Zoanthamine-Type Alkaloids from the Zoanthid Zoanthus kuroshio

J. Nat. Prod. , 2016, 79 (10), pp 2674–2680. DOI: 10.1021/acs.jnatprod.6b00625. Publication Date (Web): October 19, 2016. Copyright © 2016 The Amer...
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Zoanthamine-Type Alkaloids from the Zoanthid Zoanthus kuroshio Collected in Taiwan and Their Effects on Inflammation Yu-Ming Hsu,†,# Fang-Rong Chang,†,‡,∥,§,⊥,# I-Wen Lo,† Kuei-Hung Lai,†,▽ Mohamed El-Shazly,○ Tung-Ying Wu,□ Ying-Chi Du,† Tsong-Long Hwang,△,⬡,¶ Yuan-Bin Cheng,*,†,‡,■ and Yang-Chang Wu*,†,□,▼,●,▲ †

Graduate Institute of Natural Products, College of Pharmacy, ‡Center for Infectious Disease and Cancer Research, ∥Research Center for Environmental Medicine, and ■Research Center for Natural Products & Drug Development, Kaohsiung Medical University, Kaohsiung 80708, Taiwan § Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan ⊥ Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan ▽ Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, 75105 Uppsala, Sweden ○ Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Cairo 11566, Egypt □ Chinese Medicine Research and Development Center and ▼Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan △ Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan ⬡ Research Center for Industry of Human Ecology, Research Center for Chinese Herbal Medicine, and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan 33302, Taiwan ¶ Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 33302, Taiwan ● School of Pharmacy, College of Pharmacy, and ▲Research Center for Chinese Herbal Medicine, China Medical University, Taichung 40402, Taiwan S Supporting Information *

ABSTRACT: Zoanthus kuroshio is a colorful zoanthid with a fluorescent pink oral disc and brown tentacles, which dominates certain parts of the Taiwanese and Japanese coasts. This sea anemone is a rich source of biologically active alkaloids. In the current investigation, two novel halogenated zoanthamines [5αiodozoanthenamine (1) and 11β-chloro-11-deoxykuroshine A (2)], along with four new zoanthamines [18-epi-kuroshine A (3), 7α-hydroxykuroshine E (4), 5α-methoxykuroshine E (5), and 18-epi-kuroshine E (6)], and six known compounds were isolated from Z. kuroshio. Compounds 1 and 2 are the first examples of halogenated zoanthamine-type alkaloids isolated from nature. Compounds 3 and 6 are the first zoanthamine stereoisomers with a cis-junction of the A/B rings. All isolated compounds were evaluated for their anti-inflammatory activities by measuring their effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP.

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oanthamine-type alkaloids, isolated from Zoanthus sea anemone species, belong to a specific family of marine alkaloids.1 The framework of zoanthamine-type alkaloids is recognized by its densely functionalized fragments, an 8-oxa-6azabicyclo[3.2.1] ring and a β-methyl enone functionality, within the octacyclic ring system. These zoanthamines are divided into two structural groups depending on the presence of a methyl group at C-19 (type I) or not (type II).2 Type I structures are further categorized into two subgroups, a group with a six-membered lactone ring constructed by the connection of C9−C10−C22−C23-C24 (Ia) and another group with a five-membered lactone ring formed by the connection of C22−C23−C24−C25 (Ib). So far, only 28 zoanthamines have been reported since the first isolate was identified.3 The densely functionalized structures of the zoanthamines along with their © 2016 American Chemical Society and American Society of Pharmacognosy

multiple stereogenic centers hindered their synthesis for almost 20 years after their first isolation.4 The main attractiveness of zoanthamine-type alkaloids is not only the unique skeleton but also their potential pharmacological properties. The pharmacological activities of zoanthamines have been studied extensively, demonstrating antiplatelet aggregation,5 antiosteoporotic,6,7 anti-inflammatory,8 antibacterial,9 and cytotoxic activities.10 However, the mechanisms of action for most zoanthamines remain undefined except for norzoanthamine, a promising antiosteoporotic agent, Received: July 7, 2016 Published: October 19, 2016 2674

