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Marine Drugs 2017, 15(9), 280; doi:10.3390/md15090280

Article
Bioactive Steroids with Methyl Ester Group in the Side Chain from a Reef Soft Coral Sinularia brassica Cultured in a Tank
Chiung-Yao Huang 1Orcid, Jui-Hsin Su 2,3, Chih-Chuang Liaw 1Orcid, Ping-Jyun Sung 1,2Orcid, Pei-Lun Chiang 4, Tsong-Long Hwang 5,6,7, Chang-Feng Dai 8 and Jyh-Horng Sheu 1,9,10,11,*
1
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
2
National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan
3
Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan
4
Department of Biochemistry, University of Toronto, Toronto, ON M5G2R3, Canada
5
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
6
Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, and Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan
7
Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
8
Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan
9
Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan
10
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
11
Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan
*
Correspondence: Tel.: +886-7-525-2000 (ext. 5030); Fax: +886-7-525-5020
Received: 11 August 2017 / Accepted: 30 August 2017 / Published: 1 September 2017

Abstract

:
A continuing chemical investigation of the ethyl acetate (EtOAc) extract of a reef soft coral Sinularia brassica, which was cultured in a tank, afforded four new steroids with methyl ester groups, sinubrasones A–D (14) for the first time. In particular, 1 possesses a β-d-xylopyranose. The structures of the new compounds were elucidated on the basis of spectroscopic analyses. The cytotoxicities of compounds 14 against the proliferation of a limited panel of cancer cell lines were assayed. The anti-inflammatory activities of these new compounds 14 were also evaluated by measuring their ability to suppress superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophils. Compounds 2 and 3 were shown to exhibit significant cytotoxicity, and compounds 3 and 4 were also found to display attracting anti-inflammatory activities.
Keywords:
soft coral; Sinularia brassica; steroid; cytotoxic activity; anti-inflammatory activity

1. Introduction

Soft corals of the genus Sinularia have yielded series of natural products of differing chemical types [1]. Many of these compounds have been shown to exhibit interesting bioactivities, such as cytotoxic [2,3,4,5,6,7,8], antifouling [9], and anti-inflammatory [10,11,12,13,14,15,16,17,18] activities. Previous chemical investigation of the wild-type soft coral Sinularia brassica (May 1898) has led to the isolation of only two new steroids from this marine organism [19]. In contrast, during the course of our investigation of the bioactive substances obtained from this reef soft coral which was later cultured in a tank, 12 new withanolide-based steroidal metabolites, sinubrasolides A–L, were discovered [20,21]. As most withanolides were isolated from terrestrial plants, and have attracted considerable attention due to their versatile bioactivities [22,23], marine withanolides discovered from reef soft corals Minabea sp. [24] and Paraminabea acronocephala [25], as well as the cultured S. brassica, have also been found to possess unusual withanolide-based structures and/or exhibit interesting biological activities, we further continued our exhaustive investigation of the cultured S. brassica with the aim of discovering new bioactive metabolites for further biomedical application. This study led to the discovery of four new non-withanolidal steroids, sinubrasones A–D (14) (Figure 1). The structure of 1 is unusual as it possesses a β-d-xylopyranose at C-22 of the side-chain. The structures of 14 were established by extensive spectroscopic analyses, including 2D nuclear magnetic resonance (NMR) spectroscopy. The in vitro cytotoxicities of 14 against four cancer cell lines, murine macrophage-like (P388D1), human T-lymphoid (MOLT-4), human erythroleukemia (K-562), and human colon carcinoma (HT-29), were measured. The abilities of new compounds 14 to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced neutrophils were also assayed. Herein, we report the isolation, structure elucidation and biological activities of these compounds.

