Halogenated Sesquiterpenoids from the Red Alga ... - ACS Publications

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Halogenated Sesquiterpenoids from the Red Alga Laurencia tristicha Collected in Taiwan Jia-Yu Chen,† Chiung-Yao Huang,† Yun-Sheng Lin,‡ Tsong-Long Hwang,§ Wei-Lung Wang,⊥ Shu-Fen Chiou,† and Jyh-Horng Sheu*,†,∥,#,¶ †

Department of Marine Biotechnology and Resources and ¶Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan ‡ Department of Biological Science & Technology, Mei Ho University, Pingtung 912, Taiwan § Graduate Institute of Natural Products, College of Medicine, Chang Gung University; Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology; and Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan ⊥ Department of Biology, National Changhua University of Education, Changhua 500, Taiwan ∥ Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan # Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan S Supporting Information *

ABSTRACT: Chemical investigation of the red alga Laurencia tristicha led to the discovery of eight new halogenated chamigrane-type sesquiterpenoids (1−8) and one new bromocuparane-type sesquiterpene (9), along with nine known related metabolites (10−18). Their structures were elucidated on the basis of extensive spectroscopic analyses, and the absolute configurations of 1−8 were proposed by comparison to the biosynthetically related known compound 12. Cytotoxicity, antibacterial, and anti-inflammatory activities of these isolates were also investigated. The results showed that compound 11 exhibited good antibacterial activity against Serratia marcescens compared to the positive control ampicillin at a dosage of 100 μg/disk. Compound 17 showed strong inhibition toward elastase release generation at 10 μM.

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and by comparison of the spectroscopic data with those of related known compounds. In order to discover bioactive compounds for future medical application, the cytotoxic, antibacterial, and anti-inflammatory activities of the isolates were also evaluated and are reported herein.

he chemical constituents of marine red algae species of the genus Laurencia have been extensively investigated, and these algae have been found to be rich sources of halogenated sesquiterpenes,1−5 diterpenes,3,6−8 acetogenins,3,5 and indoles.9−11 Secondary metabolites discovered from these algae are predominantly sesquiterpenoids and are usually chlorinated or brominated.12−15 In our continuing investigation of the chemical constituents of marine algae of this genus,11 L. tristicha was collected and studied in order to discover bioactive marine natural products from Taiwanese red algae. This study led to the isolation of 18 compounds, including eight new chamigrane-type sesquiterpenes (1−8) and one cuparane-type sesquiterpene (9), along with nine known compounds, halogenated chamigrene derivatives 9-(E)-bromomethylidene1,5,5-trimethylspiro[5.5]undeca-1,7-dien-3-one (10) and 9(Z)-bromomethylidene-1,5,5-trimethylspiro[5.5]undeca-1,7dien-3-one (11),16−19 ma’ilione (12),20 [1(15)Z,2Z,4S,8R,9S]8,15-dibromochamagra-1(15),2,11 (12)-trien-9-ol (13),16,21 [1(15)E,2Z,4S,8R,9S]-8,15-dibromochamagra-1(15),2,11(12)trien-9-ol (14),16,18,21 ma’iliohydrin (15),22 isorigidol (16),23,24 allo-isoobtusol (17),20,25,26 and majusculone (18).17 The structures of the compounds were determined on the basis of extensive spectroscopic analyses (IR, MS, 1D and 2D NMR) © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The frozen specimens of L. tristicha were minced and extracted sequentially with n-hexane and MeOH. The organic extracts were filtered and concentrated under reduced pressure. The organic extracts were subjected to silica gel gravity column chromatography, and the resolved fractions were further purified by reversed-phase HPLC to yield compounds 1−18. Tristichone A (1) was obtained as a colorless oil. The LREIMS spectrum of 1 exhibited an isotopic cluster of ions at m/z 337 [M + Na]+ and 339 [M + 2 + Na]+ in the ratio 1:1, indicating the presence of a bromine atom, and with the molecular formula C14H19BrO3 as determined by HRESIMS, appropriate for five degrees of unsaturation. The infrared Received: May 19, 2016

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DOI: 10.1021/acs.jnatprod.6b00452 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 13C NMR Spectroscopic Data for Compounds 1−9a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 a

1 57.8, 55.5, 204.2, 32.8, 18.1, 48.4, 141.5, 39.0, 71.7, 69.3, 43.0, 21.6, 25.5, 116.2,

2 b

CH CH C CH2 CH2 C C CH2 CH CH C CH3 CH3 CH2

57.8, 54.8, 204.2, 33.6, 23.8, 43.8, 60.3, 36.5, 71.0, 68.8, 42.7, 21.9, 27.6, 47.3,

3 CH CH C CH2 CH2 C C CH2 CH CH C CH3 CH3 CH2

135.3, 130.9, 72.4, 30.4, 24.5, 50.5, 143.9, 38.0, 71.8, 69.7, 42.8, 21.8, 26.7, 116.3, 56.3,