DOI: 10.1021/acs.jnatprod.6b00625 J. Nat. Prod. 2016, 79, 2674−2680

Journal of Natural Products

Article

NMR fingerprints encouraged us to combine the extracts of batches 1 and 2, hoping to isolate more zoanthamine-type alkaloids with the Ib-type skeleton. Thus, the combined extracts were partitioned between EtOAc and H2O to yield two layers. The organic layer was further partitioned between n-hexane and 80% aqueous MeOH. The aqueous methanolic extract was subjected to column chromatography using Sephadex LH-20 and Si-60 silica gel, as well as reversed-phase HPLC, to yield six new (1−6) and six known (7−12) zoanthamines. Compound 1 was obtained as a white, amorphous powder, and its molecular formula (C30H38INO6) was confirmed by the analysis of its 13C NMR and HRESIMS data (m/z 658.1637 [M + Na]+). The IR spectrum showed hydroxy (3419 cm−1), γlactone (1769 cm−1), and α,β-unsaturated ketone (1714 cm−1) vibration bands. Together with the maximum UV absorption at 237 nm, compound 1 was suggested to be a zoanthamine-type alkaloid.3 Furthermore, the detailed analysis of the characteristic NMR signals indicated that 1 was a derivative of zoanthenamine (10) (Tables 1 and 2).8 The only differences were the methylene signal of 10 at C-5 was replaced by a methine and the carbon signal was deshielded from δC 44.5 to 54.1. Regarding the molecular formula, a fragment peak at m/z 509 [M − I + H]+ was observed in the ESIMS spectra, suggesting the presence of an iodine atom. The connection of the iodine atom to C-5 was suggested by the correlations between H-1/H-2/H-3 in the COSY spectrum and H-5/C-30 and H3-30/C-3, C-4, and C-5 in the HMBC spectrum. The remaining planar structure of 1 was confirmed by the key COSY and HMBC correlations (Figure 1). The relative configuration of 1 was assigned on the basis of a NOESY experiment, and the NOESY correlations (Figure 1) were compared to those of 3β-hydroxyzoanthenamide (11).12 The same NOESY correlations between H-21/H-13, H-25a, H3-26, and H3-29 in 1 and 11 indicated that they were located on the β-face. Simultaneously, the cross-peaks between H-18/ H-19 and H-28b supported the α-orientation of these protons. In addition, the strong NOESY correlations between H3-29/H7b and H-5/H-7a and H3-30 as well as the coupling constant between H-4 and H-5 (7.2 Hz) suggested a diaxial arrangement of these hydrogens, which revealed the orientation of the iodine atom at C-5 as α. Thus, the structure of 1 was elucidated and named 5α-iodozoanthenamine. Compound 2 was purified as a white, amorphous powder. It was found to possess a chloride atom as inferred from the ESIMS protonated molecule peaks [M + H]+ at m/z 543 and 545 in a 3:1 ratio, which are characteristic peaks of a monochlorinated compound. The exact masses of [M + Na]+ at m/z 566.2278 and 568.2249 matched the molecular formulas of C30H3835ClNO6Na and C30H3837ClNO6Na, respectively, which were confirmed with the 13C NMR data. The absorption bands of the IR spectrum, maximum UV absorption, and the characteristic NMR signals indicated that 2 was a chlorinated derivative of kuroshine A (7) (Tables 1 and 2).11 However, the shielded signal of C-11 from δC 73.1 to 62.2 and the HMBC correlations between H-11/C-10, C-12, C-21, and C-28, H-21/ C-12 and C-22, and H-29/C-9 and C-10 revealed that the hydroxy group at C-11 was replaced by a chlorine atom (Figure 2). The NOE correlations between H-18/H-28a and H-28b, H11/H-1a, H-1b, H-14eq, and H-28b, and H-13/H-14eq suggested the β-orientation of the chloride substitution at C11. The other stereochemical details were confirmed by similar cross-peaks shown in the NOESY spectra of 2 and 7 (Figure 2). The absolute configuration of 2 was established as 2R, 4S, 6S,

which suppressed the loss of bone weight through the inhibition of IL-6 secretion.6 In previous studies, most of the isolated zoanthamine-type alkaloids were found to belong to a type Ia or type II skeleton.2 However, all of the nine zoanthamine-type alkaloids (kuroshines A−G, 3β-hydroxyzoanthenamide, and 7α-hydroxyzoanthenamide) isolated from Zoanthus kuroshio by our group possessed the less common Ib-type skeleton.11,12 This unique structural feature encouraged us to continue investigating other zoanthamine-type alkaloids from Z. kuroshio. In the current study, we isolated 12 zoanthamines, including six new alkaloids [5α-iodozoanthenamine (1), 11β-chloro-11-deoxykuroshine A (2), 18-epi-kuroshine A (3), 7α-hydroxykuroshine E (4), 5αmethoxykuroshine E (5), and 18-epi-kuroshine E (6)] and six known components [kuroshines A, D, and E (7, 8, and 9),11,12 zoanthenamine (10),8 3β-hydroxyzoanthenamide (11),12 and 28-deoxyzoanthenamide (12)5]. Among those isolates, 5αiodozoanthenamine (1) and 11β-chloro-11-deoxykuroshine A (2) are the first halogenated examples of zoanthamines isolated from nature. In addition, 18-epi-kuroshine A (3) and 18-epikuroshine E (6) are two 18-epimeric zoanthamines with a cisjunction of the A/B rings, which are also reported for the first time. In the current study, the isolation, purification, structure elucidation, and anti-inflammatory activities of zoanthaminetype alkaloids from Z. kuroshio were carried out.