2. Results and Discussion

A reef soft coral S. brassica further cultured in a tank was collected by hand from a cultivation pool of at the National Museum of Marine Biology and Aquarium, Taiwan, in January 2010. The organisms were further stored in a freezer until extraction. The frozen bodies were minced and extracted exhaustively with CH2Cl2 and MeOH (0.5 L × 6), as previously described [20]. Fractions 12 and 18, which contained terpenoids, as revealed by 1H-NMR spectra, were further purified by column chromatography and reversed-phase high-performance liquid chromatography (HPLC) to afford 14 (see Section 3).
Sinubrasone A (1) was isolated as an amorphous solid. The high-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum of 1 exhibited a [M + Na]+ peak at m/z 611.35545, indicating the molecular formula C34H52O8, requiring nine degrees of unsaturation. The infrared (IR) spectrum revealed the presence of a hydroxy group (3396 cm−1), an ester (1735 cm−1) group, and a conjugated enone (1664 cm−1). The 13C NMR spectroscopic data of 1 exhibited 34 carbon signals (Table 1), which were assigned by the assistance of a distortionless enhancement by polarization transfer (DEPT) spectra to six methyls (including a methoxy), eight sp3 methylenes, 12 sp3 methines (including five oxymethines), three sp2 methines, three sp2, and two sp3 non-protonated carbons (including an ester carbonyl and a ketone). The above data accounted for four of the nine degrees of unsaturation, indicating a pentacyclic structure for 1. Proton signals (Table 1) resonating at δH 7.05 (1H, d, J = 10.0 Hz), 6.23 (1H, d, J = 10.0 Hz), and 6.07 (1H, s), as well as carbon signals appearing at δC 186.5 (C), 169.3 (C), 156.0 (CH), 127.5 (CH), and 123.8 (CH), indicated the presence of a 1,4-dien-3-one structural unit in ring A of the steroids [25,26]. The molecular framework of 1 was further established by correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations (Figure 2). By comparison of the NMR data of 1 with those of cladophenol glycoside A [12], it was found that the presence of an anomeric proton at δH 4.30 (1H, d, J = 7.0 Hz) to arise from a β-xylopyranose moiety prossessing carbon signals at δC 104.5 (CH, C-1′), 73.4 (CH, C-2′), 75.9 (CH, C-3′), 69.5 (CH, C-4′), and 64.9 (CH, C-5′) (Table 1), as confirmed by HMBC correlation from H-5′ to C-1′ and the NOE correlations of H-1′/H-3′ and H-2′/H-4′. Moreover, the β-xylopyranose residue attached at C-22 was assigned according to an HMBC correlation from the anomeric proton (H-1′) to C-22. Thus, the planar structure of 1 was established.
The configuration of 1 was further confirmed by analysis of their nuclear Overhauser effect (NOE) correlations. In the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of 1, NOE correlations of H-20 with H3-18 and H-22, but H-22 not with H3-21, revealed the β-orientation of H-22. Moreover, correlations of H-25 with H-22 and H-24, and H3-27 with H3-28, revealed the 20S, 22R, 24S, 25R configuration of 1. Finally, the anomeric proton (H-1′) was found to show NOE correlations with H-20 and H-22 as shown 1 in Figure 3, and H-2′ was also found to show weak NOE correlations with H-24, but H2-5′ did not show NOE correlation with H-24, revealed the β-d-xylopyranose residue of the cholesterol. On the basis of the above findings and other detailed NOE correlations, the structure of 1 was established to be that of formula 1.
