the structure of lobocrasol D (4) as shown in Fig. 1. In vitro anti-inflammatory activity of compounds 1–4 was evaluated through the inhibition of TNFa-induced NF-jB transcriptional activation in HepG2 cells (see Supplementary data). The NF-jB luciferase assay is designed to monitor the activity of NF-jB-regulated signal transduction pathways in cultured cells. The NF-jB-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal cytomegalovirus (CMV) promoter and tandem repeats of the NF-jB transcriptional response element. Using this assay, the inhibitory activity of compounds (1–4) on NF-jB activation was readily monitored. Pro-inflammatory agents, such as TNFa, activate the NF-jB pathway.7 HepG2 cells transfected with the NF-jB luciferase reporter plasmid exhibited an approximately fourfold increase in the luciferase signal after treatment with 10 ng/mL TNFa, indicating an increase in transcriptional activity compared with untreated cells. Modulation in the expression of genes underlies most cellular responses, including inflammation. Thus, molecules that interfere with factors involved in the modulation of gene expression, such as NF-jB, must also be considered potential anti-inflammatory agents.27 Among isolated compounds, 1 and 2 exhibited significant inhibitory effect on NF-jB activation with IC50 values of 6.30 ± 0.42, and 6.63 ± 0.11 lM, respectively. In contrast, compounds 3 and 4 showed moderate and/or weak activities (IC50 > 20 lM), comparing to the positive control, sulfasalazine (IC50 = 0.90 ± 0.20 lM, see Fig. 4). To confirm the transcriptional inhibitory function of compounds 1 and 2, we further investigated their effects on

iNOS and COX-2 gene expression in TNFa-stimulated HepG2 cells using RT-PCR. Consistent with their inhibitory activity toward NF-jB, compounds 1 and 2 moderately inhibited the induction of iNOS and COX-2 mRNA in a dose-dependent manner, indicating that these compounds reduced transcription of these genes. Moreover, the expression of the gene encoding the house keeping protein b-actin was unaltered in the presence of compounds 1 and 2 at the same concentration (see Fig. 5). Our data suggested that compounds 1 and 2 isolated from the soft coral L. crassum have therapeutic potential as anti-inflammatory, anti-atherosclerotic, and anti-arthritic agents. However, further investigation is required to elucidate more information on the mechanism underlying the inhibition of the TNFa-induced NF-jB pathway and the subsequent decreases in iNOS and COX-2 gene expression by compounds 1 and 2. The structure–activity relationships of compounds 1–4 indicated that the presence of an epoxy group at C-1/C-15 is necessary for the anti-inflammatory activity of these compounds. This information may facilitate identification of other anti-inflammatory lead compounds from diterpenoids and provides support for further studies. Acknowledgments This study was supported by a grant from the Vietnam National Foundation for Science & Technology Development (Project No: 104.01-2012.37) and the framework of International Cooperation Program managed by National Research Foundation of Korea (2012K2A1A2032970), and Basic Science Research Program

Fig. 5. Effects of compounds 1 and 2 on iNOS and COX-2 mRNA expression in HepG2 cells. Suf: Sulfasalazine (1.0 lM) was used as a positive control.

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through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20120006681), Republic of Korea. The authors are grateful to the Institute of Chemistry, VAST for the provision of the spectroscopic instrument. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 11.033. References and notes 1. Li, Q.; Verma, I. M. Nat. Rev. Immunol. 2002, 2, 725. 2. Baldwin, A. S. J. Clin. Invest. 2001, 107, 3. 3. Surh, Y. J.; Chun, K. S.; Cha, H. H.; Han, S. S.; Keum, Y. S.; Park, K. K.; Lee, S. S. Mutat. Res. 2001, 480–481, 243. 4. Baldwin, A. S. Annu. Rev. Immunol. 1996, 14, 649. 5. Baldwin, A. S. J. Clin. Invest. 2001, 107, 241. 6. Perkins, N. D. Nat. Rev. Mol. Cell Biol. 2007, 8, 49. 7. Paul, A. G. Eukaryon 2005, 1, 4. 8. Villa, F. A.; Gerwick, L. Immunopharmacol. Immunotoxicol. 2010, 32, 228. 9. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2012, 29, 144. 10. Gross, H.; Kehraus, S.; Nett, M.; König, G. M.; Beil, W.; Wright, A. D. Org. Biomol. Chem. 2003, 1, 944. 11. Coll, J. C. Chem. Rev. 1992, 92, 613. 12. Rashid, M. A.; Gustafson, K. R.; Boyd, M. R. J. Nat. Prod. 2000, 63, 531. 13. Su, J.-H.; Ahmed, A. F.; Sung, P.-J.; Chao, C.-H.; Kuo, Y.-H.; Sheu, J.-H. J. Nat. Prod. 2006, 69, 1134. 14. Cheng, S.-Y.; Wang, S.-K.; Chiou, S.-F.; Hsu, C.-H.; Dai, C.-F.; Chiang, M. Y.; Duh, C.-Y. J. Nat. Prod. 2010, 73, 197. 15. Lin, W.-Y.; Lu, Y.; Chen, B.-W.; Huang, C.-Y.; Su, J.-H.; Wen, Z.-H.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. Mar. Drugs 2012, 10, 617. 16. Wanzola, M.; Furuta, T.; Kohno, Y.; Fukumitsu, S.; Yasukochi, S.; Watari, K.; Tanaka, C.; Higuchi, R.; Miyamoto, T. Chem. Pharm. Bull. 2010, 58, 1203. 17. Chao, C.-H.; Wen, Z.-H.; Wu, Y.-C.; Yeh, H.-C.; Sheu, J.-H. J. Nat. Prod. 2008, 71, 1819. 18. Thao, N. P.; Nam, N. H.; Cuong, N. X.; Tai, B. H.; Quang, T. H.; Ngan, N. T. T.; Luyen, B. T. T.; Yang, S. Y.; Choi, C. H.; Kim, S.; Chae, D.; Koh, Y. S.; Kiem, P. V.; Minh, C. V.; Kim, Y. H. Bull. Korean Chem. Soc. 2013, 34, 949. 19. Quang, T. H.; Ha, T. T.; Minh, C. V.; Kiem, P. V.; Huong, H. T.; Ngan, N. T. T.; Nhiem, N. X.; Tung, N. H.; Thao, N. P.; Thuy, D. T. T.; Song, S. B.; Boo, H.-J.; Kang, H.-K.; Kim, Y. H. Bioorg. Med. Chem. Lett. 2011, 21, 2845.