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RESULTS AND DISCUSSION The ethanolic extracts of the freeze-dried zoanthid materials (Z. kuroshio) collected in Kaohsiung City (batch 1) and Taitung County (batch 2) were analyzed by NMR to identify if their major secondary metabolites belong to the rare Ib-type skeleton. The results showed similar NMR fingerprints, with the characteristic signals of the five-membered lactone ring of type Ib at δH 4.00−5.00 ppm (Figures S1 and S2). The small quantity obtained in each collection along with the similar 2675

DOI: 10.1021/acs.jnatprod.6b00625 J. Nat. Prod. 2016, 79, 2674−2680

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Table 1. 1H NMR Data of Compounds 1−6 in C5D5Na no. 1a 1b 2 3a 3b 4 5a 5b 7a 7b 8a 8b 11 13 14a 14b 16 18 19 21 23a 23b 25a 25b 26 27 28a 28b 29 30 OCH3 a

1 3.15, 3.43, 4.55, 1.53, 1.59, 1.33, 3.93,

t (5.6) d (5.6) m m m m d (7.2)

2.35, 2.46, 1.64, 2.92, 4.41, 2.23, 2.06, 2.25, 6.04, 2.83, 2.65, 2.46, 3.27, 4.22, 4.46, 5.51, 1.42, 1.92, 3.74, 4.14, 1.25, 1.02,

t (7.6) d (8.8) m td (10.0, 4.0) s s ddd (14.8, 4.8, 1.8) ddd (24.0, 12.0, 6.0) s dd (8.8, 3.6) t (4.8) s d (12.0) d (12.0) d (6.4) d (6.4) d (4.8) s d (5.6) d (5.6) s d (4.4)

2

3

4

5

3.11, 3.39, 4.52, 1.38,

d (6.4) t (6.4) m m

3.32, 3.65, 4.51, 1.46,

d (6.8) t (6.8) m m

2.86, 3.80, 4.57, 1.47,

dd (4.8, 0.4) t (4.8) m m

2.73, 3.96, 4.49, 1.49,

2.13, 1.09, 2.24, 1.66, 1.87, 1.24, 2.28, 4.63, 3.02, 2.61,

m t (12.0) dd (12.0, 5.2) dt (12.0, 3.2) td (13.2, 3.2) m td (13.4, 3.2) s m m

2.21, 1.13, 2.31, 1.70, 1.96, 1.23, 2.29, 4.47, 2.68, 2.11,

m t (11.6) m dt (12.0, 3.2) td (13.2, 3.2) dt (12.8, 3.2) m d (4.8) m m

2.35, 2.10, 2.21, 4.27,

m t (9.2) dd (9.2, 3.2) d (8.0)

2.21, m 2.89, d (10.2)

6.07, 2.64, 3.52, 3.43, 3.58,

s d (9.6) m d (1.2) s

4.43, 4.51, 1.21, 1.92, 4.12, 4.27, 1.34, 0.84,

d d d s d d s d

5.91, 2.59, 3.31, 4.01, 3.54, 3.98, 4.17, 4.56, 1.30, 1.78, 3.83,

s d (13.6) m s d (18.4) d (18.4) d (10.0) d (10.0) d, (7.6) s q (9.2)

(9.6) (9.6) (7.2) (8.8) (8.8) (6.4)

1.50, s 0.90, d (3.2)

2.02, dd (12.6, 4.8) 2.67, t (13.2) 3.28, 2.53, 2.71, 6.02, 2.66, 3.47, 3.63, 3.83, 3.92, 4.26, 4.52, 1.24, 1.89, 4.51, 4.64, 1.20, 0.89,

ddd (14.1, 11.7, 4.8) dd (18.0, 12.9) dd (18.0, 4.2) s dd (14.4, 4.8) dd (7.8, 4.8) d (0.8) d (12.8) d (12.8) d (6.8) d (6.8) d (4.8) s d (6.0) d (6.0) s d (4.4)

6

d (6.6) t (6.6) m m

1.83, 2.01, 1.46, 2.97,

m dt (11.2, 3.6) m td (12.9, 3.6)

3.24, 2.47, 2.71, 6.01, 2.60, 3.44, 3.60, 3.87, 4.09, 4.28, 4.42, 1.23, 1.87, 4.39, 4.60, 1.09, 0.96, 3.42,

ddd (14.1, 11.7, 4.8) m dd (18.0, 4.8) s dd (13.2, 4.2) dd (7.2, 4.2) d (1.2) d (19.2) d (19.2) d (10.2) d (10.2) d (7.2) s d (9.6) dd (9.6, 1.2) s d (6.6) s

