MD Marine Drugs MD 1660-3397 Molecular Diversity Preservation International 10.3390/md9060934 marinedrugs-09-00934 Article Discovery of New Eunicellins from an Indonesian Octocoral Cladiella sp. ChenYung-Husan1 TaiChia-Ying12 SuYin-Di13 ChangYu-Chia14 LuMei-Chin12 WengChing-Feng5 SuJui-Hsin126 HwangTsong-Long7 WuYang-Chang89* SungPing-Jyun12356* National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan; E-Mails: tony_chen72001@yahoo.com.tw (Y.-H.C.); j19851214@hotmail.com (C.-Y.T.); gobetter04@yahoo.com.tw (Y.-D.S.); jay0404@gmail.com (Y.-C.C.); jinx6609@nmmba.gov.tw (M.-C.L.); x2219@nmmba.gov.tw (J.-H.S.) Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan; E-Mail: cfweng@mail.ndhu.edu.tw (C.-F.W.) Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan; E-Mail: htl@mail.cgu.edu.tw (T.-L.H.) Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 404, Taiwan Authors to whom correspondence should be addressed; E-Mails: yachwu@mail.cmu.edu.tw (Y.-C.W.); pjsung@nmmba.gov.tw (P.-J.S.); Tel.: +886-8-8825037 (P.-J.S.); Fax: +886-8-8825087 (P.-J.S.). 2011 26 5 2011 9 6 934 943 14 4 2011 18 5 2011 25 5 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland. 2011

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Two new 11-hydroxyeunicellin diterpenoids, cladieunicellin F (1) and (–)-solenopodin C (2), were isolated from an Indonesian octocoral Cladiella sp. The structures of eunicellins 1 and 2 were established by spectroscopic methods, and eunicellin 2 was found to be an enantiomer of the known eunicellin solenopodin C (3). Eunicellin 2 displayed inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. The previously reported structures of two eunicellin-based compounds, cladielloides A and B, are corrected in this study.

 cladieunicellin eunicellin octocoral Cladiella superoxide anion elastase Introduction

As part of our ongoing investigation into the isolation of new natural substances from octocorals collected in the tropical West Pacific Ocean, a series of interesting diterpenoids, including cembrane-type [1], eunicellin-type (2,11-cyclized cembranoid) [2–4], and briarane-type (3,8-cyclized cembranoid) [5–7] diterpenoids, were isolated from various octocorals belonging to the genera Briareum, Cladiella, Ellisella, and Junceella. The chemical constituents of an Indonesian octocoral identified as Cladiella sp. were examined, and nine new eunicellin-based diterpenoids, including cladieunicellins A–E [2] and cladielloides A–D [3,4], were isolated. Further study of this coral yielded two new eunicellins, cladieunicellin F (1) and (–)-solenopodin C (2). In this paper, we report the isolation, structure determination, and bioactivity of new eunicellins 1 and 2 (Figure 1).

 Results and Discussion

Cladieunicellin F (1) was isolated as a colorless oil and the molecular formula for this compound was determined using HRESIMS to be C20H34O3 (four degrees of unsaturation) (m/z 345.2404 [M + Na]+, calculated for 345.2406). Comparison of the 13C NMR and DEPT data with the molecular formula indicated that there must be two exchangeable protons, which required the presence of two hydroxyl groups. This deduction was supported by a broad absorption in the IR spectrum at 3414 cm−1. The 13C NMR data for 1 confirmed the presence of twenty carbon signals (Table 1), which were characterized by DEPT as four methyls, an sp2 methylene, six sp3 methylenes, six sp3 methines (including two oxymethines), two sp3 oxygenated quaternary carbons, and an sp2 quaternary carbon. Based on the 1H and 13C NMR spectra (Table 1), 1 was determined to possess an exocyclic carbon-carbon double bond (δH 5.06, 2H, br s, H2-16; δC 150.6, s, C-7; 111.8, t, C-16). The presence of a trisubstituted epoxide containing a methyl substituent was established from the signals of an oxygenated quaternary carbon (δC 62.9, s, C-3) and an oxymethine (δH 2.81, 1H, d, J = 9.6 Hz; δC 64.8, d, CH-2) and confirmed by the proton signal of a methyl singlet at δH 1.36 (3H, s, H3-15). In the 1H NMR spectrum of 1, two doublets at δH 0.88 and 0.81 (each 3H, d, J = 6.4 Hz, H3-19 and H3-20) were indicative of the two methyls of an isopropyl group. A tertiary methyl group bonded to an oxygenated carbon was evident from the singlet signal at δH 1.24 (3H, s, H3-17). Thus, from the reported data, the proposed skeleton of 1 was suggested to be an eunicellin-based diterpenoid with three rings.