4 CH CH C CH2 CH2 C C CH2 CH CH C CH3 CH3 CH2 CH

135.3, 130.6, 69.3, 29.1, 22.0, 51.7, 143.5, 37.9, 72.0, 70.3, 42.7, 21.5, 26.7, 116.7, 70.0,

5 CH CH C CH2 CH2 C C CH2 CH CH C CH3 CH3 CH2 CH2

40.0, 68.8, 151.5, 29.8, 30.4, 49.7, 144.4, 39.9, 72.7, 75.9, 43.5, 18.8, 23.7, 116.6, 103.5,

6 CH2 CH C CH2 CH2 C C CH2 CH CH C CH3 CH3 CH2 CH2

129.9, 137.3, 67.5, 36.0, 28.2, 47.6, 142.4, 122.4, 72.9, 71.6, 43.7, 18.9, 26.8, 21.5, 28.7,

7 CH CH C CH2 CH2 C C CH CH CH C CH3 CH3 CH3 CH3

36.8, 59.4, 72.6, 39.3, 24.1, 48.4, 166.8, 127.4, 197.8, 50.0, 40.4, 27.8, 27.8, 22.4, 33.0,

8 CH2 CH C CH2 CH2 C C CH C CH2 C CH3 CH3 CH3 CH3

35.4, 61.5, 73.3, 31.5, 23.3, 48.4, 167.6, 126.9, 198.2, 50.0, 40.3, 27.5, 27.5, 22.4, 24.3, 49.3,

9 CH2 CH C CH2 CH2 C C CH C CH2 C CH3 CH3 CH3 CH2 CH3

48.7, 47.6, 70.2, 78.0, 44.5, 143.9, 128.1, 128.2, 135.5, 128.2, 128.1, 23.1, 21.7, 25.5, 20.8,

C C CH CH CH2 C CH CH C CH CH CH3 CH3 CH3 CH3

Spectrum recorded at 100 MHz in CDCl3. bAttached protons were deduced by DEPT experiments.

spectrum of 1 showed absorptions of hydroxy (νmax 3504 cm−1) and carbonyl groups (νmax 1710 cm−1). The 13C NMR spectrum along with DEPT and HSQC experiments demonstrated the presence of 14 carbon signals, which were assigned with the assistance of the DEPT spectrum as two methyls, three sp3 and one sp2 methylene, four oxygenated and halogenated methines (δC 71.7, 69.3, 57.8, and 55.5), one olefinic (δC 141.5) and two sp3 (δC 48.4 and 43.0) nonprotonated carbons, and one carbonyl carbon (δC 204.2) (Table 1). The presence of two tertiary methyls and two sp2 exomethylene protons was confirmed from 1H NMR data (Table 2). The planar structure of 1 was further established by COSY and HMBC spectra (Figure 1). From the COSY spectrum of 1, it was possible to establish three proton sequences from H-1 to H-2; H2-4 to H2-

5; and H2-8 to H-10. The HMBC correlations from both H3-12 and H3-13 to C-6, C-10, and C-11; H2-14 to C-6, C-7, and C-8; both H-1 and H2-5 to C-6; and both H-2 and H2-4 to C-3 established the carbon skeleton of a chamigrane-type norsesquiterpene. The NMR signals of CH-1 (δH 3.68; δC 57.8) and CH-2 (δH 3.35; δC 55.5) revealed the presence of an epoxy group at C-1 and C-2.27 Placement of a hydroxy group at C-9 and a bromine atom at C-10 in compound 1 was confirmed on the basis of comparison of the chemical shifts of H-9 and H-10 (δH 4.19 and 4.57) with those of known compounds.18 The relative configuration of 1 was established by analysis of the NOE correlations. In the NOESY spectrum of 1 (Figure 2), H-10 (δH 4.57, d, J = 3.2 Hz) was found to show NOE correlations with H-5b (δH 1.71, m), H-9 (δH 4.19, m), and H3B



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Table 2. 1H NMR Spectroscopic Data (δH, mult., J in Hz) for Compounds 1−5a position

1

2

3

3.68, d (4.4)

3.69, br s

6.02, d (12.0)

5.98, dd (10.4, 1.6)

2 4

3.35, 2.34, 1.99, 2.31, 1.71, 2.61, 2.55, 4.19, 4.57, 1.36, 1.23, 5.22, 5.01,

3.35, 2.54, 2.34, 2.08, 1.99, 2.40, 1.76, 4.27, 4.54, 1.47, 1.01, 3.14, 2.42,

5.99, 2.25, 1.78, 1.92, 1.86, 2.73, 2.61, 4.16, 4.57, 1.21, 1.03, 5.09, 4.88, 5.80,

5.91, 1.64, 1.47, 1.99, 1.80, 2.71, 2.58, 4.17, 4.66, 1.22, 1.06, 4.79, 5.07, 3.41, 3.46, 3.50,

5 8 9 10 12 13 14

d (4.4) m ddd (14.0, 11.6, 6.8) m m dd (14.8, 3.2) d (14.8) m d (3.2) s s s s

d (4.0) ddd (19.2, 10.4, 2.0) ddd (19.2, 9.6, 8.0) ddd (14.0, 10.4, 8.0) dd (14.0, 9.6) dd (14.8, 4.4) d (14.8) m d (3.2) s s d (4.4) d (4.4)