2.76, 3.73, 4.78, 1.37,

d (7.2) t (7.2) m m

2.12, 1.10, 2.16, 1.68, 1.80, 1.36, 2.25,

m d (6.0) t (6.0) dt (12.6, 3.6) td (12.0, 3.6) m td (16.2, 4.2)

3.53, 2.30, 2.64, 6.00, 2.94, 3.40, 3.52, 3.64,

m dd (19.8, 4.8) dd (14.4, 4.8) s t (4.2) dd (7.8, 4.2) s d (3.0)

4.13, 4.38, 1.27, 1.75, 4.03, 4.05, 1.07, 0.81,

d d d s d d s d

(10.2) (10.2) (7.8) (14.0) (14.0) (6.0)

1, 4, 5, and 6 were measured at 600 MHz NMR; 2 and 3 were measured at 400 MHz NMR.

9S, 10S, 11R, 12S, 13R, 15Z, 18R, 19R, 21R, 22R by comparing the specific rotation data of 2 with those of 7, the absolute configuration of which was determined by X-ray singlecrystallographic analysis using Cu Kα radiation in our previous study.11 Therefore, the structure of 2 was determined, and the trivial name 11β-chloro-11-deoxykuroshine A was given. Compound 3 was also obtained as a white, amorphous powder. The molecular formula (C30H39NO7) was deduced from HRESIMS and 13C NMR data, indicating that 3 possessed the same molecular formula as 7.11 Furthermore, similar signals in the 1D NMR spectra (Tables 1 and 2) and close correlations in the COSY and HMBC experiments between 3 and 7 (Figure 2) indicated that they both possessed the same planar structure. Originally, we assumed that 3 and 7 are one compound; however, they were separated into two peaks in the HPLC chromatogram. Through comparing the NOESY spectra of 3 and 7, we found that the only difference between these compounds was the orientation of H-18. Significant NOESY correlations between H-18/H-13 and H3-26 and H-21/H-25a, H3-26, and H3-29 were observed in 3, which were different from those in 7 (correlations between H-21/H-13, H-25, H326, and H3-29 and H-18/H-19), indicating a cis-junction of the A/B rings in 3 (Figure 2). Therefore, the orientation of H-18 in 3 was assigned as β rather than α as in 7. On the basis of the above-mentioned NOESY correlations and by comparing the specific rotation data with those of 7, the absolute configuration of 3 was determined as 2R, 4S, 6S, 9S, 10S, 11R, 12S, 13R, 15Z,

18S, 19R, 21R, 22R. Thus, compound 3 was identified as an epimer of 7 and named 18-epi-kuroshine A. Compound 4 was isolated as a white, amorphous powder, and its molecular formula (C30H37NO8) was confirmed by the analysis of its 13C NMR, DEPT, and HRESIMS data. Compound 4 was proposed to be a hydroxylated derivative of kuroshine E (9) by the closely similar NMR signals (Tables 1 and 2) and the replacement of a methylene group (δH 1.71; δC 30.0) in 9 by an oxymethine (δH 4.27; δC 69.0) in 4, which showed 16 mass units more than 9 in the HRESIMS spectrum.12 The hydroxy group at C-7 in 4 was confirmed by the COSY correlations between H-7/H2-8 and the HMBC correlations between H-7/C-6, H2-8/C-7 and C-9, and H3-29/ C-8 (Figure 3). The orientation of OH-7 was assigned as α based on the NOESY correlations between the β-oriented H8a/H-7, H-25b, and H3-29 and H-8b/H-23b (Figure 3). Thus, 4 was assigned as 7α-hydroxykuroshine E. Compound 5 was suggested to be another analogue of 9 based on the spectroscopic data. HRESIMS and 13C NMR data of 5 suggested the molecular formula C31H39NO8Na. The assignments of the 1H and 13C NMR signals, which were assisted by extended 2D NMR methods, indicated that 5 is an analogue of kuroshine E (9)12 bearing one secondary methoxy group (δH 3.42; δC 61.3) (Tables 1 and 2). The methoxy group was confirmed at C-5 by the key COSY [H-3/H-4/H-5 and H330/H-4] and HMBC [OCH3/C-5] correlations, and the αorientation was supported by the NOESY correlations between H3-29/H-21 and H-7a and between H-5/H-7b and H3-30 2676

DOI: 10.1021/acs.jnatprod.6b00625 J. Nat. Prod. 2016, 79, 2674−2680

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Table 2. 13C NMR Data of Compounds 1−6 in C5D5Na no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCH3 a

1 58.1, 73.4, 39.2, 35.7, 54.1, 89.7, 32.6, 26.7, 45.5, 148.5, 96.2, 30.0, 45.0, 31.0, 159.9, 127.2, 199.8, 48.7, 44.4, 112.1, 44.2, 41.3, 34.8, 178.5, 72.3, 13.3, 24.1, 73.2, 24.2, 22.4,