20. Thao, N. P.; Nam, N. H.; Cuong, N. X.; Quang, T. H.; Tung, P. T.; Dat, L. D.; Chae, D.; Kim, S.; Koh, Y. S.; Kiem, P. V.; Minh, C. V.; Kim, Y. H. Bioorg. Med. Chem. Lett. 2013, 23, 228. 21. Thao, N. P.; Nam, N. H.; Cuong, N. X.; Quang, T. H.; Tung, P. T.; Tai, B. H.; Luyen, B. T. T.; Chae, D.; Kim, S.; Koh, Y. S.; Kiem, P. V.; Minh, C. V.; Kim, Y. H. Chem. Pharm. Bull. 2012, 60, 1581. 22. Extraction and isolation: Freeze-dried bodies of the soft coral L. crassum (1.0 kg) were well grinded and extracted three times with hot MeOH (at 50 °C for 5 h each time). The obtained solutions were filtered, combined, and concentrated under reduced pressure to yield a dark brown viscous residue (75.0 g, A). This residue was suspended in water (1 L) and partitioned in turn with n-hexane and CH2Cl2 (3  1 L). The combined CH2Cl2 soluble portions were evaporated under reduced pressure to afford CH2Cl2 fraction (6.02 g, B). Fraction B was crudely separated by silica gel CC using gradient concentrations of ethyl acetate in n-hexane from 0% to 100% to yield five fractions, B-1 to B-5. Fraction B-4 (0.12 g) was fractionated into three subfractions, B-4.1 to B-4.3, by YMC RP-18 CC using stepwise elution with acetone–H2O (1:2 to 1.5:1). Subfraction B-4.2 (0.03 g) afforded compounds 1 (6.6 mg) and 2 (4.5 mg) after subjecting it to silica gel CC eluting with CH2Cl2–MeOH (12:1). Similarly, subfraction B-4.3 (0.08 g) was chromatographed on silica gel CC eluting with CHCl3–MeOH– acetone (10:1:0.3) and further separated by YMC RP-18 CC with acetone–H2O (1.2:1) to obtain compounds 3 (6.1 mg), and 4 (5.4 mg). 23. Physical and spectroscopic data of new compounds: Lobocrasol A (1): ½a25 D 12.6° (c 0.15, CHCl3); IR (KBr) mmax 3441, 2931, 2868, 1736, 1715, 1669, 1448, 1383, 1250, 1063, 1014, and 909 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) are given in Table 1; FTICRMS m/z 353.23282 [M+H]+ (calcd for C20H33O5, 353.23280). Lobocrasol B (2): ½a25 D +5.0° (c 0.15, CHCl3); IR (KBr) mmax 3375, 2926, 2864, 1758, 1660, 1452, 1382, 1061, and 971 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) are given in Table 1; FTICRMS m/z 353.23272 [M+H]+ (calcd for C20H33O5, 353.23280). Lobocrasol C (3): ½a25 D +8.4° (c 0.15, MeOH); IR (KBr) mmax 3456, 2926, 2862, 1760, 1712, 1451, 1383, 1250, and 1074 cm1; 1H NMR (CD3OD, DMSO-d6, 500 MHz) and 13C NMR (CD3OD, DMSO-d6, 125 MHz) are given in Table 2; FTICRMS m/z 353.23246 [M+H]+ (calcd for C20H33O5, 353.23280). Lobocrasol D (4): ½a25 D 4.6° (c 0.15, CHCl3); IR (KBr) mmax 3442, 2921, 2822, 1760, 1710, 1452, 1363, 1250, and 1074 cm1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) are given in Table 2; FTICRMS m/z 353.23272 [M+H]+ (calcd for C20H33O5, 353.23280). 24. Quang, T. H.; Ha, T. T.; Minh, C. V.; Kiem, P. V.; Huong, H. T.; Ngan, N. T. T.; Nhiem, N. X.; Tung, N. H.; Tai, B. H.; Thuy, D. T. T.; Song, S. B.; Kang, H.-K.; Kim, Y. H. Bioorg. Med. Chem. 2011, 19, 2625. 25. Cuong, N. X.; Tuan, T. A.; Kiem, P. V.; Minh, C. V.; Choi, E. M.; Kim, Y. H. Chem. Pharm. Bull. 2008, 56, 988. 26. Sayed, K. A. E.; Hamann, M. T.; Waddling, C. A.; Jensen, C.; Lee, S. K.; Dunstan, C. A.; Pezzuto, J. M. J. Org. Chem. 1998, 63, 7449. 27. Terracciano, S.; Aquino, M.; Rodriquez, M.; Monti, M. C.; Casapullo, A.; Riccio, R.; Paloma, L. G. Curr. Med. Chem. 2006, 13, 1947.