15

d (12.0) m ddd (12.0,12.0, 3.6) m m dd (15.2, 2.0) dd (15.2, 2.0) m d (2.8) s s br s br s s

15-OH a

4

1

dd (10.4, 1.6) m ddd (13.6,13.6, 2.8) ddd (13.6, 13.6, 2.8) m dddd (15.2,7.2, 2.0) dd (15.2, 2.8) ddd (7.2, 2.8, 2.8) d (2.8) s s d (2.0) d (2.0) dd (10.8, 5.2) dd (10.8,5.2) d (5.2)

5 2.32, 1.46, 4.50, 2.23, 1.88, 2.11, 1.53, 2.62, 2.35, 3.79, 4.39, 1.00, 1.13, 5.34, 5.06, 4.88, 4.77,

dd (12.8, 5.2) dd (12.8, 11.6) dd (11.6, 5.2) ddd (13.6, 3.6, 3.6) ddd (13.6, 13.6, 3.6) ddd (13.6, 3.6, 3.6) ddd (13.6, 13.6, 3.6) dd (13.2, 6.0) dd (13.2,10.4) ddd (10.4, 10.4, 6.0) d (10.4) s s s s s s

Spectrum recorded at 400 MHz in CDCl3.

2. Further, NOE interactions of H3-12 with H-1 (δH 3.68, d, J = 4.4 Hz) and H-1 with both H-2 (δH 3.35, d, J = 4.4 Hz) and H14b (δH 5.01, m), but not with H3-13, revealed the βorientations of H-1 and H-2. Thus, the epoxy oxygen could be placed on the α-face. The absolute configuration of 1 was proposed by comparison with that of 12, which was confirmed by single-crystal X-ray diffraction analysis, and assigned as 1S,2S,6S,9R,10S. An earlier X-ray study had established the same absolute configuration for 12.24 Tristichone B (2) was obtained as a colorless oil. The IR spectrum of 2 displayed absorption bands at 3502 and 1710 cm−1, corresponding to hydroxy and carbonyl groups, respectively. The molecular formula C14H19BrO4 was determined by HRESIMS, indicating five degrees of unsaturation. Furthermore, the 1H and 13C NMR data of 2 (Tables 1 and 2) showed high similarity to those of 1, with the exception that the signals of the Δ7,14 double bond of 1 disappeared, and instead a second epoxy group was identified in 2. Similar to 1, one of the epoxy groups was located between C-1 (δC 57.8) and C-2 (δC 54.8), and the other was found to be at C-7 (δC 60.3) and C-14 (δC 47.3) on the basis of the COSY and HMBC correlations,

Figure 1. Selected COSY and HMBC correlations of 1−3 and 5.

13 (δH 1.23, s), but not with H3-12 (δH 1.36, s), suggesting the α-orientations of H-9, H-10, and CH3-13 and the β-orientation of CH3-12.25 From the J values, H-9 and H-10 were placed in axial and equatorial positions, respectively, as shown in Figure

Figure 2. Selected NOE correlations of compounds 1 and 2. C



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Figure 3. Selected NOE correlations of compounds 3−5.

in 4. The HMBC correlations from H2-15 to C-2, C-3, and C-4 further supported the proposed planar structure of 4. The relative configuration of compound 4 was determined from analysis of correlations observed in the NOESY spectrum, which indicated NOE correlations of CH3-13 with H-5α and H4α with both H-5α and H2-15, revealing that the CH2OH group at C-3 should be α-oriented. By comparison of the NMR spectroscopic data of 3 and 4 and by detailed analysis of other key NOE correlations (Figure 3), the structure of compound 4 was determined unambiguously. Compound 5 was isolated as a colorless oil. Its molecular formula, C15H23BrO2, was established by HRESIMS, appropriate for four degrees of unsaturation. The 13C NMR and DEPT spectra showed signals of two methyls, six methylenes (including two terminal methylene groups), three methines (including two bearing hydroxy groups), and four nonprotonated carbons. The planar structure of 5 was similar to that of 4, but showed some significant differences in the signals of C-1, C-2, and C-3. The differences observed were that C-3 of 5 now has an exomethylene group instead of a C-3/C-15 diol in 4 and the C-1/C-2 double bond in 4 has been formally hydrated, with a C-2 hydroxy group in 5. In the HMBC spectrum, the observed correlations from the terminal methylene protons H2-15 to C-2, C-3, and C-4 and from the other terminal methylene protons H2-14 to C-6 and C-8 confirmed the planar structure of 5. The relative configuration of 5 was elucidated by analysis of NOE correlations and the coupling constant of 1H NMR data. H-10 (δH 4.39, d, J = 10.4 Hz) was found to show an NOE correlation with H3-13, suggesting the α-orientations of H-10 and CH3-13 as per compounds 1−4. The β-oriented CH3-12 (δH 1.00, s) was found to exhibit NOE correlations with both H-1β (δH 2.32, dd, J = 12.8, 5.2 Hz) and H-9 (δH 3.79, ddd, J = 10.4, 10.4, 6.0 Hz), while H-10 was not found in the NOE