From the 1H–1H COSY spectrum of 1 (Table 1), it was possible to differentiate among the separate spin systems of H-1/H-2, H2-4/H2-5/H-6, H2-8/H2-9/H-10/H-1, H2-12/H2-13/H-14/H-1, and H-14/H-18/H3-19 (H3-20), which was accomplished with the assistance of an HMBC experiment (Table 1). The key HMBC correlations between the protons and quaternary carbons of 1, including H-1, H-2, H2-4, H2-5, H3-15/C-3; H2-5, H-8b, H-9b, H2-16/C-7; and H-1, H-10, H2-12, H2-13, H3-17/C-11, permitted the elucidation of the carbon skeleton. An exocyclic carbon-carbon double bond at C-7 was confirmed by the HMBC correlations between H2-16/C-6, -7, -8; H-6/C-16; and H-8b/C-16 and further supported by the allylic coupling between H-8b/H2-16. The presence of the C-2/3 epoxide group was confirmed by the HMBC correlations between H-1/C-2, -3; H-2/C-1, -3, -10, -15; H2-4/C-2, -3; and H3-15/C-2, -3, -4. Thus, the remaining hydroxyl groups should be positioned at C-6 and C-11, as indicated by the key 1H–1H COSY correlations and characteristic NMR signals, although the hydroxyl protons for OH-6 and OH-11 were not observed in the 1H NMR spectrum of 1.

The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment (Figure 2): H-1 correlated with H-10 and H3-20, indicating that H-1, H-10, and the isopropyl group are situated on the same face and assigned as β protons. H-2 exhibited interactions with H-14 and Me-17, and no correlation was found between H-1/H-2, H-10/Me-17, and H-2/Me-15, indicating that H-2, H-14, and Me-17 should be α-oriented and Me-15 should be β-oriented. Furthermore, H-6 correlated with two protons of the C-9 methylene and Me-15. Consideration of molecular models found that H-6 was reasonably close to H2-9 and Me-15 when H-6 was β-oriented in 1. Based on the above findings, the structure of 1, including its relative configuration, was established, and the chiral centers for 1 were assigned as 1R*, 2S*, 3S*, 6R*, 10R*, 11R*, and 14R*. To the best of our knowledge, cladieunicellin F (1) is the first eunicellin derivative possessing a C-2/3 epoxy group.

Eunicellin 2 was isolated as a colorless oil, and the molecular formula of this compound was determined using HRESIMS to be C20H34O2 (m/z 329.2455 [M + Na]+, calculated for 329.2456). Thus, four degrees of unsaturation were determined for 2. Detailed analysis of the NMR data showed that the data for 2 were similar to those of a known eunicellin analogue, solenopodin C (3) (Figure 1), which was isolated from the gorgonian Solenopodium stechei [8]. However, the optical rotation value of 2 ( [ α ] D 22 −51 (c 0.17, CHCl3)) was substantially different from that of 3 ( [ α ] D 22 +105.6 (c 0.36)), indicating that eunicellin 2 is an enantiomer of 3 and should be designated (–)-solenopodin C. The 1H and 13C NMR data for 2 (Table 2) were assigned using 2D NMR data analysis and comparison to the NMR data of 3. The proton chemical shifts for C-8, C-9, C-12, and C-13 methylene protons and the carbon chemical shifts for C-1, C-4, C-12, and C-14 of compound 3 should be revised (Table 2).