Sinubrasone B (2) had the molecular formula C30H44O5 as determined by HRESIMS and from 13C-NMR data. Thus, nine degrees of unsaturation were determined for 2. The IR absorption band at 1735 and 1662 cm−1 indicated the presence of an ester carbonyl group and a conjugated enone, which was further supported by NMR signals resonating at δC 176.8, C in 2. The 1H and 13C-NMR spectra (Table 2) displayed resonances for a carbonyl (δC 186.3, C), three sp2 methine double bonds (δH 7.04, 1H, d, J = 10.0 Hz, δC 155.7; δH 6.23, 1H, d, J = 10.0 Hz, δC 127.6; δH 6.07, 1H, s, δC 123.9), an sp2 non-protonated carbon (δC 168.9), an oxygenated non-protonated carbon (δC 117.9), two methoxy groups (δC 51.4, δH 3.67, s; and δC 49.3, δH 3.20, s), and an oxymethine (δC 86.9, δH 3.69, m). The remaining five degrees of unsaturation identified 2 as a pentacyclic triterpane. Proton signals (Table 2) resonating at δH 7.04 (1H, d, J = 10.0 Hz), 6.23 (1H, d, J = 10.0 Hz), and 6.07 (1H, s), as well as carbon signals appearing at δC 186.3 (C), 155.7 (CH), 127.6 (CH), 123.9 (CH), and 168.9 (C), indicated the presence of a 1,4-dien-3-one structural unit in ring A of the steroids [25,26]. Analysis of COSY correlations (Figure 2) of 2 indicated three consecutive proton sequences. The connection of the three partial structures was subsequently resolved by HMBC. Methoxyl and methyl ester groups were assigned at C-16 and C-25, respectively, based on HMBC correlations from the methoxyl proton (δH 3.20, s) to C-16, and from H3-27 to C-24, C-25, and C-26. In consideration of the degrees of unsaturation and molecular formula, an ether linkage was placed between C-16 and C-22, which further confirmed the presence of a tetrahydrofuran (THF) ring fused to ring D (Figure 2). Accordingly, the planar structure of 2 was established.
The configuration of 2 was further confirmed by NOE correlations (Figure 3). It was found that H3-18 (δH 0.83, s) showed NOE interactions with H-20 (δH 1.74, m) and one of the methylene protons at C-23 (δH 1.48, m); therefore, assuming the β-orientation of H3-18, H-20 and the above H-23 should also be positioned on the β-face, while the other (δH 1.53, m) was assigned as H-23α. Furthermore, H-22 was found to show NOE correlations with H3-21 (δH 1.02, d, J = 7.0 Hz), H-23α and H-24 (δH 2.07, dddq, J = 6.5, 6.5, 6.5, 6.5 Hz), and H3-27 showed correlation with H3-28 (δH 0.93, d, J = 6.5 Hz), reflecting the α-orientations of H-22, H-24 and H-25, and the 25R configuration of 2. On the basis of the above findings and other observed correlations (Figure 3), the structure of sinubrasone B (2) was suggested.
The molecular formula of sinubrasone C (3) was found to be C29H42O4, as deduced from HRESIMS and 13C-NMR data, appropriate for nine degrees of unsaturation. The IR spectrum of 3 showed the presence of carbonyl groups of an ester and a conjugated enone (νmax 1736 and 1662 cm−1), and the 13C-NMR (Table 2) and DEPT spectra also showed a signal of an ester carbonyl group at δC 175.6, C. Inspection of the 1H and 13C-NMR data of 3 suggested the presence of a 1,2-disubstituted epoxide (δC 63.9, CH; 59.1, CH; δH 2.59, dd, J = 4.8, 2.4 Hz; 2.52, dd, J = 7.6, 2.4 Hz). These data suggested that 3 possessed a 22,23-epoxide, which was corroborated by the HMBC correlations from H3-21 to C-22, and H3-28 to C-23, as well as the COSY correlation between H-22 and H-23. Additionally, H3-27 and H3-OMe displayed HMBC correlation to carbonyl carbon C-26 (Figure 2).