which were used to establish the molecular skeleton of 2 (Figure 1). In the NOESY spectrum of 2, H-10 showed NOE interactions with H-5b (δH 1.99, dd, J = 14.0, 9.6 Hz), H-9, and H3-13, suggesting that H-9, H-10, and CH3-13 are all αoriented. H-14a (δH 3.14, d, J = 4.4 Hz) was found to show an NOE correlation with H-5a (δH 2.08, ddd, J = 14.0, 10.4, 8.0 Hz), but not with CH3-12, indicating that the epoxy oxygen at C-1 and C-2 should be placed on the β-face. Moreover, NOE correlations of H3-12 with H-1, H-1 with H-2, and H-2 with H3-13 revealed that the epoxy oxygen at C-7 is β-oriented. The HRESIMS spectrum of 3 showed a sodium adduct ion [M + Na]+ peak at m/z 492.89808 and a ratio of 1:3: 3:1 for signals of [M + Na]+, [M + 2 + Na]+, [M + 4 + Na]+, and [M + 6 + Na]+, suggesting the molecular formula C15H21Br3O2 for this metabolite. The IR signal at 3444 cm−1 indicated the presence of hydroxy groups. The NMR spectroscopic data of 3 were quite similar to those of the tribromochamigrene ma’iliohydrin,22 which has the same molecular formula as that of 3. Compound 3 was confirmed to be a diastereomer of ma’iliohydrin by COSY and HMBC correlation analysis (Figure 1). Further, assuming the β-orientation of 10-Br, H-10 was found to exhibit NOE correlations with H-9 and H3-13 (δH 1.03, s), and H3-13 with dibromomethine proton H-15 (δH 5.80, s), revealing that hydroxy groups at C-3 and C-9 should be β-oriented and CH3-13 and the CHBr2 substituent at C-3 should be α-oriented. Furthermore, vinylic proton H-1 showed an NOE interaction with the β-oriented CH3-12, revealing the β-orientation of the 1,2-double bond at spiro carbon C-6. Thus, the structure of 3 was established. The HRESIMS spectrum of tristichol B (4) showed a sodium adduct ion [M + Na]+ peak at m/z 353.07212, establishing a molecular formula of C15H23BrO3. The NMR data of 4 were similar to those of 3, the only difference being that the C-15 substituent changed from CHBr2 in 3 to CH2OH D



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correlation with H-9, reflecting the β-orientation of H-9. Further, the H-9 and H-10 were assigned to be trans, consistent with their large coupling constant (J = 10.4 Hz). On the other hand, H3-13 was found to exhibit NOE correlations with both H-10 and H-5α (δH 1.53, ddd, J = 13.6, 13.6, 3.6 Hz), H-5α with H-4α (δH 2.23, ddd, J = 13.6, 3.6, 3.6 Hz), but not with H4β (δH 1.88, ddd, J = 13.6, 13.6, 3.6 Hz), and H-2 (δH 4.50, dd, J = 11.6, 5.2 Hz) was found to show NOE correlations with H1β, H-4β, and H-14b (δH 5.06, s), indicating that the hydroxy group at C-2 was α-oriented (Figure 3). On the basis of the above findings, the structure of compound 5 was determined unambiguously. Tristichol D (6) was also obtained as a colorless oil, which possesses the molecular formula C15H23BrO2, as established by HRESIMS and NMR data, implying four degrees of unsaturation. The structure of this compound was deduced from its 13C NMR and DEPT spectra, which showed that the compound has 15 carbons, including four methyls, two methylenes, three sp2 and two sp3 methines, and one sp2 and three sp3 nonprotonated carbons. Detailed analysis of HMBC correlations from H3-14 to C-6, C-7, and C-8 and from H3-15 to C-2, C-3, and C-4 established the planar structure of 6, as shown in Figure 4. The relative configuration of 6, deduced