In a previous study, we reported the isolation and structure determination of two eunicellins, cladielloides A (4) and B (5) (Figure 3) [3]. However, based on detailed spectral data analysis, we found that the structures for these two compounds should be revised. 1D and 2D NMR spectral data analysis, particularly 1H–1H COSY and HMBC experiments, of cladielloide A (Table 3), showed that the main carbon skeleton of cladielloide A was established correctly. However, in the HMBC experiment for cladielloide A, key correlations between H-4 (δH 5.14) and an ester carbonyl at δC 171.4 (s, C-1′) and between H-2′ (δH 4.86) and two ester carbonyls at δC 171.4 (s, C-1′) and 171.1 (s, acetate carbonyl) were detected, and these findings indicated that the 2′-acetoxybutyrate group should be positioned at C-4. Thus, the remaining hydroxyl groups are attached at C-3 and C-6 in cladielloide A, respectively. Furthermore, in the NOESY spectrum of cladielloide A, H-6 (δH 4.21) correlated with H2-5 (δH 2.97 and 1.75) and H2-8 (δH 2.35), but no correlation was found between H-6 and H3-15. Consideration of molecular models found that H-6 was reasonably close to H2-5 and H2-8 when it was β-oriented. Based on the above findings, the structure, including the relative configuration, of cladielloide A should be revised as presented in eunicellin 6. Cladielloide B was found by HRESIMS to be an isomer of cladielloide A [3]. These two compounds were found to possess the same planar structure by NMR data analysis (Tables 3 and 4). In the NOESY experiment of cladielloide B, H-6 (δH 4.66) exhibited correlations with H3-15 (δH 1.33); a proton of C-5 methylene (δH 2.48); and a proton of C-8 methylene (δH 2.65), indicating that the 6-hydroxyl group in cladielloide B should be β-oriented as presented as eunicellin 7. Based on the above findings, the structures of cladielloides A and B should be revised as structures 6 and 7, respectively. The authors apologize for any inconvenience caused by these errors.

The in vitro anti-inflammatory effects of eunicellins 1 and 2 were tested. Eunicellin 2 displayed significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils at a concentration of 10 μg/mL (Table 5).

 Experimental Section General Experimental Procedures

Optical rotation values were measured with a JASCO P-1010 digital polarimeter. Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrophotometer. The NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR at 400 MHz and 100 MHz for 1H and 13C spectra, respectively, in CDCl3 at 25 °C. Proton chemical shifts were referenced to the residual CHCl3 signal (δH 7.26 ppm). 13C NMR spectra were referenced to the center peak of CDCl3 at δC 77.1 ppm. ESIMS and HRESIMS data were recorded on a BRUKER APEX II mass spectrometer. Column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck), and spots were visualized by spraying with 10% H2SO4 solution followed by heating. HPLC was performed using a system comprised of a HITACHI L-2130 pump, a HITACHI photodiode array detector L-2455, and a RHEODYNE 7725 injection port. A reverse phase column (Polaris 5 C18-A 250 × 10.0 mm, Varian, silica gel 60, 5 μm) was used for HPLC.

 Animal Material

The octocoral Cladiella sp. was collected and imported legitimately by the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan from Indonesia in 2004. The material was stored in a freezer until extraction procedures were applied. A voucher specimen (NMMBA-IND-SC-001) was deposited in the NMMBA, Taiwan. This organism was identified by comparison with previous descriptions [10,11].

 Extraction and Isolation

Sliced bodies of Cladiella sp. (wet weight, 924 g) were extracted with a mixture of MeOH and CH2Cl2 (1:1), and the residue collected after solvent evaporation was partitioned between EtOAc and H2O. The EtOAc layer was subjected to silica gel column chromatography and eluted using a mixture of n-hexane and EtOAc (stepwise from 100:1 to 0:100 n-hexane:EtOAc) to obtain 19 fractions, labeled A–S. Fractions F and I were separated by reverse phase HPLC using a mixture of MeOH and water to afford eunicellins 2 (3.3 mg, 1/1) and 1 (1.5 mg, 1/1), respectively.