The stereochemistry of 3 was determined on the basis of NOE correlations and by comparison of NMR spectroscopic data. A small coupling constant (2.4 Hz) between H-22 and H-23 suggested a trans conformation for both protons [27]. The NOE interactions of H3-18 with H-20, H-17 with H3-21, H-22 with H-20 and H-24, and H-23 with H3-28, revealed the β-orientations of H-22 and H-24. Further, the 22R and 23R configurations, as opposed to 22S and 23S, were confirmed by comparing the δ values of H-20 (δH 1.32), H3-21 (δH 1.00), H-22 (δH 2.59) and H-23 (δH 2.46) of known compound (22R,23R,24R)-3β-acetoxy-24-methyl-22,23-epoxy-5α-holestan-6-one [28] with the corresponding H-20 (δH 1.30), H3-21 (δH 0.99), H-22 (δH 2.59) and H-23 (δH 2.52) of 3, while (22S,23S,24R)-3β-acetoxy-24-methyl-22,23-epoxy-5α-cholestan-6-one [28] with 22S and 23S configurations showed corresponding NMR signals for H-20 (δH 1.17), H3-21 (δH 1.09), H-22 (δH 2.39) and H-23 (δH 2.67). In addition, the NOESY spectrum of 3 showed NOE correlations between H3-28 with H-25, and H-24 with H3-27, but not between H3-28 and H3-27, revealing the 24R and 25S configurations of 3 (Figure 4). Thus, the absolute configuration of 3 was determined.
Sinubrasone D (4) had the molecular formula of C23H32O3, as determined by HRESIMS. It was also found to possess the same A–D rings as compounds 13 by comparison of NMR spectroscopic data (Table 1 and Table 2). The gross structure of 4 was determined by detailed analysis of COSY and HMBC correlations (Figure 2). The HMBC experiment of 4 further revealed the connectivity from H3-21 (δH 1.18, d, J = 7.0 Hz) and the methoxyl (δH 3.65, s) to the carbonyl carbon (δC 177.1). The relative configurations at C-8, C-9, C-10, C-13, C-14, C-17, and C-20 in 4 were found to be the same as those of compounds 13 by comparison of NMR data and NOE correlations.
As previous studies revealed that steroids from soft corals might possess attracting biological activities [29,30,31,32,33], we further evaluated the biological activities of these isolated steroids. Compounds 14 were evaluated in terms of their cytotoxic activities against P388D1, MOLT-4, K-562, and HT-29 cell lines using the Alamar Blue assay. Compounds 2 and 3 were found to show significant cytotoxicity against all cell lines. Compounds 1 and 4 exhibited only weak cytotoxic activity against P388D1, MOLT-4, K-562, and HT-29 cell lines (Table 3).
The anti-inflammatory activities of new compounds 14 on neutrophil pro-inflammatory responses were evaluated by measuring their ability to suppress fMLP/CB-induced superoxide anion (O2−•) generation and elastase release in human neutrophils, and the results are shown in Table 4. From the results, 4 showed significant inhibitory effect (53.6 ± 1.8%) against superoxide anion generation at 10 μM. Compounds 3 and 4 also exhibited inhibitory activities against elastase release, with the inhibition rate of 58.8 ± 4.0 and 66.3 ± 6.0% in the fMLP/CB-stimulated cells at the same concentration. The IC50 values of compound 4 for the superoxide anion generation and compounds 3 and 4 for inhibition of elastase release were also measured and were found to be lower than 10 μM.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations of the isolates were measured on a JASCO P1020 digital polarimeter (JASCO Corporation, Tokyo, Japan) and on a Horiba High Sensitivity Polarimeter SEPA-300 (Horiba Ltd., Kyoto, Japan). Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer (JASCO Corporation). IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer (JASCO Corporation). NMR spectra were recorded on a Varian 400MR FT-NMR or Varian Unity INOVA500 FT-NMR (Varian Inc., Palo Alto, CA, USA) instrument at 400 MHz (or 500 MHz) for 1H and 100 MHz (or 125 MHz) for 13C in CDCl3, and the chemical shifts were referenced to residual signals of TMS (δH 0.00 ppm) and the CDCl3 (δC 77.0 ppm). ESIMS and HRESIMS data were obtained with a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Silica gel (230–400 mesh, Merck, Darmstadt, Germany) was used for column chromatography. Pre-coated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC apparatus (Hitachi Ltd., Tokyo, Japan) with a Supelco C18 column (250 × 21.2 mm, 5 μm).