(δH 1.90, m), and H-2 with H3-15, revealing the β-orientations of 2-Cl and 3-Br. Thus, the structure of sesquiterpenoid 7 was established. Compound 8, obtained as a colorless oil, was assigned the molecular formula C16H25BrO2, as deduced from HRESIMS data. The 1H and 13C NMR data of 8 were similar to those of 7 and showed the presence of an additional methoxy group at C3, and the C-2 position was replaced by bromine. Analysis of the NOE correlations of 8 revealed the same relative configurations at C-2, C-3, and C-6 as those in 7. The similar 1 H NMR (in terms of both chemical shift and coupling constant), COSY, and HMBC data (Figure 4) and the analysis of NOE correlations of 8 further revealed the same relative configuration of both compounds. Thus, the structure of 8 was established. The molecular formula of metabolite 9 was assigned as C15H21BrO from the HRESIMS and NMR data (Tables 1 and 3). The 13C NMR and DEPT spectra of 9 indicated the presence of four methyls, one methylene, six methines, and four nonprotonated carbons. The 1H and 13C NMR spectra displayed resonances for one aromatic ring (δC 128.1 and 128.2; δH 7.37 and 7.12). From the COSY spectrum of 9, it was possible to establish three proton sequences from H-3 to H2-5; H-7 to H-8; and H-10 to H-11. The connectivity of nonprotonated carbons at C-1, C-2, C-6, and C-9 was established by key HMBC correlations from H-7 to C-1; from H3-12 and H3-13 to C-1, C-2, and C-3; from H3-14 to C1, C-5, and C-6; and from H3-15 to C-8, C-9, and C-10. From these data, the carbon skeleton of a bromocuparane-type sesquiterpene 9 was elucidated. In the NOESY spectrum of 9, the correlations of H3-13 with H-4 and H3-14 suggested that H4, CH3-13, and CH3-14 are β-oriented. In addition, correlations of H3-12 with H-3 suggested that H-3 and CH3-12 are αoriented. On the basis of the above findings and other detailed NOE correlations (Figure 5), the structure of compound 9 was determined. The cytotoxicities of compounds 1−18 against the proliferation of a limited panel of cancer cell lines, including human lung adenocarcinoma (A549), human colorectal (DLD1), and human prostatic carcinoma (LNCaP) cell lines, were evaluated using the Alamar Blue assay. However, none of the compounds showed notable cytotoxicity (IC50 ≥ 10 μM). In addition, the antibacterial activities of the isolated compounds 1−18 were evaluated against Enterobacter aerogenes (ATCC13048), Serratia marcescens (ATCC25419), and Yersinia enterocolitica (ATCC23715). Compound 11 exhibited good antibacterial activity (Table 4) against S. marcescens at a dosage of 100 μg/disk (inhibition zone: 8 mm) when compared to that of the standard antibiotic ampicillin at a dosage of 100 μg/disk (inhibition zone: 5 mm). Furthermore, compounds 3, 12, 14, 15, and 17 also displayed measurable antibacterial activities against E. aerogenes, S. marcescens, and Y. enterocolitica, as shown in Table 4. The other compounds did not exhibit antibacterial activity. The anti-inflammatory activities of compounds 1−18 were evaluated by measuring their ability to suppress fMLP/CBinduced superoxide anion (O2− •) generation and elastase release in human neutrophils, and the results are shown in Table 5. At a concentration of 20 μM, compounds 3 and 17 exhibited significant inhibition (32 ± 1% and 48 ± 4%, respectively) toward N-formylmethionyl-leucyl-phenylalanine/ cytochalasin B (fMLP/CB)-induced superoxide anion (O2− •) generation. At the same concentration, 3, 13, and 17 exhibited

Figure 4. Selected COSY and HMBC correlations of 6−9.

from a NOESY spectrum (Figure 5), was similar to that of 5. In addition, H3-13 was found to exhibit NOE correlations with H5α (δH 2.06, ddd, J = 12.0, 7.2, 4.4 Hz) and H-10, and H-5β (δH 1.16, m) was found to exhibit an NOE correlation with H3-15, revealing the α-orientation of the hydroxy group at C-3. Therefore, the structure of 6 was found to possess the configuration (3R,6S,9S,10S,1Z,7Z). HRESIMS of tristichone C (7) revealed a sodium adduct ion peak at m/z 355.04332 [M + Na]+ and a ratio of 3:4:1 for signals of [M + Na]+, [M + 2 + Na]+, and [M + 4 + Na]+, indicating the molecular formula C15H22BrClO. Thus, four degrees of unsaturation were determined for 7. The 13C NMR and DEPT spectra of 7 showed signals of 15 carbons, including four methyls, four methylenes, two methines (including one sp2 methine), and two sp2 and three sp3 nonprotonated carbons. The COSY correlations shown in Figure 4 and HMBC correlations of H-8 with C-6 and C-10; H2-10 with C-9; H3-14 with C-6, C-7, and C-8; and H3-15 with C-2, C-3, and C-4 were used to establish the planar structure. In the NOESY experiment of 7, H3-13 was found to exhibit strong NOE correlations with H-2 (δH 4.46, dd, J = 12.4, 4.4 Hz) and H-5α E



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Figure 5. Selected NOE correlations of compounds 6−9.

Table 3. 1H NMR Spectroscopic Data (δH, mult., J in Hz) for Compounds 6−9a position

5.51, d (10.4)

2 3 4 5 7 8 9 10 11 12 13 14 15 OMe a

6

1

7

8

5.90, d (10.4)

2.55, dd (12.4, 12.4) 2.38, dd (12.4, 4.4) 4.46, dd (12.4, 4.4)

2.61, dd (13.6, 13.6) 2.28, dd (13.6, 4.4) 4.40, dd (13.6, 4.4)

1.85, 1.76, 2.06, 1.16,

2.26, 2.06, 2.24, 1.90,

2.16, ddd (14.8, 4.8, 3.2) 1.63, ddd (14.8, 14.8, 4.8) 1.78, m

ddd (13.6, 10.4, 4.4) ddd (13.6, 7.2, 4.8) ddd (12.0, 7.2, 4.4) m

m m m m

5.46, q (1.6) 4.30, d (8.8) 4.47, d (8.8)

5.86, s

5.82, s

2.36, m

2.35, br s

1.06, 1.14, 1.65, 1.33,

1.17, 1.15, 2.10, 1.76,

1.17, 1.14, 2.04, 1.32, 3.30,

s s d (1.6) s

s s s s

Spectrum recorded at 400 MHz in CDCl3.