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

46.3, 73.7, 39.1, 23.3, 44.4, 90.5, 30.0, 28.8, 44.7, 96.8, 62.2, 52.0, 32.9, 28.9, 161.5, 126.9, 196.5, 45.8, 46.3, 212.4, 49.6, 47.6, 35.2, 178.6, 74.4, 13.4, 24.4, 66.0, 25.8, 24.4,

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

47.0, 73.9, 39.5, 23.5, 44.8, 90.6, 30.5, 28.0, 46.8, 96.4, 81.1, 50.4, 39.1, 30.2, 161.5, 124.9, 197.5, 44.5, 50.3, 215.6, 45.3, 44.8, 34.5, 178.9, 74.0, 16.4, 23.8, 66.6, 23.9, 22.2,

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

47.2, 74.5, 39.2, 23.0, 37.4, 94.4, 69.0, 35.8, 47.4, 94.6, 206.1, 56.1, 32.1, 30.9, 162.0, 126.7, 196.2, 47.0, 45.8, 210.2, 57.5, 51.6, 34.8, 178.1, 74.0, 13.5, 24.3, 65.7, 22.2, 22.5,

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

47.2, 73.9, 39.3, 31.2, 94.3, 90.3, 28.9, 26.8, 47.4, 94.3, 206.6, 56.2, 32.2, 30.8, 162.0, 126.7, 196.3, 46.9, 45.8, 210.3, 57.2, 48.9, 34.3, 178.7, 73.7, 13.5, 24.2, 65.2, 21.5, 18.3, 61.3,

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

46.8, 74.2, 39.0, 23.2, 43.6, 90.0, 30.0, 26.4, 46.8, 94.6, 206.1, 56.1, 31.4, 31.9, 160.6, 126.4, 196.7, 49.6, 43.3, 210.3, 56.3, 49.9, 34.4, 178.0, 73.8, 17.0, 23.5, 66.7, 20.6, 22.1,

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

1, 4, 5, and 6 were measured at 150 MHz NMR; 2 and 3 were measured at 100 MHz NMR.

Figure 1. Key COSY (heavy black lines), HMBC (blue arrows), and NOESY (red arrows) correlations of 1.

(Figure 3). Thus, the structure of 5 was determined as 5αmethoxykuroshine E. Compound 6 was isolated as a white, amorphous powder. It was also found to be an isomer of kuroshine E (9)12 showing the same molecular formula of C30H37NO7 from HRESIMS and 13C NMR data. Through comparing the COSY and HMBC correlations in 6 and 9, it was found that both compounds possessed the same planar structure (Figure 3). The stereoconfiguration of 6 was almost the same as that of 9 except at C18. The NOESY correlations between H-13/H-18 and H3-26 and between H-21/H-25a, H3-26, and H3-29 indicated that H18 in 6 was β-oriented (Figure 3). The structure of 6 was determined as 18-epi-kuroshine E. In our previous study on zoanthamine-type alkaloids, kuroshine A (7) was the only compound to be studied by X-

Figure 2. Key COSY (heavy black lines), HMBC (blue arrows), and NOESY (red arrows) correlations of 2 and 3.

ray single-crystallographic analysis using Cu Kα radiation, and its absolute configuration was established based on this analysis.11 In the current study, the isolated zoanthamine-type alkaloids possessed very similar structures and were isolated from the same organism, and thus it was proposed to share a similar biogenetic pathway. On the basis of this proposal, compounds 4−6 were suggested to possess the same absolute 2677

DOI: 10.1021/acs.jnatprod.6b00625 J. Nat. Prod. 2016, 79, 2674−2680

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Table 3. Inhibitory Effects of Z. kuroshio Isolates on Superoxide Anion Generation and Elastase Release in FMLP/CB-Induced Human Neutrophils percentage of inhibition (Inh%)a compound

superoxide anion

elastase release

5α-iodozoanthenamine (1) 11β-chloro-11-deoxykuroshine A (2) 18-epi-kuroshine A (3) 7α-hydroxykuroshine E (4) 5α-methoxykuroshine E (5) 18-epi-kuroshine E (6) kuroshine A (7) kuroshine D (8) kuroshine E (9) zoanthenamine (10) 3β-hydroxyzoanthenamide (11) 28-deoxyzoanthenamide (12) LY294002b

24 ± 6** 23 ± 6* 15 ± 6 13 ± 3* 20 ± 5* 14 ± 5* 9±4 11 ± 3* 7±3 17 ± 4* 14 ± 5* 18 ± 6* 99 ± 1***

43 ± 2*** 25 ± 3** 13 ± 7 15 ± 4* 33 ± 7** 23 ± 6** 6 ± 2* 27 ± 5** 27 ± 7* 17 ± 5* 25 ± 6* 30 ± 6** 73 ± 1***

Percentage of inhibition (Inh%) at 10 μM concentration. Results are presented as mean ± SEM (n = 3, 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control value. bLY294002 was used as the positive control. a

six known zoanthamines. The halogenated zoanthamines (1 and 2) and 18-epi-zoanthamines (3 and 6) were isolated for the first time from natural sources. The anti-inflammatory activities of 1−12 were evaluated by superoxide anion and elastase release assays. Among the tested compounds, the iodinated zoanthamine 1 showed the most significant anti-inflammatory effect on inhibiting superoxide anion generation and elastase release at 10 μM (24 ± 6% and 43 ± 2%, respectively).