Cladieunicellin F (1): colorless oil; [ α ] D 23 −194 (c 0.07, CHCl3); IR (neat) νmax 3414 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; ESIMS m/z 345 [M + Na]+; HRESIMS m/z 345.2404 (Calcd for 345.2406).

(–)-Solenopodin C (2): colorless oil; [ α ] D 22 −51 (c 0.17, CHCl3); IR (neat) νmax 3427 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 2; ESIMS m/z 329 [M + Na]+; HRESIMS m/z 329.2455 (Calcd for 329.2456).

 Superoxide Anion Generation and Elastase Release by Human Neutrophils

Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [12,13]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

The authors would like to thank I-Chu Huang, the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan for her contribution to identify the marine organism. This research was supported by grants from the National Museum of Marine Biology and Aquarium (Grant No. 100100101 and No. 100200311); National Dong Hwa University; the Asia-Pacific Ocean Research Center, National Sun Yat-sen University (98C031702); and the National Science and Technology Program for Biotechnology and Pharmaceuticals, National Science Council (Grant No. NSC 99-2323-B-291-001 and No. 98-2320-B-291-001-MY3), Taiwan, awarded to P.-J.S.

Samples Availability: Not available.

 References and Notes ChangY-CHuangI-CChiangMYHwangT-LKungT-HLinC-SSheuJ-HSungP-JBriaviodiol A, a new cembranoid from a soft coralBriareum violacea Chem Pharm Bull2010581666166810.1248/cpb.58.1666 ChenY-HTaiC-YKuoY-HKaoC-YLiJ-JHwangT-LFangL-SWangW-HSheuJ-HSungP-JCladieunicellins A–E, new eunicellins from an Indonesian soft coral Cladiella spChem Pharm Bull20115935335810.1248/cpb.59.35321372417 ChenY-HTaiC-YHwangT-LWengC-FLiJ-JFangL-SWangW-HWuY-CSungP-JCladielloides A and B: new eunicellin-type diterpenoids from an Indonesian octocoral Cladiella spMar Drugs201082936294510.3390/md812293621339957 TaiC-YChenY-HHwangT-LFangL-SWangW-HLiuM-CSuJ-HWuY-CSungP-JCladielloides C and D: novel eunicellin-based diterpenoids from an Indonesian octocoral Cladiella spBull Chem Soc Jpn20118453153610.1246/bcsj.20100348 SungP-JChangP-CFangL-SSheuJ-HChenW-CChenY-PLinM-RSurvey of briarane-related diterpenoids-Part IIHeterocycles200565195204and references cited therein10.3987/REV-04-589 SungP-JSheuJ-HWangW-HFangL-SChungH-MPaiC-HSuY-DTsaiW-TChenB-YLinM-RLiG-YSurvey of briarane-type diterpenoids–Part IIIHeterocycles20087526272648and references cited therein10.3987/REV-08-633 SungP-JSuJ-HWangW-HSheuJ-HFangL-SWuY-CChenY-HChungH-MSuY-DChangY-CSurvey of briarane-type diterpenoids–Part IVHeterocycles20118312411258and references cited therein BloorSJSchmitzFJHossainMBvan der HelmDDiterpenoids from the gorgonianSolenopodium stechei J Org Chem1992571205121610.1021/jo00030a031 Diphenylene indonium (DPI) and elastatinal were used as reference compounds in anti-inflammatory activity testing. DPI displayed an inhibitory effect on superoxide anion generation (IC50 = 0.8 μg/mL), and elastatinal exhibited an inhibitory effect on elastase release (IC50 = 31 μg/mL) by human neutrophils, respectively. BayerFMKey to the genera of octocorallia exclusive of Pennatulacea (Coelenterata: anthozoa), with diagnoses of new taxaProc Biol Soc Wash198194902947 FabriciusKAldersladePSoft Corals and Sea Fans–A Comprehensive Guide to the Tropical Shallow-Water Genera of the Central-West Pacific, the Indian Ocean and the Red Sea1st edAustralian Institute of Marine ScienceQueensland, Australia2001498485 HwangT-LLiG-LLanY-HChiaY-CHsiehP-WWuY-HWuY-CPotent inhibitors of superoxide anion production in activated human neutrophils by isopedicin, a bioactive component of the Chinese medicinal herbFissistigma oldhamii Free Radic Biol Med20094652052810.1016/j.freeradbiomed.2008.11.014 HwangT-LSuY-CChangH-LLeuY-LChungP-JKuoL-MChangY-JSuppression of superoxide anion and elastase release by C18 unsaturated fatty acids in human neutrophilsJ Lipid Res2009501395140810.1194/jlr.M800574-JLR20019295184 Figures and Tables