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significant inhibitory activity against elastase release (33 ± 3%, 69 ± 1%, and 60 ± 4%, respectively). In conclusion, our investigation demonstrated that the red alga L. tristicha could be a good source of bioactive substances. Eight new halogenated chamigrane-type sesquiterpenoids (1− 8) and one new bromocuparane-type sesquiterpene (9), along with nine known related metabolites (10−18) were isolated. Compound 11 showed potent inhibition against S. marcescens compared with ampicillin. Moreover, compounds 13 and 17 might be useful agents for future anti-inflammatory drug development.

s s s s s

9

4.13, d (8.0) 4.57, ddd (8.0, 8.0, 4.4) 2.31, 2.45, 7.37, 7.12,

m dd (14.8, 4.4) d (8.4) d (8.4)

7.12, 7.37, 0.58, 1.09, 1.43, 2.33,

d (8.4) d (8.4) s s s s

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2100 polarimeter. Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were obtained on a JASCO FT/IR-4100 infrared spectrophotometer. NMR spectra were recorded on a Varian 400MR FT-NMR instrument at 400 MHz for 1H and 100 MHz for 13C in CDCl3. All NMR experiments were performed at room temperature, using CDCl3 as the solvent. Chemical shifts were referenced to residual solvent signals for CDCl3 (δH 7.27 and δC 77.0 ppm). ESIMS and HRESIMS data were recorded on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230−400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical F



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purified over silica gel using n-hexane−acetone (4:1) to afford four subfractions (H12-1−H12-4). Subfraction H12-3 was then separated by reversed-phase HPLC using MeOH−H2O (3:4) to afford 2 (6.2 mg). Fraction H13, eluted with acetone−n-hexane (1:2), was rechromatographed over silica gel, using acetone−n-hexane (1:1) as the mobile phase, to afford four subfractions (H13A1−H13A4). Subfraction H13A1 was further purified by reversed-phase HPLC using MeOH−H2O (9:5) to obtain 1 (5.6 mg) and 12 (106.3 mg) and MeOH−H2O (2:1) to obtain 3 (5.5 mg). Fraction E2, eluted with EtOAc−n-hexane (1:4), also was rechromatographed over a column packed with RP-18 gel, using MeOH−H2O (8:1) as the mobile phase. The subfraction E2-1 was further purified by reversed-phase HPLC using MeOH−H2O (3:1) to obtain 9 (0.6 mg) and 17 (67.7 mg), and subfraction E2-2 was further purified by silica gel using EtOAc−nhexane (1:10) to obtain 10 (0.7 mg) and 11 (1.8 mg). Fraction E3, eluted with EtOAc−n-hexane (1:3), was further purified over silica gel using acetone−n-hexane (1:7). Subfraction E3-1 was separated by reversed-phase HPLC using MeOH−H2O (2:1) to afford 8 (2.6 mg) and 15 (33.2 mg) and MeOH−H2O (3:1) to afford 7 (3.1 mg). Fractions E4 and E5, eluted with EtOAc−n-hexane (1:2), were further purified by reversed-phase HPLC using MeOH−H2O (6:5) to afford 5 (2.2 mg), 6 (2.6 mg), and 16 (4.7 mg). Fraction E6, eluted with EtOAc−n-hexane (2:1), was further purified by reversed-phase HPLC using MeOH−H2O (11:10) to afford 18 (2.9 mg). Fraction E8, eluted with EtOAc−n-hexane (4:1), was further purified by reversed-phase HPLC using MeOH−H2O (13:10) to afford 4 (1.8 mg). Tristichone A (1): colorless oil; [α]25 D −74 (c 1.40, CHCl3); IR (KBr) νmax 3504, 2974, 1710, 1640, and 521 cm−1; 13C and 1H NMR data, Tables 1 and 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.04112 [M + Na]+ (calcd for C14H19BrO3Na, 337.04098). Tristichone B (2): colorless oil; [α]25 D +12 (c 1.55, CHCl3); IR (KBr) νmax 3502, 2926, 1710, 878, and 517 cm−1; 13C and 1H NMR data, Tables 1 and 2; ESIMS m/z 353 [M + Na]+; HRESIMS m/z 353.03571 [M + Na]+ (calcd for C14H19BrO4Na, 353.03589). Tristichol A (3): colorless oil; [α]25 D −43 (c 1.38, CHCl3); IR (KBr) νmax 3444, 2969, 1640, 1064, 542 cm−1; 13C and 1H NMR data, Tables 1 and 2; ESIMS m/z 492 [M + Na]+; HRESIMS m/z 492.89808 [M + Na]+ (calcd for C15H21Br3O2Na, 492.89839). Tristichol B (4): colorless oil; [α]24 D −23 (c 0.43, CHCl3); IR (KBr) νmax 3396, 2966, 1639, 1046, and 536 cm−1; 13C and 1H NMR data,

Table 4. Antibacterial Activities (Zone of Inhibition in mm) of Compounds 3, 11, 12, 14, 15, and 17a inhibition zone (mm) compound

Enterobacter aerogenes

Serratia marcescens

Yersinia enterocolitica

3 11 12 14 15 17 ampicillin

−b − 3 − 1 2 11

2 8 − 4 2 2 5

4 − − 3 2 2 8

a

Dosage: 100 μg/disk. bNot determined.