3.2. Animal Material

The soft coral Sinularia brassica used in this study was originally collected from a reef and cultured in an 80-ton cultivation tank (height 1.6 m) located in the National Museum of Marine Biology and Aquarium, Taiwan, for five years. The soft coral organisms were collected in January 2010 and were stored in a −20 °C freezer until extraction, while the voucher specimen (specimen no. 201001C1) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University. The soft coral was identified by one of the authors (C.-F.D.).

3.3. Extraction and Isolation

The frozen bodies of S. brassica (0.4 kg, wet weight) were minced and extracted exhaustively with the 1:1 mixture of CH2Cl2 and MeOH (0.5 L × 6). The combined extract was evaporated under reduced pressure and the residue was partitioned between EtOAc and H2O to give the EtOAc-soluble fraction. The EtOAc extract (3.7 g) was subjected to separation using a silica gel column with a gradient of EtOAc and n-hexane in an increasing polarity (0–100%, stepwise), and then with MeOH in EtOAc (5–50%, stepwise) to yield 24 fractions. Fraction 12 eluting with EtOAc–n-hexane (1:9) was further purified with acetone–n-hexane (1:7) to give five subfractions (12A–12F). Subfraction 12B was further separated by reversed-phase HPLC using MeOH–H2O (4:1) to yield 2 (0.8 mg), 3 (1.1 mg), and 4 (0.9 mg). Fraction 18 eluting with EtOAc–n-hexane (1:3) was separated by silica gel column chromatography with acetone–n-hexane (1:3) to afford four subfractions (18A–18D). Subfraction 18C was further purified by reversed-phase HPLC using MeOH–H2O (2:1) to yield 1 (1.3 mg).
Sinubrasone A (1): amorphous solid; [ α ] D 23 −30 (c 0.50, CHCl3); UV (MeOH) λmax (log ε) 243 (4.2); IR (neat) νmax 3396, 2941, 1735 and 1664 cm−1; 13C and 1H-NMR data, see Table 1; ESIMS m/z 611 [M + Na]+; HRESIMS m/z 611.35545 [M + Na]+ (calcd. for C34H52O8Na, 611.35544).
Sinubrasone B (2): amorphous solid; [ α ] D 23 −128 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 244 (4.3); IR (neat) νmax 2930, 1735 and 1662 cm−1; 13C and 1H-NMR data, see Table 2; ESIMS m/z 507 [M + Na]+; HRESIMS m/z 507.3089 [M + Na]+ (calcd. for C30H44O5Na, 507.3086).
Sinubrasone C (3): amorphous solid; [ α ] D 23 −68 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 245 (4.2); IR (neat) νmax 2936, 2856, 1736 and 1662 cm−1; 13C and 1H-NMR data, see Table 2; ESIMS m/z 477 [M + Na]+; HRESIMS m/z 477.2978 [M + Na]+ (calcd. for C29H42O4Na, 477.2981).
Sinubrasone D (4): amorphous solid; [ α ] D 24 −65 (c 0.035, CHCl3); UV (MeOH) λmax (log ε) 244 (4.0); IR (neat) νmax 2928, 2853, 1735 and 1662 cm−1; 13C and 1H-NMR data, see Table 2; ESIMS m/z 379 [M + Na]+; HRESIMS m/z 379.2250 [M + Na]+ (calcd. for C23H32O3Na, 379.2249).

3.4. Cytotoxicity Assay

Cytotoxicity assays were performed as previous reported, using an Alamar Blue assay [34,35]. Cell lines (P388D1, MOLT-4, K-562, and HT-29) were purchased from the American Type Culture Collection (ATCC). Cancer cells were plated onto 96-well microtiter plates possessing clear flat bottoms (Thermo Scientific Nunc MicroWell plate) with densities of 5 × 103 to 1 × 104 cells per well and incubated in a humidified 5% CO2 atmosphere at 37 °C. After 15 h of culture, the solutions of compounds in DMSO were added. After 72 h, attached cells were incubated with Alamar Blue (10 μL/well, 4 h). The absorbance at 595 nm was recorded using the ELISA reader. The IC50 values represented the concentrations of the compounds tested that could reduce cell growth by 50% under the experimental conditions.