Figure 3. Key COSY (heavy black lines), HMBC (blue arrows), and NOESY (red arrows) correlations of 4−6.

configuration as 7. Thus, the absolute configurations for the main skeleton of compounds 4 and 5 were proposed as 2R, 4S, 6S, 9S, 10S, 12S, 13R, 15Z, 18R, 19R, 21R, 22R, and those at C7 of 4 and C-5 of 5 were deduced as S and R, respectively. Furthermore, the absolute configuration of 6, the 18-epimer of 7, was suggested as 2R, 4S, 6S, 9S, 10S, 12S, 13R, 15Z, 18S, 19R, 21R, 22R. Anti-inflammatory Effects of Zoanthamines 1−12 on Superoxide Anion and Elastase Release Assays. All isolated compounds in this study were evaluated for their anti-inflammatory activities by measuring their effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP (Table 3). LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive control. Among the tested compounds, 1 showed the most potent inhibitory activity on both superoxide anion generation (24 ± 6%) and elastase release (43 ± 2%). In addition, most of the tested zoanthamines, except for 2, 3, 4, 7, and 10, showed greater inhibition of elastase release. Interestingly, compound 1, the 5-iodo-substituted derivatives of 10, showed more potent inhibitory activity on elastase release than 10. Also, the inhibitory activity on elastase release of 2, the chlorosubstituted derivatives of 7, was 3-fold more than 7. According to literature reviews, halogenated derivatives of marine natural products usually possess more potent effects on different bioassays compared with the nonhalogenated analogues.13 A similar phenomenon was also observed in our anti-inflammatory screening assay. Thus, the role of halogen atoms in promoting the anti-inflammatory activity of the zoanthamines is worthy of further detailed investigation. Several factors should be considered upon studying the effect of the halogen atoms on the activity of zoanthamines such as halogen position and orientation and electrostatic, steric, and lipophilic effects. In this study, six new zoanthamine-type alkaloids were isolated from the Taiwanese zoanthid Z. kuroshio, together with

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter, whereas IR spectra were obtained on a JASCO FT/IR-4600 spectrometer. 1H and 13C NMR spectra were recorded on Varian VNMR-600 instruments (600 and 150 MHz for 1H and 13C, respectively) and Varian INOVA 400 spectrometers (400 and 100 MHz for 1H and 13C, respectively). All NMR experiments were performed at room temperature, using C5D5N or CDCl3 as the solvent. Chemical shifts were referenced to residual solvent signals for C5D5N (δH 7.21 and δC 123.5) and CDCl3 (δH 7.26 and δC 77.0 ppm). LRESIMS were measured on a VG Biotech Quattro 5022 mass spectrometer. HRESIMS data were collected using a Bruker Daltonics APEX II mass spectrometer. Silica gel 60 (Merck, 70−230 and 230−400 mesh) and Sephadex LH-20 were used for column chromatography (CC), while TLC analyses were carried out on silica gel F254 and RP-18 F254s precoated plates, and the spots were detected using 50%(aq) H2SO4 and Dragendorff’s reagent followed by heating on a hot plate. HPLC analyses were performed with Shimadzu SPDM10A DAD and Shimadzu SPD-20A UV instruments. Animal Material. Specimens of Zoanthus kuroshio were collected in Kaohsiung City (September 2012) and Taitung County (July 2014), Taiwan. The animal material was identified by Dr. Wei-Chun Liu. A voucher specimen (code no. KMU-Z7-1) was deposited in the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Separation and Isolation of Compounds 1−12. The freezedried animal materials of Z. kuroshio were extracted with 95% EtOH at room temperature three times. The collected extracts were combined and concentrated under reduced pressure to obtain the ethanolic extracts. The concentrated EtOH extract (23.9 g) was partitioned between EtOAc and H2O to yield two layers. The EtOAc layer was then partitioned between n-hexane and MeOH/H2O (8:2) layers. The MeOH/H2O extract (3.4 g) was subjected to Sephadex LH-20 CC, 2678