TLC. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm). Algal Material. The red alga Laurencia tristicha was collected by hand using scuba off the coast of Hsiao Liuchiu Island, located off Taiwan’s southwestern coast, in October 2011, at a depth of 10−15 m, and stored in a freezer until extraction. The red alga was identified by one of the authors (W.-L.W.). A voucher sample was deposited at the Department of Marine Biotechnology and Marine Resources, National Sun Yat-sen University. Extraction and Isolation. The frozen specimens of L. tristicha (219 g, dry wt) were sliced and exhaustively extracted with n-hexane (2 × 2 L), then MeOH (3 × 1.5 L), at 25 °C for 24 h. The n-hexane extract (2.34 g) was subjected to silica gel column chromatography, eluting with acetone−n-hexane (1:10, stepwise), to yield 18 fractions (H1−H18). The MeOH extract (109.4 g) was partitioned between H2O and EtOAc. The EtOAc layer obtained was chromatographed over silica gel eluting with EtOAc−n-hexane (1:5, stepwise) then with acetone−MeOH (1:1) to yield 15 fractions (E1−E15). Fraction H10, eluted with acetone−n-hexane (1:3), was further purified over silica gel using acetone−n-hexane (1:1) to afford four subfractions (H10-1− H10-4). Subfraction H10-2 was further separated by reversed-phase HPLC using MeOH−H2O (4:1) to afford 13 (26.0 mg) and 14 (13.3 mg). Fraction H12, eluting with acetone−n-hexane (1:1), was further

Table 5. Inhibitory Effects of Compounds 1−18 on Superoxide Anion Generation and Elastase Release by Human Neutrophils superoxide anion a

compound

inh %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 idelalisibc

6±4 9±4 19 ± 2*** 7 ± 2* 10 ± 2** 13 ± 1*** 12 ± 5 12 ± 5 17 ± 2** 7±4 6±4 7 ± 2* 18 ± 2*** 24 ± 4** 13 ± 1*** 11 ± 1*** 25 ± 6* 4 ± 1*** 103 ± 2***

elastase release inh %

b

6±3 8±4 32 ± 1*** 14 ± 3** 14 ± 2** 12 ± 6 14 ± 5* 10 ± 6 26 ± 3** 10 ± 4* 12 ± 5 13 ± 6 10 ± 4 10 ± 5 16 ± 6* 23 ± 2*** 48 ± 4*** 16 ± 4** −d

a

inh %

inh %b

3±1 2±1 9 ± 2** 9±4 5±2 1±3 12 ± 6 5±2 7±3 5±3 3±4 9±5 34 ± 6** 8±6 16 ± 1*** 9 ± 1*** 44 ± 4*** 18 ± 4* 100 ± 4***

14 ± 4* 18 ± 4* 33 ± 3*** 29 ± 4** 24 ± 4** 12 ± 3* 19 ± 2** 24 ± 7* 28 ± 7** 30 ± 6** 14 ± 5* 14 ± 7 69 ± 1*** 12 ± 5 22 ± 2*** 12 ± 6 60 ± 4*** 32 ± 4** −d

Percentage of inhibition (inh %) at a concentration of 10 μM. bPercentage of inhibition (inh %) at a concentration of 20 μM. cPositive control at a concentration of 10 μM. d−: not tested. *P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control value.

a

G



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μg/μL) with different volumes at various dosages and DMSO served as positive and negative controls, respectively. All the plates were incubated at 37 °C 24 h prior to the evaluation of antibacterial activity.