3.5. Human Neutrophil Superoxide Anion Generation and Elastase Release

The human neutrophils were isolated through dextran sedimentation and Ficoll centrifugation. As previously described procedures, the assay of superoxide anion generation was measured from the SOD-inhibitable reduction of ferricytochrome C. The elastase release experiment was performed according to MeO–Suc–Ala–Ala–Pro–Val–p-nitroanilide as the enzyme substrate [36,37].

4. Conclusions

Our continuing investigations demonstrated that the cultured soft coral Sinularia brassica is a good source of bioactive withanolides and non-withanolidal steroids with methyl ester groups. Moreover, it is worthwhile to note here that 14 were found to be the novel steroids with a methyl ester group, and 1 with a β-d-xylopyranose on the side chain is quite rare. Metabolites 2 and 3 possessing a methyl ester at C-25 were shown to exhibit significant cytotoxic activities against P388D1, MOLT-4, K-562, and HT-29 cancer cell lines. Compounds 3 and 4 also exhibited notable anti-inflammatory activities in inhibition of elastase release in fMLP/CB-induced human neutrophils, and 4 could inhibit the generation of superoxide anion, too. Owing to these attractive biological activities, 24 might be useful lead compounds for future drug discoveries.

Supplementary Materials

HRESIMS, 1H, and 13C spectra of new compounds 14 are available online at www.mdpi.com/1660-3397/15/9/280/s1. Figure S1. HRESIMS spectrum of 1; Figure S2. 1H-NMR spectrum (500 MHz) of compound 1 in CDCl3; Figure S3. 13C-NMR spectrum (100 MHz) of compound 1 in CDCl3; Figure S4. HRESIMS spectrum of 2; Figure S5. 1H-NMR spectrum (500 MHz) of compound 2 in CDCl3; Figure S6. 13C-NMR spectrum (125 MHz) of compound 2 in CDCl3; Figure S7. HRESIMS spectrum of 3; Figure S8. 1H-NMR spectrum (400 MHz) of compound 3 in CDCl3; Figure S9. 13C-NMR spectrum (100 MHz) of compound 3 in CDCl3; Figure S10. HRESIMS spectrum of 4; Figure S11. 1H-NMR spectrum (500 MHz) of compound 4 in CDCl3; Figure S12. 13C-NMR spectrum (125 MHz) of compound 4 in CDCl3.

Acknowledgments

This work was supported by grants from Ministry of Science and Technology (MOST 104-2113-M-110-006, MOST 105-2113-M-110-002 and 104-2320-B-110-001-MY2), and the National Sun Yat-sen University-Kaohsiung Medical University (NSYSU-KMU) Joint Research Project (NSYSUKMU 105-I008) awarded to J.-H.S.