The structures of cladieunicellin F (1), (−)-solenopodin C (2), and solenopodin C (3).

Selective NOESY correlations for 1.

The previously reported structures of cladielloides A (4) and B (5) and their revised structures 6 and 7, respectively.

1H and 13C NMR data, 1H–1H COSY, and HMBC correlations for diterpenoid 1.

C/H 1Ha 13Cb 1H–1H COSY HMBC (H→C)
1 2.12 m 36.0 (d)d H-2, H-10, H-14 C-2, C-3, C-9, C-10, C-11, C-14, C-18
2 2.81 d (9.6)c 64.8 (d) H-1 C-1, C-3, C-10, C-15
3 62.9 (s)
4a 1.27 m 27.3 (t) H-4b, H2-5 C-3, C-5, C-15
 b 1.81 m H-4a, H2-5 C-2, C-3, C-5, C-6, C-15
5 2.01 m 30.3 (t) H2-4, H-6 C-3, C-4, C-6, C-7
6 4.20 dd (10.4, 4.0) 68.4 (d) H2-5 C-16
7 150.6 (s)
8a 2.16 m 37.5 (t) H-8b, H2-9 n.o.e
 b 2.61 m H-8a, H2-9, H2-16 C-7, C-9, C-10, C-16
9a 1.44 m 22.9 (t) H2-8, H-9b, H-10 n.o.
 b 1.96 m H2-8, H-9a, H-10 C-1, C-7, C-8
10 1.68 dd (10.8, 6.0) 46.1 (d) H-1, H2-9 C-1, C-2, C-8, C-9, C-11, C-12, C-17
11 72.9 (s)
12 1.59 m 37.2 (t) H2-13 C-10, C-11, C-13, C-14, C-17
13a 1.55 m 21.8 (t) H2-12, H-13b, H-14 C-11, C-12, C-14
 b 1.71 m H2-12, H-13a, H-14 C-11, C-12, C-14
14 1.32 m 41.5 (d) H-1, H2-13, H-18 C-13
15 1.36 s 21.8 (q) C-2, C-3, C-4
16 5.06 br s 111.8 (t) H-8b C-6, C-7, C-8
17 1.24 s 24.4 (q) C-10, C-11, C-12
18 1.81 m 26.5 (d) H-14, H3-19, H3-20 C-14, C-19, C-20
19 0.88 d (6.4) 20.6 (q) H-18 C-14, C-18, C-20
20 0.81 d (6.4) 21.7 (q) H-18 C-14, C-18, C-19

Spectra were measured at 400 MHz in CDCl3.

Spectra were measured at 100 MHz in CDCl3.

J values (in hertz) are in parentheses.

Attached protons were deduced by DEPT and HMQC experiments.

n.o. = not observed.

1H and 13C NMR data for diterpenoids 2 and 3.