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eluting with MeOH, to yield five subfractions (Fr. S1 to S5). After checking with Dragendorff’s reagent, fraction S2 (1.5 g), the alkaloidrich fraction, was further chromatographed on Si-60 silica gel using CH2Cl2/MeOH (30:1) to obtain six fractions (Fr. S2-1 to S2-6). Fraction S2-1 (75.6 mg) was purified by reversed-phase HPLC (Phenomenex C18, 10 mm × 250 mm, flow rate = 2 mL/min, 75% MeOH/H2O, isocratic elution), and four subfractions were collected (Fr. S2-1-H1 to S2-6-H4). Compound 9 (48.2 mg) was obtained from the subfraction S2-1-H1. Subfraction S2-1-H4 (3.4 mg) was further purified by reversed-phase HPLC (Phenomenex C18, 10 mm × 250 mm, flow rate = 2 mL/min, 70% MeOH/H2O, isocratic elution) to afford 2 (2.1 mg). Compound 9 (105.5 mg) also precipitated from Fr. S2-2 (575.9 mg). The rest of Fr. S2-2 was subjected to SPE, eluting with a stepped gradient of MeOH/H2O (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0; 3 × 100 mL fractions were collected for each solvent system), and six subfractions were obtained (Fr. S2-2-E1 to S2-2-E2). Fraction S2-2-E1 (83.7 mg) was purified by reversed-phase HPLC (Phenomenex C18, 10 mm × 250 mm, flow rate = 2 mL/min, 50% CH3CN/H2O, isocratic elution) to afford 5 (2.6 mg) and 6 (2.8 mg). Fraction S2-3 (424.6 mg) was purified by reversed-phase HPLC (Phenomenex phenyl-hexyl, 10 mm × 250 mm, flow rate = 2 mL/min, 80% MeOH/H2O, with 0.05% NH4OH, isocratic elution), and five fractions (Fr. S2-3-H1 to S2-3-H5) were obtained. From Fr. S2-3-H2, S2-3-H3, and S2-3-H4, compounds 10 (227.1 mg), 12 (3.0 mg), and 1 (3.4 mg) were purified, respectively. Fraction S2-3-H1 (79.3 mg) was further purified by reversed-phase HPLC (Phenomenex C18, 10 mm × 250 mm, flow rate = 2 mL/min, 60% MeOH/H2O, isocratic elution), and compounds 3 (11.6 mg) and 7 (36.4 mg) were obtained. Fraction S2-4 (275.2 mg) was subjected to SPE, eluting with a stepped gradient of CH3CN/H2O (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0; 3 × 100 mL fractions were collected for each solvent system), to afford six subfractions (Fr. S2-4-E1 to S24-E6). Fraction S2-4-E3 (64.4 mg) was then purified by reversed-phase HPLC (Phenomenex phenyl-hexyl, 10 mm × 250 mm, flow rate = 2 mL/min, 33% CH3CN/H2O with 0.05% NH4OH, isocratic elution), and 10 fractions (Fr. S2-4-E3-H1 to S2-4-E3-H10) were obtained. Among these fractions, S2-4-E3-H3 and S2-4-E3-H6 were pure compounds 6 (1.9 mg) and 11 (1.4 mg), respectively. Fraction S2-4E3-H9 (6.2 mg) was further purified by reversed-phase HPLC (Phenomenex C18, 10 mm × 250 mm, flow rate = 2 mL/min, 40% CH3CN/H2O, isocratic elution) to provide 4 (2.6 mg). 5α-Iodozoanthenamine (1): amorphous white powder; [α]25 D −78 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 237 (4.3) nm; IR (neat) νmax 3419, 2925, 2853, 1769, 1714, 1696, 1658, 1590, 1457, 1394 cm−1; 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz) data, Tables 1 and 2; ESIMS m/z 636 [M + H]+; HRESIMS m/ z 658.1637 [M + Na]+ (calcd for C30H38INO6Na, 658.1636). 11β-Chloro-11-deoxykuroshine A (2): amorphous, white powder; [α]25 D −150 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 238 (4.0) nm; IR (neat) νmax 3418, 2893, 1771, 1706, 1658, 1588, 1382, 1268, 1206 cm−1; 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data, Tables 1 and 2; ESIMS m/z 544 [M + H]+; HRESIMS m/ z 566.2278 [M + Na]+ (calcd for C30H3835ClNO6Na, 566.2280). 18-epi-Kuroshine A (3): amorphous, white powder; [α]25 D −110 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 239 (3.8) nm; IR (neat) νmax 3430, 2926, 1756, 1698, 1658, 1592, 1430, 1369, 1263, 1206, 1149 cm−1; 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data, Tables 1 and 2; ESIMS m/z 526 [M + H]+; HRESIMS m/ z 548.2618 [M + Na]+ (calcd for C30H39NO7Na, 548.2619). 7α-Hydroxykuroshine E (4): amorphous, white powder; [α]25 D −8.4 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 236 (3.8) nm; IR (neat) νmax 3431, 2954, 1766, 1714, 1655, 1588, 1382, 1347, 1317, 1258, 1181 cm−1; 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz) data, Tables 1 and 2; ESIMS m/z 540 [M + H]+; HRESIMS m/ z 562.2410 [M + Na]+ (calcd for C30H37NO8Na, 562.2411). 5α-Methoxykuroshine E (5): amorphous, white powder; [α]D25 −110 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 237 (4.1) nm; IR (neat) νmax 3432, 2942, 1766, 1712, 1656, 1594, 1380, 1314, 1258, 1186, 1094 cm−1; 1H NMR (C5D5N, 600 MHz) and 13C NMR