Author Contributions

Jyh-Horng Sheu designed and guided the whole experiment. Chiung-Yao Huang isolated the compounds, and performed the data acquisition, structure elucidation, and cytotoxicity assay. Jui-Hsin Su, Chih-Chuang Liaw, and Ping-Jyun Sung performed the structure elucidation. Pei-Lun Chiang and Tsong-Long Hwang performed the inhibition of superoxide anion generation and elastase release assays. Chang-Feng Dai contributed to the species identification of soft coral. Chiung-Yao Huang and Jyh-Horng Sheu wrote the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Structures of compounds 14.
Figure 1. Structures of compounds 14.
Marinedrugs 15 00280 g001
Figure 2. Selected correlation spectroscopy (COSY) and heteronuclear multiple bond correlations (HMBC) of 14.
Figure 2. Selected correlation spectroscopy (COSY) and heteronuclear multiple bond correlations (HMBC) of 14.
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Figure 3. Selected nuclear Overhauser effect (NOE) correlations for 1.
Figure 3. Selected nuclear Overhauser effect (NOE) correlations for 1.
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Figure 4. Selected NOE correlations for 2 and 3.
Figure 4. Selected NOE correlations for 2 and 3.
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Table 1. 1H and 13C-NMR spectroscopic data of 1.
Table 1. 1H and 13C-NMR spectroscopic data of 1.
1
PositionδC a (Mult.) bδH c (J in Hz)PositionδC (Mult.)δH (J in Hz)
1156.0, CH7.05 d (10.0)1′104.5, CH4.30 d (7.0)
2127.5, CH6.23 d (10.0)2′73.4, CH3.42 dd (8.5, 7.0)
3186.5, C 3′75.9, CH3.52 dd (8.5, 8.5)
4123.8, CH6.07 s4′69.5, CH3.75 ddd (9.5, 8.5, 5.0)
5169.3, C 5′64.9, CH24.01 dd (12.0, 5.0);
632.8, CH22.47 ddd (12.5, 12.5, 4.5) 3.30 d (12.0, 9.5)
2.36 br d (12.5)26-OMe52.1, CH33.71 s
733.6, CH21.95 m; 1.04 m
835.5, CH1.63 m
952.3, CH1.04 m
1043.6, C
1122.8, CH21.70 m
1239.4, CH22.04 m; 1.16 m
1343.0, C
1455.0, CH0.99 m
1524.5, CH21.66 m; 1.20 m
1627.5, CH21.68 m; 1.34 m
1752.6, CH1.06 m
1811.9, CH30.76 s
1918.7, CH31.23 s
2039.5, CH2.04 m
2112.6, CH30.92 d (6.5)
2281.5, CH3.63 br d (11.0)
2332.3, CH21.43 ddd (14.5, 11.0, 3.5);
1.28 m
2431.5, CH2.29 m
2540.9, CH2.62 qd (6.0, 4.0)
26177.7, C
279.9, CH31.02 d (7.0)
2816.3, CH30.85 d (7.0)
a Spectrum recorded at 100 MHz in CDCl3. b Attached protons were deduced by distortionless enhancement by polarization transfer (DEPT) experiment. c Spectrum recorded at 500 MHz in CDCl3.
Table 2. 1H and 13C-NMR spectroscopic data of 24.
Table 2. 1H and 13C-NMR spectroscopic data of 24.
234
PositionδC a (Mult.) bδH c (J in Hz)δC d (Mult.)δH e (J in Hz)δC a (Mult.) δH c (J in Hz)
1155.7, CH7.04 d (10.0)156.0, CH7.05 d (10.4)155.8, CH7.05 d (10.0)
2127.6, CH6.23 d (10.0)127.5, CH6.23 d (10.4)127.5, CH6.23 d (10.0)
3186.3, C 186.5, C 186.4, C