C/H 2
3
1Ha 13Cb 1He 13Ce
1 2.73 ddd (7.6, 6.0, 6.0)c 36.6 (d)d 2.73 ddd (7.5, 6, 6) 44.8 (d)
2 5.35 d (7.6) 130.4 (d) 5.34 br d (7.5) 130.4 (d)
3 134.3 (s) 134.3 (s)
4a 2.47 ddd (13.2, 13.2, 2.8) 28.0 (t) 2.47 ddd (13.4, 13, 2.2) 35.2 (t)
 b 1.95 m 1.9 m
5a 2.13 m 25.3 (t) 2.1 m 25.3 (t)
 b 1.37 m 1.3 m
6 3.10 dd (10.8, 3.6) 65.9 (d) 3.10 dd (11.2, 3.5) 65.9 (d)
7 61.2 (s) 61.1 (s)
8a 2.14 m 38.6 (t) 2.3 m 38.6 (t)
 b 0.93 m
9a 1.58 m 22.5 (t) 1.2–1.7 m 22.2 (t)
 b 0.90 m
10 1.95 m 47.3 (d) 1.95 ddd (6, 6, 6) 47.3 (d)
11 73.2 (s) 73.2 (s)
12a 1.52 m 35.2 (t) 28.1 (t)
  b 1.49 m
13a 1.56 m 20.0 (t) 20.0 (t)
  b 1.45 m
14 1.02 m 44.7 (d) 1.0 m 36.6 (d)
15 1.71 s 24.9 (q) 1.71 s 24.9 (q)
16 1.18 s 18.2 (q) 1.22 s 18.2 (q)
17 1.22 s 26.7 (q) 1.18 s 26.8 (q)
18 1.95 m 26.6 (d) 1.90 m 26.6 (d)
19 0.97 d (6.8) 22.0 (q) 0.78 d (7) 22.0 (q)
20 0.78 d (6.8) 17.6 (q) 0.98 d (7) 17.7 (q)

Spectra were measured at 400 MHz in CDCl3.

Spectra were measured at 100 MHz in CDCl3.

J values (in hertz) are in parentheses.

Attached protons were deduced by DEPT and HMQC experiments.

Data were reported by Bloor et al. [8]. These data were measured at 300 MHz for 1H and 75 MHz for 13C in CDCl3.

1H and 13C NMR data, 1H–1H COSY, and HMBC correlations for cladielloide A (6).

C/H 1Ha 13Cb 1H–1H COSY HMBC (H→C)
1 2.74 ddd (8.0, 8.0, 4.0)c 39.7 (d)d H-2, H-10, H-14 C-2, C-3, C-10, C-11, C-14
2 3.86 d (8.0) 87.1 (d) H-1 C-3, C-4, C-14, C-15
3 74.1 (s)
4 5.14 dd (4.4, 4.4) 74.6 (d) H2-5 C-3, C-6, C-15, C-1′
5α 2.97 ddd (16.0, 4.4, 2.8) 37.2 (t) H-4, H-5β, H-6 C-3
 β 1.75 ddd (16.0, 5.6, 3.6) H-4, H-5α, H-6 C-3, C-4, C-7
6 4.21 br s 72.6 (d) H2-5, OH-6 n.o.e
7 147.6 (s)
8 2.35 br d (2.4) 40.0 (t) H-9 C-6, C-7, C-9, C-10, C-16
9 4.16 ddd (3.6, 3.2, 3.2) 81.3 (d) H2-8, H-10 n.o.
10 2.63 br s 44.6 (d) H-1, H-9 C-11
11 132.1 (s)
12 5.43 m 122.2 (d) H2-13, H3-17 n.o.
13α 2.10 m 22.8 (t) H-12, H-13β, H-14 n.o.
  β 1.98 m H-12, H-13α, H-14 n.o.
14 1.58 m 39.0 (d) H-1, H2-13, H-18 n.o.
15 1.37 s 22.4 (q) C-2, C-3, C-4
16a 5.21 s 115.2 (t) H-16b C-6, C-8
  b 5.58 s H-16a C-6, C-7, C-8
17 1.68 d (0.8) 22.0 (q) H-12
18 1.15 m 28.8 (d) H-14, H3-19, H3-20
19 0.92 d (6.4) 21.3 (q) H-18 C-14, C-18, C-20
20 0.83 d (6.4) 20.5 (q) H-18 C-14, C-18, C-19
OH-6 2.84 d (7.2) H-6 n.o.
1′ 171.4 (s)
2′ 4.86 dd (6.8, 6.0) 74.4 (d) H2-3′ C-1′, C-3′, C-4′, acetate carbonyl
3′ 1.91 m 24.3 (t) H-2′, H3-4′ C-1′, C-2′, C-4′
4′ 1.03 t (7.2) 9.3 (q) H2-3′ C-2′, C-3′
2′-OAc 171.1 (s)
2.14 s 20.6 (q) Acetate carbonyl

Spectra were measured at 400 MHz in CDCl3.