Tables 1 and 2; ESIMS m/z 353 [M + Na]+; HRESIMS m/z 353.07212 [M + Na]+ (calcd for C15H23BrO3Na, 353.07228). Tristichol C (5): colorless oil; [α]25 D −51 (c 0.15, CHCl3); IR (KBr) νmax 3390, 2959, 1699, 1046, and 521 cm−1; 13C and 1H NMR data, Tables 1 and 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.07759 [M + Na]+ (calcd for C15H23BrO2Na, 337.07736). Tristichol D (6): colorless oil; [α]26 D −10 (c 0.65, CHCl3); IR (KBr) νmax 3388, 2970, 1650, 1018, and 513 cm−1; 13C and 1H NMR data, Tables 1 and 3; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.07724 [M + Na]+ (calcd for C15H23BrO2Na, 337.07736). Tristichone C (7): colorless oil; [α]24 D +39 (c 0.78, CHCl3); IR (KBr) νmax 2965, 1669, 775, and 558 cm−1; 13C and 1H NMR data, Tables 1 and 3; ESIMS m/z 355 [M + Na]+; HRESIMS m/z 355.04332 [M + Na]+ (calcd for C15H22BrClONa, 355.04348). Tristichone D (8): colorless oil; [α]25 D +56 (c 0.43, CHCl3); UV (MeOH) λmax (log ε) 242 (3.6) and 201 (3.3); IR (KBr) νmax 2963, 1669, 1078, and 532 cm−1; 13C and 1H NMR data, Tables 1 and 3; ESIMS m/z 351 [M + Na]+; HRESIMS m/z 351.09289 [M + Na]+ (calcd for C16H25BrONa, 351.09301). 4α-Hydroxybromocuparene (9): colorless oil; [α]23 D +16 (c 0.20, CHCl3); IR (KBr) νmax 3417, 2923, 1514, 1456, 1081, and 570 cm−1; 13 C and 1H NMR data, Tables 1 and 3; ESIMS m/z 319 [M + Na]+; HRESIMS m/z 319.06708 [M + Na]+ (calcd for C15H21BrO3Na, 319.06680). 9-(E)-Bromomethylidene-1,5,5-trimethylspiro[5.5]undeca-1,720 dien-3-one (10): colorless oil; [α]25 D +50 (c 0.45, CHCl3); lit. [α]D +69.7 (c 1.25, CHCl3).17 9-(Z)-Bromomethylidene-1,5,5-trimethylspiro[5.5]undeca-1,7dien-3-one (11): colorless oil; [α]25 D +61 (c 0.18, CHCl3); lit. [α]D +91 (c 1.17, CHCl3).18,19 Ma’ilione (12): colorless oil; [α]25 D −102 (c 0.20, CHCl3); lit. [α]D −100 (c 0.20, CHCl3).24 [1(15)Z,2Z,4S,8R,9S]-8,15-Dibromochamagra-1(15),2,11(12)trien-9-ol (13): colorless oil; [α]25 D −58 (c 2.98, CHCl3); lit. [α]D −40.0 (c 0.01).21 [1(15)E,2Z,4S,8R,9S]-8,15-Dibromochamagra-1(15),2,11(12)trien-9-ol (14): colorless oil; [α]24 D −4 (c 1.90, CHCl3); lit. [α]D −4 (c 1.90, CHCl3).18 Ma’iliohydrin (15): colorless oil; [α]25 D −9 (c 0.26, CHCl3); lit. [α]D −9.6 (c 0.26, CHCl3).22 25 Isorigidol (16): colorless oil; [α]25 D −12 (c 0.32, CHCl3); lit. [α]D 23 −12 (c 0.32, CH2Cl2). allo-Isoobtusol (17): colorless oil; [α]24 D −37 (c 1.50, CHCl3); lit. [α]D −33.8 (c 1.50, CHCl3).20 Majusculone (18): colorless oil; [α]25 D +109 (c 0.20, CHCl3); lit. 17 [α]19 D +145 (c 0.965, CHCl3). Cytotoxicity Testing. Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicities of compounds 1−18 were evaluated using the Alamar Blue assay.28,29 Doxorubicin, employed as a positive control, exhibited cytotoxic activity toward A549, LNCaP, and DLD-1 cancer cell lines, with IC50 values of 0.4 ± 0.2, 1.4 ± 0.3, and 2.6 ± 0.3 μM, respectively. Additional Bioassays. The preparation methods of human neutrophils, measurement of superoxide generation, measurement of elastase release, and statistical analysis protocols have been previously published.30 In Vitro Antibacterial Assay. The antibacterial activities of the isolated compounds were evaluated against Enterobacter aerogenes (ATCC13048), Serratia marcescens (ATCC25419), and Yersinia enterocolitica (ATCC23715), based on previous reports.31,32 Bacterial strains were grown in LB (Luria−Bertani) broth medium for 24 h at 37 °C. Then, 17 mL of LB hard agar (1.5% agar) was poured into sterile Petri dishes (90 mm) and allowed to set. When testing the bacterial sample, 1000 μL of inoculum suspension was poured into the molten LB soft agar plates using a sterile micropipet. After the temperature reached around 55−60 °C, the mixture was homogenized thoroughly by mixing in a circular motion (pour-plate technique). Sterile paper disks (Advantec, 8 mm in diameter) were placed onto the top layer of the LB agar plates. Test compounds (2 μg/μL) of different volumes were applied on each of the filter paper disks. Ampicillin (5

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00452. 1D NMR, 2D NMR, and ESIMS of compounds 1−9; Xray crystal structure and crystallographic analysis of 12 (PDF) X-ray data of compound 12 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +886-7-525-2000 (ext. 5030). Fax: +886-7-525-5020. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Science Council of Taiwan (MOST104-2113-M-110-006, 104-2811-M110-026, and 104-2320-B-110-001-MY2) awarded to J.-H.S.

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