4123.9, CH6.07 s123.8, CH6.07 s123.9, CH6.07 s
5168.9, C 169.4, C 169.2, C
632.7, CH22.46 ddd (12.0, 12.0, 4.0)32.9, CH22.47 m32.8, CH22.46 ddd (13.0, 13.0, 4.0)
2.36 br d (12.0) 2.37 m 2.36 m
733.5, CH21.90 m; 1.07 m33.6, CH21.96 m; 1.05 m33.5, CH21.94 m; 1.05 m
835.1, CH1.76 m35.5, CH1.60 m35.5, CH1.64 m
952.2, CH1.09 m52.4, CH1.06 m52.2, CH1.08 m
1043.6, C 43.6, C 43.5, C
1122.4, CH21.69 m22.8, CH21.66 m22.8, CH21.71 m
1238.8, CH21.71 m39.3, CH21.99 m39.2, CH21.97 ddd (13.0, 3.0, 3.0)
1.21 m 1.21 m 1.28 m
1340.9, C 43.0, C 42.7, C
1454.6, CH1.36 dd (12.0, 5.5)55.0, CH1.02 m55.0, CH1.08 m
1533.5, CH21.96 dd (12.0, 5.5)24.6, CH21.63 m24.4, CH21.62 m
1.31 dd (12.0, 12.0) 1.17 m 1.19 m
16117.9, C 26.9, CH21.93 m; 1.60 m27.0, CH21.70 m; 1.30 m
1770.9, CH1.65 m55.8, CH1.30 m52.7, CH1.60 m
1815.3, CH30.83 s12.2, CH30.73 s12.2, CH30.76 s
1919.2, CH31.24 s18.7, CH31.23 s18.7, CH31.23 s
2038.1, CH1.74 m38.5, CH1.30 m42.4, CH2.43 m
2118.8, CH31.02 d (7.0)15.9, CH30.99 d (7.2)17.0, CH31.18 d (7.0)
2286.9, CH3.69 m63.9, CH2.59 dd (4.8, 2.4)177.1, C
2338.5, CH21.53 m; 1.48 m59.1, CH2.52 dd (7.6, 2.4)
2433.3, CH2.07 dddq (6.5, 6.5, 6.5, 6.5)39.2, CH1.54 m
2543.6, CH2.49 dq (6.5, 6.5)42.9, CH2.48 m
26176.8, C 175.6, C
2711.9, CH31.08 d (6.5)14.5, CH31.21 d (7.2)
2816.3, CH30.93 d (6.5)14.8, CH31.02 d (7.2)
16-OMe49.3, CH33.20 s
22-OMe 51.4, CH33.65 s
26-OMe51.4, CH33.67 s51.5, CH33.69 s
a Spectrum recorded at 125 MHz in CDCl3. b Attached protons were deduced by DEPT experiment. c Spectrum recorded at 500 MHz in CDCl3. d Spectrum recorded at 100 MHz in CDCl3. e Spectrum recorded at 400 MHz in CDCl3.
Table 3. Cytotoxicity (IC50 μM) of compounds 14.
Table 3. Cytotoxicity (IC50 μM) of compounds 14.
Cell lines IC50 (μM)
CompoundP388D1MOLT-4K-562HT-29
137.2 ± 4.037.8 ± 5.6bb
29.7 ± 1.26.0 ± 0.45.2 ± 0.87.6 ± 2.3
35.7 ± 1.85.3 ± 1.312.1 ± 2.410.4 ± 2.2
424.4 ± 4.831.2 ± 7.021.3 ± 3.736.5 ± 7.9
5-Fluorouracil a6.2 ± 0.76.9 ± 1.333.1 ± 8.97.7 ± 0.8
a Clinical anticancer drug used as a positive control. b ―: IC50 > 40 μM.
Table 4. Inhibitory (% Inh) effects of compounds 14 on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils at 10 μM.
Table 4. Inhibitory (% Inh) effects of compounds 14 on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils at 10 μM.
CompoundsSuperoxide AnionElastase Release
IC50 (μM) aInh % b IC50 (μM) aInh % b
1>1024.8 ± 6.5*>1035.6 ± 1.3***
2>1019.4 ± 5.0*>1039.0 ± 2.3***
3>1027.7 ± 1.3***6.6 ± 1.758.8 ± 4.0***
48.4 ± 1.153.6 ± 1.8***6.5 ± 1.166.3 ± 6.0***
Idelalisib0.07 ± 0.01102.8 ± 2.2***0.3 ± 0.199.6 ± 4.2***
a Concentration necessary for 50% inhibition (IC50). b Percentage of inhibition (Inh%) at 10 μM concentration. Results are presented as mean ± S.E.M. (n = 3–4). * p < 0.05, *** p < 0.001 compared with the control value.
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