Spectra were measured at 100 MHz in CDCl3.

J values (in hertz) are in parentheses.

Attached protons were deduced by DEPT and HMQC experiments.

n.o. = not observed.

1H and 13C NMR data, 1H–1H COSY, and HMBC correlations for cladielloide B (7).

C/H 1Ha 13Cb 1H–1H COSY HMBC (H→C)
1 2.51 m 40.6 (d)d H-2, H-10, H-14 C-10
2 3.90 d (3.6)c 88.1 (d) H-1 C-1, C-3, C-4, C-10
3 74.8 (s)
4 5.21 dd (8.0, 4.0) 73.8 (d) H2-5 C-5, C-6, C-1′
5α 2.48 m 34.2 (t) H-4, H-5β, H-6 C-6, C-7
 β 1.97 m H-4, H-5α, H-6 n.o.e
6 4.66 dd (8.8, 3.2) 83.8 (d) H2-5 C-4, C-7, C-16
7 144.2 (s)
8α 2.65 dd (14.0, 4.8) 41.4 (t) H-8β, H-9, H-16a C-7, C-9, C-10, C-16
 β 2.46 dd (14.0, 2.0) H-8α, H-9 C-6, C-7, C-16
9 4.06 br s 82.4 (d) H2-8, H-10 n.o.
10 2.58 br s 44.7 (d) H-1, H-9 C-8, C-9, C-11
11 131.1 (s)
12 5.49 m 123.1 (d) H2-13, H3-17 n.o.
13α 2.01 m 22.9 (t) H-12, H-13β, H-14 n.o.
  β 1.80 m H-12, H-13α, H-14 n.o.
14 1.39 m 39.8 (d) H-1, H2-13, H-18 C-1, C-2
15 1.33 s 22.8 (q) C-2, C-3, C-4
16a 5.26 s 117.7 (t) H-8α, H-16b C-6, C-8
  b 5.47 s H-16a C-6, C-7, C-8
17 1.69 d (1.2) 22.8 (q) H-12 C-10, C-11, C-12
18 1.80 m 27.8 (d) H-14, H3-19, H3-20 C-14, C-19, C-20
19 0.94 d (6.8) 21.7 (q) H-18 C-14, C-18, C-20
20 0.77 d (6.8) 17.5 (q) H-18 C-14, C-18, C-19
1′ 170.2 (s)
2′ 4.87 dd (6.8, 6.0) 74.3 (d) H2-3′ C-1′, C-3′, C-4′, acetate carbonyl
3′ 1.91 m 24.5 (t) H-2′, H3-4′ C-1′, C-2′, C-4′
4′ 1.02 t (7.2) 9.3 (q) H2-3′ C-2′, C-3′
2′-OAc 171.6 (s)
2.14 s 20.6 (q) Acetate carbonyl

Spectra were measured at 400 MHz in CDCl3.

Spectra were measured at 100 MHz in CDCl3.

J values (in hertz) are in parentheses.

Attached protons were deduced by DEPT and HMQC experiments.

n.o. = not observed.

Inhibitory effects of eunicellins 1 and 2 on the generation of superoxide anion and the release of elastase by human neutrophils in response to FMLP/CB.

Superoxide anion
Elastase release
Compounds Inh% Inh%
1 6.46 ± 1.28 ** 12.91 ± 3.56 *
2 45.82 ± 2.49 *** 40.45 ± 5.80 **

Percentage of inhibition (Inh%) at 10 μg/mL concentration of 1 and 2. Results are presented as the mean + S.E.M. (n = 3).

P < 0.05,

P < 0.01,

P < 0.001, as compared with the control value [9].