(C5D5N, 150 MHz) data, Tables 1 and 2; ESIMS m/z 554 [M + H]+; HRESIMS m/z 576.2566 [M + Na]+ (calcd for C31H39NO8Na, 576.2568). 18-epi-Kuroshine E (6): amorphous, white powder; [α]25 D −120 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 237 (4.1) nm; IR (neat) νmax 3397, 2954, 1768, 1712, 1658, 1429, 1373, 1319, 1268, 1211, 1144, 1101 cm−1; 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz) data, Tables 1 and 2; ESIMS m/z 524 [M + H]+; HRESIMS m/ z 546.2462 [M + Na]+ (calcd for C30H37NO7Na, 546.2462). Kuroshine A (7): [α]25 D −40 (c 0.3, MeOH), which was reported in 11 our previous study as [α]25 D −35 (c 0.5, MeOH)]. Superoxide Anion and Elastase Release Assays. Human neutrophils were prepared from healthy human donors (20−35 years old) by venipuncture, using a protocol approved by the institutional review board at Chang Gung Memorial Hospital (102-1595A3). Neutrophils were isolated using a standard method as previously described.14 Measurement of Superoxide Generation. Inhibition ratio (%) of superoxide dismutase (SOD) was measured by the reduction of ferricytochrome c as previously described.14 Neutrophils were equilibrated in 0.5 mg/mL ferricytochrome c and 1 mM Ca2+ ion solution at 37 °C for 2 min and then incubated with the test compounds for 5 min. Cells were activated with formyl-methionylleucyl-phenylalanice (fMLP, 100 nM)/cytochalasin B (CB, 1 μg/mL) for 10 min. The absorbance was continuously monitored at 550 nm using a Hitachi U-3010 double-beam six-cell positioned spectrophotometer with constant stirring. Calculations were based on the differences in absorbance with and without SOD (100 U/mL), and the extinction coefficient for reduction of ferricytochrome c to ferrocytochrome c (ε = 21.1/mM/10 mm) was used. Measurement of Elastase Release. Elastase release was measured by the degranulation of azurophilic granules.14 Neutrophils were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), an elastase substrate, at 37 °C for 2 min and then incubated with the test compounds for 5 min. Cells were activated by 100 nM fMLP and 0.5 μg/mL CB, and changes in the absorbance at 405 nm were continuously monitored to detect the elastase release. The results are expressed as the percent of the initial rate of elastase release in the fMLP/CB-activated, drug-free control system.

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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.6b00625. 1 H NMR spectra for the extracts of batches 1 and 2; 1D and 2D NMR and HRESIMS spectra of compounds 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +886-7-3121101, ext. 2162. Fax: +886-7-3114773. Email: [email protected] (Y.-B. Cheng). *Tel: +886-4-22057153. Fax: +886-4-22060248. E-mail: [email protected] (Y.-C. Wu). Author Contributions #

Y.-M. Hsu and F.-R. Chang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support for the projects from the Ministry of Science and Technology of Taiwan (NSC 102-2628-B-037-003-MY3, MOST 103-2320-B-037-005-MY2, awarded to F.-R.C., MOST103-2628-B-037-001-MY3 award to 2679

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Y.-B.C., and MOST 105-2911-I-002-302, 103-2911-I-002-303, 103-2325-B-039-008, 103-2325-B-039-007-CC1, and 102-2320B-037-012-MY2, awarded to Y.-C.W.). This study is also partially supported by Kaohsiung Medical University (Aim for the Top Universities Grant, grant no. KMU-TP104E39, KMUTP104A26), Ministry of Health and Welfare of Taiwan (MOHW105-TDU-B-212-134007), and Health and Welfare Surcharge of Tobacco Products. This work was also in-kind supported by grants from the National Health Research Institutes (NHRI-EX103-10241BI) and a grant from the Chinese Medicine Research Center, China Medical University (Ministry of Education, Aim for the Top University Plan). HRESIMS was supported by the Joint Center for High Valued Instrument, National Sun Yat-sen University; 400 and 600 MHz NMR as well as ESIMS were supported by Center for Research Resources and Development, Kaohsiung Medical University.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00625 J. Nat. Prod. 2016, 79, 2674−2680